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-keratinocyte contact and loss of the 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 center dot 1 (GJB5) expression and identify any association with BRAF mutational status, prognosis of patients with melanoma and mitogen-activated protein kinase (MAPK) inhibitor (MAPKi) treatment. 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 (MEKi), as well as in cell lines which developed MAPKi 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 with primary ones and in BRAF-mutated vs. BRAF-wildtype (BRAF(WT)) melanomas. Likewise, GJB5 expression is significantly lower in BRAF(V600E) compared with BRAF(WT) cell lines and increases on MAPKi treatment. MAPKi-resistant melanoma cells display a similar expression pattern compared with BRAF(WT) cells, with increased GJB5 expression associated with morphological changes. Enhancement of BRAF(V600E) expression in BRAF(WT) 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 BRAF(WT) cell lines confirms specific GJB5 protein downregulation. Reverse transcriptase quantitative polymerase chain reaction analysis also revealed upregulation of miR-335 in BRAF(V600E) melanoma cells, which is significantly downregulated in cells resistant to MEKi. 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. Conclusions 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.