HCC是一种具有不良临床预后的侵袭性癌症。了解推动肿瘤起始的机制对改善治疗策略非常重要。本研究旨在识别促进HCC肿瘤起始的功能性细胞膜蛋白。对在人HCC中上调表达的所有膜蛋白编码基因（n = 134）进行定制siRNA文库筛选，以球体形成作为肿瘤起始的间接指标。通过免疫荧光确认其膜定位和限制稀释法在体内确认其肿瘤起始能力之后，选择了LanC样蛋白1（LANCL1）进行进一步的表征。LANCL1抑制细胞内活性氧（ROS）生成，并且促进了体外和体内的肿瘤形成能力。机制上，质谱法鉴定出FAM49B为LANCL1的下游结合伴侣。LANCL1通过阻断FAM49B与特定的E3泛素连接酶TRIM21的相互作用来稳定FAM49B，从而保护FAM49B不被泛素蛋白酶体降解。LANCL1-FAM49B轴抑制Rac1-NADPH氧化酶驱动的ROS生成，但这种ROS抑制与LANCL1的谷胱甘肽转移酶功能无关。临床上，LANCL1和FAM49B高共表达的HCC与更晚期肿瘤阶段、较差的总生存和无疾病生存相关。此外，针对LANCL1细胞外N-末端域的抗体能够抑制HCC细胞球体形成能力，表明LANCL1是一个细胞表面蛋白，并且是HCC起始的关键因素。靶向LANCL1-FAM49B-Rac1-NADPH氧化酶-ROS信号轴可能是一个有前景的HCC治疗策略。 版权所有 © 2023 The Author(s). Wolters Kluwer Health, Inc. 发表

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 T initiation.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.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.