RESUMO

BACKGROUND: 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 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. METHODS: LNCaP 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. RESULTS: We 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. CONCLUSIONS: Overall, 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. Video Abstract.

Assuntos

Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , RNA Longo não Codificante/genética , Androgênios , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Humanos , Estimativa de Kaplan-Meier , Masculino , Neoplasias da Próstata/mortalidade , Receptores Androgênicos/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/genética

RESUMO

Background: 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 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. Methods: LNCaP 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 specifc GapmeRs (or a control with scrambled GapmeR) was followed by diferentially expressed genes (DEGs) determination with Agilent microarrays and with Signifcance Analysis of Microarrays statistical test. DEGs were tested as a tumor risk classifer 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. Results: We show that PVT1 knockdown in androgen-stimulated LNCaP cells caused statistically signifcant expression upregulation/downregulation of hundreds of genes. Interestingly, PVT1 knockdown caused upregulation of 160 genes that were repressed by androgen, including a signifcantly 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 classifcation 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 signifcant 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. Conclusions: Overall, we provide frst evidence that PVT1 was involved in signaling a genome-wide androgendependent transcriptional repressive program of tumor suppressor protein-coding genes in prostate cancer cells. Identifcation of transcriptional inhibition of tumor suppressor genes by PVT1 highlights the pathway to the investiga‑tion of mechanisms that lie behind the oncogenic role of PVT1 in cancer.