WO2002031209A2 - Genes related to development of refractory prostate cancer - Google Patents

Genes related to development of refractory prostate cancer Download PDF

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Publication number
WO2002031209A2
WO2002031209A2 PCT/US2001/031932 US0131932W WO0231209A2 WO 2002031209 A2 WO2002031209 A2 WO 2002031209A2 US 0131932 W US0131932 W US 0131932W WO 0231209 A2 WO0231209 A2 WO 0231209A2
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hrpc
protein
molecule
nucleic acid
prostate cancer
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PCT/US2001/031932
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French (fr)
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WO2002031209A3 (en
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Spyro Mousses
Olli P. Kallioniemi
Lukas Bubendorf
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The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services
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Priority to AU2002214576A priority Critical patent/AU2002214576A1/en
Publication of WO2002031209A2 publication Critical patent/WO2002031209A2/en
Publication of WO2002031209A3 publication Critical patent/WO2002031209A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present disclosure is generally related to diagnosing, prognosing, staging, preventing, and treating disease, particularly hormone refractory prostate cancer.
  • prostate cancer like normal prostate, is often androgen dependent and androgen withdrawal induces growth regression.
  • Various modes of androgen ablation therapy have since been used as the major treatment of advanced prostate cancer.
  • chemical or physical reduction in serum androgens, or chemical blockage of their action effectively arrests growth of local and metastatic prostate cancer in vivo.
  • the regressed tumors eventually develop resistance to hormonal therapy and recur as aggressive androgen independent tumors that are hormone refractory and currently incurable.
  • CWR22 is an androgen dependent human prostate carcinoma that grows rapidly as a xenograft in male nude mice, regresses after castration, and eventually (in three to ten months) becomes recurrent and re-grows independently of androgens in castrated mice.
  • Several groups have previously looked for differences in gene expression between the primary and recurrent CWR22 prostate cancer xenografts, and identified some candidate genes that can be used as biomarkers. It remains critical to make sure that findings from model systems are applicable in the clinical situation.
  • Embodiments of this disclosure provide a set of nucleic acid molecules the expression of which is altered in prostate cancer, more particularly nucleic acid molecules that show temporal expression changes during prostate cancer hormonal therapy and regression.
  • HPRC hormone-refractory prostate cancer
  • nucleic acid molecules and polypeptides useful for the detection/diagnosis/staging and treatment of neoplasms, particularly prostate and more specifically hormone-refractory prostate cancers.
  • FIG 1A, IB, 1C, and ID are schematic diagrams that show relative expression and expression patterns of 2333 genes measured using cDNA microarray technology at various time points during prostate cancer development.
  • FIG 2 shows a scatter plot that was generated for one primary and one recurrent tumor. This plot demonstrates the correlation between the samples (low variance, as indicated by lack of scatter in the majority of the genes), and highlights the most differentially expressed genes (which are circled and named).
  • FIG 3 shows lists of genes that were differentially expressed between primary and recurrent xenografts, along with related relative expression information. A set of 30 genes were most consistently differentially expressed (out of a total of 164 genes that changed 2-fold or more) in two independent experiments.
  • FIG.3A shows the 30 genes, ordered by degree of differential expression.
  • the grey-shade coding reflects the relative gene expression ratio (normalized to the mean ratio for four primary tumors) for each of six different recurrent xenografts tumors (arranged in columns). For the six recurrent tumors, the mean expression ratio relative to the mean expression levels of the primary tumors is indicated in the "Mean” column. Additionally, the maximum ratio (Max.) for the upregulated genes, and the minimum ratio (Min.) for the downregulated genes is also indicated (left column).
  • the "pool” column depicts the ratios of a direct cDNA microarray experiment where four primary tumors were pooled and compared to four recurrent tumors.
  • FIG.3B shows eight PI3/AKT FRAP pathway-related genes, the expression of which was associated with hormone-refractory cell growth (based on >two-fold induction of in the recurrent tumors relative to the primary level). Grey-shade-coded gene expression ratios as well as the mean are shown as in FIG. 3 A. The criteria for selecting these genes were i) a >two-fold change in the average ratio between primary and recurrent tumors (or during therapy) and ii) evidence from the literature suggesting the interaction of these gene products with macrolide drugs or their involvement in a rapamycin-sensitive pathway.
  • FIG. 3C shows four FK506-binding protein genes, which were associated with hormone refractory tumor growth (based on at least a two-fold response to therapy and were restored to greater than 80 % of primary levels in the recurrent tumors).
  • Grey-shade-coded gene expression ratios are shown for each of four primary (P) tumors, four tumors regressing following therapy (T), and six recurrent tumors (R). The mean ratio of gene expression (relative to the primary tumors) is shown for tumors undergoing therapy.
  • FIG.3D is a graph showing cell viability after treatment with Rapamycin (solid lines) and FK506 (dotted lines).
  • the recurrent CWR22R cell line was highly sensitive to rapamycin (IC 50 - 0.1 nM) and underwent cell death.
  • LNCap showed partial growth arrest without cell death, even at higher doses of rapamycin.
  • FK506 did not have an effect on either cell line.
  • ED 50 in CWR22R cells for rRapamycin was 0.3 ⁇ M.
  • FIG 4 shows SI OOP mRNA levels measured by three different methods (cDNA microarray, mRNA ISH, and Northern hybridization analyses) in nine xenografts.
  • the amount of S 1 OOP detected in each of these three methods was quantified and plotted in a line graph above the corresponding images. Absolute values were normalized to the three primary tumors with the lowest Northern hybridization levels.
  • FIG 5 is a bar graph showing the level of SI OOP protein expression in 440 human prostate cancer specimens at various stages of progression, measured by IHC staining.
  • An SI OOP antibody was used to stain prostate tissue sections on a tissue microarray containing hundreds of prostate specimens from different steps of cancer development (from normal epithelium, BPH, and localized cancer to metastases and hormone refractory prostate cancer). The staining was scored by two pathologists, using a scale of 0 to 4. The results show the percentage of cancers at each progression stage that had strong (score of 3 or 4) IHC staining.
  • FIG 6 shows the results of analyses of specific gene targets involved in drug response.
  • the top graphs (FIGs 6A and 6B) illustrate the dose response of CWR22R cell line viability in vitro with various emerging therapies (TSA, FR901464, rapamycin, RSD, FK506, and androgen withdrawal therapy); the levels of FKBP5 (FIG 6A) and VDUP1 (FIG 6B) are shown.
  • TSA TSA
  • FKBP5 FKBP5
  • VDUP1 VDUP1
  • FIG 6C shows representative quantitative analyses for expression of the indicated genes at 0, 1, 3, 9, and 24 hours after treatment with the indicated drugs (0.3 ⁇ M TSA, 10 mM FR901464, 1 ⁇ M rapamycin, 1 ⁇ M FK506, and 1 ⁇ M RSD).
  • FIG 6D shows the expression levels for the same genes in primary, regressing, and recurrent tumors.
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • SEQ ID NOs: 1 and 2 also referred to as S100PF and S100PR respectively, are examples of oligonucleotides useful for amplifying an SlOOp probe sequence.
  • SEQ ID NOs: 3-10 AntiSlOOP-A, -B, -C, -D, -E, -F, -G, and -H respectively, are examples of oligonucleotides useful for mRNA in situ hybridization.
  • Analog, derivative or mimetic An analog is a molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure, such as a difference in the length of an alkyl chain, though the change need not be an incremental change in length of a chain), a molecular fragment, a structure that differs by one or more functional groups, a change in ionization. Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in Remington (The Science and Practice of Pharmacology, 19th Edition (1995), chapter 28).
  • a derivative is a biologically active molecule derived from the base (parental) structure.
  • a mimetic is a biomolecule that mimics the activity of another biologically active molecule.
  • Biologically active molecules can include chemical structures that mimic the biological activities of a compound, for instance rapamycin or more generally macrolides (the basic tri-cyclic structural group that includes rapamycin). Rapamycin derivatives (including metabolic derivatives), analogs, and mimetics are disclosed, for instance, in USPN 5,508,398; Kuhn et al., J. Med. Chem. 44:2027-2034, 2001; Dickman etal, Bioorg. Med. Chem. Lett 10:1405-1408, 2000; Streit etal, Drug Metab. Dispos. 24:1272-1278, 1996; and Wong et al, J. Antibiot.
  • Double-stranded DNA has two strands, a 5' -> 3' strand, referred to as the plus strand, and a 3' -> 5' strand (the reverse complement), referred to as the minus strand. Because RNA polymerase adds nucleic acids in a 5' -> 3' direction, the minus strand of the DNA serves as the template for the RNA during transcription. Thus, the RNA formed will have a sequence complementary to the minus strand and identical to the plus strand (except that U is substituted for T).
  • Antisense molecules are molecules that are specifically hybridizable or specifically complementary to either RNA or the plus strand of DNA.
  • Sense molecules are molecules that are specifically hybridizable or specifically complementary to the minus strand of DNA.
  • Antigene molecules are either antisense or sense molecules directed to a dsDNA target.
  • siRNAs small inhibitory RNA molecules
  • Methods of using siRNAs to inhibit eukaryotic and more particularly mammalian gene expression are known to those of ordinary skill in the art; see, for instance, Caplen et al, Proc. Natl. Acad. Sci. 98(17):9742-9747, 2001, and Elbashir et al. , Nature 411 :494-498, 2001.
  • Array An arrangement of molecules, particularly biological macromolecules (such as polypeptides or nucleic acids) or cell or tissue samples, in addressable locations on or in a substrate.
  • the array may be regular (arranged in uniform rows and columns, for instance) or irregular.
  • the number of addressable locations on the array can vary, for example from a few (such as three) to more than 50, 100, 200, 500, 1000, 10,000, or more.
  • a "microarray” is an array that is miniaturized so as to require or be aided by microscopic examination for evaluation or analysis.
  • each arrayed sample (feature) is addressable, in that its location can be reliably and consistently determined within the at least two dimensions of the array.
  • the location of each sample is assigned to the sample at the time when it is applied to the array, and a key may be provided in order to correlate each location with the appropriate target or feature position.
  • ordered arrays are arranged in a symmetrical grid pattern, but samples could be arranged in other patterns (e.g., in radially distributed lines, spiral lines, or ordered clusters).
  • Addressable arrays usually are computer readable, in that a computer can be programmed to correlate a particular address on the array with information about the sample at that position (e.g, hybridization or binding data, including for instance signal intensity).
  • the individual features in the array are arranged regularly, for instance in a Cartesian grid pattern, which can be correlated to address information by a computer.
  • the sample application location on an array (the "feature") may assume many different shapes.
  • the te ⁇ n “spot” may be used herein, it refers generally to a localized placement of molecules or tissue or cells, and is not limited to a round or substantially round region.
  • substantially square regions of application can be used with arrays encompassed herein, as can be regions that are, for example substantially rectangular, triangular, oval, irregular, or another shape.
  • one or more features will occur on the array a plurality of times (e.g., twice) to provide internal controls.
  • Binding or stable binding An oligonucleotide binds or stably binds to a target nucleic acid if a sufficient amount of the oligonucleotide forms base pairs or is hybridized to its target nucleic acid, to permit detection of that binding. Binding can be detected by either physical or functional properties of the targetoligonucleotide complex. Binding between a target and an oligonucleotide can be detected by any procedure known to one skilled in the art, including both functional and physical binding assays. Binding may be detected functionally by determining whether binding has an observable effect upon a biosynthetic process such as expression of a gene, DNA replication, transcription, translation, and the like.
  • Physical methods of detecting the binding of complementary strands of DNA or RNA are well known in the art, and include such methods as DNase I or chemical footprinting, gel shift and affinity cleavage assays, Northern blotting, dot blotting and light absorption detection procedures.
  • one method that is widely used involves observing a change in light absorption of a solution containing an oligonucleotide (or an analog) and a target nucleic acid at 220 to 300 ran as the temperature is slowly increased. If the oligonucleotide or analog has bound to its target, there is a sudden increase in absorption at a characteristic temperature as the oligonucleotide (or analog) and target disassociate from each other, or melt.
  • T m The binding between an oligomer and its target nucleic acid is frequently characterized by the temperature (T m ) at which 50% of the oligomer is melted from its target.
  • T m the temperature at which 50% of the oligomer is melted from its target.
  • a higher (T m ) means a stronger or more stable complex relative to a complex with a lower (T m ).
  • a cancer is a biological condition in which a malignant tumor or other neoplasm has undergone characteristic anaplasia with loss of differentiation, increased rate of growth, invasion of surrounding tissue, and which is capable of metastasis.
  • the term cancer includes prostate cancer, such as prostate adenocarcinoma, transitional cell carcinomas, squamous cell carcinomas, and sarcomas. However, about 95% of prostate cancers are adenocarcinomas. Also included are different stages of a single cancer, for instance both primary and recurrent (hormone-refractory) prostate cancer.
  • cDNA complementary DNA: A piece of DNA lacking internal, non-coding segments (introns) and transcriptional regulatory sequences. cDNA may also contain untranslated regions (UTRs) that are responsible for translational control in the corresponding RNA molecule. cDNA is usually synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.
  • Comparative genomic hybridization A technique of differential labeling of test DNA and normal reference DNA, which are hybridized simultaneously to chromosome spreads, as described in Kallioniemi et al. (Science 258:818-821, 1992), incorporated by reference.
  • Complementarity and percentage complementarity Molecules with complementary nucleic acids form a stable duplex or triplex when the strands bind, (hybridize), to each other by forming Watson-Crick, Hoogsteen or reverse Hoogsteen base pairs. Stable binding occurs when an oligonucleotide remains detectably bound to a target nucleic acid sequence under the required conditions.
  • Complementarity is the degree to which bases in one nucleic acid strand base pair with the bases in a second nucleic acid strand. Complementarity is conveniently described by percentage, i.e. the proportion of nucleotides that form base pairs between two strands or within a specific region or domain of two strands. For example, if 10 nucleotides of a 15-nucleotide oligonucleotide form base pairs with a targeted region of a DNA molecule, that oligonucleotide is said to have 66.67% complementarity to the region of DNA targeted.
  • sufficient complementarity means that a sufficient number of base pairs exist between the oligonucleotide and the target sequence to achieve detectable binding, and in the case of the binding of an antigen, disrupt expression of gene products (such as cartilage glycoprotein-39 (CHI3L1), S-100P PROTEIN (S100P), CX3C chemokine/fractalkine (SCYD1), adenylate kinase 1 (AK1), forkhead transcription factor HFH-4 (HFH-4) (FKHL13), UDP glucuronosyltransferase precursor (UGT2B15) , Pleiotrophin (heparin binding growth factor 8)
  • gene products such as cartilage glycoprotein-39 (CHI3L1), S-100P PROTEIN (S100P), CX3C chemokine/fractalkine (SCYD1), adenylate kinase 1 (AK1), forkhead transcription factor HFH-4 (HFH-4) (FKHL13), UDP
  • PPN heat shock 27kD protein 2/Alpha-B-crystallin
  • HSP27 Proteasome (prosome, macropain) subunit, beta type, 5 (PSMB5), Inhibitor of NFKB (NFKBIA), interferon-induced 17 kD protein (ISG15), MAP kinase activated protein kinase 2 (MAPKAPK2), signal transduction protein (SH3 containing) (EFS2), hkf-1 Zinc finger protein (ZFP103), chromosome condensation 1 (CHC1), CDP- diacylglycerol synthase (CDSl), gap junction protein, alpha 1, 43kD (connexin 43) (GJAl), cyclin Dl (CCNDl), Inhibitor of DNA binding 3, helix-loop-helix protein (ID3), HI histone family, member2 (H1F2), Cytochrome B561 (CYB561), Cathepsin H (CTSH), calcin
  • the percentage complementarity that fulfills this goal can range from as little as about 50% complementarity to full (100%) complementary. In general, sufficient complementarity is at least about 50%, about 75% complementarity, about 90% or 95% complementarity, and or about 98% or even 100% complementarity.
  • DNA deoxyribonucleic acid
  • DNA is a long chain polymer which comprises the genetic material of most living organisms (some viruses have genes comprising ribonucleic acid (RNA)).
  • the repeating units in DNA polymers are four different nucleotides, each of which comprises one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached.
  • Triplets of nucleotides (referred to as codons) code for each amino acid in a polypeptide, or for a stop signal.
  • codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.
  • any reference to a DNA molecule is intended to include the reverse complement of that DNA molecule. Except where single-strandedness is required by the text herein, DNA molecules, though written to depict only a single strand, encompass both strands of a double-stranded DNA molecule. Thus, a reference to the nucleic acid molecule that encodes a specific protein, or a fragment thereof, encompasses both the sense strand and its reverse complement. Thus, for instance, it is appropriate to generate probes or primers from the reverse complement sequence of the disclosed nucleic acid molecules.
  • Deletion The removal of a sequence of DNA, the regions on either side of the removed sequence being joined together.
  • Gene amplification or genomic amplification An increase in the copy number of a gene or a fragment or region of a gene or associated 5' or 3' region, as compared to the copy number in normal tissue.
  • An example of a genomic amplification is an increase in the copy number of an oncogene.
  • a "gene deletion” is a deletion of one or more nucleic acids normally present in a gene sequence and, in extreme examples, can include deletions of entire genes or even portions of chromosomes.
  • Gene expression fingerprint A distinct or identifiable pattern of gene expression, for instance a pattern of high and low expression of a defined set of genes; in some instances, as few as one or two genes may provide a profile, but often more genes are used in a profile, for instance at least three, at least 5, at least 10, at least 20, at least 25, or at least 50 or more.
  • Gene expression fingerprints also referred to as profiles
  • Gene expression fingerprints can be linked to a tissue or cell type, to a particular stage of normal tissue growth or disease progression, or to any other distinct or identifiable condition that influences gene expression in a predictable way.
  • Gene expression fingerprints can include relative as well as absolute expression levels of specific genes, and often are best viewed in the context of a test sample compared to a baseline or control sample fingerprint.
  • a gene expression profile may be read on an array (e.g, a polynucleotide or polypeptide array).
  • arrays are now well known, and for instance gene expression arrays have been previously described in published PCT application number US99/06860 ("Hypoxia-Inducible Human Genes, Proteins, and Uses Thereof), incorporated herein by reference in its entirety.
  • Genomic target sequence A sequence of nucleotides located in a particular region in the human genome that corresponds to one or more specific genetic abnormalities, such as a nucleotide polymorphism, a deletion, or an amplification.
  • the target can be for instance a coding sequence; it can also be the non-coding strand that corresponds to a coding sequence.
  • HRPC-related molecule A molecule that is involved in, or influenced by, hormone- refractory prostate cancer. Such molecules include, for instance, nucleic acids (e.g., DNA, cDNA, or mRNAs) and proteins. Specific examples of HRPC-related molecules include the nucleic acid molecules listed in Table 1, and proteins or protein fragments encoded thereby. HRPC-related molecules may be involved in or influenced by hormone-refractory prostate cancer in many different ways, including causative (in that a change in an HRPC-related molecule leads to development of or progression to hormone-refractory prostate cancer) or resultive (in that development of or progression to hormone-refractory prostate cancer causes or results in a change in the HRPC-related molecule).
  • causative in that a change in an HRPC-related molecule leads to development of or progression to hormone-refractory prostate cancer
  • resultive in that development of or progression to hormone-refractory prostate cancer causes or results in a change in the HRPC-related molecule.
  • nucleic acid consists of nitrogenous bases that are either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)).
  • base pairing a bonding of the pyrimidine to the purine
  • base pairing More specifically, A will hydrogen bond to T or U, and G will bond to C.
  • “Complementary” refers to the base pairing that occurs between to distinct nucleic acid sequences or two distinct regions of the same nucleic acid sequence.
  • “Specifically hybridizable” and “specifically complementary” are terms that indicate a sufficient degree of complementarity such that stable and specific binding occurs between the oligonucleotide (or its analog) and the DNA or RNA target. The oligonucleotide or oligonucleotide analog need not be 100% complementary to its target sequence to be specifically hybridizable.
  • An oligonucleotide or analog is specifically hybridizable when binding of the oligonucleotide or analog to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide or analog to non-target sequences under conditions where specific binding is desired, for example under physiological conditions in the case of in vivo assays or systems. Such binding is referred to as specific hybridization.
  • Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially the Na + concentration) of the hybridization buffer will determine the stringency of hybridization, though waste times also influence stringency. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, chapters 9 and 11, herein incorporated by reference.
  • stringent conditions encompass conditions under which hybridization will only occur if there is less than 25% mismatch between the hybridization molecule and the target sequence.
  • Stringent conditions may be broken down into particular levels of stringency for more precise definition.
  • “moderate stringency” conditions are those under which molecules with more than 25% sequence mismatch will not hybridize; conditions of “medium stringency” are those under which molecules with more than 15% mismatch will not hybridize, and conditions of “high stringency” are those under which sequences with more than 10% mismatch will not hybridize.
  • Conditions of "very high stringency” are those under which sequences with more than 6% mismatch will not hybridize.
  • In vitro amplification Techniques that increase the number of copies of a nucleic acid molecule in a sample or specimen.
  • An example of amplification is the polymerase chain reaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to nucleic acid template in the sample.
  • the primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid.
  • the product of in vitro amplification may be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and or nucleic acid sequencing, using standard techniques.
  • Other examples of in vitro amplification techniques include strand displacement amplification (see U.S. Patent No. 5,744,311); transcription-free isothermal amplification (see U.S. Patent No. 6,033,881); repair chain reaction amplification (see WO 90/01069); ligase chain reaction amplification (see EP-A-320308); gap filling ligase chain reaction amplification (see U.S. Patent No. 5,427,930); coupled ligase detection and PCR (see U.S. Patent No. 6,027,889); and NASBATM RNA transcription-free amplification (see U.S. Patent No. 6,025,134).
  • Injectable composition A pharmaceutically acceptable fluid composition including at least one active ingredient.
  • the active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition can additionally include minor amounts of one or more non-toxic auxiliary substances, such as emulsifying agents, preservatives, and pH buffering agents and the like.
  • non-toxic auxiliary substances such as emulsifying agents, preservatives, and pH buffering agents and the like.
  • Isolated An "isolated" biological component (such as a nucleic acid molecule, protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, proteins and organelles.
  • Nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified by standard purification methods. The tenn also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • Neoplasm A new and abnormal growth, particularly a new growth of tissue or cells in which the growth is uncontrolled and progressive. A tumor is an example of a neoplasm.
  • Nucleotide includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA).
  • a nucleotide is one monomer in a polynucleotide.
  • a nucleotide sequence refers to the sequence of bases in a polynucleotide.
  • Oligonucleotide is a plurality of joined nucleotides joined by native phosphodiester bonds, between about 6 and about 300 nucleotides in length.
  • An oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non-naturally occurring portions.
  • oligonucleotide analogs can contain non-naturally occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorofhioate oligodeoxynucleotide.
  • Functional analogs of naturally occurring polynucleotides can bind to RNA or DNA, and include peptide nucleic acid (PNA) molecules.
  • PNA peptide nucleic acid
  • Particular oligonucleotides and oligonucleotide analogs can include linear sequences up to about 200 nucleotides in length, for example a sequence (such as DNA or RNA) that is at least 6 bases, for example at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases long, or from about 6 to about 50 bases, for example about 10-25 bases, such as 12, 15 or 20 bases.
  • a sequence such as DNA or RNA
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
  • Open reading frame A series of nucleotide triplets (codons) coding for amino acids without any internal termination codons. These sequences are usually translatable into a peptide.
  • Ortholog Two nucleic acid or amino acid sequences are orthologs of each other if they share a common ancestral sequence and diverged when a species carrying that ancestral sequence split into two species. Orthologous sequences are also homologous sequences.
  • Parenteral Administered outside of the intestine, e.g., not via the alimentary tract. Generally, parenteral formulations are those that will be administered through any possible mode except ingestion. This term especially refers to injections, whether administered intravenously, intrathecally, intramuscularly, intraperitoneally, or subcutaneously, and various surface applications including intranasal, intradermal, and topical application, for instance.
  • PNA Peptide Nucleic Acid
  • compositions and formulations suitable for pharmaceutical delivery of the nucleotides and proteins herein disclosed are conventional. Martin, Remington 's Pharmaceutical Sciences, published by Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the nucleotides and proteins herein disclosed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions e.g., powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Probes and primers Nucleic acid probes and primers can be readily prepared based on the nucleic acid molecules provided in this disclosure as indicators of disease or disease progression. It is also appropriate to generate probes and primers based on fragments or portions of these nucleic acid molecules. Also appropriate are probes and primers specific for the reverse complement of these sequences, as well as probes and primers to 5' or 3' regions.
  • a probe comprises an isolated nucleic acid attached to a detectable label or other reporter molecule.
  • Typical labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, e.g., in Sambrook et al. (In ' Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).
  • Primers are short nucleic acid molecules, for instance DNA oligonucleotides 10 nucleotides or more in length. Longer DNA oligonucleotides may be about 15, 20, 25, 30 or 50 nucleotides or more in length. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then the primer extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.
  • PCR polymerase chain reaction
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, ⁇ 1991, Whitehead Institute for Biomedical Research, Cambridge, MA).
  • probes and primers can be selected that comprise at least 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of a HRPC-related protein-encoding nucleotide sequences.
  • the disclosure thus includes isolated nucleic acid molecules that comprise specified lengths of the disclosed HRPC-related nucleotide sequences.
  • Such molecules may comprise at least 10, 15, 20, 23, 25, 30, 35, 40, 45 or 50 consecutive nucleotides of these sequences or more, and may be obtained from any region of the disclosed sequences (e.g, a HRPC-related nucleic acid may be apportioned into halves or quarters based on sequence length, and isolated nucleic acid molecules may be derived from the first or second halves of the molecules, or any of the four quarters, etc.).
  • a HRPC-related cDNA also can be divided into smaller regions, e.g. about eighths, sixteenths, twentieths, fiftieths and so forth, with similar effect.
  • Another mode of division is to select the 5' (upstream) and/or 3' (downstream) region associated with a HRPC-related gene.
  • Nucleic acid molecules may be selected that comprise at least 10, 15, 20, 25, 30, 35, 40, 50 or 100 or more consecutive nucleotides of any of these or other portions of a HRPC-related nucleic acid molecule, such as those disclosed herein, and associated flanking regions.
  • representative nucleic acid molecules might comprise at least 10 consecutive nucleotides of a human coding sequence the expression of which is influenced by prostate cancer progression, such as those listed in Table 1.
  • Protein A biological molecule expressed by a gene and comprised of amino acids.
  • purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified protein preparation is one in which the protein referred to is more pure than the protein in its natural environment within a cell or within a production reaction chamber (as appropriate).
  • Recombinant A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • Representational difference analysis A PCR-based subtractive hybridization technique used to identify differences in the mRNA transcripts present in closely related cell lines.
  • Serial analysis of gene expression The use of short diagnostic sequence tags to allow the quantitative and simultaneous analysis of a large number of transcripts in tissue, as described in Velculescu et al. (Science 270:484-487, 1995).
  • Sequence identity The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identify. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or orthologs of the HRPC-related protein, and the corresponding cDNA or gene sequence, will possess a relatively high degree of sequence identity when aligned using standard methods. This homology will be more significant when the orthologous proteins or genes or cDNAs are derived from species that are more closely related (e.g., human and chimpanzee sequences), compared to species more distantly related (e.g., human and C. elegans sequences).
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties).
  • Stringent conditions are sequence- dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5° C to 20° C lower than the thennal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence remains hybridized to a perfectly matched probe or complementary strand. Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al.
  • Nucleic acid molecules that hybridize under stringent conditions to a specific human HRPC-related protein-encoding sequence will typically hybridize to a probe based on either an entire human HRPC-related protein-encoding sequence or selected portions of the encoding sequence under wash conditions of 2x SSC at 50° C.
  • nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein.
  • protein [X] specific binding agent An agent that binds substantially only to a defined target.
  • a protein-specific binding agent binds substantially only the specified protein.
  • protein [X] specific binding agent includes anti-[X] protein antibodies (and functional fragments thereof) and other agents (such as soluble receptors) that bind substantially only to the [X] protein.
  • [X] refers to any specific or designated protein, for instance a HRPC-related protein such as those listed in Table 1.
  • Anti-[X] protein antibodies may be produced using standard procedures described in a number of texts, including Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988). The determination that a particular agent binds substantially only to the specified protein may readily be made by using or adapting routine procedures.
  • One suitable in vitro assay makes use of the Western blotting procedure (described in many standard texts, including Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988)).
  • Western blotting may be used to detennine that a given protein binding agent, such as an anti-[X] protein monoclonal antibody, binds substantially only to the [X] protein.
  • Shorter fragments of antibodies can also serve as specific binding agents.
  • Fabs, Fvs, and single-chain Fvs (SCFvs) that bind to a specified protein would be specific binding agents.
  • These antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab 1 , the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab')2, a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed
  • Subject Living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals
  • Target sequence is a portion of ssDNA, dsDNA or RNA that, upon hybridization to a therapeutically effective oligonucleotide or oligonucleotide analog, results in the inhibition of expression of a specified protein, such as a HRPC-related protein.
  • a specified protein such as a HRPC-related protein.
  • an antisense or a sense molecule can be used to target a portion of dsDNA, since both will interfere with the expression of that portion of the dsDNA.
  • the antisense molecule can bind to the plus strand, and the sense molecule can bind to the minus strand.
  • target sequences can be ssDNA, dsDNA, and RNA.
  • tissue microarray is a microarray wherein the samples are samples of tissue, for instance animal tissue such as human tissue.
  • tissue microarrays are assembled by aligning tissue cylinders (taken, for instance, from tissue blocks or biopsies) in a recipient block, such as a block of paraffin, to create a matrix of columns of sample within the block. Individual slices are cut from the surface of the block, substantially perpendicular to the axis of the cylinders, thereby yielding flat, thin arrays of tissue samples embedded in the block material. Such thin arrays are often transferred to a microscope slide or other supporting member.
  • tissue microarrays is described in, for instance, Kononen et al, Nature Medicine, 4:844-847, 1998 and PCT International Patent Publication WO99/44063A2, both of which are incorporated herein by this reference.
  • Tissue samples contained in a tissue microarray may be any set of tissues, but often a tissue microarray has a theme so to speak, for instance containing samples from a collection of different tumors, tumors from different tissues, tumors from different stages of progression, or from different treatment regimens.
  • a transformed cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques.
  • transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • Tumor A neoplasm that may be either malignant or non-malignant.
  • Tumors of the same tissue type refers to primary tumors originating in a particular organ (such as breast, prostate, bladder or lung). Tumors of the same tissue type may be divided into tumor of different sub-types (an example being prostate cancer, which can be an adenocarcinoma, transitional cell, squamous cell tumor, or sarcoma).
  • a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
  • a vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker genes and other genetic elements known in the art.
  • kits for diagnosing or prognosing development or progression of prostate cancer in a subject which methods involve detecting an abnormality in at least one HRPC- related molecule of the subject (e.g., an HRPC related nucleic acid molecule such as one listed in Table 1 or Table 4, or genes, cDNAs or other polynucleotide molecules comprising one of the listed sequences, or a fragment thereof, or an HRPC-related protein such as one encoded by such a nucleic acid molecule, or a fragment of such protein).
  • an HRPC- related molecule of the subject e.g., an HRPC related nucleic acid molecule such as one listed in Table 1 or Table 4, or genes, cDNAs or other polynucleotide molecules comprising one of the listed sequences, or a fragment thereof, or an HRPC-related protein such as one encoded by such a nucleic acid molecule, or a fragment of such protein.
  • abnormalities are detected in more than one HRPC-related molecule, for instance in at least 5, at least 10, 15, 25, 50, or 100 or more HRPC-related nucleic acid molecules listed in Table 1 or elsewhere herein, or encoded for by a nucleic acid molecule listed in Table 1 or elsewhere herein.
  • no more than the molecules listed in Table 1 or Table 4, or corresponding to (represented by) those listed in Table 1 or Table 4 are included in such analysis.
  • certain of the described methods employ detecting no more than 600, no more than 500, no more than 400, no more than 300, or no more than 200 of such molecules.
  • arrays containing two or more HRPC-related molecules are also encompassed herein.
  • Certain of such arrays are nucleic acid arrays, which contain at least one HRPC-related nucleic acid molecule, for instance at least one of the HRPC-related nucleic acid molecules listed in Table 1 or Table 4, or genes, cDNAs or other polynucleotide molecules comprising one of the listed sequences, or a fragment thereof.
  • arrays are protein (polypeptide) arrays, which contain at least one HRPC-related protein such as one encoded by a nucleic acid molecule listed in Table 1 or Table 4 (or genes, cDNAs or other polynucleotide molecules comprising one of the listed sequences, or a fragment thereof), or a fragment of such protein, or an antibody specific to such a protein or protein fragment.
  • HRPC-related protein such as one encoded by a nucleic acid molecule listed in Table 1 or Table 4 (or genes, cDNAs or other polynucleotide molecules comprising one of the listed sequences, or a fragment thereof), or a fragment of such protein, or an antibody specific to such a protein or protein fragment.
  • Such arrays can also contain any particular subset of the nucleic acids (or corresponding molecules) listed in Table 1 or Table 4. Certain of such arrays (as well as the methods described herein) also may include HRPC-related molecules that are not listed in Table 1 or Table 4.
  • HRPC-related molecule includes HRPC-related nucleic acid molecules (such as DNA or RNA or cDNA) and HRPC-related proteins, though in specific embodiments the term may be specific for any one of tliese types of molecules.
  • the term is not limited to those molecules listed in Table 1 or Table 4 (and molecules that correspond to those listed), but also includes other nucleic acids and/or proteins that are influenced (e.g., as to level, activity, localization) by or during prostate cancer progression, including all of such molecules listed herein.
  • Specific encompassed embodiments include diagnostic and/or prognostic methods in which a mutation, duplication or deletion of a HRPC-related nucleic acid in cells of the individual is detected.
  • HRPC-related molecules that can be examined for an abnormality include molecules represented by a subset of the sequences referred to in Table 1 or Table 4, such as the more than 200 sequences represented by Image Clone ID numbers: 1475595, 1460110, 50794, 78294, 190491, 66731, 143287, 754600, 754509, 308041, 70827, 361974, 503097, 796646, 41650, 841641, 724615, 839101, 504226, 810711, 435330, 773567, 431296, 345232, 756405, 256907, 415817, 366541, 223350, 366067, 724831, 814353, 236034, 809910, 1470048, 1323448, 1456424, 453689, 135221, 340734, 180864, 768562, 179276, 44505, 293104, 243343, 66317, 812251, 245920, 265874, 770
  • Molecules represented by (or corresponding to) these Image Clone IDs include the nucleic acid fragments found in the respective clones (and variants thereof), complete nucleic acids (such as cDNAs, mRNAs, or genes) encompassing such fragments, fragments and variants of these complete nucleic acid molecules, proteins encoded by such nucleic acids, and fragments and variants of such proteins.
  • Certain of the encompassed methods involve measuring an amount of the HRPC-related molecule in a sample (such as a serum or tissue sample) derived or taken from the subject, in which a difference (for instance, an increase or a decrease) in level of the HRPC-related molecule relative to that present in a sample derived or taken from the subject at an earlier time, is diagnostic or prognostic for development or progression of prostate cancer.
  • a sample such as a serum or tissue sample
  • Abnormalities in HRPC-related nucleic acid molecules can be detected using, for instance, in vitro nucleic acid amplification and/or nucleic acid hybridization.
  • the results of such detection methods can be quantified, for instance by determining the amount of hybridization or the amount of amplification.
  • detecting an abnormality includes contacting a sample from the subject with a HRPC protein-specific binding agent; and detecting whether the binding agent is bound by the sample and thereby measuring the levels of the HRPC-related protein present in the sample, in which a difference in the level of HRPC-related protein in the sample, relative to the level of HRPC-related protein found an analogous sample from a subject not having the disease or disorder, or a standard HRPC-related protein level in analogous samples from a subject not having the disease or disorder or not having a predisposition for developing the disease or disorder, is an abnormality in that HRPC- related molecule.
  • detecting the abnormality involves determining whether a HRPC- related gene expression profile from the subject indicates development or progression of prostate cancer, for instance by comparing the HRPC-related gene expression profile from the subject to at least one control gene expression fingerprint or profile for a specific stage of prostate cancer.
  • at least one control gene expression profile is a fingerprint for a normal prostate tissue, a primary prostate cancer tissue, a prostate cancer tissue responding to androgen ablation therapy, or a hormone refractory prostate cancer tissue.
  • profiles also referred to herein as fingerprints
  • arrays are nucleotide (e.g., polynucleotide) or protein (e.g, peptide, polypeptide, or antibody) arrays.
  • an array may be contacted with polynucleotides or polypeptides (respectively) from (or derived from) a sample from a subject. The amount and/or position of binding of the subject's polynucleotides or polypeptides then can be determined, for instance to produce a gene expression profile for that subject.
  • Such gene expression profile can be compared to another gene expression profile, for instance a control gene expression profile from a subject having a known prostate-related condition.
  • the subject's gene expression profile also known as a gene expression fingerprint
  • protein arrays can give rise to protein expression profiles. Both protein and gene expression profiles can more generally be referred to as expression profiles.
  • inventions are methods that involve providing nucleic acids from the subject; amplifying the nucleic acids to fo ⁇ n nucleic acid amplification products; contacting the nucleic acid amplification products with an oligonucleotide probe that will hybridize under stringent conditions with a nucleic acid encoding a HRPC-related protein; detecting the nucleic acid amplification products which hybridize with the probe; and quantifying the amount of the nucleic acid amplification products that hybridize with the probe.
  • the sequence of such oligonucleotide probes may be selected to bind specifically to a nucleic acid molecule listed in Table 1 or Table 4, or a nucleic acid molecule represented by those listed in Table 1 or Table 4.
  • the probes are attached to a solid surface, such as an array.
  • the primers may be selected to amplify a nucleic acid molecule listed in Table 1 or Table 4, or represented by tiiose listed in Table 1 or Table 4.
