lncRNA-dependent mechanisms of androgen-receptor-regulated gene activation programs

Abstract

Although recent studies have indicated roles of long non-coding RNAs (lncRNAs) in physiological aspects of cell-type determination and tissue homeostasis, their potential involvement in regulated gene transcription programs remains rather poorly understood. The androgen receptor regulates a large repertoire of genes central to the identity and behaviour of prostate cancer cells, and functions in a ligand-independent fashion in many prostate cancers when they become hormone refractory after initial androgen deprivation therapy. Here we report that two lncRNAs highly overexpressed in aggressive prostate cancer, PRNCR1 (also known as PCAT8) and PCGEM1, bind successively to the androgen receptor and strongly enhance both ligand-dependent and ligand-independent androgen-receptor-mediated gene activation programs and proliferation in prostate cancer cells. Binding of PRNCR1 to the carboxy-terminally acetylated androgen receptor on enhancers and its association with DOT1L appear to be required for recruitment of the second lncRNA, PCGEM1, to the androgen receptor amino terminus that is methylated by DOT1L. Unexpectedly, recognition of specific protein marks by PCGEM1-recruited pygopus 2 PHD domain enhances selective looping of androgen-receptor-bound enhancers to target gene promoters in these cells. In 'resistant' prostate cancer cells, these overexpressed lncRNAs can interact with, and are required for, the robust activation of both truncated and full-length androgen receptor, causing ligand-independent activation of the androgen receptor transcriptional program and cell proliferation. Conditionally expressed short hairpin RNA targeting these lncRNAs in castration-resistant prostate cancer cell lines strongly suppressed tumour xenograft growth in vivo. Together, these results indicate that these overexpressed lncRNAs can potentially serve as a required component of castration-resistance in prostatic tumours.

Figure 1. Signal-Dependent Interaction between AR and Prostate Specific LncRNAs

a and b, RIP assay performed in paired benign prostatic hyperplasia (BPH) and prostate tumor (T) tissues. c, RIP assay in DHT-treated LNCaP cells (100 nM) for indicated time points. d, RIP assay in LNCaP cells transfected with indicated ASO followed by DHT treatment (100 nM). e, Global changes in DHT-induced AR target genes in PCGEM1 or PRNCR1 depleted LNCaP cells. f, Heatmap showing the distribution of PCGEM1 and AR binding sites in DHT-stimulated LNCaP cells. g, Average tag profile analysis of the aligned 2,142 PCGEM1 ChIRP peaks. Mean ± SEM for panel c and e (n=3).

Figure 2. Mechanistic Study of LncRNA with Associated Transcription Factors/Co-activators

a and b, RIP assay in DHT-treated LNCaP cells (100 nM) expressing indicated plasmids. c. In vitro methylation assay for AR. d and e, LNCaP cells expressing Myc-tagged AR fragments (d) or co-transfect with Flag-tagged DOT1L (e) were subjected to RNA pulldown assay. f, In vitro transcribed PCGEM1 full-length, Δ411-490, or Δ1191-1270 were incubated with cell lysates extracted from DHT-treated LNCaP cell (100 nM, 1 hr) for in vitro RNA pulldown assay. Mean ± SEM for panel a and b (n=3).

Figure 3. PCGEM1 and PRNCR1 Promote Hormone-independent Activation of the AR Transcriptional Program in Castration Resistant Prostate Cancer (CRPC)

a, qRT-PCR analyses of AR targets in LNCaP cells co-transfected with indicated vectors followed by Doxycycline induction (160 ng/ml, 2d). b, RIP assay in CWR22Rv1 cells treated with or without DHT (100 nM, 1hr) using indicated antibodies. Detection of immunoprecipitated full-length AR and AR-V7 were shown. c, Cell proliferation assay in CWR22Rv1 cells stably expressing indicated shRNAs followed by Doxycycline induction (160 ng/ml) for indicated times. d, Xenografts of CWR22Rv1 cell lines harboring Doxycycline-induced shRNAs were monitored for tumor growth for the indicated time, with or without Doxycycline intake (4 mice/group). Mean ± SEM (n=6, *p<0.05 and **p<0.01).

Figure 4. Regulation of Enhancer: Promoter Interaction by PRNCR1 and PCGEM1

a, ChIP-3C in LNCaP cells with indicated ASO transfection and treatment. b, ChIP–qPCR showing Pygo2 occupancy in DHT-treated LNCaP cells (100 nM) at indicated times. c, ChIP-qPCR indicate the Pygo2 recruitment in LNCaP cells with indicated transfection and treatment. d, ChIP-3C in LNCaP cells with indicated siRNA transfection and treatment. e, Global changes in DHT-induced AR targets in LNCaP cells with indicated siRNA transfection and treatment. f, 3C assay on FASN locus in LNCaP-cds2 cells transduced with indicated shRNA harboring lentivirus. Red box indicate PCR product sequenced in a and f. Mean ± SEM (n=3, *p<0.05 and **p<0.01).