WO2008112283A2 - Microrna profiling of androgen responsiveness for predicting the appropriate prostate cancer treatment - Google Patents

Abstract

The invention relates to methods of predicting responsiveness of prostate cancer patients to androgen ablation therapy by determining an androgen responsiveness microRNA expression profile of a tumor cell, and correlating the androgen responsiveness microRNA expression profile to the therapeutic treatment (androgen responsiveness).

Description

ANDROGEN RESPONSIVENESS PROFILING

RELATED APPLICATIONS

This application claims priority to US Provisional Patent Application Serial No. 60/906,742 filed on March 12, 2007 which is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This work described herein was supported by the National Institutes of Health. The U.S. Government has certain rights in the invention.

BACKGROUND

MicroRNAs (miRNAs) are small (~22 nts), non-coding ribonucleic acids (RNAs) that regulate gene expression by translational repression, messenger RNA (mRNA) degradation, or gene silencing by methylation (for reviews, see 1; 3 and 4). Since the original discovery of a few microRNAs in Caenorhabditis elegans which regulate the timing of embryonic development (12, 1, 7), hundreds of microRNAs have been identified using cDNA cloning and bioinformatic prediction strategies (9, 8, 27, 26). Nearly a third of human genes are estimated to be regulated by a few hundred miRNAs (3). In animals, the predominant known mode of miRNA action is by binding with imperfect base-pairing to the 3'-UTR of target RNAs, thereby modulating translation (28, 29). In plants, most miRNAs function by binding with perfect or near complementarity to their mRNA targets and cleaving the mRNA vis a vis the RISC (RNA-induced silencing) complex (5). At least eight animal miRNAs and two viral miRNAs also act by causing mRNA degradation (1, 36, 37, 38, 39, 40). In a few instances, so far observed only in plants, miRNAs can cause gene silencing by directing DNA methylation (12, 3).

MicroRNAs are polymerase II transcripts that are synthesized as long RNA precursors (pri-miRNAs) that contain a stem-loop structure of about 80 bases (1, 30, 31). The pri -miRNAs are processed in the nucleus by the RNAse III enzyme Drosha and DCGR8/Pasha, which excise the stem-loop to form the pre-miRNA (22). Upon transport out of the nucleus with the help of Exportin-5 (23), the pre-miRNAs are further truncated into the short mature miRNA by the RNAseIII enzyme Dicer (32, 35, 34, 33). The microRNA biogenesis pathway may be regulated at each step, and the altered expression of the miRNA processing enzyme Dicer has been shown in lung cancer (24). miRNAs play regulatory roles in a wide range of biological processes including cellular proliferation and differentiation, early development and cell death (6, 7). The involvement of miRNAs in these biological processes suggested that miRNAs could contribute to oncogenesis. In fact, Calin et al (2002) found that more than half of the miRNAs (98 out of the 186 known at the time) are in cancer- associated regions of the genome - break-point regions, fragile sites, minimal regions of loss of heterozygosity and minimal regions of amplifications. More recently, altered expression of miRNAs has been observed in various cancers (8). Certain miRNAs (let-7 and miR- 15-a/miR 16-1) have been demonstrated to regulate genes involved in cancer pathways such as RAS (oncogene) and BcI 2 (anti-apoptotic gene), respectively (10, 11). In addition, it has been reported that the targets of miRNAs with altered expression in tumor samples are composed of a disproportionate number of oncogenes and tumor suppressors (20). Changes of expression of mRNAs have been observed in many cancers and other pathological conditions.

SUMMARY

Described herein, according to one aspect, are methods of predicting responsiveness of a tumor to therapeutic treatment comprising determining an androgen responsiveness microRNA expression profile of a tumor cell, and correlating the androgen responsiveness microRNA expression profile to the therapeutic treatment (androgen responsiveness).

In one embodiment, the androgen responsiveness microRNA expression profile comprises determination of the expression profile of one or more of the sequences listed in Table 1 or Table 2 in a tumor cell.

In an embodiment, the androgen responsiveness microRNA expression profile comprises determination of the expression profile of one or more of sequences identified in a comparison between an androgen sensitive prostate tumor cell and an androgen insensitive prostate tumor cell. In an embodiment, the prostate tumor cell can be isolated or obtained directly from a prostate tumor (e.g., biopsy or tissue sample). In another embodiment, the prostate tumor cell can be grown in culture. In an embodiment, the prostate tumor cell can change androgen status in culture. In an embodiment, the expression profile comprises determination of the expression profile of one or more of the following microRNAs: let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i, miR-15a, miR-16, miR-17-5p, miR-19a, miR-19b, miR-20a, miR-21, miR-22, miR-23a, miR-26a, miR-27a, miR-27b, miR-29a, miR- 29b, miR-30, miR-30a-5p, miR-30d, miR-93, miR-98, miR-99a, miR-101, miR-102, miR-103, miR-106a, miR-106b, miR-107, miR-125b, miR-146a, miR-146b, mi-155, miR-181a, miR-181b, miR-181c, miR-181d, miR-185, miR-191, miR-195, miR-196a, miR-196b, miR-200b, miR-320, miR-374, miR-429, miR449b, miR-491, and miR- 494. In an embodiment, the expression profile comprises determination of the expression profile of one or more of the following microRNAs: miR-146a, miR-200b, and miR-449b. In an embodiment, the expression profile comprises determination of the expression profile of one or more of the following microRNAs: let-7g, miR-16, miR-99a, miR-195, and miR-491.

In another embodiment, the androgen responsiveness microRNA expression profile comprises determination of the expression profile of one or more of the miRNAs of a BD3/DH3 comparison, an E006A/BD3 comparison, an AH9/DH3 comparison, an AH9/ E006A comparison, a BD3/AH9 comparison, or a DH3/E006A comparison.

In an embodiment, an androgen responsiveness mRNA expression profile is determined for one or more of the mRNAs identified in a comparison between an androgen sensitive prostate tumor cell and an androgen insensitive prostate tumor cell. In an embodiment, the prostate tumor cell can be isolated or obtained directly from a prostate tumor. In another embodiment, the prostate tumor cell can be grown in culture. In an embodiment, the prostate tumor cell can change androgen status in culture. In an embodiment, the androgen responsiveness mRNA expression profile comprises determination of the expression profile of one or more of the mRNAs of a BD3/DH3 comparison, an E006A/BD3 comparison, an AH9/DH3 comparison, an AH9/ E006A comparison, a BD3/AH9 comparison, or a DH3/E006A comparison. In an embodiment, the androgen responsiveness mRNA expression profile comprises determination of the expression profile of one or more mRNAs in Table 3 or Table 4.

In an embodiment, the androgen responsiveness profile is determined for tumors derived from subjects or tumors having specific characteristics in addition to androgen sensitivity, such as age of diagnosis, race, cellular differentiation, tumor grade, family history of prostate cancer or genetic predisposition, tumor size, and/ or degree of lymphovascular invasion.

In another embodiment, the androgen responsiveness microRNA expression profile or mRNA expression profile is determined by array methods, PCR methods, immunological methods, sequencing methods, expression profile methods, or kinetic profiles.

In one embodiment, PCR methods are one or more of real-time PCR, PCR, reverse transcriptase PCR, rtPCR, allele-specific PCR. In another embodiment, correlating comprises correlating an androgen- sensitive profile with an indication that a subject may respond to anti -androgen therapies.

In one embodiment, correlating comprises correlating an androgen-insensitive profile with an indication that a subject may not respond to anti-androgen therapies. In one embodiment, the methods may further comprise administering a therapeutic amount of an anti-androgen therapy to the subject correlating to an androgen-sensitive profile. In one embodiment, the anti-androgen therapy comprises one or more of luteinizing hormone-releasing hormone agonists (e.g., leuprolide, goserelin, and buserelin). Antiandrogens (e.g., flutamide and nilutamide); adrenal gland blockers (e.g., ketoconazole and aminoglutethimide); orchiectomy (a surgical procedure to remove one or both testicles to decrease hormone production); and/or estrogen.

In one embodiment, the methods may further comprise administering a therapeutic amount of one or more anti-neoplastic agents to the subject. In one embodiment, the anti-neoplastic agent comprises docetaxel, cisplatin, cyclophosphamind, doxorubicin, prednisone, 5-FU, trastuzumab, 3G4, travacin, gemcitabine, estramustine, carboplatin, or radioimmuno therapy agents. In one embodiment, the methods may further comprise co-administering one or more additional therapeutic agents to the subject.

In another embodiment, the therapeutic agents are one or more of an immunomodulatory agent, anti-inflammatory agents, glucocorticoid, steroid, non- steriodal anti-inflammatory drug, leukotreine antagonist, β 2-agonist, anticholinergic agent, sulphasalazine, penicillamine, dapsone, antihistamines, anti-malarial agents, anti-viral agents, prednisone or antibiotics.

In some embodiments, the methods include identification of a subject suspected of having or having prostate cancer. In some embodiments, the tumor is androgen dependent, androgen independent, or of unknown androgen status.

Described herein, according to one aspect, are methods of selecting a subject for anti-androgen therapy for prostate cancer, comprising determining an androgen responsiveness microRNA expression profile of a subject or preferably a tumor cell of a subject, and/or determining the mRNA expression profile of a subject or preferably a tumor cell of a subject, and correlating the expression profile results to treatment options.

In an embodiment, correlating comprises correlating an androgen-sensitive profile to treatment with anti-androgen therapies.

In another embodiment, correlating comprises correlating an androgen- insensitive profile to treatment with surgical, chemotherapeutic, and/or radiological therapies.

In one embodiment, the methods may further comprise administering a therapeutic amount of an anti-androgen therapy to a subject having an androgen- sensitive profile. Described herein, according to one aspect, are methods of assessing the risk of cancer in a subject, comprising determining the androgen responsiveness microRNA expression profile, and correlating the androgen responsiveness microRNA expression profile to cancer risk.

According to one embodiment, the cancer is prostate cancer. Described herein, according to one aspect, are methods for monitoring treatment for a subject with prostate cancer, comprising determining an androgen responsiveness microRNA expression profile of a subject and/or an mRNA expression profile of a subject; determining a pre-treatment tumor status in the subject; administering a therapeutically effective amount of an anti-androgen therapy if the pre-treatment tumor status correlates with androgen-dependent prostate cancer; and determining a post-treatment tumor status in the subject. According to another embodiment, a modulation of tumor status indicates that the tumor has become androgen-independent.

In one embodiment, the pre-treatment and post-treatment tumor statuses are determined in a diseased tissue.

Described herein, according to one aspect, are methods for screening for a prostate cancer drug target, comprising providing a population of tumor cells; contacting the cells with a candidate microRNA composition listed in Table 1 or Table 2, and the comparisons described below and determining effect of the candidate microRNA composition on cell proliferation, wherein a decrease in cell proliferation indicates that the candidate composition may be efficacious. Another aspect of the includes a method of screening for a prostate cancer drug target, comprising providing a population of tumor cells; contacting the cells with a candidate mRNA composition listed in Table 3 or 4, and the comparisons described below and determining effect of the candidate microRNA composition on cell proliferation, wherein a decrease in cell proliferation indicates that the candidate composition may be efficacious. In one embodiment, the methods may further comprise correlating the effect with the genotype.

