Long noncoding RNA-mediated activation of androgen receptor in prostate cancer.

Lnc RNAs can function through a variety of mechanisms, including chromatin remodeling (Xist, Hotair, Anril), transcriptional co-activation/repression (H19, lincRNA-p21, SRA), posttranscriptional modifications (MALAT1), protein inhibition (TERRA) and decoy elements (PTENP1).45 SRA has been identified as a steroid receptor coactivator lncRNA.6 However, the function and mechanism of most lnc RNAs remain unclear.

Surprisingly enough, early discoveries using differential display technologies described two lncRNAs, DD3/PCA37 and PCGEM1,8 which not only exhibited high prostate tissue specificity but also showed prostate tumor associated overexpression. Recent evaluations of PCa transcriptome have described several lncRNAs, including PCAT-1, a transcriptional repressor and target of Polycomb Repressive Complex 2, implicated in PCa progression9 and PRNCR1, a PCa susceptibility associated lncRNA.10 Interestingly, both PCAT-1 and PRNCR1 reside in the 8q24 PCa susceptibility locus, less than a Mb from the CMYC locus which is often amplified in PCa. While functions of many of these lncRNAs remain to better understood in PCa biology, overexpression of PCA3 in virtually all PCas has led to a recently FDA approved diagnostic test.1112

The focus of this Nature report13 is on two PCa-associated lncRNAs: PCGEM1 and PRNCR1. They cooperate in regulating the function of the male hormone receptor, the androgen receptor (AR), which plays central role in PCa onset and progression. AR pathway is activated in advanced CaPs including castration-resistant prostate cancer (CRPC). PCGEM1, a prostate specific androgen regulated gene is expressed in approximately half of all PCas with significantly increased association in tumors of African American patients. PCGEM1 also exhibits oncogenic activity in cancer cell biology experiments.1415 PRNCR1 is transcribed from the ‘gene desert’ region of chromosome 8q24, strongly associated with susceptibility to PCa. It was described as a 13 kb intron-less lncRNA that affects transactivation activity of AR.10

The Yang et al.13 Nature report connects the AR-regulated gene activation program with these two PCa-associated lncRNAs, PCGEM1 and PRNCR1. In a series of experiments physical association between these PCa-associated lncRNAs and AR was established both in human prostate tumor tissue and in LNCaP cell line model. Hormonal induction of these associations was revealed by DHT treatment of LNCaP cells. Of note, previous studies have shown that PCGEM1 itself can be induced by androgen,8 which may further cooperate with AR activation especially when it is overexpressed in PCa. Antisense oligonucleotide based knockdown of PRNCR1 abolished both its own interaction with AR, and the association of PCGEM1 with AR. However, antisense oligonucleotide targeting of PCGEM1 abolished only the PCGEM1-AR association, suggesting for a PRNCR1 dependent recruitment of PCGEM1 to AR. In vitro binding studies mapped the PRNCR1 binding site to AR 549–623 region, and the PCGEM1 binding site to the N-terminal region of AR. The lncRNA-bound AR had specific posttranslational modifications: acetylation was required for association with PRNCR1 and methylation for the PCGEM1 binding. These promising novel observations will lead to further refinement of these complex interactions.

Chromatin isolation by RNA purification (ChIRP) revealed over 2000 PCGEM1 occupancy sites in the genome, about 80% of them colocalize with AR-bound sites. Global run-on sequencing (GRO-seq) revealed that knockdown of either lncRNAs by antisense oligonucleotide decreased AR target gene expression (about 600 genes). Similarly, shRNA against either PCGEM1 or PRNCR1 reduced the DHT-induced activation of AR targets without affecting AR expression levels. Significantly, the truncated AR-V7 (75 kDa) splice variant, which can activate AR-regulated genes without ligand (hormone), associated with both lncRNAs. Knockdown of either PCGEM1 or PRNCR1 inhibited AR-regulated gene activation by AR-V7.

Finally, the biological roles of these lncRNAs were investigated in stable cell lines of CWR22Rv1 harboring dox-induced shRNA against PCGEM1 or PRNCR1. In addition to reduced expression of canonical AR target genes a reduction of cell growth and inhibition of tumor growth in a xenograft model (CRPC) was demonstrated.

Taken together, novel findings from this report, along with previous studies of PCGEM1, AR and PRNCR1, reveal biological importance of prostate associated lncRNAs (PCGEM1 and PRNCR1) in full length and truncated AR-dependent gene activation in PCa (). As PCGEM1 and PRNCR1 strongly enhance AR activity in PCa, they may be explored as potential new therapeutic targets in CRPC.

Figure 1

Schematic model illustrating AR (red dimer on DNA) activation by overexpressed lncRNAs PCGEM1 and PRNCR1(blue ribbons) in prostate tumors. The increased thickness of the red arrow represents elevated mRNA expression of AR-regulated genes. AR: androgen receptor.