  • the primers are selected to amplify a nucleic acid product encoding cartilage glycoprotein-39 (CHI3L1), S-100P PROTEIN (S100P), CX3C chemokine/fractalkine (SCYD1), adenylate kinase 1 (AK1), forkhead transcription factor HFH-4 (HFH-4) (FKHL13), UDP glucuronosyltransferase precursor (UGT2B15), Pleiotrophin (heparin binding growth factor 8) (PTN), heat shock 27kD protein 2/Alpha-B-crystallin (HSP27), Proteasome (prosome, macropain) subunit, beta type, 5 (PSMB5), Inhibitor of NFKB (NFKBIA), interferon- induced 17 kD protein (ISG 15), MAP kinase activated protein kinase 2 (MAPKAPK2), signal transduction protein (SH3 containing) (EF
  • the subject then can be treated in accordance with this selection.
  • the treatment selected in specific and tailored for the subject, based on the analysis of that subject's profile for one or more HRPC-related molecules.
  • a further embodiment is a method of modifying a level of expression or function of a HRPC-related protein in a subject.
  • Such methods may involve expressing in the subject a recombinant genetic construct that includes a promoter operably linked to a nucleic acid molecule, and wherein expression of the nucleic acid molecule changes expression of the HRPC-related protein.
  • the nucleic acid molecule may, for instance, include at least 10 consecutive nucleotides of a HRPC- related nucleic acid sequence.
  • the nucleic acid molecule is in antisense orientation relative to the promoter; in other examples, the nucleic acid molecule is in sense orientation relative to the promoter.
  • kits for measuring the level or function of one or more HRPC- related molecules may include a binding molecule that selectively binds to the HRPC- related molecule that is the target of the kit.
  • the binding molecule provided in the kit may be an antibody or antibody fragment that selectively binds to the target HRPC-related protein.
  • the binding molecule provided in the kit may be an oligonucleotide capable of hybridizing to the HRPC-related nucleic acid molecule.
  • Further embodiments are methods of screening for a compound useful in treating, reducing, or preventing prostate cancer or development or progression of prostate cancer. Such methods involve determining if application of a test compound alters a HRPC-related gene expression profile so that the profile more closely resembles a prostate-linked profile than it did prior to such treatment, and selecting a compound that so alters the HRPC-related gene expression profile.
  • the test compound is applied to a test cell.
  • the profile is determined or measured in an array format.
  • Examples of disclosed methods involve contacting test cells with a test compound, then measuring at least one HRPC-related molecule level and/or activity in the test cells.
  • a difference in HRPC-related molecule e.g., a HRPC-related nucleic acid molecule listed in Table 1, or a molecule encoded for by a nucleic acid molecule listed in Table 1
  • the test compound is useful in treating, reducing, or preventing prostate cancer or development or progression of prostate cancer.
  • HRPC-related molecule(s) level and/or activity may include creating a HRPC-related gene expression profile for the test cell after contacting the cell with the test compound, and comparing the test cell HRPC-related gene expression profile to at least one control gene expression profile for a specific stage of prostate cancer.
  • Representative control gene expression profile can include a profile for a normal prostate tissue, a primary prostate cancer tissue, a prostate cancer tissue responding to androgen ablation therapy, and/or a hormone refractory prostate cancer tissue.
  • HRPC-related molecules for the development of antibodies, including therapeutic antibodies that affect an HRPC-related pathway. It is also envisioned that the disclosed HRPC-related molecules can be used as vaccines, for instance as "cancer vaccines" to elicit an immune response from a subject that renders the subject more resistant to developing or progressing through a stage of prostate cancer. IV. Temporal gene expression changes during prostate cancer hormonal therapy The present disclosure concerns gene expression profiling of the regression and progression of the CWR22 human prostate cancer xenografts using cDNA microarrays. Highly quantitative two- color fluorescent microarray technology was used to examine differences in gene expression between primary and recurrent CWR22 prostate cancer xenografts.
  • cDNA microarrays containing 6605 gene fragments were constructed and applied to analyze the mRNA expression profile of thirteen CWR22 xenografts at different stages in the time course of treatment and progression.
  • New statistical tools including template-based gene clustering
  • were then used to mine this data e.g., to differentiate temporal gene expression profiles associated with in vivo therapy response from the noise in the data, enabling a comprehensive genomics-based analysis of the temporal mRNA expression profiles during the entire course of CWR22 treatment and progression.
  • a large set of genes and other encoding sequences e.g., expressed sequence tags, ESTs
  • Table 1 the expression of which varies during prostate cancer progression.
  • the Image Clone ID numbers of these sequences are: 843249, 796904, 399604, 788205, 260303, 287745, 796542, 243653, 486787, 134495, 796680, 448386, 298417, 124578, 141562, 713145, 815284, 51408, 22012, 344589, 839101, 511091, 365515, 144042, 153411, 853809, 884783, 75254, 109314, 293925, 949938, 132835, 773567, 415529, 809910, 142326, 139660, • 435855, 243816, 489839, 130895, 258120, 39920, 343867, 142139, 1472735, 503083, 796147, 813179, 741891, 461727, 756968, 814636, 897667, 297392, 322723, 66322, 295729, 265645, 143756, 195340,
  • Image ID Clones can be obtained from Research Genetics (2130 Memorial Parkway, Huntsville, AL 35801, US or Canada: 1- 800-533-4363). The sequences of these Image ID Clones are hereby expressly incorporated by reference, and are clearly identified based on their multiple identification listings given in Table 1.
  • HRPC-related molecules Other confidence levels could be used to select HRPC-related molecules, such as 98%, 95%, 90%, 85%, and so forth. Higher confidence levels, such as 99.99%, could also be used.
  • Molecules identified as being linked to prostate cancer referred to generally herein as HRPC-related molecules
  • HRPC-related molecules using the methods described herein can be arranged on arrays for use in diagnostic and prognostic methods. Specific arrays are contemplated that are constructed using molecules identified at such different confidence levels.
  • the techniques disclosed herein have uncovered many genes not previously associated with prostate cancer progression, and particularly not previously associated with HRPC. These newly correlated genes include those represented by the following Image ID Clones: 781047, 785778, 842968, 769921, 898286, 204214, 814701, 435076, 531319, 415089, 898062, 453107, 785707, 795936, 700792, 34778, 46182, 769921, 783697, 451907, 711768, 416833, 810711, 789204, 789182, 725454, 951142, 49352, 273546, 46717, 855487, 41117, 26578, 684655, 45233, 814117, 810552, 739511, 283315, and 897774. Of the 59 androgen-dependent sequences whose expression decreased most after castration
  • Tissue microarrays consisting of 50 xenografts and 440 clinical specimens from all stages of prostate cancer progression were utilized to validate potential drug target genes using mRNA in situ hybridization and protein immunohistochemistry.
  • S 1 OOP encoding a calcium-binding protein
  • the temporal gene expression changes identified herein facilitate identification of candidate drugs for hormone-refractory prostate cancer.
  • FKBP5 for example was identified and its utility as a therapeutic target was validated using tissue microarray analysis (see Example 3). Based on such leads, Rapamycin, MS-275, and TSA were tested for their effectiveness in influencing prostate cancer cell growth. These drugs target some of the candidate genes described herein. As described in Example 4, the inventors found that these drugs inhibit CWR22R prostate cancer cell growth in vitro.
  • tissue microarray analysis provides a new paradigm to identify, prioritize, and validate novel diagnostic and drug targets, as herein described for hormone-refractory prostate cancer.
  • the identified HRPC-related genes represent putative mediators of hormone therapy response and resistance, and as such are candidate targets for the development of novel therapeutics to maintain prostate cancer in regression following hormone ablation therapy.
  • the utility of tliese genes as candidate drug targets and biomarkers is demonstrated herein by first using tissue microarrays for high throughput translation to clinical samples, and then selecting drugs that might target these genes.
  • Analysis of cDNA microarray data with template based gene clustering and high throughput translation using tissue microarrays introduces a new, generally applicable paradigm for applying functional genomics to identify genetic programs that mediate a responses to a variety of in vivo therapies.
  • HRPC-related genes identified herein encode or correspond to soluble proteins, while other encode or correspond to membrane associated or membrane integral proteins, some of which are exposed at least to a certain extent on the exterior of a cell in which they are expressed.
  • those HRPC-related molecules that are expressed at or on the surface of a cell are selected as therapeutic targets, for instance for targeting with an antibody-based therapy, which is facilitated by the access of the HRPC-related molecule to the extracellular matrix.
  • HRPC-related molecules may be described as being "drug accessible.”
  • CWR22 is a serially transplantable, prostate cancer xenograft that was derived from a Gleason score 9 primary human prostate cancer with osseous metastasis (Wainstein et al, Cancer Res. 54:6049-6052, 1994).
  • CWR22 is highly responsive to androgen deprivation, with marked tumor regression after castration (Cheng et al., J. Natl. Cancer Inst. 88:607- 611, 1996; Nagabhushan et ⁇ /., Cancer Res. 56:3042-3046, 1996; Myers et al., J Urol. 161 :945-949, 1999).
  • About half of the treated animals develop recurrent tumors (CWR22R) over a time frame of from a few weeks to several months.
  • CWR22R is not dependent on androgen and is able to grow in castrated animals.
  • cDNA Microarrays The cDNA microarrays consisted of 6605 elements representing different (non-redundant) genes. PCR products from sequence-verified clones (Research Genetics, Huntsville AB) were prepared and printed at high density onto glass slides according to previously described protocols (Mousses et al, "Gene Expression Analysis by cDNA Microarrays," in Differential Gene Expression: A practical approach, Livesey and Hunt (eds.), Oxford University Press, 2000).
  • Labeled cDNA was made with 4-16 ⁇ g of mRNA in an oligo(dT)-primed polymerization using Superscript II reverse transcriptase (LifeTechnologies, Rockville , MD) in the presence of either Cy3 or Cy5 labeled dUTP (Amersham Pharmacia, Piscataway, NJ) as described (Mousses et al, "Gene Expression Analysis by cDNA Microarrays," in Differential Gene Expression: A practical approach, Livesey and Hunt (eds.), Oxford University Press, 2000).
  • Imaging and Image Analysis Fluorescence intensities at the immobilized targets were measured by using a custom-designed laser con-focal microscope scanner, with intensity data integrated over 15-micron square pixels and recorded at 16 bits. Image analysis was performed by using DEARRAY software. Details of the fabrication of the microarray slides, and image generation and analysis are available on the Internet at the NHGRI Microarray Website, and software is currently and freely available for Sybase UNIX and is in the process of being ported to Oracle/UNIX. Detailed information about the program itself can be found on the ArrayDB Web site at the NHGRI. A complete description of the gene clustering used is also described at the NHGRI Microarray Website.
  • clustering analysis is a powerful tool that partitions biological samples or genes into well-separated and homogeneous groups based on their statistical behaviors.
  • the main objective of clustering analysis is to find out the similarities between experiments or between each genes, given their expression ratios across all genes or samples, respectively, and then group the similar samples or genes together for the convenience of understanding and visualization.
  • the clustering methods have been heavily studied for many years and widely applied in many areas.
  • Hierarchical Clustering methods Assume there are m expression experiments containing n genes in each every experiment. After performing microarray image analysis and data integration, a m x n matrix of gene expression ratios is obtained, where each column of ratios represents the result from one expression experiment comparing the test sample to a common reference sample of choice. To simplify the discussion, the algorithm is considered only in terms of the sample clustering.
  • a distance matrix can be constructed as shown below (Table 3a).
  • the hierarchical algorithm proceeds as follows: First a pair of experiments with shortest distance or most similarity in gene expression pattern are identified (Expl and Exp2 in Table 3a). A "composite experiment” is then constructed by averaging (thus the term average-linkage algorithm) all gene expression ratios (log-transformed) from two experiments. This is referred to as Expl-2 in the example. All distances from this composite experiment to all other experiments are then examined, and used to construct a smaller matrix, as shown in Table 3b. This procedure is repeated until the distance matrix is reduced to single element.
  • each merger is represented by a binary tree, and the length of each branch is indicative of the distance between two samples, such as those given in Table 3a and 3b.
  • ⁇ and ⁇ are means and standard deviations, respectively, for Mi gene expression profile and z ' th template pattern across iVtime points.
  • ⁇ * indexes to somewhere near the best-fit template index I , but adjusted according to the similarity of its neighboring templates given the pre-defined order.
  • the aforementioned template-based algorithm provides three parameters for each gene for a given order of template sequence. They are ⁇ fo p k , I * , and F k for template index, best Pearson's similarity measure, the best fit template, and the predicted fold-change derived from the best fitted template, respectively.
  • a data-mining tool was developed to investigate the experimental model of hormone tumor therapy in vivo.
  • This model consists of independent clones of the same tumor undergoing different fates.
  • a method for comparing the cDNA microarray data across the independent clones that undergo different fates was therefore needed to identify genes with temporal expression profiles associated with the response / phenotype of therapy in vivo. Filtering variables at different stringencies was used to mine the data to identify the genes that change most significantly in a manner that best reflects the temporal nature of the phenotypic changes observed during androgen ablation therapy.
  • Variables assigned to each profile to facilitate data mining and clustering the maximum correlation coefficient; the cluster location; and the fold change in ratio.
  • the link to phenotype was accomplished by filtering the "maximum correlation coefficient" to templates that best describe the temporal profile of the phenotype. This also allows noise to be filtered out.
  • Clustering was accomplished by sorting the "cluster location". This organized the genes/templates so genes with similar profiles are clustered together. The cluster position was calculated by the weighted average of the three template positions that had the best correlation. For example, two different gene-expression profiles may have a maximum correlation coefficient for template number 5, but have different cluster locations such as 4.6 and 5.4, allowing for a continuum of locations between the templates.
  • the data can be mined further by filtering the data for "Fold Change” and "Fold Change to Recerience.” In this way, the amplitude of the change can be used to increase the stringency of the filter and identify genes that change most significantly.
  • By filtering for Fold Change to Recurrence genes were isolated that not only have a kinetic that fits the regression phenotype, but that are also restored in recurrent tumors.
  • the prostate tissue microarray was constructed from paraffin embedded tumor tissue and benign control specimens.
  • the tissue microarray permits analysis of up to 600 specimens simultaneously, greatly facilitating high throughput analysis of molecular markers in cancer tissue.
  • the prostate tissue specimens were obtained from the Institutes of Pathology, University of Basal (Switzerland) and Tampere University Hospital (Finland).
  • One pathologist reviewed all original tissue sections.
  • the tissue microarray representing prostate neoplasm progression was constructed with 0.6 mm tissue cores and precisely arranged on a standard glass slide as described by Kononen et al. (Nat. Med 4:844-847, 1998).
  • the microarray tissue samples represented 45 Benign Prostate Hypertrophy (BPH), 60 prostate intraepithelial neoplasia (PIN), 264 primary tumors, 134 hormone refractory tumors, and 41 metastatic tumors. Additionally, the tissue microarray contained 28 xenograft CRW tumor specimens and several other xenografts. Protocols for preparing prostate tissue microarrays are provided for instance in Bubendorf et al. , JNatl Cancer Inst 91:1758-1764, 1999 and Bubendorf et al. , Cancer Res. 59:803-806, 1999.
  • the tissue microarray facilitates simultaneous application of molecular diagnostic techniques, such as immunohistochemistry.
  • Antigen retrieval was performed by treatment in a pressure cooker for 30 minutes.
  • Standard indirect immunoperoxidase procedures were used for immunohistochemistry (Envision Plus, DAKO).
  • a monoclonal mouse antibody (1:1000, Transduction Laboratories, Lexington, KY) was used for detection of S100P.
  • the reactions were visualized with diaminobenzidine as a chromagen.
  • the nuclear and cytoplasm staining intensity were classified into three groups (negative, weak and strong staining) in duplicate by two pathologists (Hostetter, G. and Ferhle, W.).
  • Other antibodies used for IHC on tissue arrays included Ki67, SI OOP, FKBP5, PCNA, PSA, AR.
  • AntiSlOOP-A C ATGCCCATGGCTGTCTCTAGTTCCGTCATGGTGCTAG (SEQ ID NO: 3);
  • AntiSlOOP-B CGTGCTGCCCTCGCTGCCCGAATATCGGGAAAAGACGTCTATGAT (SEQ ID NO: 4);
  • AntiSlOOP-C TTATCCACGGCATCCTTGTCTTTTCCACTCTGCAGG (SEQ ID NO: 5); AntiSlOOP-D: TCCACCTGGGCATCTCCATTGGCGTCCAGGTCCTTGAGCA (SEQ ID NO: 6); AntiSlOOP-E: AGACGTGATTGCAGCCACGAACACGATGAACTCACTGAAG (SEQ ID NO:7); AntiSlOOP-F: CATTTGAGTCCTGCCTTCTCAAAGTACTTGTGACAGGC (SEQ ID NO: 8); AntiSlOOP-G: GGGACCATGGCTCTGCAGGAATCTGTGACATCTCCAGGGC (SEQ ID NO: 9); and AntiSlOOP-H: GCTCAGCCTAGGGGAATAATTGCCAACAAACACTTTTGGGAAGCC (SEQ ID NO: 10).
  • the scatter plot shown in FIG 2 was generated for one primary and one recurrent tumor, to demonstrate the correlation between the samples (low variance as indicated by lack of scatter) and to
  • Results Therapy-associated phenotype The CWR22 xenografts were serially passed in nude mice.
  • Ki67 staining of tliese tissues indicated that the number of cells that were proliferating (Ki67 staining positive) decreased gradually, reaching a minimum at day eight, where almost all cells were negative for Ki67 staining (FIG ID). All the recurrent tumors had Ki67 staining that was higher than the primary tumors and approached 100% of the cells. Similar results were seen with PCNA (Myers et al. , J Urol. 161 :945-949, 1999), indicating that proliferation was shut off in the recurrent tumors but the entire tumor did not respond fully to the therapy until four to eight days.
  • AR Androgen receptor
  • IHC immunohistochemical staining
  • cDNA microarrays containing sequence-verified clones that represent 6605 unique genes were constructed. Fluorescence intensity ratios relative to the standard reference (LNCap) were generated for all the genes for each experiment. Only genes that were expressed at a significant level above the background in all the experiments were used in the analysis. This intensity cutoff across all the samples (4 primary tumors, 7 time points, and 4 recurrent tumors) resulted in 2648 genes that were expressed at a sufficient level to give reliable data.
  • LNCap standard reference
  • a hierarchical, unsupervised approach was first used to cluster the data. Although it was difficult to identify clusters with the small number of samples in the time course, a cluster containing 135 genes that decreased in gene expression after therapy and then were re-expressed after recurrence was identified (listed in Table 5; 143 sequences are listed, eight of which are duplicates). Other genes that decreased and were subsequently re-expressed were also observed, but did not cluster together because of differences in kinetics.
  • Template clustering (as described above) was developed and used to organize the cDNA microarray data according to expression kinetics during the course of therapy from primary to 16 days following castration. This is a supervised clustering approach, in which a correlation of each gene to a set of templates selected to reflect the temporal nature of the phenotype is calculated. Templates with the best (maximum) expression profile were then utilized to calculate a ranking (cluster location), to sort the genes based on their kinetics. The recurrent (R) time point was not used to calculate correlation to the templates. Color-coding was used to reflect the change that occurs in expression (not ratio) during the therapy, red being the maximum point and green the minimum point.
  • a database of the gene identification information and template clustering parameters such as cluster location (sorting rank), maximum correlation coefficient, and calculated fold change from lowest to highest point of expression during the first six time points was constructed.
  • This database facilitates mining for HRPC-related genes by variable stringency query-based searches based on (1) amplitude of change during therapy, (2) specifics of kinetics such as early or late increase or decrease, and (3) extents of correlation to the phenotype templates. Filtering for higher "maximum correlation coefficient" allows the (1) selection of profiles that are more strongly associated with the therapy and (2) the elimination of noise in the data generated by large expression ratio differences due to tumor differences or experimental artifact.
  • the genes were sorted and plotted according to the template that their expression most closely resembled.
  • Unsupervised gene clustering was first used to find genes with similar gene expression profiles.
  • the plot shown in FIG 1A illustrates all 2648 genes that showed sufficiently high expression levels to be used in this analysis, organized in hierarchical clusters, as demonstrated by the dendogram on the left.
  • Therapy time points are (from left to right) numbered 1 through 7 and represent primary (P), 0.5 days, 2 days, 4 days, 8 days, 16 days post castration and recurrent (R), respectively; genes are stacked vertically.
  • a group of 139 genes that decrease after castration and then are re-expressed in the recurrent tumors forms a definable cluster (as indicated by the close branching in the dendogram); this group is outlined by a rectangle.
  • Grey-coding/shading in FIG 1A reflects actual ratio to the reference as indicated by the key below the cluster.
  • Template clustering followed by filtering for greater than two-fold ratio difference during therapy response and for profiles that have at least a 0.7 maximum correlation coefficient to any of the 12 templates resulted in 604 genes (listed in Table 1).
  • the plot in FIG IB illustrates supervised- template based clustering of these 604 genes.
  • the order of sorting is determined by the template for which each genes' expression is best correlated to, as indicated on the left of the cluster and described in the methods.
  • Color-coding is usually used to represent the relative transcript expression ratio, as measured by cDNA microarray analysis. Red customarily indicates the maximum point in gene expression, green the minimum, and levels closer to the mean approach black. These colors have been converted to shades of grey, as shown in the key below the cluster.
  • the 604 genes are stacked/clustered vertically for each of the time points in the experiment, organized from left to right and labeled 1 through 7 as for FIG 1A. For each of the first six time points, a correlation coefficient to each of the 12 templates was calculated for the expression profiles of the 2648 sufficiently expressed genes. The average of the three maximum correlation coefficients was used to calculate a precise cluster location that reflects the association of that gene to a particular profile, represented by a continuum of templates guided by the 12 shown in FIB IB.
  • each gene expression profile color plot is a number that corresponds to the fold change ( ⁇ ) in ratio between the first six time points.
  • the gene cluster order was determined by the order of templates and cluster location, as described.
  • Gene identifiers shown in FIG 1C include IMAGE clone ID and the current unigene cluster number, name and description.
  • Genes that have previously been reported to be direct targets of the androgen receptor are shown in bold text, and include the following: malate dehydrogenase 1, NAD (soluble) (MDH1), proliferating cell nuclear antigen (PCNA), brain-specific alpha tubulin (TUBA3), ornithine decarboxylase 1 (ODC1), lactate dehydrogenase A (LDHA), a disintegrin and metalloproteinase domain (ADAM9), v-fos FBJ murine osteosarcoma viral (FOS), and andromedulin (ADM).
  • MDH1 malate dehydrogenase 1
  • PCNA proliferating cell nuclear antigen
  • TUBA3 brain-specific alpha tubulin
  • ODC1 ornithine decarboxylase 1
  • LDHA lactate dehydrogenase A
  • ADAM9 a disintegrin and metalloproteinase domain
  • ADAM9 v-fos
  • the template based, supervised cluster of 59 genes (filtered for greater than three-fold change; greater than 0.8 maximum correlation coefficient; only decreasing templates) (listed in Table 2, and labeled "Decreasing"), representing the genes with the largest decrease after castration, had extensive overlap (51 of 59 genes in common, Image Clone ID numbers 767817, 840567, 785778, 283315, 814117, 45233, 29063, 684655, 134719, 24145, 898062, 855487, 42059, 53316, 273546, 293727, 49352, 725454, 856427, 789182, 531319, 149013, 47833, 626716, 789204, 453107, 416833, 711768, 451907, 66406, 129865, 796646, 204214, 898286, 814701, 416833, 43550, 435076, 207358, 769921, 204257, 207288, 811015
  • the unsupervised cluster was not inclusive of all the genes that responded to the therapy (since it only contained 139 of the 305 genes with a profile that fit a decreasing template with a minimum two-fold difference and >0.7 max. correlation coefficient). Furthermore, although supervised clustering did identify that at least 74 genes increased by more than three-fold and that fit an increasing template with more than 0.8 correlation coefficient, it was difficult to identify a coherent unsupervised cluster of increasing genes.
  • a temporal gene expression program (fingerprint), or cluster of genes, was identified that had the largest expression decrease after castration and the best correlation to a decreasing temporal template (FIG 2).
  • the genes are plotted from early repressed genes on the top, and gradually being repressed at later time points down the list to the bottom genes that had a late onset repression.
  • Investigation of the genes in this list of 59 revealed at least eight genes previously known to be stimulated by androgens, and probably direct targets of the AR.
  • the identification of these AR responsive genes in this cluster further substantiates the utility of template based gene clustering in identifying therapy response associated genes and suggests that other genes in this list may be previously unknown AR responsive genes.
  • genes in this cluster revealed that it is very rich in several important cell cycle regulators. These include genes known to be associated with cell growth of prostate cancer including PCNA, ornithine decarboxylase 1, c-fos, and tubulin. Most of the genes in this cluster however, are novel cell cycle regulators that were not previously associated with androgen ablation in prostate cancer. These include the following (Image ID Clone numbers in parentheses):
  • UBCH10 a cyclin-selective ubiquitin carrier that regulates the destruction of mitotic cyclins (769921); CDKN3, a CDK-2 associated dual phosphatase (700792);
  • CKS2 a kinase that activates CDC28 (725454); MAD2L1, which regulates mitotic checkpoints especially sensitive to kinetochore and spindle loss (814701);
  • CENPF centromere/kinetichore cell cycle protein (435076);
  • STK12 a chromosome associated kinase that plays an important role in centrosome duplication regulation, aneuploidy, and amplification (531319);
  • NEK2 a protein kinase that regulates G2-M transition (415089);
  • FKBP5 has been associated with the glucocorticoid receptor, and targeting of FKBP proteins has been shown to lead to deregulation of several signal transduction pathways.
  • transmembrane 4 superfamily member 1 Another gene that showed a large amplitude change after castration, with unknown consequence, is transmembrane 4 superfamily member 1 (7.1 fold decrease). Conversely, transmembrane 4 superfamily member 3 increased after castration (3.2 fold). Putative signaling molecule serine/threonine kinase 12 (7.0 fold decrease) and insulin induced gene 1 (8.1 fold decrease) also showed substantial expression level changes after castration. Like the known cell cycle regulators, the expression of all these other genes is restored in the recurrent tumors. It is likely that these genes mediate growth arrest after therapy, and tumor re-growth after development of therapy resistance, and therefore these genes are ideal drug target candidates. In addition, some important genes that changed but did not make the top 59 list include
  • S100P, ID3, PSA and c-myc mRNA which decreased by 5.2, 2.85, 2.77 and 3.01 fold respectfully during regression (FIG 2). These were not included in the primary list either because they did not meet the 3 fold cut-off, or because the maximum correlation coefficients were less than 0.8 (0.51 0.67, 0.71 and 0.50).
  • S100P and ID3 are especially good candidate drug targets because they are also over-expressed in recurrent CWR22R relative to their primary counterparts (Table 2).
  • This group includes: the UDP glycosyltransferase 2 family, polypeptide B15 and UGT2B4, sialyltransferase 1 (beta-galactoside alpha-2,6-sialytransferase), fatty-acid- Coenzyme A ligase, human metallothionein (MT)I-F gene, tumor suppressor PTEN, cadherin 3, placental-cadherin, gelsolin (amyloidosis, Finnish type), TAP binding protein (tapasin), and several other transcripts.
  • the increase in PTEN indicates that the AKT S6 kinase pathway may be inhibited following castration, suggesting that therapeutic intervention with rapamycin may mimic this inhibition in recurrent tumors.
  • Example 1 additional HRPC-related nucleic acid molecules were identified and further characterized. These HRPC-related nucleic acid molecules also show differences in expression during prostate cancer development, and particularly during hormone ablation therapy and subsequent progression to a hormone-refractory condition.
  • Methods and Material were essentially as described in Example 1, except that additional custom cDNA microarrays were used, constituting 6605 to 8000 elements (sequence verified clones from Research Genetics, Huntsville, Alabama), representing different (non-redundant) transcripts including 4032 to 7700 known (named) genes (Mousses et al, Funtional Genomics: Gene Expression Analysis by cDNA Microarrays Livesey and Hunt (eds). Oxford University Press: Oxford, pp. 113- 137, 2000.). All xenografts were analyzed at least twice. Either LNCap or CWR22R were used as a reference and labeled with Cy5.
  • the reference cDNA was simultaneously hybridized with Cy3 labeled test specimens on a cDNA microarray as previously described (Mousses et al, Funtional Genomics: Gene Expression Analysis by cDNA Microarrays Livesey FJ and Hunt SP (eds). Oxford University Press: Oxford, pp. 113-137, 2000.). Fabrication of the microarray slides, image generation, and the software used for the ratio analysis, and bioinformatics were as described above. Mousses et al, Funtional Genomics: Gene Expression Analysis by cDNA Microarrays Livesey and Hunt (eds). Oxford University Press: Oxford, pp. 113-137, 2000.
  • FIG 3A shows 30 genes (out of a total of 164 differentially expressed genes) that were most systematically altered in the recurrent tumors.
  • genes include SCYD1, S100P, CCND1, CRIP1, ISG15, SCNN1A, ZFP103, MAPKAPK2, UTG2B15, RABGGTA, NFKBIA, SLCYA5, AP3B2, PTPN2, FOXJl, and APOC1 (all upregulated) and FLJ23538, OXCT, PFKP, TNRC3, HXB, PFKP, OAT, PFKP, RFP, THBS 1 , LM04, MLD, CRYM, MME, HMGCS2, and SLC 12A2 (all downregulated).
  • macrolide drugs such as rapamycin and FK506
  • Rapamycin is a known inhibitor of the PI 3 K/AKT/FRAP pathway (Kunz et al, Cell, 73:585-596, 1993; Brunn et al, EMBOJ., 15:5256- 5267, 1996; Sekulic et al, Cancer Res., 60:3504-3513, 2000), and FK506 targets many of the same intracellular proteins as rapamycin.
  • Cancer cells exhibit greater than a 1000-fold (IC 50 ranging from ⁇ 1 nM to >10 mM) variability in their sensitivity to rapamycin, possibly reflecting mechanisms of intrinsic resistance (Hosoi et al, Mol. Pharmacol, 54:815-824, 1998). Cancer cells that have activated genes and pathways that signal through the PI3K/AKT/FRAP pathway may be particularly sensitive. For example, IGF-1 receptor activation is associated with the efficacy of rapamycin treatment in childhood sarcomas (Dilling et al., Cancer Res. 54:903-907, 1994).
  • Several growth factors and related genes that we observed to be overexpressed in the recurrent prostate cancers relative to the primary tumors could have similarly contributed to the activation of the PI3K/AKT/FRAP pathway and alterations in the rapamycin target expression.
  • rapamycin and FK506 bind similar intracellular targets but have different biological effects in hormone-refractory prostate cancer. These differences may be informative in elucidating those molecular pathways that are most critical for progression of prostate cancer. Rapamycin and FK506 both bind to FKBP12 (FK506-binding protein 12) (Sabers et al, J. Biol. Chem., 270:815-822, 1995; Liu et al, Cell 66:807-815, 1991).
  • Rapamycin-FKBP12 but not the FK-506-FKBP12 complex, inhibits FRAP (FKBP-Rapamycin Associated Protein), a member of the phosphoinositide-3 -kinase related kinases that regulate translation following mitogenic activation of the PI3K/AKT/FRAP pathway.
  • FK506, but not rapamycin inhibits calcineurin activity (Liu et al, Cell 66:807-815, 1991). This suggests that, of the many known and unknown targets of rapamycin and FK506, FRAP and the activity of the PI3K AKT pathway is a more likely candidate than calcineurin as a drug target in hormone-refractory prostate cancer.
  • transcriptional profiling can be used to identify candidate drugs for treatment of prostate cancer, and this approach generally, as well as the present findings more specifically, can be used for a basis of such treatment decisions.
  • SI OOP calcium binding protein
  • FKBP5 immunophilin-like proteins
  • FKBP5 in addition to being overexpressed by about two-fold on average, is one of the most repressed genes after castration. During recurrence, its expression is restored to higher levels than in the primary.
  • FKBP5 is a member of the large immunophilin chaperone proteins, which have been shown to interact with HSP90 and several steroid receptors. The expression of this protein not only appears to be regulated by the androgen receptor function, but also may affect androgen receptor activity by protein folding of the nascent receptor or by modulating its binding affinity to ligands.
  • inhibitors e.g, FK506 and rapamycin
  • immunophilins bind to immunophilins, resulting in either calcineurin inactivation and or the inhibition the phosphotidylinositol 3- kinase/PTEN/AKT/FRAP pathway (Zhong et al, Cancer Res. 60:1541-1545, 2000).
  • the phosphorylated substrates of this pathway include calcineurin and ties into the calcium signaling pathway.
  • HcB which regulates NFkB
  • NFAT NFAT
  • BAD are each substrates for this pathway and are all involved in regulation of cell survival.
  • Differential gene expression data disclosed herein indicate that such drugs such as rapamycin and FK506 could have a dual role in preventing androgen independent progression of prostate cancer, by both (1) blocking signal transduction from the phosphotidylinositol 3- kinasePTEN/AKT/FRAP pathway and (2) interfering with androgen receptor protein folding and assembly. This is an example of the differential gene expression discussed herein, to assist in selecting new therapies for treatment of primary and recurrent (ho ⁇ none-refractory) prostate cancer.
  • This example provides in-depth analysis of several HRPC-related genes, including illustrations of the clinical relevance of these genes in prostate cancer progression and staging.
  • High throughput molecular validation of candidate genes in clinical specimens was accomplished by using tissue microarray technology to assess the utility of these HRPC-related genes as biomarkers and drug targets.
  • tissue microarray in this fashion represents an important method to cross- validate data from experimental systems and human cancer, specimens.
  • Tissue microarray methods were carried out essentially as described above, and as known in the art; see, for instance, Kononen et al, Nat Med. 4(7):844-847, 1998) Clinical translation of novel gene products where an antibody does not exist can be detected on tissue microarrays using isotopic in situ hybridization (ISH) (Kononen et al. Nat Med. 4(7):844-847, 1998; Frantz et al, JPathol. 195(1):87-96, 2001)
  • ISH isotopic in situ hybridization
  • SI OOP protein overexpression was investigated by immunohistochemistry, in 440 human prostate cancer specimens at various stages of progression.
  • prostate cancer progression tissue microarray (Bubendorf et al, J Natl Cancer Inst 91:1758-1764, 1999 and Bubendorf et al, Cancer Res. 59:803-806, 1999). This array also contained about 50 different prostate cancer xenograft samples, including those used in the cDNA microarray experiments.
  • SI OOP mRNA was measured by three different methods in nine xenografts.
  • cDNA microarray ratios measure the expression of SI OOP transcript by the amount of cDNA hybridized relative to the standard reference.
  • Northern analysis with a PCR amplified fragment of the SI OOP against a blot of the same RNA used in the cDNA microarray analysis produced a fragment of expected size (-0.5 kb).
  • Northern hybridization bands were quantified using ImageQuant software from a scanned autoradiogram.
  • ISH mRNA in situ hybridization
  • FIG 4 shows that in at least the xenograft samples there is good concordance between Northern hybridization, cDNA microarray, and mRNA in situ on tissue microarray quantitation of SI OOP transcript levels. These mostly but not always correlate with immunohistochemical staining, hi at least a few cases, higher protein expression was observed with moderate levels of mRNA, indicating possible post-transcriptional regulation.
  • In situ mRNA hybridization was also used to quantitatively measure transcript levels on tissue microarray sections.
  • Immunohistochemical analysis of SI OOP protein expression in 440 human prostate cancer specimens at various stages of progression is shown in FIG 5.
  • An SI OOP- specific antibody was used to stain prostate tissue sections on a tissue microarray.
  • the staining intensity was scored by two pathologists, using a scale of from 0 to 4.
  • the results in FIG 5 show the percentage of cancers at each stage of prostate cancer progression that had strong staining (score of 3 or 4).
  • FIG 5 shows that the high expression of SI OOP protein is associated with progression in clinical prostate cancers, with increasing expression in refractory and metastatic disease.
  • FKBP5 was found to be expressed specifically in secretory cells of the normal prostate and in prostate cancer cells, but not in supporting stromal cells. Analysis of FKBP5 protein expression by IHC on the same prostate cancer tissue microarray as discussed above indicated that FKBP5 is expressed in the majority of prostate cancers, but an association with progression was not observed.
  • FKBP5 farnesoid protein
  • FKBP5 down-regulation does appear to be associated with therapeutic response, making it a candidate for therapeutic targeting in a large percentage of clinical tumors.
  • LM04 and CRYM genes were substantially down-regulated in the CWR22R tumors relative to primary CWR22, for mRNA ISH studies.
  • mRNA ISH on TMAs validated the relative expression levels seen by cDNA microarrays in the CWR22 xenograft specimens.
  • This analysis revealed a lower level of LM04 and CRYM expression in 17 recurrent CWR22R xenografts (pO.OOl) as compared to 19 primary CWR22 xenografts.
  • the xenografts on the tissue microarrays were also used to compare the measurement of mRNA by cDNA microarray and mRNA ISH on a tissue microarray.
  • mRNA ISH mRNA ISH-based detection of transcripts by inserting into the TMAs specimens that were originally used in the cDNA microarray analyses.
  • mRNA ISH was performed with several radioactively labeled oligonucleotide probes for different regions of the target genes.
  • CRYM and LM04 were down-regulated in clinical specimens from hormone-refractory tumors, which is in line with the cDNA microarray results in the CWR22 xenograft model system.
  • LM04 is a member of the LIM-only (LMO) subfamily of LIM domain-containing transcription factors that is expressed during embryonic development (Kenny et al, Proc. Natl. Acad. Sci. 95: 11257-11262, 1998) and Crystallin mu (CRYM) codes for a thyroid hormone binding protein (Kim et al, Proc. Natl. Acad. Sci. 89:9292-9296, 1992; Aoki et al, J. Invest. Dermatol. 115:402-405, 2000). Both had transcript levels that were negatively associated with clinical progression. A role in prostate cancer progression has previously not been reported for either of these genes. It is believed that the observations presented herein indicate that perturbation of these genes has a functional role in clinical prostate cancer progression and pathogenesis.
  • LMO LIM-only
  • CYM Crystallin mu
  • This example illustrates tissue microarray technology validation of the in vivo involvement of four new prostate cancer related genes.
