Understanding Wnt5a regulation of protein depalmitoylation during cell migration

了解细胞迁移过程中蛋白质去棕榈酰化的 Wnt5a 调节

• 批准号: 9379075
• 负责人: Eric S. Witze
• 金额: $ 6.94万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-09 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9379075
• 关键词: Actins; Acute; Alpha Cell; Behavior; Biochemical; Biological Assay; Biological Process; Breast Cancer Cell; CD44 Antigens; Carbon; Cell Adhesion Molecules; Cell Culture System; Cell Polarity; Cell division; Cell physiology; Cells; Collagen; Complex; Cysteine; Data; Disease; Disease Progression; Embryonic Development; Event; Fatty Acids; Glycine; Human; Image; Impairment; In Vitro; Light; MCAM gene; Malignant Neoplasms; Malignant neoplasm of ovary; Mass Spectrum Analysis; Mediating; Melanoma Cell; Molecular; Movement; Mutation; Neoplasm Metastasis; Nonmuscle Myosin Type IIB; Palmitates; Pathway interactions; Patients; Pharmacology; Play; Post-Translational Protein Processing; Proteins; Regulation; Role; Sampling; Signal Pathway; Signal Transduction; Skin; Structure; Testing; Time; Transferase; WNT Signaling Pathway; Wound Healing; Xenograft Model Antitumor Assays; Xenograft procedure; cancer cell; cancer type; cell behavior; cell motility; in vivo; inhibitor/antagonist; insight; malignant breast neoplasm; melanoma; migration; overexpression; palmitoylation; polarized cell; protein acyltransferase; protein function; public health relevance; response; small hairpin RNA; therapeutic target; tumor; tumor progression

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.

描述（由申请人提供）：该提案的中心问题是：Wnt5a如何调节细胞极性以及这些机制如何影响癌症转移？该建议将建立在最近的发现基础上，这些发现揭示了通过Wnt5a信号激活促进癌细胞侵袭来调节细胞粘附分子脱棕榈酰化。棕榈酰化是将16碳脂肪酸棕榈酸酯添加到蛋白质的半胱氨酸残基上。黑素瘤细胞的Wnt5a处理诱导黑素瘤细胞中细胞粘附分子MCAM的脱棕榈酰化。阻断棕榈酰化的MCAM突变在不存在外源性Wnt5a的情况下引起MCAM的极化定位，以及在3D胶原蛋白和异种移植肿瘤测定中增加的细胞侵袭。Wnt5a信号通过需要酰基蛋白硫酯酶APT1的机制降低棕榈酰化，并且使用shRNA或药理学抑制剂抑制APT1阻断细胞迁移和侵袭。这些研究将开始探索Wnt 5a信号如何调控肿瘤转移，并阐明Wnt 5a在极化细胞迁移过程中调控蛋白棕榈酰化的分子机制。在第一个目标的假设，一个已知的下游组件的noncanoncial Wnt途径接口与核心棕榈酰化机制，以调节depalmitoylation。Wnt5a诱导蛋白质脱棕榈酰化的机制将采用候选方法来研究，以鉴定Wnt5a诱导蛋白质脱棕榈酰化所需的Wnt信号传导组分。第二个目标是确定蛋白质棕榈酰化周期在体外和体内细胞迁移和侵袭过程中的作用。在人类肿瘤样本中鉴定的棕榈酰转移酶突变损害了棕榈酰化MCAM和抑制胶原中细胞侵袭的能力。这些突变在体内的影响将通过异种移植肿瘤测定来确定。将使用体外迁移和侵袭测定研究细胞行为，并将通过时间推移成像分析蛋白质定位和定向迁移。其他棕榈酰化的蛋白质，由Wnt5a的调节将进行生物化学纯化和鉴定，使用质谱法提供了一个广泛的观点，由Wnt5a诱导的脱棕榈酰化调节的细胞途径。在该途径中鉴定的蛋白质正在患者肿瘤样本中进行检查。