Novel mechanisms of resistance to vemurafenib in melanoma – V600E B-Raf reversion and switching VEGF-A splice isoform expression

Beazley Long, Nicholas; Gaston, Kevin; Harper, Steven J.; Orlando, Antonio; Bates, David O.

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-RafV600E), 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-RafV600E, 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-RafV600E 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-RafV600E melanomas, which promotes tumor-associated angiogenesis and melanoma-genesis. We have also identified the genetic reversal of B-RafV600E 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-RafWT activity.

Citation

Beazley Long, N., Gaston, K., Harper, S. J., Orlando, A., & Bates, D. O. (2015). Novel mechanisms of resistance to vemurafenib in melanoma – V600E B-Raf reversion and switching VEGF-A splice isoform expression. American Journal of Cancer Research, 5(1), 433--441

• Journal Article Type: Article
• Acceptance Date: Nov 28, 2014
• Online Publication Date: Dec 15, 2014
• Publication Date: Jan 1, 2015
• Deposit Date: Oct 30, 2018
• Publicly Available Date: Oct 30, 2018
• Journal: American Journal of Cancer Research
• Electronic ISSN: 2156-6976
• Publisher: e-Century Publishing
• Peer Reviewed: Peer Reviewed
• Volume: 5
• Issue: 1
• Pages: 433--441
• Keywords: Melanoma, vemurafenib, A375, 92.1, VEGF-A, VEGF-Axxxb, mechanism of resistance
• Public URL: https://nottingham-repository.worktribe.com/output/1037589
• Publisher URL: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4300704/