B-RAF is a therapeutic target in melanoma

Abstract

B-RAF is a serine/threonine-specific protein kinase that is mutated in approximately 70% of human melanomas. However, the role of this signalling molecule in cancer is unclear. Here, we show that ERK is constitutively activated in melanoma cells expressing oncogenic B-RAF and that this activity is required for proliferation. B-RAF depletion by siRNA blocks ERK activity, whereas A-RAF and C-RAF depletion do not affect ERK signalling. B-RAF depletion inhibits DNA synthesis and induces apoptosis in three melanoma cell lines and we show that the RAF inhibitor BAY43-9006 also blocks ERK activity, inhibits DNA synthesis and induces cell death in these cells. BAY43-9006 targets B-RAF signalling in vivo and induces a substantial growth delay in melanoma tumour xenografts. Our data demonstrate that oncogenic B-RAF activates ERK signalling, induces proliferation and protects cells from apoptosis, demonstrating that it is an important therapeutic target and thus provides novel strategies for clinical management of melanoma and other cancers.

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

“…These results argue against the possibility that alterations in cyclin D1 and p27 Kip1 are due to off-target siRNA effects. Consistent with previous publications (Hingorani et al, 2003;Karasarides et al, 2004;Sumimoto et al, 2004), B-RAF knockdown in WM793 cells inhibited cell growth (Figure 1g). Furthermore, B-RAF knockdown cells showed decreased phosphorylation of Rb protein and expression of cyclin A, two markers of G1 cell cycle progression (Figure 1h) (Figure 2d).…”

“…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.…”

Sorafenib in advanced melanoma: a Phase II randomised discontinuation trial analysis

“…An analysis focussing on melanoma patients was performed in the light of evidence supporting a role for increased signalling through RAF/MEK/ ERK in the onset and progression of melanoma Chang et al, 2004;Garnett and Marais, 2004), and the prevalence of oncogenic V600E BRAF mutations in melanoma biopsies (Brose et al, 2002). Recent preclinical evidence, showing that blocking V600E BRAF expression promotes apoptosis in human melanoma cells, provided a rationale for targeting signalling through RAF/ MEK/ERK in the treatment of melanoma (Hingorani et al, 2003;Karasarides et al, 2004). Despite this rationale, the data in this study at the 400 mg b.i.d.…”

“…The most prevalent oncogenic BRAF mutation is the V600E BRAF mutation (previous terminology, V599E), which is present in 63% of melanomas (Brose et al, 2002). The increased apoptosis, observed in human melanoma cell lines when BRAF expression is downregulated using RNA interference, supports a role for oncogenic BRAF-driven MEK/ERK overactivation in maintaining the transformed phenotype of malignant melanoma cells (Hingorani et al, 2003;Karasarides et al, 2004). This observation also suggests that BRAF is a rational target for the design of targeted agents to treat melanoma.…”

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

“…Expression of B-Raf V600E has been shown to transform fibroblasts and melanocytes as well as to induce haematopoietic dysplasia in mice and invasive melanomas in p53À/À zebrafish (Davies et al, 2002;Ikenoue et al, 2004;Wellbrock et al, 2004b;Mercer et al, 2005;Patton et al, 2005). Likewise, suppression of BRAF V600E expression in melanoma lines abrogates their transformed phenotype (Hingorani et al, 2003;Karasarides et al, 2004). However, little is known about the requirements of the B-Raf V600E oncoprotein to display biological activity.…”