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

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.

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

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.

Effect of lincRNA PVT1 associated with Enhancer of Zeste homolog 2 (EZH2) and with androgen receptor (AR) on the large-scale gene expression in LNCaP prostate cancer cells

O lincRNA PVT1 (Plasmacytoma Variant Translocation 1) é um RNA longo não codificador de proteínas (ncRNA) descrito como um oncogene sendo superexpresso em vários tipos de cânceres. LincRNA PVT1 está localizado na região genômica 8q24, também conhecida como 'gene desert'. O nível de expressão do lincRNA PVT1 está associado ao aumento do risco de câncer de próstata (PCa) e está correlacionado com os níveis de expressão do receptor de andrógeno (AR). No entanto, o mecanismo do envolvimento do lincRNA PVT1 com o AR no desenvolvimento de câncer de próstata ainda não está bem esclarecido. Aqui, nós testamos a hipótese que a formação do complexo AR-EZH2-PVT1 participa na regulação da expressão gênica em câncer de próstata, nas células LNCaP. A imunoprecipitação de ribonucleoproteínas seguida de PCR quantitativo (RIP-qPCR) revelou que o lincRNA PVT1 está associado fisicamente ao AR (12% do input) e à metiltransferase EZH2, proteína componente do complexo repressor Polycomb 2 (36% do input) sob condições suplementadas com andrógeno (+R1881). O lincRNA PVT1 também está associado fisicamente ao AR (10% de input) e à EZH2 (42% de input) em condições de privação de andrógeno (-R1881). Assim, a associação física entre lincRNA PVT1, AR e EZH2 é independente do hormônio andrógeno. Usando uma abordagem de estudo em larga-escala de perda e ganho de função, nossos resultados mostraram que o silenciamento do lincRNA PVT1 em células LNCaP na presença de andrógeno restaura a expressão parcialmente, totalmente ou causa superexpressão de 160 genes que tiveram a expressão inibida por andrógeno. Entre esses genes, destacamos genes envolvidos na regulação da diferenciação celular, em componentes da junção célula-célula, na inibição da migração e invasão celular e no desencadeamento da via apoptótica. Imunoprecipitação da cromatina seguida de PCR quantitativo (ChIP-qPCR), em cultura de células LNCaP suplementada com andrógeno sob silenciamento do lincRNA PVT1, mostrou aumento significativo na ocupação pela marca de histona ativadora H3K27Ac do promotor do gene NOV, um dos genes que tiveram sua expressão aumentada com o silenciamento de PVT1. O ChIP-qPCR também mostrou, após o silenciamento do lincRNA PVT1, um aumento significativo da marca H3K27me3 na região enhancer do gene NOV, uma característica de enhancers poised (prontos para ativação). Em conclusão, nós fornecemos a primeira evidência experimental para um mecanismo de ação do oncogene lincRNA PVT1 em células de câncer de próstata e demonstramos que sua ação inibidora da expressão afeta genes alvo que facilitam a proliferação e migração de células do câncer de próstata, sugerindo que o lincRNA PVT1 é um novo agente no complexo mecanismo de repressão transcricional envolvendo um RNA silenciador, o receptor de andrógeno (AR) e o potenciador de Zeste homólogo 2 (EZH2) no remodelamento da cromatina em células LNCaP

Long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) is an oncogene known to be overexpressed in various types of cancer. PVT1 lincRNA is located in the wellknown cancer-related genomic region 8q24, also known as 'gene desert. PVT1 lincRNA level of expression is associated with increased prostate cancer (PCa) risk and is correlated with androgen receptor (AR) expression levels. However, the mechanism of PVT1 and AR involvement in the development of prostate cancer is still unclear. Here, we tested the hypothesis that formation of the complex AR-EZH2-PVT1 participates in the regulation of gene expression in prostate cancer, in LNCaP cells. Ribonucleoprotein immunoprecipitation followed by quantitative PCR (RIP-qPCR) revealed that PVT1 lincRNA binds both the AR (12 % of PVT1 input) and the methyltransferase EZH2 from the Polycomb repressive complex 2 (36 % of input) under androgen-supplemented conditions (+R1881). PVT1 also binds both AR (10 % of input) and EZH2 (42 % of input) under androgen-deprived conditions (-R1881). Thus, PVT1 binding to AR and EZH2 is independent of the androgen hormone. Using a large-scale loss and gain of function approach, our results show that PVT1 knockdown (KD) in LNCaP in the presence of androgen restores the expression partially, fully or causes overexpression of 160 genes that are inhibited by androgen. Among these genes, we highlight genes involved in regulation of cell differentiation, in components of cell-cell junction, in inhibition of cell migration and invasion and in triggering of the apoptotic pathway. Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) with LNCaP cells in androgen-supplemented cultures under PVT1 lincRNA knockdown showed a significant increase in occupancy by the histone activation mark H3K27Ac of the promoter region of the NOV gene, one of the genes that had an increased expression upon PVT1 silencing. ChIPqPCR also showed a significant increase upon PVT1 lincRNA silencing of the H3K27me3 histone mark in the enhancer region of the NOV gene, a distinct feature of poised enhancers. In conclusion, we provide first experimental evidence for a mechanism of action of PVT1 lincRNA oncogene in prostate cancer cells, and show that its inhibitory action affects targetgenes that facilitate proliferation and migration of prostate cancer cells, thus suggesting PVT1 lincRNA as a novel lncRNA player in the complex mechanism of transcriptional repression involving a silencer RNA, the androgen receptor (AR) and the Enhancer of zeste homolog 2 (EZH2) in chromatin remodeling in LNCaP cells