WO2016019311A1 - Methods for the detection and treatment of prostate cancer - Google Patents

Abstract

Methods for detecting and treating prostate cancers are provided. In some aspects, methods for detecting a prostate cancer comprise measuring a level of PRUNE2 expression and/or PCA3 expression in a sample from a subject. In further aspects, a method for treating prostate cancer is provided comprising administering a PRUNE2 tumor suppressor polypeptide or a nucleic acid molecule that inhibits expression of PC A3 RNA.

Description

DESCRIPTION

METHODS FOR THE DETECTION AND TREATMENT OF PROSTATE CANCER

[0001] This application claims the benefit of United States Provisional Patent Application No. 62/032,103, filed August 1, 2014, the entirety of which is incorporated herein by reference.

[0002] The invention was made with government support under Grant Nos. CA90270 and P01-CA95616 awarded by the National Institutes of Health. The government has certain rights in the invention.

INCORPORATION OF SEQUENCE LISTING [0003] The sequence listing that is contained in the file named

"STCUP0002_ST25.txt", which is 59 KB (as measured in Microsoft Windows®) and was created on July 31, 2015 is filed herewith by electronic submission and is incorporated by reference herein.

BACKGROUND OF THE INVENTION 1. Field of the Invention

[0004] The present invention relates generally to the field of oncology and molecular biology. More particularly, it concerns methods for detecting and treating prostate cancers.

2. Description of Related Art

[0005] Prostate cancer is the most common malignancy in males. Although most prostate cancer patients have indolent disease progression, some have aggressive tumors that ultimately metastasize to bone and soft tissues resulting in substantial morbidity and mortality (1). Treatment options are limited for late-stage disease, thus there is an urgent need for the development of new therapeutic approaches combined with alternatives for early detection.

[0006] Serum level of prostate-specific antigen (PSA) is the most commonly used diagnostic biomarker for prostate cancer, but it has low specificity resulting in many false positives. The most specific tumor biomarker identified to date (2) is the long noncoding RNA (IncRNA) prostate cancer antigen 3 (PCA3), which is up-regulated in prostate cancer. PCA3 has been extensively investigated over the past decade (3, 4) and has been approved for clinical applications to aid the diagnosis of prostate cancer in both the European Union (5) and the United States (6). However, the biological function of PC A3 has proved elusive.

SUMMARY OF THE INVENTION

[0007] In a first embodiment, the invention provides a method of treating a subject having prostate cancer comprising administering to the subject a pharmaceutical composition comprising an effective amount of a PRU E2 tumor suppressor. In some aspects, the PRUNE2 tumor suppressor may comprise an expression vector encoding a PRUNE2 coding sequence or a PRUNE2 -coding mRNA. In some aspects, the expression vector comprises a plasmid or viral expression vector. In other aspects, the expression vector encoding a PRUNE2 coding sequence or a PRUNE2-coding mRNA is provided in a nanoparticle or liposome.

[0008] In some aspects, the PRUNE2 tumor suppressor comprises a PRUNE2 polypeptide, optionally, conjugated to or fused with a cell-targeting or a cell internalization moiety. The cell internalization moiety may be at the N-terminus or at the C-terminus of the PRUNE2 tumor suppressor polypeptide. In certain aspects, the cell internalization moiety may be a polypeptide, an aptamer, an antibody or an avimer. The antibody may be, for example, an IgA, an IgM, an IgE, an IgG, a Fab, a F(ab')2, a single chain antibody, or a paratope peptide. In one aspect, the cell internalization moiety comprises internalization sequences selected from the group consisting of an HIV TAT protein transduction domain, HSV VP22 protein transduction domain, or Drosophila Antennapedia homeodomain. In another aspect, the cell internalization moiety comprises a poly-arginine, a poly-methionine and/or a poly-glycine polypeptide.

[0009] Thus, certain aspects of the embodiments concern a PRUNE2 polypeptide, such as the polypeptide provided as NCBI accession no. ACY78253 incorporated herein by reference (SEQ ID NO: 1), or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0010] In a further embodiment, the invention provides a method of treating a subject having prostate cancer comprising administering to the subject a pharmaceutical composition comprising an effective amount of an inhibitor of prostate cancer associated 3 (PCA3), adenosine deaminase, RNA-specific (ADAR); non-POU domain containing, octamer-binding (NONO) and/or drosha, ribonuclease type III (DROSHA). In some aspects, the inhibitor of PCA3, ADAR, NONO or DROSHA is an inhibitory nucleic acid molecule. For example, the inhibitory nucleic acid molecule may comprise a sequence complimentary to all or part of a PCA3, ADAR, NONO or DROSHA RNA. In certain aspects, the inhibitory nucleic acid molecule is a RNA, such as a miRNA, siRNA or shRNA. In further aspects, the inhibitory nucleic acid molecule may comprise an expression vector encoding an inhibitory RNA molecule. The expression vector may comprise a plasmid or viral expression vector. In some aspects, the inhibitory nucleic acid molecule is provided in a nanoparticle or a liposome.

[0011] Accordingly, aspects of the embodiments concern inhibitory nucleic acid molecules complementary to all or part of a human PCA 3 RNA. For example, an inhibitory nucleic acid molecule can be complimentary to all or part of a PC A3 RNA provided as NCBI accession no. NR_015342, incorporated herein by reference (SEQ ID NO: 2). In still further aspects, an inhibitory nucleic acid molecule can be complimentary to all or part of a PCA3 RNA provided as NCBI accession nos. DQ374659 (splice variant 1); DQ374660 (splice variant 2); DQ374661 (splice variant 3); or DQ374662 (splice variant 3), each of which is incorporated herein by reference .

[0012] Further aspects of the embodiments concern inhibitory nucleic acid molecules that are complementary to all or part of a human drosha, ribonuclease type III (DROSHA) RNA. For example, an inhibitory nucleic acid molecule can be complimentary to all or part of a DROSHA RNA provided as NCBI accession nos. NM 013235.4 (isoform 1; SEQ ID NO: 5) or NM 001100412.1 (isoform 2), each of which is incorporated herein by reference. In yet further aspects, embodiments of the invention concern inhibitory nucleic acid molecules that are complementary to all or part of a human adenosine deaminase, RNA- specific (ADAR) RNA. For example, an inhibitory nucleic acid molecule can be complimentary to all or part of a ADAR RNA provided as NCBI accession nos. NM_001025107.2 (isoform d); NM_001193495.1 (isoform d); NM_0011 1 1.4 (isoform a); NM_015840.3 (isoform b; SEQ ID NO: 4); or NM_015841.3 (isoform c), each of which is incorporated herein by reference. In certain aspects, an inhibitory nucleic acid molecule can be complimentary to all or part of a NONO RNA provided as NCBI accession no. NM 007363 (SEQ ID NO: 3), incorporated herein by reference.

[0013] A pharmaceutical composition of the embodiments may be administered to the subject systemically or locally. In some aspects, the pharmaceutical composition may be administered two, three, four or more times. In further aspects, a second anti-cancer therapy is administered to the subject. The second anti-cancer therapy may be, for example, chemotherapy, radiotherapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy or cytokine therapy. Such a second anticancer therapy may be administered before, after or essentially simultaneously with a pharmaceutical composition of the embodiments. [0014] In yet a further embodiment, the invention provides a composition for use in treating a subject having prostate cancer the composition comprising an effective amount of a PRUNE2 tumor suppressor or an inhibitory nucleic acid molecule that comprises sequence complimentary to all or part of a PCA3, ADAR, NONO or DROSHA RNA. For example, the an inhibitory nucleic acid molecule may comprise a sequence complimentary to 18, 19, 20, 21 , 22, 23, 24 or 25 consecutive nucleotides of a PCA3, ADAR, NONO or DROSHA mRNA. In certain aspects, an inhibitory nucleic acid of the embodiments is an RNA molecule, such as a double stranded RNA (dsRNA) molecule. In still further aspects, an inhibitory nucleic acid is a dsRNA having a 1 or 2 nucleotide 3' overhang on one or both strands. For example, the dsRNA can comprise a 2 nucleotide 3 ' overhang (on one or both strands) selected from the group consisting of AA, AC, AG, AU, CA, CC, CG, CU, dAdA, dAdC, dAdG, dAdT, dCdA, dCdC, dCdG, dCdT, dGdA, dGdC, dGdG, dGdT, dTdA, dTdC, dTdG, dTdT, GA, GC, GG, GU, UA, UC, UG and UU.

[0015] Thus, in certain aspects, a composition for use in treating a subject having prostate cancer comprises an effective amount of an inhibitory nucleic acid sequence complimentary to all or part of a PCA3 RNA of SEQ ID NO: 2 (e.g., a sequence complimentary to 18, 19, 20, 21 , 22, 23, 24 or 25 consecutive nucleotides of SEQ ID NO: 2). For example, in some aspects, an inhibitory nucleic acid molecule of the embodiments comprises a sequence complimentary to any one of SEQ ID NOs: 6-19. In still further aspects, an inhibitory nucleic acid molecule comprises the sequence of any one of SEQ ID NOs: 6-19 and a sequence complementary thereto. In certain aspects, an inhibitory nucleic acid of the embodiments is an RNA molecule. In still further aspects, an inhibitory nucleic acid molecule of the embodiments is formulated in a pharmaceutically acceptable delivery vehicle, such as in a liposome or nanoparticle formulation.

[0016] In further aspects, a composition for use in treating a subject having prostate cancer comprises an effective amount of an inhibitory nucleic acid sequence complimentary to all or part of a NONO mRNA of SEQ ID NO: 3 (e.g., a sequence complimentary to 18, 19, 20, 21 , 22, 23, 24 or 25 consecutive nucleotides of SEQ ID NO: 3). For example, in some aspects, an inhibitory nucleic acid molecule of the embodiments comprises a sequence complimentary to any one of SEQ ID NOs: 20-23. In still further aspects, an inhibitory nucleic acid molecule comprises the sequence of any one of SEQ ID NOs: 20-23 and a sequence complementary thereto. In certain aspects, an inhibitory nucleic acid of the embodiments is an NA molecule. In still further aspects, an inhibitory nucleic acid molecule of the embodiments is formulated in a pharmaceutically acceptable delivery vehicle, such as in a liposome or nanoparticle formulation.

[0017] In still further aspects, a composition for use in treating a subject having prostate cancer comprises an effective amount of an inhibitory nucleic acid sequence complimentary to all or part of a ADAR mRNA of SEQ ID NO: 4 (e.g., a sequence complimentary to 18, 19, 20, 21 , 22, 23, 24 or 25 consecutive nucleotides of SEQ ID NO: 4). For example, in some aspects, an inhibitory nucleic acid molecule of the embodiments comprises a sequence complimentary to any one of SEQ ID NOs: 24-37. In still further aspects, an inhibitory nucleic acid molecule comprises the sequence of any one of SEQ ID NOs: 24-37 and a sequence complementary thereto. In certain aspects, an inhibitory nucleic acid of the embodiments is an RNA molecule. In still further aspects, an inhibitory nucleic acid molecule of the embodiments is formulated in a pharmaceutically acceptable delivery vehicle, such as in a liposome or nanoparticle formulation.

[0018] Thus, in certain aspects, a composition for use in treating a subject having prostate cancer comprises an effective amount of an inhibitory nucleic acid sequence complimentary to all or part of a DROSHA mRNA of SEQ ID NO: 5 (e.g., a sequence complimentary to 18, 19, 20, 21 , 22, 23, 24 or 25 consecutive nucleotides of SEQ ID NO: 5). For example, in some aspects, an inhibitory nucleic acid molecule of the embodiments comprises a sequence complimentary to any one of SEQ ID NOs: 38-59. In still further aspects, an inhibitory nucleic acid molecule comprises the sequence of any one of SEQ ID NOs: 38-59 and a sequence complementary thereto. In certain aspects, an inhibitory nucleic acid of the embodiments is an RNA molecule. In still further aspects, an inhibitory nucleic acid molecule of the embodiments is formulated in a pharmaceutically acceptable delivery vehicle, such as in a liposome or nanoparticle formulation. [0019] In still a further embodiment, the invention provides a method for determining whether a subject has or is at risk for developing prostate cancer. In some aspects, a sample is obtained from the subject (or from a third party) and the expression level of PRUNE2 is measured in the sample. The sample may be, for example, blood, urine, semen or a tissue biopsy. In some aspects, an elevated expression of PCA3 and a decreased expression of PRUNE2 relative to a reference indicates that the subject has or is at risk for developing prostate cancer. In further aspects, the method further comprises reporting whether the subject has or is at risk for developing prostate cancer. For example, reporting may comprise preparing an oral, written or electronic report. In some aspects, the report is provided to the subject or to a healthcare worker.

[0020] In yet still a further embodiment, the invention provides an assay method comprising obtaining a sample from a subject suspected of having prostate cancer and selectively measuring the expression level of PRUNE2 in the sample. The sample may be, for example, blood, urine, semen or a tissue biopsy. In some aspects, the method further comprises reporting the expression level.

[0021] In certain aspects of the embodiments, the expression levels of PRU E2 and PCA3 are selectively measured in the sample. In one aspect, an elevated expression of PCA3 and a decreased expression of PRU E2 relative to a reference indicates that the subject has or is at risk for developing prostate cancer. In some aspects, measuring or selectively measuring the expression level comprises measuring a protein expression level. Measuring protein expression levels may comprise, for example, performing an ELISA, a Western blot or immunohistochemistry. In other aspects, measuring or selectively measuring the expression level comprises measuring a RNA expression level. Measuring RNA expression levels may comprise, for example, performing RT-PCR, Northern blot or an array hybridization.

[0022] As used herein, the phrase "selectively measuring" refers to methods wherein only a finite number of protein (e.g., phosphoprotein) or nucleic acid (e.g., mRNA) markers are measured rather than assaying essentially all proteins or nucleic acids in a sample. For example, in some aspects "selectively measuring" nucleic acid or protein markers can refer to measuring no more than 100, 75, 50, 25 or 10 different nucleic acid or protein markers.

[0023] As used herein, the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. [0024] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more. [0025] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0026] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [0028] The patent or application file contains at least one drawing executed in color.

Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0029] FIGS. 1A-1F. Identification, cloning, genomic structure, and co- localization of PRUNE2/PCA3. (A) Genomic context, intron and exon boundaries of PCA3 and canonical PRUNE2 (GenBank FJ808772) and previously annotated transcripts mapped to chromosome 9q21 (GenBank AB050197 and BC019095). Stars indicate missing or new exons; downward arrowheads indicate initiation (open arrow) or stop (solid arrow) codons. Horizontal arrows indicate transcript orientation for PCA3 and PRUNE2, as indicated. (B) RT-PCR with R A from the patient-derived xenograft MDA-PCa-133 tumor xenograft used to clone and sequence of canonical PRUNE2 and PCA3. (C) Immunoblotting analysis of PRUNE2 in LNCaP cells stably expressing ectopic PCA3, PCA 3 -silenced, PRUNE2- silenced, or control (non-targeting construct). (D) Reverse-transcription quantitative PCR (RT-qPCR) assays with primers amplifying PCA3 or different regions of PRUNE '2 in prostate cancer LNCaP cells with silenced or ectopic PRUNE2 and PCA3. (E) Combined RNase- resistance and RNA-FISH analysis. Prior to hybridization, prostate cancer LNCaP cells were pretreated with either RNase A or RNase III. Hybridization was performed with specific probes against PCA3 and PRUNE2 transcripts. Nuclei are stained with DAPI. Arrows indicate foci. Representative confocal images are shown (size bar corresponds to 10 μηι). Fig. IE panels represent 40-fold magnifications of the same data depicted in FIG. 9A with additional negative controls. (F) Expression effects of intron6-PRUNE2 on nuclear and cytoplasmic PCA3 and PRUNE2 levels in human prostate cancer LNCaP cells. Data represent means + standard deviation (SD). *P<0.05; ** <0.01; ***P<0.001.

[0030] FIGS. 2A-2G. PRUNE2/PCA3 cellular co-localization with ADAR proteins. (A) RNA-chromatin immunoprecipitation (RNA-ChIP) and analysis of PCA3 and PRUNE2 binding by RT-qPCR in prostate cancer LNCaP cells. (B) Combined RNase- resistance and RNA-FISH analysis. Prior to hybridization, prostate cancer LNCaP cells were pretreated with RNase A. Hybridization and immunostaining were subsequently performed with specific probes against an antibody against ADAR1. (C) Analysis for PCA3 and PRUNE2 binding to ADAR1 by RNA-chromatin immunoprecipitation (RNA-ChIP). (D) Hybridization with biotin-labeled oligomers against PC A3 and PRUNE 2 in LNCaP cells after an ultraviolet (UV)-induced RNA-protein cross-linking. Immunoblot against ADAR1 is shown. (E and F) Evaluation of PCA3 and PRUNE2 expression in LNCaP cells stably expressing two independent lentiviral ADAR1 -shRNA constructs: immunoblots against PRUNE2, ADAR1, and an unrelated control protein (YY1) (panel E) and RT-qPCR (panel F) are shown. (G) Cytosolic (C) and nuclear (N) RNA fractionation followed by RT-qPCR analysis, specific oligonucleotides served for amplification of nuclear pre-mRNA and cytosolic niRNA o(PCA3 and PRUNE2. Data represent means ± SD. *P<0.05; **P<0.01.

[0031] FIGS. 3A-3E. Functional roles of RNA editing ADAR-mediated, and P54 in PRUNE2/PCA3 regulation. (A and B) Identification, quantification, and distribution of A>G/T>C changes (features pathognomonic of A-to-I editing in both strands of the PRUNE2/PCA3 dsRNA) analyzed RNA-capture followed by high-throughput sequencing. Reads were aligned against the reference human genome (hgl9) of the same region. Only non-dbSNP variations indicated by at least three reads and located out of repetitive elements were considered. (A) Distribution and percentage of all possible alteration pairs observed for the PCA3 genomic coordinates in human prostate cancer LNCaP cells are depicted. (B) RNA editing map for LNCaP cells showing the precise location of each A>G (gray circles) or T>C (black circles) sites over PCA3 and mtr n6-PRUNE2 pre-mRNA species. Each square represents one individual base from the PCA3 locus (23, 1 12 nt). Black borders delimit the bases of the four annotated exons (3,923 nt). Repeats given by RepeatMasker are marked as gray squares (B). (C to E) Evaluation of PCA3 and PRUNE 2 levels in LNCaP cells stably expressing two independent P54^NRB-shRNA clones (CI and C2) or non-targeting-shRNA control constructs (NT). Detection of PC A3 and PRUNE2 mRNA cytosolic levels by RNA- FISH (C) and by RT-qPCR (D) are shown. Immunoblot analysis of PRUNE2 expression in LNCaP P54^NRB-silenced cells or negative control is shown (E).

[0032] FIGS. 4A-4E. Analysis of PRUNE2/PCA3 expression upon androgen receptor activation. (A) Immunoblot analysis for PRUNE2, androgen receptor (AR) and phosphorylated AR (pAR) expression in human prostate cancer cells after concentration- dependent androgen stimulation with the testosterone analogue R1881 (solid arrowheads indicate the shorter forms of PRUNE2 -related proteins and the outlined arrowhead indicates the canonical PRUNE2 product). A representative gel (PAGE 3-8%) is shown. (B) Relative mRNA expression levels of PCA3 and N£2-related transcripts (5 ^'-PRUNE2, Ύ-BMCCl, or canonical PRUNE2) under R1881 stimulation. (C) Relative mRNA expression of canonical PRUNE2, PCA3, and PSA (positive control) measured by RT-qPCR in LNCaP cells after concentration-dependent R1881 stimulation. (D) Immunoblot analysis of PRUNE2 levels in LNCaP cells under steroid-depleted conditions and after androgen stimulation (soild arrowheads indicate the shorter forms of PRUNE2-related proteins and outlined arrowheads indicate the canonical PRUNE2 product). A representative gel (PAGE 4-12%) is shown. (E) RNA-FISH analysis for PCA3 and pre-mRNA of PRUNE in LNCaP cells under steroid-depleted conditions or after androgen stimulation.

[0033] FIGS. 5A-5N. Function of PRUNE2/PCA3 in prostate mouse tumor models. (A to D) Cohorts of male SCID mice that received subcutaneous (SC) administration of 5xl0⁶ LNCaP cells stably expressing ectopic PCA3, PC45-silenced, PRUNE 2 -silenced, or negative control shRNA. Tumor xenograft growth was monitored and volume was measured (A). (B to D) Mice were killed at the experimental endpoint (4 weeks). Representative experimental tumor xenografts are depicted B) and tumor mass (C) and serum PSA concentration (D) were determined. (E and F) Tumor growth in mice bearing tumor xenografts from LNCaP cells stably expressing mtron6-PRUNE2 (antisense sequence to PCA3) or control constructs. (G and H) Tumor growth in SCID mice bearing tumor xenografts of PC3 cells stably expressing either ectopic PRUNE 2 or negative control construct. (I and J) Tumor growth in mice bearing tumor xenografts of LNCaP cells stably expressing ADARl-shKNA or negative control construct. (K to N) PC45-silencing in vivo by targeting SCID mice bearing tumor xenografts of LNCaP cells stably expressing ectopic PCA3 constructs. Two cohorts of SCID mice with size-matched tumors («=10 per each group) received 8 μg of either stealth chemically-modified PC43-siRNA or negative control- siRNA per dose; treatments and controls received a series of doses («=9) through alternating intratumoral or intraperitoneal administration every-other-day. Tumor volumes, measured before each dose administration, were plotted over time (K) and representative tumors at the experimental endpoint are shown (L). Final tumor xenograft mass (M) and serum PSA concentration (N) were determined at the experimental endpoint. In each experiment, cohorts of 6-10 mice per group were treated. Data represent means ± SD. *P<0.05, **P<0.01. N.G. indicates no growth.

[0034] FIGS. 6A-6K. PRUNE2/PCA3 expression and RNA editing in clinical samples from prostate cancer patients. (A) Analysis of PRUNE 2 and PC A3 mRNA levels in human prostate cancer samples (n=4S) versus non-malignant prostate tissue («=9). Malignant (M) versus non-malignant (NM) control is indicated. PSA served as a positive control for the RT-qPCR. Mean ± SD is shown. In each case, -values are for M versus NM. (B) PCA3 and PRUNE2 expression levels from cDNA microarrays (34) were downloaded from Oncomine^®. For non-malignant gland «=29 and for prostate tumor «=115. Individual lines link the expression levels of both genes for each sample. Black lines depict the calculated slopes linking to average intensity values. (C) RNA Seq by Expectation Maximization (RSEM) expression values for PCA3 and PRUNE2 mRNA in non-malignant or cancer in prostate tissue samples from TCGA. Lines are used to connect PCA3 and PRUNE2 expression values each patient sample. Black lines are linking the mean RSEM- values for each group. (D) Representative images of a human tissue microarray (TMA) analysis of prostate cancer samples showing high-abundance of PRUNE2 in non-malignant adjacent prostate tissue control compared to tumor. In each case, IHC staining (i.e., percentage extent of expression in cells) was analyzed. Original magnification, 20-fold. (E and F) PRUNE2 estimated expression in the epithelium (E) or stromal (F) component of the tumor samples («=145) with low-grade («=50) and high-grade (n=95) versus non-malignant adjacent control tissues («=145) in human prostate cancer specimens. Mean + SD is shown.

(G) Summary statistical analysis of the TMA for PRUNE2 expression. A fitted mixed model of PRUNE2 epithelium and stromal estimated expression was used. In each case, mean + SD is shown. (H to K) RNA editing in clinical samples from three independent patients with prostate cancer (denominated Pts #1-3). All possible alterations, including putative editing sites, were determined as described for LNCaP cells and are shown for each tumor sample

(H) . A subset of editing sites suggested by large-scale sequencing was confirmed by PCR (by using gDNA or cDNA as templates) followed by Sanger-sequencing (I). Intersections of the putative edited sites among each independent patient sample are depicted (J). Individual RNA editing maps of three prostate cancer patients are shown: Distribution of A>G (gray circles)/T>C (black circles) sites over PCA3 and intron6-PRUNE2 pre-mRNA are shown ( ). A larger version (Zoom) of the RNA editing map for the human tumor sample from each of the three prostate cancer patients shown is also provided (FIGS. 23-25). [0035] FIGS. 7A-7H. Expression of PRUNE2 and PCA3 transcripts and related constructs in human cells. (A) PRUNE2 -related mRNA levels in a representative panel of human tumor cell lines. Transcripts were amplified through RT-qPCR with a set of specific primers. (B) Genomic scheme depicting intron and exon boundaries of the locus. Arrows indicate orientation of transcripts for PRUNE2 d PCA3, as indicated. Representation of PRUNE2 pre-mRNA with the location of primer sets targeted to different regions encompassing the canonical PRUNE2 and PCA3 sequences. Shaded boxes represent Northern blot (NB) probes (NB probe 1, 5'-PRUNE2; NB probe 2, canonical PRUNE2) shown in (H). (C) Relative mRNA expression of canonical PRUNE 2 and PCA3 in a panel of non-malignant prostate-derived cells and prostate cancer-derived cells. Relative expression levels were compared against a panel of standard endogenous controls (see Methods). Mean ± SD is shown. (D) Immunoblots probed with anti-PRUNE2 or anti-V5 epitope tag antibodies showing endogenous PRUNE2 expressed in prostate cancer LNCaP cells and ectopic expression of PRUNE2 in prostate cancer PC3 cells (PRUNE2-deficient). A GFP- expressing vector served as a negative control. (E) Immunoblotting analysis of whole cell extracts from LNCaP cells stably transduced with lentiviral shRNA constructs: two independent shRNA clones against PRUNE2 along with a negative control (non-targeting) shRNA were used. (F) Changes in PCA3 mRNA levels in prostate cancer LNCaP cells, from baseline (control shRNA), ectopic PCA3 expression, or endogenous C45-silencing by two independent shRNA constructs (termed G45-shRNA-Cl and PG43-shRNA-C2. (G) Relative expression of PRUNE2 and PCA3 pre-mRNA levels in LNCaP cells stably expressing PC43-shRNA or non-targeting shRNA control. (H) Northern blot analysis of RNA extracted from LNCaP cells stably expressing constructs as indicated. Corresponding primers depicted in (A) and (B) are color-coded.

