WO2004077942A2 - Modele de cancer prostatique inductible - Google Patents
Modele de cancer prostatique inductible Download PDFInfo
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- WO2004077942A2 WO2004077942A2 PCT/US2004/006118 US2004006118W WO2004077942A2 WO 2004077942 A2 WO2004077942 A2 WO 2004077942A2 US 2004006118 W US2004006118 W US 2004006118W WO 2004077942 A2 WO2004077942 A2 WO 2004077942A2
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- oncogene
- cell
- prostate
- cancer
- mammal
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Definitions
- Prostate cancer is the most common noncutaneous malignancy diagnosed in men in the United States. As with all malignancies, prostate cancer is a disease of genes. Thus, the biology of nonnal prostate development and the mechanisms underlying the initiation, progression and metastasis of prostate cancer must be understood at the molecular level to develop effective prevention and intervention strategies. For review, see, e.g., Isaacs, Cancer Surveys 25:357- 79 (1995); Isaacs et al., Cancer Cell 2:113-6 (2002); Trapman, Eur. J. Cancer 37 Suppl 7:S119-25 (2001); Abate-Shen et al., Trends Genet 18(5):Sl-5 (2002).
- This invention provides an inducible genetic model for studying the development (e.g., initiation, progression, maintenance, metastasis, regression, minimal residual disease, recurrence, and any other developmental stages) of prostate cancer and identifying anti-cancer therapeutics.
- a prostate cell of a non-human mammal e.g., a mouse, a rat, a hamster, a rabbit or a non-human primate
- a mammalian cell which has a genome comprising: (a) a prostate cancer related oncogene; and (b) a cancer-prone genetic predisposition, wherein, e.g., expression of the oncogene causes prostate cancer in the mammal or cell proliferation, wherein the cancer regresses or cell proliferation is inhibited when, e.g., expression of the oncogene is reduced.
- a cancer-prone genetic predisposition may be a known cancer-prone genetic mutation. Either or both of the prostate related oncogene and the cancer-prone genetic mutation may be inducible. Alternatively, the mammal comprising a prostate cancer related oncogene is predisposed to developing cancer. Any animal known to have a predisposition to developing cancer may be employed, including, without limitation, A/J, C3H, C57BL/6, FVB, 129, and Balb/C strains of mice. The prostate cancer related oncogene may further be operably linked to a reporter gene to facilitate detection of cells and animal s having the oncogene construct.
- the genome of the mammal or cell of the invention comprises (a) a first expression construct comprising a nucleic acid encoding a reverse tetracycline traiisactivator operably linked to a prostate-specific transcnptional element; (b) a second expression construct comprising a nucleic acid encoding an oncogene operably linked to a transcnptional element that can be regulated by the reverse tetracycline transactivator and tetracycline or a tetracycline analogue; and (c) a cancer-prone genetic predisposition, wherein induced expression of the oncogene causes prostate cancer in the mammal or cell proliferation, and wherein reduced expression of the oncogene results in cancer regression or inhibited cell proliferation.
- the genome of the mammal of the cell of the invention comprises (a) a first expression construct comprising a nucleic acid encoding a Cre recombinase operably linked to a prostate-specific transcriptional element; (b) a second expression construct comprising a nucleic acid encoding an oncogene, wherein a Lox-STOP-Lox cassette is placed upstream of the transcription or translation initiation site to prevent transcription of the oncogene or translation of the onco gene's transcript; and (c) a cancer-prone genetic predisposition, wherein upon recombination between the two LoxP sites in the presence of the Cre recombinase, transcription can proceed through the nucleic acid encoding the oncogene to produce the translated oncoprotein; and wherein induced expression of the oncogene causes prostate cancer in the mammal or cell proliferation; and wherein reduced expression of the oncogene results in cancer progression or inhibited cell proliferation.
- the prostate-specific transcriptional element is a prostate-specific promoter or enhancer from, e.g., probasin, prostate-specific antigen (PSA), prostate stem cell antigen, C3(l), hK2, PSMA, PSP94, or PacP.
- PSA prostate-specific antigen
- PSMA prostate stem cell antigen
- C3(l) prostate stem cell antigen
- hK2 prostate stem cell antigen
- PSMA prostate stem cell antigen
- PSP94 PSP94
- PacP a prostate-specific promoter or enhancer from, e.g., probasin, prostate-specific antigen (PSA), prostate stem cell antigen, C3(l), hK2, PSMA, PSP94, or PacP.
- the genome of the mammal or cell of the invention comprises (a) a nucleic acid encoding an oncogene operably linked to a nucleic acid encoding an estrogen receptor polypeptide; and (b) a cancer-prone genetic predisposition, wherein administration of estrogen or a nonhormone analogue of estrogen to the mammal or the cell allows the correct folding of the oncogene polypeptide into a functional protein; and wherein induced expression of the oncogene causes prostate cancer or cell proliferation; and wherein reduced expression of the oncogene results in cancer regression or inhibited cell proliferation.
