Abstract
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 V600EB-RAF by 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 V600EB-RAF mutation have retained the wild-type (WT) B-RAF allele, and that these cells can be rescued from the effects of V600EB-RAF knockdown by stimulation with growth factors. Ectopic expression of short hairpin RNAs specifically suppressing V600EB-RAF in melanoma cell lines reduced colony formation by ∼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 WTB-RAF. Single-cell clones with efficient knockdown of V600EB-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 V600EB-RAF knockdown in melanoma cells may have both experimental and therapeutic implications.
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Acknowledgements
We thank Vibeke Ahrenkiel for expert technical assistance, Nicole Fehrenbacher, Mikkel Rohde and Jesper Nylandsted Larsen for valuable advice, and Reuven Agami for kindly providing the pSUPER plasmid. This study was supported by grants from the Danish Cancer Society, the Neye Foundation, and the Danish Cancer Research Foundation.
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Christensen, C., Guldberg, P. Growth factors rescue cutaneous melanoma cells from apoptosis induced by knockdown of mutated (V600E) B-RAF. Oncogene 24, 6292–6302 (2005). https://doi.org/10.1038/sj.onc.1208758
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DOI: https://doi.org/10.1038/sj.onc.1208758
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