Abstract 3518: Frequent mutation and deletion of GATA4 in hepatocellular carcinoma represses proliferation terminating late-maturation genes CEBPD and HNF4A by reversible epigenetic mechanisms.
Authors: Enane Francis O. , Teo Marissa , Makishima Hideki , Hu Zhenbo , Toh Han Chong , Saunthararajah Yogen , Ng Joanna , Lai Chit , Ang Soo Fan , Hon Lim Kiat 
Published: August 2014

Abstract
One reason that hepatocellular carcinoma (HCC) is resistant to therapy is frequent mutation or deletion of master regulators of apoptosis (e.g., TP53) that mediate the actions of multiple cancer drugs. Maturation genes are another family of genes which mediate cell cycle exit. If unlike key apoptosis genes these proliferation-terminating genes are genetically intact but epigenetically repressed in HCC, then understanding and reversing the mechanisms of aberrant repression could form the basis for novel treatment. Hence, with integrated microarray analyses and Sanger sequencing of 46 primary HCC samples (25 HepB, 16 non-viral) and adjacent non-malignant liver controls, complemented by in vitro studies and confirmatory analyses of public databases, this study sought to identify pathways by which hepatocyte late-maturation MYC-antagonist genes are suppressed in HCC, and to examine the translational implications. The hepatocyte late-maturation genes CEBPD and HNF4A are known to antagonize MYC and terminate proliferation. CEBPD and HNF4A expression were significantly decreased in HCC compared to adjacent normal liver (p&lt;0.0001) even though the CEBPD and HNF4A loci were not deleted in any cases. Consistent with epigenetic repression, there was significant hypermethylation of CpG in the promoters for these genes. Two master drivers of hepatocyte commitment that regulate these maturation genes are FOXA2 and GATA4. FOXA2, and also FOXA1/3, were expressed at higher levels in HCC than in adjacent normal liver. However, GATA4 expression was significantly decreased. In 39 HCC cases with karyotype analyzed by SNP array, there were no instances of FOXA2 deletion. In contrast, GATA4 was deleted in 14/39 cases (36%) and mutated (non-synonymous missense or frameshift mutations in exons 4 or 6, e.g., 11614516G&gt;A:S357T) in 17/46 cases (37%) (total GATA4 genetic abnormality 25/43 [58%]). Although GATA4 was not mutated/deleted in every case, all 46 HCC demonstrated decreased expression of HNF4A and highly conserved HNF4A target genes, indicating that this pathway is targeted in most if not all HepB pos or neg HCC. Extrinsic expression of GATA4 in HCC cells (HepG2, PLC) renewed HNF4A and CEBPD expression, decreased MYC and SKP2, increased p27/CDKN1B, and induced cell cycle exit. GATA4 may cooperate with FOXA2 to regulate corepressor/coactivator exchange at target genes. Accordingly, disrupting corepressor function with non-cytotoxic, DNMT1 depleting concentrations of decitabine (0.2-0.5 microM) compensated for GATA4 deficiency and activated CEBPD, HNF4A, decreased MYC, increased p27/CDKN1B, and induced cell cycle exit. GATA4 mutation and deletion in HCC represses HNF4A and CEBPD by therapeutically reversible mechanisms, offering a p53-independent alternative to ineffective apoptosis-based therapy.
Citation Format: Francis O. Enane, Marissa Teo, Hideki Makishima, Zhenbo Hu, Han Chong Toh, Yogen Saunthararajah, Joanna Ng, Chit Lai, Soo Fan Ang, Lim Kiat Hon. Frequent mutation and deletion of GATA4 in hepatocellular carcinoma represses proliferation terminating late-maturation genes CEBPD and HNF4A by reversible epigenetic mechanisms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3518. doi:10.1158/1538-7445.AM2013-3518