- the nucleic acid encoding the prostate cancer related oncogene is operably linked to a nucleic acid encoding a progesterone receptor polypeptide, and where administration of progesterone or a nonhormone analogue of progesterone allows corcect folding of the oncoprotein.
- the prostate cancer related oncogene can be, for example, Aktl, Akt2, Akt3, or activating mutants thereof such as those having a myristolation inducing sequence at the 5' end of the oncogene (Pellman et al, Nature 314:374-7 (1985)), genes in the PI3K-AKT signal transduction pathways, ras (e.g., ⁇ L-ras or K-ras) un,fos, myc, src, abl, mos, sis, Erb B, Erb B2, /S-catenin, IGF-I, CDK2, CDK4, DCL2, and viral proteins such as SV40 T and PyMT.
- ras e.g., ⁇ L-ras or K-ras
- ras un,fos, myc, src, abl, mos, sis, Erb B, Erb B2, /S-catenin, IGF-I,
- the cancer-prone genetic mutation can be, for example, (a) a disabling mutation in a tumor suppressor gene (e.g., INK4a, P53, PTEN, APC, DCC, Rb, DPC4, KLF6, GSTP1, ELAC2/HPC2 and NKX3.1), (b) an activating mutation in an endogenous proto- oncogene (e.g., CTNNBl, myc, ras and her2), (c) a disabling mutation in a DNA repair gene (e.g., MSH2, MSH3, MSH6, PMS2, Ku70, Ku80, DNA/PK, ATR, ATM, XRCC4 and MLH1), or (d) a disabling or activating mutation in a prostate cancer related gene (e.g., ⁇ 27Kipl, MXI1 and androgen receptor gene).
- the disabling mutations can be accomplished using post-transcriptional silencing using, e.g., RNAi, antis
- the mammal of the invention may be transgenic, chimeric, or mosaic.
- the percentage of chimerism may be, e.g., at least 5%, 10%, 20%, 30%, 40% or 50%.
- the cell of this invention may be an ES cell, a tumor cell, a prostate cell, a tissue-specific stem cell, or a mouse cell.
- the cell may be derived or obtained from the mammal of this invention.
- explants derived from the mammal of this invention. This invention further provides methods of making the mammal and cells of this invention.
- the transgenic mammal is obtained by mating mammals chimeric for the transgene.
- a first mammal comprising a germ cell having a genome comprising a prostate cancer related oncogene is mated to a second mammal comprising a germ cell having a genome comprising a cancer-prone genetic predisposition.
- a first mammal comprising a germ cell having a genome comprising a nucleic acid encoding reverse tetracycline transactivator under the control of a prostate-specific transcriptional element is mated to a second mammal comprising a germ cell having a genome comprising an oncogene operably linked to a transcriptional element regulated by said reverse tetracycline transactivator and tetracycline or a tetracycline analogue; and wherein the gemi cell of said first mammal or of said second mammal or both further comprises a cancer-prone genetic predisposition.
- a first mammal comprising a germ cell having a genome comprising a first expression construct comprising a nucleic acid encoding a Cre recombinase operably linked to a prostate-specific transcriptional element is mated to a second mammal comprising a genn cell having a genome comprising a second expression construct comprising a nucleic acid encoding an oncogene, wherein a Lox-STOP-Lox cassette is placed upstream of the transcription or translation initiation site to prevent transcription of the oncogene or translation of the oncogene's transcript; and wherein the germ cell of said first mammal or of said second mammal or both further comprises a cancer-prone genetic predisposition.
- the prostate-specific transcriptional element is a prostate-specific promoter or enhancer from probasin, prostate- specific antigen, prostate stem cell antigen, C3(l), hK2, PSMA, PSP94 or PacP.
- a first mammal comprising a germ cell having a genome comprising an expression construct comprising a nucleic acid encoding an oncogene operably linked to an estrogen receptor polypeptide is mated to a second mammal comprising a germ cell having a genome comprising a cancer- prone genetic predisposition.
- the prostate cancer related oncogene is operably linked to a progesterone receptor polypeptide.
- the mammal of this invention may be produced by introducing a construct comprising a prostate cancer related oncogene into a zygote comprising a cancer-prone genetic predisposition; and developing the zygote into the mammal.
- the mammal may be produced by introducing a construct comprising a prostate cancer related oncogene into an ES cell comprising a cancer-prone genetic predisposition; injecting the ES cell into a blastocyst or a tetraploid blastocyst; and generating the mammal.
- This invention further provide methods of using the non-human mammals or the mammalian cells of this invention to identify new prostate related genes, surrogate biomarkers for diagnosis or monitor prostate cancer progression, and therapeutic agents to treat or prevent prostate cancer or minimal residual prostate cancer.
- This invention provides a method for determining whether an oncogene contributes to prostate cancer maintenance, comprising the steps of: (a) providing a non-human mammal having prostate cancer, wherein said mammal comprises a genome comprising: (i) said oncogene operably linked to an inducible promoter; and (ii) a cancer-prone genetic predisposition wherein said mammal comprises a tumor fomied in said mammal during expression of said oncogene; and (b) determining whether or not said prostate cancer regresses when expression of said oncogene is reduced, wherein prostate cancer regression is indicative of said oncogene contributing to prostate cancer maintenance.
