A novel c-Kit/phospho-prohibitin axis enhances ovarian cancer stemness and chemoresistance via Notch3-PBX1 and β-catenin-ABCG2 signaling

Abstract

Background: The underlying mechanism involved in ovarian cancer stemness and chemoresistance remains largely unknown. Here, we explored whether the regulation of c-Kit and plasma membrane prohibitin (PHB) affects ovarian cancer stemness and chemotherapy resistance.

Methods: Mass spectrum analysis and an in vitro kinase assay were conducted to examine the phosphorylation of PHB at tyrosine 259 by c-Kit. The in vitro effects of c-Kit on membrane raft-PHB in ovarian cancer were determined using tissue microarray (TMA)-based immunofluorescence, western blotting, immunoprecipitation, colony and spheroid formation, cell migration and cell viability assays. In vivo tumor initiation and carboplatin treatment were conducted in nude mice.

Results: We found that c-Kit and PHB colocalized in the raft domain and were positively correlated in human ovarian serous carcinoma. c-Kit interacted with PHB and facilitated the phosphorylation of PHB at tyrosine 259 (phospho-PHB^Y259) in the membrane raft to enhance ovarian cancer cell motility. The generation of SKOV3GL-G4, a metastatic phenotype of SKOV3 green fluorescent protein and luciferase (GL) ovarian cancer cells, in xenograft murine ascites showed a correlation between metastatic potential and stem cell characteristics, as indicated by the expression of c-Kit, Notch3, Oct4, Nanog and SOX2. Further study revealed that after activation by c-Kit, raft-phospho-PHB^Y259 interacted with Notch3 to stabilize Notch3 and increase the downstream target PBX1. Downregulation of raft-phospho-PHB^Y259 increased the protein degradation of Notch3 through a lysosomal pathway and inhibited the β-catenin-ABCG2 signaling pathway. Moreover, raft-phospho-PHB^Y259 played an important role in ovarian cancer stemness and tumorigenicity as well as resistance to platinum drug treatment in vitro and in vivo.

Conclusions: These findings thus reveal a hitherto unreported interrelationship between c-Kit and PHB as well as the effects of raft-phospho-PHB^Y259 on ovarian cancer stemness and tumorigenicity mediated by the Notch3 and β-catenin signaling pathways. Targeting the c-Kit/raft-phospho-PHB^Y259 axis may provide a new therapeutic strategy for treating patients with ovarian cancer.

Keywords: Cancer stem cells; Lipid raft domain of plasma membrane; Notch3; Prohibitin; c-Kit; β-catenin.