WITHDRAWN: Alpha-fetoprotein Promotes Hepatocellular Carcinoma Progression by Negatively Regulating Hippo Signaling Pathway Running title: AFP Regulate the Hippo Signaling Pathway

Abstract

Background: Alpha-fetoprotein (AFP) is a specific tumor marker for hepatocellular carcinoma (HCC) and promotes tumor cell proliferation in HCC, but the mechanism is not completely clear. This research aimed to supplement the mechanism of AFP in HCC. Methods: Lentivirus transfected HCC cells for AFP knockdown and overexpression, CCK-8, clone formation, scratch assay, and 3D multicellular tumor spheroids（MCTS） formation assay verified the positive regulation of AFP on HCC. Co-immunoprecipitation, liquid chromatography-mass spectrometry (LC-MS/ MS), and bioinformatics techniques were used to identify proteins interacting with AFP and related pathways in HCC cells, and western blot, real-time quantitative PCR(RT-qPCR), and immunofluorescence were used to detect the expression changes of proteins associated with AFP pathways. The effect of AFP on the HCC cell cycle was detected by flow cytometry. Results: AFP promotes the proliferation, migration, and the formation of MCTS of HCC cells. In HCC, AFP interacts with large tumor suppressor 2 (LATS2) and negatively regulates Hippo signaling pathway. AFP inhibits the phosphorylation of LATS2, which in turn inhibits the phosphorylation of yes associated protein (YAP). In addition, AFP was positively correlated with the expression of CyclinD1. Conclusion: AFP negatively regulates Hippo signaling pathway through the LATS2/YAP axis and promotes the proliferation, migration, and formation of MCTS of HCC cells.