  • Alterations in SI OOP, FKBP5, CRYM and LM04 genes are not only involved in the acquisition of androgen-independent growth and failure of therapy in prostate cancer xenografts but also with the progression of cancer in patients.
  • EXAMPLE 5 Targeting Candidate Genes with Known Drugs This example demonstrates the clinical effectiveness of selecting drug targets and genetic markers, indeed entire metabolic pathways, using the herein-disclosed HRPC-related genes. Several drugs were identified based on their known interaction with one or more of the HRPC-related genes or implicated pathways, and the activities of these drugs in controlling prostate cancer cell growth was examined.
  • LNCaP or CWR22R cells were trypsinized and plated at 0.5 x 10 5 cell/ml or 1 x 10 5 cell/ml respectively in 96-well culture plates. After 24 hours, cells were treated for 72 hours with serial twofold dilutions of compound. DMSO was added to the control wells. Cell viability was measured by the WST-8 assay (Dojindo Molecular Technologies Inc.).
  • the WST-8 [2- (2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonyl)-2H tetrazolium, monosodium salt] assay is based on the conversion of the tetrazolium salt WST-8 to highly water soluble formazan by viable cells (Tominaga et al, Anal. Commun. 36, 47-50, 1999).
  • the WST-8 reagent solution was added to each well. After incubation for three hours at 37 °C, the absorbance was measured at 450 nm with a reference wavelength at 630 nm. The experiments were performed in triplicate. The data are representative of three separate experiment.
  • a literature search coupled with a search of previous drug treatment data, was used to identify known compounds that could be used to target one or more of the 604 genes that changed at least two-fold following therapy response (FIG 1 and Table 1), or one or more of the 251 genes that were differentially expressed between primary and recurrent tumors (Table 6).
  • ID3 (clone: 756405) has recently been shown to be required for angiogenesis (Lyden et al, Nature 401:670-677, 1999). The inventors have also observed a decreased in thrombospondin (clone: 810512) (an angiogenesis inhibitor) during prostate cancer progression, suggesting that the expression of these two genes is changed in opposite directions in recurrence to achieve the same biological outcome, increased angiogenesis.
  • thrombospondin (clone: 810512) (an angiogenesis inhibitor) during prostate cancer progression, suggesting that the expression of these two genes is changed in opposite directions in recurrence to achieve the same biological outcome, increased angiogenesis.
  • ID3 (clone: 756405), but the inventors have observed in an independent set of cDNA microarray experiments that TSA, induced thrombospondin (clone: 810512) by as much as 8.6 fold in PC3M cells in vitro.
  • TSA works by an unknown mechanism, possibly by histone deacetylase inhibition resulting in altering transcription of a large number of genes.
  • TSA treated PC3M cells revealed targets that were similarly affected in the direction of the growth arrested xenografts. Both drugs reduced kallikrein 3 (prostate specific antigen) (clone 824568) by two-fold, possibly reflecting an inhibition of AR-dependent transcriptional activation.
  • Histone acetyltransferase 1 (clone: 745360)
  • acetyl- Coenzyme A acyltransferase (clone: 27848) are both decreased by about two-fold after castration, indicating that histone deacetylase inhibition might mimic this effect (growth suppression).
  • Rapamycin andFK506 Sirolimus are bacterial macrolides that are produced by fungi to suppress the growth of competing organisms. These drugs are immunosuppressants used extensively to prevent organ rejection. Although the two drugs are very similar both in structure and in their cellular targets, known as immunophilins (also called FKBP for FK506 binding proteins), the mechanism by which they cause immunosuppression is different. FK506 binds to immunophilins and the complex inhibits calcineurin in T-cells.
  • immunophilins also called FKBP for FK506 binding proteins
  • rapamycin-immunophilin complex inhibits signaling of the S6-kinase (clone: 204148, which also responds to castration) causing cell cycle arrest in T-cells.
  • S6-kinase clone: 204148
  • macro immunophilins that have been found to interact with steroid receptors, which may work though yet another mechanism to inhibit growth when complexed with these drugs.
  • Several drug targets identified in this study are involved in immunophilin pathways, suggesting that either FK506 or rapamycin may cause a growth inhibition of hormone refractory prostate cancer.
  • the first such candidate is a macroimmunophilin called FKBP5 (clone: 416833), one of the most strongly repressed genes in primary prostate CWR22 tumors after castration (FIG 2).
  • FKBP5 clone: 416833
  • FKBP5 mRNA expression is restored to levels higher than found in the primary tumors.
  • the availability of FKBP5 as a drug target was also confirmed using tissue microarray analysis. It is not clear if FKBP5 is required for the proliferation of CWR22R cells, but the expression of the FKBP5 transcript is associated with the proliferation phenotype.
  • FKBP5 is a large protein that associates with steroid receptors, such as the glucocorticoid receptor, through binding to HSP90. It is also possible that FKBP5 interacts with the AR.
  • Cyclin D mRNA was 2.5-fold higher in a pool of four recurrent tumors compared to a pool of four recurrent tumors.
  • Rapamycin has been shown to target and down-regulate cyclin D protein at both a transcriptional and post-transcriptional level (Hashemolhosseini et al, J. Biol. Chem. 273:14424-14429, 1998). Also, p27 had increased after castration by about 2-fold by day 8, and then went back down in the recurrent tumor. Rapamycin can increase p27 levels, making it a candidate for reversing the decrease seen in the recurrent CWR22R.
  • rapamycin effects on both cyclin D and p27 may be direct, but also may be mediated by inhibition of the phosphotidylinositol 3- kinase/PTEN/AKT/FRAP pathway.
  • Several gene expression changes have been identified herein that could converge to activate this pathway in recurrent tumors, further suggesting that this is a pathway necessary for androgen independent growth. For example, an increased was observed in expression of CDP-diacylglycerol synthase 1 (levels up to 2.77-fold higher in recurrent tumors).
  • CDP- diacylglycerol synthase 1 is a rate luniting enzyme in phosphotidylinositol 3 (PI3) production that has been shown to increase the amplitude and duration of PI3 signaling when overexpressed in model systems.
  • PTEN which is an inhibitor of this pathway, is increased during regression and re- expressed in the recurrent tumors further illustrating the importance of this pathway for proliferation of recurrent tumors. It has also been shown that rapamycin inhibits the translation of ornithine decarboxylase
  • ODC ODC transcripts by about 50% in epithelial cells.
  • ODC was repressed (3.8 fold) during CWR22 regression, but then re-expressed in the recurrent CWR22R (FIG 2).
  • FK506 has no effect on ODC transcript levels.
  • ODC and cyclin D are important stimulators of proliferation, indicating that rapamycin can be used to target these molecules and cause growth arrest in androgen independent CWR22R cells.
  • Rapamycin effectively arrested the CWR22R cells in vitro, however a complete inhibition was not accomplished at the highest concentration of FK506 (lO ⁇ M) (FIG 3D). It is believed that the interaction of rapamycin with FKBP5 and its other cellular receptor immunophilins blocks a pathway necessary for growth, while the interaction of FK506 and FKBP5 does not. It is difficult to predict the mechanism by which these drugs exert an effect on a cell, because they bind multiple cellular targets. In this case, several putative cellular targets are known for these two macrolide drugs and at least one, FKBP5, was both associated with the HRPC phenotype, and available in the relevant cells (FKBP5 protein is expressed in most clinical recurrent tumors). More specific inhibitors of FKBP5 activity can be used to elucidate the role FKBP5 plays in the growth of hormone refractory tumors.
  • This example illustrates the involvement of gene targets in pharmacological response to various emerging therapies.
  • Xenografts and Cell lines Fresh frozen tissue from CWR22 human prostate cancer xenografts (Pretlow et al, J. Natl. Cancer Inst. 85:394-398, 199)) was obtained from thirteen different mice at different stages of hormonal therapy and tumor progression (four primary untreated CWR22, five CWR22 therapy time points after 0.5 days, 2 days, 4 days, 8 days and 16 days after castration, and four independent hormone-refractory CWR22R strains). LNCaP (ATCC) and CWR22R (kindly provided by Dr.
  • FR901464 Flujisawa Pharmaceutical Co., Ltd., Ibaraki, Japan
  • Trichostatin A - TSA a histone deacetylase inhibitor; Sigma Chemical co. St. Louis, Missouri
  • DMSO a histone deacetylase inhibitor
  • cDNA Microarrays Custom cDNA microarrays were constructed consisting of 6605 to 8000 elements (sequence verified clones from Research Genetics, Huntsville, Alabama), representing different (non-redundant) transcripts including 4032 to 7700 known (named) genes (Mousses et al. in Functional Genomics, (eds. Livesey & Hunt) 113-137, Oxford University Press, Oxford, 2000).
  • All xenografts were analyzed at least twice using either LNCap or CWR22R Cy 5 labeled reference cDNA simultaneously hybridized with Cy 3 labeled CWR22 xenograft or CWR22R cell line cDNA on a cDNA microarray according to a previously described protocol (Mousses et al. in Functional Genomics, (eds. Livesey & Hunt) 113-137, Oxford University Press, Oxford, 2000). Fabrication of the microarray slides, image generation, and the software used for the ratio analysis, and bioinformatics was carried out essentially as described above.
  • Trichostatin A and FR901464 (an experimental drug found to inhibit the growth of a human solid tumor grown in mice and murine solid tumors; Nakajima et al, J. Antibiot. 49: 1204- 1211, 1996) were selected for in vitro testing in CWR22R cells based on previous pharmacogenomics analysis on PC3M cells, which suggested targeting of androgen independent growth associated genes. Rapamycin and FK506 were selected as drugs that also might target some of these candidates. To prioritize candidate gene targets that were not only associated with androgen independent growth but also involved in eliciting an effective drug response, cDNA microarray analysis of CWR22R gene expression was conducted during a time course of drug treatment in vitro.
  • the disclosed HRPC-related proteins can be expressed by standard laboratory technique. After expression, the purified HRPC-related protein or polypeptide may be used for functional analyses, antibody production, diagnostics, prognostics, and patient therapy, e.g., for prevention or treatment of prostate cancer (including hormone-refractory or metastatic prostate cancer). Furthermore, the DNA sequences encoding the disclosed HRPC-related proteins can be manipulated in studies to understand the expression of these genes and the function of their products, in particular how these HRPC-related proteins function in the control of or response to hormone- refractory prostate cancer.
  • Mutant forms of human HRPC-related proteins may be isolated based upon information contained herein, and may be studied in order to detect alteration in expression patterns in terms of relative quantities, tissue specificity and functional properties of the encoded mutant HRPC-related protein.
  • Partial or full-length cDNA sequences, which encode the subject protein, may be ligated into bacterial expression vectors.
  • E. coli Escherichia coli
  • Methods for expressing large amounts of protein from a cloned gene introduced into Escherichia coli (E. coli) or other prokaryotes may be utilized for the purification, localization, and functional analysis of proteins.
  • fusion proteins consisting of amino terminal peptides encoded by a portion of the E. coli lacZ or trpE gene linked to an HRPC-related protein may be used to prepare polyclonal and monoclonal antibodies against these proteins. Thereafter, these antibodies may be used to purify proteins by immunoaffinity chromatography, in diagnostic assays to quantitate the levels of protein and to localize proteins in tissues and individual cells by immunofluorescence.
  • Intact native protein may also be produced in E. coli in large amounts for functional studies. Methods and plasmid vectors for producing fusion proteins and intact native proteins in bacteria are described in Sambrook et al. (Sambrook et al, In Molecular Cloning: A Laboratory Manual, Ch. 17, CSHL, New York, 1989). Such fusion proteins may be made in large amounts, are easy to purify, and can be used to elicit antibody response. Native proteins can be produced in bacteria by placing a strong, regulated promoter and an efficient ribosome-binding site upstream of the cloned gene. If low levels of protein are produced, additional steps may be taken to increase protein production; if high levels of protein are produced, purification is relatively easy.
  • Fusion proteins for instance fusions that incorporate a portion of a HRPC-related protein, may be isolated from protein gels, lyophilized, ground into a powder and used as an antigen.
  • the DNA sequence can also be transferred from its existing context to other cloning vehicles, such as other plasmids, bacteriophages, cosmids, animal viruses and yeast artificial chromosomes (YACs) (Burke et al., Science 236:806-812, 1987).
  • other cloning vehicles such as other plasmids, bacteriophages, cosmids, animal viruses and yeast artificial chromosomes (YACs) (Burke et al., Science 236:806-812, 1987).
  • vectors may then be introduced into a variety of hosts including somatic cells, and simple or complex organisms, such as bacteria, fungi (Timberlake and Marshall, Science 244:1313-1317, 1989), invertebrates, plants (Gasser and Fraley, Science 244:1293, 1989), and animals (Pursel et al, Science 244:1281-1288, 1989), which cell or organisms are rendered transgenic by the introduction of the heterologous HRPC-related cDNA.
  • somatic cells such as bacteria, fungi (Timberlake and Marshall, Science 244:1313-1317, 1989), invertebrates, plants (Gasser and Fraley, Science 244:1293, 1989), and animals (Pursel et al, Science 244:1281-1288, 1989)
  • the cDNA sequence may be ligated to heterologous promoters, such as the simian virus (SV) 40 promoter in the pSV2 vector (Mulligan and Berg, Proc. Natl. Acad Sci. USA 78:2072-2076, 1981), and introduced into cells, such as monkey COS-1 cells (Gluzman, Cell 23:175-182, 1981), to achieve transient or long-term expression.
  • SV simian virus
  • the stable integration of the chimeric gene construct may be maintained in mammalian cells by biochemical selection, for example with neomycin (Southern and Berg, J. Mol. Appl. Genet. 1:327-341, 1982) or mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981).
  • DNA sequences can be manipulated with standard procedures such as restriction enzyme digestion, fill-in with DNA polymerase, deletion by exonuclease, extension by terminal deoxynucleotide transferase, ligation of synthetic or cloned DNA sequences, site-directed sequence- alteration via single-stranded bacteriophage intermediate or with the use of specific oligonucleotides in combination with PCR.
  • the cDNA sequence (or portions derived from it) or a mini gene (a cDNA with an intron and its own promoter) may be introduced into eukaryotic expression vectors by conventional techniques. These vectors are designed to permit the transcription of the cDNA in eukaryotic cells by providing regulatory sequences that initiate and enhance the transcription of the cDNA and ensure its proper splicing and polyadenylation. Vectors containing the promoter and enhancer regions of the SV40 or long terminal repeat (LTR) of the Rous Sarcoma virus and polyadenylation and splicing signal from SV40 are readily available (Mulligan et al, Proc. Natl. Acad. Sci.
  • LTR long terminal repeat
  • the level of expression of the cDNA can be manipulated with this type of vector, either by using promoters that have different activities (for example, the baculovirus pAC373 can express cDNAs at high levels in S. frugiperda cells (Summers and Smith, In Genetically Altered Viruses and the Environment, Fields et al.
  • some vectors contain selectable markers such as the gpt (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981) or neo (Southern and Berg, J. Mol. Appl. Genet. 1:327-341, 1982) bacterial genes. These selectable markers permit selection of transfected cells that exhibit stable, long-term expression of the vectors (and therefore the cDNA).
  • the vectors can be maintained in the cells as episomal, freely replicating entities by using regulatory elements of viruses such as papilloma (Sarver et al, Mol. Cell Biol. 1 :486, 1981) or Epstein-Barr (Sugden et al, Mol. Cell Biol.
  • the transfer of DNA into eukaryotic, in particular human or other mammalian cells is now a conventional technique.
  • the vectors are introduced into the recipient cells as pure DNA (transfection) by, for example, precipitation with calcium phosphate (Graham and vander Eb, Virology 52:466, 1973) or strontium phosphate (Brash et al, Mol. Cell Biol. 7:2013, 1987), electroporation (Neumann et al, EMBO J 1 :841, 1982), lipofection (Feigner et al, Proc. Natl. Acad. Sci USA 84:7413, 1987), DEAE dextran (McCuthan et al, J. Natl. Cancer Inst.
  • the cDNA, or fragments thereof can be introduced by infection with virus vectors.
  • Systems are developed that use, for example, retroviruses (Bernstein et al, Gen. Engr'g 7:235, 1985), adenoviruses (Ahmad et al, J. Virol. 57:267, 1986), or Herpes virus (Spaete et al, Cell 30:295, 1982).
  • MB1 encoding sequences can also be delivered to target cells in vitro via non-infectious systems, for instance liposomes.
  • eukaryotic expression systems can be used for studies of HRPC-related nucleic acids (such as those listed in Table 1) and mutant forms of these molecules, as well as HRPC-related proteins and mutant forms of these protein. Such uses include, for example, the identification of regulatory elements located in the 5' region of HRPC-related genes on genomic clones that can be isolated from human genomic DNA libraries.
  • the eukaryotic expression systems may also be used to study the function of the normal HRPC-related proteins, specific portions of these proteins, or of naturally occurring or artificially produced mutant versions of HRPC-related proteins.
  • the expression vectors containing HRPC-related gene sequence or cDNA, or fragments or variants or mutants thereof can be introduced into human cells, mammalian cells from other species or non-mammalian cells as desired.
  • the choice of cell is determined by the purpose of the treatment.
  • monkey COS cells Gluzman, Cell 23:175- 182, 1981
  • Chinese hamster ovary CHO
  • mouse NIH 3T3 fibroblasts or human fibroblasts or lymphoblasts may be used.
  • the present disclosure thus encompasses recombinant vectors that comprise all or part of a HRPC-related gene or cDNA sequence, for expression in a suitable host.
  • the HRPC-related nucleic acid sequence is operatively linked in the vector to an expression control sequence to form a recombinant DNA molecule, so that the HRPC-related polypeptide can be expressed.
  • the expression control sequence may be selected from the group consisting of sequences that control the expression of genes of prokaryotic or eukaryotic cells and their viruses and combinations thereof.
  • the expression control sequence may be specifically selected from the group consisting of the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, the early and late promoters of SV40, promoters derived from polyoma, adenovirus, retrovirus, baculovirus and simian virus, the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, the promoter of the yeast alpha-mating factors and combinations thereof.
  • the host cell which may be transfected with the vector of this disclosure, may be selected from the group consisting of E. coli, Pseudomonas, Bacillus subtilis, B. stear other moph ⁇ lus or other bacilli; other bacteria; yeast; fungi; insect; mouse or other animal; or plant hosts; or human tissue cells.
  • HRPC-related DNA sequences similar systems are employed to express and produce the mutant product.
  • fragments of a HRPC-related protein can be expressed essentially as detailed above. Such fragments include individual HRPC- related protein domams or sub-domains, as well as shorter fragments such as peptides. HRPC-related protein fragments having therapeutic properties may be expressed in this manner also.
  • a reduction of HRPC-related protein expression in a transgenic cell may be obtained by introducing into cells an antisense construct based on a HRPC-related protein encoding sequence, such as a cDNA or gene sequence or flanking regions thereof of any one of the proteins listed in Table 1, Table 4, or elsewhere herein.
  • a nucleotide sequence encoding a HRPC-related protein e.g.
  • cartilage glycoprotein-39 (CHI3L1), S-100P PROTEIN (S100P), CX3C chemokine/fractalkine (SCYD1), adenylate kinase 1 (AK1), forkhead transcription factor HFH-4 (HFH-4) (FKHL 13), UDP glucuronosyltransferase precursor (UGT2B 15), Pleiotrophin (heparin binding growth factor 8) (PTN), heat shock 27kD protein 2/Alpha-B-crystallin (HSP27), Proteasome (prosome, macropain) subunit, beta type, 5 (PSMB5), Inhibitor of NFKB (NFKBIA), interferon-induced 17 kD protein (ISG15), MAP kinase activated protein kinase 2 (MAPKAPK2), signal transduction protein (SH3 containing) (EFS2), hkf-1 Zinc finger protein (ZFP103), chromosome
  • the introduced sequence need not be a full-length human HRPC-related cDNA or gene, and need not be exactly homologous to the equivalent sequence found in the cell type to be transformed. Generally, however, where the introduced sequence is of shorter length, a higher degree of homology to the HRPC-related sequence likely will be needed for effective antisense suppression.
  • the introduced antisense sequence in the vector may be at least 30 nucleotides in length, and improved antisense suppression will typically be observed as the length of the antisense sequence increases.
  • the length of the antisense sequence in the vector advantageously may be greater than 100 nucleotides.
  • antisense RNA molecules bind to the endogenous mRNA molecules and thereby inhibit translation of the endogenous mRNA.
  • Ribozymes are synthetic RNA molecules that possess highly specific endoribonuclease activity. The production and use of ribozymes are disclosed in U.S. Patent No. 4,987,071 to Cech and U.S. Patent No. 5,543,508 to Haselhoff. The inclusion of ribozyme sequences within antisense RNAs may be used to confer RNA cleaving activity on the antisense RNA, such that endogenous mRNA molecules that bind to the antisense RNA are cleaved, which in turn leads to an enhanced antisense inhibition of endogenous gene expression.
  • dominant negative mutant forms of the disclosed HRPC-related sequences may be used to block endogenous activity of the corresponding gene products.
  • siRNAs small inhibitory RNA molecules
  • this disclosure also encompasses siRNAs that correspond to an HRPC- related nucleic acid, which siRNA is capable of suppressing the expression or function of its cognate (target) HRPC-related protein.
  • methods of suppressing the expression or activity of an HRPC-related molecule using an siRNA are also encompassed.
  • Suppression of expression of an HRPC-related gene can be used, for instance, to treat, reduce, or prevent cell proliferative and other disorders caused by over-expression or unregulated expression of the corresponding HRPC-related gene.
  • suppression of expression of sequences disclosed herein as being up-regulated in hormone-refractory prostate cancer can be used to treat, reduce, or prevent progression to hormone-refractory prostate cancer.
  • Monoclonal or polyclonal antibodies may be produced to any of the disclosed HRPC-related proteins, or mutant forms of these proteins. Optimally, antibodies raised against these proteins, or peptides from within such proteins, would specifically detect the protein or peptide with which the antibodies are generated. That is, an antibody generated to the SI OOP protein (or another specified protein) or a fragment thereof would recognize and bind that protein and would not substantially recognize or bind to other proteins found in human cells.
  • an antibody specifically detects a designated protein can be made by any one of a number of standard immunoassay methods; for instance, the Western blotting technique (Sambrook et al, In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989).
  • Western blotting technique Standard Immunoblotting technique
  • total cellular proteins are extracted from cells (for example, human prostate) and electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel.
  • the proteins are then transferred to a membrane (for example, nitrocellulose) by Western blotting, and the antibody preparation is incubated with the membrane. After washing the membrane to remove non-specifically bound antibodies, the presence of specifically bound antibodies is detected by the use of an anti-mouse antibody conjugated to an enzyme such as alkaline phosphatase.
  • an enzyme such as alkaline phosphatase.
  • Application of an alkaline phosphatase substrate 5-bromo-4- chloro-3-indolyl phosphate/nitro blue tetrazolium results in the production of a dense blue compound by immunolocalized alkaline phosphatase.
  • Antibodies that specifically detect the designated protein will, by this technique, be shown to bind to the designated protein band (which will be localized at a given position on the gel determined by its molecular weight).
  • Non-specific binding of the antibody to other proteins may occur and may be detectable as a weak signal on the Western blot.
  • the non-specific nature of this binding will be recognized by one skilled in the art by the weak signal obtained on the Western blot relative to the strong primary signal arising from the specific antibody-protein binding.
  • Substantially pure HRPC-related protein or protein fragment (peptide) suitable for use as an immunogen may be isolated from transfected or transformed cells, as described above. Concentration of protein or peptide in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms per milliliter. Monoclonal or polyclonal antibody to the protein can then be prepared as follows:
  • Monoclonal Antibody Production by Hybridoma Fusion Monoclonal antibody to epitopes of a designated protein (such as a HRPC-related protein, including any one of those listed in Table 1) identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (Nature 256:495-497, 1975) or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein over a period of a few weeks. The mouse is then sacrificed, and the antibody-producing cells of the spleen isolated.
  • a designated protein such as a HRPC-related protein, including any one of those listed in Table 1
  • the spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess un-fused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media).
  • HAT media aminopterin
  • the successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued.
  • Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall (Meth. Enzymol. 70:419-439, 1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988).
  • Polyclonal antiserum containing antibodies to heterogeneous epitopes of a single protein can be prepared by ⁇ nmunizing suitable animals with the expressed protein (Example 7), which can be unmodified or modified to enhance immunogenicity.
  • Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than others and may require the use of carriers and adjuvant.
  • host animals vary in response to site of inoculations and dose, with either inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intrade ⁇ nal sites appear to be most reliable.
  • Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony et al. (In Handbook of Experimental Immunology, Wier (ed.) chapter 19. Blackwell, 1973). Plateau concentration of antibody is usually in the range of about 0.1 to 0.2 mg/ml of serum (about 12 ⁇ M). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher (Manual of Clinical Immunology, Ch. 42, 1980).
  • a third approach to raising antibodies against the subject HRPC-related proteins or peptides is to use one or more synthetic peptides synthesized on a commercially available peptide synthesizer based upon the predicted amino acid sequence of the desired HRPC-related protein or peptide.
  • Antibodies also may be raised against proteins and peptides related to HRPC as described herein by subcutaneous injection of a DNA vector that expresses the desired HRPC-related protein, or a fragment thereof, into laboratory animals, such as mice. Delivery of the recombinant vector into the animals may be achieved using a hand-held form of the Biolistic system (Sanford et al, Paniculate Sci. Technol. 5:27-37, 1987) as described by Tang et al. (Nature 356:152-154, 1992).
  • Expression vectors suitable for this purpose may include those that express the HRPC-related sequence under the transcriptional control of either the human ⁇ -actin promoter or the cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • Antibody preparations prepared according to these protocols are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they also can be used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample; or for immunolocalization of the corresponding HRPC-related protein.
  • antibodies for administration to human patients, antibodies, e.g., HRPC-related protein specific monoclonal antibodies (such as antibodies to the proteins encoded by the encoding sequences referred to in Table 1), can be humanized by methods known in the art. Antibodies with a desired binding specificity can be commercially humanized (Scotgene, Scotland, UK; Oxford Molecular, Palo Alto, CA). Alternatively, human antibodies can be produced. Methods for producing human antibodies are known in the art; see, for instance, Canevari et al, Int J Biol Markers 8:147-150, 1993 and Green, J Immunol Methods 231 : 11-23, 1999, for instance.
  • the HRPC-related nucleic acid molecules provided herein can be used in methods of genetic testing for neoplasms (e.g., prostate or other cancers) or predisposition to neoplasms owing to HRPC- related nucleic acid molecule deletion, genomic amplification or mutation, or over- or under- expression in comparison to a control or baseline.
  • a biological sample of the subject which biological sample contains either DNA or RNA derived from the subject, is assayed for a mutated, amplified or deleted HRPC-related nucleic acid molecule, or for over- or under expression of a HRPC-related nucleic acid molecule.
  • Suitable biological samples include samples containing genomic DNA or RNA (including mRNA), obtained from body cells of a subject, such as those present in peripheral blood, urine, saliva, tissue biopsy, surgical specimen, amniocentesis samples and autopsy material.
  • the detection in the biological sample of a mutant HRPC-related nucleic acid molecule, a mutant HRPC-related RNA, an amplified or homozygously or heterozygously deleted HRPC-related nucleic acid molecule, or over- or under-expression of a HRPC-related nucleic acid molecule may be performed by a number of methodologies.
  • HRPC-related nucleic acid molecules can be identified through polymerase chain reaction amplification of reverse transcribed RNA (RT-PCR) or DNA isolated from breast or other tissue, followed by direct DNA sequence determination of the products; single-strand conformational polymorphism analysis (SSCP) (for instance, see Hongyo et al, Nucleic Acids Res. 21:3637-3642, 1993); chemical cleavage (including HOT cleavage) (Bateman etal, Am. J. Med. Genet. 45:233-240, 1993; reviewed in Ellis et al, Hum. Mutat.
  • SSCP single-strand conformational polymorphism analysis
  • Oligonucleotides specific to normal or mutant MB1 sequences can be chemically synthesized using commercially available machines. These oligonucleotides can then be labeled radioactively with isotopes (such as 32 P) or non-radioactively, with tags such as biotin (Ward and Langer et al, Proc. Natl. Acad. Sci.
  • Gene dosage can be important in neoplasms; it is therefore advantageous to determine the number of copies of HRPC-related nucleic acids in biological samples of a subject, e.g., serum or prostate samples.
  • Probes generated from the disclosed encoding sequence of in HRPC- related nucleic acid molecules can be used to investigate and measure genomic dosage of the corresponding HRPC-related genomic sequence.
  • interphase FISH analysis of breast cancer cell lines can be carried out as previously described (Barlund et al, Genes Chromo. Cancer 20:372-376, 1997).
  • the hybridizations can be evaluated using a Zeiss fluorescence microscope.
  • the FISH can be performed as described in Kononen et al, Nat. Med. 4:844-847, 1998. Briefly, consecutive sections of the array are deparaffinized, dehydrated in ethanol, denatured at 74° C for 5 minutes in 70% formamide/2 x SSC, and hybridized with test and reference probes. The specimens containing tight clusters of signals or >3-fold increase in the number of test probe as compared to chromosome 17 centromere in at least 10% of the tumor cells may be considered as amplified. Microarrays can be constructed as described in WO99/44063 A2 and WO99/44062A1. C. Detection of mRNA Expression Levels
  • HRPC-related molecule Over- or under-expression of a HRPC-related molecule can also be detected by measuring the cellular level of HRPC-related nucleic acid molecule-specific mRNA.
  • mRNA can be measured using techniques well known in the art, including for instance Northern analysis, RT-PCR and mRNA in situ hybridization. Details of such procedures can be found, for instance, in Examples 1 and 3.
  • the nucleic acid-based diagnostic methods of this disclosure are predictive of proliferation, metastatic potential, cancer progression, and response to treatment in patients suffering from prostate carcinomas including hormone-refractory prostate carcinomas, and other solid tumors, carcinomas, sarcomas, and cancers.
  • Cells of any tumors that demonstrate abnormal levels (e.g., through genomic amplification, deletion, mutation, or other over- or under-expression) of nucleotide sequences that share homology with the HRPC-related nucleic acids disclosed herein are aggressive tumor cells, and result in decreased survival, increased metastasis, increased rates of clinical recurrence (such as recurrence after hormone ablation therapy), and overall worsened prognosis.
  • An alternative method of diagnosing, staging, detecting, or predicting hormone-related prostate cancer is to quantitate the level of one or more HRPC-related proteins in a subject, for instance in the cells of the subject.
  • This diagnostic tool is useful for detecting reduced or increased levels of HRPC-related proteins. Localization and/or coordinated expression (temporally or spatially) of HRPC-related proteins can also be examined using well known techniques.
  • the determination of reduced or increased HRPC-related protein levels, in comparison to such expression in a normal subject e.g. , a subject not having hormone-related prostate cancer or not having a predisposition developing this condition, disease or disorder, would be an alternative or supplemental approach to the direct determination of HRPC-related nucleic acid levels by the methods outlined above and equivalents.
  • Any standard immunoassay format e.g., ELISA, Western blot, or RIA assay
  • ELISA ELISA
  • Western blot or RIA assay
  • a difference in HRPC-related polypeptide levels is indicative of an abnormal biological condition such as neoplasia. Whether the key difference is an increase or a decrease is dependent on the specific HRPC-related protein under examination, as discussed herein.
  • Immunohistochemical techniques may also be utilized for HRPC-related polypeptide or protein detection and quantification.
  • a tissue sample may be obtained from a subject, and a section stained for the presence of a HRPC-related protein using the appropriate HRPC-related protein specific binding agent and any standard detection system (e.g., one which includes a secondary antibody conjugated to horseradish peroxidase).
  • any standard detection system e.g., one which includes a secondary antibody conjugated to horseradish peroxidase.
  • a biological sample of the subject which sample includes cellular proteins, is required.
  • a biological sample may be obtained from body cells, such as those present in peripheral blood, urine, saliva, tissue biopsy, amniocentesis samples, surgical specimens and autopsy material, particularly breast cells.
  • Quantitation of a HRPC- related protein can be achieved by immunoassay and the amount compared to levels of the protein found in healthy cells. A significant difference (either increase or decrease) in the amount of HRPC- related protein in the cells of a subject compared to the amount of the same HRPC-related protein found in normal human cells is usually about a 30% or greater difference.
  • Substantial under- or over- expression of one or more HRPC-related protein(s) may be indicative of neoplasia or a predilection to neoplasia or metastasis, and especially hormone-refractory prostate cancer.
  • the protein-based diagnostic methods as described herein are predictive of proliferation, metastatic potential, cancer progression, and response to treatment in patients suffering from prostate carcinomas including hormone-refractory prostate carcinomas, and other solid tumors, carcinomas, sarcomas, and cancers.
  • Cells of any tumors that demonstrate abnormal levels (e.g., through genomic amplification, deletion, mutation, or other over- or under-expression) of nucleotide sequences that share homology with the HRPC-related nucleic acids disclosed herein are aggressive tumor cells, and result in decreased survival, increased metastasis, increased rates of clinical recurrence (such as recurrence after hormone ablation therapy), and overall worsened prognosis.
  • Retroviruses have been considered a preferred vector for experiments in gene therapy, with a high efficiency of infection and stable integration and expression (Orkin et al, Prog. Med. Genet. 7: 130-142, 1988).
  • a full-length HRPC-related gene or cDNA can be cloned into a retroviral vector and driven from either its endogenous promoter or from the retroviral LTR (long terminal repeat).
  • Other viral transfection systems may also be utilized for this type of approach, including adenovirus, adeno-associated virus (AAV) (McLaughlin et al, J. Virol.
  • Vaccinia virus Moss et al, Annu. Rev. Immunol. 5:305-324, 1987
  • Bovine Papilloma virus Bovine Papilloma virus
  • Epstein-Barr virus Margolskee et al, Mol. Cell. Biol. 8:2837-2847, 1988
  • Recent developments in gene therapy techniques include the use of RNA-DNA hybrid oligonucleotides, as described by Cole-Strauss, et al. (Science 273:1386-1389, 1996). This technique may allow for site-specific integration of cloned sequences, thereby permitting accurately targeted gene replacement.
  • Such cationic liposomes can also be targeted to specific cells through the inclusion of, for instance, monoclonal antibodies or other appropriate targeting ligands (Kao et al, Cancer Gene Ther. 3:250-256, 1996).
  • gene therapy can be carried out using antisense or other suppressive constructs, the construction of which is discussed above (Example 8).
  • Kits are provided to determine the level (or relative level) of expression of one or more species of HRPC-related mRNA (/. e. , kits containing probes) or one or more HRPC-related protein (i.e., kits containing antibodies or other HRPC-related protein specific binding agents). Kits are also provided that contain the necessary reagents for determining gene copy number (genomic amplification or deletion), such as probes or primers specific for a HRPC-related nucleic acid sequence. These kits can each include instructions, for instance instructions that provide calibration curves or charts to compare with the determined (e.g., experimentally measured) values.
  • the nucleotide sequence of HRPC-related nucleic acid molecules disclosed herein, and fragments thereof, can be supplied in the form of a kit for use in detection of HRPC-related genomic amplification/deletion and/or diagnosis of progression to or predilection to progress to hormone- refractory prostate cancer.
  • a kit for use in detection of HRPC-related genomic amplification/deletion and/or diagnosis of progression to or predilection to progress to hormone- refractory prostate cancer an appropriate amount of one or more oligonucleotide primer specific for an HRPC-related-sequence is provided in one or more containers.
  • the oligonucleotide primers may be provided suspended in an aqueous solution or as a freeze-dried or lyophilized powder, for instance.
  • the container(s) in which the oligonucleotide(s) are supplied can be any conventional container that is capable of holding the supplied fo ⁇ n, for instance, microfuge tubes, ampoules, or bottles.
  • pairs of primers may be provided in pre-measured single use amounts in individual, typically disposable, tubes, or equivalent containers. With such an arrangement, the sample to be tested for the presence of HRPC-related genomic amplification/deletion can be added to the individual tubes and in vitro amplification carried out directly.
  • each oligonucleotide primer supplied in the kit can be any amount, depending for instance on the market to which the product is directed. For instance, if the kit is adapted for research or clinical use, the amount of each oligonucleotide primer provided likely would be an amount sufficient to prime several in vitro amplification reactions. Those of ordinary skill in the art know the amount of oligonucleotide primer that is appropriate for use in a single amplification reaction. General guidelines may for instance be found in Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990), Sambrook et al.
  • a kit may include more than two primers, in order to facilitate the in vitro amplification of
  • HRPC-related genomic sequences for instance a HRPC-related nucleic acid listed in Table 1, or the 5 ' or 3' flanking region thereof.
  • kits may also include the reagents necessary to carry out in vitro amplification reactions, including, for instance, DNA sample preparation reagents, appropriate buffers (e.g., polymerase buffer), salts (e.g., magnesium chloride), and deoxyribonucleotides (dNTPs). Written instructions may also be included.
  • appropriate buffers e.g., polymerase buffer
  • salts e.g., magnesium chloride
  • dNTPs deoxyribonucleotides
  • Kits may in addition include either labeled or unlabeled oligonucleotide probes for use in detection of the in vitro amplified sequences.
  • the appropriate sequences for such a probe will be any sequence that falls between the annealing sites of two provided oligonucleotide primers, such that the sequence the probe is complementary to is amplified during the in vitro amplification reaction (if it is present in the sample). It may also be advantageous to provided in the kit one or more control sequences for use in the in vitro amplification reactions.
  • the design of appropriate positive control sequences is well known to one of ordinary skill in the appropriate art.
  • kits for Detection of mRNA Expression similar to those disclosed above for the detection of HRPC-related genomic amplification/deletion can be used to detect HRPC-related mRNA expression levels (including over- or under-expression, in comparison to the expression level in a control sample).
  • kits include an appropriate amount of one or more of the oligonucleotide primers for use in, for instance, reverse transcription PCR reactions, similarly to those provided above, with art-obvious modifications for use with RNA.
  • kits for detection of HRPC-related mRNA expression may also include reagents necessary to carry out RT-PCR or other in vitro amplification reactions, including, for instance, RNA sample preparation reagents (including e.g, an RNAse inhibitor), appropriate buffers (e.g., polymerase buffer), salts (e.g., magnesium chloride), and deoxyribonucleotides (dNTPs).
