Activating mutations of the B-RAF gene are observed in >60% of human
melanomas. Approximately 90% of these mutations occur in the activation segment of the
kinase domain as a single-base substitution that converts a
valine to
glutamic acid at
codon 599 (V599E) in exon 15. This mutation causes activation of the
kinase as well as downstream effectors of the
mitogen-activated protein kinase-signaling cascade, leading to
melanoma tumor development by an as yet unknown mechanism. In this study, we have identified the role of (V599E)B-Raf in
melanoma tumor development by characterizing the mechanism by which this
mutant protein promotes
melanoma tumorigenesis.
Small interfering RNA targeted against B-Raf or
a Raf kinase inhibitor (BAY 43-9006) was used to reduce expression and/or activity of (V599E)B-Raf in
melanoma tumors. This inhibition led to reduced activity of the
mitogen-activated protein kinase-signaling cascade and inhibited
tumor development in animals. Targeted reduction of mutant (V599E)B-Raf expression (activity) in
melanoma cells before
tumor formation inhibited
tumorigenesis by reducing the growth potential of
melanoma cells. In contrast, reduction of mutant (V599E)B-Raf activity in preexisting
tumors prevented further vascular development mediated through decreased
vascular endothelial growth factor secretion, subsequently increasing apoptosis in
tumors. These effects in combination with reduced proliferative capacity halted growth, but did not shrink the size of preexisting
melanoma tumors. Thus, these studies identify the mechanistic underpinnings by which mutant (V599E)B-RAF promotes
melanoma development and show the effectiveness of targeting this
protein to inhibit
melanoma tumor growth.