Understanding Wnt5a regulation of protein depalmitoylation during cell migration

描述（由申请人提供）：本提案的中心问题是：Wnt5a如何调节细胞极性以及这些机制如何影响癌症转移？这一建议将建立在最近发现的Wnt5a信号激活对细胞粘附分子去棕榈酰化的调节促进癌细胞侵袭的基础上。棕榈酰化是在蛋白质的半胱氨酸残基上添加16碳脂肪酸棕榈酸酯。Wnt5a处理黑色素瘤细胞可诱导黑色素瘤细胞粘附分子MCAM的去棕榈酰化。在缺乏外源Wnt5a的情况下，阻断棕榈酰基化的MCAM突变会导致MCAM的极化定位，并在3D胶原和异种移植肿瘤实验中增加细胞的侵袭。Wnt5a信号通过需要酰基蛋白硫酯酶APT1的机制降低棕榈酰化，使用shRNA或药物抑制剂抑制APT1可阻断细胞迁移和侵袭。拟开展的研究将开始探索Wnt5a信号通路如何调控肿瘤转移，阐明Wnt5a调控的蛋白棕榈酰化在极化细胞迁移过程中的分子机制。在第一个目标中，假设已知的非规范Wnt途径的下游组分与核心棕榈酰化机制接口以调节去棕榈酰化。Wnt5a诱导蛋白去棕榈酰化的机制将通过两种候选方法进行研究，以确定Wnt5a诱导蛋白去棕榈酰化所需的Wnt信号成分。第二个目标将确定蛋白棕榈酰化周期在细胞迁移和入侵过程中的作用，在体外和体内。在人类肿瘤样本中发现的棕榈酰转移酶突变损害了棕榈酰化MCAM和抑制胶原细胞侵袭的能力。这些突变在体内的影响将通过异种移植肿瘤检测来确定。细胞行为将通过体外迁移和侵袭试验进行研究，蛋白质定位和定向迁移将通过延时成像进行分析。其他受Wnt5a调控的棕榈酰化蛋白将被生化纯化，并使用质谱法进行鉴定，从而为Wnt5a诱导的去棕榈酰化调控的细胞途径提供一个广阔的视角。目前正在患者肿瘤样本中检测该途径中发现的蛋白质。

DESCRIPTION (provided by applicant): The central question of this proposal is: How is cell polarity regulated by Wnt5a and how do these mechanisms impact cancer metastasis? This proposal will build on recent findings revealing regulation of cell adhesion molecule depalmitoylation by Wnt5a signal activation promotes cancer cell invasion. Palmitoylation is the addition of the 16 carbon fatty acid palmitate to proteins on cysteine residues. Wnt5a treatment of melanoma cells induces depalmitoylation of the cell adhesion molecules MCAM in melanoma cells. MCAM mutations that block palmitoylation cause polarized localization of MCAM in the absence of exogenous Wnt5a as well as increased invasion of cells in 3D collagen and in xenograft tumor assays. Wnt5a signaling decreases palmitoylation through a mechanism requiring the acyl protein thioesterase APT1 and inhibition of APT1 using shRNA or pharmacological inhibitors blocks cell migration and invasion. The proposed studies will begin to explore how cancer metastasis is regulated by Wnt5a signaling and elucidating the molecular mechanism of protein palmitoylation regulated by Wnt5a during polarized cell migration. In the first aim the hypothesis that a known downstream component of the noncanoncial Wnt pathway interfaces with the core palmitoylation machinery to regulate depalmitoylation. The mechanism by which Wnt5a induces protein depalmitoylation will be investigated taking both a candidate approach to identify the Wnt signaling components required for Wnt5a induced protein depalmitoylation. The second aim will determine the role of the protein palmitoylation cycle during cell migration and invasion in vitro and in vivo. Mutations in palmitoyl transferases identified in human tumor samples have impaired ability to palmitoylation MCAM and to suppress cell invasion in collagen. The impact of these mutations in vivo will be determined by xenograft tumor assays. Cell behavior will be studied using in vitro migration and invasion assays and protein localization and directional migration will be analyzed by time lapse imaging. Other palmitoylated proteins that are regulated by Wnt5a will be biochemically purified and identified using mass spectrometry providing a broad view of the cellular pathways regulated by Wnt5a induced depalmitoylation. The proteins identified in the pathway are being examined in patient tumor samples.