In one embodiment, the methods may further comprise determining the androgen responsiveness microRNA expression profile and/or an mRNA expression profile of the tumor cells prior to or after providing the cells with androgen ablation therapy or other therapetutic intervention.

Described herein, according to one aspect, are methods of treating a subject suffering from cancer, comprising determining an androgen responsiveness microRNA expression profile of a subject or of a tumor cell of a subject, and/or determining the androgen responsiveness mRNA profile up a subject, and administering a therapeutic amount of a prostate cancer treatment based on the profile.

According to one embodiment, an androgen-sensitive profile indicates treatment with an anti-androgen therapy. According to one embodiment, an androgen-insensitive profile indicates treatment with surgical, chemotherapeutic, and/or radiological, therapies.

According to another embodiment, the subject is a mammal. In a related embodiment, the mammal is a human. Described herein, according to one aspect, are kits for the assessment of cancer treatment options, comprising a microRNA array that differentiates androgen dependent and androgen independent tumors and instructions for use. In another aspect, kits can further include an mRNA array that differentiates androgen dependent and androgen independent tumors and instructions for use. Described herein, according to one aspect, are kits for the assessment of cancer treatment options, comprising a microarray comprising microRNA probes that differentiates androgen dependent and androgen independent tumors, and instructions for use. In another aspect, kits can further include an mRNA microarray probes that differentiates androgen dependent and androgen independent tumors and instructions for use.

Presented herein, in accordance with one aspect are arrays comprising the microRNAs or complements thereof or fragments thereof of or one or more of sequences identified in a comparison between an androgen sensitive prostate tumor cell and an androgen insensitive prostate tumor cell, a BD3/DH3 comparison, a E006A/BD3 comparison, a AH9/DH3 comparison, a AH9/ E006A comparison, a BD3/AH9 comparison or a DH3/E006A comparison or the microRNAs listed in Table 1 or Table 2.

Presented herein, in accordance with one aspect are arrays comprising the mRNAs or complements thereof or fragments thereof of or one or more of sequences identified in a comparison between an androgen sensitive prostate tumor cell and an androgen insensitive prostate tumor cell, a BD3/DH3 comparison, a E006A/BD3 comparison, a AH9/DH3 comparison, a AH9/ E006A comparison, a BD3/AH9 comparison or a DH3/E006A comparison or the microRNAs listed in Table 3 or Table 4. Other embodiments of the invention are disclosed infra. BRIEF DESCRIPTION OF THE DRAWING

Figure 1 depicts a heat map of a microRNA prostate cancer array.

DETAILED DESCRIPTION Disclosed herein are methods of diagnosing androgen responsiveness in prostate cancer by expression profiling, e.g., microRNA expression profiling and/or mRNA expression profiling. Also disclosed are methods of selecting treatment for subjects suffering from prostate cancer based on microRNA and/or mRNA expression profiling of tumor cells. Further disclosed are are methods of discovering drug targets for treating androgen independent and androgen dependent prostate cancer. Further disclosed are kits that can be used for practicing the methods of the invention.

The invention is based in part on the identification of miRNAs involved in cancer, particularly in the shift from androgen sensitive to androgen insensitive status in prostate cancer, thus described herein is the development of a highly sensitive and reproducible microarray of miRNAs to profile the expression levels of miRNAs in cells (e.g., cancer cells). A number of microarray-based methods for profiling miRNA expression have recently been developed {Shingara, 2005 #41 } {Barad, 2004 #42} {Liu, 2004 #43 } {Baskerville, 2005 #44}. The first generation LMT arrays described herein, for example, contains 1140 probes synthesized in situ representing predicted and confirmed mature miRNA sequences. Each probe which contains 19, 20, 21, or 22-nucleotides of the mature miRNA sequence or complement to the mature miRNA sequence is tethered to the array surface by a 41, 40, 39, or 38- nucleotide linker with non-genomic sequences to produce a probe having a total length of 60 nucleotides. The mature microRNAs targets are purified using mirVana miRNA isolation kit (Ambion/ABI) and flashPAGE fractionator system

(Ambion/ABI). The purified miRNAs are directly 3 '-end labeled (without any amplification) using amine-modified nucleotides, coupled with Cy3 dye and hybridized onto the array.

Microarrays and methods for identification of mRNAs differentially expressed in cells are well known in the art and commercially available, e.g., from Affymetrix.

Also described herein are methods of identifying and predicting inter-patient differences in prognostic prediction for survival in prostate cancer. It further related to methods for determining and exploiting such differences to improve medical outcomes.

As used herein, the term "phenotype" refers to a trait which can be compared between subjects, such as presence or absence of a condition (e.g., androgen dependent and androgen independent tumors), a visually observable difference in appearance between subjects, metabolic variations, physiological variations, variations in the function of biological molecules, and the like. An example of a phenotype is length of survival. For example, a phenotype of a variant is longer survival of androgen dependent prostate cancer, whereas a phenotype of an androgen independent subject is correlated with a shorter survival of prostate cancer.

The terms "disease" or "condition" are commonly recognized in the art and designate the presence of signs and/or symptoms in a subject or patient that are generally recognized as abnormal. Diseases or conditions may be diagnosed and categorized based on pathological changes. Signs may include any-objective evidence of a disease such as changes that are evident by physical examination of a patient or the results of diagnostic tests that may include, among others, laboratory tests to determine the presence of DNA sequence expression profiles or variant forms of certain genes in a patient. Symptoms are subjective evidence of disease or a patients condition, e.g., the patients perception of an abnormal condition that differs from normal function, sensation, or appearance, which may include, without limitations, physical disabilities, morbidity, pain, and other changes from the normal condition experienced by a subject. Various diseases or conditions include, for example, those categorized in standard textbooks of medicine including, without limitation, textbooks of nutrition, allopathic, homeopathic, and osteopathic medicine. In certain aspects, the disease or condition is selected from the group consisting of the types of diseases listed in standard texts such as Harrison's Principles of Internal Medicine (14th Ed) by Anthony S. Fauci, Eugene Braunwald, Kurt J. Isselbacher, et al. (Editors), McGraw Hill, 1997, or Robbins Pathologic Basis of Disease (6th edition) by Ramzi S. Cotran, Vinay Kumar, Tucker Collins & Stanley L. Robbins, W B Saunders Co., 1998, or the Diagnostic and Statistical Manual of Mental Disorders: DSM-IV (4th edition), American Psychiatric Press, 1994, or other texts described below. The phrase "suffering from a disease or condition" means that a subject is either presently subject to the signs and symptoms, or is more likely to develop such signs and symptoms than a normal subject in the population (e.g., suffering from prostate cancer). Thus, for example, a subject suffering from a condition can include a developing fetus, a subject to a treatment or environmental condition which enhances the likelihood of developing the signs or symptoms of a condition, or a subject who is being given or will be given a treatment which increase the likelihood of the subject developing a particular condition. Thus, methods of the present invention which relate to treatments of patients (e.g., methods for selecting a treatment, selecting a patient for a treatment, and methods of treating a disease or condition in a patient) can include primary treatments directed to a presently active disease or condition, secondary treatments which are intended to cause a biological effect relevant to a primary treatment, and prophylactic treatments intended to delay, reduce, or prevent the development of a disease or condition, as well as treatments intended to cause the development of a condition different from that which would have been likely to develop in the absence of the treatment.

In certain embodiments, the disease or condition is prostate cancer. Prostate cancer may be, for example, androgen dependent or androgen independent. Treatment choices for the two type generally different and thus there is a need in the art for methods to determine which type of prostate cancer is present or if there is a shift from one type to the other. Androgen dependent prostate cancer may be treated by anti-androgen therapies (e.g., therapies that block, inhibit, inhibit production, block activity, competitors, etc.).

The term "therapy" refers to a process that is intended to produce a beneficial change in the condition of a mammal, e.g., a human, often referred to as a patient. A beneficial change can, for example, include one or more of restoration of function, reduction of symptoms, limitation or retardation of progression of a disease, disorder, or condition or prevention, limitation or retardation of deterioration of a patient's condition, disease or disorder. Such therapy can involve, for example, nutritional modifications, administration of radiation, administration of a drug, behavioral modifications, and combinations of these, among others. Therapies include, for example, anti-androgen therapies for hormore dependent prostate cancer. Anti- androgen therapy may comprise one or more of luteinizing hormone-releasing hormone agonists (e.g., leuprolide, goserelin, and buserelin), antiandrogens (e.g., flutamide and nilutamide); adrenal gland blockers (e.g., ketoconazole and aminoglutethimide); orchiectomy (a surgical procedure to remove one or both testicles to decrease hormone production); and/or estrogen. Other therapies include, for example, androgen-insensitive therapies, including for example, surgical, chemotherapeutic, and/or radiological therapies

The terms "drug" and "therapeutic agent," as used herein refer to a chemical entity or biological product, or combination of chemical entities or biological products, administered to a subject to treat or prevent or control a disease or condition, e.g., an anti-neoplastic agent. The chemical entity or biological product is preferably, but not necessarily a low molecular weight compound, but may also be a larger compound, for example, an oligomer of nucleic acids, amino acids, or carbohydrates including without limitation proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, lipoproteins, and modifications and combinations thereof. A biological product is preferably a monoclonal or polyclonal antibody or fragment thereof such as a variable chain fragment or single chain antibody, nanobody; cells; or an agent or product arising from recombinant technology, such as, without limitation, a recombinant protein, recombinant vaccine, or DNA construct developed for therapeutic, e.g., human therapeutic, use. The term "drug" may include, without limitation, compounds that are approved for sale as pharmaceutical products by government regulatory agencies (e.g., U.S. Food and Drug Administration (FDA), European Medicines Evaluation Agency (EMEA), and a world regulatory body governing the International Conference of Harmonization (ICH) rules and guidelines), compounds that do not require approval by government regulatory agencies, food additives or supplements including compounds commonly characterized as vitamins, natural products, and completely or incompletely characterized mixtures of chemical entities including natural compounds or purified or partially purified natural products. The term "drug" as used herein is synonymous with the terms "medicine," "pharmaceutical product," or "product." Most preferably the drug is approved by a government agency for treatment of a specific disease or condition. Included are "candidate compounds," which refers to a drug, agent or compound that is under investigation, either in laboratory or human clinical testing for a specific disease, disorder, or condition.

The term "probe," as used herein, refers to a molecule that detectably distinguishes between target molecules differing in structure. Detection can be accomplished in a variety of different ways depending on the type of probe used and the type of target molecule. Thus, for example, detection may be based on discrimination of activity levels of the target molecule, but preferably is based on detection of specific binding. Examples of such specific binding include antibody binding and nucleic acid probe hybridization (e.g., a microRNA to a complementary probe). Thus, for example, probes can include enzyme substrates, antibodies and antibody fragments, and nucleic acid hybridization probes. Thus, in preferred embodiments, the detection of the presence or absence of the at least one microRNA expression profile involves contacting a nucleic acid sequence, preferably a nucleic acid probe, where the probe preferentially hybridizes to the complimentary nucleic acid sequence, where the hybridization is carried out under selective hybridization conditions. Unless otherwise specified, a nucleic acid probe can include one or more nucleic acid analogs, labels or other substituents or moieties so long as the base- pairing function is retained. For example, techniques such as OLA, TAQMAN, and methods described in US Patent Application Publication No. 2004/0121371, which is hereby incorporated by reference, are also useful detection methods according to the methods disclosed herein.

