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

基本信息

批准号：
8759864
负责人：
Eric S. Witze
金额：
$ 33.2万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
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/8759864
关键词：
Actins Acute Behavior Biological Assay Biological Process Breast Cancer Cell CD44 gene 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 In Vitro Light MCAM gene Malignant Neoplasms Malignant neoplasm of ovary Mass Spectrum Analysis Mediating Melanoma Cell Molecular Movement Mutation Myosin ATPase Neoplasm Metastasis Palmitates Pathway interactions Patients Play Post-Translational Protein Processing Proteins Regulation Role Sampling Signal Pathway Signal Transduction Skin Structure Testing Time Transferase 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 function public health relevance response small hairpin RNA therapeutic target tumor tumor progression

项目摘要

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（申请人提供）：该提案的核心问题是：Wnt5a如何调节细胞极性以及这些机制如何影响癌症转移？该提案将建立在最近的发现之上，该发现揭示了 Wnt5a 信号激活对细胞粘附分子去棕榈酰化的调节促进了癌细胞的侵袭。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。 Wnt5a 处理黑色素瘤细胞会诱导黑色素瘤细胞中细胞粘附分子 MCAM 的去棕榈酰化。在缺乏外源性 Wnt5a 的情况下，阻断棕榈酰化的 MCAM 突变会导致 MCAM 极化定位，并在 3D 胶原蛋白和异种移植肿瘤测定中增加细胞侵袭。 Wnt5a 信号传导通过需要酰基蛋白硫酯酶 APT1 的机制来减少棕榈酰化，并且使用 shRNA 或药理学抑制剂抑制 APT1 可阻止细胞迁移和侵袭。拟议的研究将开始探索Wnt5a信号如何调节癌症转移，并阐明极化细胞迁移过程中Wnt5a调节的蛋白质棕榈酰化的分子机制。第一个目标是假设非经典 Wnt 通路的已知下游组件与核心棕榈酰化机制相互作用以调节去棕榈酰化。将采用候选方法来研究 Wnt5a 诱导蛋白质去棕榈酰化的机制，以鉴定 Wnt5a 诱导蛋白质去棕榈酰化所需的 Wnt 信号传导成分。第二个目标将确定蛋白质棕榈酰化循环在体外和体内细胞迁移和侵袭过程中的作用。在人类肿瘤样本中发现的棕榈酰转移酶突变损害了棕榈酰化 MCAM 和抑制胶原蛋白细胞侵袭的能力。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来研究细胞行为，并通过延时成像来分析蛋白质定位和定向迁移。受 Wnt5a 调节的其他棕榈酰化蛋白将使用质谱进行生化纯化和鉴定，从而为 Wnt5a 诱导的去棕榈酰化调节的细胞途径提供广泛的视野。正在患者肿瘤样本中检查该途径中鉴定的蛋白质。