Epigenetic control of the metabolic tumor suppressor FBP1 in liver cancer

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Abstract Hepatocellular carcinoma (HCC) is the primary form of liver cancer and afflicts over half a million people worldwide. Alarmingly, mortality rates have steadily increased in recent years, highlighting the importance of identifying new therapies. One promising area of treating HCC is in targeting cellular metabolism. Specifically, aerobic glycolysis is a common feature of liver cancer, which is an otherwise genetically and pathologically heterogeneous disease. Neoplastic hepatocyte cells attain this glycolytic phenotype in part by decreasing physiological gluconeogenesis. Indeed, the mRNA and protein levels of a rate-limiting gluconeogenic enzyme, fructose-1,6-bisphosphatase 1 (FBP1), are significantly depleted in HCC. Our lab has found that ectopically expressing FBP1 in HCC xenografts reduces tumor growth, whereas deleting Fbp1 accelerates autochthonous models of liver cancer. These results suggest a strong tumor suppressive role for FBP1, and substantiate the therapeutic potential of FBP1 activity. Therefore, my overall goal is to elucidate the mechanism of FBP1 repression to discover new therapeutic strategies for HCC. Three key pieces of preliminary data indicate that the histone 3 lysine 27 (H3K27) methyltransferase, Enhancer of Zeste Homolog 2 (EZH2), contributes to FBP1 suppression: (1) publicly available ChIP-seq data reveals EZH2 enrichment sites at the FBP1 gene locus in human embryonic stem cells. (2) EZH2 mRNA levels progressively increase with HCC stage and negatively correlate with FBP1 expression. (3) Functionally, depleting EZH2 in HCC cells increases FBP1 mRNA, whereas elevating EZH2 expression in normal hepatocytes diminishes FBP1 protein abundance. I hypothesize that EZH2 directly catalyzes H3K27 trimethylation at the FBP1 promoter to suppress its transcription, and inhibiting EZH2 will reduce HCC growth, in part, through metabolic consequences of FBP1 activation. To address these ideas, I will first characterize the physical interaction of EZH2 with the FBP1 locus in HCC by ChIP. Additionally, I will determine how EZH2 is recruited to the FBP1 promoter by performing co-IP and mass spectrometry, followed by validation of candidate binding partners. Second, to determine biological effects relevant to clinical response, I will perform comprehensive metabolite analyses of HCC cells under conditions of EZH2 loss. Importantly, I expect to see both FBP1-dependent and independent alterations in metabolism upon EZH2 reduction, which will have implications for metabolite biomarkers, synthetic lethality, and resistance mechanisms. This work will decipher the complex nature of FBP1 epigenetic silencing in HCC, and outline new treatment paradigms that reactivate gluconeogenesis in HCC.