[0036] FIG. 8. Canonical PRUNE2 protein expression in human prostate- and prostate cancer-derived cells. Evaluation of canonical PRUNE2 protein expression by immunoblot in a representative panel (n=9) of human prostate- and prostate cancer-derived cells (see Materials and methods) stably transduced with either PRUNE2-s KNA, ectopic PC AS, or control constructs.

[0037] FIGS. 9A-9C. Cellular co-localization of PRUNE2/PCA3 RNA duplex. (A to C) Prostate cancer LNCaP cells were subjected to either RNase A or RNase III treatment followed by labeled oligonucleotide hybridization as described (Methods). PCA3-cy3 (located either in PCA3 exon 4 for mature mRNA or in PC A3 intron 1 for pre-mRNA; as indicated) and PR UNE2-cy5 (located in PR UNE2 intron 6) oligomers were used. Pre-mRNA of PRUNE 2 and mRNA of PCA3 are also shown. Enhanced signal in merged panels indicates PCA3 and PRUNE2 co-localization within the nucleus (DAPI is also shown where indicated). Labeled oligonucleotide-cy3 and -cy5 GFP probes served as negative controls (A). Ribonuclease digestion controls of the pre-mRNA or mRNA for PCA3 and PRUNE2 are shown (B, C).

[0038] FIG. 10. Effects of PC A3 expression on PRUNE2 coding sequence.

Analysis of combinatorial transfections in human prostate cancer PC3 cells (PRUNE2- deficient). Empty vector, V5-PRUNE2, mtmn6-PRUNE2, and ectopic PCA3 were used as indicated. Immunoblots with either a V5-tag antibody or an anti-PRUNE2 antibody demonstrate that PCA3 levels have no detectable effect on exogenous canonical PRUNE2 expression compared to controls.

[0039] FIGS. 11A-11D. Nuclear co-localization of PRUNE2 pre-mRNA and

PC A3. (A and B) LNCaP cells stably overexpressing PC A3. RNAse-resistance assay (A) on nuclear RNA was subjected to followed by RT-PCR (B) by using specific oligonucleotides for PC A3 and PRUNE2 pre-mRNA. (C) Cells were subjected to RNA-FISH using labeled oligonucleotides for PCA3 (located in exon 4) and PRUNE2 (located in intron 6 of PRUNE2). (D) HeLa cells stably co-expressing ectopic PC A3 and intmn6-PRUNE2 RNA were subjected to RNA-FISH using labeled oligonucleotides for PCA3 (located in exon 4) and PRUNE2 (located in intron 6 οΐ PRUNE2).

[0040] FIGS. 12A-12B. Co-localization of PRUNE2 and PCA3 with Drosha and Dicer by RNA-FISH. (A and B) LNCaP cells were subjected to RNase pre-treatment as indicated followed by oligonucleotide hybridization and immunofluorescence as described (Methods). FISH (PCA3-cy3, located in exon 4; PRUNE2-cy5, located in intron 6) and combined RNA immunofluorescence with an either anti-Drosha (A) or anti-Dicer (B) antibody. Co-localization analysis of PC A3 and PRUNE 2 with Drosha or Dicer within the nucleus was indicated from merge with DAPI. Representative images are shown. Scale bar,

[0041] FIGS. 13A-13E. Cellular co-localization of PRUNE2/PCA3 RNA duplex with ADARl/2. (A to D) LNCaP cells were subjected to either DNAase or RNase treatment as indicated, followed by oligonucleotide hybridization. FISH and combined RNA immunofluorescence with an anti-ADARl (A to C) or anti-ADAR2 (D) antibodies, as indicated. Labeled oligonucleotides PC A 3 -cy3 (located in exon 4) and PRUNE2-cy5 (located in PRUNE2 intron 6) were used. Signal in merge panels indicates either PCA3 and ADARl or ADAR2 co-localization or PRUNE 2 and ADARl or ADAR2 co-localization within the nucleolus (DAPI staining is shown where indicated). Labeled oligonucleotide-cy3 and -cy5 GFP probes served as negative controls (C). (E) HeLa cells stably co-expressing PCA3 and introu6-PRUNE2 RNA were subjected to RNase A treatment followed by oligonucleotide hybridization. FISH and combined RNA immunofluorescence with an anti-ADARl antibody. Labeled oligonucleotides PCA3 (located in exon 4) and PRUNE2 (located in PRUNE2 intron 6) were used, as indicated. Merge panels indicate either PCA3 and ADARl co-localization or PRUNE2 and ADARl co-localization within the nucleolus (DAPI staining is shown where indicated). Representative images are shown. Scale bars, 10 μιη.

[0042] FIGS. 14A-14C. Effects of Drosha and Dicer on the regulation of PCA3 and PRUNE2 levels. (A to C) LNCaP cells stably expressing Drosha, Dicer, or non- targeting shRNA lentiviral constructs were used as indicated. Two independent silencing constructs (shRNACl and shRNAC2) for each, Drosha and Dicer are shown (A), immunoblotting analysis of Drosha, Dicer, PRUNE2, GAPDH, and tubulin (A and B). RT- qPCR analysis for PCA3 and PRUNE2 mRNA (C). [0043] FIGS. 15A-15H. Analysis PRUNE 2 and PCA3 regulation through ADAR- mediated mechanisms. (A) T-qPC on fractionated RNA from cytosol (C) or nucleus (N) from LNCaP cells stably expressing ADARl-shRNA or negative control constructs. (B) Quality control of cytosolic and nuclear R A-fractionation by agarose gel electrophoresis after RT-PCR amplification. PRUNE2 and PCA3 pre-mRNA species are detected mostly in the nucleus as well as the controls HYOU1 and SON (control nuclear mRNAs); in contrast, GAPDH mRNA (control cytosolic mRNA) is detected mostly in the cytosol. (C to F) HeLa cells stably expressing intron6-PRUNE2-GFP were transduced with either PCA3 or control constructs. Cells were analyzed for reporter GFP expression by FACS (C) and by immunob lotting with anti-GFP antibodies (D) after 24, 48, and 100 h. (E) HeLa cells stably expressing intron6-PRUNE2-GFP, PCA3, ADARl-shRNA, non-targeting shR A control, or negative control (empty) lentivirus were analyzed after 48 hours for the reporter GFP expression. (F) Human tumor cell lines stably co-expressing C^ -luciferase (Luc) were transduced with intron6-PRUNE2-GFP or negative control expression vector. Tumor cells were lysed and luciferase activity was measured at 36 hours post-transduction. *P < 0.05, **P < 0.01 , < 0.001. (G and H) Evaluation of PRU E2 levels in LNCaP cells stably expressing ADAR1- or ADAR2-shRNA, and control lentivirus. Quality control of two individual ADAR1- and ADAR2-shRNA constructs (G). Whole cell extracts were probed with antibodies against PRU E2 and tubulin (H). [0044] FIGS. 16A-16F. Co-localization of PRUNE2 and PCA3 with P54^NRB and its partners (PSF and PSPC1). LNCaP cells were subjected to RNase pre-treatment as indicated followed by oligonucleotide hybridization and immunofluorescence as described (Materials and methods). (A to C) Detection of PCA3-cy3 (located in exon 4) and PRUNE 2- cy5 (located in intron 6) by RNA-FISH and immunofluorescence with an anti-P54^NRB antibody. Merge panels indicate PC A3 and pre-mRNA PRUNE 2 co-localization with P54^NRB; labeled oligonucleotide-cy3 against Actin mRNA served as a negative control (A to C). (D and E) FISH and combined RNA immunofluorescence with an anti-PSF antibody. Labeled oligonucleotides PCA3-cy3 and PRUNE 2-cy5 (located in PRUNE2 intron 6) were used. PCA3 and pre-mRNA of PRUNE2 and PSF are shown. Either PCA3 and PSF or PRUNE2 and PSF co-localize within the nucleus (DAPI staining shown where indicated). (F) RNA-FISH and combined immunofluorescence with an anti-PSPC-1 antibody. A labeled PCA3 oligonucleotide (located in exon 4) and a PRUNE2 oligonucleotide (located in intron 6) and an anti-PSPC-1 antibody were used as indicated. Merge panels indicate either PCA3 and PSPC-1 co-localization or PRUNE2 and PSPC-1 co-localization within the nucleus (DAPI staining shown where indicated). Scale bars, 10 μηι. Representative images are shown.

[0045] FIGS. 17A-17B. Function of PRUNE2/PCA3 in prostate cancer cells. (A) LNCaP cell proliferation in vitro after alteration of PCA3 and PRUNE2 levels or treatment with the androgen analogue R1881. (B) Colony formation assay in soft agar medium of LNCaP cells transduced with ectopic PC A3 or PRUNE2, PCA3- or PRUNE2-&KNA&, or controls as indicated. In each experiment, mean ± SD is shown. *P < 0.05; **P < 0.01.

[0046] FIGS. 18A-18E. Epistasis analysis of the functional interplay between PRUNE2 and PC A3 in human prostate cancer cells. (A) Tumor cell growth analysis in PC3 cells stably expressing iatron6-PRUNE2, ectopic PRUNE2, ectopic PCA3, intron6- PRUNE2 plus ectopic PCA3, C45-shRNA, PRUNE2-shRNA, or control shRNA constructs. (B) Tumor cell growth analysis in LNCaP cells stably expressing C^-shRNA, PRUNE2- shRNA,

canonical V5-PRUNE2, or control constructs. (C) Anchorage- independent cell colony growth in soft agar medium of PC3 cells stably expressing canonical V5-PRUNE2 or control vector. In each experiment, mean + SD is shown. *P < 0.05, **P < 0.01 , ***P < 0.001. (D) Immunoblots of extracts from LNCaP cells stably expressing PRUNE2-s RNA, G45-snRNA, ectopic PCA3, trox\6-PRUNE2 , or control shRNA constructs. (E) Immunoblots of whole extracts from LNCaP cells stably transduced with canonical V5-PRUNE2, ectopic PCA3, mtron6-PRUNE2, or control shRNA constructs. Antibodies against V5-tag or actin were used.

[0047] FIGS. 19A-19E. Effects of ADAR1- and ^^-silencing in prostate cancer cells. (A and B) Adhesion of LNCaP cells stably expressing ADARl-sKKNA, P54^NRB- shRNA, ectopic PCA3 PC45-shRNA, or control shRNA was evaluated (A). Representative microscopy images are shown (B). (C to E) Cell growth of LNCaP cells stably expressing ADARl-shRNA or control shRNA. Cell proliferation (C), cell doubling time (D), and anchorage-independent colony formation in soft agar (E) are shown. In each experiment, mean ± SD is shown. *P < 0.05, **P < 0.01.

