WO2016019311A1 - Méthodes pour la détection et le traitement d'un cancer de la prostate - Google Patents

Méthodes pour la détection et le traitement d'un cancer de la prostate Download PDF

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WO2016019311A1
WO2016019311A1 PCT/US2015/043240 US2015043240W WO2016019311A1 WO 2016019311 A1 WO2016019311 A1 WO 2016019311A1 US 2015043240 W US2015043240 W US 2015043240W WO 2016019311 A1 WO2016019311 A1 WO 2016019311A1
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prune2
rna
nucleic acid
pca3
acid molecule
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PCT/US2015/043240
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English (en)
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Ahmed SALAMEH
Alessandro LEE
Diana N. NUNES
Emmanuel DIAD-NETO
Renata PASQUALINE
Wadih Arap
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Stc.Unm
Board Of Regents, University Of Texas System
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Publication of WO2016019311A1 publication Critical patent/WO2016019311A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides

Definitions

  • the present invention relates generally to the field of oncology and molecular biology. More particularly, it concerns methods for detecting and treating prostate cancers.
  • 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.
  • PSA prostate-specific antigen
  • IncRNA long noncoding RNA
  • PCA3 prostate cancer antigen 3
  • 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).
  • the biological function of PC A3 has proved elusive.
  • 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.
  • the PRUNE2 tumor suppressor may comprise an expression vector encoding a PRUNE2 coding sequence or a PRUNE2 -coding mRNA.
  • the expression vector comprises a plasmid or viral expression vector.
  • the expression vector encoding a PRUNE2 coding sequence or a PRUNE2-coding mRNA is provided in a nanoparticle or liposome.
  • 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.
  • 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.
  • 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.
  • the cell internalization moiety comprises a poly-arginine, a poly-methionine and/or a poly-glycine polypeptide.
  • 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.
  • 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).
  • PCA3, ADAR, NONO or DROSHA is an inhibitory nucleic acid molecule.
  • the inhibitory nucleic acid molecule may comprise a sequence complimentary to all or part of a PCA3, ADAR, NONO or DROSHA RNA.
  • the inhibitory nucleic acid molecule is a RNA, such as a miRNA, siRNA or shRNA.
  • 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.
  • the inhibitory nucleic acid molecule is provided in a nanoparticle or a liposome.
  • aspects of the embodiments concern inhibitory nucleic acid molecules complementary to all or part of a human PCA 3 RNA.
  • 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).
  • 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 .
  • inhibitory nucleic acid molecules that are complementary to all or part of a human drosha, ribonuclease type III (DROSHA) RNA.
  • 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.
  • 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.
  • ADAR RNA-specific
  • 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.
  • 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.
  • a pharmaceutical composition of the embodiments may be administered to the subject systemically or locally.
  • the pharmaceutical composition may be administered two, three, four or more times.
  • 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.
  • 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.
  • 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.
  • an inhibitory nucleic acid of the embodiments is an RNA molecule, such as a double stranded RNA (dsRNA) molecule.
  • an inhibitory nucleic acid is a dsRNA having a 1 or 2 nucleotide 3' overhang on one or both strands.
  • 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.
  • 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).
  • an inhibitory nucleic acid molecule of the embodiments comprises a sequence complimentary to any one of SEQ ID NOs: 6-19.
  • an inhibitory nucleic acid molecule comprises the sequence of any one of SEQ ID NOs: 6-19 and a sequence complementary thereto.
  • an inhibitory nucleic acid of the embodiments is an RNA molecule.
  • an inhibitory nucleic acid molecule of the embodiments is formulated in a pharmaceutically acceptable delivery vehicle, such as in a liposome or nanoparticle formulation.
  • 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).
  • an inhibitory nucleic acid molecule of the embodiments comprises a sequence complimentary to any one of SEQ ID NOs: 20-23.
  • an inhibitory nucleic acid molecule comprises the sequence of any one of SEQ ID NOs: 20-23 and a sequence complementary thereto.
  • an inhibitory nucleic acid of the embodiments is an NA molecule.
  • an inhibitory nucleic acid molecule of the embodiments is formulated in a pharmaceutically acceptable delivery vehicle, such as in a liposome or nanoparticle formulation.
  • 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).
  • an inhibitory nucleic acid molecule of the embodiments comprises a sequence complimentary to any one of SEQ ID NOs: 24-37.
  • an inhibitory nucleic acid molecule comprises the sequence of any one of SEQ ID NOs: 24-37 and a sequence complementary thereto.
  • an inhibitory nucleic acid of the embodiments is an RNA molecule.
  • an inhibitory nucleic acid molecule of the embodiments is formulated in a pharmaceutically acceptable delivery vehicle, such as in a liposome or nanoparticle formulation.
  • 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).
  • an inhibitory nucleic acid molecule of the embodiments comprises a sequence complimentary to any one of SEQ ID NOs: 38-59.
  • an inhibitory nucleic acid molecule comprises the sequence of any one of SEQ ID NOs: 38-59 and a sequence complementary thereto.
  • an inhibitory nucleic acid of the embodiments is an RNA molecule.
  • an inhibitory nucleic acid molecule of the embodiments is formulated in a pharmaceutically acceptable delivery vehicle, such as in a liposome or nanoparticle formulation.
  • the invention provides a method for determining whether a subject has or is at risk for developing prostate cancer.
  • 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.
  • 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.
  • the method further comprises reporting whether the subject has or is at risk for developing prostate cancer.
  • reporting may comprise preparing an oral, written or electronic report.
  • the report is provided to the subject or to a healthcare worker.
  • 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.
  • the method further comprises reporting the expression level.
  • the expression levels of PRU E2 and PCA3 are selectively measured in the sample.
  • 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.
  • 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.
  • 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.
  • protein e.g., phosphoprotein
  • nucleic acid e.g., mRNA
  • “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.
  • 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.
  • 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.
  • prostate cancer LNCaP cells 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 ⁇ ). 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.
  • 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).
  • 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.
  • 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).
  • RNA- FISH C
  • D RT-qPCR
  • E Immunoblot analysis of PRUNE2 expression in LNCaP P54 NRB -silenced cells or negative control is shown (E).
  • 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, ⁇ -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.
  • 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 5xl0 6 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.
  • FIGS. 6A-6K PRUNE2/PCA3 expression and RNA editing in clinical samples from prostate cancer patients.
  • 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.
  • NB Northern blot
  • (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.
  • 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.
  • 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
  • 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).
  • FIG. 10 Effects of PC A3 expression on PRUNE2 coding sequence.
  • FIGS. 11A-11D Nuclear co-localization of PRUNE2 pre-mRNA
  • 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).
  • 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,
  • 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.
  • Labeled oligonucleotides PC A 3 -cy3 (located in exon 4) and PRUNE2-cy5 (located in PRUNE2 intron 6) were used.
  • PCA3 located in exon 4
  • PRUNE2 located in PRUNE2 intron 6
  • 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 ⁇ .
  • 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.
  • RT- qPCR analysis for PCA3 and PRUNE2 mRNA C.
  • 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.
  • 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).
  • 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 ⁇ . Representative images are shown.
  • 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.
  • 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.
  • 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.
  • 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 . conserveed 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.