As used herein the term "chemical class" refers to a group of compounds that share a common chemical scaffold but which differ in respect to the substituent groups linked to the scaffold. Examples of chemical classes of drugs include, for example, phenothiazines, piperidines, benzodiazepines and aminoglycosides. Members of the phenothiazine class include, for example, compounds such as chlorpromazine hydrochloride, mesoridazine besylate, thioridazine hydrochloride, acetophenazine maleate trifluoperazine hydrochloride and others, all of which share a phenothiazine backbone. Members of the piperidine class include, for example, compounds such as meperidine, diphenoxylate and loperamide, as well as phenylpiperidines such as fentanyl, sufentanil and alfentanil, all of which share the piperidine backbone. Chemical classes and their members are recognized by those skilled in the art of medicinal chemistry. A preferred chemical class is that to which docetaxel belongs, e.g., the taxoid family. For example, see US Patent Nos: 4814470, 5438072, 5698582, and 5714512, which are hereby incorporated by reference in their entirety.

"Predicting prognostic outcome," refers to, for example, the determination or forecasting of length of survival and/or probability of survival. For example, this may be based on one or more of the microRNA expression profiles disclosed herein. This may be based on one or more of the mRNA expression profiles disclosed herein.

"Predicting responsiveness of a tumor," refers to, for example, the determination or forecasting of whether a tumor may react to a particular treatment. For example, this may be based on the androgen responsiveness microRNA expression profile or mRNA expression profile (e.g., whether the profile reveals if the tumor is androgen dependent or independent).

"Androgen responsiveness microRNA expression profile," as used herein refers to the particular expression profile of a subject, a tissue of a subject and/or of a cell of a subject. The expression profile may be of just one microRNA, or may be of many microRNAs. For example, the androgen responsiveness microRNA expression profile may be of one or more of the microRNAs listed in Table 1 or Table 2; or one or more microRNAs of the comparisons and may be determined by an array method of other detection method discussed herein (e.g., one or more of the BD3/DH3 comparison, E006A/BD3 comparison, AH9/DH3 comparison, AH9/ E006A comparison, BD3/AH9 comparison, the DH3/E006A comparison, or the prostate tumor sample comparison.) The expression profile may also be determined, for example, by biochemical methods, e.g., array based methods, PCR based methods, and other method now known or later developed in the art. "Androgen responsiveness mRNA expression profile," as used herein refers to the particular expression profile of a subject, a tissue of a subject and/or of a cell of a subject. The expression profile may be of just one mRNA, or may be of many mRNAs. For example, the androgen responsiveness mRNA expression profile may be of one or more of the microRNAs listed in Table 3 or Table 4; or one or more mRNAs of the comparisons and may be determined by an array method of other detection method discussed herein (e.g., one or more of the BD3/DH3 comparison, E006A/BD3 comparison, AH9/DH3 comparison, BD3/DH3 comparison, AH9/ E006A comparison, BD3/AH9 comparison, the DH3/E006A comparison, or the prostate tumor sample comparison.) The expression profile may also be determined, for example, by biochemical methods, e.g., array based methods, PCR based methods, and other method now known or later developed in the art.

"Anti-neoplastic agent," as used herein is an agent that will halt tumor growth, slow tumor growth, kill tumor cells, cause tumor cells to enter apoptosis, limit the blood supply to tumors and the like. Examples include, docetaxel, cisplatin, cyclophosphamind, doxorubicin, prednisone, 5-FU, trastuzumab (Herceptin ), 3G4 (travacin equivalent) travacin, gemcitabine, estramustine, carboplatin, radiation.

"Co-administering," as used herein refers to the administration with another agent, either at the same time, in the same composition, at alternating times, in separate compositions, or combinations thereof.

"One or more additional anti-neoplastic agents," refers to the selection of additional therapeutic agents that may be co-administered with the anti-neoplastic agent are selected from cisplatin, mitomycin, capecitabine, irinotecan, topotecan, estramustine, vinorelbine, cyclophosphamide, ifosfamide, doxorubicin, epirubicin, 5- FU, trastuzumab (Herceptin ™), 3G4 (travacin equivalent) travacin, gemcitabine, estramustine, carboplatin, imatinib, gefitinib, erlotinib, cetuximab (Erbitux), bevacizumab (Avastin), thalidomide, or radiation.

As used herein, the terms "tumor" or "cancer" refer to a condition characterized by anomalous rapid proliferation of abnormal cells, or the slow rate of death of abnormal cells in a subject. The abnormal cells often are referred to as "neoplastic cells," which are transformed cells that can form a solid tumor. The term "tumor" refers to an abnormal mass or population of cells (e.g., two or more cells) that result from excessive or abnormal cell division or insufficient cell death or apoptosis, whether malignant or benign, and pre-cancerous and cancerous cells.

Malignant tumors are distinguished from benign growths or tumors in that, in addition to uncontrolled cellular proliferation, they can invade surrounding tissues and can metastasize. In prostate cancer, neoplastic cells may be identified in the prostate one or more adjacent tissues or organs (e.g., lymph node), or in a prostate and one or more non-adjacent tissues or organs to which the cancer cells have metastasized. As used herein, "tumor" or "cancer," refers prostate cancer.

The term "invasion" as used herein refers to the spread of cancerous cells to adjacent surrounding tissues. The term "invasion" often is used synonymously with the term "metastasis," which as used herein refers to a process in which cancer cells travel from one organ or tissue to another non-adjacent organ or tissue.

Prostate cancer, along with lung and colon cancer, are the three most common causes of death from cancer in men in the U.S., but prostate is by far the most prevalent of all human malignancies with the exception of skin cancer (Scott R. et al., J. Urol., 101:602,1969; Sakr W A et al., J. Urol., 150: 379, 1993). It is one of the top three causes of death from cancer in men in the United States (Greenlee R T et al., CA Cancer J. Clin. VoI 15, 2001). Currently, treatments available for prostate cancer require not only an early detection of the malignancy and a reliable assessment of the severity of the cancer.

As used herein, "assessing the risk of cancer in a subject," refers to, for example, the determination of the clinical outcome based on percentages of, for example, survival given their expression profile and treatment options.

"Providing," refers to obtaining, by for example, buying or making the, e.g., polypeptide, drug, polynucleotide, probe, and the like. The material provided may be made by any known or later developed biochemical or other technique. For example, polypeptides may be obtained from cultured cells. The cultured cells, for example, may comprise an expression construct comprising a nucleic acid segment encoding the polypeptide. Cells and/or subjects may be treated and/or contacted with one or more antineoplastic treatments including, surgery, chemotherapy, radiotherapy, gene therapy, immune therapy or hormonal therapy, or other therapy recommended or proscribed by self or by a health care provider.

"Obtaining" refers to purchase, procure, manufacture, or otherwise come into possession of.

As used herein, "treating, preventing or alleviating cancer," refers to the prophylactic or therapeutic use of the therapeutic agents described herein.

"Substantially purified" when used in the context of a polypeptide or polynucleotide, or fragment or variant thereof that are at least 60% free, preferably 75% free and more preferably 90% free from other components with which they are naturally associated. An "isolated polypeptide" or "isolated polynucleotide" is, therefore, a substantially purified polypeptide or polynucleotide, respectively.

The term "subject" includes organisms which are capable of suffering from cancer or who could otherwise benefit from the administration of a compound or composition of the invention, such as human and non-human animals. Preferred human animals include human patients suffering from or prone to suffering from cancer or associated state, as described herein. The term "non-human animals" of the invention includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, and non- mammals, such as non-human primates, e.g., sheep, dog, cow, chickens, amphibians, reptiles, etc. A human subject can be referred to as a patient.

A method for "predicting" or "diagnosing" as used herein refers to a clinical or other assessment of the condition of a subject based on observation, testing, or circumstances.

"Determining a level of expression" or "determining an expression profile," may be by any now known or hereafter developed assay or method of determining expression level, for example, immunological techniques, PCR techniques, immunoassay, quantitative immunoassay, Western blot or ELISA, quantitative (RT- )PCR, and/or Northern blot. The level may be of RNA or protein may be determined, for example, by sequencing, real-time PCR, PCR, allele-specific PCR, Pyrosequencing, SNP Chip technology, or RFLP.

A sample or samples may be obtained from a subject, for example, by swabbing, biopsy, lavage or phlebotomy. Samples include tissue samples, blood, sputum, bronchial washings, biopsy aspirate, or ductal lavage.

"Therapeutically effective amount," as used herein refers to an amount of an agent which is effective, upon single or multiple dose administration to the cell or subject, in prolonging the survivability of the patient with such a disorder beyond that expected in the absence of such treatment. Compositions described herein may be administered, for example, systemically, intratumorally, intravascularally, to a resected tumor bed, orally, or by inhalation.

As used herein, the term "primer" refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (e.g., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Boil. Chem. 260:2605-2608 (1985); Rossolini et al., MoI. Cell Probes, 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

As used herein, the term "polymerase chain reaction" (PCR) refers to the methods of U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, all of which are hereby incorporated by reference, directed to methods for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. As used herein, the terms "PCR product" and "amplification product" refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.

As used herein, the term "recombinant DNA molecule" as used herein refers to a DNA molecule, which is comprised of segments of DNA joined together by means of molecular biological techniques.

As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends. In either a linear or circular DNA molecule, discrete elements are referred to as being "upstream" or 5' of the "downstream" or 3' elements. This terminology reflects the fact that transcription proceeds in a 5' to 3' fashion along the DNA strand. The promoter and enhancer elements which direct transcription of a linked gene are generally located 5' or upstream of the coding region. However, enhancer elements can exert their effect even when located 3' of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.

The term "detectable label" is understood as a chemical modification, binding agent, or other tag that can be readily observed, preferably in a quantitative manner, such as a fluorescent tag that has a specific wavelengths of absorbtion and emission to allow detection of the compound associated with the detectable label. The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to the compliment of a test sequence. Optionally, the identity exists over a region that is at least about 50 amino acids or nucleotides in length, or more preferably over a region that is 75-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. A "comparison window," as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math., 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. MoI Biol., 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. U.S.A., 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Unless specifically stated or obvious from context, as used herein, the term "or " is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms

"a", "an", and "the" are understood to be singular or plural.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Methods of Predicting Responsiveness, Selecting Subjects, and Assessing Risks of Treatments In one aspect, the invention provides a method for determining prognosis of a subject suffering from prostate cancer by determining whether or not the tumor is androgen dependent or independent (e.g., responsive or unresponsive). The methods disclosed herein may be used with other genotyping or tumor marker methods if necessary. Preferably the at least one expression profile includes a plurality of expression profiles.

In another aspect, methods of predicting prognosis of a subject comprise identifying a subject having or suspected of having prostate cancer, determining a microRNA expression profile and/or an mRNA expression profile, and correlating the profile to the prognosis. The determining may comprise methods including, for example, array based methods, PCR based methods, immunological methods (antibodies, western blots, RIAs, etc), sequencing methods (direct and indirect sequencing of oligonucleotides or nucleic acids and peptides or proteins or Pyrosequencing), expression level of microRNAs, kinetics of microRNAs, PCR methods (real-time PCR, allele-specific PCR, reverse-transcriptase PCR, PCR), SNP Chip technology, and/or other assays described herein. The expression profile, refers to, for example, the expression analysis of one or more microRNAs. The assays may be informative if only one microRNA expression is determined. "Correlating," "correlation," "correlates," as used herein refer to the establishment of mutual or reciprocal relationship between expression profile and therapeutic efficacy of certain treatments as described herein or the status of the prostate tumor, e.g., hormone dependent or independent status. That is, correlating refers to relating the expression profile to risk, treatment, tumor type or other parameter described herein.