[0048] FIGS. 20A-20G. PRUNE2 domains, interactions and co-localization with RhoA and Nm23-Hl. (A), Predicted protein domains of PRUNE2 and putative interaction sites with RhoA and Nm23-Hl . Conserved domains are indicated as boxes labeled as PPX1 , DHH, DHHA2, and BCH relative to the protein sequences. (B) PRU E2 co- immunoprecipitation with RhoA and Nm23-Hl . Starved LNCaP cell lysates were obtained after 10 min stimulation with a growth factor admixture (GF+; see methods) or BSA (GF-). Immunoprecipitation was performed with an anti-PRUNE2 antibody. Both RhoA and Nm23- HI co-precipitated with PRU E2 upon growth factor stimulation. Total cell extracts prior to immunoprecipitation served as loading controls. (C and D) Immunostaining and confocal microscopy analysis of the entire LNCaP-derived spheroids were reconstructed by merging the full Z-section series. Co-localization of PRUNE2 with RhoA (C) and Nm23-Hl (D) is shown. DAPI is also shown where indicated. Arrows indicate protein co-localization. (E) RLWE2-silencing affects ERK and AKT phosphorylation. LNCaP stably expressing PRUNE2 -shRNA or control shRNA constructs were stimulated with GF+ for 10 min. Whole cell lysates were subjected to immunoblot analysis for ERK1/2 and AKT. (F) Immunoblots probed for PRUNE2 in LNCaP cell lysates grown in either non-adherent or adherent conditions as indicated. (G) Immunostaining and confocal microscopy analysis of the entire LNCaP-derived spheroids were reconstructed by merging the full Z-section series. Co- localization of PRUNE2 and tubulin along with DAPI staining are shown as indicated. Arrows indicate protein co-localization.

[0049] FIGS. 21A-21L. Biological function of PRUNE2 In prostate cancer cells.

(A) Cell cycle analysis by BrdU incorporation. (B) Cell-doubling time. (C to F), PRUNE2 complementation assay in LNCaP cells stably expressing either control shRNA (C) or PRUNE2-s KNA (D) were transduced with an shRNA-resistant PRUNE2 (PRUNE2-RNAQ) construct. In both cases, PRUNE2-RNAQ expression resulted in a decrease in LNCaP cell adhesion and spreading. LNCaP cells stably expressing either control shRNA (E) or PRUNE2 shRNA (F) were transduced with PRUNE2-RNAQ . In both cases, ectopic expression of PRUNE2-RNAQ did not result in detectable changes in cell death. (G to L), Effects of PRUNE2 overexpression on prostate cancer cell adhesion and migration. (G and H) LNCaP cells stably expressing PRUNE2-KNAQ., control shRNA, or two independent PRUNE2-shRNA constructs per gene were evaluated for their capacity to adhere (G) and migrate (H) in the presence of RPMI containing 2.5% FBS plus a growth factor admixture. (I) LNCaP cells stably co-expressing PRUNE2-s RNA and PRUNE2-RNAQ, or control shRNA constructs were analyzed for cell adhesion. (J) LNCaP, DU145, and PC3 prostate cancer cells were transfected with PRUNE2, an unrelated tumor suppressor gene (RASSF1A) as a positive control, or vector alone as a negative control and analyzed for cell adhesion. (K and L) Effect of PCA3 overexpression on cell adhesion in LNCaP cells. Cells stably expressing PCA3, control shRNA, or two independent C45-shRNA constructs per gene were evaluated for their capacity to adhere ( ) and migrate (L) in the presence of RPMI containing 2.5% FBS plus a growth factor admixture. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

I. The Present Invention

[0050] Prostate cancer is the most common malignancy in males and methods for early diagnosis and effective treatment of the disease are still lacking. The long noncoding (lnc) RNA PCA3 can be used as a specific prostate cancer biomarker but its biological function was not previously clear. Studies herein characterize an new tumor suppressor gene, PRUNE2, which harbors the PCA3 locus as an antisense intronic lncRNA. It is shown that PCA3 expression controls PRUNE2 levels through the formation of a PRUNE2/PCA3 double-stranded RNA that undergoes ADAR-dependent adenosine-to-inosine RNA-editing. PRUNE2 expression or silencing in prostate cancer cells decreased and increased cell proliferation, respectively. Moreover, functional characterization in immuno deficient tumor- bearing mice revealed that PRUNE2 and PCA3 elicit opposite effects on tumor growth. Finally, the co-regulation and RNA editing of PRUNE2 and PCA3 were confirmed in human prostate cancer patients, supporting the medical relevance of our findings. These results establish PCA3 as a dominant negative oncogene and PRUNE2 as a previously unrecognized tumor suppressor gene in prostate cancer, and reveal an alternative regulatory mechanism for an antisense intronic lncRNA via ADAR-mediated RNA editing. This PCA3/PRUNE2 regulatory axis represents a molecular target for diagnostic and therapeutic intervention in prostate cancer.

[0051] Thus, embodiments of the invention, provide diagnostic and treatment methods that leverage the tumor suppressor activity of PRUNE2. For example, in some aspects, a prostate cancer may be treated by restoring the activity of PRUNE2 in the cancer cells. For example, PRUNE2 protein (or protein coding sequence) can be delivered to prostate cancer cells to inhibit tumor cell growth. Alternatively or additionally, an inhibitory nucleic acid can be delivered to down-regulate PCA3 RNA expression and thereby increase endogenous PRUNE2 expression in the cancer cells. By restoring tumor suppressor activity in tumor cells, the cell growth can be effectively inhibited. [0052] Likewise, the identification of PRU E2 as a key regulator of prostate cancer growth provides new diagnostic methods for early cancer detection. For example, in some aspects, PRUNE2 expression levels from a sample of a subject can be used to detect prostate cancer (or a risk of developing prostate cancer). In still further aspects, PRU E2 expression can be measured in conjunction of PCA3 expression thereby enhancing the sensitivity and specificity of prostate cancer diagnostic testing.

II. Examples

[0053] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 - Identification and characterization of PRUNE ^'2

Relationship with PCA3

[0054] Certain mammalian lncRNAs are embedded in the intronic-antisense regions of protein-coding genes (Cabili et al., 2011; Nakaya et al., 2007; Esteller, 2011; Geisler and Coller, 2013; Mercer and Mattick, 2013). PC A3 is a spliced IncRNA transcribed from chromosome 9q21 (Bussemakers et al., 1999; Auprich et al., 2011) and, within the same locus, two protein-coding mRNA transcripts (5'-PRUNE2 and 2>'-BMCCl) have been annotated as distinct genes flanking PCA3 in the antisense orientation (FIG. 1A) (Strausberg et al., 2002; Machida et al., 2006). However, subsequent in silico analysis suggested that these sequences could be part of a single transcriptional unit covering a large region where PCA3 is embedded (Clarke et al., 2009; Clarke et al., 2010; Lavin et al., 2009), thereby identifying it as an antisense intronic IncRNA. To investigate this possibility, RNA samples were screened from human prostate cancers (cell lines and tumor samples) by RT-PCR. From MDA-PCa-133, a patient-derived xenograft (PDX) of a prostate cancer bone metastasis (Lee et al., 2011; Brenner et al., 2011), two transcripts encoded from the same locus were cloned and sequenced: a splicing variant of a large transcriptional unit (-300 kb) merging 5'- PRUNE2 and V-BMCC1 (termed "canonical" PRUNE2) along with a splicing variant of an antisense transcriptional unit (~23 kb) from PCA 3 (FIGS. 1A and IB).

[0055] The quantitative gene expression profile of canonical PRUNE 2 in representative panels of human tumor and non-malignant cell lines was analyzed (FIGS. 7A and 7B). Canonical PRUNE2, along with 5 '-PRUNE2 and Ύ-BMCCl, was detectable in prostate cancer cell lines using transcript-specific primers (FIG. 7B), with the highest levels in the androgen-dependent (LNCaP) cells, as well as in several brain and breast cell lines. Canonical PRUNE2 levels were analyzed alongside PCA3 IncR A in the prostate cancer cell lines and differential expression of the two genes was observed: androgen-dependent (LNCaP) cells displayed the highest levels of both PRUNE2 and PCA3, compared to androgen-independent (DU145 and PC3) cells (FIG. 7C). Expression of native or recombinant V5 -tagged PRUNE2 was confirmed by immunoblot analysis and the predicted endogenous protein (-337 kDa) was observed in LNCaP cells, but not in PC3 cells (FIGS. 7D, 7E and 7F). PCA3 IncRNA Binds PRUNE2 pre-mRNA and Regulates its Levels

[0056] Prostate cancer cell lines (LNCaP and PC3) stably transduced with ectopic PCA3, C43-shRNA, ectopic PRUNE2, PRUNE2-shRNA, or the corresponding control constructs, were generated. Levels of endogenous PRUNE2 protein, pre-mRNA and mRNA increased with C4i-silencing and decreased with ectopic PC A3 expression (FIGS. 1C, ID, 7G and 7H); importantly this regulatory effect was more pronounced for canonical PRUNE 2 than for 5 '-PRUNE2 or 3 ' -BMCC1 mRNA, or their corresponding protein levels (FIGS. 1C, ID, 7B and 7H). These results were confimer in several prostate- and prostate cancer-derived cells, where ectopic PCA3 expression induced down-regulation of endogenous PRUNE2 expression (FIG. 8). [0057] To determine whether PRUNE2/PCA3 form a dsRNA, RNA fluorescence in situ hybridization (FISH) was used. PCA3 and PRUNE2 hybridized in the same nuclear foci (FIGS. IE and 9A). These foci were completely depleted upon treatment with RNase III, which degrades only dsRNA, but not with RNase A, which degrades only single-stranded (ss)RNA (FIGS. IE and 9A-9C), indicating the formation of a dsRNA derived from the physical association of PCA3 and PRUNE2 pre-mRNA. [0058] To determine whether binding of PRUNE2 mR A to PC AS was required for the regulation of PRU E2 levels, the effect of PCA3 on exogenous mature PRUNE2 cDNA, which has no sequence complementarity to PCA3 and therefore would be unable to form a dsRNA, was assessed. Indeed, PCA3 down-regulated endogenous PRUNE2 pre-mR A expression, but did not affect the exogenous expression of canonical PRUNE2 mRNA and protein (FIGS. ID, 8 and 10). To complement this, a PRUNE2 construct was designed and expressed that contains no protein-coding sequence but is fully complementary to PC A3 (termed intron6-PR UNE2) and should therefore be able to bind PC A3 and possibly sequester it from canonical PRUNE2. Consistent with this, overexpression of iniron6-PRUNE2 caused an increase in endogenous canonical PRUNE 2 mRNA in the cytoplasm, and a concomitant reduction in the nucleus (FIG. IF). A direct interaction between PCA3 and its correspondent anti-sense sequence (intron6-PRUNE2) was confirmed by nuclear digestion of RNA expressing both sequences in tumor cells (FIG. 1 1C). These data suggest that binding of canonical PRUNE2 pre-mRNA to PCA3 is required for the regulation of PRUNE2 levels. ADAR and Drosha Bind PRUNE2/PCA3 dsRNA and Regulate PRUNE2 Levels

[0059] Notably, dsRNA species are targets of enzymes involved in RNA degradation pathways, including the RNA interference (RNAi) machinery and the adenosine deaminase acting on RNA (ADAR) family of proteins. RNAi-based regulation of dsRNAs occurs via the RNase enzymes Drosha and Dicer. It was found that Drosha, but not Dicer, immunoprecipitates (Fig. 2A) and co-localizes with the PRUNE2/PCA3 dsRNA in the nucleus (FIGS. 12A-12B). ADAR members are key regulatory enzymes for RNA-editing and sequestering of noncoding RNA sequences, such as introns and untranslated mRNAs (Hundley and Bass, 2010; Fatica and Bozzoni, 2014; Chen et al., 2008; Peters et al., 2003; Naganuma and Hirose, 2013; Keegan et al., 2004; Bass, 2002), derived from the hybridization of retroinverted Alu-elements (Fatica and Bozzoni, 2014; Chen et al., 2008). Conversion of adenosine -to-inosine (A-to-I) RNA-editing occurs after nuclear dsRNA formation. Using RT-qPCR, co-RNA FISH and RNA-chromatin immunoprecipitation (RNA-ChIP), it was found that endogenous PCA3 and pre-mRNAs of PRUNE2 co-localize to nuclear foci associated with ADAR proteins, which were sensitive to RNase III treatment (FIGS. 2A-2C and 13A-13E). PRUNE2/PCA3 dsRNA and ADAR1 formed a complex only when both RNA species were co-expressed (FIG. 13E); the corresponding signals for PRUNE2/PCA3 dsRNA decreased after PCA3- or PRt/N£2-silencing and increased with ectopic expression of PCA3 (FIG. 2D).

