DNA methylation variations are required for epithelial-to-mesenchymal transition induced by cancer-associated fibroblasts in prostate cancer cells.

Widespread genome hypo-methylation and promoter hyper-methylation of epithelium-specific genes are hallmarks of stable epithelial-to-mesenchymal transition (EMT), which in prostate cancer (PCa) correlates with castration resistance, cancer stem cells generation, chemoresistance and worst prognosis. Exploiting our consolidated 'ex-vivo' system, we show that cancer-associated fibroblasts (CAFs) released factors have pivotal roles in inducing genome methylation changes required for EMT and stemness in EMT-prone PCa cells. By global DNA methylation analysis and RNA-Seq, we provide compelling evidence that conditioned media from CAFs explanted from two unrelated patients with advanced PCa, stimulates concurrent DNA hypo- and hyper-methylation required for EMT and stemness in PC3 and DU145, but not in LN-CaP and its derivative C4-2B, PCa cells. CpG island (CGI) hyper-methylation associates with repression of genes required for epithelial maintenance and invasion antagonism, whereas activation of EMT markers and stemness genes correlate with CGI hypo-methylation. Remarkably, methylation variations and EMT-regulated transcripts almost completely reverse qualitatively and quantitatively during MET. Unsupervised clustering analysis of the PRAD TCGA data set with the differentially expressed (DE) and methylated EMT signature, identified a gene cluster of DE genes defined by a CAF+ and AR- phenotype and worst diagnosis. This gene cluster includes the relevant factors for EMT and stemness, which display DNA methylation variations in regulatory regions inversely correlated to their expression changes, thus strongly sustaining the ex-vivo data. DNMT3A-dependent methylation is essential for silencing epithelial maintenance and EMT counteracting genes, such as CDH1 and GRHL2, that is, the direct repressor of ZEB1, the key transcriptional factor for EMT and stemness. Accordingly, DNMT3A knock-down prevents EMT entry. These results shed light on the mechanisms of establishment and maintenance of coexisting DNA hypo- and hyper-methylation patterns during cancer progression, the generation of EMT and cell stemness in advanced PCa, and may pave the way to new therapeutic implications.

The SRA protein UHRF1 promotes epigenetic crosstalks and is involved in prostate cancer progression.

Epigenetic silencing of tumour suppressor genes is an important mechanism involved in cell transformation and tumour progression. The Set and RING-finger-associated domain-containing protein UHRF1 might be an important link between different epigenetic pathways. Here, we report that UHRF1 is frequently overexpressed in human prostate tumours and has an important role in prostate cancer pathogenesis and progression. Analysis of human prostate cancer samples by microarrays and immunohistochemistry showed increased expression of UHRF1 in about half of the cases. Moreover, UHRF1 expression was associated with reduced overall survival after prostatectomy in patients with organ-confined prostate tumours (P < 0.0001). UHRF1 expression was negatively correlated with several tumour suppressor genes and positively with the histone methyltransferase (HMT) EZH2 both in prostate tumours and cell lines. UHRF1 knockdown reduced proliferation, clonogenic capability and anchorage-independent growth of prostate cancer cells. Depletion of UHRF1 resulted in reactivation of several tumour suppressor genes. Gene reactivation upon UHRF1 depletion was associated with changes in histone H3K9 methylation, acetylation and DNA methylation, and impaired binding of the H3K9 HMT Suv39H1 to the promoter of silenced genes. Co-immunoprecipitation experiments showed direct interaction between UHRF1 and Suv39H1. Our data support the notion that UHRF1, along with Suv39H1 and DNA methyltransferases, contributes to epigenetic gene silencing in prostate tumours. This could represent a parallel and convergent pathway to the H3K27 methylation catalyzed by EZH2 to synergistically promote inactivation of tumour suppressor genes. Deregulated expression of UHRF1 is involved in the prostate cancer pathogenesis and might represent a useful marker to distinguish indolent cancer from those at high risk of lethal progression.

UHRF1 coordinates peroxisome proliferator activated receptor gamma (PPARG) epigenetic silencing and mediates colorectal cancer progression.

Peroxisome proliferator-activated receptor gamma (PPARG) inactivation has been identified as an important step in colorectal cancer (CRC) progression, although the events involved have been partially clarified. UHRF1 is emerging as a cofactor that coordinates the epigenetic silencing of tumor suppressor genes, but its role in CRC remains elusive. Here, we report that UHRF1 negatively regulates PPARG and is associated with a higher proliferative, clonogenic and migration potential. Consistently, UHRF1 ectopic expression induces PPARG repression through its recruitment on the PPARG promoter fostering DNA methylation and histone repressive modifications. In agreement, UHRF1 knockdown elicits PPARG re-activation, accompanied by positive histone marks and DNA demethylation, corroborating its role in PPARG silencing. UHRF1 overexpression, as well as PPARG-silencing, imparts higher growth rate and phenotypic features resembling those occurring in the epithelial-mesenchymal transition. In our series of 110 sporadic CRCs, high UHRF1-expressing tumors are characterized by an undifferentiated phenotype, higher proliferation rate and poor clinical outcome only in advanced stages III-IV. In addition, the inverse relationship with PPARG found in vitro is detected in vivo and UHRF1 prognostic significance appears closely related to PPARG low expression, as remarkably validated in an independent dataset. The results demonstrate that UHRF1 regulates PPARG silencing and both genes appear to be part of a complex regulatory network. These findings suggest that the relationship between UHRF1 and PPARG may have a relevant role in CRC progression.