Prostate Cancer Genetic-Susceptibility Locus on Chromosome 20Q13 Is Amplified and Coupled to Androgen Receptor-Regulation in Metastatic Tumors

Published OnlineFirst December 30, 2013; DOI: 10.1158/1541-7786.MCR-13-0477

Molecular Cancer Research

Prostate Cancer Genetic-susceptibility Locus on Chromosome 20q13 is Ampliﬁed and Coupled to Androgen Receptor-regulation in Metastatic Tumors

David P. Labbe1,2, Dawid G. Nowak4, Genevieve Deblois1,3, Laurent Lessard5, Vincent Giguere 1,2,3, Lloyd C. Trotman4, and Michel L. Tremblay1,2,3

Abstract The 20q13 chromosomal region has been previously identified as the hereditary prostate cancer genetic- susceptibility locus on chromosome 20 (HPC20). In this study, the 20q13 region was shown to be frequently co-amplified with the androgen receptor (AR) in metastatic prostate cancer. Furthermore, the AR signaling axis, which plays an essential role in the pathogenesis of prostate cancer, was demonstrated to be central to the regulation of the 20q13 common amplified region (CAR). High-resolution mapping analyses revealed hot spots of AR recruitment to response elements in the vicinity of most genes located on the 20q13 CAR. Moreover, amplification of AR significantly co-occurred with CAR amplification on 20q13 and it was confirmed that the majority of AR-bound genes on the 20q13 CAR were indeed regulated by androgens. These data reveal that amplification of the AR is tightly linked to amplification of the AR-regulated CAR region on 20q13. These results suggest that the cross-talk between gene amplification and gene transcription is an important step in the development of castration-resistant metastatic disease. Implications: These novel results are a noteworthy example of the cross-talk between gene amplification and gene transcription in the development of advanced prostate cancer. Visual Overview: http://mcr.aacrjournals.org/content/early/2014/02/07/1541-7786.MCR-13-0477/F1.large.jpg. Mol Cancer Res; 12(2); 184–9. 2013 AACR.

Introduction of developing the disease (1). In fact, a positive family history Prostate cancer is the most frequent cancer in North is one of the strongest risk factors for prostate cancer and it is American men and the second leading cause of cancer- linked to approximately 10% to 15% of cases (2). related deaths. Age, African ancestry, and diet are among At least 15 different loci located on 10 distinct chromo- the known risk factors contributing to prostate cancer somes have been linked to hereditary prostate cancer (3), but – there is no single highly penetrant prostate cancer suscepti- development. In addition, evidence based on case control, fi cohort, twin, and family studies demonstrates that prostate bility gene identi ed to date. Instead, the heredity of prostate cancer is also a genetic disease. Men with a history of familial cancer is attributable to a large number of genes that have small or hereditary prostate cancer have a 2- to 7-fold increased risk effect(s) on their own, further illustrating the heterogeneity of the disease (2). In addition, recent genome-wide association studies revealed a minimum of 30 common genetic loci associated with prostate cancer risk, making this disease the Authors' Affiliations: 1Goodman Cancer Research Centre; 2Department fi of Medicine, Division of Experimental Medicine; 3Department of Biochem- most proli c of all cancers in term of common susceptibility istry and Oncology, McGill University, Montreal, Quebec, Canada; 4Cold loci. However, there is no clear evidence that the various loci 5 Spring Harbor Laboratory, NY; and Department of Molecular Oncology, associated with the risk of developing prostate cancer are also John Wayne Cancer Institute at St. John's Health Center, Santa Monica, California associated with either aggressiveness or mortality (4). Current techniques enable the detection and treatment of Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). most early stage tumors. Still, androgen-deprivation therapy Corresponding Authors: Michel L. Tremblay, Goodman Cancer Research targeting androgen receptor (AR) transcriptional activity has Centre, McGill University, 1160 Pine Avenue, Room 617, Montreal, QC, remained the first line of treatment for advanced disease since Canada H3A 1A3. Phone: 514-398-8280; Fax: 514-398-6769; E-mail: its description in 1941, although it ultimately leads to [email protected]; and Lloyd C. Trotman, Cold Spring Harbor Laboratory, 1 Bungtown Road, James Building, Room 210, Cold Spring incurable castration-resistant metastatic prostate cancer Harbor, NY 11724. Phone: 516-367-5054; Fax: 516-367-8454; E-mail: (CRMPC). Strikingly, most CRMPCs still rely on the AR [email protected] transcriptional activity due to different adaptive mechanisms, doi: 10.1158/1541-7786.MCR-13-0477 such as AR mutation, ligand-independent AR activation, 2013 American Association for Cancer Research. endogenous androgen synthesis, or even AR amplification (5).