[0060] Because both ADAR and Drosha proteins associate to the PRUNE2 '/PC A3 dsRNA, the functional role for these pathways in the regulation of PRUNE2 was evaluated. Conditional Drosha-silencing caused an up-regulation of PRUNE2 but not PCA3 (FIGS. 14A-14C). A physical association between Dicer and the PRUNE2/PCA3 dsRNA was not observed. Conditional Dicer-silencing increased the levels of PRUNE2 but not PCA3, perhaps suggesting an independent Drosha/Dicer-mediated mechanism in the regulation of PRUNE2 that do not require PCA3. Silencing ADARl in human tumor cells also increased PRUNE2 mRNA and protein levels (FIGS. 2E-2G). It was also found that ADAR-depleted prostate cancer cells have increased cytosolic PRUNE2 and PCA3 levels (FIGS. 2G and 15A- 15B), revealing the importance of ADAR members in the regulation of both genes. This is consistent with recent reports demonstrating novel functions of A-to-I editing in the regulation of noncoding RNA species (Mallela and Nishikura, 2012). Taken together, these results indicate a functional role for the regulation of PRU E2 by both Drosha/Dicer and ADAR members, and support the existence of crosstalk between the RNAi and RNA editing pathways (Ota, et al., 2013; Ganesan and Rao, 2008).

[0061] To gain further functional insight into the regulation of PRUNE2 and PCA3, sensor/reporter assays were established in which PCA3 or the PCA3 antisense sequence (i.e.,

was fused to reporter genes to generate PCA3-luciferase and intron6- PRUNE2-GFP . Reporter expression (FACS and luminescence assays) demonstrated that the co-expression of intmn6-PRUNE2-GFP plus PCA3 or intron6-PRUNE2 plus PCA3- luciferase resulted in reduction of the corresponding reporter signals compared to controls (FIGS. 15C-15F). Thus, in addition to PC A3 regulating canonical PRUNE2 levels, PRUNE2 pre-mRNA can also downregulate PCA3 (FIG. IF). Silencing of either ADARl or ADAR2 increased the reporter signals as well as PRUNE2 expression, confirming that these enzymes are required for this co-regulatory effect on both RNAs (FIGS. 15A, 15B and 15E-15H).

RNA Editing of PRUNE 2 and PC A3 RNAs

[0062] The results thus far have shown that both ADAR and Drosha proteins associate with PRUNE2/PCA3 dsRNA and regulate PRUNE2 levels. The results also illustrate that ADAR proteins regulate the levels of PCA3, and likely function via A-to-I RNA editing. To test this directly, the presence of A-to-I editing was evaluated throughout the genomic coordinates of PCA3 and its corresponding antisense pre-mRNA intron6- PRUNE2 by RNA-capture followed by next-generation sequencing. Although in general RNA editing is found mostly in Alu-elements, repetitive elements (including Alu-sequences) were filtered to avoid erroneous alignments. The results show that A>G/T>C changes, which are characteristic of A-to-I editing, were the most frequent substitutions. Editing sites were distributed in intronic as well as exonic regions, suggesting that a dsRNA hybrid is also formed between pre-mRNA species of both genes (FIGS. 3A-3B), as observed in the co- localization experiments (FIGS. 9B-9C).

[0063] The Drosophila behavior human splicing (DBHS) protein P54^NRB preferentially binds to inosine containing RNA (RNA-I) and regulates gene expression (Naganuma and Hirose, 2013; Nishikura, 2010). Therefore, a potential role for P54^NRB and for other DBHS proteins in regulating PRUNE2/PCA3 was investigated. Both, PCA3 and PRUNE2 pre-mRNA species associated with P54^NRB and the other two known mammalian family -members (PSF and PSPC-1) compared to a negative control RNA, as determined by RNA-ChIP (Fig. 2A) and immunofluorescence (FIGS. 16A-16F). In addition, P54^NRB- silenced prostate cancer cells had increased levels of PCA3 and PRUNE2 mRNA (FIGS. 3C- 3D) and concomitant increase of PRUNE2 protein levels relative to controls (FIG. 3E). These results further support the observations that PRUNE2 and PCA3 RNAs undergo A-to-I editing, and also reveal a functional role for DBHS proteins in their regulation. The PRUNE2/PCA3 Regulatory Axis Functions in Prostate Cancer Growth

[0064] Androgen-dependence and resistance to androgen-deprivation therapy are central to the biological and clinical features of human prostate cancer. Thus, it was investigated whether stimuli elicited by androgen receptor (AR) activation would regulate PCA3 and PRUNE2 expression in prostate cancer cells. Androgen-dependent LNCaP cells (grown in steroid-depleted serum) had lower PCA3 and higher PRUNE2 levels than androgen-independent PC3 cells (FIGS. 4A-4B and 7C). Consistently, androgen stimulation with a synthetic testosterone homologue induced a concomitant increase of PCA3 and decrease of PRUNE2 levels (FIGS. 4B-4C). Again, canonical PRUNE2 was the most down- regulated transcript when compared to either 5 ^'-PRUNE 2 or 3 ^'-BMCCl transcripts (FIGS. 4B and 4D). An increase in nuclear localization of PRUNE2 and PCA3 along with androgen- induced AR responses was also observed (FIG. 4E). Therefore, PRUNE2/PCA3 regulation appears to be sensitive to AR activation. [0065] To further assess the functional role(s) of the PRUNE2/PCA3 regulatory axis in prostate cancer, human prostate cancer cell lines (LNCaP, PRU E2-expressing; PC3, PRUNE2-deficient) stably expressing lentiviral constructs designed to either silence or ectopically express PRUNE 2 and PCA3 were generated (FIGS. 7D-7F and 10). PCA3- silencing or ectopic PRUNE2 expression decreased tumor cell proliferation in vitro; in contrast, RLWis2-silencing or ectopic PCA3 expression increased tumor cell proliferation (FIGS. 17 and 18A-18C). In addition, ectopic expression of mtx n6-PRUNE2 and PCA3, respectively increased and decreased endogenous, but not exogenous PRU E2 expression in prostate cancer cell lines (FIGS. 18D-18E). These results are consistent with the proposed negative regulation of R UNE2 by PC A 3.

[0066] To extend these results to human tumor xenograft models, LNCaP prostate cancer cells stably expressing PRUNE2-s RNA, ectopic PCA3, G43-shRNA, or controls were subcutaneously injected into severe combined immunodeficiency (SCID) mice. Ri/NE2-silencing and ectopic PCA3 expression yielded markedly larger tumor xenografts than controls; in contrast, tumor growth was greatly diminished relative to controls when PCA3 was silenced (FIGS. 5A-5C). Of note, increased serum prostate-specific antigen (PSA) concentrations were observed in SCID mice that received LNCaP cells with ectopic PC A3 expression or R UNE2 -silencing compared to controls (FIG. 5D). In vitro, and also in human tumor xenograft models, there was a decrease in tumor growth in LNCaP, but not in PC3 cells upon mtrou6-PRUNE2 expression (FIGS. 5E, 5F, and 18A). These results support a critical role for the PRUNE2/ C43 regulatory axis in prostate cancer growth. Moreover, the tumor suppressor activity of PRUNE2 was illustrated in PRUNE2-deficient PC3 cells stably expressing ectopic PRUNE2 administered in SCID mice relative to controls (FIGS. 5G-5H, 18A and 18C). Finally, silencing ADAR1 in LNCaP cells clearly increased PRUNE2 levels (FIGS. 2F, 2G, 3D and 3E) and reduced cell adhesion (FIGS. 19A-19B), as well as both cell proliferation in vitro and tumor growth in vivo (FIGS. 5K-5L and 19C-19E).

[0067] To explore the possibility of clinical translation of these findings, the PCA3 sense-strand was specifically targeted with a modified small-interfering (si)RNA (stealth RNAi™_pC43) serially administered to tumor-bearing mice with established xenografts. Tumor growth inhibition and serum PSA concentration reduction was observed relative to scrambled siRNA control (FIGS. 5K-5N). Taken together, these results support the working hypothesis that PRUNE2 expression has a functional tumor suppressive role in prostate cancer and suggest that the regulatory mechanism of PRUNE2 by PCA3 might be a suitable molecular target for intervention.

Molecular mechanism(s) through which PRUNE2 suppresses tumor growth

[0068] PRUNE2 has three functional domains (Lee, 2010): BCH, DHHA2, and PPX1 (FIG. 20A). BCH inhibits RhoA, a small GTPase that regulates the cytoskeleton, cell adhesion and migration (Soh and Low, 2008) while DHHA2 interacts with Nm23-Hl, a metastasis suppressor (Galasso and Zollo, 2009). It was found that endogenous PRUNE2 co- immunoprecipitates and co-localizes with RhoA and Nm23-Hl (FIGS. 20B-20D). PRUNE2- silencing increased phosphorylation of ERKl/2 and AKT, which are established downstream effectors of RhoA-mediated signaling pathways (FIG. 20E). Consistent with an inhibitory role for PRUNE2 in RhoA signaling, PRUNE2 levels increased up to three-fold when LNCaP cells were grown in non-adherent culture conditions (FIG. 20F), and the distribution of PRUNE2 was inversely correlated with focal adhesion sites in LNCaP-derived spheroids (FIGS. 20C, 20D and 20G). To uncover the functional effects of PRU E2 elevation on prostate cancer cells, stable tumor cells expressing PRUNE2-s KNA or ectopic PRUNE2 constructs showed an increase and decrease in cell proliferation (doubling-time), respectively (FIGS. 21A-21B). Moreover, alterations in tumor cell adhesion and spreading were observed, but no detectable effect on apoptosis (FIGS. 21C-21F). Consistently, reciprocal effects of PRUNE2 on adhesion, spreading, and migration of human prostate cancer cells were noted: decreased upon PRUNE2 expression or RhoA down-regulation, but increased with ectopic expression of PCA3- or RLW£2-silencing (FIGS. 21G-21L). Collectively, these results and the established functions of interacting proteins (Soh and Low, 2008; Galasso and Zollo, 2009; Basile et al., 2007) suggest that PRU E2 primarily decreases tumor growth by inhibiting cell proliferation, but also affects cell adhesion, spreading, and migration.

Levels of PCA3 and PRUNE2 Inversely Correlate in Human Prostate Cancer Patient Samples

[0069] The expression of PCA3 and PRUNE2 in human prostate cancer samples was examined. First, reverse-transcription quantitative PCR (RT-qPCR) analysis on tumor RNA samples from prostate cancer patients («=48) and non-malignant areas of the prostate (n=9) was performed. PRUNE2 mRNA expression was detected more often in non-tumor compared to the tumor-containing areas of the prostate, supporting a tumor growth suppressive function. In contrast, PCA3 mRNA levels showed the opposite pattern (FIG. 6A), with high expression levels more frequently detected in tumors relative to non-tumors, consistent with its role in the negative regulation of PRUNE2. These clinical findings were independently validated in silico. PRUNE2 and PCA3 expression levels were evaluated through the Oncomine^® Research Premium Edition (Rhodes, 2004) in a large sample («=144) subset (Taylor 2010) of primary non-treated prostate malignant tumors (n=1 15) and non- malignant prostate tissue («=29). Samples from prostate cancer-derived cell lines, from metastatic lesions, and from patients that received neoadjuvant therapy were excluded from the analysis to minimize variation due to other factors. Moreover, The Cancer Genome Atlas (TCGA) was also used as an unrelated large dataset (n=50 non-malignant control samples; «=333 prostate cancer samples) to validate the opposite expression between PRUNE2 and PC A3. It was found that low PC A3 levels correlate to high PRUNE 2 levels in non-malignant prostate samples, and vice- versa in prostate cancer samples (FIGS. 6B-6C). In yet another independent patient dataset, the protein expression pattern of PRU E2 in a large series of clinically well-annotated primary prostate cancer specimens (n=145), matched to adjacent histologically normal prostate tissue (n=145) was analyzed. In each case, immunohistochemical (IHC) staining was compared between the epithelial and stromal cells within tumors to the non-malignant epithelial and stromal cells from adjacent non-malignant areas of the same specimen (FIGS. 6D-6G). A higher abundance of PRU E2 in non-tumor areas compared to those of tumors was found. The inverse correlation between the native expression of PRUNE2 and PC A3 in clinical samples further supports a functional role for their regulation in human prostate cancer.

RNA Editing of PC A3 and PRUNE2 in Human Prostate Cancer Patients

[0070] Samples obtained from a cohort of prostate cancer patients by RNA-capture followed by next-generation sequencing were analyzed, and the presence of RNA editing was detected (FIG. 6H), which was also observed by classic Sanger-sequencing (FIG. 61) of genomic and cDNA clones from the same patients. Bioinformatic analyses again showed A>G/T>C alterations to be the most frequent substitutions, indicating A-to-I editing in both PCA3 and PRUNE2 pre-mRNA strands, with no clear editing hotspots identified in this limited set of human tumor samples (FIG. 6J). The editing maps provided for all patients show a similar distribution of alterations for both RNA strands, suggesting the interaction of the pre-mRNAs of both PCA3 and PRUNE2 transcripts (FIG. 6K). Example 2 - Materials and Methods

Bioinformatics and sequence analysis

[0071] Chromosomal locations, annotated transcripts, spliced expressed sequence tags, and sequence mapping were visualized on the Genome Browser from the University of California-Santa Cruz (available of the world wide web at genome.uscs.edu), by using the latest version of the human genome assembly (hgl9). Conserved domain analyses were performed through the Conserved Domain Database

(www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and sequence alignments were made with ClustalW (available of the world wide web at ebi.ac.uk/clustalw) or CLC bio (available of the world wide web at clcbio.com). PCA3 and PRUNE2 expression were also evaluated by using RNA-Seq data from the TCGA Research Network (http://cancergenome.nih.gov/). These data were downloaded and expression values were calculated through RSEM (54).

