Chenodeoxycholic acid (CDCA) through TGR5-dependent CREB-signaling activation enhances Cyclin D1 expression and promotes human endometrial cancer cell proliferation

Abstract

Increasing epidemiologic data in humans as well as many in vitro investigative reports suggest that overweight and obesity are important risk factors for type-I EC which is the most common malignancy in women and accounts for 80% of all ECs. The strongest support for mechanisms to link obesity and cancer risk involves the metabolic and endocrine effects of obesity and the alterations that they induce in the production of peptide and steroid hormones. One way in which fat might exert its effect is stimulation of bile acids (BAs) synthesis and secretion. Bile acids (BAs) are amphipathic detergents that are synthesized in the liver and stored in the gallbladder. An important physiological role of BAs is to facilitate the uptake of lipids together with the fat-soluble vitamins A, D, E and K from the intestine. BAs facilitate these absorptive processes through their detergent properties, which allow the emulsification of lipids. Moreover, BAs have evolved over the past few years from being considered as simple lipid solubilizers to complex metabolic integrators. Beyond the orchestration of bile acids, lipid and glucose metabolism by the nuclear receptor farnesoid X receptor (FXR), BAs also act as signaling molecules through the non-genomic pathway activated by a BAdedicated G protein-coupled receptor (GPCR) TGR5 (also known as BG37 or MBAR). Despite the efficient enterohepatic recirculation of bile acids, a small amount of them can spill over into the systemic circulation particularly during high fat diet. Since in the obese women a strong correlation with endometrial cancer does exist, we investigated the biological effects of different concentrations of the primary bile acid CDCA in a human endometrial type-I cancer cell line, Ishikawa. Ishikawa cell proliferation was determined by [3H]-thymidine incorporation assay after 72 hours of treatment with different concentrations of CDCA. The effects of CDCA on the expression of the cell cycle regulatory proteins were evaluated by RT-PCR and Western blotting assays. Transient transfection method and mutagenesis studies were performed to functionally characterize the Cyclin D1 promoter while ChIP assay was to highlight the direct involvement of the ciselements in CDCA-dependent Cyclin D1 up-regulation. In addition, the effects of impaired expression and function of the proteins involved in CDCA activated signaling were assessed by using small interfering RNAs (siRNAs) technology. Our results indicate that low concentrations (< 30μM) of CDCA were able to stimulate Ishikawa cell growth as evidenced by [3H]Thymidine incorporation and cell cycle analysis by inducing a significant increase in Cyclin D1(CD1) protein and mRNA expression. In contrast, according to previous findings, high doses (> 50μ) of CDCA showed cytotoxic effects concomitantly with an increase of CDK inhibitor p21WAF1/CIP1 expression through a p53-indipendent pathway. Moreover, mutagenesis studies and ChIP analysis revealed that the CDCA-induced CD1 expression requires a cyclic AMP-responsive element (CRE) binding protein motif within the CD1 proximal promoter. Silencing of a cell surface bile acid sensor TGR5 and/or CREB gene expression by RNA interference reversed the CDCA-dependent induction of CD1 expression and proliferation in Ishikawa cells. In conclusion, extrahepatic spillover of BAs following high fat diet particularly in overweight and obese women, could be involved in the onset and/or maintenance of endometrial cancer by stimulation of TGR5 that activates ERK signaling that in turn induces the recruitment of the transcription factor CREB to the Cyclin D1 gene promoter enhancing cell proliferation