Ras-induced Epigenetic Inactivation of the RRAD (Ras-related Associated with Diabetes) Gene Promotes Glucose Uptake in a Human Ovarian Cancer Model

Yan Wang,Guiling Li,Fengbiao Mao,Xianfeng Li,Qi Liu,Lin Chen,Lu Lv,Xin Wang,Jinyu Wu,Wei Dai,Guan Wang,Enfeng Zhao,Kai-Fu Tang,Zhong Sheng Sun

doi:10.1074/jbc.m113.527671

Abstract

RRAD (Ras-related associated with diabetes) is a small Ras-related GTPase that is frequently inactivated by DNA methylation of the CpG island in its promoter region in cancer tissues. However, the role of the methylation-induced RRAD inactivation in tumorigenesis remains unclear. In this study, the Ras-regulated transcriptome and epigenome were profiled by comparing T29H (a Ras(V12)-transformed human ovarian epithelial cell line) with T29 (an immortalized but non-transformed cell line) through reduced representation bisulfite sequencing and digital gene expression. We found that Ras(V12)-mediated oncogenic transformation was accompanied by RRAD promoter hypermethylation and a concomitant loss of RRAD expression. In addition, we found that the RRAD promoter was hypermethylated, and its transcription was reduced in ovarian cancer versus normal ovarian tissues. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine resulted in demethylation in the RRAD promoter and restored RRAD expression in T29H cells. Additionally, treatment with farnesyltransferase inhibitor FTI277 resulted in restored RRAD expression and inhibited DNA methytransferase expression and activity in T29H cells. By employing knockdown and overexpression techniques in T29 and T29H, respectively, we found that RRAD inhibited glucose uptake and lactate production by repressing the expression of glucose transporters. Finally, RRAD overexpression in T29H cells inhibited tumor formation in nude mice, suggesting that RRAD is a tumor suppressor gene. Our results indicate that Ras(V12)-mediated oncogenic transformation induces RRAD epigenetic inactivation, which in turn promotes glucose uptake and may contribute to ovarian cancer tumorigenesis.

Highlights

Increased glucose uptake is essential for carcinogenesis
Using a well defined human ovarian cancer model, we have demonstrated that RRAD expression was lower in T29H than in T29 cells due to promoter hypermethylation (Fig. 1)
We demonstrated that RRAD inhibited glucose uptake in ovarian epithelial cells (Fig. 5)

Summary

Background

Increased glucose uptake is essential for carcinogenesis. Results: RasV12-induced epigenetic inactivation of RRAD promotes glucose uptake and tumor formation. Our results indicate that RasV12-mediated oncogenic transformation induces RRAD epigenetic inactivation, which in turn promotes glucose uptake and may contribute to ovarian cancer tumorigenesis. Ilany et al [27] generated mice that overexpress RRAD in muscle and found that on a high fat diet, the transgenic mice developed more severe glucose intolerance than wild-type mice due to increased insulin resistance, and there was a further reduction in plasma triglyceride levels in the transgenic mice, which was associated with increased levels of lipoprotein lipase These observations led us to speculate that RRAD may be involved in cancer aerobic glycolysis by regulating glucose uptake. We investigated the role of RRAD in glucose uptake and the oncogenic potential of Ras in ovarian epithelial cells

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION