Role of Connexin43 in Tumour Prograssion and Drug Resistance in BRAF-driven Tumors

Abstract

Mutations in the oncogenic serine/threonine protein kinase BRAF are casually involved in the onset and progression of several cancers, including more than 50% of cases of melanoma. Malignant melanoma is the most lethal form of skin cancer, with an increasing incidence worldwide. The most common BRAF mutation is a valine-to-glutamate transition (V600E), which results in constitutive activation of the kinase activity. BRAF and MEK inhibitors (BRAF/MEKi) combination have been approved for use in different cancers with BRAF mutations. Despite BRAF/MEK inhibition have shown efficacy for metastatic disease, acquired resistance remains the main barrier to increase progression-free survival and overall survival. Connexins (Cxs) are a family of transmembrane proteins that form hemichannels and gap junctions that permit the paracrine and direct intercellular communication by allowing the diffusion of ions, small molecules and metabolites. Alterations in connexin43 (Cx43) and gap junction intercellular communication (GJIC) have been implicated in tumour development and progression. However, due to the controversial role of Cx43 in melanoma progression, the aims of this project were to investigate the role of Cx43 in tumours with BRAF mutations and the mechanisms involved in acquired drug resistance to BRAF/MEKi. The results of this study demonstrate that Cx43 is poorly expressed in human melanoma and it is mainly localized in the cytoplasm. Cx43 overexpression, using a vector or small extracellular vesicles (sEVs) containing Cx43 significantly decreases in cell growth and proliferation and increases senescence and cell death by apoptosis in in vitro assays. We find that the effects on cell growth are independent of the channel activity. Post-translational modification such as SUMOylation of Cx43 detected in all human melanoma cell lines may affect protein stability, subcellular localization or protein-protein interactions. In fact, the presence of Cx43 in sEVs including exosomes, radically changes the content of proteins and small molecules of RNA (sRNA) in these vesicles, indicating that Cx43 may participate in the recruitment of proteins and sRNA. Indeed, the presence of Cx43 changes the composition and function of the released sEVs. In addition, the restoration of Cx43 in tumour cells with a mutation in BRAF significantly increases the efficay of BRAF/MEKi, and prevents drug resistance by reinforcing cellular senescence and increasing cell death by apoptosis. Importantly, double resistant cell lines do not contain Cx43, and the transfection with a vector to overexpress Cx43 or treatment with sEVs containing Cx43, re-sensitizes resistant cells to BRAF/MEKi. In this study we have identified Cx43 as a new therapeutic target for the treatment of tumours with BRAF mutations and to overcome drug resistance to BRAF/MEKi.