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：
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
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/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

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（由申请人提供）：该提案的主要问题是：Wnt5a的细胞极性如何调节，这些机制如何影响癌症转移？该建议将基于最新发现，揭示了通过WNT5A信号激活对细胞粘附分子depalmitoylation的调节，从而促进了癌细胞侵袭。棕榈酰化是在半胱氨酸残基上添加16个碳脂肪酸棕榈酸酯与蛋白质的添加。 WNT5A治疗黑色素瘤细胞可诱导黑色素瘤细胞中细胞粘附分子的去山膜叶胶化。在没有外源WNT5A的情况下，棕榈酰化的MCAM突变会导致MCAM的极化定位，并增加3D胶原蛋白和异种移植肿瘤分析中细胞的侵袭。 Wnt5a信号传导通过需要酰基蛋白硫酯酶APT1的机制降低棕榈酰化，并使用SHRNA或药理抑制剂阻止细胞迁移和侵袭。拟议的研究将开始探讨如何通过WNT5A信号传导调节癌症转移，并阐明在极化细胞迁移期间由Wnt5a调节的蛋白质棕榈酰化的分子机制。在第一个目标中，假设是非统一Wnt途径的已知下游成分与核心棕榈酰化机制接口以调节depalmitoylation。将研究WNT5A诱导蛋白质去氨木酰化的机制，采用两种候选方法来识别Wnt5a诱导的蛋白质depalmitoylation所需的Wnt信号传导成分。第二个目标将决定蛋白质棕榈酰化周期在细胞迁移和体外侵袭过程中的作用。在人类肿瘤样品中鉴定出的棕榈酰转移酶的突变具有棕榈酰化MCAM和抑制胶原蛋白细胞浸润的能力。这些突变在体内的影响将由异种移植肿瘤分析确定。将使用体外迁移和侵袭测定法研究细胞行为，蛋白质定位，定向迁移将通过时间衰变成像进行分析。其他受Wnt5a调节的棕榈酰化蛋白将使用质谱法进行生化纯化和鉴定，从而提供了由Wnt5a诱导的deplmitoylation调节的细胞途径的广阔视野。在途径中鉴定出的蛋白质正在患者肿瘤样品中检查。