Table1_Autophagic Inhibition of Caveolin-1 by Compound Phyllanthus urinaria L. Activates Ubiquitination and Proteasome Degradation of β-catenin to Suppress Metastasis of Hepatitis B-Associated Hepatocellular Carcinoma.docx

Compound Phyllanthus urinaria L. (CP) is a traditional Chinese medicine (TCM) formula for cancer treatment in the clinic, particularly during progression of hepatitis B-associated hepatocellular carcinoma (HBV-associated HCC). Nevertheless, its anti-metastatic action and mechanisms are not well elucidated. In this study, CP was found to exert remarkable inhibitory effects on the proliferation, migration and invasion of HBV-associated HCC cells. The following network and biological analyses predicted that CP mainly targeted Caveolin-1 (Cav-1) to induce anti-metastatic effects, and Wnt/β-catenin pathway was one of the core mechanisms of CP action against HBV-associated HCC. Further experimental validation implied that Cav-1 overexpression promoted metastasis of HBV-associated HCC by stabilizing β-catenin, while CP administration induced autophagic degradation of Cav-1, activated the Akt/GSK3β-mediated proteasome degradation of β-catenin via ubiquitination activation, and subsequently attenuated the metastasis-promoting effect of Cav-1. In addition, the anti-cancer and anti-metastatic action of CP was further confirmed by in vivo and ex vivo experiments. It was found that CP inhibited the tumor growth and metastasis of HBV-associated HCC in both mice liver cancer xenograft and zebrafish xenotransplantation models. Taken together, our study not only highlights the novel function of CP formula in suppressing metastasis of HBV-associated HCC, but it also addresses the critical role of Cav-1 in mediating Akt/GSK3β/β-catenin axis to control the late-phase of cancer progression.

复合扶桑叶（CP）系传统中药（TCM）配方，用于临床癌症治疗，尤其是在乙型肝炎病毒（HBV）相关肝细胞癌（HBV-associated HCC）进展期间。尽管如此，其抗转移作用及机制尚未得到充分阐明。本研究发现，CP对HBV相关HCC细胞的增殖、迁移和侵袭表现出显著的抑制作用。随后进行的网络和生物分析预测，CP主要通过靶向Caveolin-1（Cav-1）诱导抗转移效应，而Wnt/β-catenin通路是CP对抗HBV相关HCC作用的核心机制之一。进一步的实验验证表明，Cav-1的过表达通过稳定β-catenin促进HBV相关HCC的转移，而CP的施加则诱导Cav-1的自噬降解，通过泛素化激活激活Akt/GSK3β介导的蛋白酶体降解β-catenin，从而减弱Cav-1的促转移效应。此外，CP的抗肿瘤和抗转移作用通过体内和体外实验得到了进一步证实。研究发现，CP在HBV相关HCC小鼠肝肿瘤异种移植和斑马鱼异种移植模型中均抑制了肿瘤的生长和转移。综上所述，本研究不仅突出了CP配方在抑制HBV相关HCC转移中的新颖功能，而且揭示了Cav-1在调节Akt/GSK3β/β-catenin轴以控制癌症进展后期中的关键作用。