Abstract 4943: Regulation of human Cripto-1 gene expression by nuclear receptors and DNA promoter methylation

Human Cripto-1 (CR-1) is a membrane bound protein that belongs to the epidermal growth factor (EGF)-CFC family. CR-1 plays an important role in regulating embryonic development while also having significant implications in various forms of carcinogenesis. Previous studies have shown that germ cell nuclear factor (GCNF), a member of the nuclear receptor superfamily of transcription factors, inhibits CR-1 expression in human embryonal carcinoma NTERA-2 cells following retinoic acid-induced differentiation due to direct binding to a DR0 element within the CR-1 promoter. In the present study, we investigated the effects on CR-1 gene expression of another member of the nuclear receptor superfamily, liver receptor homologue-1 (LRH-1), which can bind to the same GCNF-binding site within the promoter of several genes. NTERA-2 cells transfected with a CR-1 promoter luciferase reporter vector showed substantially increased CR-1 promoter activity in the presence of LRH-1 and decreased CR-1 promoter activity in the presence of GCNF. Retinoic acid treatment of NTERA-2 cells induced downregulation of CR-1 and LRH-1 mRNA and protein expression. In contrast, a strong increase in GCNF was detected in NTERA-2 cells that have been treated with retinoic acid. Additionally, chromatin immunoprecipitation assay displayed direct binding of LRH-1 to the CR-1 promoter in NTERA-2 cells. Real-time PCR and Western blot analysis of NTERA-2 cells transfected with LRH-1 siRNA revealed a significant decrease in CR-1 mRNA and protein expression, suggesting that LRH-1 is required for mantaining high levels of CR-1 expression in embryonal carcinoma cells. Real time PCR analysis for GCNF and LRH-1 in human embryonal carcinoma cells and in a panel of human breast cancer cell lines, expressing very low levels of CR-1, revealed that GCNF is highly expressed in MCF7, T47D and ZR75-1 breast cancer cells as compared to NTERA-2 and NCCIT embryonal carcinoma cells. Since one of the mechanisms used by GCNF to repress gene expression is through promoter DNA methylation, we investigated the role of epigenetic modification of the CR-1 promoter in embryonal and breast carcinoma cells. While NTERA-2 and NCCIT cells showed very low levels of CR-1 promoter methylation, in human breast cancer cells CR-1 promoter was highly methylated in agreement with low levels of CR-1 expression in these cell lines. Finally, treatment of various breast cancer cell lines with the demethylating agent 5-aza-2′-deoxycytidine alone or in combination with the histone deacetylase inhibitors trichostatin A or valproic acid induced a significant increase in CR-1 expression, thus overcoming the repressive effects of GCNF. Collectively, these findings offer promising insight into the transcriptional regulation of CR-1 gene expression and its critical role in the pathogenesis of human cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4943.