Gap Junction Protein Connexin43 (Cx43) Promotes Glioma Invasion via the Annexin A2/MMP3 Signaling Axis

Glioblastoma is among the most aggressive primary brain tumors, with the invasion of surrounding tissue driven by coordinated remodeling of the extracellular matrix (ECM) by proteolytic enzymes, including the matrix metalloproteinase (MMP) family. The gap junction protein connexin43 (Cx43) has emerged as an unexpected promoter of this invasive phenotype. However, the downstream molecular mechanisms linking Cx43 to ECM proteolysis remain poorly understood. We previously demonstrated selective upregulation of MMP3 within the secretome of Cx43-overexpressing glioma cells, implicating Cx43 in the regulation of ECM protease activity. To identify the molecular intermediaries responsible, we carried out a proteomic survey of gap junction (GJ)-associated signaling networks. Amongst these, we identified a Cx43 protein-protein interaction involving annexin A2 (ANXA2), a known regulator of ECM proteases. SDS-PAGE/Western blot and confocal imaging demonstrated that elevated Cx43 expression drives selective recruitment of ANXA2 to gap junction membranes, forming a junctional signaling platform rather than increasing total ANXA2 abundance. This recruitment coincided with co-localization of S100A10, suggesting assembly of an intact ANXA2/S100A10 complex at gap junctions. Using ANXA2-mimetic peptides targeting the S100A10-binding domain, we demonstrate that disruption of the ANXA2/S100A10 complex leads to a significant reduction in MMP3 activity. Furthermore, in 3D spheroids grown in ECM-replicating gels, ANXA2 peptide treatment significantly reduced the invasive potential of C6-13 (Cx43-overexpressing) cells. Together, these data define a novel Cx43[->]ANXA2[->]S100A10[->]MMP3 signaling axis, advancing our understanding of ECM remodeling in glioblastoma and establishing the ANXA2/S100A10 complex as a promising therapeutic target for disrupting Cx43-driven glioblastoma progression.