- Also provided is a method of identifying a prostate cancer related gene comprising the steps of: (a) establishing a first and a second molecular profile of a prostate tumor cell of the non-human mammal of claim 1 or the cell of claim 28 at two different stages of prostate cancer; and (b) comparing the first and second molecular profiles, wherein an alteration in expression or activity pattern of a candidate gene is indicative of said gene being a prostate cancer related gene.
- Also provided is a method of identifying a biomarker to diagnose or monitor prostate cancer progression comprising the steps of: (a) establishing a first molecular profile of a prostate cell of the non-human mammal of claim 1 or the cell of claim 28 wherein expression of the prostate cancer related oncogene is not induced; (b) establishing a second molecular profile of said cell wherein expression of said oncogene is induced and wherein the cell becomes cancerous; and (c) comparing the first and second molecular profiles, wherein an alteration in expression or activity pattern of a candidate gene is indicative of said gene being a biomarker for prostate cancer.
- This invention also provide a method of identifying a therapeutic agent to treat prostate cancer, comprising the steps of: (a) administering a candidate compound to the non-human mammal of claim 1 that has developed prostate cancer; and (b) observing the effect of said compound on cancer development, wherein a decrease in tumor size, metastasis, angiogenesis or growth rate, or apoptosis of the cancer is indicative of said compound being a therapeutic agent to treat prostate cancer.
- the method of identifying a therapeutic agent to prevent prostate cancer comprises the steps of: (a) administering a candidate compound to the non-human mammal of claim 1 that has not yet developed prostate cancer; (b) inducing expression of the prostate cancer related oncogene to cause cancer; and (c) observing the effect of said compound on cancer development, wherein absence of cancer formulation is indicative of said compound being a therapeutic agent to prevent prostate cancer. Also provided is a method of identifying a therapeutic agent to treat prostate cancer, comprising the steps of: (a) contacting the cell of claim 28 with a candidate compound wherein said cell is a prostate tumor cell; and (b)observing the effect of said compound on cell proliferation, wherein inhibition of cell proliferation is indicative of said compound being a therapeutic agent to treat prostate cancer.
- Also provided is a method of identifying a therapeutic agent to treat prostate cancer comprising the steps of: (a) administering a candidate compound to the non-human mammal of claim 1 that has developed prostate cancer or to cultured prostate cancer cells derived from said mammal; and (b) observing the effect of said compound on expression or activity level of a biomarker for prostate cancer in said mammal or said cells, wherein an alteration of biomarker expression or activity is indicative of said compound being a therapeutic agent to treat prostate cancer.
- Also provided is a method of identifying a therapeutic agent to prevent prostate cancer comprising the steps of: (a) administering a candidate compound to the non-human mammal of claim 1 that has not yet developed prostate cancer or to cultured prostate cancer cells derived from said mammal; (b) inducing expression of the prostate related oncogene to cause cancer; and (c) observing the effect of said compound on expression or activity level of a biomarker for prostate cancer in said mammal or said cells, wherein absence of alteration of biomarker expression or activity is indicative of said compound being a therapeutic agent to treat prostate cancer.
- Also provided is a method of identifying a therapeutic agent to treat prostate cancer comprising the steps of: (a) contacting the cell of claim 28 with a candidate compound wherein the cell is a prostate tumor cell; and (b) observing the effect of said compound on expression or activity level of a biomarker for prostate cancer in said cells, wherein an alteration of biomarker expression or activity is indicative of said compound being a therapeutic agent to treat prostate cancer.
- Also provided is a method of identifying a therapeutic agent to prevent prostate cancer comprising the steps of: (a) establishing a first molecular profile of a non-cancerous prostate cell of the non-human mammal of claim 1, or a prostate cell derived from said mammal, by identifying a plurality of biomarkers whose patterns of expression or biological activity correspond to the non-cancerous stage of said prostate cell, and wherein expression of the prostate cancer related oncogene is not induced; (b) contacting said prostate cell with a candidate compound; (c) establishing a second molecular profile of said contacted prostate cell, wherein said second pattern of expression or biological activity of said biomarkers correspond to said prostate cell, wherein expression of said oncogene is induced to cause cancer; and (d) comparing the first and second profiles, wherein substantial similarity of the first and second profiles is indicative of said compound being a therapeutic agent to prevent prostate cancer.
- This invention also provides a method of identifying a gene involved in minimal residual prostate cancer, comprising the steps of: (a) establishing a first molecular profile for a non-cancerous prostate cell of the non- human mammal of claim 1 ; and (b) establishing a second molecular profile for a prostate cell of the non-human mammal of claim 1 having minimal residual prostate cancer; (c) establishing a third molecular profile for a cancerous prostate cell of the non-human mammal of claim 1 wherein cancer is induced by expression of the prostate cancer related oncogene; and (d) comparing the first, second and third profiles, wherein an alteration in expression patterns of said gene in the first and second profiles while substantial similarity of expression patterns of said gene in the second and third profiles is indicative of said gene being involved in minimal residual prostate cancer.