Correlation can be based on a change in the level of one or more markers. Correlation can also be based on a change in the level of at least a specific number of markers from a specific subset of those shown in Tables 1 through 4, or one or more of the pairwise comparisons from cells obtained directly from prostate tumors (e.g., biopsies, tissue sections) or prostate tumors grown in culture prior to pairwise comparisons. For example, a change to androgen insensitivity can be correlated with a change in expression levels of at least 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 of a predetermined set of miRs and/or mRNAs having 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or more members. Methods described herein may further comprise administering a therapeutic amount of an anti-neoplastic treatment to the subject.

As used herein, the terms "effective" and "effectiveness" includes both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of the treatment to result in a desired biological effect in the patient. Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and/or organism level (often referred to as side-effects) resulting from administration of the treatment. On the other hand, the term "ineffective" indicates that a treatment does not provide sufficient pharmacological effect to be therapeutically useful, even in the absence of deleterious effects, at least in the unstratifϊed population. (Such a treatment may be ineffective in a subgroup that can be identified by the expression profile or profiles.) "Less effective" means that the treatment results in a therapeutically significant lower level of pharmacological effectiveness and/or a therapeutically greater level of adverse physiological effects, e.g., greater liver toxicity.

Thus, in connection with the administration of a drug, a drug which is "effective against" a disease or condition indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease load, reduction in tumor mass or cell numbers, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or condition.

In another aspect, methods of predicting responsiveness of a tumor to therapeutic treatment comprise determining the androgen responsiveness microRNA expression profile, and correlating the expression profile to the treatment. The determining may comprise methods including, for example, array based methods, PCR based methods, immunological methods (antibodies, western blots, RIAs, etc), sequencing methods (direct and indirect sequencing of oligonucleotides or nucleic acids and peptides or proteins or Pyrosequencing), PCR methods (real-time PCR, allele-specific PCR, reverse-transcriptase PCR, PCR), nucleic acid chip technology, or other assays described herein. The androgen responsiveness microRNA expression profile, refers to, for example, the expression profile of one or more microRNAs differentially expressed in androgen dependent and androgen independent tumors and/or normal tissue.

In one embodiment, the correlation of patient responses to therapy is carried out in a clinical trial, e.g., as described herein according to any of the variations described. As indicated above, in aspects of this invention involving selection of a patient for a treatment, the selection may be positive selection or negative selection. Thus, the methods can include eliminating a treatment for a patient or elimination of a patient for a treatment.

The term "differential" or "differentially" generally refers to a statistically significant different level in the specified property or effect. Preferably, the difference is also functionally significant. Thus, "differential binding or hybridization" is a sufficient difference in binding or hybridization to allow discrimination using an appropriate detection technique. Likewise, "differential effect" or "differentially active" in connection with a therapeutic treatment or drug refers to a difference in the level of the effect or activity that is distinguishable using relevant parameters and techniques for measuring the effect or activity being considered. Preferably the difference in effect or activity is also sufficient to be clinically significant, such that a corresponding difference in the course of treatment or treatment outcome would be expected, at least on a statistical basis. Differentially expressed, in connection with a microRNA refers, for example, to a difference in presence or level of expression of a microRNA. Differentially expressed, in connection with an mRNA refers, for example, to a difference in presence or level of expression of an mRNA.

The polymerase chain reaction (PCR) is a widely known method for amplifying nucleic acids. Of the PCR techniques, RT-PCR (Reverse Transcription- PCR), competitive RT-PCR and the like are used for detecting and quantifying a trace amount of mRNA, and show their effectiveness.

In recent years, a real-time quantitative detection technique using PCR has been established (TaqMan PCR, Genome Res., 6 (10), 986 (1996), ABI PRISM.TM. Sequence Detection System, Applied Biosystems). This technique measures the amount of nucleic acids using a particular fluorescent-labeled probe (TaqMan probe). More specifically, this technique utilizes the following principles: For example, a fluorescent-labeled probe having a reporter dye at the 5' end and a quencher dye at the 3' end is annealed to the target DNA, and the DNA is subjected to normal PCR. As the extension reaction proceeds, the probe is hydrolyzed from the 5' end by the 5'-3' exonuclease activity possessed by DNA polymerase. As a result, the reporter dye at the 5' end is separated from the quencher dye at the 3' end, thereby eliminating the FRET (Fluorescence Resonance Energy Transfer, the reduction in fluorescence intensity owing to the decrease in the energy level of the reporter dye caused by the resonance of the two fluorescent dyes) effect produced by the spatial proximity between the two dyes, and increasing the fluorescence intensity of the reporter dye that has been controlled by the quencher dye. The target nucleic acid can be selectively quantified and detected in real-time by measuring the increase of the fluorescence intensity.

This technique is advantageous in that it can test various samples simultaneously in a short time, since, unlike the detection and quantification technique using conventional PCR it does not involve complicated steps, such as agarose gel electrophoresis of the amplified product after PCR and analysis of the electrophoresis pattern.

Determining the presence of a particular expression profile or plurality of expression profiles in a patient can be performed in a variety of ways, many of which are described infra. Other techniques will be known by those of skill in the art having the benefit of this disclosure. In preferred embodiments, the detection of the presence or absence of the at least one microRNA of an expression profile involves contacting a nucleic acid sequence corresponding to one of the microRNAs with a probe. The probe is able to distinguish the presence or a particular microRNA, e.g., by differential binding or hybridization. Thus, exemplary probes include nucleic acid hybridization probes, peptide nucleic acid probes, nucleotide-containing probes which also contain at least one nucleotide analog, and antibodies. Those skilled in the art are familiar with the preparation of probes with particular specificities. Those skilled in the art will recognize that a variety of variables can be adjusted to optimize the discrimination of the presence or absence of a micrRNA, including changes in salt concentration, temperature, pH and addition of various compounds that affect the differential affinity of GC vs. AT base pairs, such as tetramethyl ammonium chloride. (See Current Protocols in Molecular Biology by F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. D. Seidman, K. Struhl, and V. B. Chanda (editors, John Wiley & Sons).

In the context of this invention, the term "analyzing a sequence" refers to determining at least some sequence information about the sequence, e.g., determining the nucleotides present at a particular site or sites in the sequence, particularly sites that are known to vary in a population, or determining the base sequence of all of a portion of the particular sequence. In one embodiment, the probe is a nucleic acid probe 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, preferably at least 17 nucleotides in length, more preferably at least 20, 21, 22, 23, 24, or 25, preferably 500 or fewer nucleotides in length, more preferably 200 or 100 or fewer, still more preferably 70, 60, 50 or fewer, and most preferably 30 or fewer. In preferred embodiments, the probe has a length in a range from any one of the above lengths to any other of the above lengths (including endpoints). The probe specifically hybridizes under selective hybridization conditions to a nucleic acid sequence corresponding to all or a portion of one of the microRNAs identified in connection with above aspects. Also in preferred embodiments, the probe has a detectable label, preferably a fluorescent label. A variety of other detectable labels are known to those skilled in the art. Such a nucleic acid probe can also include one or more nucleic acid analogs.

In connection with nucleic acid probe hybridization, the term "specifically hybridizes" indicates that the probe hybridizes to a sufficiently greater degree to the target sequence than to a sequence having a mismatched base. The term "specifically hybridizes," thus refers to the probe hybridizing to the target sequence, and not to non-target sequences, at a level which allows ready identification of probe/target sequence hybridization under selective hybridization conditions. Specific hybridization does not need to occur over the full length of the probe or the target. The target may hybridize to only a portion of the probe, or vice versa. Thus,

"selective hybridization conditions" refer to conditions that allow such differential binding. Similarly, the terms "specifically binds" and "selective binding conditions" refer to such differential binding of any type of probe. Likewise, provided herein are isolated, purified or enriched nucleic acid sequences of 15 to 500 nucleotides in length, preferably 15 to 100 nucleotides in length, more preferably 15 to 50 nucleotides in length, and most preferably 15 to 30 nucleotides in length, which has a sequence which corresponds to a portion of one of the microRNAs identified for aspects above. Preferably the lower limit for the preceding ranges for the portion of the probe that specifically hybridizes to the microRNA is 17, 18, 19, 20, 21 22, 23, 24, or 25 nucleotides in length. In other embodiments, the nucleic acid sequence is 30 to 300 nucleotides in length, or 45 to 200 nucleotides in length, or 45 to 100 nucleotides in length. Likewise, the invention provides a set of primers or amplification oligonucleotides (e.g., 2, 3, 4, 6, 8, 10 or even more) adapted for binding to or extending through at least one gene identified herein.

In reference to nucleic acid sequences which "correspond" to a microRNA or mRNA, the term "correspond" refers to a nucleotide sequence relationship, such that the nucleotide sequence has a nucleotide sequence which is the same as the microRNA or mRNA, or an indicated portion thereof, or has a nucleotide sequence which is exactly complementary in normal Watson-Crick base pairing to at least a portion thereof, or is an RNA equivalent of such a sequence, e.g., an mRNA, or is a cDNA derived from an mRNA of the gene.

The term "effective amount" refers to a dosage or amount that is sufficient to reduce, halt, or slow tumor progression to result in alleviation, lessening or amelioration of symptoms in a patient or to achieve a desired biological outcome, e.g., slow or stop tumor growth or reduction or disappearance of a tumor. "Pharmaceutically acceptable excipients or vehicles" include, for example, water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

In the therapeutic methods of the invention, a treatment compound can be administered to a subject in any of several ways. For example, an anticancer agent can be administered as a prophylactic to prevent the onset of or reduce the severity of a targeted condition. Alternatively, an anticancer agent can be administered during the course of a targeted condition.

In some cases, the selection of a method of treatment or assessment, e.g., a therapeutic regimen, may incorporate selection of one or more from a plurality of medical therapies. Thus, the selection may be the selection of a method or methods which is/are more effective or less effective than certain other therapeutic regimens (with either having varying safety parameters). Likewise or in combination with the preceding selection, the selection may be the selection of a method or methods, which is safer than certain other methods of treatment in the patient.

