Abstract
BackgroundThe 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.MethodsMass 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.ResultsWe 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-PHBY259) 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-PHBY259 interacted with Notch3 to stabilize Notch3 and increase the downstream target PBX1. Downregulation of raft-phospho-PHBY259 increased the protein degradation of Notch3 through a lysosomal pathway and inhibited the β-catenin—ABCG2 signaling pathway. Moreover, raft-phospho-PHBY259 played an important role in ovarian cancer stemness and tumorigenicity as well as resistance to platinum drug treatment in vitro and in vivo.ConclusionsThese findings thus reveal a hitherto unreported interrelationship between c-Kit and PHB as well as the effects of raft-phospho-PHBY259 on ovarian cancer stemness and tumorigenicity mediated by the Notch3 and β-catenin signaling pathways. Targeting the c-Kit/raft-phospho-PHBY259 axis may provide a new therapeutic strategy for treating patients with ovarian cancer.
Highlights
The underlying mechanism involved in ovarian cancer stemness and chemoresistance remains largely unknown
Results mast/stem cell factor receptor (c-Kit) phosphorylates PHB at the Tyr259 residue To identify which tyrosine kinase increases the phosphorylation of PHB to form phospho-PHBY259, we used the online software Group-based Prediction System (GPS) for prediction
Since c-Kit and Notch3 have been reported to play a role in the regulation of ovarian cancer stem cells (CSCs) [19, 34, 35] and CSC-driven metastasis [36, 37], we examined their expression in these different phenotypes of SKOV3GL cells
Summary
The underlying mechanism involved in ovarian cancer stemness and chemoresistance remains largely unknown. We explored whether the regulation of c-Kit and plasma membrane prohibitin (PHB) affects ovarian cancer stemness and chemotherapy resistance. Exploring the molecular mechanism to develop approaches to prevent ovarian tumor recurrence is an unmet clinical need. One promising approach is to study the survival signaling pathways of tumor initiating cancer stem cells (CSCs) that possess the ability to self-renew, differentiate and regenerate tumors in vivo. These CSCs are generally resistant to conventional cancer therapies and are positively regulated in tumor metastasis and recurrence [3,4,5]. Pluripotent stem cell markers of normal stem cells, notably OCT4, NANOG and SOX2, have been found to play an important role in ovarian cancer development and metastasis and can be used to verify cell stemness [12]
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