- a method of identifying a therapeutic agent to treat or prevent minimal residual prostate cancer comprising the steps of: (a) administering a candidate compound to the non-human mammal of claim 1, wherein the mammal has minimal residual prostate cancer; and (b) observing the effect of said compound on expression or activity level of a gene involved in minimal residual prostate cancer, wherein an alteration in expression or activity level of said gene is indicative of said compound being a therapeutic agent to treat or prevent minimal residual prostate cancer.
- the profiles can be established by any standard technique, e.g., suppression subtraction, differential display, proteomic analysis, serial analysis of gene expression and comparative genomic hybridization.
- Fig. 1 is a drawing illustrating an expression construct of a reverse tetracycline transactivator (rtTA) under the control of a probasin promoter.
- rtTA reverse tetracycline transactivator
- pA denotes polyA sequence.
- Fig. 2 is a drawing illustrating an expression construct of an Akt oncogene whose expression is inducible by a reverse tetracycline transactivator and tetracycline.
- TetO denotes Tet operator sequence.
- Akt denotes an Akt oncogene, e.g., Aktl, Akt2, Akt3 or an activating mutant thereof.
- SV40 pA denotes a polyA sequence from a SV40 virus.
- This invention features a nonhuman mammal (e.g., a mouse, a rat, a hamster, a rabbit or a non-human primate), in which the genome of at least some of its cells contains a constitutive or inducible system for expressing an oncogene (e.g., a nucleotide sequence encoding an oncoprotein) that is related to prostate cancer.
- an oncogene e.g., a nucleotide sequence encoding an oncoprotein
- the genome of the cells further contains a genetic predisposition that renders the mammal even more susceptible to cancer than it would otherwise be; in these embodiments, the mammal develops spontaneous prostate cancer much sooner than a mammal that does not contain the genetic predisposition.
- the mammal upon induced expression of the oncogene in the prostate, develops spontaneous prostate hyperplasia (e.g., benign prostate hyperplasia and prostate intraepithelial neoplasm I - III) that may progress to tumors (e.g., epithelial tumors and/or neuroendocrine tumors).
- the genetic predisposition is one or more genetic mutation such as disabling mutations in a tumor suppressor gene, a disabling mutation in a prostate cancer related gene, a disabling mutation in a DNA repair gene, or an activating mutation in an endogenous proto-oncogene.
- the genetic mutation may also be controlled by an inducible system.
- the prostate cancer related oncogene is constitutively expressed and the genetic mutation that renders the mammal more susceptible to developing cancer is mducible, wherein induction of the mutation results in development of prostate cancer (e.g., benign prostate hyperplasia and prostate intraepithelial neoplasm I - III).
- prostate cancer e.g., benign prostate hyperplasia and prostate intraepithelial neoplasm I - III.
- the tumors may be androgen-dependent or androgen-independent. Metastasis may eventually occur. The tumors may regress when expression of the oncogene is turned off or reduced. Induction of tumorigenicity can be determined, for example, by monitoring the mammal for development of a tumor. Alternatively, a soft agar assay or any of the other assays described herein can be employed.
- the mammal of this invention can be a transgenic animal all of whose germ and somatic cells contain the inducible oncogene and the genetic predisposition.
- the mammal is a chimeric or mosaic animal in which only some of its somatic and/or germ cells contain the oncogene and the genetic predisposition.
- the percentage of chimerism is at least 5%, 10%, 20%, 30%, 40% or 50%.
- the inducible prostate cancer model of this invention provides an advantageous way to study prostate cancer as compared to the conventional tumor explant models. In this model, prostate cancer occurs only upon expression of the introduced oncogene in the animal host. Further, a prostate related oncogene may be repeatedly inducible, reducible and re-inducible.
- each host represents a distinct tumorigenesis event.
- each tumor originates from implanted cells of the same tumor cell line.
- the inducible model better resembles clinical development of prostate cancer, where each patient and/or each tumor represents a distinct tumorigenic event.
- tumors in this model arise de novo in a natural prostate environment and their development hence better resembles clinical conditions.
- Prostate cancer related oncogenes useful in this invention include, without limitation, Aktl, Akt2, Akt3, or activating mutants thereof such as those having a myristolation inducing sequence at the 5' end of the oncogene (Pellman et al., Nature 314:374-7 (1985)), genes in the PI3K-AKT signal transduction pathways, ras (e.g., H-ras or K-ras),jun,fos, myc, src, abl, mos, sis, Erb B, Erb B2, j3-catenin, IGF-I, CDK2, CDK4, DCL2, and viral proteins such as SV40 T and PyMT.
