542 BRAFV600E-inhibition drives EMT gene expression enhancing invasiveness and metastasis in a bioluminescent murine model of BRAFV600E/NRASQ61K melanoma

Abstract

BRAF inhibitor (BRAFi) resistance compromises long term survivorship of malignant melanoma patients. Mutant NRASQ61K with constitutive activation of PI3K/AKT/mTOR signaling is a major mediator of BRAFi resistance, and BRAFi therapy can accelerate pre-existing RAS-mutant malignancies including NRAS-mutant leukemias in melanoma patients. Here, employing NanoString transcriptomic analysis of isogenic (A375-BRAFV600E versus A375-BRAFV600E/NRASQ61K) malignant melanoma cells we demonstrate that BRAFi treatment selectively targets BRAFV600E/NRASQ61K cells with induction of Epithelial to Mesenchymal Transition (EMT) gene expression, paradoxically promoting invasiveness and metastasis in vivo. Cancer progression nCounterTM pathway analysis identified ‘EMT’ and ‘Proliferative Control’ gene expression networks specific to BRAFV600E/NRASQ61K status. Strikingly, in contrast to BRAFi (vemurafenib, VEM)-induced antiproliferative and antiinvasive effects in BRAFV600E cells, VEM enhanced proliferation and invasiveness of BRAFV600E/NRASQ61K cells. RT2Profiler PCR array analysis confirmed VEM-upregulation of genes promoting EMT and proliferation [AKT1, MMP3, PDGFRB, RAC1, SPARC, ZEB1, ZEB2 (≤ 350-fold; p<0.05)] detectable only in BRAFV600E/NRASQ61K cells, while causing the expected downregulation of EMT-driver genes [CDH2, FN1, FOXC2, IGFBP4, MMP9, VIM, WNT5A (≤ 40-fold; p<0.05)] only in the BRAFV600E isogenic variant. Phenotypic transwell migration assays confirmed the seemingly opposing effects of VEM treatment on melanoma cell invasiveness [achieving blockade (BRAFV600E) or enhancement (BRAFV600E/NRASQ61K)]. In a bioluminescent SCID mouse metastasis model using A375-luc isogenic variants, VEM treatment (50 mg/kg; p.o., q.d.) enhanced lung tumor burden imposed by BRAFV600E/NRASQ61K cells, while blocking metastasis of BRAFV600E cells. Our data provide preclinical evidence that identifies a BRAFi-driven upregulation of EMT-related gene expression potentially enhancing invasiveness and metastasis in human BRAFV600E/NRASQ61K melanoma.