SNX18 inhibits melanoma migration by modulating the activation ratio of Cdc42 & RhoA [microarray]

To identify novel proteins that control melanoma development and progression, we integrated high-throughput proteomics, mRNA and miRNAs datasets from isogenic primary (WM115) and metastatic (WM266-4) melanoma cell-lines. We focused on differentially expressed proteins that had non-differential mRNA levels and were putatively targeted by a high number of miRNAs. SNX18, a sub-family member of sorting-nexins that participates in endocytosis and autophagy was down-regulated by 6-fold in WM266-4, and predicted to be targeted by 17 miRNAs. Over-expression of SNX18 in several melanoma cell-lines significantly reduced migration rate, while silencing of SNX18 resulted in the opposite effect. Notably, progression tissue microarrays demonstrated a trend for SNX18 downregulation in early metastasis. Luciferase assays confirmed the direct regulation of SNX18 by multiple miRNAs. Indeed, over-expression of these miRNAs repressed endogenous SNX18 expression at the protein but not mRNA level. Further, protein but not mRNA expression levels correlated with better overall survival as determined in a cohort of 48 metastatic melanoma patients and in the TCGA database. Mechanistically, SNX18-mediated reduced migration coincided with RhoA activation and Cdc42 inactivation. Both play a role, as RhoA inhibitor and constitutively activated Cdc42 mutants abrogated this effect. Moreover, phosphorylation-defective SNX18 mutants (S233A,S233D) migrated faster, with S233A inactivating RhoA and activating Cdc42, while S233D activated both. The downstream phosphorylation of the p38>MAPKAPK2>HSP27 signaling pathway was concordant. Both mutants were more abundant in the plasma membrane. Taken together, phosphorylation of S233 is crucial for SNX18 activation, but de-phosphorylation dynamics, which may occur at the membrane, is required for functional effect. mRNA expression profile of primary (WM115) and metastatic (WM266-4) melanoma cell lines. A total of 3 biological replicates of WM115 and WM266-4 were compared.

为筛选调控黑色素瘤发生与进展的新型蛋白，我们整合了来自同基因原代（WM115）与转移性（WM266-4）黑色素瘤细胞系的高通量蛋白质组学、mRNA及microRNAs（miRNAs）数据集。我们重点关注mRNA水平无差异表达，但被大量miRNAs潜在靶向的差异表达蛋白。分选连接蛋白（sorting-nexin）亚家族成员SNX18参与内吞作用与自噬过程，在WM266-4细胞中表达下调6倍，且被预测可被17种miRNAs靶向。在多株黑色素瘤细胞系中过表达SNX18可显著降低细胞迁移速率，而沉默SNX18则会产生相反的生物学效应。值得注意的是，进展期组织微阵列实验显示，SNX18在早期转移灶中呈现表达下调趋势。双荧光素酶报告基因实验证实，多种miRNAs可直接调控SNX18的表达。具体而言，这些miRNAs的过表达会在蛋白层面抑制内源性SNX18的表达，但对其mRNA水平无显著影响。进一步分析显示，在48例转移性黑色素瘤患者队列及癌症基因组图谱（TCGA）数据库中，SNX18的蛋白表达水平（而非mRNA水平）与更好的总生存期呈显著正相关。从分子机制来看，SNX18介导的迁移抑制与RhoA激活及Cdc42失活密切相关。二者均发挥关键调控作用：RhoA抑制剂与组成型激活的Cdc42突变体均可抵消这一迁移抑制效应。此外，磷酸化缺陷型SNX18突变体（S233A、S233D）的细胞迁移速率更快：其中S233A可使RhoA失活并激活Cdc42，而S233D则可同时激活RhoA与Cdc42。p38>MAPKAPK2>HSP27信号通路的下游磷酸化水平也与之相符。两种突变体在细胞膜上的蛋白丰度均更高。综上，S233位点的磷酸化对SNX18的激活至关重要，但发挥功能效应所必需的是可能发生于细胞膜上的去磷酸化动态过程。本数据集包含原代（WM115）与转移性（WM266-4）黑色素瘤细胞系的mRNA表达谱数据，共对3份生物学重复的WM115与WM266-4细胞进行了对照分析。