- ras e.g., H-ras or K-ras
- jun,fos myc
- src abl
- mos mos
- sis Erb B, Erb B2, j3-catenin
- IGF-I CD
- RNA polymerase II e.g., the tetracycline transactivator systems, reverse tetracycline transactivator systems, ecdysone systems, methallothionine systems, LacO/LPTG systems, and TetO/tetracycline systems.
- RNA polymerases I and III also can be used with or without modifications.
- a Lox-STOP-Lox cassette is placed upstream to the transcription or translation initiation site of a transgene that is an oncogene, preventing transcription of the gene or translation of the gene's transcript.
- the genome of the host cell also contains the coding sequence for a Cre recombinase under the transcriptional control of one or more prostate-specific elements (e.g., promoters and/or enhancers).
- the prostate-specific element may be stage-specific.
- Useful prostate-specific transcription control elements may be those from genes specifically or preferentially expressed in, e.g., prostate epithelial cells or terminally differentiated prostate epithelia cells, including, without limitation, probasin (e.g., minimal probasin promoters or long probasin promoters), prostate-specific antigen (PSA), prostate stem cell antigen, C3(l) gene, hK2, PSMA, PSP94 and PacP.
- probasin e.g., minimal probasin promoters or long probasin promoters
- PSA prostate-specific antigen
- PSMA prostate stem cell antigen
- a prostate-specific enhancer may be used in combination with a minimal promoter.
- a portion of the prostate-specific promoter sufficient to direct expression in prostate tissue may be used.
- the Cre recombinase is thus specifically or preferentially expressed in the prostate, causing deletion of the STOP signal flanked by the Lox sites and allowing expression of the oncogene.
- the promoter for the oncogene is an inducible promoter such that expression of the oncogene in the prostate is inducible.
- the oncogene is operably linked to a promoter activatable by a reverse tetracycline transactivator and tetracycline (or an analogue thereof such as doxycycline).
- this promoter contains a Tet operator sequence.
- the genome of the host cell also contains a reverse tetracycline transactivator transgene regulated by prostate-specific transcription control elements as described above. Expression of the oncogene can thus be turned on (i.e., induced) or off (i.e., non-induced) specifically in the prostate upon administration or withdrawal of tetracycline or its analogue. See also Gossen et al., Science 268:1766-9 (1995).
- Expression of the oncogene may also be inducibly switched on or off by fusing the oncogene to, e.g., a coding sequence for an estrogen receptor polypeptide sequence, where administration of estrogen or a nonhormone estrogen analogue (e.g., hydroxytamoxifen) will allow the correct folding of the oncogene polypeptide into an functional protein.
- a similar inducible system involves fusion of the oncogene with a coding sequence for a progesterone receptor polypeptide sequence wherein expression of the oncogene is induced by progesterone or a nonhormone progesterone analogue (e.g., RU486).
- vectors can be used for the oncogene expression. These vectors can be based on plasmids, transposons or viruses such as retroviruses, adenoviruses, and lentiviruses. The vectors can be introduced into zygotes, embryonic stem (ES) cells, tissue-specific stem cells, organ explants or the prostate in situ as required via a variety of methods, including but not limited to, liposome fusion (transposom.es), routine nucleic acid transfection methods such as electroporation, calcium phosphate precipitation, and microinjection, and infection by viral vectors.
- ES embryonic stem
- transposom.es routine nucleic acid transfection methods
- electroporation calcium phosphate precipitation
- microinjection infection by viral vectors.
- a reporter gene may be fused to the oncogene.
- a reporter gene can be, for example, a fluorescent protein such as a green fluorescent protein, a yellow fluorescent protein, a blue fluorescent protein, a red fluorescent protein (e.g., dsRed), or any variation thereof; or a luminescent protein such as luciferase and jS-galactosidase.
- the mammal of the invention may further comprise one or more genetic predispositions rendering it even more susceptible to tumorigenesis.
- Animals with such genetic predispositions include, without limitations, tumor-prone mouse strains (e.g., A/J, C3H, C57BL/6, FVB, 129 and Balb/C).
- the genetic predispositions may be due to one or more genetic mutations, including, without limitations, disabling (e.g., null, conditionally null, or dominant negative) mutations in a tumor suppressor gene (e.g., LNK4a, P53, APC, DCC, PTEN, Rb (Jacks et al., Nature 359:295-300 (1992), DPC4, KLF6, GSTP1, ELAC2/HPC2, orNKX3.1), disabling or activating mutations in certain prostate cancer related genes (e.g., p27Kipl, MXI1, the androgen receptor gene), disabling mutations in a DNA repair gene (e.g., MSH2, MSH3, MSH6, PMS2, Ku70, Ku80, DNA/PK, ATR, ATM, XRCC4, or MLH1), and activating mutations in an endogenous proto-oncogene (e.g., CTNNBl, myc, ras and her2).
- RNA interference RNA interference
- RNAi constructs may be introduced into the host genome to inhibit expression of the target gene (e.g., the tumor suppressor gene, prostate cancer related gene, or DNA repair gene).
- RNAi is a sequence-specific posttranscriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA). It causes degradation of mRNAs homologous in sequence to the dsRNA.
- dsRNA double-stranded RNA
- RNAi constructs With the use of RNAi constructs, one can further control tumorigenesis in the animal by inducibly expressing RNAi molecules that interrupt activity of the targeted gene.
- antisense or ribozyme constructs wherein the antisense or ribozyme is inducibly expressed, can be used to inducibly target expression, function or activity of targeted gene
- the cancer-prone mutations may be mducible while the prostate related oncogene is or is not inducible.
- transgenic, chimeric, or mosaic mammals harboring oncogene expression constructs are inter-crossed with mammals having a cancer-prone genetic predisposition described in Part II, supra, to generate a mammal predisposed to developing cancer and having the inducible oncogene.
- two mammal lines are prepared for mating, where one line contains a reverse tetracycline transactivator gene under the control of a prostate-specific promoter (e.g., the probasin-rtTA construct shown in Fig. 1), and the other line harbors an oncogene linked to a promoter containing a tetO sequence (e.g., the tetO-Akt construct shown in Fig. 2).
- a prostate-specific promoter e.g., the probasin-rtTA construct shown in Fig. 1
- an oncogene linked to a promoter containing a tetO sequence e.g., the tetO-Akt construct shown in Fig. 2
- each of these two lines may be intercrossed with Rb+/- or Rb-/- mammals to generate transgenic, chimeric or mosaic mammals whose genome contains the oncogene under the inducible control of tetracycline in the prostate (e.g., containing both probasin-rtTA and tetO-Akt) in a heterozygous or homozygous Rb null background.
- each line may be intercrossed with mammals with a heterozygous or homozygous null mutation , in another tumor suppressor gene (e.g., P53 or INK4a).
- constructs for the inducible oncogene are introduced into zygotes derived from animals already containing a cancer-prone genetic predisposition.
- the constructs are injected into the two-cell stage of an embryo or injected into a zygote.
- the embryo or zygote can then be developed into a transgenic or mosaic animal.
- the constructs are stably integrated into ES cell lines derived from animals containing the genetic predisposition.
- the ES cells are then injected to blastocysts to generate chimeric or mosaic animals containing the oncogene expression constructs in the background of the genetic predisposition.
- ES cells can also be injected into tetraploid blastocysts to generate transgenic animals whose genome contains the oncogene expression constructs in the background of the genetic predisposition.
- chimeric and mosaic mammals it is desirable to determine whether their prostates contain the oncogene transgene. To better identify these mammals, one can incorporate a reporter gene into the oncogene construct.
- the mammals of this invention may develop prostate cancer (e.g., benign prostate hyperplasia and/or prostate intraepithelial neoplasm I - III) spontaneously within a few months of the induction of oncogene expression.
- the mammals may be treated with carcinogens (e.g., 9,10-dimethyl-l,2- benzanthracene, ENU, urethane, Dimethylhydrazone and Azoxymethane) to expedite this process.
- carcinogens e.g., 9,10-dimethyl-l,2- benzanthracene, ENU, urethane, Dimethylhydrazone and Azoxymethane
- Prostate tumor explants may be obtained from the mammals of this invention. 4. MAMMALIAN CELLS OF THE INVENTION
- Mammalian cells of this invention comprise an oncogene and a cancer-prone genetic predisposition where induced expression of the oncogene or of the genetic predisposition causes the cell to become cancerous or tumorigenic where cancer or tumorigenesis is inhibited when expression of the oncogene or genetic predisposition is reduced.
- Tumorigenesis or cancer development may be assayed using standard techniques, e.g., by assaying cell proliferation, invasive capability, immortalization, anchorage independence. These properties may be determined using any method including those described herein.
- the cells may be obtained or derived from the mammals of the invention.
- the mammalian cells are ES cells, tumor cells, tissue-specific stem cells, or prostate cells. 5. EXEMPLARY USES
- the mammals of this invention and prostate cells derived from the mammals can be used to delineate the initiation, progression, maintenance, regression, minimal residual disease, recurrence, or any other developmental stages of prostate cancer. They can also be used to develop and validate anti-cancer therapeutics. The following describes a few such uses.
- the mammals or cells of this invention may be used to identify new prostate cancer related genes, e.g., prostate cancer suppressor genes or a gene suspected of being required for tumor initiation, progression, maintenance, metastasis, regression, minimal residual disease, recurrence, and/or any other developmental stages.
- a mammal may be obtained as having an expression construct comprising a candidate oncogene, which is operably linked to an inducible promoter so that expression of the oncogene can be repeatedly inducible, reducible and re-inducible, and a cancer-prone genetic predisposition. Then, if the prostate cancer regresses when expression of the oncogene is reduced, the oncogene is involved in prostate cancer development or maintenance.
- Forward genetic screens may also be used to identify new prostate cancer related genes.
