V599EB-RAF is an Oncogene in Melanocytes

Abstract

The oncogenic version of B-RAF, V599E B-RAF, is found in approximately 70% of human melanomas. However, the role that this oncogene plays in melanoma is unclear because V559E B-RAF is also found in approximately 80% of benign nevi. We have examined the role of oncogenic B-RAF in the early stages of melanoma by expressing V599E B-RAF in cultured melanocytes. In these cells, V599E B-RAF induced constitutive mitogen activated ERK-activating kinase (MEK) and extracellular signalregulated kinase (ERK) signaling, 12-O-tetradecanoylphorbol-13-acetateindependent growth, and tumorigenicity in nude mice. Intriguingly, in RAS-transformed melanocytes, B-RAF depletion did not block MEK-ERK signaling or cell cycle progression. Similarly, B-RAF depletion blocked MEK-ERK signaling in human melanoma cells harboring oncogenic B-RAF, but not in melanoma cells harboring oncogenic RAS. Thus, although B-RAF can act as a potent oncogene in the early stages of melanoma by signaling through MEK and ERK, it is not required for this signaling in RAS-transformed melanocytes due to innate redundancy within the pathway. These findings have important implications for future therapeutic strategies.

Mutant B-RAF signaling and cyclin D1 regulate Cks1/S-phase kinase-associated protein 2-mediated degradation of p27Kip1 in human melanoma cells

“…These data complement our previous studies showing that expression of mutant B-RAF is sufficient to upregulate cyclin D1 and downregulate p27 Kip1 in human melanocytes, and that pharmacological inhibition of the B-RAF effector, MEK, has the opposite effects in melanoma cells (Bhatt et al, 2005). Our findings also provide mechanistic details that underlie others' studies in melanoma cells showing that B-RAF-MEK signaling is necessary for S-phase entry, proliferation and anchorage-independent growth in vitro (Collisson et al, 2003;Hingorani et al, 2003;Karasarides et al, 2004;Sumimoto et al, 2004), and growth in vivo (Collisson et al, 2003;Sumimoto et al, 2004;Sharma et al, 2005), and the report that B-RAF V600E transforms mouse melanocytes (Wellbrock et al, 2004b). It is possible that wild-type B-RAF, which is activated by autocrine growth factor signaling (Satyamoorthy et al, 2003), may also contribute to the regulation of cyclin D1 and p27 Kip1 in our experiments utilizing siRNA that targets both mutant and wild-type B-RAF.…”

Oncogenic BRAF induces chronic ER stress condition resulting in increased basal autophagy and apoptotic resistance of cutaneous melanoma

“…Expression vectors for BRAF V600E mutant, 12 GFP or pcDNA4 (Invitrogen, Carlsbad, CA, USA), wt/mutant Bcl-2, 39 JNK-DN 51 and p38-DN 52 (Addgene plasmids 8768, 13340, 13846, 20356, respectively; Cambridge, MA, USA) vectors were transiently transfected using lipofectamine LTX, according to the manufacturer's recommendations.…”

“…4,5 Surprisingly, p53 mutations are very rare in melanoma, but activity is, however, impaired through direct or indirect inactivation of key elements of this pathway, including through the suppression of APAF-1 expression, 6 loss of PTEN function, 7 dysregulation of Bcl-2 expression, 8 upregulation of the anti-apoptotic protein Mcl-1 together with its altered slice variant expression 9,10 and the ER chaperone GRP78. [11][12][13] Oncogenic mutations, however, in the Ras/Raf pathway are the most well-described genetic mutations associated with melanoma development and progression. 14 Indeed, up to 90% of all melanomas harbour activating NRAS or BRAF mutations, with BRAF V600E representing more than 90% of BRAF mutations, 15,16 the consequence of which is the constitutive activation of RAF-extracellular signal-regulated kinase/ERK signalling promoting melanoma proliferation and resistance to apoptosis.…”

Mutant B-RAF mediates resistance to anoikis via Bad and Bim

“…The most common mutation in B-RAF is a valine to glutamic acid substitution at residue 600 in the activation loop rendering B-RAF and the downstream Mitogenactivated protein/extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway constitutively active. Mutant B-RAF can transform NIH3T3 fibroblasts and immortalized mouse melanocytes (Wellbrock et al, 2004) but may not be sufficient to initiate melanoma since benign nevus cells harbor B-RAF mutations (Dong et al, 2003). However, B-RAF mutations correlate with melanoma progression as they have been detected in 75% of vertical growth phase tumors compared to 10% of radial growth phase tumors (Dong et al, 2003).…”