Prostate cancer genetic-susceptibility locus on chromosome 20q13 is androgen receptor-regulated and amplified in metastatic tumors

Abstract

The 20q13 chromosomal region has been previously identified as the hereditary prostate cancer genetic-susceptibility locus on chromosome 20 (HPC20). In this study, we demonstrate that this region is also frequently co-amplified with the androgen receptor (AR) in metastatic prostate cancer. We show that the AR signaling axis, which plays a key role in the pathogenesis of prostate cancer, is central to the regulation of the 20q13 common amplified region (CAR). High-resolution location analyses revealed hot spots of AR recruitment to response elements in the vicinity of most genes located on the 20q13 CAR. We found that amplification of AR significantly co-occurred with amplification of the CAR on 20q13 and confirmed that most AR-binding 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.

Introduction

Prostate cancer (PCa) is the most frequent cancer in North American men and the second leading cause of cancer-related death. Age, African ancestry and diet are among the known risk factors contributing to PCa development. Additionally, evidence from case-control, cohort, twin and family-based studies demonstrate that PCa is also a genetic disease. Men with a history of familial or hereditary PCa have a two- to seven-fold increased risk of developing the disease (1). In fact, a positive family history is one of the strongest risk factors for PCa and it is linked to approximately 10 to 15% of cases (2).

At least 15 different loci located on 10 distinct chromosomes have been linked to hereditary PCa (3) but there is no single highly penetrant PCa susceptibility gene identified to date. Instead, the heredity of PCa is attributable to a large number of genes that have small effect(s) on their own, further illustrating the heterogeneity of the disease (2). Additionally, recent genome-wide association studies revealed a minimum of 30 common genetic loci associated with PCa risk, making this disease the most prolific of all cancers in term of common susceptibility loci. However, there is no clear evidence that the various loci associated with the risk of developing PCa are also associated with either aggressiveness or mortality (4).

Current techniques enable the detection and treatment of most early stage tumors. Still, androgen deprivation therapy targeting androgen receptor (AR) transcriptional activity has remained the first line of treatment for advance disease since its description in 1941, although it ultimately leads to an incurable castration-resistant metastatic PCa (CRMPC). Strikingly, most CRMPC still rely on the AR transcriptional activity due to different adaptive mechanisms such as AR mutation, ligand-independent AR activation, endogenous androgen synthesis or even AR amplification (5).

In this study, we report that a region of the previously identified hereditary PCa genetic-susceptibility locus located on chromosome 20 (HPC20) (6) is frequently co-amplified with the AR in metastatic PCa tumors. Interestingly, we found that this region is also a hot spot for AR recruitment to chromatin. We show that AR binds and regulates most genes within the common amplified region (CAR) suggesting that the coordinated copy number gain and increased transcriptional output of the 20q13 CAR might be an important event leading to the development of CRMPC.

Material and Methods

Copy number alteration in prostate cancer

Analysis of copy number alteration on the 20q chromosomal arm is based on the published copy number profiles from 181 primary and 37 metastatic prostate tumors (7) using the Nexus Copy Number software v6.0 (Biodiscovery Inc.). Circo graph was performed using the R software and Rcircos package (8).

Cell culture

LNCaP cells were purchased from ATCC (Manassas, VA) and maintained in RPMI 1640 medium (Wisent, St-Bruno, Qc) supplemented with 10% FBS, L-glutamine, and 50 µg/ml gentamycin. The synthetic androgen analog R1881 was obtained for Perkin Elmer (Waltham, MA). For androgenic stimulation assays, cells were first androgen-deprived in phenol-free RPMI 1640 supplemented with 5% charcoal-stripped FBS, L-glutamine, and 50 µg/ml gentamycin. After 48 hours, medium was refreshed and R1881 or ethanol (vehicle) was added for the indicated period.

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

Chromatin was prepared from LNCaP cells exposed to 1 nM R1881 or vehicle for 4hrs. Chromatin-immunoprecipitation (ChIP) was performed as described previously (9) using antibodies specific to AR (mouse monoclonal anti-AR from Lab Vision, Fremont, CA and BD Biosciences, San Jose, CA). Amplification and labeling of AR-bound ChIP fragments was performed as described previously (9). Hybridization was carried out on a custom-designed tiled array from Agilent covering the q-arm of chr.20 at a resolution of 150 bp and analyzed from assembly hg18, using the Feature Extraction 10 alignment program and ChIP Analytics 3.1 program for peak detection (Agilent).

Analysis of gene expression

Total RNA extraction, reverse-transcription and quantitative real-time PCR (qPCR) were performed as already described (9). For MIR645 reverse transcription, the qScript microRNA cDNA synthesis kit was used (Quanta Biosciences, Gaithersburg, MD). The primer sequences used can be found in Supplementary Table 1. Threshold cycle numbers were calculated using the second derivative maximum obtain with the LightCycler^®480 software version 3.5 (Roche, Laval, Qc). Data was normalized according to RPLP0 levels (Supplementary Table 1). mRNA expression Z-Scores were obtained from the cBIO portal (www.cbioportal.org) using the Taylor et al. data set (7). mRNA expression was represented as the Z-Score of PCa samples versus normal prostate samples. Then, the average Z-Scores from primary samples was subtracted from the average Z-Scores from metastatic samples.

