Epigenetic Silencing of Peroxisome Proliferator-Activated Receptor γ Is a Biomarker for Colorectal Cancer Progression and Adverse Patients' Outcome

Abstract

The relationship between peroxisome proliferator-activated receptor γ (PPARG) expression and epigenetic changes occurring in colorectal-cancer pathogenesis is largely unknown. We investigated whether PPARG is epigenetically regulated in colorectal cancer (CRC) progression. PPARG expression was assessed in CRC tissues and paired normal mucosa by western blot and immunohistochemistry and related to patients' clinicopathological parameters and survival. PPARG promoter methylation was analyzed by methylation-specific-PCR and bisulphite sequencing. PPARG expression and promoter methylation were similarly examined also in CRC derived cell lines. Chromatin immunoprecipitation in basal conditions and after epigenetic treatment was performed along with knocking-down experiments of putative regulatory factors. Gene expression was monitored by immunoblotting and functional assays of cell proliferation and invasiveness. Methylation on a specific region of the promoter is strongly correlated with PPARG lack of expression in 30% of primary CRCs and with patients' poor prognosis. Remarkably, the same methylation pattern is found in PPARG-negative CRC cell lines. Epigenetic treatment with 5′-aza-2′-deoxycytidine can revert this condition and, in combination with trichostatin A, dramatically re-activates gene transcription and receptor activity. Transcriptional silencing is due to the recruitment of MeCP2, HDAC1 and EZH2 that impart repressive chromatin signatures determining an increased cell proliferative and invasive potential, features that can experimentally be reverted. Our findings provide a novel mechanistic insight into epigenetic silencing of PPARG in CRC that may be relevant as a prognostic marker of tumor progression.

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

“…This segment is highly enriched in CpG motifs and is selectively methylated in PPARG-negative cells, thus having a crucial role in silencing this gene. Figure 3c illustrates these characteristics along with the results of the bisulphite sequencing of the same region; part of these results have previously been reported (Pancione et al, 2010b). UHRF1 was engaged at very low levels in HT29 cells, more in HCT116 and even more in RKO cells, confirming the inverse relationship with PPARG expression (Figure 3a).…”

“…We also showed that PPARG silencing involves DNA methylation at a specific promoter region and recruitment of MeCP2, HDAC1 and EZH2 responsible for H3K27 methylation for the maintenance of the repressed status (Pancione et al, 2010b). MeCP2 and EZH2 levels inversely correlate with PPARG expression, as MeCP2 and EZH2 silencing re-activates PPARG, confirming their role in PPARG epigenetic repression (Pancione et al, 2010b).…”

“…Consistently, variations in PPARG expression or gene mutations have been associated with tumorigenesis (Sarraf et al, 1999;Grommes et al, 2004;Sabatino et al, 2005;Wang and Dubois, 2008;Capaccio et al, 2010). We recently showed that PPARG epigenetic silencing has a key role in sporadic colorectal cancer (CRC) and significantly associates with a more aggressive course and patients' worse prognosis; in the same tumors, PPARG has been shown to be an independent prognostic factor (Ogino et al, 2009;Pancione et al, 2010b). We also showed that PPARG silencing involves DNA methylation at a specific promoter region and recruitment of MeCP2, HDAC1 and EZH2 responsible for H3K27 methylation for the maintenance of the repressed status (Pancione et al, 2010b).…”

“…We have shown that PPARG has an important role in colon tumorigenesis serving as tumor suppressor gene and is often epigenetically repressed (Pancione et al, 2010b). As UHRF1 coordinates epigenetic silencing of TSGs (Bostick et al, 2007;Alhosin et al, 2011), we investigated whether it modulates PPARG expression by screening a series of human CRC cell lines, HT29, HCT116 and RKO, stratified on the basis of a stable or unstable microsatellite system, and a positive or negative CpG island methylator phenotype (Issa, 2004).…”

“…We recently showed that PPARG epigenetic silencing has a key role in sporadic colorectal cancer (CRC) and significantly associates with a more aggressive course and patients' worse prognosis; in the same tumors, PPARG has been shown to be an independent prognostic factor (Ogino et al, 2009;Pancione et al, 2010b). We also showed that PPARG silencing involves DNA methylation at a specific promoter region and recruitment of MeCP2, HDAC1 and EZH2 responsible for H3K27 methylation for the maintenance of the repressed status (Pancione et al, 2010b). MeCP2 and EZH2 levels inversely correlate with PPARG expression, as MeCP2 and EZH2 silencing re-activates PPARG, confirming their role in PPARG epigenetic repression (Pancione et al, 2010b).…”