Resistance to BRAF Inhibitor PLX-4720 Resulting in Dramatic EGFR Gene Expression Changes in BRAFV600E-Positive Melanoma Cell Lines

摘要

Somatic mutations of BRAF and NRAS oncogenes are early steps in the development of human malignant melanoma. BRAF directed therapy is one of the most promising approaches for melanoma patients with unresectable or metastatic melanoma carrying the BRAF mutation. PLX-4720, a highly selective inhibitor of the mutant BRAFV600E oncogene has been recently approved by FDA with limitations due to early drug resistance. The goal of our study is to define the molecular alterations associated with BRAF inhibitor resistance. We developed PLX-4720 resistant melanoma cell lines, defined the growth inhibitory effect of PLX-4720 on BRAFmut/NRASWT, BRAFWT/NRASWT and BRAFWT/NRASmut cell lines. Using high throughput techniques (Affymetrix Human Gene 1.0 ST microarray) copy number changes and gene expression profiles of the resistant and original cell lines were compared. Matrigel Invasion Assays were also used to define the invasive potential of melanoma cells. Only cell lines with BRAFV600E mutation showed resistance to PLX-4720. More than 2200 genes were differently expressed in the resistant cell lines including the overexpression of the MMPs, ABC transporters, EGFR, PDGFR genes and downregulation of PMEL, MLANA, TYRP1 genes related to melanosomes, genes involved in cell adhesion CDH1, 3, 7, 18, 19 and several tumor suppressor genes. The most altered pathways in the resistant cell lines included AMPK and EGFR. We observed that PLX-4720 inhibit growth of BRAFV600E cell lines, whereas it has a growth promoting effect on cell lines harboring NRAS mutations. WM- 983Bres showed decreased EGFR copy number, but increased mRNA level when compared to the WM983Aresc1.