PVT1 signals an androgen-dependent transcriptional repression program in prostate cancer cells and a set of the repressed genes predicts high-risk tumors

Lucas F. daSilva,Alexandre Videira,Felipe C. Beckedorff,Sergio Verjovski-Almeida

doi:10.1186/s12964-020-00691-x

Abstract

BackgroundAndrogen receptor (AR) and polycomb repressive complex 2 (PRC2) are known to co-occupy the loci of genes that are downregulated by androgen-stimulus. Long intergenic non-coding RNA (lincRNA) PVT1 is an overexpressed oncogene that is associated with AR in LNCaP prostate cancer cells, and with PRC2 in HeLa and many other types of cancer cells. The possible involvement of PVT1 in mediating androgen-induced gene expression downregulation in prostate cancer has not been explored.MethodsLNCaP cell line was used. Native RNA-binding-protein immunoprecipitation with anti-AR or anti-EZH2 was followed by RT-qPCR with primers for PVT1. Knockdown of PVT1 with specific GapmeRs (or a control with scrambled GapmeR) was followed by differentially expressed genes (DEGs) determination with Agilent microarrays and with Significance Analysis of Microarrays statistical test. DEGs were tested as a tumor risk classifier with a machine learning Random Forest algorithm run with gene expression data from all TCGA-PRAD (prostate adenocarcinoma) tumors as input. ChIP-qPCR was performed for histone marks at the promoter of one DEG.ResultsWe show that PVT1 knockdown in androgen-stimulated LNCaP cells caused statistically significant expression upregulation/downregulation of hundreds of genes. Interestingly, PVT1 knockdown caused upregulation of 160 genes that were repressed by androgen, including a significantly enriched set of tumor suppressor genes, and among them FAS, NOV/CCN3, BMF, HRK, IFIT2, AJUBA, DRAIC and TNFRSF21. A 121-gene-set (out of the 160) was able to correctly predict the classification of all 293 intermediate- and high-risk TCGA-PRAD tumors, with a mean ROC area under the curve AUC = 0.89 ± 0.04, pointing to the relevance of these genes in cancer aggressiveness. Native RIP-qPCR in LNCaP showed that PVT1 was associated with EZH2, a component of PRC2. PVT1 knockdown followed by ChIP-qPCR showed significant epigenetic remodeling at the enhancer and promoter regions of tumor suppressor gene NOV, one of the androgen-repressed genes that were upregulated upon PVT1 silencing.ConclusionsOverall, we provide first evidence that PVT1 was involved in signaling a genome-wide androgen-dependent transcriptional repressive program of tumor suppressor protein-coding genes in prostate cancer cells. Identification of transcriptional inhibition of tumor suppressor genes by PVT1 highlights the pathway to the investigation of mechanisms that lie behind the oncogenic role of PVT1 in cancer.DiTiwbuLh6zGF35UTdvYfAVideo

Highlights

Androgen receptor (AR) and polycomb repressive complex 2 (PRC2) are known to co-occupy the loci of genes that are downregulated by androgen-stimulus
We performed native RNA‐binding protein immunoprecipitation (RIP)-Quantitative PCR (qPCR) in order to test if PVT1 associated with AR and with PRC2 in LNCaP cells
PVT1 was detected as significantly enriched in the antiAR fraction relative to the non-specific IgG control in hormone-starved or androgen-stimulated cells (Fig. 1b, blue and red bars), similar to the positive control Long non-coding RNA (lncRNA) PCGEM [17] (Fig. 1c, blue and red bars)

Summary

Introduction

Androgen receptor (AR) and polycomb repressive complex 2 (PRC2) are known to co-occupy the loci of genes that are downregulated by androgen-stimulus. Long intergenic non-coding RNA (lincRNA) PVT1 is an over‐ expressed oncogene that is associated with AR in LNCaP prostate cancer cells, and with PRC2 in HeLa and many other types of cancer cells. Development of PCa is highly dependent on androgen receptor (AR) [2, 3], a transcription factor that induces expression activation or repression of prostate-specific genes through AR interaction with hundreds of coactivators or corepressors [4]. The ability of AR to repress transcription in LNCaP prostate cancer cells is related among other factors to cooperation with EZH2 histone-modifying enzyme, a component of the polycomb repressor complex 2 (PRC2) [5]. Specific AR-associated coregulators provide expression fine-tuning of hundreds of androgen-responsive target genes [6, 7], the full complement of factors underlying expression activation or repression in the prostate have not yet been fully defined. Understanding the complex network of gene regulation involving lncRNAs, and identifying their gene targets, will allow for their use in new strategies for diagnosis, prognosis and cancer therapy [14, 15]

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