Aberrant TGFβ/SMAD4 signaling contributes to epigenetic silencing of a putative tumor suppressor,RunX1T1in ovarian cancer

Abstract

Aberrant TGFβ signaling pathway may alter the expression of down-stream targets and promotes ovarian carcinogenesis. However, the mechanism of this impairment is not fully understood. Our previous study has identified RunX1T1 as a putative SMAD4 target in an immortalized ovarian surface epithelial cell line, IOSE. In this study, we report that transcription of RunX1T1 was confirmed to be positively regulated by SMAD4 in IOSE cells and epigenetically silenced in a panel of ovarian cancer cell lines by promoter hypermethylation and histone methylation at H3 lysine 9. SMAD4 depletion increased repressive histone modifications of RunX1T1 promoter without affecting promoter methylation in IOSE cells. Epigenetic treatment can restore RunX1T1 expression by reversing its epigenetic status in MCP3 ovarian cancer cells. When transiently treated with a demethylating agent, the expression of RunX1T1 was partially restored in MCP3 cells, but gradual re-silencing through promoter re-methylation was observed after the treatment. Interestingly, SMAD4 knockdown accelerated this re-silencing process, suggesting that normal TGF-beta signaling is essential for the maintenance of RunX1T1 expression. In vivo analysis confirmed that hypermethylation of RunX1T1 was detected in 35.7% (34/95) of ovarian tumors with high clinical stages (P=0.035) and in 83% (5/6) of primary ovarian cancer-initiating cells. Additionally, concurrent methylation of RunX1T1 and another SMAD4 target, FBXO32 which was previously found to be hypermethylated in ovarian cancer was observed in this same sample cohort (P< 0.05). Restoration of RunX1T1 inhibited cancer cell growth. Taken together, dysregulated TGFβ/SMAD4 signaling may lead to epigenetic silencing of a putative tumor suppressor, RunX1T1, during ovarian carcinogenesis.

C/EBPβ regulates homeostatic and oncogenic gastric cell proliferation

“…Methylation analysis of the RUNX1t1 promoter was determined by methylation-specific PCR (MSP), as previously described [30]. MSP method distinguishes unmethylated from methylated alleles in a given gene based on sequence changes produced after bisulfite treatment of DNA, which converts unmethylated but not methylated cytosines to uracil.…”

The roles of TGFβ in the tumour microenvironment

“…This seems to be essential to overcome the tumour-suppressor actions of TGFβ signalling in epithelial cells. Numerous cancer types show hypermethylation of runt-related transcription factor 3 ( RUNX3 ), which results in attenuation of its growth-suppressive functions 137 . Active TGFβ signalling is required for the maintenance of this methylation, as tumours overexpressing SMAD7 (which inhibits TGFβ signalling), lose methylation of promoters and reverse the effects of gene silencing 138 .…”

Involvement of p300/CBP and epigenetic histone acetylation in TGF-β1-mediated gene transcription in mesangial cells

“…In contrast, histone deacetylation by HDACs promotes chromatin compaction and gene repression (17,25,27). Evidence shows that TGF-␤1 can regulate epigenetic modifications to regulate target genes in diverse cell types, including vascular smooth muscle cells (42), ovarian (63), and mammary epithelial cells (61), immune cells (66), and fibroblasts (15). Reports show that histone modifications including H3K9Ac and H3K4 methylation (H3K4me) play a role in PMA-induced laminin gene expression in rat mesangial cells (35) and in inflammatory gene expression in tubular cells from acute renal injury models (34,65).…”