Recent studies revealed that EpCAM is one of the molecular markers for hepatic cancer initiating cells. However, the mechanism how the EpCAM expression in the liver cancer cell is regulated still remains to be delineated. In this study, we identified candidate genes critical for the maintenance of EpCAM positive hepatocellular carcinoma (HCC) cells using a global RNAi screening method. HCC cells were infected with pools of ~200,000 lentiviral shRNA library, then they were sorted to EpCAM positive or negative group. The abundance of shRNA in each group was analyzed using a microarray hybridization method. As a result, 76 candidate shRNAs with significantly different expression between EpCAM positive and negative populations were identified by class comparison analysis. Unsupervised clustering analysis of these genes in 247 HCC samples clearly separated EpCAM positive HCC cases from negative cases. Furthermore, we found that the EpCAM-dependent gene signature can predict survival in epithelial-like HCC cases. We focused on shRNA for PMPCB gene, which has an important role for processing mitochondrial protein, since it ranks as the top differentially altered shRNA-targeted gene between EpCAM positive and negative cells. PMPCB shRNA efficiently reduced EpCAM positive population and effectively inhibited cell proliferation both in vitro and in vivo. Interestingly, PMPCB shRNA specifically induced cell death in EpCAM positive HCC cells, but not in EpCAM negative cells. These data suggest that EpCAM positive cells functionally depend on PMPCB expression. On the other hand, we confirmed that PMPCB shRNA induced physiological dysregulation of mitochondrial function, leading to the increase of reactive oxygen species (ROS) production. We found that PMPCB shRNA significantly reduced TCF4 activity as well as the expression level of c-myc and cyclin D1, which are the downstream molecules of Wnt/β-catenin pathway. Although total β-catenin levels in the cytosol and nuclei fractions were not affected by PMPCB knockdown, overexpression of constitutively activated mutant β-catenin partially rescued the cells from PMPCB-induced apoptosis. Forkhead box O (FOXO) is known to be induced by the ROS production, bind β-catenin and decrease TCF activity, leading to the induction of apoptosis. We found that the nuclear translocation of FOXO family molecules, such as FOXO1 and FOXO3, were facilitated by PMPCB shRNA in EpCAM positive cells. These results suggest that the activation of FOXOs induced by PMPCB knockdown-mediated ROS production suppressed the activity of Wnt/β-catenin pathway, leading to the death of EpCAM positive HCC cells. In conclusion, we discovered a novel candidate target molecule of EpCAM positive HCC cells using a genome-wide RNAi screening method. Our study shed light on the mechanisms how EpCAM positive HCC cells are maintained and identified a new target molecule that has therapeutic vulnerability in EpCAM positive HCC cells.

Citation Format: Atsushi Takai, Hien Dang, Naoki Oishi, Xin Wei Wang. Identification of novel cancer driver genes in hepatocellular carcinoma cells by RNAi screening method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4391. doi:10.1158/1538-7445.AM2017-4391