Connexin43 functions as a non-canonical phenotypic stability factor in promoting hybrid Epithelial/Mesenchymal phenotype in glioblastoma cells

Glioblastoma (GBM) is a highly malignant and aggressive brain tumor with patients typically experiencing a median survival of 15–18 months after diagnosis. Gap junction protein Connexin43 (Cx43) plays a crucial role in GBM by having both tumor-suppressing and tumor-promoting roles. Here, we identify a critical tumor-promoting role of Cx43 in GBM by functioning as a non-canonical phenotypic stability factor and driving partial EMT, which enhances the acquisition of stemness properties in the cells. Using high-grade mouse astrocytoma cell lines, we found that CT2A cells had higher Cx43 gap junction assembly as compared to KR158 cells. The increased Cx43 assembly in CT2A cells activates the NF-κB signaling pathway, promoting a hybrid E/M phenotype and thereby enhancing self-renewal properties. CT2A cells also exhibited collective cell migration, a characteristic feature of hybrid E/M phenotype, stress resistance, and proliferative properties. To verify Cx43′s role in NF-κB pathway activation, DBT and DBT-Erp-29 cells (with higher Cx43 expression) were studied, showing increased NF-κB activation in DBT-Erp-29 cells. Interestingly, KR158 cells formed longer tunneling nanotubes to expedite alternative cellular communication due to reduced gap junctional intercellular communication (GJIC). These results offer valuable insights into targeting Cx43-mediated signaling pathways due to the potential tumor-promoting role of Cx43.