  • 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.
  • FIGS. 21A-21L Biological function of PRUNE2 In prostate cancer cells.
  • 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.
  • 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.
  • a prostate cancer may be treated by restoring the activity of PRUNE2 in the cancer cells.
  • PRUNE2 protein or protein coding sequence
  • an inhibitory nucleic acid can be delivered to down-regulate PCA3 RNA expression and thereby increase endogenous PRUNE2 expression in the cancer cells.
  • the identification of PRU E2 as a key regulator of prostate cancer growth provides new diagnostic methods for early cancer detection.
  • PRUNE2 expression levels from a sample of a subject can be used to detect prostate cancer (or a risk of developing prostate cancer).
  • PRU E2 expression can be measured in conjunction of PCA3 expression thereby enhancing the sensitivity and specificity of prostate cancer diagnostic testing.
  • 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.
  • RNA samples were screened from human prostate cancers (cell lines and tumor samples) by RT-PCR.
  • 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).
  • 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).
  • RNA fluorescence in situ hybridization FISH was used to determine whether PRUNE2/PCA3 form a dsRNA.
  • 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.
  • 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.
  • 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).
  • 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.
  • A-to-I adenosine -to-inosine
  • RNA-ChIP RNA-chromatin immunoprecipitation
  • 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).
  • 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).
  • DBHS Drosophila behavior human splicing 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).
  • Androgen-dependence and resistance to androgen-deprivation therapy are central to the biological and clinical features of human prostate cancer.
  • AR androgen receptor
  • PRUNE2 was the most down- regulated transcript when compared to either 5 ' -PRUNE 2 or 3 ' -BMCCl transcripts (FIGS. 4B and 4D).
  • human prostate cancer cell lines LNCaP, PRU E2-expressing; PC3, PRUNE2-deficient
  • 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).
  • 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).
  • LNCaP prostate cancer cells stably expressing PRUNE2-s RNA, ectopic PCA3, G43-shRNA, or controls were subcutaneously injected into severe combined immunodeficiency (SCID) mice.
  • SCID severe combined immunodeficiency
  • 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).
  • PSA serum prostate-specific antigen
  • 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).
  • PCA3 and PRUNE2 The expression of PCA3 and PRUNE2 in human prostate cancer samples was examined.
  • RT-qPCR reverse-transcription quantitative PCR
  • 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.
  • 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.
  • TCGA Cancer Genome Atlas
  • Anti-bromodeoxyuridine (BrdU; Millipore), anti- ⁇ Actin, anti- ⁇ 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.
  • VEGF Vascular endothelial growth factor
  • basic fibroblast growth factor (bFGF) basic fibroblast growth factor (bFGF)
  • EGF epidermal growth factor
  • IGF insulin-like growth factor
  • An admixture i.e., 10 ng EGF, 10 ng bFGF, 10 ng IGF, 20 ng VEGF
  • heparin 5 units/ml
  • Methyltrienolone R1881 ; Perkin Elmer was used for androgenic stimulation in steroid-deprived conditions as indicated.
  • RNaseA and RNase III were commercially obtained.
  • Secondary antibodies were purchased (Jackson ImmunoResearch or Invitrogen).
  • 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.
  • PrEC Human epithelial
  • RWPE-1 epithelial
  • RWPE-2 epithelial
  • WPMY-1 stromal transformed prostate cells
  • VaP, 22Rv prostate cancer cells
  • 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
  • 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 ⁇ shRNA was created by site-directed mutagenesis.
  • 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, ⁇ - 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 LNATM oligonucleotides (Exiqon) were used for PC A3 and PRUNE2.
  • 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 TRCN000005
  • P54 NRB -shRNA 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).
  • 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 ⁇ 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.
  • 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 ⁇ lysis buffer A [10 mM Tris (pH8.0), 140 mM NaCl, 1.5 mM MgCl 2 , 0.1% IGEPAL, 2 mM vanadyl ribonucleoside complex], and incubated on ice for 5 min.
  • PBS ice-cold phosphate-buffered saline
  • RNA fluorescence in situ hybridization and confocal microscopy 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.
