Sema6A and Mical1 control cell growth and survival of BRAFV600E human melanoma cells

Abstract

// Rossella Loria 1 , Giulia Bon 1 , Valentina Perotti 2 , Enzo Gallo 3 , Ilaria Bersani 2 , Paola Baldassari 2 , Manuela Porru 4 , Carlo Leonetti 4 , Selene Di Carlo 1 , Paolo Visca 3 , Maria Felice Brizzi 5 , Andrea Anichini 2 , Roberta Mortarini 2 and Rita Falcioni 1 1 Department of Experimental Oncology, Regina Elena National Cancer Institute, Rome, Italy 2 Human Tumors Immunobiology Unit, Dept. of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3 Department of Pathology, Regina Elena National Cancer Institute, Rome, Italy 4 Department of Chemotherapy, Regina Elena National Cancer Institute, Rome, Italy 5 Department of Medical Sciences, University of Turin, Turin, Italy Correspondence: Rita Falcioni, email: // Roberta Mortarini, email: // Keywords : BRAF V600E melanoma, NRAS Q61R melanoma, Sema6A, Mical1, cell survival Received : August 28, 2014 Accepted : December 12, 2014 Published : December 18, 2014 Abstract We used whole genome microarray analysis to identify potential candidate genes with differential expression in BRAF V600E vs NRAS Q61R melanoma cells. We selected, for comparison, a peculiar model based on melanoma clones, isolated from a single tumor characterized by mutually exclusive expression of BRAF V600E and NRAS Q61R in different cells. This effort led us to identify two genes, SEMA6A and MICAL1, highly expressed in BRAF-mutant vs NRAS-mutant clones. Real-time PCR, Western blot and immunohistochemistry confirmed preferential expression of Sema6A and Mical1 in BRAF V600E melanoma. Sema6A is a member of the semaphorin family, and it complexes with the plexins to regulate actin cytoskeleton, motility and cell proliferation. Silencing of Sema6A in BRAF-mutant cells caused cytoskeletal remodeling, and loss of stress fibers, that in turn induced cell death. Furthermore, Sema6A depletion caused loss of anchorage-independent growth, inhibition of chemotaxis and invasion. Forced Sema6A overexpression, in NRAS Q61R clones, induced anchorage-independent growth, and a significant increase of invasiveness. Mical1, that links Sema/PlexinA signaling, is also a negative regulator of apoptosis. Indeed, Mical-1 depletion in BRAF mutant cells restored MST-1-dependent NDR phosphorylation and promoted a rapid and massive NDR-dependent apoptosis. Overall, our data suggest that Sema6A and Mical1 may represent new potential therapeutic targets in BRAF V600E melanoma.