title: Novel mechanisms of resistance to vemurafenib in melanoma - V600E B-Raf reversion and switching VEGF-A splice isoform expression.
abstract: Targeting activating mutations in the proto-oncogene B-Raf, in melanoma, has led to increases in progression free survival. Treatment with vemurafenib, which inhibits the most common activating-mutated form of B-Raf (B-Raf(V600E)), eventually results in resistance to therapy. VEGF-A is the principal driver of angiogenesis in primary and metastatic lesions. The bioactivity of VEGF-A is dependent upon alternative RNA splicing and pro-angiogenic isoforms of VEGF-A are upregulated in many disease states dependent upon angiogenesis, including cancers. Using techniques including RT-PCR, Western blotting, ELISA and luciferase reporter assays, the effect of vemurafenib on proliferation, ERK1/2 phosphorylation and the levels of pro- and anti-angiogenic VEGF-A isoforms was investigated in melanoma cell types expressing either wild-type B-Raf or B-Raf(V600E), including a primary melanoma culture derived from a highly vascularised and active nodule taken from a patient with a V600E mutant melanoma. The primary melanoma culture was characterised and found to have reverted to wild-type B-Raf. In B-Raf(V600E) A375 cells ERK1/2 phosphorylation, pro-angiogenic VEGF-A mRNA, total VEGF-A protein expression and VEGF-A 3'UTR activity were all decreased in a concentration-dependent manner by vemurafenib. Conversely vemurafenib treatment of wild-type B-Raf cells significantly increased ERK1/2 phosphorylation, pro-angiogenic VEGF-A mRNA and total VEGF-A expression in a concentration-dependent manner. A switch to pro-angiogenic VEGF-A isoforms, with a concomitant upregulation of expression by increasing VEGF-A mRNA stability, may be an additional oncogenic and pathological mechanism in B-Raf(V600E) melanomas, which promotes tumor-associated angiogenesis and melanoma-genesis. We have also identified the genetic reversal of B-Raf(V600E) to wild-type in an active melanoma nodule taken from a V600E-positive patient and continued vemurafenib treatment for this patient is likely to have had a detrimental effect by promoting B-Raf(WT) activity.
authors: Microvascular Research Laboratories, University of Bristol Bristol, UK, BS2 8EJ ; School of Life Sciences, University of Nottingham, Queen's Medical School Nottingham, UK, NG7 2UH.
authors: Nicholas
authors: N
authors: Beazley-Long
authors: School of Biochemistry, University of Bristol Bristol, UK, BS8 1TD.
authors: Kevin
authors: K
authors: Gaston
authors: Microvascular Research Laboratories, University of Bristol Bristol, UK, BS2 8EJ.
authors: Steven J
authors: SJ
authors: Harper
authors: Department of Plastic Surgery, Southmead Hospital Brunel Building, North Bristol Trust, UK, BS10 5NB.
authors: Antonio
authors: A
authors: Orlando
authors: Microvascular Research Laboratories, University of Bristol Bristol, UK, BS2 8EJ ; Cancer Biology, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Queen's Medical Centre Nottingham, UK, NG7 2UH.
authors: David O
authors: DO
authors: Bates
keywords: 92.1; A375; Melanoma; VEGF-A; VEGF-Axxxb; mechanism of resistance; vemurafenib
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