The selection may involve either positive selection or negative selection or both, meaning that the selection can involve a choice that a particular method would be an appropriate method to use and/or a choice that a particular method would be an inappropriate method to use. Thus, in certain embodiments, the presence of an androgen independent profile is indicative that certain treatments will be effective or otherwise beneficial (or more likely to be beneficial) in the patient. Stating that the treatment will be effective means that the probability of beneficial therapeutic effect is greater than in a subject not having the appropriate presence or absence of particular variances. In other embodiments, the presence of a certain profile is indicative that the treatment will be ineffective or contra-indicated for the patient. For example, a treatment may be contra-indicated if the treatment results, or is more likely to result, in undesirable side effects, an excessive level of undesirable side effects, and/or no beneficial results. A determination of what constitutes excessive side-effects will vary, for example, depending on the disease or condition being treated, the availability of alternatives, the expected or experienced efficacy of the treatment, and the tolerance of the patient. As for an effective treatment, this means that it is more likely that desired effect will result from the treatment administration in a patient with a particular profile than in a patient who has a different profile. Also in preferred embodiments, the presence of certain profiles is indicative that the treatment is both effective and unlikely to result in undesirable effects or outcomes, or vice versa (is likely to have undesirable side effects but unlikely to produce desired therapeutic effects). In reference to response to a treatment, the term "tolerance" refers to the ability of a patient to accept a treatment, based, e.g., on deleterious effects and/or effects on lifestyle. Frequently, the term principally concerns the patients perceived magnitude of deleterious effects such as nausea, weakness, dizziness, and diarrhea, among others. Such experienced effects can, for example, be due to general or cell- specific toxicity, activity on non-target cells, cross-reactivity on non-target cellular constituents (non-mechanism based), and/or side effects of activity on the target cellular substituents (mechanism based), or the cause of toxicity may not be understood. In any of these circumstances one may identify an association between the undesirable effects and variances in specific genes. Also in other embodiments, the method of selecting a treatment includes eliminating a treatment, where the presence or absence of certain profiles is indicative that the treatment will be ineffective or contra-indicated, e.g., would not reduce tumor size, would not stop tumor growth, and/or metastasis. In other preferred embodiments, in cases in which undesirable side-effects may occur or are expected to occur from a particular therapeutic treatment, the selection of a method of treatment can include identifying both a first and second treatment, where the first treatment is effective to treat the disease or condition, and the second treatment reduces a deleterious effect of the first treatment. The phrase "eliminating a treatment" refers to removing a possible treatment from consideration, e.g., for use with a particular patient based on the presence or absence of certain profiles as described herein in one or more cells of that patient, or to stopping the administration of a treatment which was in the course of administration. Usually, the treatment will involve the administration of a compound preferentially active or safe in patients with certain profiles, where the profile is identified herein. The administration may involve a combination of compounds. Thus, in preferred embodiments, the method involves identifying such an active compound or combination of compounds, where the compound is less active or is less safe or both when administered to a patient having a different profile.

Also in some embodiments, the method of selecting a treatment involves selecting a method of administration of a compound, combination of compounds, or pharmaceutical composition, for example, selecting a suitable dosage level and/or frequency of administration, and/or mode of administration of a compound. The method of administration can be selected to provide better, preferably maximum therapeutic benefit. In this context, "maximum" refers to an approximate local maximum based on the parameters being considered, not an absolute maximum.

Also in this context, a "suitable dosage level" refers to a dosage level that provides a therapeutically reasonable balance between pharmacological effectiveness and deleterious effects. Often this dosage level is related to the peak or average serum levels resulting from administration of a drug at the particular dosage level.

Described herein, according to one aspect, are methods of predicting responsiveness of a tumor to therapeutic treatment comprising determining an androgen responsiveness microRNA expression profile of a tumor cell, and correlating the androgen responsiveness microRNA expression profile to the therapeutic treatment (androgen responsiveness).

Correlating, as used herein, comprises for example, relating the microRNA expression profile of a subject to one or more of an androgen-sensitive profile or an androgen-insensitive profile. These profiles are based on clinical data and associations of expression with a subject condition. These relations indicate whether a subject may respond to a certain therapy.

Correlating may comprise relating an androgen-sensitive profile with an indication that a subject may respond to anti-androgen therapies. Correlating may also comprises relating an androgen-insensitive profile with an indication that a subject may not respond to anti-androgen therapies.

In other therapeutic methods of the invention, provided are methods of treating a subject suffering from cancer, comprising determining an expression profile of a subject or a cell of a subject, and administering an anticancer agent to the subject. The expression profile may be determined as described herein.

A treatment compound can be administered to a subject, either alone or in combination with one or more therapeutic agents, as a pharmaceutical composition in mixture with conventional excipient, e.g., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral or intranasal application which do not deleteriously react with the active compounds and are not deleterious to the recipient thereof. Suitable pharmaceutically acceptable carriers include for example, water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously react with the active compounds.

The pharmaceutical agents may be conveniently administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical arts, e.g., as described in Remington 's Pharmaceutical Sciences (Mack Pub. Co., Easton, PA, 1980). Formulations for parenteral administration may contain as common excipients such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. In particular, biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release of certain agents.

The concentration of one or more treatment compounds in a therapeutic composition will vary depending upon a number of factors, including the dosage of the agent to be administered, the chemical characteristics (e.g., hydrophobicity) of the composition employed, and the intended mode and route of administration. In general terms, one or more than one of the agents or activators may be provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v of a compound for parenteral administration. It will be appreciated that the actual preferred amounts of active compounds used in a given therapy will vary according to e.g., the specific compound being utilized, the particular composition formulated, the mode of administration and characteristics of the subject, e.g., the species, sex, weight, general health and age of the subject. Optimal administration rates for a given protocol of administration can be readily ascertained by those skilled in the art using conventional dosage determination tests conducted with regard to the foregoing guidelines. Suitable dose ranges may include from about lμg/kg to about lOOmg/kg of body weight per day.

Described herein, according to one aspect, are methods of assessing the risk of cancer in a subject, comprising determining the androgen responsiveness microRNA expression profile, and correlating the androgen responsiveness microRNA expression profile to cancer risk.

According to one embodiment, comprises correlating an androgen-sensitive profile to treatment with anti-androgen therapies.

In another embodiment, correlating comprises correlating an androgen- insensitive profile to treatment with surgical, chemotherapeutic, and/or radiological therapies.

According to one embodiment, the cancer is prostate.

Described herein, according to one aspect, are methods for monitoring treatment for a subject with prostate cancer, comprising determining an androgen responsiveness microRNA expression profile of a subject and/or mRNA expression profile of a subject; determining a pre-treatment tumor status in the subject; administering a therapeutically effective amount of an anti-androgen therapy if the pre-treatment tumor status correlates with androgen-dependent prostate cancer; and determining a post-treatment tumor status in the subject.

According to another embodiment, a modulation of tumor status indicates that the tumor has become androgen-independent. In one embodiment, the pre-treatment and post-treatment tumor statuses are determined in a diseased tissue.

Described herein, according to one aspect, are methods for screening for a prostate cancer drug target, comprising providing a population of tumor cells; contacting the cells with a candidate microRNA composition listed in Table 1 , Table 2, one or more of a BD3/DH3 comparison, a E006A/BD3 comparison, a AH9/DH3 comparison, a AH9/ E006A comparison, a BD3/AH9 comparison, a DH3/E006A comparison, or a prostate tumor cell comparison, and determining effect of the candidate microRNA composition on cell proliferation, wherein a decrease in cell proliferation indicates that the candidate composition may be efficacious. The method can also be practiced using mRNAs identified in Table 3 or Table 4, or one or more of a BD3/DH3 comparison, a E006A/BD3 comparison, a AH9/DH3 comparison, a AH9/ E006A comparison, a BD3/AH9 comparison, a DH3/E006A comparison, or a prostate tumor cell comparison, either alone or in conjunction with microRNAs. In one embodiment, the methods may further comprise correlating the effect with the genotype.

In one embodiment, the methods may further comprise correlating the effect with one or more specific characteristic of the patient or the tumor including age of diagnosis, race, cellular differentiation, tumor grade, family history of prostate cancer or genetic predisposition, tumor size, and/ or degree of lymphovascular invasion.

In one embodiment, the methods may further comprise determining the androgen responsiveness microRNA expression profile of the tumor cells prior to or after providing a therapeutic agent to the cells. In one embodiment, the methods may further comprise determining the androgen responsiveness mRNA expression profile of the tumor cells prior to or after providing a therapeutic agent to the cells.

Described herein, according to one aspect, are methods of treating a subject suffering from cancer, comprising determining an androgen responsiveness microRNA expression profile of a subject or of a tumor cell of a subject and/or the mRNA expression profile of a subject, and administering a therapeutic amount of a prostate cancer treatment based on the profile.

According to one embodiment, an androgen-sensitive profile indicates treatment with an anti-androgen therapy. According to one embodiment, an androgen-insensitive profile indicates treatment with surgical, chemotherapeutic, and/or radiological therapies.

According to another embodiment, the subject is a mammal. In a related embodiment, the mammal is a human.

In one aspect, presented herein are methods for screening for a prostate cancer therapeutic compound. This may be done, for example, by providing a population of tumor cells and contacting the cells with a candidate compound targeted to a microRNA listed in Table 1 or Table 2, one or more of a BD3/DH3 comparison, a E006A/BD3 comparison, a AH9/DH3 comparison, a AH9/ E006A comparison, a BD3/AH9 comparison, a DH3/E006A comparison, or a prostate tumor cell comparison; and determining effect of the candidate microRNA composition on cell proliferation, wherein a decrease in cell proliferation and/or an increase in cell apoptosis indicates that the candidate composition may be efficacious. This may be done by a standard in vitro cell proliferation assay. Methods and kits for cell proliferation and apoptosis assays are well known to those in the art. Compounds targeted to microRNAs include, for example, antisense nucleic acids, siRNAs, and ribozymes when the miR is overexpressed in the prostate tumor cell and/or the androgen insensitive line. Compounds can be tartgeted to either miRs or miR precursors such as unprocessed pri-miRNAs. In an alternative embodiment wherein the miR expression is reduced in the prostate tumor cell or the androgen insensitive tumor cell, the miR or pri-miRNA can be used as a therapeutic compound. The screening method can also be performed by determining the expression profile of one or more of the mRNAs identified in Table 3 or Table 4, or in one or more of a BD3/DH3 comparison, a E006A/BD3 comparison, a AH9/DH3 comparison, a AH9/ E006A comparison, a BD3/AH9 comparison, a DH3/E006A comparison, or a prostate tumor cell comparison.

Methods for determining the therapeutic capacity of a candidate compound to reduce, halt, or otherwise modify tumor growth in a subject comprise determining pre-treatment levels or size of tumor masses in a subject; administering a therapeutically effective amount of a candidate agent to the subject; and determining a post-treatment levels or size of tumor masses in subject. In one embodiment, a decrease in the tumor size indicated that the candidate agent is efficacious. In a related embodiment, the stabilization of tumor size in a subject indicates that the agent is efficacious.

A method of assessing the therapeutic capacity or efficacy of the treatment in a subject includes determining the pre-treatment tumor status (e.g., by visual inspection of tissue, measurement of tumor regression or growth at various times before, during and after treatment, wherein the measurement is with, for example, a caliper) and then administering a therapeutically effective amount of an anti- microRNA compound to the subject. After an appropriate period of time (e.g., after an initial period of treatment) after the administration of the compound, e.g., 2 hours, 4 hours, 8 hours, 12 hours, or 72 hours, the level of tumor growth or cell proliferation is determined again. The modulation of the cell proliferation indicates efficacy of the treatment. The tumor status may be determined periodically throughout treatment. For example, the tumor status may be checked every few hours, days or weeks to assess the further efficacy of the treatment. A decrease in tumor growth, for example, indicates that the treatment with an agent is efficacious.

A control experiment is generally tailored for use in a particular assay. For example, most control experiments involve subjecting a test sample (e.g., a population of cells or lysate thereof) to medium, saline, buffer or water instead of a potential therapeutic compound in parallel to the cells receiving an amount of test compound.

