Abstract 3740: Role of phosphatidylcholine in β-catenin-driven hepatocellular carcinoma

Introduction: This study aimed to clarify the role of phosphatidylcholine (PC) and choline metabolites in β-catenin-driven hepatocellular carcinoma (HCC) using human HCC cell lines and transgenic zebrafish expressing hepatocyte-specific activated β-catenin. Background: β-catenin is widely believed to be a major oncogene in HCC. PC is one of the major constituents of biological membranes and is synthesized via the phosphatidylethanolamine N-methyltransferase (PEMT) and cytidine diphosphate (CDP)-choline pathways in the liver. Several studies have reported that changes in gene expression and enzyme activity lead to altered PC synthesis in cancer, which contributes to malignant growth. However, the role of altered PC synthesis in HCC and the contribution of activated β-catenin to changes in PC metabolism have not been fully explored. Methods and Results: Transgenic zebrafish expressing hepatocyte-specific activated β-catenin develop HCC as adults and show significant liver enlargement during larval development due to increased cell proliferation. We measured PC levels in transgenic zebrafish HCC and non-transgenic control livers using liquid chromatography-mass spectrometry (LC-MS). PC levels in transgenic zebrafish HCC were significantly higher than in controls. Using a colorimetric assay kit, we found that PC levels were significantly higher in human HCC cell lines (SNU398, C3A and PLC/PRF/5) than in immortalized human hepatocytes (THLE2). To clarify the role of PC and choline metabolites in β-catenin-driven liver overgrowth, we treated larval zebrafish with bezafibrate (PEMT inhibitor) or meclofenoxate (CPT/CEPT inhibitor). We found that both of these inhibitors of PC lipid synthesis reduced larval liver size and decreased the number of EdU-labeled proliferative cells in transgenic zebrafish. Histologically, nonalcoholic steatohepatitis (NASH)-like changes, such as steatosis, ballooning, inflammation, and hepatocellular necrosis, were detected in the livers of transgenic larvae treated with bezafibrate or meclofenoxate. To characterize the role of PC in human HCC cell lines, we treated SNU398, C3A and PLC/PRF/5 cells with bezafibrate or meclofenoxate and measured PC levels by colorimetric assay and viability by CellTiter-Glo Assay. We found that both drugs decreased PC levels and viability in these HCC cell lines. Furthermore, SNU398 cells overexpressing activated β-catenin using a Tet-on system demonstrated higher PC levels and greater viability than SNU398 control cells. Conclusion: Our results suggest that increased PC synthesis is associated with cell proliferation, zebrafish larval liver overgrowth, and hepatocarcinogenesis. These results also suggest that activated β-catenin may regulate choline metabolites and contribute to an increase in PC production. Citation Format: Junko Kuramoto, Richard Smith, Sharanya M. Kalasekar, Alexis Fulbright, Gregory S. Ducker, Kimberley J. Evason. Role of phosphatidylcholine in β-catenin-driven hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3740.