Melanoma is a particularly aggressive tumor type that exhibits a high level of resistance to apoptosis. The serine/ threonine kinase B-RAF is mutated in 50% to 70% of melanomas and protects melanoma cells from anoikis, a form of apoptosis induced by lack of adhesion or adhesion to an inappropriate matrix. Mutant B-RAF down-regulates two BH3-only proapoptotic proteins, Bim EL and Bad. BH3-only proteins act, at least in part, by sequestering prosurvival Bcl-2 family proteins and preventing them from inhibiting the mitochondrial apoptotic pathway. Several Bcl-2 proteins are up-regulated in melanoma; however, the mechanisms of up-regulation and their role in melanoma resistance to anoikis remain unclear. Using RNA interference, we show that depletion of Mcl-1 renders mutant B-RAF melanoma cells sensitive to anoikis. By contrast, minor effects were observed following depletion of either Bcl-2 or Bcl-XL . Mcl-1 expression is enhanced in melanoma cell lines compared with melanocytes and up-regulated by the B-RAF-MEK-extracellular signal-regulated kinase 1/2 pathway through control of Mcl-1 protein turnover. Similar to B-RAF knockdown cells, adhesion to fibronectin protected Mcl-1 knockdown cells from apoptosis. Finally, expression of Bad, which does not sequester Mcl-1, further augmented apoptosis in nonadherent Mcl-1 knockdown cells. Together, these data support the notion that BH3 mimetic compounds that target Mcl-1 may be effective for the treatment of melanoma in combinatorial strategies with agents that disrupt fibronectin-integrin signaling.

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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...

Supplementary Data from Mcl-1 Is Required for Melanoma Cell Resistance to Anoikis