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In this study, we report that a region of the previously (www.cbioportal.org) using the dataset by Taylor and collea- identified hereditary prostate cancer genetic-susceptibility gues (7). mRNA expression was represented as the Z-score of locus located on chromosome 20 (HPC20; 6) is frequently prostate cancer samples versus normal prostate samples. Then, coamplified with the AR in metastatic prostate cancer the average Z-scores from primary samples were subtracted tumors. Interestingly, we found that this region is also a from the average Z-scores from metastatic samples. hot spot for AR recruitment to chromatin. We show that AR binds and regulates most genes within the common ampli- Statistical analysis fied region (CAR), suggesting that the coordinated copy Statistical analyses were performed with the Prism 5.0 number gain and increased transcriptional output of the GraphPad Software. The significance of gene expression 20q13 CAR might be an important event leading to the modulation following R1881 treatment was assessed by the development of CRMPC. Mann–Whitney test. The differences in the risk of biochemical relapse were computed using the log-rank test. Materials and Methods Copy number alteration in prostate cancer Results Analysis of copy number alteration on the 20q chromo- Given the addiction of prostate cancer cells to AR tran- somal arm is based on the published copy number profiles scriptional activity, androgen-deprivation therapy invariably from 181 primary and 37 metastatic prostate tumors (7) results in adaptive mechanisms that maintain AR signaling. using the Nexus Copy Number software v6.0 (Biodiscovery Accordingly, AR gene amplification was observed exclusively Inc.). Circos graph was performed using the R software and in CRMPC samples (7). In addition, metastatic tumors RCircos package (8). displayed an array of DNA copy number alterations (CNA) scattered throughout the genome that can possibly synergize Cell culture with AR gene amplification. Analysis of metastatic prostate LNCaP cells were purchased from the American Type cancer samples revealed frequent coamplification (35%) Culture Collection and maintained in RPMI-1640 medium of several genomic regions on eight different chromosomes (Wisent) supplemented with 10% FBS, L-glutamine, and 50 with the AR gene (Fig. 1). Surprisingly, our analysis uncov- mg/mL gentamycin. The synthetic androgen analog R1881 ered that the AR is significantly coamplified with a region was obtained from PerkinElmer. For androgenic stimulation located on the 20q chromosome arm, previously identified as assays, cells were first androgen deprived in phenol-free HPC20 (6), uniquely in metastatic tumors (Supplementary RPMI 1640 supplemented with 5% charcoal-stripped FBS, Fig. S1). We determined that the CAR on 20q13 is located L-glutamine, and 50 mg/mL gentamycin. After 48 hours, within the HPC20, which is flanked by the markers medium was refreshed and R1881 or ethanol (vehicle) was D20S887 and D20S196 (Figs. 2A and B, orange bars). added for the indicated period.

ChIP assays and ChIP-on-chip analysis on chr.20 tiled

X array 1 22 Chromatin was prepared from LNCaP cells exposed to 1 21 AR nmol/L R1881 or vehicle for 4 hours. Chromatin immuno- 20 2 precipitation (ChIP) was performed as described previously 19 20q13 (9) using antibodies specific to AR (mouse monoclonal anti- 18 AR from Lab Vision and BD Biosciences). Amplification and 17 3 labeling of AR-bound ChIP fragments was performed as 16 described previously (9). Hybridization was carried out on 4 a custom-designed tiled array from Agilent covering the q arm 15 of chr.20 at a resolution of 150 bp and analyzed from assembly hg18, using the Feature Extraction 10 alignment program and 14 ChIP Analytics 3.1 program for peak detection (Agilent). 5 13 Analysis of gene expression 6 Total RNA extraction, reverse transcription, and quanti- 12 tative real-time PCR were performed as already described MIR645 11 7 (9). For reverse transcription, the qScript micro- 8 10 RNA cDNA synthesis kit was used (Quanta Biosciences). 9 The primer sequences used can be found in Supplementary Table S1. Threshold cycle numbers were calculated using the Figure 1. AR is coamplified with a region within the HPC20 in metastatic second derivative maximum obtain with the LightCycler prostate cancer. Circos plot of genome-wide coamplification events in AR amplified metastatic samples at P 0.0125. The 20q13, within the 480 software version 3.5 (Roche). Data were normalized HPC20 locus, is the sole region associated with AR amplification on according to RPLP0 levels (Supplementary Table S1). mRNA chromosome 20. A total of eight different chromosomes possess at least expression Z-scores were obtained from the cBIO portal one locus significantly associated with AR amplification.