  • RNA sample preparation reagents including e.g, an RNAse inhibitor
  • appropriate buffers e.g., polymerase buffer
  • salts e.g., magnesium chloride
  • dNTPs deoxyribonucleotides
  • Kits may in addition include either labeled or unlabeled oligonucleotide probes for use in detection of an in vitro amplified target sequence.
  • the appropriate sequences for such a probe will be any sequence that falls between the annealing sites of the two provided oligonucleotide primers, such that the sequence the probe is complementary to is amplified during the PCR reaction. It may also be advantageous to provided in the kit one or more control sequences for use in the in vitro amplification reactions.
  • the design of appropriate positive control sequences is well known to one of ordinary skill in the appropriate art.
  • kits may be provided with the necessary reagents to carry out quantitative or semi-quantitative Northern analysis of HRPC-related mRNA.
  • kits include, for instance, at least one HRPC-related sequence-specific oligonucleotide for use as a probe.
  • This oligonucleotide may be labeled in any conventional way, including with a selected radioactive isotope, enzyme substrate, co- factor, ligand, chemiluminescent or fluorescent agent, hapten, or enzyme.
  • Kits for Detection of HRPC-linked Protein or Peptide Expression Kits for the detection of HRPC-linked protein expression, for instance abnormal (over or under) expression of a protein encoded for by a nucleic acid molecule listed in Table 1, are also encompassed herein.
  • kits will include at least one target (HRPC-linked) protein (e.g., cartilage glycoprotein-39 (CHI3L1), S-100P PROTEIN (S100P), CX3C chemokine/fractalkine (SCYDl), adenylate kinase 1 (AK1), forkhead transcription factor HFH-4 (HFH-4) (FKHL13), UDP glucuronosyltransferase precursor (UGT2B 15), Pleiotrophin (heparin binding growth factor 8)
  • HRPC-linked protein e.g., cartilage glycoprotein-39 (CHI3L1), S-100P PROTEIN (S100P), CX3C chemokine/fractalkine (SCYDl), adenylate kinase 1 (AK1), forkhead transcription factor HFH-4 (HFH-4) (FKHL13), UDP glucuronosyltransferase precursor (UGT2B 15), Pleiotrophin (heparin
  • PPN heat shock 27kD protein 2/Alpha-B-crystallin
  • HSP27 Proteasome (prosome, macropain) subunit, beta type, 5 (PSMB5), Inhibitor of NFKB (NFKBIA), interferon-induced 17 kD protein (ISG15), MAP kinase activated protein kinase 2 (MAPKAPK2), signal transduction protein (SH3 containing) (EFS2), hkf-1 Zinc finger protein (ZFP103), chromosome condensation 1 (CHCl), CDP- diacylglycerol synthase (CDSl), gap junction protein, alpha 1, 43kD (connexin 43) (GJAl), cyclin Dl (CCND1), Inhibitor of DNA binding 3, helix-loop-helix protein (ID3), HI histone family, member2 (H1F2), Cytochrome B561 (CYB561), Cathepsin H (CTSH), calcin
  • the HRPC-linked protein specific binding agent and control may be contained in separate containers.
  • the kits may also include a means for detecting HRPC-related proteimagent complexes, for instance the agent may be detectably labeled. If the detectable agent is not labeled, it may be detected by second antibodies or protein A, for example, either of both of which also may be provided in some kits in one or more separate containers. Such techniques are well known.
  • kits include instructions for carrying out the assay. Instructions will allow the tester to determine whether HRPC-linked expression levels are elevated or reduced in comparison to a control sample. Reaction vessels and auxiliary reagents such as chromogens, buffers, enzymes, etc. may also be included in the kits.
  • the HRPC-related molecules disclosed herein can be used to identify compounds that are useful in treating, reducing, or preventing prostate cancer or development or progression of prostate cancer. These molecules can be used alone or in combination, for instance in sets of two or more that are linked to cancer or cancer progression.
  • a test compound is applied to a cell, for instance a test cell, and at least one HRPC-related molecule level and/or activity in the cell is measured and compared to the equivalent measurement from a test cell (or from the same cell prior to application of the test compound). If application of the compound alters the level and/or activity of a HRPC-related molecule (for instance by increasing or decreasing that level), then that compound is selected as a likely candidate for further characterization.
  • a test agent that opposes or inhibits an HRPC-related change is selected for further study, for example by exposing the agent to a hormone refractory prostate cancer cell in vitro, to determine whether in vitro growth is inhibited.
  • Such identified compounds may be useful in treating, reducing, or preventing prostate cancer or development or progression of prostate cancer.
  • the compound isolated will inhibit or inactivate a HRPC-related molecule, for instance those represented by the nucleic acids listed in Table 1.
  • Methods for identifying such compounds optionally can include the generation of a HRPC- related gene expression profile, as described herein.
  • Control gene expression profiles useful for comparison in such methods may be constructed from normal prostate tissue, primary prostate cancer tissue, prostate cancer tissue responding to androgen ablation therapy, and/or a hormone refractory prostate cancer tissue.
  • rapamycin has been herein identified as a compound that influences the levels of HRPC-related molecules, in particular certain of the nucleic acid molecules listed in for instance Table 1 (as discussed in more detail above). With the provision herein of this identification, the use of rapamycin as a treatment for HRPC is now enabled, as is the use of rapamycin derivatives or rapamycin-like compounds. It is believed that rapamycin can be used on its own as such a treatment, or can be used in combination with known or newly identified treatments for HRPC.
  • EXAMPLE 15 Gene Expression Profiles (Fingerprints) With the provision herein of methods for determining molecules that are linked to HRPC, and the provision of a large collection of such HRPC-linked molecules (as represented for instance by those listed in Table 1), gene expression profiles that provide information on the prostate cancer-state of a subject are now enabled.
  • HRPC-related expression profiles comprise the distinct and identifiable pattern of expression (or level) of sets of HRPC-related genes, for instance a pattern of high and low expression of a defined set of genes, or molecules that can be correlated to such genes, such as mRNA levels or protein levels or activities.
  • the set of molecules in a particular profile will usually include at least one that is represented by (or correlated to) the following Image ID Clones: 781047, 785778, 842968, 769921, 898286, 204214, 814701, 435076, 531319, 415089, 898062, 453107, 785707, 795936, 700792, 34778, 46182, 769921, 783697, 451907, 711768, 416833, 810711, 789204, 789182, 725454, 951142, 49352, 273546, 46717, 855487, 41117, 26578, 684655, 45233, 814117, 810552, 739511, 283315, and 897774.
  • Image ID Clones 781047, 785778, 842968, 769921, 898286, 204214, 814701, 435076, 531319, 415089, 898062
  • HRPC-related gene expression profiles more than one molecule corresponding to the Image ID Clones listed in Table 1 are included in the profile.
  • any subset of the molecules listed in Table 1 may be included in a single gene expression profile. Specific examples of such subsets include those molecules that show an increasing expression profile during prostate cancer progression, those that show a decreasing expression profile, those that are most highly correlated to a particular stage of prostate cancer progression, and so forth.
  • gene expression profiles may be further broken down by the manner of molecules included in the profile.
  • certain examples of profiles may mclude a specific class of HRPC-related molecules, such as those molecules involved in cell cycle control.
  • Particular profiles are specific for a particular stage of normal tissue (e.g., prostate tissue) growth or disease progression (e.g., progression of prostate cancer).
  • gene expression profiles can be established for a pre-prostate cancer tissue (i.e., normal prostate tissue), a primary prostate cancer tissue, a prostate cancer tissue responding to androgen ablation therapy, and a hormone refractory prostate cancer tissue.
  • a pre-prostate cancer tissue i.e., normal prostate tissue
  • a primary prostate cancer tissue i.e., a primary prostate cancer tissue
  • a prostate cancer tissue responding to androgen ablation therapy e.g., hormone refractory prostate cancer tissue.
  • Each of these profiles includes information on the expression level of at least one, but usually two or more, genes that are linked to prostate cancer (e.g., HRPC-related genes).
  • Such information can include relative as well as absolute expression levels of specific genes.
  • the value measured may be the relative or absolute level of protein expression, which can be correlated with a "gene expression level.”
  • Results from the gene expression profiles of an individual subject are often viewed in the context of a test sample compared to a baseline or control sample fingerprint.
  • the levels of molecules that make up a gene expression profile can be measured in any of various known ways, which may be specific for the type of molecule being measured.
  • nucleic acid levels such as direct gene expression levels, such as the level of mRNA expression
  • Protein levels may be measured using standard protein assays, using immunologic-based assays (such as ELISAs and related techniques), or using activity assays, for instance. Examples for measuring nucleic acid and protein levels are provided herein; other methods are well known to those of ordinary skill in the art.
  • HRPC-related gene expression profiles can be in array format, such as a nucleotide (e.g., polynucleotide) or protein array or microarray.
  • arrays to determine the presence and/or level of a collection of biological macromolecules is now well known (see, for example, methods described in published PCT application number US99/06860, describing hyproxia- related gene expression arrays).
  • array-based measurement methods an array may be contacted with polynucleotides (in the case of a nucleic acid-based array) or polypeptides (in the case of a protein-based array) from a sample from a subject.
  • the amount and/or position of binding of the subject's polynucleotides or polypeptides then can be determined, for instance to produce a gene expression profile for that subject.
  • Such gene expression profile can be compared to another gene expression profile, for instance a control gene expression profile from a subject having a known prostate-related condition.
  • the subject's gene expression profile can be correlated with one or more appropriate treatments, which may be correlated with a control (or set of control) expression profiles for stages of prostate cancer progression, for instance.
  • This disclosure provides the identification of HRPC-related molecules that exhibit alterations in expression during development of refractory prostate cancer, and expression 'fingerprints (profiles) specific for prostate cancer stages. It further provides methods of using these identified nucleic acid molecules, and proteins encoded thereby, and expression fingerprints or profiles, to predict and/or diagnose hormone-refractory prostate cancer, and to elect treatments for instance based on likely response.
  • These identified HRPC-related molecules also can serve as therapeutic targets, and can be used in methods for identifying, developing and testing therapeutic compounds, including for instance rapamycin derivatives, analogs, and mimetics. It will be apparent that the precise details of the methods described may be varied or modified without departing from the spirit of the described invention. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Abstract

The present disclosure provides hormone-refractory prostate cancer (HPRC)-related nucleic acid molecules and proteins useful for the detection of neoplasms, particularly prostate and more specifically hormone-refractory prostate cancers. Also provided are methods of using these biological materials in the diagnosis, staging, detection, and treatment of neoplasia, and particularly hormone-refractory prostate cancer.

Description

GENES RELATED TO DEVELOPMENT OF REFRACTORY PROSTATE CANCER
FIELD The present disclosure is generally related to diagnosing, prognosing, staging, preventing, and treating disease, particularly hormone refractory prostate cancer.
BACKGROUND
About 40 years ago it was observed that prostate cancer, like normal prostate, is often androgen dependent and androgen withdrawal induces growth regression. Various modes of androgen ablation therapy have since been used as the major treatment of advanced prostate cancer. Generally, chemical or physical reduction in serum androgens, or chemical blockage of their action, effectively arrests growth of local and metastatic prostate cancer in vivo. Unfortunately, in almost all patients, the regressed tumors eventually develop resistance to hormonal therapy and recur as aggressive androgen independent tumors that are hormone refractory and currently incurable.
Identification of the genes that regulate the therapeutically induced tumor regression, and the genes associated with resistance to therapy, are candidate targets that can be used for rational design of therapeutic interventions.
The clinical course of hormone therapy response and eventual recurrence can be modeled experimentally using CWR22 xenografts. CWR22 is an androgen dependent human prostate carcinoma that grows rapidly as a xenograft in male nude mice, regresses after castration, and eventually (in three to ten months) becomes recurrent and re-grows independently of androgens in castrated mice. Several groups have previously looked for differences in gene expression between the primary and recurrent CWR22 prostate cancer xenografts, and identified some candidate genes that can be used as biomarkers. It remains critical to make sure that findings from model systems are applicable in the clinical situation.
Molecular mechanisms involved in the regression of prostate cancer after androgen deprivation, as well as in the re-growth of androgen-independent tumors, remain poorly understood. There is a need to better understand patterns of gene expression that trigger prostate tumor regression and/or re-growth, as well as downstream genes that may serve as indicators of prostate cancer progression.
BRIEF SUMMARY OF THE DISCLOSURE
Embodiments of this disclosure provide a set of nucleic acid molecules the expression of which is altered in prostate cancer, more particularly nucleic acid molecules that show temporal expression changes during prostate cancer hormonal therapy and regression.
Provided herein in various embodiments are hormone-refractory prostate cancer (HPRC)- related nucleic acid molecules and polypeptides useful for the detection/diagnosis/staging and treatment of neoplasms, particularly prostate and more specifically hormone-refractory prostate cancers. Also provided are methods of using these biological materials in the diagnosis, staging, detection, and treatment of neoplasia, and particularly hormone-refractory prostate cancer.
The foregoing and other features and advantages of these and other embodiments will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
FIG 1A, IB, 1C, and ID are schematic diagrams that show relative expression and expression patterns of 2333 genes measured using cDNA microarray technology at various time points during prostate cancer development.
FIG 2 shows a scatter plot that was generated for one primary and one recurrent tumor. This plot demonstrates the correlation between the samples (low variance, as indicated by lack of scatter in the majority of the genes), and highlights the most differentially expressed genes (which are circled and named). FIG 3 shows lists of genes that were differentially expressed between primary and recurrent xenografts, along with related relative expression information. A set of 30 genes were most consistently differentially expressed (out of a total of 164 genes that changed 2-fold or more) in two independent experiments.
FIG.3A shows the 30 genes, ordered by degree of differential expression. The grey-shade coding reflects the relative gene expression ratio (normalized to the mean ratio for four primary tumors) for each of six different recurrent xenografts tumors (arranged in columns). For the six recurrent tumors, the mean expression ratio relative to the mean expression levels of the primary tumors is indicated in the "Mean" column. Additionally, the maximum ratio (Max.) for the upregulated genes, and the minimum ratio (Min.) for the downregulated genes is also indicated (left column). The "pool" column depicts the ratios of a direct cDNA microarray experiment where four primary tumors were pooled and compared to four recurrent tumors.
FIG.3B shows eight PI3/AKT FRAP pathway-related genes, the expression of which was associated with hormone-refractory cell growth (based on >two-fold induction of in the recurrent tumors relative to the primary level). Grey-shade-coded gene expression ratios as well as the mean are shown as in FIG. 3 A. The criteria for selecting these genes were i) a >two-fold change in the average ratio between primary and recurrent tumors (or during therapy) and ii) evidence from the literature suggesting the interaction of these gene products with macrolide drugs or their involvement in a rapamycin-sensitive pathway.
FIG. 3C shows four FK506-binding protein genes, which were associated with hormone refractory tumor growth (based on at least a two-fold response to therapy and were restored to greater than 80 % of primary levels in the recurrent tumors). Grey-shade-coded gene expression ratios are shown for each of four primary (P) tumors, four tumors regressing following therapy (T), and six recurrent tumors (R). The mean ratio of gene expression (relative to the primary tumors) is shown for tumors undergoing therapy.
FIG.3D is a graph showing cell viability after treatment with Rapamycin (solid lines) and FK506 (dotted lines). The effects of these drugs on the viability of the hormone refractory CWR22R cell line (marked with a "C") and LNCap (marked with an "L") was tested in vitro. The recurrent CWR22R cell line was highly sensitive to rapamycin (IC50 - 0.1 nM) and underwent cell death. In contrast, LNCap showed partial growth arrest without cell death, even at higher doses of rapamycin. FK506 did not have an effect on either cell line. ED50 in CWR22R cells for rRapamycin was 0.3 μM.
FIG 4 shows SI OOP mRNA levels measured by three different methods (cDNA microarray, mRNA ISH, and Northern hybridization analyses) in nine xenografts. The amount of S 1 OOP detected in each of these three methods was quantified and plotted in a line graph above the corresponding images. Absolute values were normalized to the three primary tumors with the lowest Northern hybridization levels.
FIG 5 is a bar graph showing the level of SI OOP protein expression in 440 human prostate cancer specimens at various stages of progression, measured by IHC staining. An SI OOP antibody was used to stain prostate tissue sections on a tissue microarray containing hundreds of prostate specimens from different steps of cancer development (from normal epithelium, BPH, and localized cancer to metastases and hormone refractory prostate cancer). The staining was scored by two pathologists, using a scale of 0 to 4. The results show the percentage of cancers at each progression stage that had strong (score of 3 or 4) IHC staining.
FIG 6 shows the results of analyses of specific gene targets involved in drug response. The top graphs (FIGs 6A and 6B) illustrate the dose response of CWR22R cell line viability in vitro with various emerging therapies (TSA, FR901464, rapamycin, RSD, FK506, and androgen withdrawal therapy); the levels of FKBP5 (FIG 6A) and VDUP1 (FIG 6B) are shown. A time course of treatment for each drug was analyzed by cDNA microarray and a database of the resulting data was mined to find genes that are involved in more than one therapeutic response. Specific examples are shown, including CRYM ATP1B2, OAT, QSCN6, GSN, PLU-1, GFPT2, ZCYTOR7, and VDUP1.
FIG 6C shows representative quantitative analyses for expression of the indicated genes at 0, 1, 3, 9, and 24 hours after treatment with the indicated drugs (0.3 μM TSA, 10 mM FR901464, 1 μM rapamycin, 1 μM FK506, and 1 μM RSD).
FIG 6D shows the expression levels for the same genes in primary, regressing, and recurrent tumors. SEQUENCE LISTING
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:
SEQ ID NOs: 1 and 2, also referred to as S100PF and S100PR respectively, are examples of oligonucleotides useful for amplifying an SlOOp probe sequence. SEQ ID NOs: 3-10 (AntiSlOOP-A, -B, -C, -D, -E, -F, -G, and -H respectively, are examples of oligonucleotides useful for mRNA in situ hybridization.
DETAILED DESCRIPTION
I. Abbreviations AR androgen receptor
LNCap prostate cancer cell line (developed by Dr. Leland Chung)
HRPC hormone refractory prostate cancer
ED50 50% Effective Dose
ISH in situ hybridization IHC immunohistochemical
MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide)
TSA trichostatin A
PCNA proliferating cell nuclear antigen
II. Terms
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182- 9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
In order to facilitate review of the various embodiments, the following explanations of certain terms are provided:
Analog, derivative or mimetic: An analog is a molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure, such as a difference in the length of an alkyl chain, though the change need not be an incremental change in length of a chain), a molecular fragment, a structure that differs by one or more functional groups, a change in ionization. Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in Remington (The Science and Practice of Pharmacology, 19th Edition (1995), chapter 28). A derivative is a biologically active molecule derived from the base (parental) structure. A mimetic is a biomolecule that mimics the activity of another biologically active molecule. Biologically active molecules can include chemical structures that mimic the biological activities of a compound, for instance rapamycin or more generally macrolides (the basic tri-cyclic structural group that includes rapamycin). Rapamycin derivatives (including metabolic derivatives), analogs, and mimetics are disclosed, for instance, in USPN 5,508,398; Kuhn et al., J. Med. Chem. 44:2027-2034, 2001; Dickman etal, Bioorg. Med. Chem. Lett 10:1405-1408, 2000; Streit etal, Drug Metab. Dispos. 24:1272-1278, 1996; and Wong et al, J. Antibiot. (Tokyo) 51:487-491, 1998. Antisense, Sense, and Antigene: Double-stranded DNA (dsDNA) has two strands, a 5' -> 3' strand, referred to as the plus strand, and a 3' -> 5' strand (the reverse complement), referred to as the minus strand. Because RNA polymerase adds nucleic acids in a 5' -> 3' direction, the minus strand of the DNA serves as the template for the RNA during transcription. Thus, the RNA formed will have a sequence complementary to the minus strand and identical to the plus strand (except that U is substituted for T).
Antisense molecules are molecules that are specifically hybridizable or specifically complementary to either RNA or the plus strand of DNA. Sense molecules are molecules that are specifically hybridizable or specifically complementary to the minus strand of DNA. Antigene molecules are either antisense or sense molecules directed to a dsDNA target.
A gene-suppressive technology that is similar to antisense technology involves the use of small inhibitory RNA molecules (siRNAs) to inhibit a target gene. Methods of using siRNAs to inhibit eukaryotic and more particularly mammalian gene expression are known to those of ordinary skill in the art; see, for instance, Caplen et al, Proc. Natl. Acad. Sci. 98(17):9742-9747, 2001, and Elbashir et al. , Nature 411 :494-498, 2001.
Array: An arrangement of molecules, particularly biological macromolecules (such as polypeptides or nucleic acids) or cell or tissue samples, in addressable locations on or in a substrate. The array may be regular (arranged in uniform rows and columns, for instance) or irregular. The number of addressable locations on the array can vary, for example from a few (such as three) to more than 50, 100, 200, 500, 1000, 10,000, or more. A "microarray" is an array that is miniaturized so as to require or be aided by microscopic examination for evaluation or analysis.
Within an array, each arrayed sample (feature) is addressable, in that its location can be reliably and consistently determined within the at least two dimensions of the array. Thus, in ordered arrays the location of each sample is assigned to the sample at the time when it is applied to the array, and a key may be provided in order to correlate each location with the appropriate target or feature position. Often, ordered arrays are arranged in a symmetrical grid pattern, but samples could be arranged in other patterns (e.g., in radially distributed lines, spiral lines, or ordered clusters). Addressable arrays usually are computer readable, in that a computer can be programmed to correlate a particular address on the array with information about the sample at that position (e.g, hybridization or binding data, including for instance signal intensity). In some examples of computer readable formats, the individual features in the array are arranged regularly, for instance in a Cartesian grid pattern, which can be correlated to address information by a computer. The sample application location on an array (the "feature") may assume many different shapes. Thus, though the teπn "spot" may be used herein, it refers generally to a localized placement of molecules or tissue or cells, and is not limited to a round or substantially round region. For instance, substantially square regions of application can be used with arrays encompassed herein, as can be regions that are, for example substantially rectangular, triangular, oval, irregular, or another shape.
In certain example arrays, one or more features will occur on the array a plurality of times (e.g., twice) to provide internal controls.
Binding or stable binding: An oligonucleotide binds or stably binds to a target nucleic acid if a sufficient amount of the oligonucleotide forms base pairs or is hybridized to its target nucleic acid, to permit detection of that binding. Binding can be detected by either physical or functional properties of the targetoligonucleotide complex. Binding between a target and an oligonucleotide can be detected by any procedure known to one skilled in the art, including both functional and physical binding assays. Binding may be detected functionally by determining whether binding has an observable effect upon a biosynthetic process such as expression of a gene, DNA replication, transcription, translation, and the like.
Physical methods of detecting the binding of complementary strands of DNA or RNA are well known in the art, and include such methods as DNase I or chemical footprinting, gel shift and affinity cleavage assays, Northern blotting, dot blotting and light absorption detection procedures. For example, one method that is widely used, because it is so simple and reliable, involves observing a change in light absorption of a solution containing an oligonucleotide (or an analog) and a target nucleic acid at 220 to 300 ran as the temperature is slowly increased. If the oligonucleotide or analog has bound to its target, there is a sudden increase in absorption at a characteristic temperature as the oligonucleotide (or analog) and target disassociate from each other, or melt.
The binding between an oligomer and its target nucleic acid is frequently characterized by the temperature (Tm) at which 50% of the oligomer is melted from its target. A higher (Tm) means a stronger or more stable complex relative to a complex with a lower (Tm).
Cancer: A cancer is a biological condition in which a malignant tumor or other neoplasm has undergone characteristic anaplasia with loss of differentiation, increased rate of growth, invasion of surrounding tissue, and which is capable of metastasis. The term cancer includes prostate cancer, such as prostate adenocarcinoma, transitional cell carcinomas, squamous cell carcinomas, and sarcomas. However, about 95% of prostate cancers are adenocarcinomas. Also included are different stages of a single cancer, for instance both primary and recurrent (hormone-refractory) prostate cancer. cDNA (complementary DNA): A piece of DNA lacking internal, non-coding segments (introns) and transcriptional regulatory sequences. cDNA may also contain untranslated regions (UTRs) that are responsible for translational control in the corresponding RNA molecule. cDNA is usually synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.
Comparative genomic hybridization: A technique of differential labeling of test DNA and normal reference DNA, which are hybridized simultaneously to chromosome spreads, as described in Kallioniemi et al. (Science 258:818-821, 1992), incorporated by reference.
Complementarity and percentage complementarity: Molecules with complementary nucleic acids form a stable duplex or triplex when the strands bind, (hybridize), to each other by forming Watson-Crick, Hoogsteen or reverse Hoogsteen base pairs. Stable binding occurs when an oligonucleotide remains detectably bound to a target nucleic acid sequence under the required conditions.
Complementarity is the degree to which bases in one nucleic acid strand base pair with the bases in a second nucleic acid strand. Complementarity is conveniently described by percentage, i.e. the proportion of nucleotides that form base pairs between two strands or within a specific region or domain of two strands. For example, if 10 nucleotides of a 15-nucleotide oligonucleotide form base pairs with a targeted region of a DNA molecule, that oligonucleotide is said to have 66.67% complementarity to the region of DNA targeted. In the present disclosure, "sufficient complementarity" means that a sufficient number of base pairs exist between the oligonucleotide and the target sequence to achieve detectable binding, and in the case of the binding of an antigen, disrupt expression of gene products (such as cartilage glycoprotein-39 (CHI3L1), S-100P PROTEIN (S100P), CX3C chemokine/fractalkine (SCYD1), adenylate kinase 1 (AK1), forkhead transcription factor HFH-4 (HFH-4) (FKHL13), UDP glucuronosyltransferase precursor (UGT2B15) , Pleiotrophin (heparin binding growth factor 8)
(PTN), heat shock 27kD protein 2/Alpha-B-crystallin (HSP27), Proteasome (prosome, macropain) subunit, beta type, 5 (PSMB5), Inhibitor of NFKB (NFKBIA), interferon-induced 17 kD protein (ISG15), MAP kinase activated protein kinase 2 (MAPKAPK2), signal transduction protein (SH3 containing) (EFS2), hkf-1 Zinc finger protein (ZFP103), chromosome condensation 1 (CHC1), CDP- diacylglycerol synthase (CDSl), gap junction protein, alpha 1, 43kD (connexin 43) (GJAl), cyclin Dl (CCNDl), Inhibitor of DNA binding 3, helix-loop-helix protein (ID3), HI histone family, member2 (H1F2), Cytochrome B561 (CYB561), Cathepsin H (CTSH), calcineurin alpha (PPP3CA), 54 kDa progesterone receptor-associated immunophilin (FKBP5), translocation protein 1 (TLOC1), Clusterin (complement lysis inhibitor; testosterone-repressed prostate message 2; apolipoprotein J) (CLU), Pulmonary surfactant-associated protein A (SFTPA1), protease inhibitor 12 (PI12; neuroserpin) (PI12), Thrombospondin 1 (THBS1), Ribophorin I (RPN1), A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 1 (ADAMTS1), Collagen, type IV, alpha 5 (Alport syndrome) (COL4A5), LIM domain only 4 / breast tumor autoantigen (LM04), bumetanide-sensitive Na-K-Cl cotransporter (NKCC1) (SLC12A2), Fibronectin (FN1), Crystallin Mu (CRYM) and "upregulated by 1,25-dihydroxyvitamin D-3" (VDUP1)). When expressed or measured by percentage of base pairs formed, the percentage complementarity that fulfills this goal can range from as little as about 50% complementarity to full (100%) complementary. In general, sufficient complementarity is at least about 50%, about 75% complementarity, about 90% or 95% complementarity, and or about 98% or even 100% complementarity.
A thorough treatment of the qualitative and quantitative considerations involved in establishing binding conditions that allow one skilled in the art to design appropriate oligonucleotides for use under the desired conditions is provided by Beltz et al. Methods Enzymol 100:266-285, 1983, and by Sambrook et al. (ed), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
DNA (deoxyribonucleic acid): DNA is a long chain polymer which comprises the genetic material of most living organisms (some viruses have genes comprising ribonucleic acid (RNA)). The repeating units in DNA polymers are four different nucleotides, each of which comprises one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached. Triplets of nucleotides (referred to as codons) code for each amino acid in a polypeptide, or for a stop signal. The term codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed. Unless otherwise specified, any reference to a DNA molecule is intended to include the reverse complement of that DNA molecule. Except where single-strandedness is required by the text herein, DNA molecules, though written to depict only a single strand, encompass both strands of a double-stranded DNA molecule. Thus, a reference to the nucleic acid molecule that encodes a specific protein, or a fragment thereof, encompasses both the sense strand and its reverse complement. Thus, for instance, it is appropriate to generate probes or primers from the reverse complement sequence of the disclosed nucleic acid molecules.
Deletion: The removal of a sequence of DNA, the regions on either side of the removed sequence being joined together.
Gene amplification or genomic amplification: An increase in the copy number of a gene or a fragment or region of a gene or associated 5' or 3' region, as compared to the copy number in normal tissue. An example of a genomic amplification is an increase in the copy number of an oncogene. A "gene deletion" is a deletion of one or more nucleic acids normally present in a gene sequence and, in extreme examples, can include deletions of entire genes or even portions of chromosomes. Gene expression fingerprint (or profile): A distinct or identifiable pattern of gene expression, for instance a pattern of high and low expression of a defined set of genes; in some instances, as few as one or two genes may provide a profile, but often more genes are used in a profile, for instance at least three, at least 5, at least 10, at least 20, at least 25, or at least 50 or more. Gene expression fingerprints (also referred to as profiles) can be linked to a tissue or cell type, to a particular stage of normal tissue growth or disease progression, or to any other distinct or identifiable condition that influences gene expression in a predictable way. Gene expression fingerprints can include relative as well as absolute expression levels of specific genes, and often are best viewed in the context of a test sample compared to a baseline or control sample fingerprint. By way of example, a gene expression profile may be read on an array (e.g, a polynucleotide or polypeptide array). Arrays are now well known, and for instance gene expression arrays have been previously described in published PCT application number US99/06860 ("Hypoxia-Inducible Human Genes, Proteins, and Uses Thereof), incorporated herein by reference in its entirety. Genomic target sequence: A sequence of nucleotides located in a particular region in the human genome that corresponds to one or more specific genetic abnormalities, such as a nucleotide polymorphism, a deletion, or an amplification. The target can be for instance a coding sequence; it can also be the non-coding strand that corresponds to a coding sequence.
HRPC-related molecule: A molecule that is involved in, or influenced by, hormone- refractory prostate cancer. Such molecules include, for instance, nucleic acids (e.g., DNA, cDNA, or mRNAs) and proteins. Specific examples of HRPC-related molecules include the nucleic acid molecules listed in Table 1, and proteins or protein fragments encoded thereby. HRPC-related molecules may be involved in or influenced by hormone-refractory prostate cancer in many different ways, including causative (in that a change in an HRPC-related molecule leads to development of or progression to hormone-refractory prostate cancer) or resultive (in that development of or progression to hormone-refractory prostate cancer causes or results in a change in the HRPC-related molecule).
Hybridization: Oligonucleotides and their analogs hybridize by hydrogen bonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary bases. Generally, nucleic acid consists of nitrogenous bases that are either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)).
These nitrogenous bases form hydrogen bonds between a pyrimidine and a purine, and the bonding of the pyrimidine to the purine is referred to as "base pairing." More specifically, A will hydrogen bond to T or U, and G will bond to C. "Complementary" refers to the base pairing that occurs between to distinct nucleic acid sequences or two distinct regions of the same nucleic acid sequence. "Specifically hybridizable" and "specifically complementary" are terms that indicate a sufficient degree of complementarity such that stable and specific binding occurs between the oligonucleotide (or its analog) and the DNA or RNA target. The oligonucleotide or oligonucleotide analog need not be 100% complementary to its target sequence to be specifically hybridizable. An oligonucleotide or analog is specifically hybridizable when binding of the oligonucleotide or analog to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide or analog to non-target sequences under conditions where specific binding is desired, for example under physiological conditions in the case of in vivo assays or systems. Such binding is referred to as specific hybridization.
Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially the Na+ concentration) of the hybridization buffer will determine the stringency of hybridization, though waste times also influence stringency. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, chapters 9 and 11, herein incorporated by reference.
For purposes of the present disclosure, "stringent conditions" encompass conditions under which hybridization will only occur if there is less than 25% mismatch between the hybridization molecule and the target sequence. "Stringent conditions" may be broken down into particular levels of stringency for more precise definition. Thus, as used herein, "moderate stringency" conditions are those under which molecules with more than 25% sequence mismatch will not hybridize; conditions of "medium stringency" are those under which molecules with more than 15% mismatch will not hybridize, and conditions of "high stringency" are those under which sequences with more than 10% mismatch will not hybridize. Conditions of "very high stringency" are those under which sequences with more than 6% mismatch will not hybridize. In vitro amplification: Techniques that increase the number of copies of a nucleic acid molecule in a sample or specimen. An example of amplification is the polymerase chain reaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. The product of in vitro amplification may be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and or nucleic acid sequencing, using standard techniques. Other examples of in vitro amplification techniques include strand displacement amplification (see U.S. Patent No. 5,744,311); transcription-free isothermal amplification (see U.S. Patent No. 6,033,881); repair chain reaction amplification (see WO 90/01069); ligase chain reaction amplification (see EP-A-320308); gap filling ligase chain reaction amplification (see U.S. Patent No. 5,427,930); coupled ligase detection and PCR (see U.S. Patent No. 6,027,889); and NASBA™ RNA transcription-free amplification (see U.S. Patent No. 6,025,134).
Injectable composition: A pharmaceutically acceptable fluid composition including at least one active ingredient. The active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition can additionally include minor amounts of one or more non-toxic auxiliary substances, such as emulsifying agents, preservatives, and pH buffering agents and the like. Such injectable compositions that are useful for use with the nucleotides and proteins of this disclosure are conventional; appropriate formulations are well known in the art.
Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The tenn also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. Neoplasm: A new and abnormal growth, particularly a new growth of tissue or cells in which the growth is uncontrolled and progressive. A tumor is an example of a neoplasm.
Nucleotide: "Nucleotide" includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.
Oligonucleotide: An oligonucleotide is a plurality of joined nucleotides joined by native phosphodiester bonds, between about 6 and about 300 nucleotides in length. An oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non-naturally occurring portions. For example, oligonucleotide analogs can contain non-naturally occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorofhioate oligodeoxynucleotide. Functional analogs of naturally occurring polynucleotides can bind to RNA or DNA, and include peptide nucleic acid (PNA) molecules.
Particular oligonucleotides and oligonucleotide analogs can include linear sequences up to about 200 nucleotides in length, for example a sequence (such as DNA or RNA) that is at least 6 bases, for example at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases long, or from about 6 to about 50 bases, for example about 10-25 bases, such as 12, 15 or 20 bases.
Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
Open reading frame: A series of nucleotide triplets (codons) coding for amino acids without any internal termination codons. These sequences are usually translatable into a peptide. Ortholog: Two nucleic acid or amino acid sequences are orthologs of each other if they share a common ancestral sequence and diverged when a species carrying that ancestral sequence split into two species. Orthologous sequences are also homologous sequences. Parenteral: Administered outside of the intestine, e.g., not via the alimentary tract. Generally, parenteral formulations are those that will be administered through any possible mode except ingestion. This term especially refers to injections, whether administered intravenously, intrathecally, intramuscularly, intraperitoneally, or subcutaneously, and various surface applications including intranasal, intradermal, and topical application, for instance.
Peptide Nucleic Acid (PNA): An oligonucleotide analog with a backbone comprised of monomers coupled by amide (peptide) bonds, such as amino acid monomers joined by peptide bonds.
Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in this disclosure are conventional. Martin, Remington 's Pharmaceutical Sciences, published by Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the nucleotides and proteins herein disclosed.
In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically- neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non- toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
Probes and primers: Nucleic acid probes and primers can be readily prepared based on the nucleic acid molecules provided in this disclosure as indicators of disease or disease progression. It is also appropriate to generate probes and primers based on fragments or portions of these nucleic acid molecules. Also appropriate are probes and primers specific for the reverse complement of these sequences, as well as probes and primers to 5' or 3' regions.
A probe comprises an isolated nucleic acid attached to a detectable label or other reporter molecule. Typical labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, e.g., in Sambrook et al. (In ' Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).
Primers are short nucleic acid molecules, for instance DNA oligonucleotides 10 nucleotides or more in length. Longer DNA oligonucleotides may be about 15, 20, 25, 30 or 50 nucleotides or more in length. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then the primer extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art. Methods for preparing and using nucleic acid probes and primers are described, for example, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989), Ausubel et al. (ed.) (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998), and Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990). PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, © 1991, Whitehead Institute for Biomedical Research, Cambridge, MA). One of ordinary skill in the art will appreciate that the specificity of a particular probe or primer increases with its length. Thus, for example, a primer comprising 30 consecutive nucleotides of a HRPC-related protein encoding nucleotide will anneal to a target sequence, such as another homolog of the designated HRPC-related protein, with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, in order to obtain greater specificity, probes and primers can be selected that comprise at least 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of a HRPC-related protein-encoding nucleotide sequences.
The disclosure thus includes isolated nucleic acid molecules that comprise specified lengths of the disclosed HRPC-related nucleotide sequences. Such molecules may comprise at least 10, 15, 20, 23, 25, 30, 35, 40, 45 or 50 consecutive nucleotides of these sequences or more, and may be obtained from any region of the disclosed sequences (e.g, a HRPC-related nucleic acid may be apportioned into halves or quarters based on sequence length, and isolated nucleic acid molecules may be derived from the first or second halves of the molecules, or any of the four quarters, etc.). A HRPC-related cDNA also can be divided into smaller regions, e.g. about eighths, sixteenths, twentieths, fiftieths and so forth, with similar effect.
Another mode of division is to select the 5' (upstream) and/or 3' (downstream) region associated with a HRPC-related gene.