Pharmaceutical Compositions The small molecule, peptide, nucleic acid, and antibody therapeutics described herein may be formulated into pharmaceutical compositions and be provided in kits. The pharmaceutical formulations may also be coated on medical devices or onto nano-particles for delivery.

The phrase "pharmaceutically acceptable carrier" is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals. The carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, .alpha.-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, intramuscular, intraperotineal, rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

Methods of preparing these formulations or compositions include the step of bringing into association an compositon of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; absorbents, such as kaolin and bentonite clay; lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluent commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert dilutents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.

Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention. Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. The compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually. Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated analgesic effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day, more preferably from about 0.01 to about 50 mg per kg per day, and still more preferably from about 1.0 to about 100 mg per kg per day. An effective amount is that amount that treats cancer or associated disease. If desired, the effective daily dose of the active compound may be administered as one dose or as, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. Additional therapeutic agents may include, but are not limited to, immunomodulatory agents, anti-inflammatory agents (e.g., adrenocorticoids, corticosteroids (e.g., beclomethasone, budesonide, flunisolide, fluticasone, triamcinolone, methlyprednisolone, prednisolone, prednisone, hydrocortisone), glucocorticoids, steroids, non-steriodal anti-inflammatory drugs (e.g., aspirin, ibuprofen, diclofenac, and COX-2 inhibitors), and leukotreine antagonists (e.g., montelukast, methyl xanthines, zafirlukast, and zileuton), beta2-agonists (e.g. , albuterol, biterol, fenoterol, isoetharie, metaproterenol, pirbuterol, salbutamol, terbutalin formoterol, salmeterol, and salbutamol terbutaline), anticholinergic agents (e.g., ipratropium bromide and oxitropium bromide), sulphasalazine, penicillamine, dapsone, antihistamines, anti-malarial agents (e.g., hydroxychloroquine), anti-viral agents, and antibiotics (e.g. , dactinomycin (formerly actinomycin), bleomycin, erythomycin, penicillin, mithramycin, anthramycin (AMC))

Kits In one aspect, kits for the assessment of androgen sensitive status are provided. The kits comprise microRNA arrays. Alternatively, or in addition, the kits comprise mRNA arrays. Optionally the kits contain instructions for use.

The arrays may be one or more of arrays comprising the microRNAs or complements thereof or fragments thereof of one or more of a BD3/DH3 comparison, a E006A/BD3 comparison, a AH9/DH3 comparison, a BD3/DH3 comparison, a AH9/ E006A comparison, a BD3/AH9 comparison or a DH3/E006A comparison, an androgen sensitive/ androgen insensitive prostate tumor cell comparison, or the microRNAs listed in Table 1 or Table 2. The arrays may be one or more of arrays comprising the mRNAs or complements thereof or fragments thereof of one or more of a BD3/DH3 comparison, a E006A/BD3 comparison, a AH9/DH3 comparison, a BD3/DH3 comparison, a AH9/ E006A comparison, a BD3/AH9 comparison or a DH3/E006A comparison, an androgen sensitive/ androgen insensitive prostate tumor cell comparison, or the mRNAs listed in Table 3 or Table 4. The kits may comprise and instructions for use.

In another aspect, kits for the assessment of cancer treatment options based on androgen sensitive status are provided and comprise an array and/or microarray, the arrays comprising the microRNAs or complements thereof or fragments thereof of one or more of a BD3/DH3 comparison, a E006A/BD3 comparison, a AH9/DH3 comparison, a AH9/ E006A comparison, a BD3/AH9 comparison or a DH3/E006A comparison, an androgen sensitive/ androgen insensitive prostate tumor cell comparison, or the microRNAs listed in Table 1 or Table 2 and instructions for use. Similarly, or in conjunction, the kits comprise an array or microarray comprising the mRNAs or complements thereof or fragments thereof of one or more of a BD3/DH3 comparison, a E006A/BD3 comparison, a AH9/DH3 comparison, a AH9/ E006A comparison, a BD3/AH9 comparison or a DH3/E006A comparison, an androgen sensitive/ androgen insensitive prostate tumor cell comparison, or the mRNAs listed in Table 3 or Table 4. Kits can include one or more probes for determining the microRNA expression profile and/or the mRNA expression profile of a cell, particularly a prostate tumor cell.

The kits described above may further contain enzymes, buffers, detectable labeling agents, and/or pharmaceutical compositions for treatment. The kits are preferably adapted and configured to be suitable for identification of the microRNA expression profiles and/or mRNA expression profiles described herein.

The kit may also optionally contain instructions for use, which can include a listing of the expressions correlating androgen sensitivity. The kits may contain arrays with one or more of the expression profiles contained thereon

Additional configurations for kits of this invention will be apparent to those skilled in the art.

All documents mentioned herein are incorporated by reference herein in their entirety.

EXAMPLES The present invention is further illustrated by the following non-limiting examples.

Example 1— Cell and Tissue Samples Prostate cell lines derived from an African-American patient {Koochekpour,

2004 #45} were profiled. Interestingly, the parental cell line (E006AA) and the cell lines derived from the parental cell line (DH3, BD3, AH9) have differing sensitivities to androgen. The parental cell line E006AA and the progeny cell line BD3 are dependent on androgen for growth, whereas the progeny cell lines DH3 and AH9 are not dependent on androgen for growth. The molecular differences correlated with differences in androgen sensitivities in prostate cancer cell lines were identified using both mRNA profiling using Affymetrix Genechip arrays and two novel miRNA profiling microarrays.

Six formalin fixed paraffin embedded (FFPE) prostate tumor samples, three of which were androgen naϊve or sensitive, three of which were androgen-refractory (i.e., insensitive), were obtatined from Dr. Jonathan Malamed throughthe NCI Cooperative Prostate Resource. No further information (e.g., race, age of diagnosis, cellular differentiation, tumor grade, family history of prostate cancer or genetic predisposition, tumor size, and/ or degree of lymphovascular invasion) was available regarding the tumors.

Example 2— Microarrays

Four different microarrays were evaluated for the analysis of cell and tissue samples. The summary of the properties of each of the microarrays is provided in the table below.

The LMT microarray having 1140 features from miRBase 7.1 was used for most of the comparisons described herein (Table 1). Later experiments were performed with a higher density array having 1668 features from miRBase 9.0 and 4 replicates of each probe per array (Table 2). Appropriate controls were also included in the microarrays. It is understood that as additional miRNAs are identified, they can be incorporated into the microarrays described herein. Higher density arrays allow for a larger number of replicates to be performed simultaneously, increasing confidence in results.

The LMT microarrays as noted in the table above have a coefficient of variation that is nearly 1%, combined with a good signal to noise ratio and a small difference between negative control and background as shown above.

Example 3— miRNA Microarray Design and Manufacturing miRNA sequences were downloaded from miRBase (http://microrna.sanger.ac.uk) (Release 7.1 for LMT_miRNA_vl microarray and

Release 9.0 for LMT_miRNA_v2 microarray). All unique mature miRNA sequences are selected. For LMT_miRNA_vl microarray, the reverse compliment sequences of mature miRNA were tailed at the 3 ' end with a linker (GGCCACATAC GGAACGCTATGTGATACGTATAGTAGGATA) to make the total length to 60 mer. The miRNA features of 60 mer oligos plus control features were printed on Agilent Custom

Microarray. For LMT_miRNA_v2 microarray, both mature miRNA sequences and the reverse compliment sequences were tailed at the 3 'end with the linker to make the total length to 60 mer. All features were represented with 4 replicates on the Agilent Custom Microarray in LMT_miRNA_v2 microarrays. Advantages of arrays such as theses include the use of very low amount of starting material (only about 0.5 micrograms), high sensitivity, high reproducibility, tissue-specific gene expression, the omission of any PCR-based amplification methods that may skew the original population of miRNAs.

Example 4- RNA Preparation

The total RNAs were extracted from prostate cell lines using acidic phenol (pH 4.5) and enriched for small RNAs (<200 nts) using the columns from the mirVana. miRNA Isolation kit (Ambion®) according to manufacturer's instructions. Total RNA was extracted from FFPEs using the RecoverAll Total Nucleic Acid Isolation Kit from Ambion® following manufacturer's instructions.

The total RNA from either source was either enriched for the short miRNAs for use with the Ambion® labeling kit or used directly in labeling reactions using the Exiqon labeling kit. Enrichment for small RNAs (< 10 -40 nts) was accomplished using the flashPAGE Fractionator (Ambion®) gel electrophoresis columns. Briefly 1- 5 μg of total RNA from each sample to be hybridized onto the microarray was resuspended in loading dye and loaded onto the top of the column containing the denaturing polyacrylamide gel matrix. The fraction that eluted before the blue dye marker (<40 nt in size) was recovered by ethanol precipitation overnight at -2O⁰C. In later experiments labeling was performed exclusively with the Exiqon kit, and no enrichment for short RNAs was performed. No differences in the microRNAs identified by eliminating the enrichment step.

Example 5~ miRNA Labeling

Two methods of labeling the miRNAs were employed. The first was using the miRVANA labeling kit (Ambion®). The purified miRNAs were labeled at the 3 '-end with amine-modified nucleotides using the poly (A) polymerase and the miRVana miRNA Labeling Kit (Ambion®). The amine-modified nucleotides were purified using glass-fiber filter spin columns in the kit and conjugated to NHS-ester modified Cy5 or Cy3 dyes (GE Healthcare). The dye-labeled miRNAs were sometimes further purified from unincorporated dyes using a second set of glass fiber filter spin columns. This further purification step had not effect on the microRNAs that were identified.

A preferred labeling method was performed using the Exiqon labeling kit directly on the total RNA without further purification for the shorter mature miRNAs. The 3 '-end of the total RNA was labeled with Hy3 dye (Exiqon) vis a vis ligation to an oligonucleotide at O⁰C for 1 hour, after which the labeled RNA was used for hybridization onto microarrays without the need for column purification.

Example 6— Microarray Hybridization and Data Analysis A 2x Hybridization Buffer and 10x blocking buffer (Agilent) were added to the fluorescently labeled miRNAs. Hybridizations were performed either with a single labeled RNA test sample; or a mixture of a test sample labeled with one dye, and a control sample (e.g., pooled total human RNA from Ambion) labeled with another dye.

The samples were then added to the microarray slides and hybridized for 16 h at 47ºC in sealed chambers inside the Agilent hybridization oven. After the 16 hour incubation the slides were washed with the Agilent wash buffer 1 and 2 and then dried with the Agilent stabilization and drying solution. Then slides were then immediately scanned using the Agilent scanner.

Example 7— Data analysis for single label microarray

A value for each feature was normalized from raw intensity data by subtracting background as determined by negative control features from the value of each feature. Normalized values were compared to positive control features and a threshold cutoff was established for positive staining of a feature. Pairwise comparisons were made between cell lines and tumor samples to determine differential expression of a microRNA or an mRNA by subtracting the normalized value of one test sample from another test sample.

Example 8— Data analysis for double label microarray

Equal amounts (0.5 μg each) of labeled test (i.e., prostate tumor cell or cell line RNA) and control RNA (i.e., pooled total human RNA) were mixed and applied to the microarray as described above. A value for each feature was normalized by subtracting background signal from the test sample signal; and subtracting the background signal of the reference or control RNA for each control RNA. Next, the background subtracted signal from the test sample signal was compared to the background subtracted signal from the reference or control RNA, and a ratio was obtained. The ratio was used to compare the miRNA expression between androgen sensitive and androgen insensitive prostate cells.