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A 20q

30 Mb 35 Mb 40 Mb 45 Mb 50 Mb 55 Mb 60 Mb

Genes Exons CNVs miRNA

20q12 – 20q13.2 B 15% 10% 5% 0% 5% Genes Exons CNVs miRNA

C 20q12 – 20q13.33

40 Mb 45 Mb 50 Mb 55 Mb

AR binding region

Significant Genes Exons CNVs miRNA hsa-mir-646

AR amplified 36% (22 samples)

AR not ampl. (15 samples) 0%

Figure 2. The 20q13 CAR is rich in AR binding sites and is coamplified with the AR. A, representation of the amplified (blue) and deleted (red) regions on the entire 20q chromosomal arm for patients demonstrating amplification of the 20q13 CAR (orange bars) in primary and metastatic tumors. Localization of the AR-bound segments identified by ChIP-on-chip analysis is also indicated (vertical red lines, P 10 5). B, zoomed representation of panel A. C, overview of the 20q12–20q13.33 locus revealed that the genomic region significantly associated with AR coamplification in metastatic cancer falls within the AR-dense binding region identified within the HPC20 (green).

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Owing to the frequent coamplification of the 20q13 static prostate cancer. Notably, this region encodes two CAR with the AR in metastatic prostate cancer, we inves- transcription factors, namely SNAI1 and CEBPB. SNAI1 tigated whether this region was subjected to AR-transcrip- is an important mediator of the epithelial-mesenchymal tional regulation. We performed genomic location analysis transition, a critical event in the metastatic process (14), of AR recruitment to chromatin in the androgen-sensitive and whose expression mediates cell survival and inhibits LNCaP cell line, using a tiled array covering the q arm of cellular senescence in metastatic prostate cancer cell lines human chromosome 20. Cells were maintained in a (15). On the other hand, CEBPB is a recognized oncogene steroid-depleted medium for 48 hours before stimulation in Ras-mediated tumorigenesis (16). In addition, the with the synthetic androgen analog R1881 for 4 hours. 20q13 CAR encodes MIR645, a member of the micro- ChIP-on-chip analyses reveal 245 segments bound by the RNA family that regulates gene expression posttranscrip- AR, mostly in nonpromoter regions (Supplementary Table tionally. Beside a single report suggesting that the coex- S2, P 10 5). Strikingly, the 20q13 CAR is a local hot pression of MIR410 and MIR645 is negatively associated spot in AR binding sites (Figs. 2A and B, vertical red lines). with overall survival in advanced serous ovarian cancer Refined analysis of this locus uncovered that AR coampli- (17), the role of MIR645 in cancer remains unknown. fication with the 20q13 is restricted to the CAR in Similarly, a recurrent lung cancer amplicon located at metastatic prostate cancer, which also corresponds to a 14q13.3 was found to encode three transcription factors, region rich in AR binding sites (Fig. 2C). namely TTF1/NKX2-1, NKX2-8,andPAX9.Remarkably, Fourteen genomic segments bound by AR in LNCaP cells although the overexpression of a single transcription factor were associated with 10 of the 16 genes (62.5%) present did not modify the proliferation of premalignant lung within the 20q13 CAR (Fig. 3A). To validate the relevance epithelial cell, overexpression of any pairwise combination of this AR binding profile, we examined the impact of AR led to a major increase in their tumorigenic potential (18). activation on the expression of amplicon-resident genes. Together, the altered transcriptional output mediated by R1881 treatment resulted in the significant modulation of the coordinated DNA copy number gain of SNAI1, 7 of the 10 genes that we found associated with AR-bound CEBPB,andMIR645 might contribute to a global tran- segments (Fig. 3B, P < 0.01). The three remaining genes scriptional rewiring process, ultimately resulting in the were either not affected by R1881 treatment (PARD6B), increased tumor aggressiveness observed in patients bear- were not expressed in LNCaP cells (LOC284751), or were ing the 20q13 CAR. impossible to distinguish from a chimeric transcript with A noteworthy feature of the 20q13 CAR is its extensive specific primers (UBE2V1 vs. TMEM189-UBE2V1; Fig. regulation by AR. ChIP-on-chip analysis using the andro- 3B). Interestingly, we found that the expression level of most gen-sensitive LNCaP cell line revealed AR binding in the of the genes within the 20q13 CAR was increased in vicinity of a large proportion of genes within the 20q13 metastatic samples (Fig. 3C). In addition, analysis of the CAR (Fig. 3A). Importantly, this result was not biased by three metastatic samples positives for AR and CAR coam- chromosomal abnormalities because LNCaP cells do not plification for which clinical survival data were collected (7) have CNA in the 20q13 region (19). Surprisingly, most demonstrate significant earlier biochemical recurrence com- protein-coding transcripts from AR-bound genes were pared with patients without AR and CAR amplification (Fig. sensitive to R1881 treatment (Fig. 3B). As previously 3D). Together, these results suggest that AR, through its described, PTPN1 mRNA expression was strongly regu- amplification/activation, could synergistically enhance the lated by the AR (9). We also identified several novel AR- expression of genes within the 20q13 CAR in advanced regulated genes within the 20q13 CAR with functions yet disease and significantly alter patient's prognosis when to be described in the prostate, including the solute carrier coamplified with the CAR. family 9, member 8 (SLC9A8), the spermatogenesis- associated protein 2 (SPATA2), the ring finger protein 114 (RNF114), and the activity-dependent neuroprotec- Discussion tor (ADNP). Interestingly, the breast cancer amplified Genetic linkage studies identified several susceptibility sequence 4 (BCAS4)wasmodestlybutsignificantly reg- loci for type II diabetes (10, 11) and obesity (12) mapping ulated by R1881. Finally, we also describe CEBPB to the D20S196 marker located on the 20q13 chromosomal (encoding C/EBPb) as an AR-regulated target. Zhang region. In addition, this region, flanked by the markers and colleagues recently demonstrated that C/EBPb can D20S196 and D20S887 (HPC20), has been described as effectively transactivate the prostate-specificantigen a hereditary prostate cancer susceptibility locus (6). Inter- (PSA) promoter, which is regulated by androgen response estingly, we and others have shown that the protein elements in the absence of androgen. However, increased tyrosine phosphatase 1B (encoded by PTPN1), which is C/EBPb expression results in a decreased transactivation located in the vicinity of the D20S196 marker and is part of of the PSA promoter in the presence of androgen. In the CAR, is indeed implicated in type II diabetes and contrast, AR activation results in an increased C/EBPb obesity (13) and plays a tumor-promoting role in prostate transcriptional activity on CCAAT enhancer binding cancer (9). protein elements (20). This interesting cross-talk, togeth- In this study, we further demonstrate that the 20q13 er with the fact that AR-regulation of the 20q13 CAR is chromosomal region is significantly amplified in meta- accompanied by AR coamplification, supports a complex

