Sema6A and Mical1 control cell growth and survival of BRAFV600E human melanomas

Therapeutic targeting of BRAFV600Eand of MEK has shown a significant impact on progression-free and overall survival in advanced melanoma, but only a fraction of patients benefit from these treatments, suggesting that additional signaling pathways involved in melanoma growth/survival need to be identified. To this end, we used whole genome microarray analysis to identify differentially expressed genes in a set of neoplastic clones, isolated from a single melanoma metastasis, and characterized by mututally exclusive expression of BRAFV600E or NRASQ61R. By this approach we identified two genes, SEMA6A and Mical-1 belonging to the semaphorin-plexin signaling pathway and higly expressed, at mRNA and protein level, in BRAF-mutant neoplastic clones. Real-time PCR, Western blot analysis and immunohistochemistry confirmed the preferential expression of SEMA-6A and Mical-1 in BRAFV600E neoplastic cells from melanoma clones, primary and metastatic cell lines and tissue sections from melanoma lesions. SEMA6A depletion, by specific RNA-interference experiments, led to cytoskeletal remodeling, loss of stress fibers, generation of actin-rich protrusion, and cell death, whereas SEMA6A overexpression, in NRASQ61R clones, promoted invasiveness. Mical-1 depletion, by siRNA, in BRAFV600E melanomas, did not alter the actin cytoskeleton organization but caused a strong NDR phosphorylation and NDR-dependent apoptosis. Overall, these results suggest that the SEMA and MICAL pathways contribute to promote survival of BRAFV600E melanomas. The human melanoma cell lines Me 4405, Me26635, Me665/2, Me10538; clones 2/33; 2/21; 2/4; 2/17; 2/14; 2/59 were established in our laboratory from a surgical specimens. Cells were routinely maintained in RPMI medium 1640 supplemented with 10% FBS and 2 mM glutamine. Cells were maintained at 37°C in a water-saturated atmosphere of 5% CO2 in air. 6x106 cells were seeded in 75 cm2 flasks; after 72h Me 4405 and Me26635 were untreated or treated with fotemustine at 300 μmol/L for 6 hours. Each treatment or combination was performed in triplicate. The cells were collected and RNA extracted.