Genome sequencing studies have recently shown that oncogenic mutations of B-Raf occur in approximately 70% of malignant melanoma tumors and cell lines (1
). Remarkably, most of these mutations specify a single amino acid substitution, V600E, which constitutively elevates B-Raf catalytic activity. Other B-Raf mutations do not affect kinase activity, but instead promote activation of c-Raf1 (4
). Melanoma stages are based on clinical and histopathological features, and include dysplastic or atypical nevi, early radial growth phase (RGP) primary melanoma, advanced vertical growth phase (VGP) primary melanoma, and metastatic melanoma (5
). High frequencies of B-Raf mutations have been observed in nevoid/RGP specimens as well as advanced tumors (6
). As a result, the MKK1/2-ERK1/2 protein kinase cascade is constitutively active in most melanoma tissues and cell lines (2
). B-Raf/MKK/ERK signaling promotes cell proliferation, survival, and vascular development in melanoma xenograft tumors, and several reports have demonstrated that tumor growth is suppressed upon blocking this pathway (9
). Thus, identifying cellular targets of this pathway provides a way to elucidate molecular determinants of melanoma.
Plexins and semaphorins are receptor and ligand families which function to regulate axon guidance and neuronal growth cone collapse (12
). Among the best characterized receptor members are plexin B1, which directly binds semaphorin 4D, and plexin A1, which complexes with class 3 semaphorins in association with neuropilins. Plexins A1 and B1 mediate cell guidance by recruiting various GTPases to distinct receptor domains. In response to ligand binding, Rho is activated by recruitment of guanine nucleotide exchange factors (GEFs) to receptors, and Rac is inhibited by direct receptor binding and sequestration (14
). Plexins A and B also inhibit R-Ras, by binding the Rnd1 GTPase, which recruits R-Ras-GTP to the receptor, followed by GTP hydrolysis catalyzed by an intrinsic receptor GAP domain (19
). These mechanisms have been proposed to underlie cell-cell attraction and repulsion, where R-Ras-GTP hydrolysis drives cell contraction and axonal growth cone collapse, Rac controls cell-cell attraction, and Rho causes localized increases in contractile force by stress fibers, leading to growth cone collapse and cell-cell repulsion (reviewed in 21
Plexin-semaphorins have also been implicated in cancer. Plexin B1-semaphorin 4D induces invasion in many cell types, and promotes endothelial cell angiogenesis (24
). Frequent missense mutations or overexpression of plexin B1 have been found in prostate cancers, and are associated with increased invasion (26
). In several cancer cell types, plexin B1 promotes invasive growth by associating with c-Met and elevating c-Met phosphorylation (27
). In other cells, plexin B1 blocks cell migration through c-Met by inhibiting RhoA, while activating cell migration through ErbB2 (29
). Semaphorin 4D is highly expressed in malignant cells from prostate, colon, breast, and lung cancer tissues, and in head and neck squamous cell cancers mediates tumor angiogenesis via
paracrine signaling to endothelial cells (30
). Likewise, semaphorin 3A, 3C, and 3E are overexpressed in breast, ovarian, and lung cancer cells, and are correlated with angiogenesis and vascularization. On the other hand, overexpression of semaphorin 3F in melanoma retards angiogenesis and metastasis of mouse xenografts, and alters the tumor microenvironment to form a benign, encapsulated tumor with well-defined borders (31
). In estrogen-receptor positive breast cancers, low plexin B1 is associated with greater malignancy and poor prognosis (33
). Thus, responses to plexin-semaphorin vary widely between cancer types.
Here, we conducted a microarray screen to identify targets of constitutive B-Raf/MKK/ERK signaling in melanoma. We report that plexin B1 is repressed by MAP kinase signaling in cells isolated from human melanomas at varying stages of progression. Further experiments revealed a novel tumor suppressor role for plexin B1 in melanoma cells which is correlated with suppression of AKT, as well as a role in inhibition of cell migration. Our results demonstrate that oncogenic B-Raf promotes tumor growth and cell migration by inhibitory cross-regulation of plexin B1 signal transduction.