Example 9— miRNA Array Analysis of Pairwise Cell Line Comparisons After global normalization of raw intensity data using the median intensity it was observed that most of the miRNAs are expressed at the same level among the four cells lines E006A (androgen sensitive), BD3 (androgen sensitive), AH9 (androgen insensitive), and DH3 (androgen insensitive). However, it was observed that the following differential expression of miRNAs between the androgen-sensitive and androgen-insensitive cell lines (Table 1). miR-27a was found to be expressed more highly in the androgen sensitive cell lines as compared to the androgen-insensitive cell lines. miR-27 a has been reported to be increased during 12-O-tetradecanoylphorbol- 13 -acetate (TPA-induced differentiation, suggesting their importance in relation to monocytic differentiation of human leukemia cells (HL-60) {Kasashima, 2004 #48}. miR-15b was more highly expressed in the androgen sensitive cell lines. In contrast, it is interesting that mir-15a (which is an isoform of the miR-15b) is more highly expressed in the androgen insensitive cell lines. miR-15 and miR-16 have been previously been reported to be involved in human cancer. There is frequent deletion or down-regulation and miR-15 and miR-16 genes in chronic lymphocytic leukemia (CLL) (9). In addition, miR-15a has been demonstrated to regulate genes which are involved in cancer pathways such as RAS (oncogene) and BcI 2 (anti-apoptotic gene) respectively (11). miR -146a and miR-146b were more highly expressed in the androgen- insensitive cell lines compared to the androgen sensitive cell lines. Finally, miR-155 is more highly expressed in the androgen insensitive cell lines.

Differentially regulated miRNAs in prostate cell lines

The expression of the first listed miRNA is higher unless otherwise noted.

E006A (androgen sensensitive) vs BD3 (androgen sensensitive)

R- 103 miR-98 let-7b miR-29b miR-31 Iet7e miR-27a let-7c miR- 19b let-7a let-7d miR-200c let-7f miR-21 miR- 181b miR-374 miR-29 miR-181d miR-26a miR-23a

AH9 (androgen insensensitive) vs DH3 (androgen insensensitive) miR- 103 miR- 107 miR- 196a miR- 19a miR- 106a miR-155 miR-29b let-7f miR-30d

Overall, androgen sensitive cell lines were found to have lower expression levels of miR-30, miR 155, miR-15a, miR-146a and b, miR-125, and miR-101 as compared to androgen insensitive cell lines; and androgen insensitive cell lines were found to have lower expression of miR-27a and b, miR-185, miR-320, miR-429, and miR-494. Example 10— Quantitative PCR confirmation of Cell Line Microarray Results

A few of the miRNAs identified in the microarray analysis of the cell lines were selected for confirmation via Q-PCR using the TaqMan® MicroRNA Assays (ABI). RNU6b, a small nuclear RNA, was used as the endogenous control. TaqMan® MicroRNA Reverse Transcription Kit (ABI) was used with specific miRNA primers provided with the TaqMan® MicroRNA Assay to convert miRNA to cDNA. 5ng of total RNA was used in a 15uL reaction according to the manufacture's instructions.

PCR products are then synthesized from the cDNA samples using miRNA specific primers and probe from the TaqMan MicroRNA Assays (ABI). The probes have a FAM reporter dye linked to the 5 'end, a minor groove binder (MGB) at the 3' end and a nonfluorescent quencher (NFQ) at the 3' end of the probe. The Q-PCR reactions were run in duplicate reaction according to manufacture's instructions in a 2OuL final volume in 384- well microtiter plates. The 384- well microtiter reaction plates were loaded onto the ABI Prism® 7900HT Sequence Detection System(ABI). The thermocycling conditions were as follows: 95°C for 10 minutes and 40 cycles of 95°C for 15 seconds and 6O⁰C for 1 minute.

A ΔΔCT analysis was then preformed to compare the androgen sensitive samples with their androgen insensitive counterparts using RNU6B as the endogenous control.

Differential expression of the following miRNAs: let-7g, miR-494, miR-146a, miR-146b, miR-15a, miR-27a and miR-27b was confirmed Q-PCR. MicroRNAs let- 7g and miR-15a were expressed more highly in the androgen sensitive cell lines than in the androgen insensitive cell lines. miR-494 was more highly expressed in the androgen insensitive lines.

Interestingly, the putative targets (from Target Scan) for miR-494 include Ras- GTPase activating protein G3BP2 (Ras-GTPase activating protein SH3 domain- binding protein 2) and testis expressed sequences 264. Ras-GTPase can enhance androgen receptor responses to low levels of circulating androgens 54.

Thus, the decreased expression of miR-494 in the androgen insensitive prostate tumor cell lines could de-repress the Ras-GTPase expression which would in turn enhance androgen receptor responses to low levels of circulating androgens, thereby turning the prostate cells insensitive to anti-androgen therapy 55.

Example 11— miRNA Array Analysis of Tumor Tissue Comparisons

Six formalin fixed paraffin embedded (FFPE) prostate tumor samples, three which were androgen naϊve or sensitive and three of which were androgen-refractory, were obtained from Dr. Jonathan Melamed through the NCI Cooperative Prostate Resource. Total RNA was isolated from the prostate tumor samples, labeled, and subjected to microarray analysis as described above. Upon Class Comparison analysis of the miRNA array data using BRB Array Tools software, the following differentially expressed miRNAs were identified: miR-195, let-7g, miR-16, miR-491, miR-99a. Significantly, let-7g had also been identified by miRNA array analysis and RT-PCR of the prostate cell line samples.

Example 12— miRNA Array Analysis using a High Density Microarray

The LMT_miRNA_v2 high density microarray contains a larger number of features; 1668 predicted and confirmed microRNAs (miRNAs) from the updated 9.0 version of the Sanger miRBase containing additional newly discovererd miRNAs (http://microrna.sanger.ac.uk). This version of the array includes four replicates of each probe, resulting in greater statistical significance from a single experiment. For LMT_miRNA_v2 microarray, mature miRNA sequences and their complements were tailed at the 3 'end with the linker to make the total length to 60 mer as was done for the LMT miRNA vl microarray. The larger number of microRNAs and the fourfold increase in the number of replicates allows for greater statistical confidence in the data derived from the high density microarray.

The array was performed using two-color staining of the test (cell line) and control samples (total pooled human RNA) as described above. A class comparison analysis using BRB array tools E006AA (androgen sensitive) vs DH3 (androgen insensitive) comparison confirmed a change in the expression profile of mIR-146a and indentified miR-200b and miR 449b as having a change in expression profile in the shift from androgen sensitive to androgen insensitive. The differential expression of these three miRs between the cell lines was confirmed by Q-PCR. Example 13- Affymetrix Genechip mRNA Analysis:

The interactions between the androgen-sensitive parental cell line E006AA were compared pairwise with the derived cell lines AH9 (androgen insensitive), DH3 (androgen insensitive), and BD3 (androgen sensitive). When it was compared the 3 derived cell lines with the (androgen sensitive) parental cell line E006AA, we found genes that were 30 genes that were down-regulated in both of the androgen insensitive cell lines AH9 and DH3, but not changed in the androgen sensitive BD3 cell lines. Notably, it was found a gene that is deleted in bladder cancer, a MAPkinase 6 gene, and MUC 18 glycoportein that is a melanoma adhesion molecule had decreased expression in the androgen insensitive lines. See Table 3.

When (androgen sensitive) E006AA was compared with the 3 derived cell lines for the up-regulated genes in the (androgen insensitive) AH9 and DH3, but unchanged in the (androgen sensitive) BD3, found were 41 genes. They are listed in Table 4 below. Notably, human connective tissue growth factor, human senescence- associated epithelial membrane protein (SEMPl) mRNA, and human tumorous imaginal discs (Drosophila) homolog (TIDl) all had increased expression in the androgen insensitive cell lines.

Example 14— miRNA Array Analysis of Tumor Tissue Comparisons using Matched Samples

Samples are obtained for a number of prostate tumor samples associated with information about the tumor sample and the man from whom it was obtained. For example, information such as age of diagnosis, race, cellular differentiation, tumor grade, family history of prostate cancer or genetic predisposition, tumor size, degree of lympho vascular invasion of the tumor, etc. Serial samples from an individual can also be obtained, for example, a biopsy from initial diagnosis of an androgen sensitive rumor and a portion of the tumor after removal after transition to androgen insensitive status. Tumor samples can be in any form as long as they were obtained and preserved under conditions to maintain the stability of the miRNAs. For example, samples may be frozen sections, fixed or unfixed, or paraffin embedded samples. Samples are paired based on information available regarding the tumor. For example, tumor samples can be grouped by size (e.g., less than 2 cm largest dimension, 2-4 cm largest dimension, 4-6 cm largest dimension, larger than 6 cm largest dimension) and androgen status (androgen sensitive or insensitive). Tumors can be grouped by family history, or age of onset, again being further stratified for androgen status. Tumor samples from a single individual can be grouped. RNA is isolated, labeled, and miRNA expression is determined using the methods and microarrays disclosed herein. Such analyses can be used to futher characterize miRNAs and their involvement in androgen status change. For example, miRs that are consistently overexpressed in androgen insensitive tumors as compared to androgen sensitive tumors are likely related to androgen sensitivity. miRs with expression levels that correlate with size regardless of androgen status, the larger the tumor, the higher the expression of the specific miR, are likely related to tumor size rather than androgen sensitivity.

Similar assays can be performed to analyze differential mRNA expression in such matched samples.

Example 15— Monitoring of Androgen Sensitive Status in a Man with Prostate Cancer Using microRNA Expression Profiling

A man is diagnosed with prostate cancer by usual methods. A biopsy is performed, and the sample is formalin fixed, sectioned, and stained using conventional methods to confirm that the tumor is cancerous. Androgen suppression therapy is initiated. Tumor size is monitored using standard imaging methods, and reduction in tumor size is observed. Moreover, the therapy is well tolerated.

Transition of prostate tumors from androgen dependent status to androgen independent status is frequently observed at about 18-24 months after the initiation of androgen supression therapy. This transition results in the need to change therapeutic regimens to a chemotherapeutic regimen rather than a hormone based regimen. Alternatively, or in conjunction with chemotherapy, surgery may be considered. As both surgery and chemotherapy are most effective when the tumor is small, it is best to initiate such an intervention as early as possible, when required. However, as chemotherapy and surgery should not be undertaken unless required, they are often delayed until tumor growth is observed after androgen suppression therapy.

Periodically after the initiation of androgen ablation therapy, a tumor biopsy can be obtained and compared to the earlier obtained sample for a change in expression of one or more miRNA known to be correlated with a decrease in androgen sensitivity. For example, a change in the level of expression of miRNAs can be determined using a microarray such as those disclosed herein. A microarray can include all known miRNAs, or preferably a limited set of miRNAs known to be associated with the transition from androgen sensistivy to androgen insensitivity such as those taught herein. A change in level of expression of one or more miRNAs can be determined, for example, by Q-PCR. RNA can be isolated from both the initial and the later tumor biopsies and compared to determine changes in miRNA expression over time. The expression of all known miRNAs can be determined, or the expression of a subset of miRNAs such as those taught herein can be performed. If no changes in miRNA expression levels are observed, androgen ablation therapy is continued as determined appropriate by the oncologist. Another biopsy can be obtained at an appropriate interval. If changes indicating a transition to androgen insensitivity are observed, alternative therapeutic interventions can be considered and tumor imaging to monitor tumor size can be performed at shorter intervals for indications of tumor growth. A specific therapy can be selected based on the modulation of expression of specific miRs.

