Musashi2 Contributes to the Maintenance of CD44v6+ Liver Cancer Stem Cells via Notch1 Signaling Pathway

Background: Liver cancer stem cells (LCSCs) contribute to hepatocellular carcinoma (HCC) development, metastasis, and drug resistance. MSI2 and Notch1 signaling are involved in the promotion and maintenance of CSCs. However, it is ambiguous if they are involved in the maintenance of CD44v6+ LCSCs. Therefore, we investigate the clinical significance and mechanism of MSI2 in the maintenance of stemness properties of CD44v6+ LCSCs. Methods: The expression and correlation of MSI2 and CD44v6 were analyzed by a tissue microarray containing 88 HCC samples. CD44v6+/- cells were isolated using microbeads sorting. We explored the roles of MSI2 and Notch1 signaling in CD44v6+ LCSCs by sphere formation assay, transwell assay, clone formation assay in vitro, and xenograft tumor models in vivo. A Notch RT² PCR Array, Co-immunoprecipitation, and RNA-immunoprecipitation were used to further investigate the molecular mechanism of MSI2 in activating Notch1 signaling. Findings: MSI2 was positively correlated with CD44v6, highly expressed in HCC tissues, and correlated with tumor differentiation and lymph node metastasis. Furthermore, MSI2 and Notch1 signaling pathway were elevated in sorted CD44v6+ cells than CD44v6- cells and played essential roles in the maintenance of self-renewal, invasion and tumorigenic capacity of CD44v6+ LCSCs. Mechanically, MSI2 could directly bind to Lunatic fringe (LFNG) mRNA and protein, and increase the stability of LFNG resulting in activating Notch1 signaling pathway. Interpretation: Our results demonstrated that MSI2 maintained the stemness of CD44v6+ LCSCs by activating Notch1 signaling pathway through LFNG. MSI2 could probably be a potential molecular target for stem cell-targeted therapy of HCC. Funding: This work was supported by the National Natural Science Foundation of China (81172063, 81372352 and 81802418). Funders had no influence on study design, data collection, data analysis, interpretation, writing of the report. Funding Statement: This work was supported by the National Natural Science Foundation of China (81172063, 81372352 and 81802418). Funders had no influence on study design, data collection, data analysis, interpretation, writing of the report. Declarations of Interests: The authors declare no conflict of interest. Ethics Approval Statement: This project was approved by the ethics committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (HUST, Wuhan, China).