Adhesion control of cyclin D1 and p27Kip1 levels is deregulated in melanoma cells through BRAF-MEK-ERK signaling

Abstract

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.

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

“…Initially, we show that mutant B-RAF expression is required for cyclin D1 expression and p27 Kip1 downregulation in 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).…”

“…Two Skp2 immunoreactive bands were observed consistent with others' reports Sutterluty et al, 1999;Garriga et al, 2003). We have previously shown that serum-starved NHEM express high levels of p27 Kip1 that are downregulated in an adhesion and growth factor-dependent manner (Bhatt et al, 2005). We next determined whether Skp2 levels were regulated by adhesion and/or growth factors in NHEM.…”

“…Expression of B-RAF V600E in human melanocytes enhances cyclin D1 levels and decreases p27 Kip1 expression (Bhatt et al, 2005). To determine if mutant B-RAF is required for expression of cyclin D1 and p27 Kip1 in melanoma cells, we utilized RNA interference (RNAi) to selectively knockdown B-RAF.…”

“…We have recently investigated the requirements for cell cycle events in human melanocytes and their dysregulation in melanoma. Human melanocytes are dependent upon adhesion for efficient growth factor activation of ERK1/2, which mediates induction of cyclin D1 and downregulation of p27 Kip1 (Conner et al, 2003;Bhatt et al, 2005). Mutations in B-RAF, a regulator of the mitogen-activated protein-kinase kinase (MEK)-ERK1/2 pathway, are associated with approximately 70% of melanomas; the most common mutant is B-RAF V600E (Davies et al, 2002;Wellbrock et al, 2004a).…”

“…Mutations in B-RAF, a regulator of the mitogen-activated protein-kinase kinase (MEK)-ERK1/2 pathway, are associated with approximately 70% of melanomas; the most common mutant is B-RAF V600E (Davies et al, 2002;Wellbrock et al, 2004a). In melanoma cells harboring B-RAF V600E , ERK1/2 activity is adhesion-independent and MEK activity is required for constitutive expression of cyclin D1 and downregulation of p27 Kip1 (Bhatt et al, 2005). Expression of either B-RAF V600E or a regulatable B-RAF-ER fusion protein in melanocytes bypasses the adhesion and growth factor requirements for ERK1/2 activation, cyclin D1 induction and p27 Kip1 suppression.…”