c-Myc Antagonises the Transcriptional Activity of the Androgen Receptor in Prostate Cancer Affecting Key Gene Networks

Abstract

Prostate cancer (PCa) is the most common non-cutaneous cancer in men. The androgen receptor (AR), a ligand-activated transcription factor, constitutes the main drug target for advanced cases of the disease. However, a variety of other transcription factors and signaling networks have been shown to be altered in patients and to influence AR activity. Amongst these, the oncogenic transcription factor c-Myc has been studied extensively in multiple malignancies and elevated protein levels of c-Myc are commonly observed in PCa. Its impact on AR activity, however, remains elusive. In this study, we assessed the impact of c-Myc overexpression on AR activity and transcriptional output in a PCa cell line model and validated the antagonistic effect of c-MYC on AR-targets in patient samples. We found that c-Myc overexpression partially reprogrammed AR chromatin occupancy and was associated with altered histone marks distribution, most notably H3K4me1 and H3K27me3. We found c-Myc and the AR co-occupy a substantial number of binding sites and these exhibited enhancer-like characteristics. Interestingly, c-Myc overexpression antagonised clinically relevant AR target genes. Therefore, as an example, we validated the antagonistic relationship between c-Myc and two AR target genes, KLK3 (alias PSA, prostate specific antigen), and Glycine N-Methyltransferase (GNMT), in patient samples. Our findings provide unbiased evidence that MYC overexpression deregulates the AR transcriptional program, which is thought to be a driving force in PCa.

Keywords: Androgen receptor; Chromatin immunoprecipitation exonuclease (ChIP-exo); DNA damage; Glycine N-Methyltransferase (GNMT); Prostate cancer; c-Myc.

Fig. 1

AR and MYC are co-enriched at enhancer-like binding sites. (a–b) LNCaP and VCaP cells were hormone-starved for 72 h and subsequently treated with 1 nM R1881 for the indicated time. (a) Transcript and (b) protein levels of MYC and KLK3 relative to b-actin were measured by real-time PCR and Western blotting analysis, respectively (n = 3). (c) Venn diagram showing the overlap between MYC and AR binding sites in LNCaP-MYC cells. (d) Density plot of AR ChIP-exo reads within ± 2 kb of common AR/MYC binding sites or all AR binding sites in LNCaP-MYC cells treated with R1881 orR1881 + doxycycline. (e) Distribution of common AR/MYC sites in LNCaP-MYC cells across the genome. (f) Motif analysis of common AR/MYC binding sites in LNCaP-MYC cells. The top 5 motifs are shown. (g) Sequence logo of the top motif for FOXA1 in the JASPAR database and as observed in this study. (h) Venn diagram showing the overlap between common AR/MYC binding sites in LNCaP-MYC and FOXA1 binding sites in LNCaP derivatives retrieved from a previously published dataset (Sahu et al., 2011). Density plots of H3K4me1, H3K4me3, H3K27ac and H3K27me3 ChIP-seq reads within ± 2 kb of (i) AR, (j) MYC and (k) common AR/MYC binding sites in LNCaP-MYC treated with R1881 (R) or R1881 + doxycycline (RD).

Fig. 2

MYC overexpression antagonises R1881-induced gene expression. (a) Gene Set Enrichment Analysis (GSEA) of hormone-starved LNCaP-MYC cells treated with R1881 (top row) or R1881 + doxycycline (bottom row) for 12 h. The c2: curated gene set compendium and a custom AR target gene signature consisting of genes upregulated by hormone treatment in both LNCaP and VCaP cells from Asangani et al. (Asangani et al., 2014) were used as input. The top up- and downregulated gene sets of the c2 compendium are shown. (b) GSEA of MYC-depleted LNCaP using the c2: curated gene set compendium as input. The top up- and downregulated gene sets are shown (Koh et al., 2011a). Venn diagrams showing (c) overlaps between R1881-induced genes and genes altered by MYC-overexpression, and (d) R1881-repressed genes and genes altered by MYC-overexpression. Heatmap of unsupervised hierarchical clustering for R1881-induced and -repressed genes for which MYC overexpression (e) enhanced or (f) antagonised AR action.

Fig. 3

Validation of selected AR targets antagonised by MYC overexpression. University of California, Santa Cruz (UCSC) genome browser visualisation of AR, MYC, H3K4me1, H3K4me3, H3K27ac, H3K27me3 and IgG binding events around the two well-established AR-target genes (a) KLK3 and (b) CAMKK2, which were antagonised by MYC overexpression. The binding events considered significant (p < 0.0001) and present in at least two biological replicates are indicated by the black boxes below the bigwig tracks provided. ChIP-qPCR validation of (c) AR and (d) MYC binding to enhancer regions of KLK3, CAMKK2, SOCS2, ERRFI1 and GNMT in LNCaP-MYC cells. (e–f) LNCaP-MYC cells were hormone-starved for 72 h and subsequently treated with 1 nM R1881, R1881 plus doxycycline (R1881 + Dox) or vehicle control for the indicated time points. (e) qRT-PCR validation of R1881-induction and repression through MYC overexpression of GNMT, SOCS2, ERRFI1, CAMKK2, and KLK3 transcripts in LNCaP-MYC cells (n = 3). (f) qRT-PCR for GNMT, SOCS2, ERRFI1, CAMKK2, and KLK3 transcripts upon knockdown of MYC in LNCaP and VCaP (n = 3). (g) Representative Western blot validation of R1881-induction and repression by MYC overexpression for CAMKK2, GNMT, and KLK3 at the indicated time points. Protein levels were normalized to GAPDH.

Fig. 4

MYC is inversely correlated with KLK3 and GNMT in prostate cancer patients with advanced disease. (a) Prognostic properties of R1881-induced genes antagonised by MYC overexpression in two publicly available clinical datasets (Glinsky et al., 2004, Taylor et al., 2010) and Kaplan-Meier survival curves for (b) SOCS2 (c) KLF6, and (d) EAF2. Proportions of specimens according to immunohistochemical staining intensities of (e) MYC, (f) KLK3 and (g) GNMT in patient samples of BPH (n = 68), localized PCa (n = 101), PCa with lymph node metastases (n = 71) and CRPC (n = 112). Nuclear staining for MYC was defined as percentage of stained nuclei multiplied by staining intensity. Staining intensities for KLK3 and GNMT were divided into four groups (negative, weak, moderate and strong). See supplemental information for details. Representative nuclear staining for (h) MYC, (i) cytoplasmic KLK3, and (j) GNMT in the indicated sample groups.