Statistics

Statistical analyses were performed with the Prism 5.0 GraphPad Software (La Jolla, CA). The significance of gene expression modulation following R1881 treatment was assessed by the Mann-Whitney test. The differences in the risk of biochemical relapse were computed using the log-rank test.

Results

Given the addiction of prostate cancer cells to AR transcriptional activity, androgen deprivation therapy invariably results in adaptive mechanisms that maintain AR signaling. Accordingly, AR gene amplification is observed exclusively in CRMPC samples (7). In addition, metastatic tumors also display an array of DNA copy-number variations (CNVs) scattered throughout the genome that can possibly synergize with AR gene amplification. Analysis of metastatic PCa samples reveals frequent co-amplification (≥35%) of several genomic regions on eight different chromosomes with the AR gene (Figure 1). Surprisingly, our analysis uncovers that the AR is significantly co-amplified with a region located on the 20q chromosome arm, previously identified as HPC20 (6), uniquely in metastatic tumors (Supplementary Figure S1). We determined that the common amplified region (CAR) on 20q13 is located within the HPC20, which is flanked by the markers D20S887 and D20S196 (Figure 2A and B, orange bars).

Figure 1. AR is co-amplified with a region within the HPC20 in metastatic prostate cancer.

Circo plot of genome-wide co-amplification events in AR amplified metastatic samples at P≤0.0125. The 20q13, within the HPC20 locus, is the sole region associated to AR amplification on chromosome 20. A total of eight different chromosomes possess at least one locus significantly associated to AR amplification.

Figure 2. The 20q13 common amplified region is rich in AR binding sites and is co-amplified with the AR.

(A) Representation of the amplified (blue) and deleted (red) regions on the entire 20q chromosomal arm for patient demonstrating amplification of the 20q13 CAR (orange bars) in primary and metastatic tumors. Localization of the AR-bound segments identified by ChIP-on-chip 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 co-amplification in metastatic cancer falls within the AR-dense binding region identified within the HPC20 (green).

Owing to the frequent co-amplification of the 20q13 CAR with the AR in metastatic PCa, we investigated whether this region is subjected to AR-transcriptional regulation. We performed genomic location analysis of AR recruitment to chromatin in the androgen-sensitive LNCaP cell line using a tiled array covering the q-arm of human chromosome 20. Cells were maintained in steroid-depleted medium for 48 hours before stimulation with the synthetic androgen analog R1881 for 4 hours. ChIP-on-chip analyses reveals 245 segments bound by the AR, mostly in non-promoter regions (Supplementary Table 2, P≤10⁻⁵). Strikingly, the 20q13 CAR is a local hot spot in AR binding sites (Figure 2A and B, vertical red lines). Refined analysis of this locus uncovers that AR co-amplification with the 20q13 is restricted to the CAR in metastatic PCa, which also corresponds to a region rich in AR binding sites (Figure 2C).

Fourteen genomic segments bound by AR in LNCAP cells were associated with 10 of the 16 genes (62.5%) present within the 20q13 CAR (Figure 3A). In order to validate the relevance of this AR binding profile, we examined the impact of AR activation on the expression of amplicon-resident genes. R1881 treatment resulted in the significant modulation of 7 out of the 10 genes that we found associated with AR bound segments (Figure 3B, P<0.01). The three remaining genes were either not affected by R1881 treatment (PARD6B), not expressed in LNCaP cells (LOC284751) or impossible to distinguish from a chimeric transcript with specific primers (UBE2V1 vs. TMEM189-UBE2V1) (Figure 3B). Interestingly, we found that the expression level of most of the genes within the 20q13 CAR was increased in metastatic samples (Figure 3C). Additionally, analysis of the three metastatic samples positives for AR and CAR co-amplification for which clinical survival data was collected (7), demonstrate significant earlier biochemical recurrence compared to patients without AR and CAR amplification (Figure 3D). Together, these results suggest that AR, through its amplification/activation, could synergistically enhance the expression of genes within the 20q13 CAR in advanced disease and significantly alter the patient’s prognosis when co-amplified with the CAR.

Figure 3. The 20q13 common amplified region is androgen regulated, upregulated in metastatic disease and associated with earlier biochemical recurrence when co-amplified with the AR.

