Abstract 418: Epigenetic regulation of glypican-3 in hepatocellular carcinoma

Abstract

Abstract Introduction: Glypican 3 (GPC3) is a cell surface heparan sulfate proteoglycan which plays critical roles in the control of cell division and growth regulation. Although repressed in most of adult tissues, GPC3 is found to be upregulated in different types of cancer, including hepatocellular carcinoma (HCC). To address whether the expression of GPC3 in HCC is regulated by epigenetic mechanisms, we studied the DNA methylation profiles of the two CpG islands in the GPC3 promoter by high resolution melting analysis (HRM) and by pyrosequencing. Histone modifications were also examined by ChIP assays to further explore how epigenetic changes contribute to GPC3 expression in HCC. Results: We observed hypomethylation in promoters of 5 different HCC cell lines (which express GPC3) but a partially hypermethylated pattern in HCO2, a HCC cell line in our panel which does not express GPC3. We measured average CpG methylation by pyrosequencing, with the average methylation values for GPC3 expressing cells at 7.74% vs. HCO2 at 43.25% (P<0.01). Similarly, the average methylation frequency in 9 different primary hepatocytes (also no GPC3 expression) is 31.85%, equals to that of HCO2 and much higher than GPC3 expressing cells (P<0.01). In tissues, the methylation profiles of GPC3 promoters are more complicated, where hypo- and hypermethylation could be found in both tumor and non-tumor adjacent tissues. In 34 pairs of tissues, 23 pairs (67.6%) have methylation patterns correlate with expression data. However, treatment of HCO2 with demethylating agent (5-aza-dC) resulted in little reactivation of GPC3 expression, suggesting that DNA methylation may not be the only factor modifying transcription of GPC3. Treatment of HCO2 with histone deacetylase inhibitor trichostatin-A (TSA) resulted in more than 10 folds of GPC3 expression, suggesting histone modification plays a critical role in regulation of GPC3. ChIP analysis in different HCC cell lines as well as primary hepatocytes revealed that the histone marker H3K9Ac (associated with transcription) is present at the GPC3 promoter region of Huh7 but absence in HCO2 and in primary hepatocytes, whereas occupancy of the promoter by repressive markers H3K9Me3 and H3K27Me3 is increased in HCO2 and primary hepatocytes but not Huh7. ChIP assay performed on two pairs of HCC tissues showed similar correlation data between GPC3 expression and histone modification. Conclusions: In this study, we showed that methylation is correlated with transcriptional repression of GPC3 in HCC cell lines, but demethylation of GPC3 promoter is not enough to reactivate the protein expression, suggesting DNA methylation is not the predominant regulatory mechanism for GPC3 gene. Histone modification, on the other hand correlates very tightly with GPC3 expression in HCC cell lines and tumors. Together, our data suggest that epigenetic alterations, including DNA methylation and histone modifications are critical for transcriptional regulation of GPC3 in HCC. Citation Format: Thu Le Trinh, William Puszyk, Chen Liu. Epigenetic regulation of glypican-3 in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 418. doi:10.1158/1538-7445.AM2014-418