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A Chr. 20 20p13 p12.3 12.2 20p12.1 20q12 13.12 20q13.2 q13.33

20q13 CAR D20S196 D20S887

SLC9A8 CEBPB PARD6B

SPATA2 AK307192 BCAS4

RNF114 LOC284751 ADNP SNAI1 PTPN1 UBE2V1 MIR645

TMEM189-UBE2V1 FAM65C Figure 3. The 20q13 CAR is AR recruitment in gene vicinity (ChIP-on-chip) TMEM189 androgen regulated, upregulated in metastatic disease, and B C associated with earlier biochemical recurrence when coampliﬁed with AR bound 3 the AR. A, binding proﬁle of the AR AR unbound from ChIP-on-chip analysis 3.0 * 2 performed in R1881-treated 2.5 LNCaP cells on a high resolution * tiled array covering the 20q 2.0 1 chromosomal arm revealed that a * majority of genes within the 20q13 1.5 * * * CAR possess AR binding sites in (normalized) 1.0 0 their vicinity. B, R1881 treatment * (10 nmol/L, 24 hours) modulates Relative gene expression 0.5 the mRNA-expression level of −1 primary mRNA expression Z -scores most genes within the 20q13 CAR 0.0

1 B 4 Difference between averaged metastatic and 8 1 1 B C R A that were associated with AR DNP ADNP A – Vehicle SNAI1 CEBPB PTPN AM65C BCAS SNAI1 CEBPPTPN1 BCAS4 binding sites in their vicinity (Mann SLC9A8SPATA2RNF114 UBE2V1 MIR645F PARD6 SLC9ASPATA2RNF114 UBE2V FAM65PARD6B TMEM189AK307192 TMEM189 LOC284751 Whitney test, P < 0.01 compared with vehicle; N ¼ 3, SEM). C, TMEM189-UBE2V1 TMEM189-UBE2V genes within the CAR are preferentially upregulated in D 100% metastatic samples compared with primary samples as demonstrated by the difference between the averages of mRNA Z-scores (primary samples, N ¼ 109; 80% metastatic samples, N ¼ 19). D, the risk of biochemical relapse is significantly higher in patients AR CARAmp harboring and CAR 60% coamplifications (N ¼ 3) compared with patients with neither regions ** amplified (N ¼ 169) (log-rank test; ARNo Amp ; CARNo Amp Amp Amp CAR , N ¼ 10; AR , N ¼ 15).