(A) Binding profile of the AR from ChIP-on-chip performed in R1881-treated LNCaP cells on a high resolution tiled array covering the 20q chromosomal arm reveals that a majority of genes within the 20q13 CAR possess AR binding sites in their vicinity. (B) R1881-treatement (10 nM, 24 hours) modulate the mRNA expression level of most genes within the 20q13 CAR that were associated to AR binding sites in their vicinity (Mann-Whitney test, *P<0.01 compared to vehicle; N=3, ±SEM). (C) Genes within the CAR are preferentially upregulated in metastatic samples compared to primary samples as demonstrated by the difference between the averages of mRNA Z-Scores (primary samples, N=109; metastatic samples, N=19). (D) The risk of biochemical relapse is significantly higher in patients harboring AR and CAR co-amplifications (N=3) compared to patients with neither regions amplified (N=169) (log-rank test, CAR^Amp, N=10; AR^Amp, N=15).

Discussion

Genetic linkage studies identified several susceptibility loci for type II diabetes (10, 11) and obesity (12) mapping to the D20S196 marker located on the 20q13 chromosomal region. Additionally, this region, flanked by the markers D20S196 and D20S887 (HPC20), has also been described as a hereditary PCa susceptibility locus (6). Interestingly, we and others have shown that the protein tyrosine phosphatase 1B (encoded by PTPN1), which is located in the vicinity of the D20S196 marker and is part of the CAR, is indeed implicated in type II diabetes, obesity (13) and plays a tumor-promoting role in PCa (9).

In this study, we further demonstrate that the 20q13 chromosomal region is significantly amplified in metastatic PCa. Importantly, this region encodes two transcription factors, namely SNAI1 and CEBPB. SNAI1 is an important mediator of the epithelial-mesenchymal transition (EMT), a critical event in the metastatic process (14), and whose expression mediates cell survival and inhibits cellular senescence in metastatic PCa cell lines (15). On the other hand, CEBPB is an important oncogene in Ras-mediated tumorigenesis (16). Additionally, the 20q13 CAR also encodes MIR645, a member of the microRNA family that regulates gene expression post-transcriptionally. Beside a single report suggesting that co-expression of MIR410 and MIR645 is negatively associated with overall survival in advanced serous ovarian cancer (17), the role of MIR645 in cancer remains unknown. Similarly, a recurrent lung cancer amplicon located at 14q13.3 was found to encode three transcription factors, namely TTF1/NKX2-1, NKX2-8 and PAX9. Remarkably, while the overexpression of a single transcription factor did not modified the proliferation of premalignant lung epithelial cell, overexpression of any pairwise combination led to a major increase in their tumorigenic potential (18). Together, the altered transcriptional output mediated by the coordinated DNA copy number gain of SNAI1, CEBPB and MIR645 might contribute to a global transcriptional rewiring process ultimately resulting in the increased tumor aggressiveness observed in patients bearing the 20q13 CAR.

A remarkable feature of the 20q13 CAR is its extensive regulation by AR. ChIP-on-chip analysis using the androgen-sensitive LNCaP cell line reveals AR binding in the vicinity of a large proportion of genes within the 20q13 CAR (Figure 3A). Importantly, this result was not biased by chromosomal abnormalities since LNCaP cells do not have copy number alterations in 20q13 region (19). Surprisingly, most protein-coding transcripts from AR-bound genes were sensitive to R1881 treatment (Figure 3B). As we previously described, PTPN1 mRNA expression was strongly regulated by the AR (9). We also identified several novel AR-regulated genes within the 20q13 CAR with functions yet to be described in the prostate, including the solute carrier family 9, member 8 (SLC9A8), the spermatogenesis-associated protein 2 (SPATA2), the ring finger protein 114 (RNF114) and the activity-dependent neuroprotector (ADNP). Interestingly, the breast cancer amplified sequence 4 (BCAS4), is modestly but significantly regulated by R1881. Finally, we also describe CEBPB (encoding C/EBPβ) as an AR-regulated target. Zhang et al. recently demonstrated that C/EBPβ can effectively trans-activate the prostate-specific antigen (PSA) promoter, which is regulated by androgen response elements, in the absence of androgen. However, increased C/EBPβ expression results in a decreased trans-activation of the PSA promoter in presence of androgen. In contrast, AR activation results in an increased C/EBPβ transcriptional activity on CCAAT enhancer binding protein elements (20). This interesting crosstalk, together with the fact that AR-regulation of the 20q13 CAR is accompanied by AR co-amplification, support a complex transcriptional rewiring in a significant subset of metastatic tumors.

In summary, we report the frequent amplification of the 20q13 chromosomal region in metastatic PCa, a region that had been previously identified as a hereditary PCa susceptibility locus. The extensive regulation of genes within the 20q13 CAR by AR, some of which already associated with oncogenic functions, suggests that the highly specific co-amplification of this chromosomal region with the AR might synergize and contribute to increased tumor aggressiveness and to the development of metastatic disease. These findings reflect the fact that AR amplification is not frequently seen at diagnosis but very common in advanced therapy-resistant disease. Thus, like AR amplification status, AR–CAR amplification status should be most informative as marker for disease progression after therapeutic intervention. These results may also reflect a potential example of the amplification of a locus after enhanced transcription activity in that locus. Finally, these results justify further investigation to address the respective roles of the different genes within the 20q13 CAR.

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.