Growth factors rescue cutaneous melanoma cells from apoptosis induced by knockdown of mutated (V 600 E) B-RAF.
Constitutive activation of the RAS-RAF-MEK-ERK signaling cascade is a hallmark of cutaneous malignant melanoma. A single activating mutation (c.1799 T>A; p.V 600 E) in the gene encoding the serine/threonine kinase B-RAF occurs in >60% of the tumors. Previous work has shown that knockdown of (V 600 E)B-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 (V 600 E)B-RAF mutation have retained the wild-type (WT) B-RAF allele, and that these cells can be rescued from the effects of (V 600 E)B-RAF knockdown by stimulation with growth factors. Ectopic expression of short hairpin RNAs specifically suppressing (V 600 E)B-RAF in melanoma cell lines reduced colony formation by approximately 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 (WT)B-RAF. Single-cell clones with efficient knockdown of (V 600 E)B-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 (V 600 E)B-RAF knockdown in melanoma cells may have both experimental and therapeutic implications.
Christensen
Claus
C
Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark.
Guldberg
Per
P
D000483:Alleles; D017209:Apoptosis / Q000502:physiology*; D001483:Base Sequence; D045744:Cell Line, Tumor; D017931:DNA Primers; D006133:Growth Substances / Q000502:physiology*; D006801:Humans; D008545:Melanoma / Q000235:genetics / Q000473:pathology*; D009154:Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D012878:Skin Neoplasms / Q000235:genetics / Q000473:pathology*
D017931:DNA Primers; D006133:Growth Substances; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf