Non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC) is an emerging malignancy due to the rising prevalence of NAFLD. However, no drug is available to target NAFLD-HCC. In this study, we aim to unravel novel therapeutic targets of NAFLD-HCC utilizing a high-throughput CRISPR/Cas9 screening strategy. We utilized the Epi-drug CRISPR/Cas9 library consisting of single-guide RNAs (sgRNAs) targeting over 1,000 genes representing the FDA-approved drug targets and epigenetic regulators to perform loss-of-function screening in two NAFLD-HCC cell lines (HKCI2 and HKCI10). CRISPR/Cas9 library screening unraveled TUBB4B as an essential gene for NAFLD-HCC cell growth. TUBB4B was overexpressed in NAFLD-HCC tumors compared with adjacent normal tissues (N = 17) and was associated with poor survival (p < 0.01). RNA-sequencing and functional assays revealed that TUBB4B knockout in NAFLD-HCC promoted cell apoptosis, cell cycle arrest, and cellular senescence, leading to suppressed NAFLD-HCC growth in vitro and in vivo. We identified that TUBB4B inhibitor mebendazole (MBZ), an FDA-approved drug, inhibited NAFLD-HCC growth by inducing apoptosis and cellular senescence. Since protein expression of pro-survival Bcl-xL was induced in TUBB4B knockout NAFLD-HCC cells, we examined combination of TUBB4B inhibition with navitoclax, a Bcl-xL inhibitor that selectively targets senescent cells. Consistent with our hypothesis, either TUBB4B knockout or MBZ synergized with navitoclax to inhibit NAFLD-HCC cell growth via the induction of intrinsic and extrinsic apoptosis pathways. In summary, TUBB4B is a novel therapeutic target in NAFLD-HCC. Inhibition of TUBB4B with MBZ in combination with navitoclax synergistically inhibited NAFLD-HCC cell growth, representing a promising strategy for the treatment of NAFLD-HCC. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Carcinoma, Hepatocellular , Liver Neoplasms , Non-alcoholic Fatty Liver Disease , Humans , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Non-alcoholic Fatty Liver Disease/drug therapy , Non-alcoholic Fatty Liver Disease/genetics , Non-alcoholic Fatty Liver Disease/metabolism , Signal Transduction
