Rab8 GTPase Regulates Klotho-Mediated Inhibition of Cell Migration and Invasiveness by Directly Modulating its Surface Expression in Human Non-Small Cell Lung Cancer

Background: The klotho (KL) gene is an anti-aging gene that has recently been shown to also function as a general tumor suppressor. However, there is currently only limited information regarding the potential molecular signals for regulation of Klotho without identifying precise molecular mechanisms or interactions. Methods: We performed a mass spectrometry (MS) assay to screen candidate proteins complexed with Klotho derived from immunoprecipitation in human non-small cell lung cancer (NSCLC) cells, and identified Rab8 to be the protein that most prominently interacts with Klotho. We further investigated whether Rab8 can regulate trafficking of Klotho and which process it would modulate using surface biotinylation assay, immunofluorescence and fluorescence ratio microscopy. Furthermore, we explored whether Rab8 is involved in Klotho-mediated function in NSCLC, and verified the results which we found in vitro using xenograft mouse model. Findings: We report discovery of Rab8 as a Klotho-interacting protein that acts as a critical modulator of Klotho surface expression in human NSCLC. In particular, we report that Rab8 is co-localized and associated with Klotho, and Klotho trafficking is regulated by Rab8. Moreover, we found that Rab8 modulates surface levels of Klotho via a post-biosynthetic pathway, as opposed to an endocytic pathway. Furthermore, we demonstrate that Rab8 is involved in Klotho-mediated regulation of cell migration, invasiveness, epithelial-mesenchymal transition (EMT), and Wnt-β-catenin signaling in NSCLC. Additionally, Rab8 overexpression was also found to increase Klotho-mediated inhibition of NSCLC tumorigenesis in vivo. Interpretation: Overall, our findings suggest that Rab8 GTPase can regulate Klotho-mediated inhibition of Wnt signaling activity by modulating translocation of Klotho onto the cell surface, which in turn affects Klotho-mediated inhibition of cell migration and invasiveness in NSCLC. These results have important implications for the development of new therapeutic targets, Klotho-related research in the context of NSCLC as well as other areas, and provide a working model for Rab8 function in the context of cancer and cancer biology. Funding Statement: This study was supported by the International Science & Technology Cooperation Program of China (No. 2014DFA31940); The National Natural Science Foundation of China (Nos. 81572259 and 81302011); The Jiangsu Province’s Youth Medical Talents Program (No. QNRC2016593); The Jiangsu Provincial Key Discipline of Medicine (No. ZDXKA2016003); The Six Talent Peaks Project in Jiangsu Province (No. 2018-WSN-003). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: Ethical approval was obtained from Jiangsu Province Hospital (The First Affiliated Hospital of Nanjing Medical University) Research Ethics Committee.