A Sharma, NR Trivedi, MA Zimmerman, DA Tuveson, CD Smith, GP Robertson
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.