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Background and Aims: HCC is an aggressive cancer with a poor clinical outcome. Understanding the mechanisms that drive tumor initiation is important for improving treatment strategy. This study aimed to identify functional cell membrane proteins that promote HCC tumor initiation., Approach and Results: Tailor-made siRNA library screening was performed for all membrane protein-encoding genes that are upregulated in human HCC (n = 134), with sphere formation as a surrogate readout for tumor initiation. Upon confirmation of membranous localization by immunofluorescence and tumor initiation ability by limiting dilution assay in vivo, LanC-like protein-1 (LANCL1) was selected for further characterization. LANCL1 suppressed intracellular reactive oxygen species (ROS) and promoted tumorigenicity both in vitro and in vivo. Mechanistically, with mass spectrometry, FAM49B was identified as a downstream binding partner of LANCL1. LANCL1 stabilized FAM49B by blocking the interaction of FAM49B with the specific E3 ubiquitin ligase TRIM21, thus protecting FAM49B from ubiquitin-proteasome degradation. The LANCL1-FAM49B axis suppressed the Rac1-NADPH oxidase-driven ROS production, but this suppression of ROS was independent of the glutathione transferase function of LANCL1. Clinically, HCCs with high co-expression of LANCL1 and FAM49B were associated with more advanced tumor stage, poorer overall survival, and disease-free survival. In addition, anti-LANCL1 antibodies targeting the extracellular N-terminal domain were able to suppress the self-renewal ability, as demonstrated by the sphere formation ability of HCC cells., Conclusions: Our data showed that LANCL1 is a cell surface protein and a key contributor to HCC initiation. Targeting the LANCL1-FAM49B-Rac1-NADPH oxidase-ROS signaling axis may be a promising therapeutic strategy for HCC., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background and Aims: Hepatitis B virus (HBV) integrations are common in hepatocellular carcinoma (HCC). In particular, alterations of the telomerase reverse transcriptase (TERT) gene by HBV integrations are frequent; however, the molecular mechanism and functional consequence underlying TERT HBV integration are unclear., Approach and Results: We adopted a targeted sequencing strategy to survey HBV integrations in human HBV-associated HCCs (n = 95). HBV integration at the TERT promoter was frequent (35.8%, n = 34/95) in HCC tumors and was associated with increased TERT mRNA expression and more aggressive tumor behavior. To investigate the functional importance of various integrated HBV components, we employed different luciferase reporter constructs and found that HBV enhancer I (EnhI) was the key viral component leading to TERT activation on integration at the TERT promoter. In addition, the orientation of the HBV integration at the TERT promoter further modulated the degree of TERT transcription activation in HCC cell lines and patients' HCCs. Furthermore, we performed array-based small interfering RNA library functional screening to interrogate the potential major transcription factors that physically interacted with HBV and investigated the cis-activation of host TERT gene transcription on viral integration. We identified a molecular mechanism of TERT activation through the E74 like ETS transcription factor 4 (ELF4), which normally could drive HBV gene transcription. ELF4 bound to the chimeric HBV EnhI at the TERT promoter, resulting in telomerase activation. Stable knockdown of ELF4 significantly reduced the TERT expression and sphere-forming ability in HCC cells., Conclusions: Our results reveal a cis-activating mechanism harnessing host ELF4 and HBV integrated at the TERT promoter and uncover how TERT HBV-integrated HCCs may achieve TERT activation in hepatocarcinogenesis., (© 2020 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)