  • RNAse inhibitor 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.
  • Immunoprecipitation assays were performed as described (49). Briefly, a total of 3 x 10 6 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.
  • 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 ⁇ 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 Na 3 VC>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.
  • 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.
  • immunoprecipitation buffer 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% IGEPAL, 1 mM phenylmethylsulfonyl fluoride (PMSF), proteinase inhibitor cocktail; (Sigma)
  • PMSF phenylmethylsulfonyl fluoride
  • PMSF proteinase inhibitor cocktail
  • R aseA-treated and non-treated cell extracts were pre-cleared with 40 ⁇ protein- A- plus protein-G-agarose beads (Roche) and 2.5 ⁇ g mouse anti-ADARl antibody or irrelevant isotype control antibody at 4°C for 2 hours followed by 40 ⁇ protein-A- plus protein-G- agarose beads for 30 min at RT.
  • 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).
  • UV ultraviolet
  • Cells were seeded in 200 ⁇ 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.
  • ELISA cell death detection enzyme-linked immunosorbent assay
  • Cells were seeded in 200 ⁇ 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.
  • a growth factor admixture described above
  • 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 ⁇ -tubulin localization. RNA-capture library preparation and large-scale sequencing analysis
  • RNA molecules derived from the PRUNE2/PCA3 locus were captured and sequenced in large-scale for a comprehensive analysis of A-to-I editing.
  • mature and immature RNA molecules derived from the PRUNE 2 locus were captured by using 120 nucleotide (nt) probes designed for a 2x tiling coverage (eArray; Agilent).
  • nt nucleotide
  • 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).
  • 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.
  • 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.
  • PC3 and LNCaP cell lines were established, each stably expressing ectopic PRUNE2, ectopic PC A3, control vector, C43-silenced, PRUNE2 -silenced, and control- shRNA.
  • stably expressing pool transduced cells and their corresponding controls were allowed to grow for 48 hours to reach 85% confluence.
  • 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.
  • 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).
  • IHC immunohistochemistry
  • 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.
  • 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.
  • 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.
  • repeated measurements e.g. , TMA cores
  • siRNA small interfering RNA
  • mRNA read messenger RNA
  • 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.
  • 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)
  • 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)
  • siADAR_ITG1769 5'-AGAATATGCCCAGTTCGCTAGTCAA-3 ' (SEQ ID NO:
  • siADAR_ITG1916 5 '-GCAGGATGCAGCTATGAAA-3 ' (SEQ ID NO: 27)
  • siADAR_ITG2092 '-CCACACTGCTTGAGTGTAT-3 '
  • siADAR_ITG2292 5 '-GCGACCAACTCCATGGCTT-3 '
  • siADAR_ITG2328 5 '-GGTATGATCTCAGAGTCACTTGATA-3 ' (SEQ ID NO:
  • siADAR_ITG2507 5 '-GCCCAAGTTCGTTTACCAA-3 '
  • siADAR_ITG2716 '-GCTTCAACACTCTGACTAA-3 '
  • siADAR_ITG3112 5 '-GCACAGAATCCCGCCACTA-3 '
  • siADAR_ITG3416 5 '-GACAAGAGATGGGAGTGCATTTGAG-3 '
  • siADAR_ITG3485 5 '-CAGAGTCAGCATATATGATTCCAAA-3 ' (SEQ ID NO:
  • siADAR_DMC 1080 5'-GACAGCAACTCCACATCTGCCTT-3 ' (SEQ ID NO: 36)
  • siADAR_DMC3779 5 ' -TATGGGCTATGGGAACTGGATT-3 ' (SEQ ID NO: 37)
  • 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:
  • siDSH_ITG2197 5 '-CC AATATTCCACTGTGTAA-3 ' (SEQ ID NO: 42)
  • siDSH_ITG2386 5 '-GCCCAAGATTTCATTTCAT-3 '
  • siDSH_ITG2768 5 '-CCTAGCAAATAGTCCCAAA-3 '
  • siDSH_ITG2875 5 '-CGGTGGAGCTAAGTAGCCAAGGATT-3 '
  • siDSH_ITG2990 5 '-GCATTTGGACAAGTTGATA-3 ' (SEQ ID NO: 46)
  • siDSH ITG3299 5 '-CAGCGTCCATTTGTACTATTTGTTT-3 ' (SEQ ID NO:
  • 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) si

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Abstract

L'invention concerne des méthodes permettant de détecter et de traiter des cancers de la prostate. Selon certains aspects, lesdites méthodes permettant de détecter un cancer de la prostate consistent à mesurer un niveau d'expression de PRUNE2 et/ou d'expression de PCA3 dans un échantillon provenant d'un sujet. Selon d'autres aspects, une méthode permettant de traiter un cancer de la prostate consiste à administrer un polypeptide PRUNE2 suppresseur de tumeur ou une molécule d'acide nucléique qui inhibe l'expression de l'ARN de PCA3.
PCT/US2015/043240 2014-08-01 2015-07-31 Méthodes pour la détection et le traitement d'un cancer de la prostate WO2016019311A1 (fr)

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