Poster presentation, P168

Official XXIst International Pigment Cell Conference website - 21-24 Sept 2011, Bordeaux - France | updated: September 04 2011

MAPK inhibitors may reverse the senescence-like phenotype associated with a low proliferation index of melanoma cells bearing the V600EBRAF mutation

BACKGROUND: The mutations of BRAF are described in about 50% of melanoma tumors with 90% of these mutations occurring at a single site, leading to the V600E substitution. This mutation leads to the constitutive activation of the MAPK pathway which is critical for cancer cell survival. OBJECTIVES: We evaluated the impact of V600EBRAF mutation on melanoma cell proliferation and response to various MAPK inhibitors. METHODS: We compared 3 V600EBRAF-mutated (MM032, MM043, MM074) with 3 wildtype (WT) melanoma cell lines (HBL, LND1, MM079) addressing: 1. ERK phosphorylation (Western blotting), 2. cell proliferation (crystal violet staining), 3. apoptosis (annexin V), 4. senescence (beta-galactosidase activity), and 5. response to a MEK inhibitor (U0126) in terms of proliferation and senescence. RESULTS AND DISCUSSION: First, we confirm that mutated cells exhibited significantly higher levels of ERK1/2 phosphorylation. But, surprisingly, these cells had a 2.5-fold lower proliferation index (d3/d1 ratio) as compared to the group of WT cells. We also found no significant difference in apoptotic cells between the two groups, but mutated cells expressed a higher level of the cell cycle inhibitor p21WAF, indicating that the lower growth rate of these cells was rather due to a reduced cell cycling and not to an increased cell death. Moreover, we observed that beta-galactosidase activity was extremely high in mutated cells along with significant increase both in cell size (2 fold) and in p53 expression, supporting that constitutive MAPK hyper-activation induces a senescence-like phenotype. Furthermore, we interfered with the MAPK signaling pathway by long-term exposure of mutated cells to non toxic but effective concentrations of various inhibitors, and we found, e.g. with a MEK inhibitor (U0126), that both beta-galactosidase activity and cell volume significant decrease together with an increase of the proliferation index. CONCLUSIONS: We confirm that in V600EBRAF melanoma cells, the hyper-activation of ERK leads to a senescence-like phenotype associated with a low proliferation. We also report that long-term (from 10 to 17 days in our study) inhibition of MAPK signaling pathway may reverse the senescence leading to a stimulation of cell proliferation. Therefore, the MAPK pathway inhibitors currently tested for the treatment of melanoma might promote proliferation in cells with high MAPK activity, especially in V600EBRAF mutated ones.

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