Nucleic acid molecules may be selected that comprise at least 10, 15, 20, 25, 30, 35, 40, 50 or 100 or more consecutive nucleotides of any of these or other portions of a HRPC-related nucleic acid molecule, such as those disclosed herein, and associated flanking regions. Thus, representative nucleic acid molecules might comprise at least 10 consecutive nucleotides of a human coding sequence the expression of which is influenced by prostate cancer progression, such as those listed in Table 1. Protein: A biological molecule expressed by a gene and comprised of amino acids.
Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein referred to is more pure than the protein in its natural environment within a cell or within a production reaction chamber (as appropriate). Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
Representational difference analysis: A PCR-based subtractive hybridization technique used to identify differences in the mRNA transcripts present in closely related cell lines. Serial analysis of gene expression: The use of short diagnostic sequence tags to allow the quantitative and simultaneous analysis of a large number of transcripts in tissue, as described in Velculescu et al. (Science 270:484-487, 1995).
Sequence identity: The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identify. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or orthologs of the HRPC-related protein, and the corresponding cDNA or gene sequence, will possess a relatively high degree of sequence identity when aligned using standard methods. This homology will be more significant when the orthologous proteins or genes or cDNAs are derived from species that are more closely related (e.g., human and chimpanzee sequences), compared to species more distantly related (e.g., human and C. elegans sequences).
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman Adv. Appl. Math. 2: 482, 1981; Needleman & Wunsch Mol. Biol. 48: 443, 1970; Pearson & Lipman Proc. Natl. Acad. Sci. USA 85: 2444, 1988; Higgins & Sharp Gene, 73: 237-244, 1988; Higgins & Sharp CABIOS 5: 151-153, 1989; Corpet et al. Nuc. Acids Res. 16, 10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al. Meth. Mol. Bio. 24, 307-31, 1994. Altschul et al. (J. Mol. Biol. 215:403-410, 1990), presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J. Mol. Biol. 215:403-
410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. By way of example, for comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties).
An alternative indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions. Stringent conditions are sequence- dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5° C to 20° C lower than the thennal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence remains hybridized to a perfectly matched probe or complementary strand. Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and Tijssen (Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes Part I, Chapter 2, Elsevier, New York, 1993). Nucleic acid molecules that hybridize under stringent conditions to a specific human HRPC-related protein-encoding sequence will typically hybridize to a probe based on either an entire human HRPC-related protein-encoding sequence or selected portions of the encoding sequence under wash conditions of 2x SSC at 50° C.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein.
Specific binding agent: An agent that binds substantially only to a defined target. Thus a protein-specific binding agent binds substantially only the specified protein. As used herein, the term "protein [X] specific binding agent" includes anti-[X] protein antibodies (and functional fragments thereof) and other agents (such as soluble receptors) that bind substantially only to the [X] protein. In this context, [X] refers to any specific or designated protein, for instance a HRPC-related protein such as those listed in Table 1.
Anti-[X] protein antibodies may be produced using standard procedures described in a number of texts, including Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988). The determination that a particular agent binds substantially only to the specified protein may readily be made by using or adapting routine procedures. One suitable in vitro assay makes use of the Western blotting procedure (described in many standard texts, including Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988)). Western blotting may be used to detennine that a given protein binding agent, such as an anti-[X] protein monoclonal antibody, binds substantially only to the [X] protein.
Shorter fragments of antibodies can also serve as specific binding agents. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bind to a specified protein would be specific binding agents. These antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab1, the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab')2, a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of making these fragments are routine.
Subject: Living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals
Target sequence: "Target sequence" is a portion of ssDNA, dsDNA or RNA that, upon hybridization to a therapeutically effective oligonucleotide or oligonucleotide analog, results in the inhibition of expression of a specified protein, such as a HRPC-related protein. Either an antisense or a sense molecule can be used to target a portion of dsDNA, since both will interfere with the expression of that portion of the dsDNA. The antisense molecule can bind to the plus strand, and the sense molecule can bind to the minus strand. Thus, target sequences can be ssDNA, dsDNA, and RNA.
Tissue microarray ("tissue chip"): A tissue microarray is a microarray wherein the samples are samples of tissue, for instance animal tissue such as human tissue. Examples of tissue microarrays are assembled by aligning tissue cylinders (taken, for instance, from tissue blocks or biopsies) in a recipient block, such as a block of paraffin, to create a matrix of columns of sample within the block. Individual slices are cut from the surface of the block, substantially perpendicular to the axis of the cylinders, thereby yielding flat, thin arrays of tissue samples embedded in the block material. Such thin arrays are often transferred to a microscope slide or other supporting member. The construction of tissue microarrays is described in, for instance, Kononen et al, Nature Medicine, 4:844-847, 1998 and PCT International Patent Publication WO99/44063A2, both of which are incorporated herein by this reference.
Tissue samples contained in a tissue microarray may be any set of tissues, but often a tissue microarray has a theme so to speak, for instance containing samples from a collection of different tumors, tumors from different tissues, tumors from different stages of progression, or from different treatment regimens.
Transformed: A transformed cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
Tumor: A neoplasm that may be either malignant or non-malignant. "Tumors of the same tissue type" refers to primary tumors originating in a particular organ (such as breast, prostate, bladder or lung). Tumors of the same tissue type may be divided into tumor of different sub-types (an example being prostate cancer, which can be an adenocarcinoma, transitional cell, squamous cell tumor, or sarcoma).
Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter disclosed herein belongs. The singular terms "a", "an", and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. III. Description of several specific embodiments
Provided herein are methods of diagnosing or prognosing development or progression of prostate cancer in a subject, which methods involve detecting an abnormality in at least one HRPC- related molecule of the subject (e.g., an HRPC related nucleic acid molecule such as one listed in Table 1 or Table 4, or genes, cDNAs or other polynucleotide molecules comprising one of the listed sequences, or a fragment thereof, or an HRPC-related protein such as one encoded by such a nucleic acid molecule, or a fragment of such protein). In certain embodiments, abnormalities are detected in more than one HRPC-related molecule, for instance in at least 5, at least 10, 15, 25, 50, or 100 or more HRPC-related nucleic acid molecules listed in Table 1 or elsewhere herein, or encoded for by a nucleic acid molecule listed in Table 1 or elsewhere herein. In certain specific embodiments, no more than the molecules listed in Table 1 or Table 4, or corresponding to (represented by) those listed in Table 1 or Table 4, are included in such analysis. For instance, certain of the described methods employ detecting no more than 600, no more than 500, no more than 400, no more than 300, or no more than 200 of such molecules.
Also encompassed herein are arrays containing two or more HRPC-related molecules. Certain of such arrays are nucleic acid arrays, which contain at least one HRPC-related nucleic acid molecule, for instance at least one of the HRPC-related nucleic acid molecules listed in Table 1 or Table 4, or genes, cDNAs or other polynucleotide molecules comprising one of the listed sequences, or a fragment thereof. Other described arrays are protein (polypeptide) arrays, which contain at least one HRPC-related protein such as one encoded by a nucleic acid molecule listed in Table 1 or Table 4 (or genes, cDNAs or other polynucleotide molecules comprising one of the listed sequences, or a fragment thereof), or a fragment of such protein, or an antibody specific to such a protein or protein fragment. Such arrays can also contain any particular subset of the nucleic acids (or corresponding molecules) listed in Table 1 or Table 4. Certain of such arrays (as well as the methods described herein) also may include HRPC-related molecules that are not listed in Table 1 or Table 4. Abnormalities detected by these methods for instance can be different for different HRPC- related molecules, and may include increases or decreases in the level (amount) or functional activity of such molecules, or in their localization or stability. As used herein, the term "HRPC-related molecule" includes HRPC-related nucleic acid molecules (such as DNA or RNA or cDNA) and HRPC-related proteins, though in specific embodiments the term may be specific for any one of tliese types of molecules. The term is not limited to those molecules listed in Table 1 or Table 4 (and molecules that correspond to those listed), but also includes other nucleic acids and/or proteins that are influenced (e.g., as to level, activity, localization) by or during prostate cancer progression, including all of such molecules listed herein. Specific encompassed embodiments include diagnostic and/or prognostic methods in which a mutation, duplication or deletion of a HRPC-related nucleic acid in cells of the individual is detected.
In certain embodiments, HRPC-related molecules that can be examined for an abnormality include molecules represented by a subset of the sequences referred to in Table 1 or Table 4, such as the more than 200 sequences represented by Image Clone ID numbers: 1475595, 1460110, 50794, 78294, 190491, 66731, 143287, 754600, 754509, 308041, 70827, 361974, 503097, 796646, 41650, 841641, 724615, 839101, 504226, 810711, 435330, 773567, 431296, 345232, 756405, 256907, 415817, 366541, 223350, 366067, 724831, 814353, 236034, 809910, 1470048, 1323448, 1456424, 453689, 135221, 340734, 180864, 768562, 179276, 44505, 293104, 243343, 66317, 812251, 245920, 265874, 770212, 784910, 839094, 712049, 669435, 841470, 782339, 297061, 429466, 300137, 487172, 343744, 795730, 268876, 742132, 755578, 502682, 510381, 140574, 135630, 278242, 742862, 1049033, 270136, 768260, 53039, 211813, 195051, 125769, 122955, 129342, 292392, 139331, 143995, 139250, 243360, 194307, 235040, 295483, 143756, 897768, 1456160, 34778, 810512, 753184, 200814, 470393, 23185, 128126, 42373, 511521, 810117, 950682, 783696, 815555, 897531, 713145, 502690, 469969, 309893, 725877, 343987, 49318, 42864, 193087, 162533,
1309620, 685801, 825740, 756708, 28469, 187147, 246304, 130280, 753587, 123980, 241985, 564621, 841507, 810703, 784772, 143306, 246722, 298417, 51582, 757222, 884783, 417424, 324891, 504791, 725877, 743230, 377048, 42627, 144797, 244955, 204735, 144747, 292749, 196109, 120375, 121981, 121715, 243403, 127409, 130053, 243291, 203514, 133130, 134495, 296552, 138601, 167076, 197323, 197637, 194906, 194985, 196125, 196303, 243784, 280122, 245235, 197856, 200604, 203400, 207448, 234469, 210548, 208940, 208434, 211951, 212098, 233399, 240138, 137396, 241097, 239835, 308231, 292312, 292391, 293421, 293306, 293785, 295044, 295590, 296102, 296602, 297110, 191572, 195132, 233274, 246546, 296562, 214331, 214043, 126230, 128245, 129616, 134312, 230613, 239711, 134537, 127646, 136984, 210610, 293457, 233299, 281125, 26184, 39093, 39884, and/or 2911545. Molecules represented by (or corresponding to) these Image Clone IDs include the nucleic acid fragments found in the respective clones (and variants thereof), complete nucleic acids (such as cDNAs, mRNAs, or genes) encompassing such fragments, fragments and variants of these complete nucleic acid molecules, proteins encoded by such nucleic acids, and fragments and variants of such proteins.
Certain of the encompassed methods involve measuring an amount of the HRPC-related molecule in a sample (such as a serum or tissue sample) derived or taken from the subject, in which a difference (for instance, an increase or a decrease) in level of the HRPC-related molecule relative to that present in a sample derived or taken from the subject at an earlier time, is diagnostic or prognostic for development or progression of prostate cancer.
Abnormalities in HRPC-related nucleic acid molecules can be detected using, for instance, in vitro nucleic acid amplification and/or nucleic acid hybridization. The results of such detection methods can be quantified, for instance by determining the amount of hybridization or the amount of amplification.
Abnormalities in HRPC-related proteins can be detected using, for instance, a HRPC protein-specific binding agent, which in some instances will be detectably labeled. In certain embodiments, therefore, detecting an abnormality includes contacting a sample from the subject with a HRPC protein-specific binding agent; and detecting whether the binding agent is bound by the sample and thereby measuring the levels of the HRPC-related protein present in the sample, in which a difference in the level of HRPC-related protein in the sample, relative to the level of HRPC-related protein found an analogous sample from a subject not having the disease or disorder, or a standard HRPC-related protein level in analogous samples from a subject not having the disease or disorder or not having a predisposition for developing the disease or disorder, is an abnormality in that HRPC- related molecule.
In other embodiments, detecting the abnormality involves determining whether a HRPC- related gene expression profile from the subject indicates development or progression of prostate cancer, for instance by comparing the HRPC-related gene expression profile from the subject to at least one control gene expression fingerprint or profile for a specific stage of prostate cancer. In specific examples of such methods, at least one control gene expression profile is a fingerprint for a normal prostate tissue, a primary prostate cancer tissue, a prostate cancer tissue responding to androgen ablation therapy, or a hormone refractory prostate cancer tissue. Examples of such profiles (also referred to herein as fingerprints) can be in an array format, such as a nucleotide (e.g., polynucleotide) or protein array or microarray, or generated from such an array.
Specific embodiments of methods for detecting an abnormality in at least one HRPC-related molecule use the arrays disclosed herein. Such arrays are nucleotide (e.g., polynucleotide) or protein (e.g, peptide, polypeptide, or antibody) arrays. In such methods, an array may be contacted with polynucleotides or polypeptides (respectively) from (or derived from) a sample from a subject. The amount and/or position of binding of the subject's polynucleotides or polypeptides then can be determined, for instance to produce a gene expression profile for that subject. Such gene expression profile can be compared to another gene expression profile, for instance a control gene expression profile from a subject having a known prostate-related condition. Optionally, the subject's gene expression profile (also known as a gene expression fingerprint) can be correlated with one or more appropriate treatments. Similarly, protein arrays can give rise to protein expression profiles. Both protein and gene expression profiles can more generally be referred to as expression profiles.
Other embodiments are methods that involve providing nucleic acids from the subject; amplifying the nucleic acids to foπn nucleic acid amplification products; contacting the nucleic acid amplification products with an oligonucleotide probe that will hybridize under stringent conditions with a nucleic acid encoding a HRPC-related protein; detecting the nucleic acid amplification products which hybridize with the probe; and quantifying the amount of the nucleic acid amplification products that hybridize with the probe. The sequence of such oligonucleotide probes may be selected to bind specifically to a nucleic acid molecule listed in Table 1 or Table 4, or a nucleic acid molecule represented by those listed in Table 1 or Table 4. In some embodiments, the probes are attached to a solid surface, such as an array. Likewise, the primers may be selected to amplify a nucleic acid molecule listed in Table 1 or Table 4, or represented by tiiose listed in Table 1 or Table 4. In specific examples of such methods, the primers are selected to amplify a nucleic acid product encoding cartilage glycoprotein-39 (CHI3L1), S-100P PROTEIN (S100P), CX3C chemokine/fractalkine (SCYD1), adenylate kinase 1 (AK1), forkhead transcription factor HFH-4 (HFH-4) (FKHL13), UDP glucuronosyltransferase precursor (UGT2B15), Pleiotrophin (heparin binding growth factor 8) (PTN), heat shock 27kD protein 2/Alpha-B-crystallin (HSP27), Proteasome (prosome, macropain) subunit, beta type, 5 (PSMB5), Inhibitor of NFKB (NFKBIA), interferon- induced 17 kD protein (ISG 15), MAP kinase activated protein kinase 2 (MAPKAPK2), signal transduction protein (SH3 containing) (EFS2), hkf-1 Zinc finger protein (ZFP103), chromosome condensation 1 (CHC1), CDP-diacylglycerol synthase (CDSl), gap junction protein, alpha 1, 43 kD (connexin 43) (GJAl), cyclin Dl (CCND1), Inhibitor of DNA binding 3, helix-loop-helix protein (ID3), HI histone family, member2 (H1F2), Cytochrome B561 (CYB561), Cathepsin H (CTSH), calcineurin alpha (PPP3CA), 54 kDa progesterone receptor-associated immunophilin (FKBP5), translocation protein 1 (TLOC1), Clusterin (complement lysis inhibitor; testosterone-repressed prostate message 2; apolipoprotein J) (CLU), Pulmonary surfactant-associated protein A (SFTPA1), protease inhibitor 12 (PI12; neuroserpin) (PI12), Thrombospondin 1 (THBSl), Ribophorin I (RPN1), A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 1 (ADAMTS 1), Collagen, type IV, alpha 5 (Alport syndrome) (COL4A5), LIM domain only 4 / breast tumor autoantigen (LM04), bumetanide-sensitive Na-K-Cl cotransporter (NKCC1) (SLC12A2), Fibronectin (FN1), Crystallin Mu (CRYM) or "upregulated by 1,25-dihydroxyvitamine D-3 (VDUP1).
Also encompassed are methods of prostate cancer therapy, in which an abnormality in at least one HRPC-related molecule of a subject is detected using a method described herein, and; if such abnormality is identified, a treatment is selected to prevent or reduce hormone-refractory prostate cancer or to delay the onset of hormone-refractory prostate cancer. The subject then can be treated in accordance with this selection. In some examples, the treatment selected in specific and tailored for the subject, based on the analysis of that subject's profile for one or more HRPC-related molecules.
A further embodiment is a method of modifying a level of expression or function of a HRPC-related protein in a subject. Such methods may involve expressing in the subject a recombinant genetic construct that includes a promoter operably linked to a nucleic acid molecule, and wherein expression of the nucleic acid molecule changes expression of the HRPC-related protein. The nucleic acid molecule may, for instance, include at least 10 consecutive nucleotides of a HRPC- related nucleic acid sequence. In specific examples of such methods, the nucleic acid molecule is in antisense orientation relative to the promoter; in other examples, the nucleic acid molecule is in sense orientation relative to the promoter.
Other embodiments are kits for measuring the level or function of one or more HRPC- related molecules, which kits may include a binding molecule that selectively binds to the HRPC- related molecule that is the target of the kit. In some examples of such kits where the HRPC-related molecule level is a HRPC-related protein level, the binding molecule provided in the kit may be an antibody or antibody fragment that selectively binds to the target HRPC-related protein. In other examples of such kits where the HRPC-related molecule level is a HRPC-related nucleic acid molecule level, the binding molecule provided in the kit may be an oligonucleotide capable of hybridizing to the HRPC-related nucleic acid molecule.
Further embodiments are methods of screening for a compound useful in treating, reducing, or preventing prostate cancer or development or progression of prostate cancer. Such methods involve determining if application of a test compound alters a HRPC-related gene expression profile so that the profile more closely resembles a prostate-linked profile than it did prior to such treatment, and selecting a compound that so alters the HRPC-related gene expression profile. In specific examples of such methods, the test compound is applied to a test cell. In some of such methods, the profile is determined or measured in an array format.
Also encompassed are compounds selected using the methods described herein, which are useful in treating, reducing, or preventing prostate cancer or development or progression of prostate cancer.
Examples of disclosed methods involve contacting test cells with a test compound, then measuring at least one HRPC-related molecule level and/or activity in the test cells. In such methods, a difference in HRPC-related molecule (e.g., a HRPC-related nucleic acid molecule listed in Table 1, or a molecule encoded for by a nucleic acid molecule listed in Table 1) level and or activity in the test cells, relative to the analogous HRPC-related molecule level and/or activity found in analogous cells not contacted with the test compound, indicates that the test compound is useful in treating, reducing, or preventing prostate cancer or development or progression of prostate cancer. Measuring the
HRPC-related molecule(s) level and/or activity may include creating a HRPC-related gene expression profile for the test cell after contacting the cell with the test compound, and comparing the test cell HRPC-related gene expression profile to at least one control gene expression profile for a specific stage of prostate cancer. Representative control gene expression profile can include a profile for a normal prostate tissue, a primary prostate cancer tissue, a prostate cancer tissue responding to androgen ablation therapy, and/or a hormone refractory prostate cancer tissue.
Also disclosed herein are use of identified target HRPC-related molecules for the development of antibodies, including therapeutic antibodies that affect an HRPC-related pathway. It is also envisioned that the disclosed HRPC-related molecules can be used as vaccines, for instance as "cancer vaccines" to elicit an immune response from a subject that renders the subject more resistant to developing or progressing through a stage of prostate cancer. IV. Temporal gene expression changes during prostate cancer hormonal therapy The present disclosure concerns gene expression profiling of the regression and progression of the CWR22 human prostate cancer xenografts using cDNA microarrays. Highly quantitative two- color fluorescent microarray technology was used to examine differences in gene expression between primary and recurrent CWR22 prostate cancer xenografts. cDNA microarrays containing 6605 gene fragments were constructed and applied to analyze the mRNA expression profile of thirteen CWR22 xenografts at different stages in the time course of treatment and progression. New statistical tools (including template-based gene clustering) were then used to mine this data (e.g., to differentiate temporal gene expression profiles associated with in vivo therapy response from the noise in the data), enabling a comprehensive genomics-based analysis of the temporal mRNA expression profiles during the entire course of CWR22 treatment and progression. For instance, a large set of genes and other encoding sequences (e.g., expressed sequence tags, ESTs) have been identified (Table 1), the expression of which varies during prostate cancer progression. The Image Clone ID numbers of these sequences are: 843249, 796904, 399604, 788205, 260303, 287745, 796542, 243653, 486787, 134495, 796680, 448386, 298417, 124578, 141562, 713145, 815284, 51408, 22012, 344589, 839101, 511091, 365515, 144042, 153411, 853809, 884783, 75254, 109314, 293925, 949938, 132835, 773567, 415529, 809910, 142326, 139660, • 435855, 243816, 489839, 130895, 258120, 39920, 343867, 142139, 1472735, 503083, 796147, 813179, 741891, 461727, 756968, 814636, 897667, 297392, 322723, 66322, 295729, 265645, 143756, 195340, 78294, 296880, 713922, 203772, 383188, 431296, 842939, 347434, 308041, 202535, 66582, 795730, 292770, 788107, 128143, 214162, 298231, 453183, 502518, 755054, 415978, 630013, 813678, 236034, 257162, 813841, 770074, 266146, 377692, 755952, 297063,
840511, 415084, 841695, 183462, 731051, 80221, 758222, 71116, 137139, 136557, 205239, 509731, 208718, 840460, 111750, 711918, 809998, 784296, 813266, 257422, 82734, 811161, 322160, 844725, 276091, 177772, 461804, 898258, 248412, 344139, 796689, 223350, 78353, 502832, 250654, 214990, 813611, 140806, 1469234, 781362, 813698, 377731, 79782, 768644, 772425, 150702, 246430, 210494, 120681, 132140, 431501, 366966, 35105, 201393, 137096, 453005,
131050, 897485, 781233, 768246, 897906, 246765, 1412481, 111150, 124071, 42070, 1455976, 66982, 362483, 382195, 196992, 564621, 814409, 504763, 143519, 82903, 234736, 812246, 125685, 436155, 898219, 773637, 269878, 427812, 868368, 81475, 784910, 859359, 809353, 753069, 377320, 1472775, 130826, 46284, 586685, 811162, 50680, 840990, 1461664, 796613, 68977, 796198, 80338, 770394, 138936, 784593, 768299, 232772, 32493, 432210, 126858, 245936, 1161775, 840942, 361974, 51406, 41650, 77728, 278570, 271670, 26249, 810331, 811088, 627306, 179890, 121454, 289447, 810104, 774471, 377252, 949928, 294881, 292364, 142788, 246619, 345935, 854899, 770059, 49630, 773301, 781139, 854338, 785967, 770388, 810960, 383175, 128460, 279970, 139217, 283023, 813823, 242062, 461327, 756595, 826142, 841008, 134229, 1323448, 300137, 843098, 51582, 128785, 839991, 783998, 130276, 214006, 840687, 66731, 272327, 197054, 814240, 121792, 809456, 145001, 685371, 345626, 136954, 143887, 120964, 842784, 1374571, 152453, 842836, 233583, 346552, 51293, 360885, 815774, 852829, 35077, 488956, 31143, 741067, 725877, 345858, 35828, 34849, 31397, 203469, 713782, 296754, 768370, 246546, 66686, 547058, 144924, 767475, 591281, 1474337, 823691, 812955, 1416782, 232670, 183602, 1343468, 289818, 486186, 784772, 491565, 897781, 1472150, 745343, 788256, 1461138, 742798, 214965, 842968, 490778, 842863, 415102, 825606, 196303, 490995, 564803, 292933, 841507, 415089, 785707, 1492304, 884867, 774446, 781047, 811046/727526, 126650, 233274, 838802, 232837, 45544, 280934, 294487, 795936, 141818, 131316, 700792, 969748, 340558, 34778, 28469, 795498, 293569, 47853, 200402, 47853, 852520, 825295, 26184, 1456160, 491001, 757489, 321661, 592359, 51448, 725284, 46182, 324891, 811015, 36393, 207288, 744417, 813648, 745138, 612274, 785616, 214614, 869538, 71101, 204257, 853368, 769921, 320509, 249603, 207358, 435076, 242578, 139705, 299360, 753457, 810899, 34355, 291057, 43550, 826211, 789147, 295483, 797016, 309288, 44975, 416833, 897567, 809588, 772304, 323404, 809603, 295986, 725188, 744047, 783697, 814615, 814701, 898286, 950690, 66564, 454339, 204214, 796646, 129865, 756401, 66406, 451907, 827144, 25584, 365641, 840894, 782679, 711768, 234237, 416833, 884718, 788185, 453107, 204148, 509887, 289551, 740554, 211747, 66728, 789204, 362926, 878676, 611150, 50506, 162775, 743230, 626716, 47833, 796278, 128243, 80946, 149013, 531319, 950482, 950473, 789182, 856427, 725454, 951142, 878130, 49352, 322914, 1472643, 293727, 273546,
772220, 53316, 42059, 810854, 768260, 626531, 471598, 44537, 769603, 376785, 760344, 840364, 856489, 490772, 46171, 855487, 281003, 509495, 43231, 487348, 898062, 795738, 24145, 40017, 429182, 825677, 755239, 971367, 129146, 825312, 384081, 26578, 814287, 787938, 857264, 813675, 134719, 626206, 684655, 29063, 433666, 42096, 325641, 246120, 80410, 1160558, 45233, 214884, 824906, 814117, 810057, 1455641, 545403, 773383, 840702, 810552, 739511, 145503,
135449, 724387, 283315, 897177, 866874, 502669, 324618, 897774, 73381, 41452, 321389, 949971, 785778, 50359, 813280, 308682, 531957, 486175, 40026, 28823, 487425, 42880, 416316, 32875, 753862, 795543, 263727, 824568, 366156, 1492412, 840567, 51666, 72050, 47647, 809535, 810725, 842825, 767817, 80500, 856454, 811150, 470930, 242698, 83279, 884993, 211275, 741958, 433474, 196650, 782439, 843121, 28410, 378502, 214906, 43241, 47542, 109863, 814765, 384015, 489823, 83011, 134544, 711552, 195051, 268727, 742132, 108265, 280837, 770837, 241988, 66555, 208413, 399532, 291880, 814731, 42313, 433350, 415145, and 2911545. These Image ID Clones can be obtained from Research Genetics (2130 Memorial Parkway, Huntsville, AL 35801, US or Canada: 1- 800-533-4363). The sequences of these Image ID Clones are hereby expressly incorporated by reference, and are clearly identified based on their multiple identification listings given in Table 1.
Up to 231 (3.5%) of the 6605 coding sequences assayed were differentially expressed between primary and recurrent xenografts. Using data from the direct hybridization of mRNA from a pool of four primary CWR22 xenografts against a pool of four CWR22R recurrent xenografts, the data was filtered for an intensity of at least greater than the mean in the maximum between the cy5 and cy3 signals. The mean intensity was 3058, which was used as a cut-off to ensure that the data was of high quality, however other intensities may be used. This intensity cut-off resulted in 2276 genes with sufficient expression. Based on the 99.9% confidence level, 231 of these 2276 coding sequences were considered differentially expressed though prostate progression. These include a set of 90 sequences that display reduced cDNA production after hormone therapy with an increase (are up-regulated) during tumor recurrence (Image ID Clones: 1475595, 1460110, 50794, 78294, 190491, 66731, 143287, 754600, 754509, 308041, 70827, 361974, 503097, 796646, 41650, 841641, 724615, 839101, 504226, 810711, 435330, 773567, 431296, 345232, 756405, 256907, 415817, 366541, 223350, 366067, 724831, 814353, 236034, 809910, 1470048, 1323448, 1456424, 453689, 135221, 340734, 180864, 768562, 179276, 44505, 293104, 243343, 66317, 812251, 245920, 265874, 770212, 784910, 839094, 712049, 669435, 841470, 782339, 297061, 429466, 300137, 487172, 343744, 795730, 268876, 742132, 755578, 502682, 510381, 140574, 135630, 278242, 742862, 1049033, 270136, 768260, 53039, 211813, 195051, 125769, 122955, 129342, 292392, 139331, 143995, 139250, 243360, 194307, 235040, 295483, and 143756), and a set of 131 sequences that display increased cDNA production after hormone therapy with a decrease (are down-regulated) during tumor recurrence (Image ID Clones: 897768, 1456160, 34778, 810512, 753184, 200814, 470393, 23185, 128126, 42373, 511521, 810117, 950682, 783696, 815555, 897531, 713145, 502690, 469969, 309893, 725877, 343987, 49318, 42864, 193087, 162533, 1309620, 685801, 825740, 756708, 28469, 187147, 246304, 130280, 753587, 123980, 241985, 564621, 841507, 810703, 784772, 143306,
246722, 298417, 51582, 757222, 884783, 417424, 324891, 504791, 725877, 743230, 377048, 42627, 144797, 244955, 204735, 144747, 292749, 196109, 120375, 121981, 121715, 243403, 127409, 130053, 243291, 203514, 133130, 134495, 296552, 138601, 167076, 197323, 197637, 194906, 194985, 196125, 196303, 243784, 280122, 245235, 197856, 200604, 203400, 207448, 234469, 210548, 208940, 208434, 211951, 212098, 233399, 240138, 137396, 241097, 239835, 308231, 292312, 292391, 293421, 293306, 293785, 295044, 295590, 296102, 296602, 297110, 191572, 195132, 233274, 246546, 296562, 214331, 214043, 126230, 128245, 129616, 134312, 230613, 239711, 134537, 127646, 136984, 210610, 293457, 233299, 281125, 26184, 39093, and 39884). Other confidence levels could be used to select HRPC-related molecules, such as 98%, 95%, 90%, 85%, and so forth. Higher confidence levels, such as 99.99%, could also be used. Molecules identified as being linked to prostate cancer (referred to generally herein as HRPC-related molecules) using the methods described herein can be arranged on arrays for use in diagnostic and prognostic methods. Specific arrays are contemplated that are constructed using molecules identified at such different confidence levels.
In particular, the techniques disclosed herein have uncovered many genes not previously associated with prostate cancer progression, and particularly not previously associated with HRPC. These newly correlated genes include those represented by the following Image ID Clones: 781047, 785778, 842968, 769921, 898286, 204214, 814701, 435076, 531319, 415089, 898062, 453107, 785707, 795936, 700792, 34778, 46182, 769921, 783697, 451907, 711768, 416833, 810711, 789204, 789182, 725454, 951142, 49352, 273546, 46717, 855487, 41117, 26578, 684655, 45233, 814117, 810552, 739511, 283315, and 897774. Of the 59 androgen-dependent sequences whose expression decreased most after castration
(labeled "Decreasing" in Table 2), 58 (98.3%) displayed restored transcript levels in the recurrent tumors, indicating re-activation of androgen-dependent genes in the absence of a ligand.
Tissue microarrays consisting of 50 xenografts and 440 clinical specimens from all stages of prostate cancer progression were utilized to validate potential drug target genes using mRNA in situ hybridization and protein immunohistochemistry. Measured by cDNA microarrays, S 1 OOP (encoding a calcium-binding protein) was among the most highly overexpressed genes in the CWR22R recurrent tumors; this gene was also highly expressed in the majority of hormone-refractory clinical prostate cancers, but rarely (<10%) in benign prostate lesions.
The temporal gene expression changes identified herein facilitate identification of candidate drugs for hormone-refractory prostate cancer. FKBP5 for example was identified and its utility as a therapeutic target was validated using tissue microarray analysis (see Example 3). Based on such leads, Rapamycin, MS-275, and TSA were tested for their effectiveness in influencing prostate cancer cell growth. These drugs target some of the candidate genes described herein. As described in Example 4, the inventors found that these drugs inhibit CWR22R prostate cancer cell growth in vitro. Thus, incorporating cDNA microarray technologies for genomics-based discovery of therapy response genes, with high throughput tissue microarray analysis, provides a new paradigm to identify, prioritize, and validate novel diagnostic and drug targets, as herein described for hormone-refractory prostate cancer.
The identified HRPC-related genes represent putative mediators of hormone therapy response and resistance, and as such are candidate targets for the development of novel therapeutics to maintain prostate cancer in regression following hormone ablation therapy. The utility of tliese genes as candidate drug targets and biomarkers is demonstrated herein by first using tissue microarrays for high throughput translation to clinical samples, and then selecting drugs that might target these genes. Analysis of cDNA microarray data with template based gene clustering and high throughput translation using tissue microarrays introduces a new, generally applicable paradigm for applying functional genomics to identify genetic programs that mediate a responses to a variety of in vivo therapies. It is contemplated that certain of the HRPC-related genes identified herein encode or correspond to soluble proteins, while other encode or correspond to membrane associated or membrane integral proteins, some of which are exposed at least to a certain extent on the exterior of a cell in which they are expressed. In some embodiments, those HRPC-related molecules that are expressed at or on the surface of a cell are selected as therapeutic targets, for instance for targeting with an antibody-based therapy, which is facilitated by the access of the HRPC-related molecule to the extracellular matrix. These HRPC-related molecules may be described as being "drug accessible."
The disclosure is further illustrated by the following non-limiting Examples.
EXAMPLE 1
Identification of Genes with Altered Expression in Hormone Refractory Prostate Cancer
This example provides a description of how the disclosed HRPC-related nucleic acid molecules were identified. These HRPC-related nucleic acid molecules show differences in expression during prostate cancer development, and particularly during hormone ablation therapy and subsequent progression to a honnone-refractory condition. Methods and Material:
Xenografts and Cell Line: CWR22 is a serially transplantable, prostate cancer xenograft that was derived from a Gleason score 9 primary human prostate cancer with osseous metastasis (Wainstein et al, Cancer Res. 54:6049-6052, 1994). CWR22 is highly responsive to androgen deprivation, with marked tumor regression after castration (Cheng et al., J. Natl. Cancer Inst. 88:607- 611, 1996; Nagabhushan et α/., Cancer Res. 56:3042-3046, 1996; Myers et al., J Urol. 161 :945-949, 1999). About half of the treated animals develop recurrent tumors (CWR22R) over a time frame of from a few weeks to several months. CWR22R is not dependent on androgen and is able to grow in castrated animals.
Thirteen fresh-frozen human prostate xenograft tissues were recovered from mice at different stages of hormonal therapy (four primary untreated CWR22, CWR22 after 0.5 days, 2 days, 4 days, 8 days and 16 days after castration, and four independent hormone-refractory CWR22R strains). LNCap (ATCC) and CWR22R (established from recurrent CWR22R xenografts) cell lines were cultured in RPMI1640 (BibcoBRL) with 10% Fetal Bovine Serum (GibcoBRL) at 37 °C and 5% C02. The tumors were flash frozen and stored at -70 °C. RNA was extracted by crushing the tumors in liquid nitrogen and used directly for mRNA isolation with the FastTrack 2.0 Kit (Invitrogen Corp., Carlsbad, CA). cDNA Microarrays: The cDNA microarrays consisted of 6605 elements representing different (non-redundant) genes. PCR products from sequence-verified clones (Research Genetics, Huntsville AB) were prepared and printed at high density onto glass slides according to previously described protocols (Mousses et al, "Gene Expression Analysis by cDNA Microarrays," in Differential Gene Expression: A practical approach, Livesey and Hunt (eds.), Oxford University Press, 2000).
Labeled cDNA was made with 4-16 μg of mRNA in an oligo(dT)-primed polymerization using Superscript II reverse transcriptase (LifeTechnologies, Rockville , MD) in the presence of either Cy3 or Cy5 labeled dUTP (Amersham Pharmacia, Piscataway, NJ) as described (Mousses et al, "Gene Expression Analysis by cDNA Microarrays," in Differential Gene Expression: A practical approach, Livesey and Hunt (eds.), Oxford University Press, 2000). The standard reference cDNA (Cy5 labeled LNCap cDNA) and the Cy3 labeled test cDNA from a xenograft mRNA were simultaneously hybridized to the microarray according to the protocol described previously (Mousses et al, "Gene Expression Analysis by cDNA Microarrays," in Differential Gene Expression: A practical approach, Livesey and Hunt (eds.), Oxford University Press, 2000).
Imaging and Image Analysis: Fluorescence intensities at the immobilized targets were measured by using a custom-designed laser con-focal microscope scanner, with intensity data integrated over 15-micron square pixels and recorded at 16 bits. Image analysis was performed by using DEARRAY software. Details of the fabrication of the microarray slides, and image generation and analysis are available on the Internet at the NHGRI Microarray Website, and software is currently and freely available for Sybase UNIX and is in the process of being ported to Oracle/UNIX. Detailed information about the program itself can be found on the ArrayDB Web site at the NHGRI. A complete description of the gene clustering used is also described at the NHGRI Microarray Website.
In brief, clustering analysis is a powerful tool that partitions biological samples or genes into well-separated and homogeneous groups based on their statistical behaviors. The main objective of clustering analysis is to find out the similarities between experiments or between each genes, given their expression ratios across all genes or samples, respectively, and then group the similar samples or genes together for the convenience of understanding and visualization. The clustering methods have been heavily studied for many years and widely applied in many areas.
Hierarchical Clustering methods: Assume there are m expression experiments containing n genes in each every experiment. After performing microarray image analysis and data integration, a m x n matrix of gene expression ratios is obtained, where each column of ratios represents the result from one expression experiment comparing the test sample to a common reference sample of choice. To simplify the discussion, the algorithm is considered only in terms of the sample clustering.
To achieve the objective of clustering, all pair-wise similarities between samples are evaluated first, and then an "average linkage algorithm" is employed to group similar samples. Typically, a Pearson correlation coefficient or Euclidean distance is used to quantify the similarity. Under certain normalization condition, these two similarity measurements are equivalent. After evaluating similarities from all pairs of samples, a distance matrix can be constructed as shown below (Table 3a). The hierarchical algorithm proceeds as follows: First a pair of experiments with shortest distance or most similarity in gene expression pattern are identified (Expl and Exp2 in Table 3a). A "composite experiment" is then constructed by averaging (thus the term average-linkage algorithm) all gene expression ratios (log-transformed) from two experiments. This is referred to as Expl-2 in the example. All distances from this composite experiment to all other experiments are then examined, and used to construct a smaller matrix, as shown in Table 3b. This procedure is repeated until the distance matrix is reduced to single element.
