Abstract
Constitutively active mutated BRAF kinase occurs in more than 40% of patients suffering from melanoma. To block its activity, a specific inhibitor, vemurafenib, is applied as a therapy. Unfortunately, patients develop resistance to this drug rather quickly. Previously, we demonstrated that pairs of inhibitors directed against EGFR (epidermal growth factor receptor) and MET (hepatocyte growth factor receptor) trigger a synergistic cytotoxic effect in human melanoma cells, and decrease their invasive abilities. In this study, we aimed to generate and characterize melanoma cells resistant to vemurafenib treatment, and then to evaluate the effectiveness of a previously developed therapy in this model. We showed that melanoma cells resistant to the BRAF inhibitor are characterized by a lower proliferation rate and they acquire a spindle-like shape. Using Western Blot, we also noticed increased levels of EGFR, MET, and selected markers of cancer stem cells in generated cell lines. Resistant cells also exhibited increased invasive abilities and elevated proteolytic activity, observed using scratch wound assays and gelatin zymography. Moreover, combination therapy reduced their viability, as measured with a colorimetric cytotoxicity test, and decreased invasiveness. The obtained results validate the application of combination therapy directed against EGFR and MET in melanoma cells resistant to treatment with inhibitors of mutated BRAF.
Highlights
Among all patients suffering from skin cancers, only 4% are diagnosed with malignant melanoma
We aimed to extend our previous work, where we tested a combination therapy directed against proteins frequently overexpressed in melanoma—EGFR and MET—in a panel of human melanoma cell lines and samples derived from patients
Genetic mechanisms of resistance apply to BRAF alone: amplification of gene copying and alternative splicing, which were detected in 20%–32% of melanoma cases [18,19], or genes encoding proteins directly, and indirectly interacting with BRAF kinase
Summary
Among all patients suffering from skin cancers, only 4% are diagnosed with malignant melanoma. One of the most prevalent drivers in melanoma progression is mutated BRAF, identified in more than 40% of patients suffering from this cancer [2,3]. BRAF is a member of the RAF serine-threonine family of kinases, which are components of the MAPK (mitogen-activated protein kinase) signaling pathway. They become active following the phosphorylation of their upstream effectors, RTKs (receptor tyrosine kinases). The majority of BRAF mutations occur in exon 15 at position 600, resulting in the substitution of valine for glutamic acid (V600E, 70–90%) or lysine (V600K, 10–30%) This aberration produces kinase, which is constitutively active independently of upstream regulators [6]. To combat emerging resistance to BRAF inhibitors, novel combination therapies have been developed, among which a treatment using inhibitors of BRAF and MEK, a downstream effector of BRAF, has shown the greatest potential so far [9]
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