GJB5 association with BRAF mutation and survival in cutaneous malignant melanoma.

Background Gap junctional intercellular communication is crucial for epidermal cellular homeostasis. Inability to establish melanocyte-keratinocytes contacts and loss of intercellular junction's integrity may contribute to melanoma development. Connexins, laminins and desmocollins have been implicated in the control of melanoma growth, where their reduced expression has been reported in metastatic lesions. Objectives The aim of this study was to investigate Connexin 31.1 (GJB5) expression and identify any association with BRAF mutational status, melanoma patient prognosis and MAPK inhibitors (MAPKi) treatment. Material and methods GJB5 expression was measured at RNA and protein level in melanoma clinical samples and established cell lines treated or not with BRAF and MEK inhibitors, as well as in cell lines which developed MAPK inhibitors resistance. Findings were further validated and confirmed by analysis of independent datasets. Results Our analysis reveals significant downregulation of GJB5 expression in metastatic melanoma lesions compared to primary ones and in BRAF mutated versus BRAF wild-type melanomas. Likewise, GJB5 expression is significantly lower in BRAFV600E compared with BRAFWT cell lines and increases upon MAPKi treatment. MAPKi-resistant melanoma cells display a similar expression pattern compared to BRAFWT cells, with increased GJB5 expression associated with morphological changes. Enhancement of BRAFV600E expression in BRAFWT melanoma cells significantly upregulates miR-335-5p expression with consequent downregulation of GJB5, one of its targets. Furthermore, overexpression of miR-335-5p in two BRAFWT cell lines confirms specific GJB5 protein downregulation. RT-qPCR analysis also revealed upregulation of miR-335 in BRAFV600E melanoma cells, which is significantly downregulated in cells resistant to MEK inhibitors. Our data were further validated using the TCGA-SKCM dataset, where BRAF mutations associate with increased miR-335 expression and inversely correlate with GJB5 expression. In clinical samples, GJB5 underexpression is also associated with patient overall worse survival, especially at early stages. Conclusion We identified a significant association between metastases / BRAF mutation and low GJB5 expression in melanoma. Our results identify a novel mechanism of Gap-junctional protein regulation, suggesting a prognostic role for GJB5 in cutaneous melanoma.