TABLE 3a TABLE 3b
Figure imgf000030_0003
Figure imgf000030_0001
The graphical visualization of the hierarchical algorithm is illustrated by a dendrogram, where each merger is represented by a binary tree, and the length of each branch is indicative of the distance between two samples, such as those given in Table 3a and 3b.
Template Based Gene Clustering algorithm:
To fully exploit the characteristics of temporal response of gene expression to a given treatment, either an instantaneous stimulation or a continuously increasing/decreasing excitation, a sequence of pre-ordered templates which reflect all possible gene expression responses for a given stimulation was employed. The objective of the template-based algorithm is, given the kth. gene's temporal expression profile, to evaluate the similarities to all of ordered templates, and then based on the similarities of all templates, to produce a template index and a best similarity measure based on the Pearson correlation coefficient. Let the temporal expression profile for Mi gene is gk(t„) (logio- transformed expression ratios), and the rth response template to be Tj(t„), n = 1, ..., _V. The similarity between the Mi gene expression profile and rth template is defined by,
Figure imgf000030_0002
where μ and σ are means and standard deviations, respectively, for Mi gene expression profile and z'th template pattern across iVtime points. For a given gene k, the best similarity pk from all templates is
A = max {A,,- } and let the I* to be the template that satisfies pw* = pk, the template index is ak for gene k,
Σ l' +2 . ak = - ,wXA,
Σ f+2 i=l'-2 Pk,i
Usually, α* indexes to somewhere near the best-fit template index I , but adjusted according to the similarity of its neighboring templates given the pre-defined order. The predicted fold-change of gene expression profile is also defined based on the best-fitted template / to be Fk = 10*A where b is the slope of the regression line. Typically, the aforementioned template-based algorithm provides three parameters for each gene for a given order of template sequence. They are αfo pk, I*, and Fk for template index, best Pearson's similarity measure, the best fit template, and the predicted fold-change derived from the best fitted template, respectively. Given the characteristics of these parameters, we can easily perform following data analysis: 1) sorting the k to order the gene expression profiles; 2) eliminating genes with small pk or small Fk since their temporal expression profiles do not resemble close enough to any of the templates, and/or simply do not respond to the stimulation; and 3) studying the template given by for the property of gene functions.
A data-mining tool was developed to investigate the experimental model of hormone tumor therapy in vivo. This model consists of independent clones of the same tumor undergoing different fates. A method for comparing the cDNA microarray data across the independent clones that undergo different fates was therefore needed to identify genes with temporal expression profiles associated with the response / phenotype of therapy in vivo. Filtering variables at different stringencies was used to mine the data to identify the genes that change most significantly in a manner that best reflects the temporal nature of the phenotypic changes observed during androgen ablation therapy.
Three criteria were used to mine the data to find genes that are associated with the phenotype: Variables assigned to each profile to facilitate data mining and clustering: the maximum correlation coefficient; the cluster location; and the fold change in ratio. The link to phenotype was accomplished by filtering the "maximum correlation coefficient" to templates that best describe the temporal profile of the phenotype. This also allows noise to be filtered out.
Clustering was accomplished by sorting the "cluster location". This organized the genes/templates so genes with similar profiles are clustered together. The cluster position was calculated by the weighted average of the three template positions that had the best correlation. For example, two different gene-expression profiles may have a maximum correlation coefficient for template number 5, but have different cluster locations such as 4.6 and 5.4, allowing for a continuum of locations between the templates.
The data can be mined further by filtering the data for "Fold Change" and "Fold Change to Recuirence." In this way, the amplitude of the change can be used to increase the stringency of the filter and identify genes that change most significantly. By filtering for Fold Change to Recurrence, genes were isolated that not only have a kinetic that fits the regression phenotype, but that are also restored in recurrent tumors.
Northern Analysis Xenograft and cell line mRNA (4 μg) was subjected to electrophoresis in a formamide containing agarose gel and blotted onto a nylon membrane (Hybond-N from Amersham) and probed according to the manufacturers protocol. An 342 bp SI OOP-specific DNA probe was PCR amplified with S100P specific primers (SEQ ID NO: 1, also referred to as S100PF, and SEQ ID NO: 2, also referred to as S100PR) from cDNA made with Superscript II reverse transcriptases using the manufacturers protocol (LifeTechnologies, Rockville , MD). The probe was radiolabeled with 32P (NEN) using the Ohgolabelling Kit (Pharmacia Biotechemicals Piscataway, NJ) according to the manufacturers specifications.
IHC and mRNA in situ using Tissue Microarray The prostate tissue microarray was constructed from paraffin embedded tumor tissue and benign control specimens. The tissue microarray permits analysis of up to 600 specimens simultaneously, greatly facilitating high throughput analysis of molecular markers in cancer tissue. The prostate tissue specimens were obtained from the Institutes of Pathology, University of Basal (Switzerland) and Tampere University Hospital (Finland). One pathologist (L. Bubendorf) reviewed all original tissue sections. The tissue microarray representing prostate neoplasm progression was constructed with 0.6 mm tissue cores and precisely arranged on a standard glass slide as described by Kononen et al. (Nat. Med 4:844-847, 1998). The microarray tissue samples represented 45 Benign Prostate Hypertrophy (BPH), 60 prostate intraepithelial neoplasia (PIN), 264 primary tumors, 134 hormone refractory tumors, and 41 metastatic tumors. Additionally, the tissue microarray contained 28 xenograft CRW tumor specimens and several other xenografts. Protocols for preparing prostate tissue microarrays are provided for instance in Bubendorf et al. , JNatl Cancer Inst 91:1758-1764, 1999 and Bubendorf et al. , Cancer Res. 59:803-806, 1999.
The tissue microarray facilitates simultaneous application of molecular diagnostic techniques, such as immunohistochemistry. Antigen retrieval was performed by treatment in a pressure cooker for 30 minutes. Standard indirect immunoperoxidase procedures were used for immunohistochemistry (Envision Plus, DAKO). A monoclonal mouse antibody (1:1000, Transduction Laboratories, Lexington, KY) was used for detection of S100P. The reactions were visualized with diaminobenzidine as a chromagen. The nuclear and cytoplasm staining intensity were classified into three groups (negative, weak and strong staining) in duplicate by two pathologists (Hostetter, G. and Ferhle, W.). Other antibodies used for IHC on tissue arrays included Ki67, SI OOP, FKBP5, PCNA, PSA, AR.
Representative primers used for SI OOP mRNA in situ hybridization were as follows: AntiSlOOP-A: C ATGCCCATGGCTGTCTCTAGTTCCGTCATGGTGCTAG (SEQ ID NO: 3); AntiSlOOP-B: CGTGCTGCCCTCGCTGCCCGAATATCGGGAAAAGACGTCTATGAT (SEQ ID NO: 4);
AntiSlOOP-C: TTATCCACGGCATCCTTGTCTTTTCCACTCTGCAGG (SEQ ID NO: 5); AntiSlOOP-D: TCCACCTGGGCATCTCCATTGGCGTCCAGGTCCTTGAGCA (SEQ ID NO: 6); AntiSlOOP-E: AGACGTGATTGCAGCCACGAACACGATGAACTCACTGAAG (SEQ ID NO:7); AntiSlOOP-F: CATTTGAGTCCTGCCTTCTCAAAGTACTTGTGACAGGC (SEQ ID NO: 8); AntiSlOOP-G: GGGACCATGGCTCTGCAGGAATCTGTGACATCTCCAGGGC (SEQ ID NO: 9); and AntiSlOOP-H: GCTCAGCCTAGGGGAATAATTGCCAACAAACACTTTTGGGAAGCC (SEQ ID NO: 10). The scatter plot shown in FIG 2 was generated for one primary and one recurrent tumor, to demonstrate the correlation between the samples (low variance as indicated by lack of scatter) and to highlight the most differentially expressed genes.
Results Therapy-associated phenotype: The CWR22 xenografts were serially passed in nude mice.
Tumor material was harvested from mice at (1) primary, (2) androgen withdrawal therapy induced regression, and (3) recurrent stages. Ki67 staining of tliese tissues indicated that the number of cells that were proliferating (Ki67 staining positive) decreased gradually, reaching a minimum at day eight, where almost all cells were negative for Ki67 staining (FIG ID). All the recurrent tumors had Ki67 staining that was higher than the primary tumors and approached 100% of the cells. Similar results were seen with PCNA (Myers et al. , J Urol. 161 :945-949, 1999), indicating that proliferation was shut off in the recurrent tumors but the entire tumor did not respond fully to the therapy until four to eight days. Androgen receptor (AR) immunohistochemical (IHC) staining (as described above), and northern analysis (as described herein) showed only a small increase in AR mRNA and protein after castration. In general, AR protein levels were about two-fold higher in the recurrent tumors. cDNA Microarray Analysis with Template Based Gene Clustering
For tliese analyses, cDNA microarrays containing sequence-verified clones that represent 6605 unique genes were constructed. Fluorescence intensity ratios relative to the standard reference (LNCap) were generated for all the genes for each experiment. Only genes that were expressed at a significant level above the background in all the experiments were used in the analysis. This intensity cutoff across all the samples (4 primary tumors, 7 time points, and 4 recurrent tumors) resulted in 2648 genes that were expressed at a sufficient level to give reliable data.
Differences in gene expression between primary CWR22 and recurrent CWR22R xenografts were measured in four separate experiments. Direct comparisons of a pool of four primary xenografts and four recurrent tumors were done by labeling one pool with Cy 5 and the other with Cy 3 and hybridizing them together (Direct P/R column). The experiment was repeated with the Cy 3 and Cy 5 dyes reversed. Each of the four primary and four recurrent xenografts were also hybridized individually against the standard reference cell line (LNCap) in eight independent experiments on a different microarray print of the same clones. In Table 4, the average of the four recurrent ratios relative to LNCap is divided by the average of the four primary recurrent ratio relative to LNCap, to give the column labeled Avg(4xL)/Avg(4xL). Finally, the pools were also hybridized separately on two different cDNA microarray slides against a standard reference (LNCap) to give the last column in Table 2, labeled Direct 4x4 Pooled. Table 2 shows genes that were selected based on consistency across all the pooled experiments and a significant difference in the average of four primary to four recurrent in the eight independent experiments. (Significance was considered at the 99 % confidence level). Table 4
Figure imgf000034_0001
A hierarchical, unsupervised approach was first used to cluster the data. Although it was difficult to identify clusters with the small number of samples in the time course, a cluster containing 135 genes that decreased in gene expression after therapy and then were re-expressed after recurrence was identified (listed in Table 5; 143 sequences are listed, eight of which are duplicates). Other genes that decreased and were subsequently re-expressed were also observed, but did not cluster together because of differences in kinetics.
Template clustering (as described above) was developed and used to organize the cDNA microarray data according to expression kinetics during the course of therapy from primary to 16 days following castration. This is a supervised clustering approach, in which a correlation of each gene to a set of templates selected to reflect the temporal nature of the phenotype is calculated. Templates with the best (maximum) expression profile were then utilized to calculate a ranking (cluster location), to sort the genes based on their kinetics. The recurrent (R) time point was not used to calculate correlation to the templates. Color-coding was used to reflect the change that occurs in expression (not ratio) during the therapy, red being the maximum point and green the minimum point. A database of the gene identification information and template clustering parameters such as cluster location (sorting rank), maximum correlation coefficient, and calculated fold change from lowest to highest point of expression during the first six time points was constructed. This database facilitates mining for HRPC-related genes by variable stringency query-based searches based on (1) amplitude of change during therapy, (2) specifics of kinetics such as early or late increase or decrease, and (3) extents of correlation to the phenotype templates. Filtering for higher "maximum correlation coefficient" allows the (1) selection of profiles that are more strongly associated with the therapy and (2) the elimination of noise in the data generated by large expression ratio differences due to tumor differences or experimental artifact. The genes were sorted and plotted according to the template that their expression most closely resembled. Since the order of each gene is not dependent of the order of other genes, filtering of the data and re-clustering does not require new calculations. Unsupervised gene clustering was first used to find genes with similar gene expression profiles. The plot shown in FIG 1A illustrates all 2648 genes that showed sufficiently high expression levels to be used in this analysis, organized in hierarchical clusters, as demonstrated by the dendogram on the left. Therapy time points are (from left to right) numbered 1 through 7 and represent primary (P), 0.5 days, 2 days, 4 days, 8 days, 16 days post castration and recurrent (R), respectively; genes are stacked vertically. A group of 139 genes that decrease after castration and then are re-expressed in the recurrent tumors forms a definable cluster (as indicated by the close branching in the dendogram); this group is outlined by a rectangle. Grey-coding/shading in FIG 1A reflects actual ratio to the reference as indicated by the key below the cluster. Template clustering followed by filtering for greater than two-fold ratio difference during therapy response and for profiles that have at least a 0.7 maximum correlation coefficient to any of the 12 templates resulted in 604 genes (listed in Table 1). The plot in FIG IB illustrates supervised- template based clustering of these 604 genes. The order of sorting is determined by the template for which each genes' expression is best correlated to, as indicated on the left of the cluster and described in the methods. Color-coding is usually used to represent the relative transcript expression ratio, as measured by cDNA microarray analysis. Red customarily indicates the maximum point in gene expression, green the minimum, and levels closer to the mean approach black. These colors have been converted to shades of grey, as shown in the key below the cluster. The 604 genes are stacked/clustered vertically for each of the time points in the experiment, organized from left to right and labeled 1 through 7 as for FIG 1A. For each of the first six time points, a correlation coefficient to each of the 12 templates was calculated for the expression profiles of the 2648 sufficiently expressed genes. The average of the three maximum correlation coefficients was used to calculate a precise cluster location that reflects the association of that gene to a particular profile, represented by a continuum of templates guided by the 12 shown in FIB IB.
Temporal Gene Expression Program Associated with the Response to, and Failure of, Hormone Therapy Filtering data from the microarray analyses at the level of a three-fold difference in the regressing time points yielded a set of 131 genes (Table 2), 59 of which clustered together due to their correlation (>0.8) to decreasing templates (labeled "Decreasing" in Table 2; Image ID Clone numbers: 788256, 196303, 415089, 785707, 774446, 781047, 126650, 795936, 131316, 700792, 324891, 811015, 207288, 204257, 769921, 249603, 207358, 435076, 43550, 416833, 814701, 898286, 204214, 796646, 129865, 66406, 451907, 711768, 416833, 453107, 509887, 66728, 789204, 626716, 47833, 149013, 531319, 789182, 856427, 725454, 49352, 293727, 273546, 53316, 42059, 855487, 281003, 898062, 24145, 134719, 684655, 29063, 45233, 814117, 283315, 785778, 840567, 767817, 742132) and FIG 1C, and 72 of which clustered together due to their correlation to increasing templates (labeled "Increasing" in Table 2; Image ID Clone numbers: 843249, 298417, 815284, 839101, 153411, 243816, 489839, 39920, 343867, 503083, 897667, 66322, 195340, 78294, 630013, 257162, 813841, 266146, 840511, 415084, 841695, 137139, 136557, 509731, 840460, 111750, 711918, 809998, 784296, 82734, 322160, 177772, 223350, 502832, 813611, 140806, 772425, 246430, 132140, 137096, 768246, 897906, 1412481, 124071, 42070, 362483, 382195, 130826, 811162, 796613, 138936, 811088, 142788, 345935, 773301, 781139, 810960, 813823, 242062, 843098, 51582, 839991, 840687, 66731, 272327, 121792, 120964, 1374571, 842836,
360885, 815774, 35828). On the right of each gene expression profile color plot is a number that corresponds to the fold change (Δ) in ratio between the first six time points. The gene cluster order was determined by the order of templates and cluster location, as described. Gene identifiers shown in FIG 1C include IMAGE clone ID and the current unigene cluster number, name and description. Genes that have previously been reported to be direct targets of the androgen receptor are shown in bold text, and include the following: malate dehydrogenase 1, NAD (soluble) (MDH1), proliferating cell nuclear antigen (PCNA), brain-specific alpha tubulin (TUBA3), ornithine decarboxylase 1 (ODC1), lactate dehydrogenase A (LDHA), a disintegrin and metalloproteinase domain (ADAM9), v-fos FBJ murine osteosarcoma viral (FOS), and andromedulin (ADM).
The template based, supervised cluster of 59 genes (filtered for greater than three-fold change; greater than 0.8 maximum correlation coefficient; only decreasing templates) (listed in Table 2, and labeled "Decreasing"), representing the genes with the largest decrease after castration, had extensive overlap (51 of 59 genes in common, Image Clone ID numbers 767817, 840567, 785778, 283315, 814117, 45233, 29063, 684655, 134719, 24145, 898062, 855487, 42059, 53316, 273546, 293727, 49352, 725454, 856427, 789182, 531319, 149013, 47833, 626716, 789204, 453107, 416833, 711768, 451907, 66406, 129865, 796646, 204214, 898286, 814701, 416833, 43550, 435076, 207358, 769921, 204257, 207288, 811015, 700792, 131316, 795936, 126650, 781047, 774446, 785707, 415089) with the hierarchical (unsupervised) cluster of 139 genes (Table 5). However, the unsupervised cluster was not inclusive of all the genes that responded to the therapy (since it only contained 139 of the 305 genes with a profile that fit a decreasing template with a minimum two-fold difference and >0.7 max. correlation coefficient). Furthermore, although supervised clustering did identify that at least 74 genes increased by more than three-fold and that fit an increasing template with more than 0.8 correlation coefficient, it was difficult to identify a coherent unsupervised cluster of increasing genes.
By template based clustering and filtering the data, a temporal gene expression program (fingerprint), or cluster of genes, was identified that had the largest expression decrease after castration and the best correlation to a decreasing temporal template (FIG 2). The genes are plotted from early repressed genes on the top, and gradually being repressed at later time points down the list to the bottom genes that had a late onset repression. Investigation of the genes in this list of 59 revealed at least eight genes previously known to be stimulated by androgens, and probably direct targets of the AR. The identification of these AR responsive genes in this cluster further substantiates the utility of template based gene clustering in identifying therapy response associated genes and suggests that other genes in this list may be previously unknown AR responsive genes.
Further examination of the genes in this cluster revealed that it is very rich in several important cell cycle regulators. These include genes known to be associated with cell growth of prostate cancer including PCNA, ornithine decarboxylase 1, c-fos, and tubulin. Most of the genes in this cluster however, are novel cell cycle regulators that were not previously associated with androgen ablation in prostate cancer. These include the following (Image ID Clone numbers in parentheses):
two BUB (budding uninhibited by benzimidazoles) genes, which regulate the cell cycle at the mitotic checkpoint by controlling chromosome segregation and responding to spindle disruption (781047, 785778, 842968);
UBCH10, a cyclin-selective ubiquitin carrier that regulates the destruction of mitotic cyclins (769921); CDKN3, a CDK-2 associated dual phosphatase (700792);
CDC2 delta T which regulates entry into S-phase and mitosis (898286);
CDC18L, which initiates replication (204214);
CKS2, a kinase that activates CDC28 (725454); MAD2L1, which regulates mitotic checkpoints especially sensitive to kinetochore and spindle loss (814701);
CENPF, a centromere/kinetichore cell cycle protein (435076);
STK12, a chromosome associated kinase that plays an important role in centrosome duplication regulation, aneuploidy, and amplification (531319); NEK2 a protein kinase that regulates G2-M transition (415089);
CDC20, responsible for nuclear movement prior to anaphase and chromosome separation (898062); and
CDC45L, required for the initiation of DNA replication (453107).
The abundance of growth regulatory genes in this cluster, and of genes known to be direct targets of the androgen receptor, provides further supports that genes in this cluster are dependent on androgens and are mediating the AR dependent growth arrest following androgen ablation.
Transcript levels of the genes in this cluster are restored when therapy fails, suggesting that these are also the genes that mediate the androgen-independent growth in recurrent tumors. These observations are also consistent with the hypothesis that resistance to therapy occurs through an androgen independent activation of the AR.
In addition, several genes were repressed that have never previously been associated with cellular proliferation. FKBP5, for example, was repressed by as much as 5.8-fold after castration. This may be a direct effect of decreased androgen receptor transcriptional activation. FKBP5 has been associated with the glucocorticoid receptor, and targeting of FKBP proteins has been shown to lead to deregulation of several signal transduction pathways.
Another gene that showed a large amplitude change after castration, with unknown consequence, is transmembrane 4 superfamily member 1 (7.1 fold decrease). Conversely, transmembrane 4 superfamily member 3 increased after castration (3.2 fold). Putative signaling molecule serine/threonine kinase 12 (7.0 fold decrease) and insulin induced gene 1 (8.1 fold decrease) also showed substantial expression level changes after castration. Like the known cell cycle regulators, the expression of all these other genes is restored in the recurrent tumors. It is likely that these genes mediate growth arrest after therapy, and tumor re-growth after development of therapy resistance, and therefore these genes are ideal drug target candidates. In addition, some important genes that changed but did not make the top 59 list include
S100P, ID3, PSA and c-myc mRNA, which decreased by 5.2, 2.85, 2.77 and 3.01 fold respectfully during regression (FIG 2). These were not included in the primary list either because they did not meet the 3 fold cut-off, or because the maximum correlation coefficients were less than 0.8 (0.51 0.67, 0.71 and 0.50). Of this group, S100P and ID3 are especially good candidate drug targets because they are also over-expressed in recurrent CWR22R relative to their primary counterparts (Table 2).
Most, but not all, of the genes that show increased expression following therapy response generally remained elevated in the recurrent tumors. This is contrary to the repressed genes, whose transcript levels were largely restored. Some genes, however, increased during therapy and then were restored in the recurrents. This group includes: the UDP glycosyltransferase 2 family, polypeptide B15 and UGT2B4, sialyltransferase 1 (beta-galactoside alpha-2,6-sialytransferase), fatty-acid- Coenzyme A ligase, human metallothionein (MT)I-F gene, tumor suppressor PTEN, cadherin 3, placental-cadherin, gelsolin (amyloidosis, Finnish type), TAP binding protein (tapasin), and several other transcripts. The increase in PTEN indicates that the AKT S6 kinase pathway may be inhibited following castration, suggesting that therapeutic intervention with rapamycin may mimic this inhibition in recurrent tumors.
EXAMPLE 2
Identification of Further Genes with Altered Expression in Hormone Refractory Prostate Cancer
Using different microarrays, and methods essentially similar to those described above in
Example 1, additional HRPC-related nucleic acid molecules were identified and further characterized. These HRPC-related nucleic acid molecules also show differences in expression during prostate cancer development, and particularly during hormone ablation therapy and subsequent progression to a hormone-refractory condition.
Methods and Material: Methods and materials were essentially as described in Example 1, except that additional custom cDNA microarrays were used, constituting 6605 to 8000 elements (sequence verified clones from Research Genetics, Huntsville, Alabama), representing different (non-redundant) transcripts including 4032 to 7700 known (named) genes (Mousses et al, Funtional Genomics: Gene Expression Analysis by cDNA Microarrays Livesey and Hunt (eds). Oxford University Press: Oxford, pp. 113- 137, 2000.). All xenografts were analyzed at least twice. Either LNCap or CWR22R were used as a reference and labeled with Cy5. The reference cDNA was simultaneously hybridized with Cy3 labeled test specimens on a cDNA microarray as previously described (Mousses et al, Funtional Genomics: Gene Expression Analysis by cDNA Microarrays Livesey FJ and Hunt SP (eds). Oxford University Press: Oxford, pp. 113-137, 2000.). Fabrication of the microarray slides, image generation, and the software used for the ratio analysis, and bioinformatics were as described above. Mousses et al, Funtional Genomics: Gene Expression Analysis by cDNA Microarrays Livesey and Hunt (eds). Oxford University Press: Oxford, pp. 113-137, 2000.
Template-based clustering was performed as described above. Most Systematically Altered Genes
Another set of genes was identified that showed differential expression between primary and recurrent tumors. Based on the mean gene expression ratios from six recurrent and four primary tumors, expression levels of 104 of the 3495 informative genes (3.0%) were significantly (2-fold or more) increased, and those of 60 genes (1.7%) decreased in the recurrent tumors. FIG 3A shows 30 genes (out of a total of 164 differentially expressed genes) that were most systematically altered in the recurrent tumors. These genes include SCYD1, S100P, CCND1, CRIP1, ISG15, SCNN1A, ZFP103, MAPKAPK2, UTG2B15, RABGGTA, NFKBIA, SLCYA5, AP3B2, PTPN2, FOXJl, and APOC1 (all upregulated) and FLJ23538, OXCT, PFKP, TNRC3, HXB, PFKP, OAT, PFKP, RFP, THBS 1 , LM04, MLD, CRYM, MME, HMGCS2, and SLC 12A2 (all downregulated).
Among the 164 genes were several genes coding for proteins that either converged on the PI3K/AKT/FRAP pathway or represented direct targets of macrolide drugs (such as rapamycin and FK506). As highlighted in FIG 3B, several genes that were androgen-responsive and re-expressed in the recurrent tumors (CCND1, ODC1, EIF1EBP1, MAPKAPK2, NFKBIA, CDSl, FKBP4, and FOXJl) met these criteria and suggested involvement of rapamycin-sensitive signaling in hormone- refractory tumors.
These findings appeared to indicate that rapamycin-sensitive gene products and signaling pathways play a role in androgen independent growth in the recurrent tumors. To further evaluate this hypothesis, the effects of rapamycin and FK506 on the growth and viability of a cell line established from the recurrent CWR22R xenografts were studied. Rapamycin is a known inhibitor of the PI3K/AKT/FRAP pathway (Kunz et al, Cell, 73:585-596, 1993; Brunn et al, EMBOJ., 15:5256- 5267, 1996; Sekulic et al, Cancer Res., 60:3504-3513, 2000), and FK506 targets many of the same intracellular proteins as rapamycin. Death of the hormone-independent CWR22R cells was observed at very low doses of rapamycin (IC50 ~ 0.1 nM) (FIG 3D), whereas hormone-responsive LNCap prostate cancer cell lines exhibited partial inhibition, even at high doses (FIG 3D). FK506 treatment did not have an inhibitory effect on either the CWR22R or LNCap cells even at the highest doses tested (greater than 80% cell survival at a dose of 10 mM). The results are based on two different cell lines that are not isogenic and may have other differences contributing to the observed effects. However, both these results of the global-scale gene expression studies and the data from the in vitro sensitivity testing, indicate that further studies are warranted to explore rapamycin as a candidate drug for the treatment of hormone refractory prostate cancers.
Cancer cells exhibit greater than a 1000-fold (IC50 ranging from <1 nM to >10 mM) variability in their sensitivity to rapamycin, possibly reflecting mechanisms of intrinsic resistance (Hosoi et al, Mol. Pharmacol, 54:815-824, 1998). Cancer cells that have activated genes and pathways that signal through the PI3K/AKT/FRAP pathway may be particularly sensitive. For example, IGF-1 receptor activation is associated with the efficacy of rapamycin treatment in childhood sarcomas (Dilling et al., Cancer Res. 54:903-907, 1994). Several growth factors and related genes that we observed to be overexpressed in the recurrent prostate cancers relative to the primary tumors (such as HGF, VEGFC, FGF2, IGFBP3, PDGFA, LTBP4, GFR, PGF, ITPKB, CDSl, and FKHL13) could have similarly contributed to the activation of the PI3K/AKT/FRAP pathway and alterations in the rapamycin target expression.
Finally, the two macrolide drugs rapamycin and FK506 bind similar intracellular targets but have different biological effects in hormone-refractory prostate cancer. These differences may be informative in elucidating those molecular pathways that are most critical for progression of prostate cancer. Rapamycin and FK506 both bind to FKBP12 (FK506-binding protein 12) (Sabers et al, J. Biol. Chem., 270:815-822, 1995; Liu et al, Cell 66:807-815, 1991). Rapamycin-FKBP12, but not the FK-506-FKBP12 complex, inhibits FRAP (FKBP-Rapamycin Associated Protein), a member of the phosphoinositide-3 -kinase related kinases that regulate translation following mitogenic activation of the PI3K/AKT/FRAP pathway. In contrast, FK506, but not rapamycin, inhibits calcineurin activity (Liu et al, Cell 66:807-815, 1991). This suggests that, of the many known and unknown targets of rapamycin and FK506, FRAP and the activity of the PI3K AKT pathway is a more likely candidate than calcineurin as a drug target in hormone-refractory prostate cancer. This example clearly illustrates that transcriptional profiling can be used to identify candidate drugs for treatment of prostate cancer, and this approach generally, as well as the present findings more specifically, can be used for a basis of such treatment decisions.
EXAMPLE 3 Analysis of Specific Genes
A direct comparison of a pool of four primary CWR22 xenografts and four recurrent CWR22R xenografts was done by labeling one pool with Cy5 and the other with Cy3 and hybridizing them together (Direct P/R column in Table 2). This resulted in 251 genes (3.8% of the 6605 genes assayed; listed in Table 6) that were differentially expressed at the 99 % confidence level. This analysis was also done against the standard reference for each tumor individually and in pools with the most consistently differentially expressed genes shown in Table 4.
One of the most highly differentially expressed genes is a calcium binding protein, SI OOP. It was found to be expressed 16 times (by cDNA microarray analysis) to 100 times (by Northern hybridization analysis) higher in one recurrent xenograft compared to the primary. The SI OOP protein has been reported to be associated with increased survival and loss of senescence in breast cancer cells. This data indicates that SI OOP expression may be androgen dependent, as would be expected if it is involved in prostate cancer progression.
Several immunophilin-like proteins were also identified as being differentially expressed. FKBP5, in addition to being overexpressed by about two-fold on average, is one of the most repressed genes after castration. During recurrence, its expression is restored to higher levels than in the primary. FKBP5 is a member of the large immunophilin chaperone proteins, which have been shown to interact with HSP90 and several steroid receptors. The expression of this protein not only appears to be regulated by the androgen receptor function, but also may affect androgen receptor activity by protein folding of the nascent receptor or by modulating its binding affinity to ligands. There are several inhibitors (e.g, FK506 and rapamycin) that bind to immunophilins, resulting in either calcineurin inactivation and or the inhibition the phosphotidylinositol 3- kinase/PTEN/AKT/FRAP pathway (Zhong et al, Cancer Res. 60:1541-1545, 2000). The phosphorylated substrates of this pathway include calcineurin and ties into the calcium signaling pathway. In addition, HcB (which regulates NFkB), NFAT, and BAD are each substrates for this pathway and are all involved in regulation of cell survival. Decreased expression of PTEN, and increased expression of CDP-DG synthase, H B, PHYH, and several other changes also converge on, and possibly alter activity of, this pathway. Drugs that target immunophilins such as FK506 and rapamycin have been shown to inhibit this pathway at the level of FRAP, leading to (1) loss of activity for kinases with mitogen activated protein kinase (MAPK) like substrates and (2) inactivation of calcineurin. Differential gene expression data disclosed herein indicate that such drugs such as rapamycin and FK506 could have a dual role in preventing androgen independent progression of prostate cancer, by both (1) blocking signal transduction from the phosphotidylinositol 3- kinasePTEN/AKT/FRAP pathway and (2) interfering with androgen receptor protein folding and assembly. This is an example of the differential gene expression discussed herein, to assist in selecting new therapies for treatment of primary and recurrent (hoπnone-refractory) prostate cancer.
EXAMPLE 4 Tissue Microarray Analysis of Candidate Biomarkers
This example provides in-depth analysis of several HRPC-related genes, including illustrations of the clinical relevance of these genes in prostate cancer progression and staging. High throughput molecular validation of candidate genes in clinical specimens was accomplished by using tissue microarray technology to assess the utility of these HRPC-related genes as biomarkers and drug targets. Using a tissue microarray in this fashion represents an important method to cross- validate data from experimental systems and human cancer, specimens.
Tissue microarray methods were carried out essentially as described above, and as known in the art; see, for instance, Kononen et al, Nat Med. 4(7):844-847, 1998) Clinical translation of novel gene products where an antibody does not exist can be detected on tissue microarrays using isotopic in situ hybridization (ISH) (Kononen et al. Nat Med. 4(7):844-847, 1998; Frantz et al, JPathol. 195(1):87-96, 2001)
SWOP
The prevalence of SI OOP protein overexpression was investigated by immunohistochemistry, in 440 human prostate cancer specimens at various stages of progression.
These specimens were arrayed in a prostate cancer progression tissue microarray (Bubendorf et al, J Natl Cancer Inst 91:1758-1764, 1999 and Bubendorf et al, Cancer Res. 59:803-806, 1999). This array also contained about 50 different prostate cancer xenograft samples, including those used in the cDNA microarray experiments.
SI OOP mRNA was measured by three different methods in nine xenografts. cDNA microarray ratios measure the expression of SI OOP transcript by the amount of cDNA hybridized relative to the standard reference. Northern analysis with a PCR amplified fragment of the SI OOP against a blot of the same RNA used in the cDNA microarray analysis produced a fragment of expected size (-0.5 kb). Northern hybridization bands were quantified using ImageQuant software from a scanned autoradiogram. An mRNA in situ hybridization (ISH) was performed by radio- labeling eight non-overlapping oligonucleotide (-45 bp) that span the coding region and hybridizing them to tissue microarrays containing hundreds of sections (including xenografts) described in the methods and materials. The signal was quantified using a Fuji phosphoimager and scanner and Bos software. The quantification of each of these there methods is plotted above the images for each of the nine xenografts. The absolute values are normalized to three of the primary tumors with the lowest Northern hybridization levels. For each of the xenograft tumors, the SI OOP protein expression is shown by IHC staining is shown below the graph (FIG 4).
FIG 4 shows that in at least the xenograft samples there is good concordance between Northern hybridization, cDNA microarray, and mRNA in situ on tissue microarray quantitation of SI OOP transcript levels. These mostly but not always correlate with immunohistochemical staining, hi at least a few cases, higher protein expression was observed with moderate levels of mRNA, indicating possible post-transcriptional regulation.
In situ mRNA hybridization was also used to quantitatively measure transcript levels on tissue microarray sections. Immunohistochemical analysis of SI OOP protein expression in 440 human prostate cancer specimens at various stages of progression is shown in FIG 5. An SI OOP- specific antibody was used to stain prostate tissue sections on a tissue microarray. The staining intensity was scored by two pathologists, using a scale of from 0 to 4. The results in FIG 5 show the percentage of cancers at each stage of prostate cancer progression that had strong staining (score of 3 or 4). FIG 5 shows that the high expression of SI OOP protein is associated with progression in clinical prostate cancers, with increasing expression in refractory and metastatic disease.
FKBP5
Translation of the observations on FKBP5 to clinical specimens is of interest because of this protein is associated with therapeutic response, and is over-expressed in recurrent tumors. Until now, it was thought that FKBP5 was only expressed in T-cells, and that it would make a good drug target for specific immunosuppression through the inhibition of glucocorticoid receptor transcriptional activation. Using prostate cancer progression tissue microarrays, FKBP5 was found to be expressed specifically in secretory cells of the normal prostate and in prostate cancer cells, but not in supporting stromal cells. Analysis of FKBP5 protein expression by IHC on the same prostate cancer tissue microarray as discussed above indicated that FKBP5 is expressed in the majority of prostate cancers, but an association with progression was not observed. Many of the primary and early lesions had common expression of this protein, thereby indicating that FKBP5 would not make a good biomarker for prostate cancer progression or the development of hormone refractory or metastatic disease. However, FKBP5 down-regulation does appear to be associated with therapeutic response, making it a candidate for therapeutic targeting in a large percentage of clinical tumors.
LM04 and CRYM
LM04 and CRYM genes were substantially down-regulated in the CWR22R tumors relative to primary CWR22, for mRNA ISH studies. In both cases, mRNA ISH on TMAs validated the relative expression levels seen by cDNA microarrays in the CWR22 xenograft specimens. This analysis revealed a lower level of LM04 and CRYM expression in 17 recurrent CWR22R xenografts (pO.OOl) as compared to 19 primary CWR22 xenografts. In addition to permitting us to validate our observations, the xenografts on the tissue microarrays were also used to compare the measurement of mRNA by cDNA microarray and mRNA ISH on a tissue microarray. As an example, there is a high correlation (r=0.96, n=16) between the levels of LM04 mRNA measured by mRNA ISH on a tissue microarray and data from cDNA microarrays.
A significant decrease (pO.OOl) of mRNA levels was observed for both LM04 and CRYM during tumor progression in cancer patients by mRNA ISH on the TMA. The mean intensity of actin mRNA was used as a negative control in the mRNA ISH. Comparison of mRNA ISH levels between primary and hormone refractory tumors on the same array revealed no significant differences between the two groups (P = 0.927).
Since antibodies are often not available for gene products discovered from cDNA microarray surveys, it remains essential to detect these transcripts on tissue microarrays using mRNA ISH. We validated here mRNA ISH-based detection of transcripts by inserting into the TMAs specimens that were originally used in the cDNA microarray analyses. There was an excellent correlation between mRNA ISH and cDNA microarray results, indicating that this method can be used to accurately measure mRNA levels in samples on a tissue microarray format. mRNA ISH was performed with several radioactively labeled oligonucleotide probes for different regions of the target genes. The use of short probes to different regions of the genes made it possible to obtain a signal even from degraded mRNAs that inevitably exist in clinical specimens. CRYM and LM04 were down-regulated in clinical specimens from hormone-refractory tumors, which is in line with the cDNA microarray results in the CWR22 xenograft model system.
LM04 is a member of the LIM-only (LMO) subfamily of LIM domain-containing transcription factors that is expressed during embryonic development (Kenny et al, Proc. Natl. Acad. Sci. 95: 11257-11262, 1998) and Crystallin mu (CRYM) codes for a thyroid hormone binding protein (Kim et al, Proc. Natl. Acad. Sci. 89:9292-9296, 1992; Aoki et al, J. Invest. Dermatol. 115:402-405, 2000). Both had transcript levels that were negatively associated with clinical progression. A role in prostate cancer progression has previously not been reported for either of these genes. It is believed that the observations presented herein indicate that perturbation of these genes has a functional role in clinical prostate cancer progression and pathogenesis.
