ERK5-dependent mechanisms regulate melanoma cell proliferation

Melanoma is the deadliest skin cancer, with a very poor prognosis in advanced stages. Available treatments for melanoma, especially in its intermediate or advanced stages, are unsatisfactory. ERK5 is a member of the Mitogen-Activated Protein kinase family and regulates cell functions critical for tumor development, such as proliferation, invasion and angiogenesis. In silico data analysis indicated that ERK5 pathway components are upregulated in 47% of human melanomas. On this basis, we hypothesized that ERK5 could play a relevant role in melanoma. To study the possible role of ERK5 in the biology of melanoma, we employed a number of human melanoma cell lines. During my PhD, we found that ERK5 is required for the growth of melanoma cells and xenografts harboring wild type (wt) or mutated BRAF (V600E). We later identified a new interplay between ERK5 and the mutated form of B-RAF (V600E) frequently present in melanoma patients. Indeed, using melanoma cells harboring wt BRAF (M26c) and HEK293T cells we found that B-RAFV600E positively regulates ERK5 expression and phosphorylation and increases ERK5 nuclear localization, including that in the chromatin-bound fraction. Accordingly, combined pharmacological inhibition of BRAFV600E and MEK5 is required to decrease nuclear ERK5, that is critical for the regulation of cell proliferation. Furthermore, the combination of MEK5 or ERK5 inhibitors with Vemurafenib is more effective than single treatments in reducing 2D colony formation and growth of BRAFV600E melanoma cells and xenografts. To deepen the knowledge of the molecular mechanisms by which ERK5 controls the growth of melanoma cells, we performed microarray analysis to quantify mRNAs following genetic silencing of ERK5, using two different ERK5-targeting shRNAs (shERK5-275 and shERK5-262), in two different BRAF-mutated melanoma cell lines (A375 and SKMel-5). In particular, by meta-analysis of microarray data, we identified 34 differentially expressed genes (DEGs) (logFC > 1.5) shared by A375 and SKMel-5 cells. Among the above-mentioned genes we further identified 16 upregulated and 18 downregulated DEGs. The idea is to use the upregulated genes in order to identify possible compensative pathways upon ERK5 genetic silencing and to use the downregulated genes to identify new functions of ERK5 in melanoma. The results of the present study shed light on the fact that ERK5 is involved in several mechanisms including, surely, mechanisms involved in melanoma proliferation.