Abstract 508: Hypoxia influences the response to vemurafenib in V600E mutant melanoma cells
Authors: Pucciarelli Daniela , Takáčová Martina , Csaderova Lucia , Juliano Filippo , Lengger Nina , Breiteneder Heimo , Pastorekova Silvia , Hafner Christine 
Published: May 2015

Melanoma is a cancer still escalating in incidence and despite advances in understanding the molecular nature of melanoma, few treatment options are available for metastatic melanoma. Hypoxia-inducible factor 1 α (HIF-1α), a transcriptional regulator of the hypoxic response has been associated with melanomas. Here we investigated the influence of hypoxia on the treatment with vemurafenib in a panel of BRAF V600E mutant melanoma cell lines, the mechanisms responsible for the prometastatic effects of HIF-1α and the influence of hypoxia on the expression of proteins involved in downstream kinase pathways. Melanoma cell lines M14, 518A2 and A375 were grown in normoxia (21% pO2) and hypoxia (2% pO2) with and without 1mM vemurafenib. Antiproliferative effects were evaluated in a real-time setting in the impedance-based x-CELLigence® system. Western blot analyses of HIF-1α and proteins involved in PI3K, JAK-STAT and MAPK pathways were performed. To evaluate the effects of hypoxia and vemurafenib on cell motility 2D migration and Matrigel® invasion assays were performed. Under hypoxic conditions M14 and A375 cells showed a reduced proliferation rate (-20% for M14 cells and -60% for A375 cells) compared to normoxic conditions. In contrast, 518A2 cells were not susceptible to hypoxic conditions and the proliferation rate did not change. After addition of vemurafenib, hypoxic M14 and 518A2 cells reduced cell growth by additional 20% and 38% compared to normoxic, vemurafenib-treated M14 and 518A2 cells, respectively. Surprisingly, hypoxic vemurafenib-treated A375 cells showed an enhanced cell proliferation rate (+34%) when compared to normoxic, vemurafenib-treated A375 melanoma cells. In Western blot analyses the expression of HIF-1α was reduced in vemurafenib-treated M14 and 518A2 cells but not in A375 cells, reflecting the data obtained by proliferation assays. The expression of pFAK, pERK and pAKT was down-regulated in normoxic and hypoxic, vemurafenib-treated M14 and A375 cells. In contrast, the expression of pERK was up-regulated in normoxic, vemurafenib-treated 518A2 cells, whereas PKCα and pAKT expression levels were up-regulated in hypoxic, vemurafenib-treated and untreated 518A2 cells and the expression of pSTAT3 was up-regulated in normoxic, vemurafenib-treated A375 cells only, showing the cell-type specific response to both, vemurafenib and hypoxia. Motility of hypoxic, vemurafenib-treated A375 cells was enhanced (+45%) when compared to normoxic, vemurafenib-treated A375 cells, whereas the invasion capacity of hypoxic, vemurafenib-treated 518A2 cells was enhanced (+22%) when compared to normoxic, vemurafenib-treated 518A2 cells. Our data indicate that the changes induced by a hypoxic microenvironment can give rise to a subpopulation of highly invasive, therapy resistant melanoma cells and furthermore can affect the response to Vemurafenib in melanoma cells in a cell-type specific manner.

Citation Format: Daniela Pucciarelli, Martina Takáčová, Lucia Csaderova, Filippo Juliano, Nina Lengger, Heimo Breiteneder, Silvia Pastorekova, Christine Hafner. Hypoxia influences the response to vemurafenib in V600E mutant melanoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 508. doi:10.1158/1538-7445.AM2014-508