Reagents

[0072] Anti-bromodeoxyuridine (BrdU; Millipore), anti-β Actin, anti-β Tubulin (ECM Biosciences), ChIP grade anti-REDl, ChIP grade anti-Dicer, anti-Nm23-Hl , anti- RhoA (Abeam), anti-PRUNE2 (ProteinTech), anti-A T, anti-pAKTl, anti-pER l/2, anti- p44/42 MAP kinase, anti-Dicer, anti-Drosha, anti-S6RP (Cell Signaling Technology), anti- ADAR1 (Sigma or Abnova) and anti-ADAR2 (Sigma) were commercially obtained. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and insulin-like growth factor (IGF) were purchased (R&D Systems). An admixture (i.e., 10 ng EGF, 10 ng bFGF, 10 ng IGF, 20 ng VEGF) supplemented with heparin (5 units/ml) to a final concentration of 50 ng/ml served for growth factor stimulation unless otherwise specified. Methyltrienolone (R1881 ; Perkin Elmer) was used for androgenic stimulation in steroid-deprived conditions as indicated. RNaseA and RNase III (Invitrogen™), DNasel-RNase free (NEB), P54^NRB, AR, pAR, charcoal-stripped fetal bovine serum (FBS; Invitrogen) were commercially obtained. Secondary antibodies were purchased (Jackson ImmunoResearch or Invitrogen).

Cell lines and tissue culture

[0073] Human tumor cell lines used (HeLa, LNCaP, PC3, DU145, SF-268, SF-539, SNB-75, U-87, BT-549, Hs587T, MCF-7, NCI-ADR-RES, NCI-H322M, A549K, EKVX, NCI-H266, SK-MEL-28, UACC-257, OVCAR-8, S -OV-3, ACHN, HEK293, TK-10, KS 1767, and COLO205) were grown in RPMI containing 5% FBS. Human epithelial (PrEC) and stromal (PRSC) primary prostate cells, epithelial (RWPE-1 , RWPE-2) and stromal (WPMY-1) transformed prostate cells, and prostate cancer cells (VCaP, 22Rv) were cultured in optimized medium (ATCC).

Cloning and cDNA generation

[0074] Total RNAs from tumor cell lines or xenografts were isolated through the RNeasy kit (Qiagen), the All-in-One kit (Norgen Biotek), or the TRIzol reagent (Life Technologies). Total RNA samples from human normal tissues (prostate, brain, liver, kidney, breast, lung, pancreas, spleen, and testis) were commercially obtained (Stratagene). cDNAs were synthesized by using the Superscript III reverse transcriptase (Invitrogen or Promega) from total RNA, with N15 random pentadecamers, oligo dT primers, or specific oligonucleotides as indicated. Canonical PRUNE2 and PCA3 were amplified by RT-PCR with KAPA HiFi DNA polymerase (KAPA Biosystems), cloned into pENTR directional TOPO (Invitrogen), and fully sequenced. Verified coding sequences were re-amplified and subcloned into a pcDNA-DEST40 expression vector (Invitrogen). shRNA-resistant PRUNE 2 (PRUNE 2 Ω shRNA) was created by site-directed mutagenesis.

Quantitative RT-PCR and northern blotting

[0075] RT-qPCR analyses were performed with SYBR-green in a 7500 Fast Real- Time PCR system (Applied Biosystems). Gene expression levels were normalized against the average Ct of 3 standard endogenous controls (P0 large ribosomal protein, β- glucuronidase, and TATA box-binding protein), and the results were analyzed according to the AACt method (48). Data were reported as fold induction; samples were normalized on to their internal housekeeping genes followed by normalization of each sample to its control. For Northern blotting, customized LNA™ oligonucleotides (Exiqon) were used for PC A3 and PRUNE2. For retro-transcription, we designed specific anti-sense primers for PCA3 or PRUNE2 that allowed generation of specific cDNAs from corresponding pre-mRNA overlapped regions that enabled the generation of strand-specific cDNAs and PCR products for either PCA3 or PRUNE2. Amplified products were confirmed by sequencing.

Small-interfering RNA and short-hairpin RNA

[0076] Custom ordered siRNAs against PCA3 were transfected into tumor cells by using the NeoFX transfection reagent (Ambion). PC45-silencing experiments were performed with retroviral pLKO. l and human GIPZ vectors from the RNAi Consortium (TRC) lentiviral shRNA library (Open Biosystems) expressing specific shRNAs for human PRUNE 2 (oligonucleotide ID TRCN0000121740, referred to as PRUNE2-Cland oligonucleotide ID TRCN0000144868, referred to as PRUNE2-C2), human PCA3 (oligonucleotide ID V2LHS_ 24225, referred to as PCA3-C1; and oligonucleotide ID V2LHS 24226, referred to as PCA3-C2), human ADAR1 (oligonucleotide ID TRCN0000050788, referred to as ADAR1-C1 ; and oligonucleotide ID TRCN0000050790, referred to as ADAR1-C2 (Sigma), human GIPZ ADAR2 (#RHS4287), human TRIPZ lentiviral inducible shRNAmir (#RHS4740); Drosha (#RHS4740-NM030621) and Dicer (#RHS4740-NM013235; Open Biosystems). Lentivirus particles for P54^NRB-shRNA (TRCN0000074558 referred to as P54^NRB-C1 and TRCN0000074559 referred to as P54^NRB- C2; Sigma). Stable clones were maintained under puromycin selection. Validated non- targeting siRNAs (Ambion) and shRNAs (Open Biosystems) sequences served as negative controls. Customized Stealth chemically modified, HPLC -purified RNAi sequences against PC A3 or scrambled controls were purchased (Invitrogen).

Lentivirus preparation

[0077] Lentiviral vectors (pCCLsin.PTT.PGK.EGFP.Wpre, pMDLg/pRRE, pRSV- Rev, and pMD2.VSVG) were used as described (49). Briefly, 293FT cells were transiently transfected (Lipofectamine 2000; Invitrogen) for 16 h, after which the lentiviruses were harvested 24 and 48 hours later and filtered through 0.22 μιτι pore cellulose acetate filters. Recombinant lentiviruses were concentrated by ultracentrifugation for 2 hours at 50,000-g. Lentiviral vector viability was confirmed by reporter gene expression and drug selection. Cells were transfected with the FuGeneHD reagent (Roche) and transgene expression analyzed at 24, 36, 48, or 100 hours post-transfection. Corresponding empty plasmids served as negative controls.

Cell fractionation and nuclear RNA analysis

[0078] Nuclear/cytoplasmic RNA fractionation was performed as described (50). Tumor cells were grown in fibronectin-coated plates. At 70% confluence, cells were harvested, centrifuged, and rinsed with ice-cold phosphate-buffered saline (PBS). In brief, cell pellets were re-suspended by gentle pipetting in 200 μί lysis buffer A [10 mM Tris (pH8.0), 140 mM NaCl, 1.5 mM MgCl₂, 0.1% IGEPAL, 2 mM vanadyl ribonucleoside complex], and incubated on ice for 5 min. The same lysate sample served for total RNA extraction by the TRIzol reagent (Invitrogen) and for centrifugation (1,000 g for 3 min at 4°C) as well as to isolate the cytoplasmic fraction and pellet the nuclei. Cell-equivalent amounts of cytoplasmic and nuclear RNA samples were used for nuclear retention analysis. Nuclear/cytoplasmic ratios were normalized to GAPDH, HYOU1, or SON RNA controls. RNA fluorescence in situ hybridization and confocal microscopy

[0079] To detect PCA3 and PRUNE2 RNAs, cells were fixed in 3.6% formaldehyde for 3 min at RT, followed by acetone:methanol 1 :1 (vol/vol) for 5 min at -20°C. Cells were permeabilized in PBS containing 0.3% Triton X-100 and 5 mM vanadyl ribonucleoside complex (Invitrogen) on ice for 5 min; vanadyl ribonucleoside complex (an RNAse inhibitor) was omitted if the RNAse enzymatic activity was to be determined. Cells were washed three times in PBS, for 10 min, and rinsed once in 2x saline-sodium citrate (SSC) buffer prior to hybridization. Hybridization was carried out by using labeled cy3, cy5, and 488 nm DNA- oligonucleotide probes in a moist chamber at 37°C overnight (ON) as described (51). For co- localization studies after in situ hybridization, cells were fixed for 5 min in PBS-containing 2% formaldehyde. Immunofluorescence and imaging were performed as described³.

Immunoprecipitation and immunoblot analysis

[0080] Immunoprecipitation assays were performed as described (49). Briefly, a total of 3 x 10⁶ subconfluent cells were starved for 36 hours in RPMI containing 0.25% BSA and 0.05%) FBS. Cells were stimulated for 15 min at 37°C with a growth factor admixture described above. After washes with ice-cold PBS containing 0.1 mM sodium orthovanadate, cells were solubilized in 1 ml lysis buffer [50 mM Tris(hydroxymethyl) aminomethane HCI (pH 7.4), 150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.25% sodium deoxycholate, 1 mM ethylene glycol tetraacetic acid (EGTA), 1 mM ethylenediaminetetraacetic acid (EDTA), 2.5 mM sodium pyrophosphate, 1 mM sodium fluoride, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate (Na₃VC>4) (pH 10.0), anti-protease and anti-phosphatase cocktail; (Sigma)], collected, and incubated on ice for 10 min. Cell lysates were centrifuged at 10,000 g for 15 min, and supernatants were pre-cleared for 1 hours at 4°C by incubation either with 15 μΐ protein A- or protein G-agarose (Roche). Pre-cleared lysates were subsequently used for immunoprecipitation with specific antibodies as indicated. After incubation the solution was centrifuged at 1,000 g for 4 min and washed thrice with 0.5 ml lysis buffer and once with ice-cold PBS containing ImM Na₃VC>4. Immunoprecipitates were separated by 3-8%, 4-12% or 4% bis-Tris NuPAGE (Invitrogen) as indicated, transferred to nitrocellulose membranes, and immunoblotted with specified antibodies.

RNA-protein complex immunoprecipitation

[0081] Tumor cells at 70% confluence were rinsed twice and scrapped into ice-cold PBS. Cell pellets were re-suspended in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% IGEPAL, 1 mM phenylmethylsulfonyl fluoride (PMSF), proteinase inhibitor cocktail; (Sigma)], subjected to two rounds of gentle sonication, and centrifuged to obtain cell extracts. For RNaseA treatment, 200 μg/ml RNaseA was added to cell extracts, and the admixture was incubated at 37°C for 30 min. For immunoprecipitation, R aseA-treated and non-treated cell extracts were pre-cleared with 40 μΐ protein- A- plus protein-G-agarose beads (Roche) and 2.5 μg mouse anti-ADARl antibody or irrelevant isotype control antibody at 4°C for 2 hours followed by 40 μΐ protein-A- plus protein-G- agarose beads for 30 min at RT.

Ultraviolet-cross-linking and chromatin immunoprecipitation

[0082] Subconfluent cells cultured in complete RPMI for 24 hours underwent ultraviolet (UV)-mediated cross-linking, extract preparation, and SDS-PAGE or immunoprecipitation as described (52). For Western blot oligo-hybridization, specific 3 ^'- biotinylated oligonucleotides were used.

Cell proliferation, cell viability, and cell death assays

[0083] Cells were seeded in 200 μΐ growth medium at a density of 5-10,000 cells per well onto E-Plates 96 (Roche). Cell attachment and growth were monitored every 15 min for 48-72 hours with real-time cell electronic sensing (RT-CES) technology (Roche). The assay system expresses impedance in arbitrary cell index (CI) units. The CI at each time point is defined as (Rn-Rb)/15; where Rn is the cell-electrode impedance of the well when it contains cells and Rb is the background impedance of the well with the medium alone. Cell proliferation comparable data were measured 72 hours later with the WST-1 cell proliferation reagent (Roche). Cell viability was evaluated by the Trypan blue-exclusion methodology. Cell death assays were performed through a standardized cell death detection enzyme-linked immunosorbent assay (ELISA) kit (Roche). In brief, cells were cultured in complete RPMI medium for 24 hours. After cell lysis, the cytoplasmic fraction was pre-diluted to 1 : 10 (vol/vol) with incubation buffer and tested for nucleosomes in the immunoassay substrate reaction.