This example illustrates tissue microarray technology validation of the in vivo involvement of four new prostate cancer related genes. Alterations in SI OOP, FKBP5, CRYM and LM04 genes are not only involved in the acquisition of androgen-independent growth and failure of therapy in prostate cancer xenografts but also with the progression of cancer in patients.
EXAMPLE 5 Targeting Candidate Genes with Known Drugs This example demonstrates the clinical effectiveness of selecting drug targets and genetic markers, indeed entire metabolic pathways, using the herein-disclosed HRPC-related genes. Several drugs were identified based on their known interaction with one or more of the HRPC-related genes or implicated pathways, and the activities of these drugs in controlling prostate cancer cell growth was examined.
Cell viability and Drug Treatment
Exponentially growing LNCaP or CWR22R cells were trypsinized and plated at 0.5 x 105 cell/ml or 1 x 105 cell/ml respectively in 96-well culture plates. After 24 hours, cells were treated for 72 hours with serial twofold dilutions of compound. DMSO was added to the control wells. Cell viability was measured by the WST-8 assay (Dojindo Molecular Technologies Inc.). The WST-8 [2- (2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonyl)-2H tetrazolium, monosodium salt] assay is based on the conversion of the tetrazolium salt WST-8 to highly water soluble formazan by viable cells (Tominaga et al, Anal. Commun. 36, 47-50, 1999). The WST-8 reagent solution was added to each well. After incubation for three hours at 37 °C, the absorbance was measured at 450 nm with a reference wavelength at 630 nm. The experiments were performed in triplicate. The data are representative of three separate experiment.
MS-275 and TSA
A literature search, coupled with a search of previous drug treatment data, was used to identify known compounds that could be used to target one or more of the 604 genes that changed at least two-fold following therapy response (FIG 1 and Table 1), or one or more of the 251 genes that were differentially expressed between primary and recurrent tumors (Table 6).
ID3 (clone: 756405) has recently been shown to be required for angiogenesis (Lyden et al, Nature 401:670-677, 1999). The inventors have also observed a decreased in thrombospondin (clone: 810512) (an angiogenesis inhibitor) during prostate cancer progression, suggesting that the expression of these two genes is changed in opposite directions in recurrence to achieve the same biological outcome, increased angiogenesis. Currently, there are no known inhibitors of ID3 (clone: 756405), but the inventors have observed in an independent set of cDNA microarray experiments that TSA, induced thrombospondin (clone: 810512) by as much as 8.6 fold in PC3M cells in vitro.
TSA works by an unknown mechanism, possibly by histone deacetylase inhibition resulting in altering transcription of a large number of genes. TSA treated PC3M cells revealed targets that were similarly affected in the direction of the growth arrested xenografts. Both drugs reduced kallikrein 3 (prostate specific antigen) (clone 824568) by two-fold, possibly reflecting an inhibition of AR-dependent transcriptional activation. Histone acetyltransferase 1 (clone: 745360) and acetyl- Coenzyme A acyltransferase (clone: 27848) are both decreased by about two-fold after castration, indicating that histone deacetylase inhibition might mimic this effect (growth suppression). Cyclin Dl (clone 841641) mRNA levels dropped to about 50 % only slightly after castration but the CWR22R recurrent tumors overexpressed it relative to the primary. Similarly chromosome condensation 1 (clone 724615) was 2.8 times higher (pooled experiment) in recurrent tumors. Both Cyclin Dl (clone 841641) and chromosome condensation 1 (clone 724615) were repressed by about three-fold by TSA treatment. The recurrent to primary ratio for protease inhibitor 12 (neuroserpin) (clone 564621) was 0.27, but TSA induced it by 8.67 fold.
Gene expression changes in response to treatment with these two drugs indicated that they might restore the expression of several genes that are associated with therapy resistance in CWR22R xenografts. TSA effectively inhibited growth of CWR22R, as indicated in FIG 3D. It is not known which of the above mentioned targets were affected, or by which mechanisms these two drugs caused growth arrest. It is possible that these drugs had a more global gene expression effect, which simultaneously restored multiple androgen responsive genes that are required for growth in the recurrent tumors.
Rapamycin andFK506 Sirolimus (Rapamycin) and Tacrolimus (FK506) are bacterial macrolides that are produced by fungi to suppress the growth of competing organisms. These drugs are immunosuppressants used extensively to prevent organ rejection. Although the two drugs are very similar both in structure and in their cellular targets, known as immunophilins (also called FKBP for FK506 binding proteins), the mechanism by which they cause immunosuppression is different. FK506 binds to immunophilins and the complex inhibits calcineurin in T-cells. In contrast, rapamycin-immunophilin complex inhibits signaling of the S6-kinase (clone: 204148, which also responds to castration) causing cell cycle arrest in T-cells. In addition, there are "macro" immunophilins that have been found to interact with steroid receptors, which may work though yet another mechanism to inhibit growth when complexed with these drugs. Several drug targets identified in this study are involved in immunophilin pathways, suggesting that either FK506 or rapamycin may cause a growth inhibition of hormone refractory prostate cancer. The first such candidate is a macroimmunophilin called FKBP5 (clone: 416833), one of the most strongly repressed genes in primary prostate CWR22 tumors after castration (FIG 2). The expression of FKBP5 (clone: 416833) is restored in hormone refractory CWR22R prostate cancer. In some tumors, FKBP5 mRNA expression (determined using cDNA microarray and RT-PCR quantitation) is restored to levels higher than found in the primary tumors. The availability of FKBP5 as a drug target was also confirmed using tissue microarray analysis. It is not clear if FKBP5 is required for the proliferation of CWR22R cells, but the expression of the FKBP5 transcript is associated with the proliferation phenotype. FKBP5 is a large protein that associates with steroid receptors, such as the glucocorticoid receptor, through binding to HSP90. It is also possible that FKBP5 interacts with the AR.
Cyclin D mRNA was 2.5-fold higher in a pool of four recurrent tumors compared to a pool of four recurrent tumors. Rapamycin has been shown to target and down-regulate cyclin D protein at both a transcriptional and post-transcriptional level (Hashemolhosseini et al, J. Biol. Chem. 273:14424-14429, 1998). Also, p27 had increased after castration by about 2-fold by day 8, and then went back down in the recurrent tumor. Rapamycin can increase p27 levels, making it a candidate for reversing the decrease seen in the recurrent CWR22R. These rapamycin effects on both cyclin D and p27 may be direct, but also may be mediated by inhibition of the phosphotidylinositol 3- kinase/PTEN/AKT/FRAP pathway. Several gene expression changes have been identified herein that could converge to activate this pathway in recurrent tumors, further suggesting that this is a pathway necessary for androgen independent growth. For example, an increased was observed in expression of CDP-diacylglycerol synthase 1 (levels up to 2.77-fold higher in recurrent tumors). CDP- diacylglycerol synthase 1 is a rate luniting enzyme in phosphotidylinositol 3 (PI3) production that has been shown to increase the amplitude and duration of PI3 signaling when overexpressed in model systems. PTEN, which is an inhibitor of this pathway, is increased during regression and re- expressed in the recurrent tumors further illustrating the importance of this pathway for proliferation of recurrent tumors. It has also been shown that rapamycin inhibits the translation of ornithine decarboxylase
(ODC) transcripts by about 50% in epithelial cells. In this study, ODC was repressed (3.8 fold) during CWR22 regression, but then re-expressed in the recurrent CWR22R (FIG 2). Interestingly, FK506 has no effect on ODC transcript levels. Both ODC and cyclin D are important stimulators of proliferation, indicating that rapamycin can be used to target these molecules and cause growth arrest in androgen independent CWR22R cells.
Rapamycin effectively arrested the CWR22R cells in vitro, however a complete inhibition was not accomplished at the highest concentration of FK506 (lOμM) (FIG 3D). It is believed that the interaction of rapamycin with FKBP5 and its other cellular receptor immunophilins blocks a pathway necessary for growth, while the interaction of FK506 and FKBP5 does not. It is difficult to predict the mechanism by which these drugs exert an effect on a cell, because they bind multiple cellular targets. In this case, several putative cellular targets are known for these two macrolide drugs and at least one, FKBP5, was both associated with the HRPC phenotype, and available in the relevant cells (FKBP5 protein is expressed in most clinical recurrent tumors). More specific inhibitors of FKBP5 activity can be used to elucidate the role FKBP5 plays in the growth of hormone refractory tumors.
EXAMPLE 6 Pharmacogenomics Analysis
This example illustrates the involvement of gene targets in pharmacological response to various emerging therapies.
Xenografts and Cell lines: Fresh frozen tissue from CWR22 human prostate cancer xenografts (Pretlow et al, J. Natl. Cancer Inst. 85:394-398, 199)) was obtained from thirteen different mice at different stages of hormonal therapy and tumor progression (four primary untreated CWR22, five CWR22 therapy time points after 0.5 days, 2 days, 4 days, 8 days and 16 days after castration, and four independent hormone-refractory CWR22R strains). LNCaP (ATCC) and CWR22R (kindly provided by Dr. Jim Jacobberger's Laboratory at Case Western University) cell lines were cultured in RPMI1640 10% fetal bovine serum (Life Technologies Rockville, MD) at 37 °C and 5% C02. mRNA was extracted with the FastTrack 2.0 Kit (Invitrogen Corporation; Carlsbad, California).
Drug Treatment and Cell viability: Exponentially growing LNCaP or CWR22R cells were trypsinized and plated at 0.5 x 105 cells/ml or 1 x 105 cells/ml respectively in 96-well culture plates. After 24 hours, cells were treated for 72 hours with serial two-fold dilutions of either FK-506 (Tacrolimus, Calbiochem Inc., San Diego, California), Rapamycin (Sirolimus) (Sigma Chemical co. St. Louis, Missouri), FR901464 (Fujisawa Pharmaceutical Co., Ltd., Ibaraki, Japan), Trichostatin A - TSA (a histone deacetylase inhibitor; Sigma Chemical co. St. Louis, Missouri) or DMSO as a control. The structure of FR901464 is as follows:
Figure imgf000048_0001
Cell viability was measured (triplicate experiments) by the WST-8 assay (Dojindo Molecular Technologies Inc., Gaithersburg, Maryland). CWR22R cells were treated with various drugs at effective doses for 1, 3, 9 and 24 hours followed by mKNA isolated for cDNA microarray experiments. Analysis of mRNA expression by cDNA Microarrays: Custom cDNA microarrays were constructed consisting of 6605 to 8000 elements (sequence verified clones from Research Genetics, Huntsville, Alabama), representing different (non-redundant) transcripts including 4032 to 7700 known (named) genes (Mousses et al. in Functional Genomics, (eds. Livesey & Hunt) 113-137, Oxford University Press, Oxford, 2000). All xenografts were analyzed at least twice using either LNCap or CWR22R Cy 5 labeled reference cDNA simultaneously hybridized with Cy 3 labeled CWR22 xenograft or CWR22R cell line cDNA on a cDNA microarray according to a previously described protocol (Mousses et al. in Functional Genomics, (eds. Livesey & Hunt) 113-137, Oxford University Press, Oxford, 2000). Fabrication of the microarray slides, image generation, and the software used for the ratio analysis, and bioinformatics was carried out essentially as described above.
Results: Trichostatin A (TSA) and FR901464 (an experimental drug found to inhibit the growth of a human solid tumor grown in mice and murine solid tumors; Nakajima et al, J. Antibiot. 49: 1204- 1211, 1996) were selected for in vitro testing in CWR22R cells based on previous pharmacogenomics analysis on PC3M cells, which suggested targeting of androgen independent growth associated genes. Rapamycin and FK506 were selected as drugs that also might target some of these candidates. To prioritize candidate gene targets that were not only associated with androgen independent growth but also involved in eliciting an effective drug response, cDNA microarray analysis of CWR22R gene expression was conducted during a time course of drug treatment in vitro.
Functional analysis of the dose response of each of these four drugs was carried out using a viability assay on CWR22R cells in vitro (FIG 6). A strong inhibition of growth and survival was seen for CWR22R cells with rapamycin, TSA, and FR901464, but not with FK506. Neither FK506 nor rapamycin were as effective at inhibiting the hormone-dependent LNCap prostate cancer cells. The global gene expression profiles indicate mechanisms of drug action that are distinctly different from androgen withdrawal response related signaling (FIG 6A and 6B). Despite this, some candidate genes that were associated with androgen independent growth of CWR22R were also involved in eliciting a response to some of these drug treatments including FKBP5, CRYM, and several others (FIG 6C and 6D; ATP1B2, OAT, QSCN6, GSN, PLU-1, GFPT2, ZCYTOR7, and VDUP1).
Pharmacogenomic analysis revealed that the two drugs TSA and FR901464 work by distinct mechanisms, which do appear not to involve androgen signaling (SM unpublished data). Although some commonly repressed genes were identified across experiments, VDUP1 (upregulated by 1,25- dihydroxyvitamin D-3 ; accession number XM_002093) was the only transcript that was up-regulated in response to therapy in vivo (maximum of 18 fold) and in vitro to Rapamycin, TSA and FR901464 (maximum of 73 fold). Based on these results, this gene acts as suppressor of tumor growth and survival.
EXAMPLE 7
Expression of HRPC-related Polypeptides
The disclosed HRPC-related proteins (and fragments thereof) can be expressed by standard laboratory technique. After expression, the purified HRPC-related protein or polypeptide may be used for functional analyses, antibody production, diagnostics, prognostics, and patient therapy, e.g., for prevention or treatment of prostate cancer (including hormone-refractory or metastatic prostate cancer). Furthermore, the DNA sequences encoding the disclosed HRPC-related proteins can be manipulated in studies to understand the expression of these genes and the function of their products, in particular how these HRPC-related proteins function in the control of or response to hormone- refractory prostate cancer. Mutant forms of human HRPC-related proteins (and corresponding encoding sequences) may be isolated based upon information contained herein, and may be studied in order to detect alteration in expression patterns in terms of relative quantities, tissue specificity and functional properties of the encoded mutant HRPC-related protein. Partial or full-length cDNA sequences, which encode the subject protein, may be ligated into bacterial expression vectors.
Methods for expressing large amounts of protein from a cloned gene introduced into Escherichia coli (E. coli) or other prokaryotes may be utilized for the purification, localization, and functional analysis of proteins. For example, fusion proteins consisting of amino terminal peptides encoded by a portion of the E. coli lacZ or trpE gene linked to an HRPC-related protein may be used to prepare polyclonal and monoclonal antibodies against these proteins. Thereafter, these antibodies may be used to purify proteins by immunoaffinity chromatography, in diagnostic assays to quantitate the levels of protein and to localize proteins in tissues and individual cells by immunofluorescence.
Intact native protein may also be produced in E. coli in large amounts for functional studies. Methods and plasmid vectors for producing fusion proteins and intact native proteins in bacteria are described in Sambrook et al. (Sambrook et al, In Molecular Cloning: A Laboratory Manual, Ch. 17, CSHL, New York, 1989). Such fusion proteins may be made in large amounts, are easy to purify, and can be used to elicit antibody response. Native proteins can be produced in bacteria by placing a strong, regulated promoter and an efficient ribosome-binding site upstream of the cloned gene. If low levels of protein are produced, additional steps may be taken to increase protein production; if high levels of protein are produced, purification is relatively easy. Suitable methods are presented in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and are well known in the art. Often, proteins expressed at high levels are found in insoluble inclusion bodies. Methods for extracting proteins from these aggregates are described by Sambrook et al. (In Molecular Cloning: A Laboratory Manual, Ch. 17, CSHL, New York, 1989). Vector systems suitable for the expression of lacZ fusion genes include the pUR series of vectors (Ruther and Muller- Hill, EMBO J. 2:1791, 1983), pEXl-3 (Stanley and Luzio, EMBO J. 3:1429, 1984) and pMRlOO (Gray et al, Proc. Natl. Acad. Sci. USA 79:6598, 1982). Vectors suitable for the production of intact native proteins include pKC30 (Shimatake and Rosenberg, Nature 292:128, 1981), pKK177-3 (Amann and Brosius, Gene 40:183, 1985) and pET-3 (Studiar and Moffatt, J. Mol. Biol. 189:113, 1986). Fusion proteins, for instance fusions that incorporate a portion of a HRPC-related protein, may be isolated from protein gels, lyophilized, ground into a powder and used as an antigen. The DNA sequence can also be transferred from its existing context to other cloning vehicles, such as other plasmids, bacteriophages, cosmids, animal viruses and yeast artificial chromosomes (YACs) (Burke et al., Science 236:806-812, 1987). These vectors may then be introduced into a variety of hosts including somatic cells, and simple or complex organisms, such as bacteria, fungi (Timberlake and Marshall, Science 244:1313-1317, 1989), invertebrates, plants (Gasser and Fraley, Science 244:1293, 1989), and animals (Pursel et al, Science 244:1281-1288, 1989), which cell or organisms are rendered transgenic by the introduction of the heterologous HRPC-related cDNA.
For expression in mammalian cells, the cDNA sequence may be ligated to heterologous promoters, such as the simian virus (SV) 40 promoter in the pSV2 vector (Mulligan and Berg, Proc. Natl. Acad Sci. USA 78:2072-2076, 1981), and introduced into cells, such as monkey COS-1 cells (Gluzman, Cell 23:175-182, 1981), to achieve transient or long-term expression. The stable integration of the chimeric gene construct may be maintained in mammalian cells by biochemical selection, for example with neomycin (Southern and Berg, J. Mol. Appl. Genet. 1:327-341, 1982) or mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981).
DNA sequences can be manipulated with standard procedures such as restriction enzyme digestion, fill-in with DNA polymerase, deletion by exonuclease, extension by terminal deoxynucleotide transferase, ligation of synthetic or cloned DNA sequences, site-directed sequence- alteration via single-stranded bacteriophage intermediate or with the use of specific oligonucleotides in combination with PCR.
The cDNA sequence (or portions derived from it) or a mini gene (a cDNA with an intron and its own promoter) may be introduced into eukaryotic expression vectors by conventional techniques. These vectors are designed to permit the transcription of the cDNA in eukaryotic cells by providing regulatory sequences that initiate and enhance the transcription of the cDNA and ensure its proper splicing and polyadenylation. Vectors containing the promoter and enhancer regions of the SV40 or long terminal repeat (LTR) of the Rous Sarcoma virus and polyadenylation and splicing signal from SV40 are readily available (Mulligan et al, Proc. Natl. Acad. Sci. USA 78:1078-2076, 1981; Gorman et al, Proc. Natl. Acad. Sci USA 78:6777-6781, 1982). The level of expression of the cDNA can be manipulated with this type of vector, either by using promoters that have different activities (for example, the baculovirus pAC373 can express cDNAs at high levels in S. frugiperda cells (Summers and Smith, In Genetically Altered Viruses and the Environment, Fields et al. (Eds.) 22:319-328, CSHL Press, Cold Spring Harbor, New York, 1 85) or by using vectors that contain promoters amenable to modulation, for example, the glucocorticoid-responsive promoter from the mouse mammary tumor virus (Lee et al, Nature 294:228, 1982). The expression of the cDNA can be monitored in the recipient cells 24 to 72 hours after introduction (transient expression).
In addition, some vectors contain selectable markers such as the gpt (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981) or neo (Southern and Berg, J. Mol. Appl. Genet. 1:327-341, 1982) bacterial genes. These selectable markers permit selection of transfected cells that exhibit stable, long-term expression of the vectors (and therefore the cDNA). The vectors can be maintained in the cells as episomal, freely replicating entities by using regulatory elements of viruses such as papilloma (Sarver et al, Mol. Cell Biol. 1 :486, 1981) or Epstein-Barr (Sugden et al, Mol. Cell Biol. 5:410, 1985). Alternatively, one can also produce cell lines that have integrated the vector into genomic DNA. Both of these types of cell lines produce the gene product on a continuous basis. One can also produce cell lines that have amplified the number of copies of the vector (and therefore of the cDNA as well) to create cell lines that can produce high levels of the gene product (Alt et al, J. Biol. Chem. 253:1357, 1978).
The transfer of DNA into eukaryotic, in particular human or other mammalian cells, is now a conventional technique. The vectors are introduced into the recipient cells as pure DNA (transfection) by, for example, precipitation with calcium phosphate (Graham and vander Eb, Virology 52:466, 1973) or strontium phosphate (Brash et al, Mol. Cell Biol. 7:2013, 1987), electroporation (Neumann et al, EMBO J 1 :841, 1982), lipofection (Feigner et al, Proc. Natl. Acad. Sci USA 84:7413, 1987), DEAE dextran (McCuthan et al, J. Natl. Cancer Inst. 41:351, 1968), microinjection (Mueller et al, Cell 15:579, 1978), protoplast fusion (Schafher, Proc. Natl. Acad. Sci. USA 77:2163-2167, 1980), or pellet guns (Klein et al, Nature 327:70, 1987). Alternatively, the cDNA, or fragments thereof, can be introduced by infection with virus vectors. Systems are developed that use, for example, retroviruses (Bernstein et al, Gen. Engr'g 7:235, 1985), adenoviruses (Ahmad et al, J. Virol. 57:267, 1986), or Herpes virus (Spaete et al, Cell 30:295, 1982). MB1 encoding sequences can also be delivered to target cells in vitro via non-infectious systems, for instance liposomes.
These eukaryotic expression systems can be used for studies of HRPC-related nucleic acids (such as those listed in Table 1) and mutant forms of these molecules, as well as HRPC-related proteins and mutant forms of these protein. Such uses include, for example, the identification of regulatory elements located in the 5' region of HRPC-related genes on genomic clones that can be isolated from human genomic DNA libraries. The eukaryotic expression systems may also be used to study the function of the normal HRPC-related proteins, specific portions of these proteins, or of naturally occurring or artificially produced mutant versions of HRPC-related proteins.
Using the above techniques, the expression vectors containing HRPC-related gene sequence or cDNA, or fragments or variants or mutants thereof, can be introduced into human cells, mammalian cells from other species or non-mammalian cells as desired. The choice of cell is determined by the purpose of the treatment. For example, monkey COS cells (Gluzman, Cell 23:175- 182, 1981) that produce high levels of the SV40 T antigen and permit the replication of vectors containing the SV40 origin of replication may be used. Similarly, Chinese hamster ovary (CHO), mouse NIH 3T3 fibroblasts or human fibroblasts or lymphoblasts may be used.
The present disclosure thus encompasses recombinant vectors that comprise all or part of a HRPC-related gene or cDNA sequence, for expression in a suitable host. The HRPC-related nucleic acid sequence is operatively linked in the vector to an expression control sequence to form a recombinant DNA molecule, so that the HRPC-related polypeptide can be expressed. The expression control sequence may be selected from the group consisting of sequences that control the expression of genes of prokaryotic or eukaryotic cells and their viruses and combinations thereof. The expression control sequence may be specifically selected from the group consisting of the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, the early and late promoters of SV40, promoters derived from polyoma, adenovirus, retrovirus, baculovirus and simian virus, the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, the promoter of the yeast alpha-mating factors and combinations thereof.
The host cell, which may be transfected with the vector of this disclosure, may be selected from the group consisting of E. coli, Pseudomonas, Bacillus subtilis, B. stear other mophϊlus or other bacilli; other bacteria; yeast; fungi; insect; mouse or other animal; or plant hosts; or human tissue cells.
It is appreciated that for mutant or variant HRPC-related DNA sequences, similar systems are employed to express and produce the mutant product. In addition, fragments of a HRPC-related protein can be expressed essentially as detailed above. Such fragments include individual HRPC- related protein domams or sub-domains, as well as shorter fragments such as peptides. HRPC-related protein fragments having therapeutic properties may be expressed in this manner also.
EXAMPLE 8 Suppression of HRPC-related Gene Expression
A reduction of HRPC-related protein expression in a transgenic cell may be obtained by introducing into cells an antisense construct based on a HRPC-related protein encoding sequence, such as a cDNA or gene sequence or flanking regions thereof of any one of the proteins listed in Table 1, Table 4, or elsewhere herein. For antisense suppression, a nucleotide sequence encoding a HRPC-related protein, e.g. all or a portion of the cartilage glycoprotein-39 (CHI3L1), S-100P PROTEIN (S100P), CX3C chemokine/fractalkine (SCYD1), adenylate kinase 1 (AK1), forkhead transcription factor HFH-4 (HFH-4) (FKHL 13), UDP glucuronosyltransferase precursor (UGT2B 15), Pleiotrophin (heparin binding growth factor 8) (PTN), heat shock 27kD protein 2/Alpha-B-crystallin (HSP27), Proteasome (prosome, macropain) subunit, beta type, 5 (PSMB5), Inhibitor of NFKB (NFKBIA), interferon-induced 17 kD protein (ISG15), MAP kinase activated protein kinase 2 (MAPKAPK2), signal transduction protein (SH3 containing) (EFS2), hkf-1 Zinc finger protein (ZFP103), chromosome condensation 1 (CHCl), CDP-diacylglycerol synthase (CDSl), gap junction protein, alpha 1, 43kD (connexin 43) (GJAl), cyclin Dl (CCNDl), Inhibitor of DNA binding 3, helix-loop-helix protein (ID3), HI histone family, member2 (H1F2), Cytochrome B561 (CYB561), Cathepsin H (CTSH), calcineurin alpha (PPP3CA), 54 kDa progesterone receptor-associated immunophilin (FKBP5), translocation protein 1 (TLOC1), Clusterin (complement lysis inhibitor; testosterone-repressed prostate message 2; apolipoprotein J) (CLU), Pulmonary surfactant-associated protein A (SFTPA1), protease inhibitor 12 (PI12; neuroserpin) (PI12), Thrombospondin 1 (THBS1), Ribophorin I (RPN1), A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 1 (ADAMTS1), Collagen, type IV, alpha 5 (Alport syndrome) (COL4A5), LIM domain only 4 / breast tumor autoantigen (LM04), bumetanide-sensitive Na-K-Cl cotransporter (NKCC1) (SLC12A2), Fibronectin (FN1), Crystallin Mu (CRYM) or "upregulated by 1,25-dihydroxyvitamin D-3" (VDUP1) cDNA or gene, is arranged in reverse orientation relative to the promoter sequence in the transformation vector. Other aspects of the vector may be chosen as for any other expression vector (see, e.g, Example 7).
The introduced sequence need not be a full-length human HRPC-related cDNA or gene, and need not be exactly homologous to the equivalent sequence found in the cell type to be transformed. Generally, however, where the introduced sequence is of shorter length, a higher degree of homology to the HRPC-related sequence likely will be needed for effective antisense suppression. The introduced antisense sequence in the vector may be at least 30 nucleotides in length, and improved antisense suppression will typically be observed as the length of the antisense sequence increases. The length of the antisense sequence in the vector advantageously may be greater than 100 nucleotides.
Although the exact mechanism by which antisense RNA molecules interfere with gene expression has not been elucidated, it is believed that antisense RNA molecules bind to the endogenous mRNA molecules and thereby inhibit translation of the endogenous mRNA.
Suppression of endogenous HRPC-related gene expression can also be achieved using ribozymes. Ribozymes are synthetic RNA molecules that possess highly specific endoribonuclease activity. The production and use of ribozymes are disclosed in U.S. Patent No. 4,987,071 to Cech and U.S. Patent No. 5,543,508 to Haselhoff. The inclusion of ribozyme sequences within antisense RNAs may be used to confer RNA cleaving activity on the antisense RNA, such that endogenous mRNA molecules that bind to the antisense RNA are cleaved, which in turn leads to an enhanced antisense inhibition of endogenous gene expression.
In addition, dominant negative mutant forms of the disclosed HRPC-related sequences may be used to block endogenous activity of the corresponding gene products.
Suppression can also be achieved using small inhibitory RNA molecules (siRNAs) (see, for instance, Caplen et al, Proc. Natl. Acad. Sci. 98(17):9742-9747, 2001, and Elbashir et al, Nature 411:494-498, 2001). Thus, this disclosure also encompasses siRNAs that correspond to an HRPC- related nucleic acid, which siRNA is capable of suppressing the expression or function of its cognate (target) HRPC-related protein. Also encompassed are methods of suppressing the expression or activity of an HRPC-related molecule using an siRNA.
Suppression of expression of an HRPC-related gene can be used, for instance, to treat, reduce, or prevent cell proliferative and other disorders caused by over-expression or unregulated expression of the corresponding HRPC-related gene. In particular, suppression of expression of sequences disclosed herein as being up-regulated in hormone-refractory prostate cancer can be used to treat, reduce, or prevent progression to hormone-refractory prostate cancer. EXAMPLE 9 Production of Protein Specific Binding Agents
Monoclonal or polyclonal antibodies may be produced to any of the disclosed HRPC-related proteins, or mutant forms of these proteins. Optimally, antibodies raised against these proteins, or peptides from within such proteins, would specifically detect the protein or peptide with which the antibodies are generated. That is, an antibody generated to the SI OOP protein (or another specified protein) or a fragment thereof would recognize and bind that protein and would not substantially recognize or bind to other proteins found in human cells.
The determination that an antibody specifically detects a designated protein (e.g., a HRPC- related protein as disclosed herein) can be made by any one of a number of standard immunoassay methods; for instance, the Western blotting technique (Sambrook et al, In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989). To determine that a given antibody preparation (such as one produced in a mouse) specifically detects a designated protein by Western blotting, total cellular proteins are extracted from cells (for example, human prostate) and electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel. The proteins are then transferred to a membrane (for example, nitrocellulose) by Western blotting, and the antibody preparation is incubated with the membrane. After washing the membrane to remove non-specifically bound antibodies, the presence of specifically bound antibodies is detected by the use of an anti-mouse antibody conjugated to an enzyme such as alkaline phosphatase. Application of an alkaline phosphatase substrate 5-bromo-4- chloro-3-indolyl phosphate/nitro blue tetrazolium results in the production of a dense blue compound by immunolocalized alkaline phosphatase. Antibodies that specifically detect the designated protein will, by this technique, be shown to bind to the designated protein band (which will be localized at a given position on the gel determined by its molecular weight). Non-specific binding of the antibody to other proteins may occur and may be detectable as a weak signal on the Western blot. The non- specific nature of this binding will be recognized by one skilled in the art by the weak signal obtained on the Western blot relative to the strong primary signal arising from the specific antibody-protein binding.
Substantially pure HRPC-related protein or protein fragment (peptide) suitable for use as an immunogen may be isolated from transfected or transformed cells, as described above. Concentration of protein or peptide in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms per milliliter. Monoclonal or polyclonal antibody to the protein can then be prepared as follows:
A. Monoclonal Antibody Production by Hybridoma Fusion Monoclonal antibody to epitopes of a designated protein (such as a HRPC-related protein, including any one of those listed in Table 1) identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (Nature 256:495-497, 1975) or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein over a period of a few weeks. The mouse is then sacrificed, and the antibody-producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess un-fused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall (Meth. Enzymol. 70:419-439, 1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988).
B. Polyclonal Antibody Production by Immunization
Polyclonal antiserum containing antibodies to heterogeneous epitopes of a single protein can be prepared by ύnmunizing suitable animals with the expressed protein (Example 7), which can be unmodified or modified to enhance immunogenicity. Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than others and may require the use of carriers and adjuvant. Also, host animals vary in response to site of inoculations and dose, with either inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradeπnal sites appear to be most reliable. An effective immunization protocol for rabbits can be found in Vaitukaitis et al. {J. Clin. Endocrinol. Metab. 33:988-991, 1971). Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony et al. (In Handbook of Experimental Immunology, Wier (ed.) chapter 19. Blackwell, 1973). Plateau concentration of antibody is usually in the range of about 0.1 to 0.2 mg/ml of serum (about 12 μM). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher (Manual of Clinical Immunology, Ch. 42, 1980).
C. Antibodies Raised against Synthetic Peptides A third approach to raising antibodies against the subject HRPC-related proteins or peptides is to use one or more synthetic peptides synthesized on a commercially available peptide synthesizer based upon the predicted amino acid sequence of the desired HRPC-related protein or peptide.
D. Antibodies Raised by Injection of Protein Encoding Sequence
Antibodies also may be raised against proteins and peptides related to HRPC as described herein by subcutaneous injection of a DNA vector that expresses the desired HRPC-related protein, or a fragment thereof, into laboratory animals, such as mice. Delivery of the recombinant vector into the animals may be achieved using a hand-held form of the Biolistic system (Sanford et al, Paniculate Sci. Technol. 5:27-37, 1987) as described by Tang et al. (Nature 356:152-154, 1992). Expression vectors suitable for this purpose may include those that express the HRPC-related sequence under the transcriptional control of either the human β-actin promoter or the cytomegalovirus (CMV) promoter.
Antibody preparations prepared according to these protocols are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they also can be used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample; or for immunolocalization of the corresponding HRPC-related protein.
For administration to human patients, antibodies, e.g., HRPC-related protein specific monoclonal antibodies (such as antibodies to the proteins encoded by the encoding sequences referred to in Table 1), can be humanized by methods known in the art. Antibodies with a desired binding specificity can be commercially humanized (Scotgene, Scotland, UK; Oxford Molecular, Palo Alto, CA). Alternatively, human antibodies can be produced. Methods for producing human antibodies are known in the art; see, for instance, Canevari et al, Int J Biol Markers 8:147-150, 1993 and Green, J Immunol Methods 231 : 11-23, 1999, for instance.
EXAMPLE 10
Nucleic Acid-Based Analysis
The HRPC-related nucleic acid molecules provided herein can be used in methods of genetic testing for neoplasms (e.g., prostate or other cancers) or predisposition to neoplasms owing to HRPC- related nucleic acid molecule deletion, genomic amplification or mutation, or over- or under- expression in comparison to a control or baseline. For such procedures, a biological sample of the subject, which biological sample contains either DNA or RNA derived from the subject, is assayed for a mutated, amplified or deleted HRPC-related nucleic acid molecule, or for over- or under expression of a HRPC-related nucleic acid molecule. Suitable biological samples include samples containing genomic DNA or RNA (including mRNA), obtained from body cells of a subject, such as those present in peripheral blood, urine, saliva, tissue biopsy, surgical specimen, amniocentesis samples and autopsy material.
The detection in the biological sample of a mutant HRPC-related nucleic acid molecule, a mutant HRPC-related RNA, an amplified or homozygously or heterozygously deleted HRPC-related nucleic acid molecule, or over- or under-expression of a HRPC-related nucleic acid molecule, may be performed by a number of methodologies.
A. Detection of Unknown Mutations:
Unknown mutations in HRPC-related nucleic acid molecules can be identified through polymerase chain reaction amplification of reverse transcribed RNA (RT-PCR) or DNA isolated from breast or other tissue, followed by direct DNA sequence determination of the products; single-strand conformational polymorphism analysis (SSCP) (for instance, see Hongyo et al, Nucleic Acids Res. 21:3637-3642, 1993); chemical cleavage (including HOT cleavage) (Bateman etal, Am. J. Med. Genet. 45:233-240, 1993; reviewed in Ellis et al, Hum. Mutat. 11:345-353, 1998); denaturing gradient gel electrophoresis (DGGE), ligation amplification mismatch protection (LAMP); or enzymatic mutation scanning (Taylor and Deeble, Genet. Anal. 14:181-186, 1999), followed by direct sequencing of amplicons with putative sequence variations. B. Detection of Known Mutations: The detection of specific known DNA mutations in HRPC-related nucleic acid molecules may be achieved by methods such as hybridization using allele specific oligonucleotides (ASOs) (Wallace et al, CSHL Symp. Quant. Biol. 51:257-261, 1986), direct DNA sequencing (Church and Gilbert, Proc. Natl. Acad. Sci. USA 81:1991-1995, 1988), the use of restriction enzymes (Flavell et al, Cell 15:25, 1978; Geever et al, 1981), discrimination on the basis of electrophoretic mobility in gels with denaturing reagent (Myers and Maniatis, Cold Spring Harbor Symp. Quant. Biol.
51:275-284, 1986), RNase protection (Myers et al, Science 230:1242, 1985), chemical cleavage (Cotton et al, Proc. Natl. Acad. Sci. USA 85:4397-4401, 1985), and the ligase-mediated detection procedure (Landegren et al, Science 241:1077, 1988). Oligonucleotides specific to normal or mutant MB1 sequences can be chemically synthesized using commercially available machines. These oligonucleotides can then be labeled radioactively with isotopes (such as 32P) or non-radioactively, with tags such as biotin (Ward and Langer et al, Proc. Natl. Acad. Sci. USA 78:6633-6657, 1981), and hybridized to individual DNA samples immobilized on membranes or other solid supports by dot-blot or transfer from gels after electrophoresis. These specific sequences are visualized by methods such as autoradiography or fluorometric (Landegren et al, Science 242:229-237, 1989) or colorimetric reactions (Gebeyehu et al, Nucleic Acids Res. 15:4513-4534, 1987). Using an ASO specific for a normal allele, the absence of hybridization would indicate a mutation in the particular region of the gene, or deleted MB1 gene. In contrast, if an ASO specific for a mutant allele hybridizes to a clinical sample then that would indicate the presence of a mutation in the region defined by the ASO. C. Detection of Genomic Amplification or Deletion
Gene dosage (copy number) can be important in neoplasms; it is therefore advantageous to determine the number of copies of HRPC-related nucleic acids in biological samples of a subject, e.g., serum or prostate samples. Probes generated from the disclosed encoding sequence of in HRPC- related nucleic acid molecules can be used to investigate and measure genomic dosage of the corresponding HRPC-related genomic sequence.
Appropriate techniques for measuring gene dosage are known in the art; see for instance, US Patent No. 5,569,753 ("Cancer Detection Probes") and Pinkel etal. (Nat. Genet. 20:207-211, 1998) ("High Resolution Analysis of DNA Copy Number Variation using Comparative Genomic Hybridization to Microarrays"). Determination of gene copy number in cells of a patient-derived sample using other techniques is known in the art. For example, amplification of a HRPC-related nucleic acid sequence in cancer-derived cell lines as well as uncultured prostate cancer or other cells can be carried out using bicolor FISH analysis. By way of example, interphase FISH analysis of breast cancer cell lines can be carried out as previously described (Barlund et al, Genes Chromo. Cancer 20:372-376, 1997). The hybridizations can be evaluated using a Zeiss fluorescence microscope.
