Sema6A and Mical1 control cell growth and survival of BRAFV600E human melanomas. Homo sapiens

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. Overall design: 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.

针对BRAFV600E（BRAFV600E）与MEK（MEK）的靶向治疗已对晚期黑色素瘤的无进展生存期与总生存期产生显著改善，但仅少数患者可从此类治疗中获益，这提示我们仍需挖掘参与黑色素瘤生长与存活的其他信号通路。
为此，我们采用全基因组微阵列分析（whole genome microarray analysis），在一组源自单一黑色素瘤转移灶的肿瘤克隆中鉴定差异表达基因（differentially expressed genes）；该组克隆均以BRAFV600E或NRASQ61R（NRASQ61R）的互斥表达为特征。
通过该策略，我们鉴定出两个属于semaphorin-plexin信号通路的基因SEMA6A与Mical-1，它们在BRAF突变型肿瘤克隆的mRNA与蛋白水平均呈高表达。
实时荧光定量PCR（Real-time PCR）、蛋白质免疫印迹（Western blot）与免疫组织化学（immunohistochemistry）实验证实，SEMA6A与Mical-1在黑色素瘤克隆、原发性及转移性细胞系的BRAFV600E突变肿瘤细胞，以及黑色素瘤病灶的组织切片中均呈优先表达。
通过特异性RNA干扰（RNA-interference）实验敲低SEMA6A，可引发细胞骨架重构、应力纤维丢失、肌动蛋白富集突起形成，并诱导细胞死亡；而在NRASQ61R克隆中过表达SEMA6A，则可促进细胞侵袭能力。
在BRAFV600E突变型黑色素瘤中通过小干扰RNA（siRNA）敲低Mical-1，虽未改变肌动蛋白细胞骨架的组织形态，但可引发强烈的NDR磷酸化及NDR依赖的细胞凋亡。
综上，本研究结果表明SEMA与MICAL通路可促进BRAFV600E突变型黑色素瘤的存活。

总体设计：
本研究使用的人类黑色素瘤细胞系包括Me 4405、Me26635、Me665/2、Me10538；克隆株包括2/33、2/21、2/4、2/17、2/14、2/59，均由本实验室从手术标本中建立。
细胞常规培养于添加10%胎牛血清（FBS）与2mM谷氨酰胺的RPMI 1640培养基中，于37℃、5%CO2的饱和湿度空气环境下培养。
以6×10^6个细胞接种于75cm²培养瓶中，培养72小时后，将Me 4405与Me26635分为未处理组与给药组：给药组以300μmol/L的福莫司汀（fotemustine）处理6小时。
每组处理均设置3次生物学重复。收集细胞并提取总RNA。