Growth factors rescue cutaneous melanoma cells from apoptosis induced by knockdown of mutated (V600E) B-RAF

Constitutive activation of the RAS-RAF-MEK-ERK signaling cascade is a hallmark of cutaneous malignant melanoma. A single activating mutation (c. 1799T > A; p. V600E) in the gene encoding the serine/threonine kinase B-RAF occurs in > 60% of the tumors. Previous work has shown that knockdown of B-V600E-RAFby RNA interference induces a variety of phenotypic changes in cultured melanoma cells, including lower proliferation rates, reduced anchorage-independent growth and apoptosis. Here, we show that the majority of melanomas harboring the B-V600E-RAF mutation have retained the wild-type (WT) B-RAF allele, and that these cells can be rescued from the effects of B-V600E-RAF knockdown by stimulation with growth factors. Ectopic expression of short hairpin RNAs specifically suppressing B-V600E-RAF in melanoma cell lines reduced colony formation by similar to 80%. This response could be rescued by basic fibroblast growth factor, hepatocyte growth factor or, to a lesser extent, endothelin-1. Rescue with growth factors was not possible in cell lines lacking B-WT-RAF. Single-cell clones with efficient knockdown of B-V600E-RAF could be propagated in the presence of basic fibroblast growth factor but underwent apoptosis or senescence-like growth arrest upon withdrawal of this growth factor. The ability of growth factors to modulate the response of B-V600E-RAF knockdown in melanoma cells may have both experimental and therapeutic implications.