- the genetic screens can be conducted using, e.g., retroviral insertion, transposon insertion, genetrap vectors, RNAi or inducible RNAi. Using these elements allows for identification of the target genes. Genetic screens are described in, e.g., US Patent Application No. 20030003478; Mikkers et al., Adv Cancer Res. 88:53-99 (2003); and Suzuki et al., Nat Genet. 32(l):166-74 (2002).
- a gene expression profile for prostate cancer undergoing different stages e.g., genesis, maintenance, progression, regression or recurrence
- due to expression or nonexpression of the introduced oncogene can be established.
- genes whose expression patterns are altered may be prostate cancer related genes.
- tumor initiation genes might be turned off during tumor maintenance. Approaches that focus on genes and pathways involved in the tumor maintenance, rather than initial tumor development, may lead to the development of better anti-cancer therapies and diagnosis for advanced disease.
- genes identified as being involved in initiation of cancer can be used in the discovery of therapies and diagnosis relating to preventive or early control of disease.
- Techniques used to establish gene expression profiles include the use of, e.g., suppression subtraction (in cell culture), differential display, proteomic analysis, serial analysis of gene expression (SAGE) and comparative genomic hybridization (CGH).
- suppression subtraction in cell culture
- differential display proteomic analysis
- SAGE serial analysis of gene expression
- CGH comparative genomic hybridization
- cDNA and/or oligonucleotide microarrays can be used.
- the mammals of the invention may also be used to identify surrogate biomarkers for diagnosis or to follow prostate cancer progression in a mammal (e.g., a mouse, a rat, a rabbit, a nonhuman primate, or a human).
- the biomarkers can be identified based on the differences between expression profiles of the induced (i.e., expression of a prostate related oncogene is induced) and non- induced (i.e., expression of the oncogene is not induced) stages (e.g., genesis, maintenance, progression, regression and recurrence) in non-human mammals or mammalian cells of this invention.
- Blood, urine or other body fluids from the mammal or the cells can be tested with ELISAs or other assays to determine which biomarkers are released from the diseased prostate tissue into circulation during genesis, maintenance, progression or regression of the cancer.
- diagnosis may involve detecting expression or activity level of the biomarker, wherein an abnormally high or low level relative to control (e.g., at least about 50%, 100%, 150%, 200%, 250%, or 300% higher or at least 10%, 25%, 50%, 75%, or 100% lower) is indicative of an abnormal condition.
- an abnormally high or low level relative to control e.g., at least about 50%, 100%, 150%, 200%, 250%, or 300% higher or at least 10%, 25%, 50%, 75%, or 100% lower
- biomarkers are particularly useful clinically in detecting or monitoring prostate cancer progression post cancer therapy. These biomarkers can also be used clinically to assess the toxicity of any prostate cancer therapy.
- the mammals of the invention may further be used to screen therapeutic agents for prostate cancer.
- One such method involves administering a candidate compound to a mammal that has developed prostate cancer or contacting prostate cancer cells derived from such a mammal with a candidate compound. Then one can observe the effect of the compound on cancer regression, as indicated by, e.g., reduction of tumor size, metastasis, angiogenesis and growth rates, or apoptosis or inhibited proliferation of the cultured cancer cells.
- Several methods may be employed to evaluate the effect of the compound.
- apoptosis in an exemplary method of determining cell proliferation, growth of cells is examined in media containing 10%, 2.5%, and 0.5% serum. Growth curves over 10 to 14 day periods can be analyzed by cell counts on days 0, 1, 3, 5, 7, 9, 11, and 13. Quantitative measure of S phase progression can be determined by BrdU incorporation. These two assays provide both static and dynamic views of the proliferative history of these cells. For example, if the cell culture has a higher S phase percentage than the Cre-excised control, as measured by BrdU incorporation, and yet their growth curves are overlapping, this suggests that, although there is increased S phase progression, there must be increased death, resulting in similar growth curves. To determine the rate of apoptosis in low and high serum conditions, Annexin V staining by FACS can be performed. Alternatively, cells can be seeded in chamber slides and fixed in methanol: acetone for TUNEL staining.
- Boyden chamber assays can be performed to measure the migration of cells (Shimizu et ah, Biochem. Biophys. Res. Comm. 264:751, 1999). Briefly, the lower well of a chamber is filled with 600 TI of medium with 10%> or 2.5% FCS, and the upper well is seeded with 400 TI of cell suspension. A cellulose acetate membrane filter is then interposed between the two chambers. The chambers are kept in a humidified atmosphere of 5%> CO 2 at 37°C for 4 hours. Filters are then washed, fixed with methanol: acetone, and stained with crystal violet. The number of cells that migrate into the filter and reach its lower side can be determined microscopically. Triplicate assays can be performed for each cell line and its controls.
- a low- density seeding assay can be used as a surrogate assay for immortalization potential.
- 2500 cells are seeded per well in a 6 well plate.
- Cells with high potential for immortalization are able to grow to form visible colonies in 14 days.
- the number of emerging colonies can be used as a quantitative measure for the immortalization potential of those cells.
