Mutations in BRAF, a component of extracellular signalregulated kinases 1 and 2 (ERK) cascade, are frequent in melanoma. It is important to understand how BRAF mutations contribute to malignant traits including anchorage-and growth factor-independence. We have previously shown that efficient activation of ERK in normal human epidermal melanocytes (NHEM) requires both adhesion to the extracellular matrix and growth factors. Mutant V599E BRAF is sufficient to promote ERK activation independent of adhesion and growth factors. Here, we analysed regulation of G1 cell cycle events in NHEM and human melanoma cells. We show that S phase entry in NHEM requires both adhesion and growth factor signaling through the MEK-ERK pathway. This control correlates with induction of cyclin D1 and downregulation of p27 Kip1 , two key G1 cell cycle events. In melanoma cells expressing V599E BRAF, cyclin D1 was constitutively expressed independent of adhesion but dependent upon MEK activation and nuclear accumulation of ERK. Reduction of cyclin D1 levels by RNA interference inhibited S phase entry in melanoma cells. Importantly, expression of V599E BRAF in NHEM was sufficient to promote cyclin D1 promoter activity in the absence of adhesion. Additionally, p27Kip1 levels were downregulated in V599E BRAF-expressing melanoma cells and active BRAF was sufficient to downregulate p27 Kip1 in serum-starved NHEM. Thus, adhesion-growth factor cooperation, leading to efficient activation of ERK, regulates cyclin D1 and p27Kip1 levels in human melanocytes and mutant BRAF overrides adhesion-growth factor control of these two G1 cell cycle proteins in melanomas. These findings provide important insight into how BRAF mutations contribute to aberrant human melanocyte proliferation.

The actin cytoskeleton controls multiple cellular functions, including cell morphology, movement, and growth. Accumulating evidence indicates that oncogenic activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) pathway is accompanied by actin cytoskeletal reorganization. However, the signaling events contributing to actin cytoskeleton remodeling mediated by aberrant ERK1/2 activation are largely unknown. Mutant B-RAF is found in a variety of cancers, including melanoma, and it enhances activation of the MEK/ERK1/2 pathway. We show that targeted knockdown of B-RAF with small interfering RNA or pharmacological inhibition of MEK increased actin stress fiber formation and stabilized focal adhesion dynamics in human melanoma cells. These effects were due to stimulation of the Rho/Rho kinase (ROCK)/LIM kinase-2 signaling pathway, cumulating in the inactivation of the actin depolymerizing/severing protein cofilin. The expression of Rnd3, a Rho antagonist, was attenuated after B-RAF knockdown or MEK inhibition, but it was enhanced in melanocytes expressing active B-RAF. Constitutive expression of Rnd3 suppressed the actin cytoskeletal and focal adhesion effects mediated by B-RAF knockdown. Depletion of Rnd3 elevated cofilin phosphorylation and stress fiber formation and reduced cell invasion. Together, our results identify Rnd3 as a regulator of cross talk between the RAF/MEK/ERK and Rho/ROCK signaling pathways, and a key contributor to oncogene-mediated reorganization of the actin cytoskeleton and focal adhesions.

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D-type cyclins regulate G 1 cell cycle progression by enhancing the activities of cyclin-dependent kinases (CDKs), and their expression is frequently altered in malignant cells. We and others have previously shown that cyclin D1 is up-regulated in melanoma cells through adhesion-independent MEK-ERK1/2 signaling initiated by mutant B-RAF. Here, we describe the regulation and role of cyclin D3 in human melanoma cells. Cyclin D3 expression was enhanced in a cell panel of human melanoma cell lines compared with melanocytes and was regulated by fibronectin-mediated phosphatidylinositol 3-kinase/Akt signaling but not MEK activity. RNA interference experiments demonstrated that cyclin D3 contributed to G 1 -S cell cycle progression and proliferation in melanoma cells. Overexpression of cyclin D1 did not recover the effects of cyclin D3 knockdown. Finally, immunoprecipitation studies showed that CDK6 is a major binding partner for cyclin D3, whereas CDK4 preferentially associated with cyclin D1. Together, these findings demonstrate that cyclin D3 is an important regulator of melanoma G 1 -S cell cycle progression and that D-type cyclins are differentially regulated in melanoma cells. G 1 cell cycle progression and entry into S phase are regulated by the activities of cyclin-dependent kinases (CDKs).2 In early G 1 CDK4 and CDK6 are activated in response to increased expression of D-type cyclins (1). Three D-type cyclins are expressed in mammalian cells: D1, D2, and D3. Activation of CDK4/6 promotes hyperphosphorylation of the retinoblastoma protein, release and derepression of E2F activity, and entry into S phase (2). Genetic depletion studies in mice have illustrated some non-overlapping roles for D-type cyclins. Cyclin D1-deficient mice display defects caused by reduced proliferation of retinal cells and mammary epithelial cells during pregnancy (3, 4), mice lacking cyclin D2 display hypoplasia in the ovaries or testes (5), and cyclin D3-deficient mice display defective thymocyte maturation (6).A hallmark characteristic of malignant cells is their aberrant G 1 -S cell cycle progression and proliferation (7). D-type cyclins are frequently overexpressed in human tumors due either to gene amplification or altered control of signaling pathways, and this overexpression likely contributes to aberrant cell cycle progression in many tumor types (5,6,8). Metastatic melanoma is an aggressive skin cancer with a rising incidence rate. Currently, it is only effectively treated by early detection and surgery. Melanoma arises from the transformation of melanocytes, the pigment-producing cells in the skin, and its progression is well characterized (9). Radial growth phase is characterized by cell growth within the epidermis (in situ) or as nests in the papillary dermal layer (microinvasion). The vertical growth phase is associated with growth perpendicular to the skin surface and the gain of tumorigenic properties. Subsequent metastasis of melanoma to distant sites is associated with a poor prognosis.Expression of cyclin D1 is enhance...

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