Abstract
The incidence of malignant melanoma, one of the deadliest cancers, continues to increase. Here we tested connexin (Cx) expression in primary melanocytes, melanoma cell lines and in a common nevus, dysplastic nevus, and thin, thick, and metastatic melanoma tumor progression series involving the tumor microenvironment by utilizing in silico analysis, qRT-PCR, immunocyto-/histochemistry and dye transfer tests. Primary melanocytes expressed GJA1/Cx43, GJA3/Cx46 and low levels of GJB2/Cx26 and GJC3/Cx30.2 transcripts. In silico data revealed downregulation of GJA1/Cx43 and GJB2/Cx26 mRNA, in addition to upregulated GJB1/Cx32, during melanoma progression. In three melanoma cell lines, we also showed the loss of GJA1/Cx43 and the differential expression of GJB1/Cx32, GJB2/Cx26, GJA3/Cx46 and GJC3/Cx30.2. The dominantly paranuclear localization of connexin proteins explained the ~10–90 times less melanoma cell coupling compared to melanocytes. In melanocytic tumor tissues, we confirmed the loss of Cx43 protein, fall of cell membrane and elevated paranuclear Cx32 with moderately increased cytoplasmic Cx26 and paranuclear Cx30.2 positivity during tumor progression. Furthermore, we found Cx43, Cx26 and Cx30 proteins upregulated in the melanoma adjacent epidermis, and Cx43 in the tumor flanking vessels. Therefore, differential connexin expression is involved in melanocytic tumor progression where varying connexin isotypes and levels reflect tumor heterogeneity-related bidirectional adaptive interactions with the microenvironment.
Highlights
Despite easy access and visibility of the skin and organized screening programs, cutaneous melanoma is one of the most fatal cancers, which shows an increasing incidence in white-skinned populations worldwide [1]
In datasets using GSE3189 arrays, melanomas showed downregulated GJA1 (Cx43; LogFC = −4.1; p < 0.001), GJB3 (Cx31; LogFC = −3.4; p < 0.001), GJB5 (Cx31.1; LogFC = −3.3; p < 0.001) and upregulated GJB1 (Cx32; LogFC = +1.5; p < 0.001) gene expression compared to nevi [26]
Our results reveal diverse Cx isotype expression during melanocytic tumor progression including the loss of Cx43 protein with moderate recurrence in metastases and elevation of diffuse cytoplasmic Cx26
Summary
Despite easy access and visibility of the skin and organized screening programs, cutaneous melanoma is one of the most fatal cancers, which shows an increasing incidence in white-skinned populations worldwide [1]. In spite of experimental data on the potential role of Cxs in controlling melanoma development and metastatic invasion, studies on melanocytic tumor progression in clinical cases are still incomplete Cxs and their channels play key roles in maintaining multicellular homeostasis in most tissues, including the skin (as has been discussed by several papers related to this issue), with particular importance of regulating cell death, proliferation [6] and migration [7]. They can interact directly with a wide range of intracellular regulatory molecules, including adhesion, proto-oncogene and oncosuppressor proteins (“gap junction proteome”), to provide channel independent functions [8]
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