- anchorage independence is evaluated in soft-agar assays. For example, in soft- agar, 10,000 cells are seeded per well in a 6-well plate. Colony formation is monitored daily by microscopic inspection. Cell clusters of greater than 0.5 mm in size are counted as a colony. The number of colonies is a quantitative marker for the tumorigenic potential of the cells.
- a candidate compound is administered to a mammal or cell of the invention wherein the inducible oncogene is expressed to cause formation of cancer. If no cancer results, the compound is a candidate prophylactic agent capable of preventing tumor formation and/or growth.
- a therapeutic agent can be identified based on the molecular profile it elicits.
- a first molecular profile (e.g., transcriptional, proteomic or genomic) of the prostate cells from the mammals of this invention or the mammalian cells of this invention is established by, e.g., identifying a plurality of biomarkers whose patterns of expression or biological function alternations correspond to the non-induced (i.e., expression of a prostate related oncogene is not induced) stage (e.g., genesis, maintenance, progression, regression and recurrence) of the mammal or the mammalian cells.
- stage e.g., genesis, maintenance, progression, regression and recurrence
- a second molecular profile of these biomarkers is established corresponding to the induced (i.e., expression of the prostate oncogene is induced in the mammal or the mammalian cells) stage (e.g., genesis, maintenance, progression, regression and recurrence) of the mammal or the mammalian cells in the presence of a candidate compound.
- stage e.g., genesis, maintenance, progression, regression and recurrence
- the two profiles are compared, wherein substantial similarity of the two profiles indicates that the test compound is a potential anti-cancer drag.
- “Substantial similarity” means that the Pearson correlation coefficient of biomarker expression activity for the two molecular profiles is statistically significant, with a p value of less than 0.1 (e.g., less than 0.05, 0.02, or 0.01).
- the non-overlapping portion between the two profiles may represent nonspecific activity of the candidate compound and allow prediction of the potential toxicity of the compound.
- the mammals of this invention may be used to study minimal residual prostate cancer and to identify therapeutic agents to treat minimal residual prostate cancer.
- a non-human mammal having a genome which comprises an inducible prostate cancer related oncogene and a cancer-prone genetic predisposition, has minimal residual diseases if tumor recurrence occurs at a site of a previous tumor, which formed when expression of the oncogene was induced, and regressed when expression of the oncogene was reduced.
- Therapeutic agents may be identified by administering a candidate compound to a mammal having residual prostate cancer.
- An alteration in expression or activity of a gene involved in minimal residual prostate cancer indicates that the compound may be useful as a therapeutic agent to treat minimal residual prostate cancer.
- compounds can be identified as being useful for preventing minimal residual prostate cancer. 6. Examples
- Transgenic mice having the constructs shown in Figs. 1 and 2 are established and bred with transgenic mice having a Rb+/- mutation.
- the resultant mice contain a rtTA inducible system for prostate Atk expression in a Rb-/- background.
- These mice are fed with doxycycline-containing drinking water (2 mg/ml sucrose water) or food pellets and observed for spontaneous tumor development in the prostate.
- Primary tumors are adapted to culture by mechanical mincing with sterilized razor blades and brief trypsinization and maintained on RPMI media containing 10% serum and supplemented with doxycycline (2 ⁇ g/ml media).
- prostate cancer tissue samples or derivative cancer cells are fixed and embedded.
- An antibody against a prostate cancer related protein e.g., antibodies to PTEN or E-cadherin
- the cells are seeded at a density of 20,000 cells per well in a 12-well plate in media with or without doxycycline. Media is changed every 3 days for all samples. Duplicate wells are typsinized, and cell numbers are counted by hemacytometer at different time points and plotted against time. Studies are conducted in media containing 10%, 1% and 0.5% serum. Growth curve determinations are performed in cells maintained on doxycycline prior to experiments as well as cells already removed from doxycycline for 3 days.
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Abstract
La présente invention a trait à des mammifères et à des cellules comportant une construction d'expression pour un acide nucléique codant pour un oncogène lié au cancer prostatique et dans lequel le mammifère et la cellule comprennent une prédisposition génétique sensible au cancer.
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| WO2001009308A1 (fr) * | 1999-07-29 | 2001-02-08 | Dana-Farber Cancer Institute, Inc. | Modele de cancer inductible permettant d'etudier la base moleculaire d'interactions de cellules tumorales in vivo |
| WO2002079419A2 (fr) * | 2001-03-28 | 2002-10-10 | Dana-Farber Cancer Institute, Inc. | Identification et caracterisation de genes |
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| WO2001009308A1 (fr) * | 1999-07-29 | 2001-02-08 | Dana-Farber Cancer Institute, Inc. | Modele de cancer inductible permettant d'etudier la base moleculaire d'interactions de cellules tumorales in vivo |
| WO2002079419A2 (fr) * | 2001-03-28 | 2002-10-10 | Dana-Farber Cancer Institute, Inc. | Identification et caracterisation de genes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US10260104B2 (en) | 2010-07-27 | 2019-04-16 | Genomic Health, Inc. | Method for using gene expression to determine prognosis of prostate cancer |
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