Example 16— Monitoring Androgen Sensitive Status in a Man with Prostate Cancer Using Gene Expression

A man is diagnosed with prostate cancer by usual methods. A biopsy is performed, and the sample is formalin fixed, sectioned, and stained using conventional methods to confirm that the tumor is cancerous. Androgen suppression therapy is initiated. Tumor size is monitored using standard imaging methods, and reduction in tumor size is observed. Moreover, the therapy is well tolerated.

Periodically after the initiation of androgen ablation therapy, a tumor biopsy can be obtained and compared to the earlier obtained sample for a change in expression of one or more mRNAs known to be correlated with a decrease in androgen sensitivity, such as those listed in Tables 3 and 4. For example, a change in the level of expression of mRNAs can be determined using a microarray such as those disclosed herein. A microarray can include all known mRNAs, or preferably a limited set of mRNAs known to be associated with the transition from androgen sensistivy to androgen insensitivity such as those taught herein. A change in level of expression of one or more mRNAs can be determined, for example, by Q-PCR. RNA can be isolated from both the initial and the later tumor biopsies and compared to determine changes in mRNAs expression over time. The expression of all known mRNAs can be determined, or the expression of a subset of mRNAs such as those taught herein can be performed. If no changes in mRNAs expression levels are observed, androgen ablation therapy is continued as determined appropriate by the oncologist. Another biopsy can be obtained at an appropriate interval. If changes indicating a transition to androgen insensitivity are observed, alternative therapeutic interventions can be considered and tumor imaging to monitor tumor size can be performed at shorter intervals for indications of tumor growth. A specific therapy can be selected based on the modulation of expression of specific mRNAs.

Example 17— Monitoring Androgen Sensitive Status in a Man with Prostate Cancer Using a Combination of microRNA Expression ProΩling and Gene Expression

A man is diagnosed with prostate cancer by usual methods. A biopsy is performed, and the sample is formalin fixed, sectioned, and stained using conventional methods to confirm that the tumor is cancerous. Androgen suppression therapy is initiated. Tumor size is monitored using standard imaging methods, and reduction in tumor size is observed. Moreover, the therapy is well tolerated.

Periodically after the initiation of androgen ablation therapy, a tumor biopsy can be obtained and compared to the earlier obtained sample for a change in expression of one or more miRNAs and one or more mRNAs known to be correlated with a decrease in androgen sensitivity. For example, a change in the level of expression of miRNAs and mRNAs can be determined using a microarray such as those disclosed herein. A microarray can include all known miRNAs and mRNAs, or preferably a limited set of miRNAs and mRNAs known to be associated with the transition from androgen sensistivy to androgen insensitivity such as those taught herein. A change in level of expression of one or more miRNAs or mRNAs can be determined, for example, by Q-PCR. RNA can be isolated from both the initial and the later tumor biopsies and compared to determine changes in miRNAs and mRNAs expression over time. The expression of all known miRNAs and mRNAs can be determined, or the expression of a subset of miRNAs and mRNAs such as those taught herein can be performed. If no changes in miRNA and mRNA expression levels are observed, androgen ablation therapy is continued as determined appropriate by the oncologist. Another biopsy can be obtained at an appropriate interval. If changes indicating a transition to androgen insensitivity are observed, alternative therapeutic interventions can be considered and tumor imaging to monitor tumor size can be performed at shorter intervals for indications of tumor growth. A specific therapy can be selected based on the modulation of expression of specific miRNAs and mRNAs.

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A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

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Table 2

Claims

What is claimed is:

1. A method of predicting responsiveness of a tumor to therapeutic treatment comprising: determining an androgen responsiveness microRNA expression profile of a tumor cell, and correlating the androgen responsiveness microRNA expression profile to the therapeutic treatment (androgen responsiveness).

2. The method of claim 1, wherein the androgen responsiveness microRNA expression profile comprises determination of the expression profile of one or more of the sequences listed in Table 1 or Table 2.

3. The method of claim 1 or 2, wherein the androgen responsiveness microRNA expression profile comprises determination of the expression profile of one or more of sequences identified in a comparison between an androgen sensitive prostate tumor cell and an androgen insensitive prostate tumor cell.

4. The method of claim 3, wherein the androgen sensitive and androgen insensitive prostate tumor cells are obtained directly from a tumor.

5. The method of claim 3, wherein the androgen sensitive and androgen insensitive prostate tumor cells are grown in culture prior to the comparison.

6. The method of any of the preceding claims, wherein the expression profile comprises determination of the expression profile of one or more microRNAs of at least one of a BD3/DH3 comparison, an AH9/E006A comparison, a BD3/AH9 comparison, or a DH3/E006A comparison.

7. The method of any of the preceding claims, wherein the expression profile comprises determination of the expression profile of one or more microRNAs from the group consisting of let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i, miR-15a, miR-16, miR-17-5p, miR-19a, miR-19b, miR-20a, miR-21 , miR-22, miR- 23a, miR-26a, miR-27a, miR-27b, miR-29a, miR-29b, miR-30, miR-30a-5p, miR- 3Od, miR-93, miR-98, miR-99a, miR-101, miR-102, miR-103, miR-106a, miR-106b, miR-107, miR-125b, miR-146a, miR-146b, mi-155, miR-181a, miR-181b, miR-181c, miR-181d, miR-185, miR-191, miR-195, miR-196a, miR-196b, miR-200b, miR-320, miR-374, miR-429, miR449b, miR-491, and miR-494.

8. The method of any of the preceding claims, wherein the expression profile comprises determination of one or more microarrays from the group consisting of miR-146a, miR-200b, and miR-449b.

9. The method of claim 1 or 2, wherein the expression profile comprises determination of the expression profile of one or more microRNAs of at least one of a E006A/BD3 comparison, or an AH9/DH3 comparison.

10. The method of any of the preceeding claims, further comprising determining an mRNA expression profile of the tumor.

1 1. The method of claim 10, wherein the mRNA expression profile comprises determination of the expression profile of one or more of the mRNAs listed in Table 3 or Table 4.

12. The method of claim 10, wherein wherein the androgen responsiveness microRNA expression profile comprises determination of the expression profile of one or more of sequences identified in a comparison between an androgen sensitive prostate tumor cell and an androgen insensitive prostate tumor cell.

13. The method of any of the preceeding claims, wherein the androgen responsiveness microRNA expression profile is determined by array methods, PCR methods, immunological methods, sequencing methods, expression profile methods, or kinetic profiles.

14. The method of claim 13, wherein PCR methods are one or more of real-time PCR, PCR, reverse transcriptase PCR, rtPCR, allele-specific PCR.

15. The method of any of the preceeding claims, wherein correlating comprises correlating an androgen-sensitive profile with an indication that a subject may respond to anti-androgen therapies.

16. The method of any of the preceeding claims, wherein correlating comprises correlating an androgen-insensitive profile with an indication that a subject may not respond to anti-androgen therapies.

17. The method of any of the preceeding claims, further comprising administering a therapeutic amount of an anti-androgen therapy to the subject correlating to an androgen-sensitive profile.

18. The method of claim 17, wherein the anti-androgen therapy comprises one or more of luteinizing hormone-releasing hormone agonists (e.g., leuprolide, goserelin, and buserelin).

19. The method of any of the preceeding claims, further comprising administering a therapeutic amount of at least one anti-neoplastic agent to the subject.

20. The method of claim 19, wherein the anti-neoplastic agent comprises docetaxel, cisplatin, cyclophosphamind, doxorubicin, prednisone, 5-FU, trastuzumab, 3G4, travacin, gemcitabine, estramustine, carboplatin, or radioimmunotherapy agents.

21. A method of selecting a subject for anti-androgen therapyfor prostate cancer, comprising: determining an androgen responsiveness microRNA expression profile of a subject or a cell of a subject, and correlating the expression profile results to treatment options.

22. The method of claim 21, futher comprising determining an androgen responsiveness mRNA expression profile of a subject or a cell of a subject.

23. The method of claim 21 or 22, wherein correlating comprises correlating an androgen-sensitive profile to treatment with anti-androgen therapies.

24. The method of claim 21 or 22, wherein correlating comprises correlating an androgen-insensitive profile to treatment with surgical, chemotherapeutic, or radiological therapies.

25. The method of claim 21 or 22, further comprising administering a therapeutic amount of an anti-androgen therapy to a subject having an androgen- sensitive profile.

26. A method for monitoring treatment for a subject with prostate cancer, comprising: determining an androgen responsiveness microRNA expression profile of a prostate tumor from a subject; determining a pre-treatment androgen status of the tumor in the subject; administering a therapeutically effective amount of an anti-androgen therapy if the pre-treatment tumor status correlates with androgen-dependent prostate cancer; and determining a post-treatment androgen status of the tumor in the subject.

27. The method of claim 26, further comprising determining an androgen responsiveness mRNA expression profile of a prostate tumor from a subject.

28. The method of claim 26 or 27, wherein a modulation of tumor status indicates that the tumor has become androgen-independent.

29. A method of treating a subject suffering from cancer, comprising: determining an androgen responsiveness microRNA expression profile of a subject or of a cell of a subject, and administering a therapeutic amount of a prostate cancer treatment based on the profile.

30. The method of claim 29, further comprising determining an androgen responsiveness mRNA expression profile of a subject or of a cell of a subject.

31. The method of claim 29 or 30, wherein an androgen-sensitive profile indicates treatment with an anti-androgen therapy.

32. The method of claim 29 or 30, wherein an androgen-insensitive profile indicates treatment with surgical, chemotherapeutic, or radiological, therapies.

33. The method of any of claims 21 to 32, wherein the subject is a mammal.

34. The method of claim 33, wherein the mammal is a human.

35. A kit for the assessment of cancer treatment options, comprising: a microRNA array that differentiates androgen dependent and androgen independent tumors and instructions for use.

36. A kit for the assessment of cancer treatment options, comprising: a microarray comprising microRNA probes that differentiates androgen dependent and androgen independent tumors, and instructions for use.

37. A kit for the assessment of cancer treatment options, comprising: an mRNA array that differentiates androgen dependent and androgen independent tumors and instructions for use.

38. A kit for the assessment of cancer treatment options, comprising: a microarray comprising mRNA probes that differentiates androgen dependent and androgen independent tumors, and instructions for use.

39. An array comprising the microRNAs or complements thereof or fragments thereof of one or more of the expression profile of one or more of sequences identified in a comparison between an androgen sensitive prostate tumor cell and an androgen insensitive prostate tumor cell, or the microRNAs listed in Table

1 or Table 2.

40. An array comprising the microRNAs or complements thereof or fragments thereof of one or more of the expression profile of one or more of sequences identified in a comparison between an androgen sensitive prostate tumor cell and an androgen insensitive prostate tumor cell, or the microRNAs listed in Table 3 or Table 4.