40%

20% ARAmp Biochemical recurence free survival Biochemical

ARAmp ; CARAmp 0% * P < 0.00005 0 25 50 75 100 125 150 Time (months)

transcriptional rewiring in a significant subset of meta- region that had been previously identified as a hereditary static tumors. prostate cancer susceptibility locus. The extensive regulation In summary, we report the frequent amplification of the of genes within the 20q13 CAR by AR, some of which 20q13 chromosomal region in metastatic prostate cancer, a already associated with oncogenic functions, suggests that

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AR Regulation of the 20q13 Amplicon

the highly specific coamplification of this chromosomal Writing, review, and/or revision of the manuscript: D.P. Labbe, D.G. Nowak, AR L. Lessard, V. Giguere, L.C. Trotman, M.L. Tremblay region with the might synergize and contribute to Administrative, technical, or material support (i.e., reporting or organizing data, increased tumor aggressiveness and to the development of constructing databases): D.P. Labbe, G. Deblois, L.C. Trotman metastatic disease. These findings reflect the fact that AR Study supervision: V. Giguere, L.C. Trotman, M.L. Tremblay amplification is not frequently seen at diagnosis but is very Acknowledgments common in advanced therapy-resistant diseases. Thus, like The authors thank Serge Hardy and Kelly-Anne Pike for helpful discussions and AR amplification status, AR–CAR amplification status Noriko Uetani for technical assistance with figure design and drawing. should be most informative as marker for disease progression fl after therapeutic intervention. These results may also re ect Grant Support a potential example of the amplification of a locus after D.P. Labbe is a recipient of a Canadian Institute of Health Research (CIHR) enhanced transcriptional activity at that locus. Finally, these Frederick Banting and Charles Best Doctoral Research Award and a CIHR/Fonds de recherche du Quebec–Sante Training Grant in Cancer Research FRN53888 of the results justify further investigation to address the respective McGill Integrated Cancer Research Training Program. G. Deblois is supported by a roles of the different genes within the 20q13 CAR. Predoctoral Traineeship Award (W81XWH-10-1-0489) from the U.S. Department of Defense Breast Cancer Research Program. This work was supported by CIHR fl operating grants to V. Giguere (MOP-64275) and M.L. Tremblay (MOP-62887) Disclosure of Potential Con icts of Interest as well as a U.S. Army Department of Defense Award (#W81XWH-09-1-0259), a fl No potential con icts of interest were disclosed. Prostate Cancer Canada Grant (#02013-33) and a Jeanne and Jean-Louis Levesque Chair in Cancer Research to M.L. Tremblay, and by grants to L.C. Trotman from the Authors' Contributions NIH (CA137050), the Department of the Army (W81XWH-09-1-0557), and the Conception and design: D.P. Labbe, L. Lessard, V. Giguere, L.C. Trotman, Robertson Research Fund of Cold Spring Harbor Laboratory. The costs of publication of this article were defrayed in part by the payment of page M.L. Tremblay advertisement Development of methodology: D.P. Labbe, D.G. Nowak, L.C. Trotman charges. This article must therefore be hereby marked in accordance with Acquisition of data (provided animals, acquired and managed patients, provided 18 U.S.C. Section 1734 solely to indicate this fact. facilities, etc.): D.P. Labbe, G. Deblois Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- Received September 9, 2013; revised November 20, 2013; accepted December 3, tational analysis): D.P. Labbe, D.G. Nowak, G. Deblois, L.C. Trotman 2013; published OnlineFirst December 30, 2013.

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www.aacrjournals.org Mol Cancer Res; 12(2) February 2014 189

Prostate Cancer Genetic-susceptibility Locus on Chromosome 20q13 is Amplified and Coupled to Androgen Receptor-regulation in Metastatic Tumors

David P. Labbé, Dawid G. Nowak, Geneviève Deblois, et al.

Mol Cancer Res 2014;12:184-189. Published OnlineFirst December 30, 2013.

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