For tissue microarrays, the FISH can be performed as described in Kononen et al, Nat. Med. 4:844-847, 1998. Briefly, consecutive sections of the array are deparaffinized, dehydrated in ethanol, denatured at 74° C for 5 minutes in 70% formamide/2 x SSC, and hybridized with test and reference probes. The specimens containing tight clusters of signals or >3-fold increase in the number of test probe as compared to chromosome 17 centromere in at least 10% of the tumor cells may be considered as amplified. Microarrays can be constructed as described in WO99/44063 A2 and WO99/44062A1. C. Detection of mRNA Expression Levels
Over- or under-expression of a HRPC-related molecule can also be detected by measuring the cellular level of HRPC-related nucleic acid molecule-specific mRNA. mRNA can be measured using techniques well known in the art, including for instance Northern analysis, RT-PCR and mRNA in situ hybridization. Details of such procedures can be found, for instance, in Examples 1 and 3.
The nucleic acid-based diagnostic methods of this disclosure are predictive of proliferation, metastatic potential, cancer progression, and response to treatment in patients suffering from prostate carcinomas including hormone-refractory prostate carcinomas, and other solid tumors, carcinomas, sarcomas, and cancers. Cells of any tumors that demonstrate abnormal levels (e.g., through genomic amplification, deletion, mutation, or other over- or under-expression) of nucleotide sequences that share homology with the HRPC-related nucleic acids disclosed herein are aggressive tumor cells, and result in decreased survival, increased metastasis, increased rates of clinical recurrence (such as recurrence after hormone ablation therapy), and overall worsened prognosis.
EXAMPLE 11
Protein-Based Analysis
An alternative method of diagnosing, staging, detecting, or predicting hormone-related prostate cancer is to quantitate the level of one or more HRPC-related proteins in a subject, for instance in the cells of the subject. This diagnostic tool is useful for detecting reduced or increased levels of HRPC-related proteins. Localization and/or coordinated expression (temporally or spatially) of HRPC-related proteins can also be examined using well known techniques. The determination of reduced or increased HRPC-related protein levels, in comparison to such expression in a normal subject (e.g. , a subject not having hormone-related prostate cancer or not having a predisposition developing this condition, disease or disorder, would be an alternative or supplemental approach to the direct determination of HRPC-related nucleic acid levels by the methods outlined above and equivalents. The availability of antibodies specific to specific HRPC-related protein(s) will facilitate the detection and quantitation of cellular HRPC-related protein(s) by one of a number of immunoassay methods which are well known in the art and are presented in Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988). Methods of constructing such antibodies are discussed above, in Example 9.
Any standard immunoassay format (e.g., ELISA, Western blot, or RIA assay) can be used to measure HRPC-related polypeptide or protein levels; comparison is to wild-type (normal) HRPC- related protein levels, and a difference in HRPC-related polypeptide levels is indicative of an abnormal biological condition such as neoplasia. Whether the key difference is an increase or a decrease is dependent on the specific HRPC-related protein under examination, as discussed herein. Immunohistochemical techniques may also be utilized for HRPC-related polypeptide or protein detection and quantification. For example, a tissue sample may be obtained from a subject, and a section stained for the presence of a HRPC-related protein using the appropriate HRPC-related protein specific binding agent and any standard detection system (e.g., one which includes a secondary antibody conjugated to horseradish peroxidase). General guidance regarding such techniques can be found in, e.g., Bancroft and Stevens (Theory and Practice ofHistological Techniques, Churchill Livingstone, 1982) and Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).
For the purposes of quantitating a HRPC-related protein, a biological sample of the subject, which sample includes cellular proteins, is required. Such a biological sample may be obtained from body cells, such as those present in peripheral blood, urine, saliva, tissue biopsy, amniocentesis samples, surgical specimens and autopsy material, particularly breast cells. Quantitation of a HRPC- related protein can be achieved by immunoassay and the amount compared to levels of the protein found in healthy cells. A significant difference (either increase or decrease) in the amount of HRPC- related protein in the cells of a subject compared to the amount of the same HRPC-related protein found in normal human cells is usually about a 30% or greater difference. Substantial under- or over- expression of one or more HRPC-related protein(s), may be indicative of neoplasia or a predilection to neoplasia or metastasis, and especially hormone-refractory prostate cancer.
The protein-based diagnostic methods as described herein are predictive of proliferation, metastatic potential, cancer progression, and response to treatment in patients suffering from prostate carcinomas including hormone-refractory prostate carcinomas, and other solid tumors, carcinomas, sarcomas, and cancers. Cells of any tumors that demonstrate abnormal levels (e.g., through genomic amplification, deletion, mutation, or other over- or under-expression) of nucleotide sequences that share homology with the HRPC-related nucleic acids disclosed herein are aggressive tumor cells, and result in decreased survival, increased metastasis, increased rates of clinical recurrence (such as recurrence after hormone ablation therapy), and overall worsened prognosis.
EXAMPLE 12:
Gene Therapy
Gene therapy approaches for combating neoplasia (particularly prostate cancer, including hormone-refractory prostate cancer) in subjects are made possible by the present disclosure. Retroviruses have been considered a preferred vector for experiments in gene therapy, with a high efficiency of infection and stable integration and expression (Orkin et al, Prog. Med. Genet. 7: 130-142, 1988). A full-length HRPC-related gene or cDNA can be cloned into a retroviral vector and driven from either its endogenous promoter or from the retroviral LTR (long terminal repeat). Other viral transfection systems may also be utilized for this type of approach, including adenovirus, adeno-associated virus (AAV) (McLaughlin et al, J. Virol. 62:1963-1973, 1988), Vaccinia virus (Moss et al, Annu. Rev. Immunol. 5:305-324, 1987), Bovine Papilloma virus (Rasmussen et al, Methods En∑ymol. 139:642-654, 1987) or members of the herpesvirus group such as Epstein-Barr virus (Margolskee et al, Mol. Cell. Biol. 8:2837-2847, 1988). Recent developments in gene therapy techniques include the use of RNA-DNA hybrid oligonucleotides, as described by Cole-Strauss, et al. (Science 273:1386-1389, 1996). This technique may allow for site-specific integration of cloned sequences, thereby permitting accurately targeted gene replacement.
In addition to delivery of HRPC-related protein encoding sequences to cells using viral vectors, it is possible to use non-infectious methods of delivery. For instance, lipidic and liposome- mediated gene delivery has recently been used successfully for transfection with various genes (for reviews, see Templeton and Lasic, Mol. Biotechnol. 11:175-180, 1999; Lee and Huang, Crit. Rev. Ther. Drug Carrier Syst. 14:173-206; and Cooper, Semin. Oncol. 23:172-187, 1996). For instance, cationic liposomes have been analyzed for their ability to transfect monocytic leukemia cells, and shown to be a viable alternative to using viral vectors (de Lima et al, Mol. Membr. Biol. 16:103-109, 1999). Such cationic liposomes can also be targeted to specific cells through the inclusion of, for instance, monoclonal antibodies or other appropriate targeting ligands (Kao et al, Cancer Gene Ther. 3:250-256, 1996).
To reduce the level of HRPC-related gene expression, gene therapy can be carried out using antisense or other suppressive constructs, the construction of which is discussed above (Example 8).
EXAMPLE 13 Kits
Kits are provided to determine the level (or relative level) of expression of one or more species of HRPC-related mRNA (/. e. , kits containing probes) or one or more HRPC-related protein (i.e., kits containing antibodies or other HRPC-related protein specific binding agents). Kits are also provided that contain the necessary reagents for determining gene copy number (genomic amplification or deletion), such as probes or primers specific for a HRPC-related nucleic acid sequence. These kits can each include instructions, for instance instructions that provide calibration curves or charts to compare with the determined (e.g., experimentally measured) values. A. Kits for Detection of HRPC-related Genomic
Amplification or Deletion
The nucleotide sequence of HRPC-related nucleic acid molecules disclosed herein, and fragments thereof, can be supplied in the form of a kit for use in detection of HRPC-related genomic amplification/deletion and/or diagnosis of progression to or predilection to progress to hormone- refractory prostate cancer. In such a kit, an appropriate amount of one or more oligonucleotide primer specific for an HRPC-related-sequence is provided in one or more containers. The oligonucleotide primers may be provided suspended in an aqueous solution or as a freeze-dried or lyophilized powder, for instance. The container(s) in which the oligonucleotide(s) are supplied can be any conventional container that is capable of holding the supplied foπn, for instance, microfuge tubes, ampoules, or bottles. In some applications, pairs of primers may be provided in pre-measured single use amounts in individual, typically disposable, tubes, or equivalent containers. With such an arrangement, the sample to be tested for the presence of HRPC-related genomic amplification/deletion can be added to the individual tubes and in vitro amplification carried out directly.
The amount of each oligonucleotide primer supplied in the kit can be any amount, depending for instance on the market to which the product is directed. For instance, if the kit is adapted for research or clinical use, the amount of each oligonucleotide primer provided likely would be an amount sufficient to prime several in vitro amplification reactions. Those of ordinary skill in the art know the amount of oligonucleotide primer that is appropriate for use in a single amplification reaction. General guidelines may for instance be found in Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990), Sambrook et al. (In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989), and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998). A kit may include more than two primers, in order to facilitate the in vitro amplification of
HRPC-related genomic sequences, for instance a HRPC-related nucleic acid listed in Table 1, or the 5 ' or 3' flanking region thereof.
In some embodiments, kits may also include the reagents necessary to carry out in vitro amplification reactions, including, for instance, DNA sample preparation reagents, appropriate buffers (e.g., polymerase buffer), salts (e.g., magnesium chloride), and deoxyribonucleotides (dNTPs). Written instructions may also be included.
Kits may in addition include either labeled or unlabeled oligonucleotide probes for use in detection of the in vitro amplified sequences. The appropriate sequences for such a probe will be any sequence that falls between the annealing sites of two provided oligonucleotide primers, such that the sequence the probe is complementary to is amplified during the in vitro amplification reaction (if it is present in the sample). It may also be advantageous to provided in the kit one or more control sequences for use in the in vitro amplification reactions. The design of appropriate positive control sequences is well known to one of ordinary skill in the appropriate art.
B. Kits for Detection of mRNA Expression Kits similar to those disclosed above for the detection of HRPC-related genomic amplification/deletion can be used to detect HRPC-related mRNA expression levels (including over- or under-expression, in comparison to the expression level in a control sample). Such kits include an appropriate amount of one or more of the oligonucleotide primers for use in, for instance, reverse transcription PCR reactions, similarly to those provided above, with art-obvious modifications for use with RNA.
In some embodiments, kits for detection of HRPC-related mRNA expression may also include reagents necessary to carry out RT-PCR or other in vitro amplification reactions, including, for instance, RNA sample preparation reagents (including e.g, an RNAse inhibitor), appropriate buffers (e.g., polymerase buffer), salts (e.g., magnesium chloride), and deoxyribonucleotides (dNTPs). Written instructions may also be included.
Kits may in addition include either labeled or unlabeled oligonucleotide probes for use in detection of an in vitro amplified target sequence. The appropriate sequences for such a probe will be any sequence that falls between the annealing sites of the two provided oligonucleotide primers, such that the sequence the probe is complementary to is amplified during the PCR reaction. It may also be advantageous to provided in the kit one or more control sequences for use in the in vitro amplification reactions. The design of appropriate positive control sequences is well known to one of ordinary skill in the appropriate art.
Alternatively, kits may be provided with the necessary reagents to carry out quantitative or semi-quantitative Northern analysis of HRPC-related mRNA. Such kits include, for instance, at least one HRPC-related sequence-specific oligonucleotide for use as a probe. This oligonucleotide may be labeled in any conventional way, including with a selected radioactive isotope, enzyme substrate, co- factor, ligand, chemiluminescent or fluorescent agent, hapten, or enzyme. C. Kits For Detection of HRPC-linked Protein or Peptide Expression Kits for the detection of HRPC-linked protein expression, for instance abnormal (over or under) expression of a protein encoded for by a nucleic acid molecule listed in Table 1, are also encompassed herein. Such kits will include at least one target (HRPC-linked) protein (e.g., cartilage glycoprotein-39 (CHI3L1), S-100P PROTEIN (S100P), CX3C chemokine/fractalkine (SCYDl), adenylate kinase 1 (AK1), forkhead transcription factor HFH-4 (HFH-4) (FKHL13), UDP glucuronosyltransferase precursor (UGT2B 15), Pleiotrophin (heparin binding growth factor 8)
(PTN), heat shock 27kD protein 2/Alpha-B-crystallin (HSP27), Proteasome (prosome, macropain) subunit, beta type, 5 (PSMB5), Inhibitor of NFKB (NFKBIA), interferon-induced 17 kD protein (ISG15), MAP kinase activated protein kinase 2 (MAPKAPK2), signal transduction protein (SH3 containing) (EFS2), hkf-1 Zinc finger protein (ZFP103), chromosome condensation 1 (CHCl), CDP- diacylglycerol synthase (CDSl), gap junction protein, alpha 1, 43kD (connexin 43) (GJAl), cyclin Dl (CCND1), Inhibitor of DNA binding 3, helix-loop-helix protein (ID3), HI histone family, member2 (H1F2), Cytochrome B561 (CYB561), Cathepsin H (CTSH), calcineurin alpha (PPP3CA), 54 kDa progesterone receptor-associated immunophilin (FKBP5), translocation protein 1 (TLOCl), Clusterin (complement lysis inhibitor; testosterone-repressed prostate message 2; apolipoprotein J) (CLU), Pulmonary surfactant-associated protein A (SFTPA1), protease inhibitor 12 (PI12; neuroserpin) (PI12), Thrombospondin 1 (THBS1), Ribophorin I (RPN1), A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 1 (ADAMTS 1), Collagen, type IV, alpha 5 (Alport syndrome) (COL4A5), LIM domain only 4 / breast tumor autoantigen (LM04), bumetanide-sensitive Na-K-Cl cotransporter (NKCC1) (SLC12A2), Fibronectin (FN1), or Crystallin Mu (CRYM)) specific binding agent (e.g., a polyclonal or monoclonal antibody or antibody fragment), and may include at least one control. The HRPC-linked protein specific binding agent and control may be contained in separate containers. The kits may also include a means for detecting HRPC-related proteimagent complexes, for instance the agent may be detectably labeled. If the detectable agent is not labeled, it may be detected by second antibodies or protein A, for example, either of both of which also may be provided in some kits in one or more separate containers. Such techniques are well known.
Additional components in some kits include instructions for carrying out the assay. Instructions will allow the tester to determine whether HRPC-linked expression levels are elevated or reduced in comparison to a control sample. Reaction vessels and auxiliary reagents such as chromogens, buffers, enzymes, etc. may also be included in the kits.
EXAMPLE 14 Identification of Therapeutic Compounds
The HRPC-related molecules disclosed herein, and more particularly the linkage of these molecules to cancer and cancer progression, can be used to identify compounds that are useful in treating, reducing, or preventing prostate cancer or development or progression of prostate cancer. These molecules can be used alone or in combination, for instance in sets of two or more that are linked to cancer or cancer progression.
By way of example, a test compound is applied to a cell, for instance a test cell, and at least one HRPC-related molecule level and/or activity in the cell is measured and compared to the equivalent measurement from a test cell (or from the same cell prior to application of the test compound). If application of the compound alters the level and/or activity of a HRPC-related molecule (for instance by increasing or decreasing that level), then that compound is selected as a likely candidate for further characterization. In particular examples, a test agent that opposes or inhibits an HRPC-related change is selected for further study, for example by exposing the agent to a hormone refractory prostate cancer cell in vitro, to determine whether in vitro growth is inhibited. Such identified compounds may be useful in treating, reducing, or preventing prostate cancer or development or progression of prostate cancer. In particular embodiments, the compound isolated will inhibit or inactivate a HRPC-related molecule, for instance those represented by the nucleic acids listed in Table 1. Methods for identifying such compounds optionally can include the generation of a HRPC- related gene expression profile, as described herein. Control gene expression profiles useful for comparison in such methods may be constructed from normal prostate tissue, primary prostate cancer tissue, prostate cancer tissue responding to androgen ablation therapy, and/or a hormone refractory prostate cancer tissue. By way of specific example, rapamycin has been herein identified as a compound that influences the levels of HRPC-related molecules, in particular certain of the nucleic acid molecules listed in for instance Table 1 (as discussed in more detail above). With the provision herein of this identification, the use of rapamycin as a treatment for HRPC is now enabled, as is the use of rapamycin derivatives or rapamycin-like compounds. It is believed that rapamycin can be used on its own as such a treatment, or can be used in combination with known or newly identified treatments for HRPC.
EXAMPLE 15 Gene Expression Profiles (Fingerprints) With the provision herein of methods for determining molecules that are linked to HRPC, and the provision of a large collection of such HRPC-linked molecules (as represented for instance by those listed in Table 1), gene expression profiles that provide information on the prostate cancer-state of a subject are now enabled.
HRPC-related expression profiles comprise the distinct and identifiable pattern of expression (or level) of sets of HRPC-related genes, for instance a pattern of high and low expression of a defined set of genes, or molecules that can be correlated to such genes, such as mRNA levels or protein levels or activities. The set of molecules in a particular profile will usually include at least one that is represented by (or correlated to) the following Image ID Clones: 781047, 785778, 842968, 769921, 898286, 204214, 814701, 435076, 531319, 415089, 898062, 453107, 785707, 795936, 700792, 34778, 46182, 769921, 783697, 451907, 711768, 416833, 810711, 789204, 789182, 725454, 951142, 49352, 273546, 46717, 855487, 41117, 26578, 684655, 45233, 814117, 810552, 739511, 283315, and 897774. In other examples of HRPC-related gene expression profiles, more than one molecule corresponding to the Image ID Clones listed in Table 1 are included in the profile. By way of example, any subset of the molecules listed in Table 1 (or corresponding to the molecules in this list) may be included in a single gene expression profile. Specific examples of such subsets include those molecules that show an increasing expression profile during prostate cancer progression, those that show a decreasing expression profile, those that are most highly correlated to a particular stage of prostate cancer progression, and so forth. Alternatively, gene expression profiles may be further broken down by the manner of molecules included in the profile. Thus, certain examples of profiles may mclude a specific class of HRPC-related molecules, such as those molecules involved in cell cycle control.
Particular profiles are specific for a particular stage of normal tissue (e.g., prostate tissue) growth or disease progression (e.g., progression of prostate cancer). Thus, gene expression profiles can be established for a pre-prostate cancer tissue (i.e., normal prostate tissue), a primary prostate cancer tissue, a prostate cancer tissue responding to androgen ablation therapy, and a hormone refractory prostate cancer tissue. Each of these profiles includes information on the expression level of at least one, but usually two or more, genes that are linked to prostate cancer (e.g., HRPC-related genes). Such information can include relative as well as absolute expression levels of specific genes. Likewise, the value measured may be the relative or absolute level of protein expression, which can be correlated with a "gene expression level." Results from the gene expression profiles of an individual subject are often viewed in the context of a test sample compared to a baseline or control sample fingerprint. The levels of molecules that make up a gene expression profile can be measured in any of various known ways, which may be specific for the type of molecule being measured. Thus, nucleic acid levels (such as direct gene expression levels, such as the level of mRNA expression) can be measured using specific nucleic acid hybridization reactions. Protein levels may be measured using standard protein assays, using immunologic-based assays (such as ELISAs and related techniques), or using activity assays, for instance. Examples for measuring nucleic acid and protein levels are provided herein; other methods are well known to those of ordinary skill in the art.
Examples of HRPC-related gene expression profiles can be in array format, such as a nucleotide (e.g., polynucleotide) or protein array or microarray. The use of arrays to determine the presence and/or level of a collection of biological macromolecules is now well known (see, for example, methods described in published PCT application number US99/06860, describing hyproxia- related gene expression arrays). In array-based measurement methods, an array may be contacted with polynucleotides (in the case of a nucleic acid-based array) or polypeptides (in the case of a protein-based array) from a sample from a subject. The amount and/or position of binding of the subject's polynucleotides or polypeptides then can be determined, for instance to produce a gene expression profile for that subject. Such gene expression profile can be compared to another gene expression profile, for instance a control gene expression profile from a subject having a known prostate-related condition. Optionally, the subject's gene expression profile can be correlated with one or more appropriate treatments, which may be correlated with a control (or set of control) expression profiles for stages of prostate cancer progression, for instance.
This disclosure provides the identification of HRPC-related molecules that exhibit alterations in expression during development of refractory prostate cancer, and expression 'fingerprints (profiles) specific for prostate cancer stages. It further provides methods of using these identified nucleic acid molecules, and proteins encoded thereby, and expression fingerprints or profiles, to predict and/or diagnose hormone-refractory prostate cancer, and to elect treatments for instance based on likely response. These identified HRPC-related molecules also can serve as therapeutic targets, and can be used in methods for identifying, developing and testing therapeutic compounds, including for instance rapamycin derivatives, analogs, and mimetics. It will be apparent that the precise details of the methods described may be varied or modified without departing from the spirit of the described invention. We claim all such modifications and variations that fall within the scope and spirit of the claims below.
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Claims

1. A method of diagnosing or prognosing development or progression of prostate cancer in a subject, comprising detecting an abnonnality in at least one HRPC-related molecule of the subject, wherein at least one such molecule is represented by Image ID Clone: 781047, 785778, 842968, 769921, 898286, 204214, 814701, 435076, 531319, 415089, 898062, 453107, 785707,
795936, 700792, 34778, 46182, 769921, 783697, 451907, 711768, 416833, 810711, 789204, 789182, 725454, 951142, 49352, 273546, 46717, 855487, 41117, 26578, 684655, 45233, 814117, 810552, 739511, 283315, 897774 or 2911545 (VDUP1).
2. The method of claim 1, comprising detecting abnormalities in more than one HRPC-related molecule.
3. The method of claim 2, wherein at least a second HRPC-related molecule is represented by a molecule listed in Table 1 or Table 4.
4. The method of claim 2, further comprising detecting an abnormality in at least one HRPC-related molecule not listed in Table 1 or Table 4.
5. The method of claim 1, comprising detecting an increase or decrease in expression or activity level of SI OOP, FKBP5, LM04, CRYM, or a combination of two or more thereof.
6. A method of diagnosing or prognosing development or progression of prostate cancer in a subject, comprising detecting an abnormality in at least 5, at least 10, at least 15, at least 25, at least 50, or at least 100 HRPC-related nucleic acid molecules listed in Table 1 or Table 4 or encoded for by a nucleic acid molecule listed in Table 1 or Table 4.
7. The method of claim 2, wherein an abnormality is detected in at least 5, 10, 15, 25, 50, or 100 HRPC-related nucleic acid molecules listed in Table 1 or Table 4 or encoded for by a nucleic acid molecule listed in Table 1 or Table 4.
8. The method of claim 1 or claim 6, where an abnormality comprises over- or under- expression of the HRPC-related molecule.
9. The method of claims 8, wherein an abnormality is over-expression.
10. The method of claim 9, where at least one HRPC-related molecule is represented by Image Clone ID number: 1475595, 1460110, 50794, 78294, 190491, 66731, 143287, 754600, 754509, 308041, 70827, 361974, 503097, 796646, 41650, 841641, 724615, 839101, 504226, 810711, 435330, 773567, 431296, 345232, 756405, 256907, 415817, 366541, 223350, 366067, 724831,
814353, 236034, 809910, 1470048, 1323448, 1456424, 453689, 135221, 340734, 180864, 768562, 179276, 44505, 293104, 243343, 66317, 812251, 245920, 265874, 770212, 784910, 839094, 712049, 669435, 841470, 782339, 297061, 429466, 300137, 487172, 343744, 795730, 268876, 742132, 755578, 502682, 510381, 140574, 135630, 278242, 742862, 1049033, 270136, 768260, 53039, 211813, 195051, 125769, 122955, 129342, 292392, 139331, 143995, 139250, 243360, 194307, 235040, 295483, or 143756.
11. The method of claim 8, wherein an abnormality is underexpression.
12. The method of claim 11 , where at least one HRPC-related molecule is represented by Image Clone ID number: 897768, 1456160, 34778, 810512, 753184, 200814, 470393, 23185, 128126, 42373, 511521, 810117, 950682, 783696, 815555, 897531, 713145, 502690, 469969, 309893, 725877, 343987, 49318, 42864, 193087, 162533, 1309620, 685801, 825740, 756708, 28469, 187147, 246304, 130280, 753587, 123980, 241985, 564621, 841507, 810703, 784772, 143306,
246722, 298417, 51582, 757222, 884783, 417424, 324891, 504791, 725877, 743230, 377048, 42627, 144797, 244955, 204735, 144747, 292749, 196109, 120375, 121981, 121715, 243403, 127409, 130053, 243291, 203514, 133130, 134495, 296552, 138601, 167076, 197323, 197637, 194906, 194985, 196125, 196303, 243784, 280122, 245235, 197856, 200604, 203400, 207448, 234469, 210548, 208940, 208434, 211951, 212098, 233399, 240138, 137396, 241097, 239835, 308231, 292312, 292391, 293421, 293306, 293785, 295044, 295590, 296102, 296602, 297110, 191572, 195132, 233274, 246546, 296562, 214331, 214043, 126230, 128245, 129616, 134312, 230613, 239711, 134537, 127646, 136984, 210610, 293457, 233299, 281125, 26184, 39093, or 39884.
13. The method of claim 1 or claim 6, comprising: measuring an amount of the HRPC-related molecule in a sample derived from the subject, in which a difference in level of the HRPC-related molecule relative to that present in a sample derived from the subject at an earlier time, is diagnostic or prognostic for development or progression of prostate cancer.
14. The method of claim 1 or claim 6, wherein detecting an abnonnality comprises: measuring a HRPC-related molecule level in a sample derived from the subject, in which a difference in the HRPC-related molecule level in the sample, relative to the HRPC-related molecule level found in an analogous sample from a subject not having the disease or disorder, or a standard HRPC-related molecule level in analogous samples from a subject not having the disease or disorder or not having a predisposition developing the disease or disorder, is an abnormality in that HRPC- related molecule.
15. The method of claim 1 or claim 6, wherein detecting an abnormality comprises: measuring a level of HRPC-related protein functional activity in a sample derived from the subject, in which a difference in the level of HRPC-related protein functional activity in the sample, relative to the level of HRPC-related protein functional activity found an analogous sample from a subject not having the disease or disorder or a standard HRPC-related protein functional activity level in analogous samples from a subject not having the disease or disorder or not having a predisposition for developing the disease or disorder, is an abnormality in that HRPC-related molecule.
16. The method of claim 1 or claim 6, where the HRPC-related molecule is a HRPC- related nucleic acid molecule (DNA or RNA or cDNA) or a HRPC-related protein.
17. The method of claim 16, wherein at least one HRPC-related molecule is a HRPC- related nucleic acid.
18. The method of claim 17, comprising in vitro nucleic acid amplification.
19. The method of claim 17, comprising nucleic acid hybridization.
20. The method of claim 19, further comprising determining the amount of hybridization.
21. The method of claim 16, wherein at least one HRPC-related molecule is a HRPC- related protein.
22. The method of claim 21, wherein detecting the abnormality comprises: contacting a sample from the subject with a HRPC protein-specific binding agent; and detecting whether the binding agent is bound by the sample and thereby measuring the levels of the HRPC-related protein present in the sample, in which a difference in the level of HRPC-related protein in the sample, relative to the level of HRPC-related protein found an analogous sample from a subject not having the disease or disorder, or a standard HRPC-related protein level in analogous samples from a subject not having the disease or disorder or not having a predisposition for developing the disease or disorder, is an abnormality in that HRPC-related molecule.
23. The method according to claim 23, wherein the specific binding agent is detectably labeled.
24. The method of claim 1 or claim 6, wherein the abnormality is detected in a sample from the subject, and the sample comprises serum.
25. The method of claim 1 or claim 6, wherein the abnormality is detected in a sample from the subject, and the sample comprises prostate tissue.
26. The method of claim 17, comprising: providing nucleic acids from the subject; amplifying the nucleic acids to form nucleic acid amplification products; contacting the nucleic acid amplification products with an oligonucleotide probe that will hybridize under stringent conditions with a nucleic acid encoding a HRPC-related protein; detecting the nucleic acid amplification products which hybridize with the probe; and quantifying the amount of the nucleic acid amplification products that hybridize with the probe.
27. The method of claim 26, where the sequence of the oligonucleotide probe is selected to bind specifically to a nucleic acid molecule listed in Table 1 or Table 4.
28. The method of claim 26, where the primers are selected to amplify a nucleic acid molecule listed in Table 1.
29. The method of claim 26, where the primers are selected to amplify a nucleic acid product encoding cartilage glycoprotein-39 (CHI3L1), S-100P PROTEIN (S100P), CX3C chemokine/fractalkine (SCYD1), adenylate kinase 1 (AK1), forkhead transcription factor HFH-4 (HFH-4) (FKHL13), UDP glueuronosyltransferase precursor (UGT2B15), Pleiotrophin (heparin binding growth factor 8) (PTN), heat shock 27kD protein 2/Alpha-B-crystallin (HSP27), Proteasome (prosome, macropain) subunit, beta type, 5 (PSMB5), Inhibitor of NFKB (NFKBIA), interferon- induced 17 kD protein (ISG15), MAP kinase activated protein kinase 2 (MAPKAPK2), signal transduction protein (SH3 containing) (EFS2), hkf-1 Zinc finger protein (ZFP103), chromosome condensation 1 (CHCl), CDP-diacylglycerol synthase (CDSl), gap junction protein, alpha 1, 43kD (connexin 43) (GJAl), cyclin Dl (CCNDl), Inhibitor of DNA binding 3, helix-loop-helix protein (ID3), HI histone family, member2 (H1F2), Cytochrome B561 (CYB561), Cathepsin H (CTSH), calcineurin alpha (PPP3CA), 54 kDa progesterone receptor-associated immunophilin (FKBP5), translocation protein 1 (TLOCl), Clusterin (complement lysis inhibitor; testosterone-repressed prostate message 2; apolipoprotein J) (CLU), Pulmonary surfactant-associated protein A (SFTPA1), protease inhibitor 12 (PI12; neuroserpin) (PI12), Thrombospondin 1 (THBSl), Ribophorin I (RPNl), A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 1 (ADAMTS 1), Collagen, type IV, alpha 5 (Alport syndrome) (COL4A5), LIM domain only 4 / breast tumor autoantigen (LM04), bumetanide-sensitive Na-K-Cl cotransporter (NKCC 1 ) (SLC 12 A2), Fibronectin (FN1), Crystallin Mu (CRYM) or "upregulated by 1,25-dihydroxyvitamin D-3" (VDUP1).
30. The method of claim 1 or claim 6, comprising detecting a mutation, duplication or deletion of a HRPC-related nucleic acid in cells of the individual.
31. The method of claim 1 or claim 6, comprising detecting decreased, increased, or mutant HRPC-related protein in cells of the individual.
32. A method of selecting a prostate cancer therapy, comprising: detecting an abnormality in at least one HRPC-related molecule of a subject; and if such abnormality is identified, selecting a treatment to prevent or reduce hormone- refractory prostate cancer or to delay the onset of hormone-refractory prostate cancer.
33. The method of claim 32, wherein the at least one HRPC-related molecule is SCYDl, S100P, CCNDl, CRIPl, ISG15, SCNNIA, ZFP103, MAPKAPK2, UTG2B15, RABGGTA, NFKBIA, SLCYA5, AP3B2, PTPN2, FOXJl, APOC1, FLJ23538, OXCT, PFKP, TNRC3, HXB, PFKP, OAT, PFKP, RFP, THBSl, LM04, MLD, CRYM, MME, HMGCS2, SLC12A2, ODC1, EIF4EBP1, CDSl, FKBP4, VDUP1, or FKBP5.
34. The method of claim 32, comprising detecting an abnormality in more than one HRPC-related molecule of a subject, wherein the more than one HRPC-related molecules are two or more of SCYDl, S100P, CCNDl, CRIPl, ISG15, SCNNIA, ZFP103, MAPKAPK2, UTG2B15, RABGGTA, NFKBIA, SLCYA5, AP3B2, PTPN2, FOXJl, APOC1, FLJ23538, OXCT, PFKP, TNRC3, HXB, PFKP, OAT, PFKP, RFP, THBSl, LM04, MLD, CRYM, MME, HMGCS2, SLC12A2, ODC1, EIF4EBP1, CDSl, FKBP4, VDUP1, or FKBP5.
35. The method of claim 32, wherein the at least one HRPC-related molecule is S100P, FKBP5, LM04, CRYM, or a combination of two or more thereof.
36. The method of claim 32, further comprising treating the subject with the selected treatment.
37. The method of claim 36, wherein the selected treatment comprises treating the subject with rapamycin, or a derivative, mimetic, or analog of rapamycin.
38. The method of claim 32, wherein detecting an abnormality in at least one HRPC- related molecule of a subject comprises quantitatively or qualitatively analyzing a DNA, mRNA, cDNA, protein, or protein modification.
39. A method of modifying a level of expression of a HRPC-related protein in a subject, comprising: expressing in the subject a recombinant genetic construct comprising a promoter operably linked to a nucleic acid molecule, wherein the nucleic acid molecule comprises at least 10 consecutive nucleotides of a HRPC-related nucleic acid sequence, wherein expression of the nucleic acid molecule changes expression of the HRPC-related protein, and wherein the HRPC-related nucleic acid sequence is represented by Image ID Clone number 781047, 785778, 842968, 769921, 898286, 204214, 814701, 435076, 531319, 415089, 898062, 453107, 785707, 795936, 700792, 34778, 46182, 769921, 783697, 451907, 711768, 416833, 810711, 789204, 789182, 725454, 951142, 49352, 273546, 46717, 855487, 41117, 26578, 684655, 45233, 814117, 810552, 739511, 283315, or 897774.
40. The method of claim 39 wherein the nucleic acid molecule is in antisense orientation relative to the promoter.
41. The method of claim 39 wherein the nucleic acid molecule is in sense orientation relative to the promoter.
42. The method of claim 39, wherein the recombinant genetic construct expresses a siRNA corresponding to a HRPC-related nucleic acid sequence.
43. A kit for measuring a HRPC-related molecule level, comprising a binding molecule that selectively binds to the HRPC-related molecule, wherein the HRPC-related molecule is represented by Image ID Clone number 781047, 785778, 842968, 769921, 898286, 204214, 814701, 435076, 531319, 415089, 898062, 453107, 785707, 795936, 700792, 34778, 46182, 769921, 783697, 451907, 711768, 416833, 810711, 789204, 789182, 725454, 951142, 49352, 273546, 46717, 855487, 41117, 26578, 684655, 45233, 814117, 810552, 739511, 283315, or 897774.
44. The kit of claim 43, wherein the levels of a plurality of HRPC-related molecules are measured.
45. The kit of claim 44, comprising an array.
46. The kit of claim 43, wherein the HRPC-related molecule level is a HRPC-related protein level, and the binding molecule is an antibody or antibody fragment that selectively binds a HRPC-related protein.
47. The kit of claim 43, wherem the HRPC-related molecule level is a HRPC-related nucleic acid molecule level, and the binding molecule is an oligonucleotide capable of hybridizing to the HRPC-related nucleic acid molecule.
48. The method of claim 1 or claim 6, wherein detecting the abnormality comprises: determining whether a HRPC-related gene expression profile from the subject indicates development or progression of prostate cancer.
49. The method of claim 48, wherein the gene expression profile comprises an array.
50. The method of claim 48, comprising: comparing the HRPC-related gene expression profile from the subject to at least one control gene expression fingerprint for a specific stage of prostate cancer.
51. The method of claim 50, where the at least one control gene expression profile is a fingerprint for a normal prostate tissue, a primary prostate cancer tissue, a prostate cancer tissue responding to androgen ablation therapy, or a hormone refractory prostate cancer tissue.
52. A method of screening for a compound useful in treating, reducing, or preventing prostate cancer or development or progression of prostate cancer, comprising determining if application of a test compound alters a HRPC-related gene expression profile so that the profile more closely resembles a prostate-linked profile than it did prior to such treatment, and selecting a compound that so alters the HRPC-related gene expression profile, wherein the HRPC-related gene expression profile includes at least one molecule represented by Image ID Clone number 781047, 785778, 842968, 769921, 898286, 204214, 814701, 435076, 531319, 415089, 898062, 453107, 785707, 795936, 700792, 34778, 46182, 769921, 783697, 451907, 711768, 416833, 810711, 789204, 789182, 725454, 951142, 49352, 273546, 46717, 855487, 41117, 26578, 684655, 45233, 814117, 810552, 739511, 283315, 897774, or 2911545.
53. The method of claim 52, wherein the compound inhibits or inactivates a molecule represented by those listed in Table 1 or Table 4.
54. The method of claim 52, wherein the test compound is applied to a test cell.
55. The method of claim 52, comprising: contacting test cells with a test compound; and measuring at least one HRPC-related molecule level and/or activity in the test cells, in which a difference in HRPC-related molecule level and/or activity in the test cells, relative to the analogous HRPC-related molecule level and/or activity found in analogous cells not contacted with the test compound, indicates that the test compound is useful in treating, reducing, or preventing prostate cancer or development or progression of prostate cancer.
56. The method of claim 55, wherein at least one HRPC-related molecule is a nucleic acid molecule listed in Table 1 or Table 4, or is encoded for by a nucleic acid molecule listed in Table 1 or Table 4.
57. The method of claim 55, in which measuring at least one HRPC-related molecule level and/or activity comprises: creating a HRPC-related gene expression profile for the test cell after contacting the cell with the test compound; and comparing the test cell HRPC-related gene expression profile to at least one control gene expression profile for a specific stage of prostate cancer. 209
138
58. The method of claim 57, where the control gene expression profile is a profile for a normal prostate tissue, a primary prostate cancer tissue, a prostate cancer tissue responding to androgen ablation therapy, or a hormone refractory prostate cancer tissue.
59. The method of claim 52, wherein the profile comprises an array.
60. A compound selected by the method of claim 52.
61. The method of claim 36, wherein the selected treatment comprises treating the subject with FR901464, or a derivative, mimetic, or analog of FR901464.
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