Cell adhesion and migration assays

[0084] Cells were seeded in 200 μΐ of RPMI medium supplemented with 2.5% FBS plus 50 ng of a growth factor admixture (described above) at a density of 5-10,000 cells per well onto E-Plates 96 (Roche). Cell adhesion was monitored every 15 min, during 4 hours through RT-CES technology (Roche). A 24-well in colorimetric format (CytoSelect; Cell Biolabs) was used for cell migration assays. Briefly, a cell suspension (1 ,000 cells) was placed in the upper chamber, and RPMI medium containing 2.5% FBS plus the growth factor admixture was placed in the lower chamber and incubated at 37°C and 5% CO2 for 4 hours. Cell migration was subsequently quantified.

Cell cycle analysis

[0085] Cells were synchronized in RPMI containing 0.2% bovine serum albumin (BSA) and stimulated with 10%> FBS for 24 hours. Proliferating cells were labeled by administration of BrdU at 10 μΜ for 1 hours (Sigma- Aldrich). Cells were harvested by trypsinization, washed in ice-cold PBS and fixed in 100% ethanol for 30 min on ice. DNA denaturation was achieved by incubation of fixed cells in 2 N HCl containing 0.05 % Triton X-100 for 30 min at RT. Residual acid was neutralized with 0.1 M sodium borate (pH8.5). Samples were incubated with an anti-BrdU monoclonal antibody, followed by cy-3- conjugated secondary antibody anti-mouse IgG (Jackson ImmunoResearch Laboratories). Total DNA content was measured with 50 μΜ propidium iodide (PI). Flow cytometry analysis was performed in a FACS Canto II System by using the FACS Diva software (Becton-Dickinson).

Soft agar colony formation assay

[0086] The analysis of anchorage-independent cell growth and colony formation was performed as described (53). In brief, cells transduced with the indicated constructs were suspended at either 5xl0³ or 10⁵ cells per well in 2 ml of 0.35% low-melting agarose in 35- mm culture dishes containing 0.7% agarose base. Triplicates were prepared and evaluated for each construct. Colonies were allowed to form at 37°C under standard tissue culture conditions for 21-28 days. After incubation, staining of the formed colonies with 0.005% crystal violet (CV) enabled visual inspection, photographs, and optical density measurements after CV solubilization.

Confocal analysis of tumor cell-derived spheroids

[0087] Tumor spheroids were prepared by growing 50-200 LNCaP cells in non- adherent 96-microwell culture dishes for 18 hours in RPMI containing 10% FBS. Spheroids were removed and cultured on fibronectin-coated slides in RPMI containing 2.5% FBS plus a growth factor admixture (described above). Six hours later, spheroids were fixed, immunostained with the appropriate antibody, and analyzed three-dimensionally for PRUNE2, RhoA, Nm23-Hl, and β-tubulin localization. RNA-capture library preparation and large-scale sequencing analysis

[0088] RNA molecules derived from the PRUNE2/PCA3 locus were captured and sequenced in large-scale for a comprehensive analysis of A-to-I editing. For the library preparation, mature and immature RNA molecules derived from the PRUNE 2 locus (-300 Kb) were captured by using 120 nucleotide (nt) probes designed for a 2x tiling coverage (eArray; Agilent). Captured RNAs were used as templates for the construction of libraries through the "SureSelect RNA Target Enrichment for Illumina Paired-end Multiplexed Sequencing" kit and protocol (Agilent). Libraries were sequenced by using a MiSeq instrument (Illumina) generating >3 million 150 nt reads per sample (i.e., -450 million bases sequenced per sample). Reads were aligned to the human reference genome (hgl9) with the Burrows- Wheeler Aligner (BWA-0.6.1). Repetitive regions from RepeatMasker were masked and >400,000 on-target reads for each sample remained. After removing variants present in dbSNP (release 137), all discrepancies between the sample reads and hgl (confirmed by at least three independent sequence reads) were considered and further analyzed through the CLC Genomics Workbench (version 6.0.3). A detailed analysis of putative ADAR1- and AD AR2 -mediated editing (A>G/ T>C substitutions) was performed over the locus of interest, including the mapping of putative editing sites, the determination of editing frequency (i.e., putative edits per Kb) and editing density (i.e., percentage of reads showing the alteration).

RNA-editing analysis by Sanger- equencing

[0089] Nuclear or total RNA samples were isolated and subjected to three pulses of sonication (300 Hertz) to produce RNA fragments of 1 ,000-2,000 nt, followed by DNasel treatment. RNA samples were added to two different mini-libraries of specific primers (designed to cover intronic, as well as exonic regions of PC A3 and the corresponding intron6- PRUNE2) and the admixtures were heated to 99°C for 10 min, and ice-chilled for 5 min. Specific cDNAs for PCA3 and PRUNE2 were produced by reverse transcription with Superscript II/III (Invitrogen) by using primers designed to bind intronic as well as exonic regions of PCA3 and the corresponding intron6- RLWE2. Resultant cDNAs were amplified by PCR (850-1 ,000 bp), subcloned in the TOPO vector, and individual single colonies were picked and amplified. The A-to-I editing (reflected by A>G/T>C changes) frequency was estimated by Sanger sequencing of >100 individual clones containing the expected inserts. As evidence of RNA editing, we considered only non-dbSNP alterations located outside of repetitive elements (including Alus), and only those represented by at least three distinct reads.

Tumor-bearing mouse assays

[0090] PC3 and LNCaP cell lines were established, each stably expressing ectopic PRUNE2, ectopic PC A3, control vector, C43-silenced, PRUNE2 -silenced, and control- shRNA. In each case, stably expressing pool transduced cells and their corresponding controls were allowed to grow for 48 hours to reach 85% confluence. Afterwards, paired test and control tumor cells were counted, washed in serum-free medium, and re-suspended to a final concentration of SxlO^l^"1 in serum- and phenol-free basic RPMI medium. Cells were subsequently mixed in 50% volume of phenol-free Matrigel™ (Becton Dickinson), and cell suspensions (final volume of 200μ1) were administered subcutaneously in the right flanks of male 6-week-old SCID mice (Charles River). Tumor xenograft growth was monitored serially over time. The MDA PCa 133 patient-derived xenograft (PDX) has been reported elsewhere (19, 20); total RNA was obtained from an early tumor passage and used to clone canonical PRUNE2. All animal experimentation was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas M. D. Anderson Cancer Center (MDACC).

Tissue microarray and immunohistochemistry

[0091] A human tissue microarray (TMA) was constructed from prostate cancer patients (n=145) and consisted of intermediate- or high-risk tumors (Gleason score > 7, locally advanced > pT2, peripheral zone tumors; n=95) and low-risk tumors (Gleason score 6, peripheral zone tumors, previously untreated, who underwent prostatectomy; η=5ϋ). Areas representative of all histologic tumor patterns of the Gleason grades were selected from the individual specimens. The TMA consisted of 1 ,500 cores, and each individual patient was represented by a set of 0.6 mm-diameter cores (median, 12; range, 18-53). For immunohistochemistry (IHC) analysis, images in each core of the TMA were acquired by the use of a BLISS imaging system (Bacus Laboratories) as described (55-57). A standard percentage system was used for assessment of involvement (percentage of tumor cells exhibiting detectable staining) as described (58). The extent of PRUNE2 protein expression was determined in tumor epithelium versus adjacent stromal tissue; TMA slides were stained with an anti-PRUNE2/BMCCl rabbit polyclonal antibody (ProteinTech Group) at a 1 :70 dilution. The intensity of staining was scored as absent, low, or high. An automated stainer (DAKO) and standard 3,3-diaminobenzidine were used. Tumor samples were clinically annotated and selected from a serum and tissue bank supported by the National Cancer Institute (NCI) Specialized Program of Research Excellence (SPORE) in prostate cancer at MDACC. All human experimentation was reviewed and approved by the Clinical Research Committee (CRC) and by the IRB at MDACC. All human specimens were obtained after the patients provided written informed consent under an IRB-approved experimental protocol. Total RNA samples purified from tumors from human prostate cancer patients were also obtained from the Tumor Bank at A.C. Camargo Cancer Center (ACCCC) after its IRB approval. Statistics

[0092] PRUNE2 expression in the samples was summarized by the use of standard descriptive statistics for continuous variables or tabulations for categorical variables. The primary analysis was based on the involvement score (extent of staining) alone, which was treated as a continuous variable. Statistical significance was determined by the appropriate tests. The non-parametric Wilcoxon-Mann-Whitney test served to assess differences in expression between high-grade, low-grade and bone metastatic cases, stromal and epithelial compartments. The Student's t-test or Fisher's exact test were used in the data analysis for categorical variables as appropriate. To incorporate repeated measurements (e.g. , TMA cores) from an individual patient, mixed-effects models were fitted to allow estimates of variability either within or among patients. The -values and standard deviations were reported in the data analysis with independently repeated experiments (59). All reported P- values are two-sided and levels <0.05 were considered statistically significant. Analyses were done with SAS for Windows (1999-2000; SAS Institute, Release 8.1) and S-PLUS 2000 (1988-2000; Insightful Corporation, Release 3).

Example 2 - Inhibitory Nucleic Acids for PCA3 Targeting

[0093] Synthetic small interfering RNA (siRNA) was generated based on the gene (read messenger RNA (mRNA)) of interest. In some cases, the siRNAs were constructed having 2 complementary small RNA strands (19-25 bp in length) with 2-nucleotide overhang at the 3' end. The sense (forward strand 5' to 3') and anti-sense (reverse strand 5' to 3') strands of a siRNA are called specifically 'passenger' and 'guide' strands, respectively. The passenger strand is essentially a copy of the target sequence present in the gene of interest. For targeting of the PCA3 mRNA (NCBI accession number NR 015342; SEQ ID NO:2), the following (passenger) sequences were used: shDD3C3_blN: 5 '-TGTTATTCTACAGTGTAGAAAGGTC-3 ' (SEQ ID NO: 6) shDD3C4_blN: 5'-GACCCTTCGTGTTGCTGCCTAATAT-3' (SEQ ID NO: 7) siPCA3_ITG2213: 5'-gcatttgtgggttctctta-3' (SEQ ID NO: 8)

siPCA3_ITG2370: 5'-gctcaggtgctttcactaa-3' (SEQ ID NO: 9)

siPCA3_ITG2458: 5'-gctcataggagagaatata-3' (SEQ ID NO: 10)

siPCA3_ITG2649: 5'-ccagtgtcatgagttgaattctcct-3' (SEQ ID NO: 11)

siPCA3_ITG2704: 5'-gctctcctcttgacacata-3' (SEQ ID NO: 12)

siPCA3_ITG2763: 5'-ccaacacatcgcttaccaa-3' (SEQ ID NO: 13)

siPCA3_ITG2909: 5'-gcctatgggctatattgctttagat-3' (SEQ ID NO: 14)

siPCA3_ITG3227: 5'-cctttctaatgaagatccatagaat-3' (SEQ ID NO: 15)

siPCA3_ITG3243: 5'-ccatagaatttgctacatt-3' (SEQ ID NO: 16)

siPCA3_DMC1 12: 5'-gatacagaggtgagaaataagaaag-3' (SEQ ID NO: 17)

siPCA3_DMC202: 5'-cagcaagatgacaatataatgtcta-3' (SEQ ID NO: 18)

siPCA3_DMC521 : 5'-gagaaaatcttgatggcttcacaag-3' (SEQ ID NO: 19)

[0094] For targeting of the NONO mRNA (NCBI accession number NR_007363; SEQ ID NO: 3), the following (passenger) sequences were used: siNONO_DMC1306: 5'-cagagaagctggttataaa-3' (SEQ ID NO: 20)

siNONO_DMC853: 5'-ctgaggaagaaatgaggaa-3' (SEQ ID NO: 21)

siNONO_DMC1378: 5'-gctcctttgagtatgaatat-3' (SEQ ID NO: 22)

siNONO_DMC1277: 5'-ggaccagttagatgatgaa-3' (SEQ ID NO: 23)

[0095] For targeting of the ADAR mRNA (NCBI accession number NR_015840; SEQ ID NO: 4), the following (passenger) sequences were used: siAD AR ITG914 : 5 ' -CCCG AGTTTGGAACCGGAA-3 ' (SEQ ID NO: 24) siADAR_ITG1431 : 5'-TCAAATGCCTCAAATAACATGGTAA-3' (SEQ ID NO:

25) siADAR_ITG1769: 5'-AGAATATGCCCAGTTCGCTAGTCAA-3 ' (SEQ ID NO:

26)

siADAR_ITG1916: 5 '-GCAGGATGCAGCTATGAAA-3 ' (SEQ ID NO: 27) siADAR_ITG2092: '-CCACACTGCTTGAGTGTAT-3 ' (SEQ ID NO: 28) siADAR_ITG2292: 5 '-GCGACCAACTCCATGGCTT-3 ' (SEQ ID NO: 29) siADAR_ITG2328: 5 '-GGTATGATCTCAGAGTCACTTGATA-3 ' (SEQ ID NO:

30)

siADAR_ITG2507: 5 '-GCCCAAGTTCGTTTACCAA-3 ' (SEQ ID NO: 31) siADAR_ITG2716: '-GCTTCAACACTCTGACTAA-3 ' (SEQ ID NO: 32) siADAR_ITG3112: 5 '-GCACAGAATCCCGCCACTA-3 ' (SEQ ID NO: 33) siADAR_ITG3416: 5 '-GACAAGAGATGGGAGTGCATTTGAG-3 ' (SEQ ID NO:

34)

siADAR_ITG3485 : 5 '-CAGAGTCAGCATATATGATTCCAAA-3 ' (SEQ ID NO:

35)

siADAR_DMC 1080: 5'-GACAGCAACTCCACATCTGCCTT-3 ' (SEQ ID NO: 36) siADAR_DMC3779: 5 ' -TATGGGCTATGGGAACTGGATT-3 ' (SEQ ID NO: 37)

[0096] For targeting of the DROSHA mRNA (NCBI accession number NR 013235; SEQ ID NO: 5), the following (passenger) sequences were used: siDSH_ITG494: 5 ' -GC AGCCTCCTGTGCAATAT-3 ' (SEQ ID NO: 38) siDSH JTG 1649 5 '-GGGAGATTCTACAGTGGTT-3 ' (SEQ ID NO: 39) siDSH JTG 1664 5 '-GGTTGGAACGAGTAGGCTT-3 ' (SEQ ID NO: 40) siDSH_ITG1799 5 '-CAGTGAATCCGAGTGTGAGTCTGAT-3 ' (SEQ ID NO:

41)

siDSH_ITG2197 5 '-CC AATATTCCACTGTGTAA-3 ' (SEQ ID NO: 42) siDSH_ITG2386 5 '-GCCCAAGATTTCATTTCAT-3 ' (SEQ ID NO: 43) siDSH_ITG2768 5 '-CCTAGCAAATAGTCCCAAA-3 ' (SEQ ID NO: 44) siDSH_ITG2875 5 '-CGGTGGAGCTAAGTAGCCAAGGATT-3 ' (SEQ ID NO:

45)

siDSH_ITG2990: 5 '-GCATTTGGACAAGTTGATA-3 ' (SEQ ID NO: 46) siDSH ITG3299: 5 '-CAGCGTCCATTTGTACTATTTGTTT-3 ' (SEQ ID NO:

47)

siDSH_ITG3543 : '-CAGTTATTTGGACGCTTGCTCTTTA-3 ' (SEQ ID NO: 48)

siDSH ITG3629: 5 '-GCCAAATACTGATCGACAA-3 ' (SEQ ID NO: 49) siDSH DMC1556 5 '-GAATGAGGAGGAAGAAGAA-3 ' (SEQ ID NO: 50) siDSH DMC1995 5 '-GGAATTAGGCACAGCATTT-3 ' (SEQ ID NO: 51) siDSH DMC2201 5 '-TATTCCACTGTGTAAAGTAATT-3 ' (SEQ ID NO: 52) siDSH DMC2566 5 '-AATGCAAAGGCATGATTGTT-3 ' (SEQ ID NO: 53) siDSH DMC2846 5 '-GCAGAAGAATACAATGAGA-3 ' (SEQ ID NO: 54) siDSH DMC3180 5 '-GGGATTAACACCTTGATAA-3 ' (SEQ ID NO: 55) siDSH DMC3329 '-TCTGGAAGAAGGAGGATTAG-3 ' (SEQ ID NO: 56) siDSH DMC3782 5 '-CCACAATCAGAGAATGGAATT-3 ' (SEQ ID NO: 57) siDSH DMC4184 5 '-AGGAAAAGAGCCAGACATT-3 ' (SEQ ID NO: 58) siDSH DMC4462 5 '-AGACTGAAGACATCAAGAAATAA-3 ' (SEQ ID NO:

59) [0097] For initial studies to demonstrate the effectiveness of the above sequences, double stranded si NAs will be transfected into cells and the effectiveness of gene expression knock-down will be assessed.

* # *

[0098] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of treating a subject having prostate cancer comprising administering to the subject a pharmaceutical composition comprising an effective amount of a PRUNE2 tumor suppressor or an inhibitory nucleic acid molecule that comprises sequence complimentary to all or part of a prostate cancer associated 3 (PCA3), adenosine deaminase, NONO, RNA-specific (ADAR); or drosha, ribonuclease type III (DROSHA) RNA.

2. The method of claim 1, comprising administering an effective amount of a PRUNE2 tumor suppressor.

3. The method of claims 1, the PRUNE2 tumor suppressor comprises an expression vector encoding a PRUNE2 coding sequence or a PRUNE2-coding mRNA.

4. The method of claim 3, wherein the expression vector comprises a plasmid or viral expression vector.

5. The method of claim 3, wherein expression vector encoding a PRUNE2 coding sequence or a PRUNE2-coding mRNA is provided in a liposome.

6. The method of claim 1, the PRUNE2 tumor suppressor comprises a PRUNE2 tumor suppressor polypeptide.

7. The method of claim 6, wherein the PRUNE2 tumor suppressor polypeptide is conjugated to or fused with a cell-targeting or a cell internalization moiety.

8. The method of claim 7, wherein the cell internalization moiety is at the N- terminus of the PRUNE2 tumor suppressor polypeptide.

9. The method of claim 7, wherein the cell internalization moiety is at the C- terminus of the PRUNE2 tumor suppressor polypeptide.

10. The method of claims 7, wherein the cell internalization moiety is a polypeptide, an aptamer, an antibody or an avimer.

11. The method of claim 10, wherein the antibody is an IgA, an IgM, an IgE, an IgG, a Fab, a F(ab')2, a single chain antibody, or a paratope peptide.

12. The method of claim 7, wherein the cell internalization moiety comprises internalization sequences selected from the group consisting of an HIV TAT protein transduction domain, HSV VP22 protein transduction domain, or Drosophila Antennapedia homeodomain.

13. The method of claim 7, wherein the cell internalization moiety comprises a poly-arginine, a poly-methionine and/or a poly-glycine polypeptide.

14. The method of claim 1, comprising administering an effective amount of an inhibitory nucleic acid molecule that comprises sequence complimentary to all or part of a PCA3; ADAR; or DROSHA RNA.

15. The method of claim 14, wherein the inhibitory nucleic acid molecule comprises sequence complimentary to all or part of a PCA3 RNA.

16. The method of claim 15, wherein the inhibitory nucleic acid molecule comprises sequence complimentary to all or part of SEQ ID NO: 2.

17. The method of claim 16, wherein the inhibitory nucleic acid molecule comprises sequence complimentary to any one of SEQ ID NOs: 6-19.

18. The method of claim 17, wherein the inhibitory nucleic acid molecule comprises the sequence of any one of SEQ ID NOs: 6-19 and a sequence complementary thereto.

19. The method of claim 14, wherein the inhibitory nucleic acid molecule comprises sequence complimentary to all or part of a NONO RNA.

20. The method of claim 19, wherein the inhibitory nucleic acid molecule comprises sequence complimentary to all or part of SEQ ID NO: 3.

21. The method of claim 20, wherein the inhibitory nucleic acid molecule comprises sequence complimentary to any one of SEQ ID NOs: 20-23.

22. The method of claim 21, wherein the inhibitory nucleic acid molecule comprises the sequence of any one of SEQ ID NOs: 20-23 and a sequence complementary thereto.

23. The method of claim 14, wherein the inhibitory nucleic acid molecule comprises sequence complimentary to all or part of an ADAR RNA.

24. The method of claim 23, wherein the inhibitory nucleic acid molecule comprises sequence complimentary to all or part of SEQ ID NO: 4.

25. The method of claim 24, wherein the inhibitory nucleic acid molecule comprises sequence complimentary to any one of SEQ ID NOs: 24-37.

26. The method of claim 25, wherein the inhibitory nucleic acid molecule comprises the sequence of any one of SEQ ID NOs: 34-37 and a sequence complementary thereto.

27. The method of claim 14, wherein the inhibitory nucleic acid molecule comprises sequence complimentary to all or part of a DROSHA RNA.

28. The method of claim 27, wherein the inhibitory nucleic acid molecule comprises sequence complimentary to all or part of SEQ ID NO: 5.

29. The method of claim 28, wherein the inhibitory nucleic acid molecule comprises sequence complimentary to any one of SEQ ID NOs: 38-59.

30. The method of claim 29, wherein the inhibitory nucleic acid molecule comprises the sequence of any one of SEQ ID NOs: 38-59 and a sequence complementary thereto.

31. The method of claim 14, wherein the inhibitory nucleic acid molecule is a RNA.

32. The method of claim 31 , wherein the RNA is a miRNA, siRNA or shRNA.

33. The method of claim 14, wherein the inhibitory nucleic acid molecule comprises an expression vector encoding an inhibitory RNA molecule.

34. The method of claim 33, wherein the expression vector comprises a plasmid or viral expression vector.

35. The method of claim 14, wherein the inhibitory nucleic acid molecule is provided in a liposome.

36. The method of claims 1, wherein the pharmaceutical composition is administered systemically.

37. The method of claim 1, wherein the wherein the pharmaceutical composition is administered locally.

38. The method of claim 1, wherein the pharmaceutical composition is administered two, three, four or more times.

39. The method of claim 1, further comprising administering at least a second anti-cancer therapy to the subject.

40. A composition for use in treating a subject having prostate cancer the composition comprising an effective amount to a PRU E2 tumor suppressor or an inhibitory nucleic acid molecule that comprises sequence complimentary to all or part of a PCA3, ADAR, or DROSHA RNA.

41. The method of claim 39, wherein the second anti-cancer therapy is chemotherapy, radiotherapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy or cytokine therapy.

42. A method for determining whether a subject has or is at risk for developing prostate cancer comprising:

(a) obtaining a sample from the subject; and

(b) measuring the expression level of PRUNE2 in the sample, wherein an elevated expression of PCA3 and a decreased expression of PRUNE2 relative to a reference indicates that the subject has or is at risk for developing prostate cancer.

43. An assay method comprising:

(a) obtaining a sample from a subject suspected of having prostate cancer; and (b) selectively measuring the expression level of PRU E2 in the sample.

44. The method of claim 42 or 43, further comprising measuring or selectively measuring the expression level of PCA3 in the sample.

45. The method of claim 42, further comprising measuring the expression level of PRUNE2 and PC A3 in the sample, wherein an elevated expression of PCA3 and a decreased expression of PRU E2 relative to a reference indicates that the subject has or is at risk for developing prostate cancer.

46. The method of claim 42 or 43, wherein the sample is a blood, urine, semen or tissue biopsy sample.

47. The method of claim 42 or 43, wherein measuring or selectively measuring the expression level comprises measuring a protein expression level.

48. The method of claim 47, wherein measuring protein expression levels comprises performing an ELISA, a Western blot or immunohistochemistry.

49. The method of claim 42 or 43, wherein measuring or selectively measuring the expression level comprises measuring a RNA expression level.

50. The method of claim 49, wherein measuring RNA expression levels comprises performing RT-PCR, Northern blot or an array hybridization.

51. The method of claim 42, further comprising reporting whether the subject has or is at risk for developing prostate cancer.

52. The method of claim 43, further comprising reporting the expression level.

53. The method of claim 51 or 52, wherein reporting comprises preparing a written or electronic report.

54. The method of claim 53, comprising providing the report to the patient, a doctor, a hospital or an insurance company.

55. A composition for use in treatment of a subject having prostate cancer comprising an inhibitory nucleic acid molecule complimentary to any one of SEQ ID NOs: 8- 19, 20-23, 24-37 or 38-59.

56. The composition of claim 55, wherein the inhibitory nucleic acid molecule is

RNA.

57. The composition of claim 55, wherein the inhibitory nucleic acid molecule is a double stranded RNA.