The role of Src on RhoGDI2 function in bladder cancer metastasis

Src对RhoGDI2功能在膀胱癌转移中的作用

基本信息

批准号：
7899799
负责人：
Courtney Pollard
金额：
$ 2.75万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-01 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7899799
关键词：
Address Affect Binding Biological Assay Bladder Neoplasm CCL4 gene Cancer Patient Cancer cell line Cell Line Cell membrane Cells Co-Immunoprecipitations Complex Data Development Disease Genes Glutamic Acid Goals Growth Guanine Nucleotide Dissociation Inhibitors Human Immigration In Vitro Literature Lung Malignant Neoplasms Malignant neoplasm of urinary bladder Manuscripts Mass Spectrum Analysis Metastasis Suppression Metastasis Suppressor Genes Metastasis Suppressor Proteins Methods Migration Assay Morbidity - disease rate Mus Muscle Mutate Mutation Nature Neoplasm Metastasis Pathway interactions Phenotype Phosphorylation Phosphorylation Site Play Preparation Protein Binding Proto-Oncogenes Regulation Role Site Staging Techniques Tertiary Protein Structure Tissues Tumor Cell Invasion Two-Hybrid System Techniques Tyrosine Yeasts cancer cell cell motility in vitro Assay in vivo migration mimetics mortality mutant overexpression paralogous gene prevent rho rho GTP-Binding Proteins sarcoma src-Family Kinases

项目摘要

DESCRIPTION (provided by applicant): A significant proportion of bladder cancer patients initially present with muscle invasive disease often having a high rate of metastasis which is frequently fatal. Understanding and preventing formation of metastases in bladder cancer is critical in decreasing its mortality and morbidity. The Theodorescu lab has shown that Rho GDP-dissociation inhibitor 2 (RhoGDI2) is a metastasis suppressor gene in bladder cancer. While target molecules that are important in the RhoGDI2 metastasis suppression pathway have been identified, regulation of the RhoGDI2 metastasis suppression pathway remains unclear. Through the use of mass spectroscopy and co-immunoprecipitation methods the Theodorescu lab has shown that cellular sarcoma tyrosine kinase (Src) is able to phosphorylate and bind RhoGDI2. The lab has also shown that Src phospho- mimetic RhoGDI2 (glutamic acid substitution for tyrosine 153 at Src phosphorylation site on RhoGDI2) suppresses experimental in vivo metastasis more than 10 fold when compared to WT RhoGD12. Additionally, it has been shown that constitutively active Src transfected into the aggressive bladder cancer cell line UMUC3 reduces migration in a cell motility assay. The latter two results are surprising considering the classic dogma of Src acting as a proto-oncogene in most cancers. The goal of this project is to elucidate the interaction of Src and RhoGDI2 in regards to the metastasis suppression pathway of bladder cancer. Two aims have been proposed to evaluate the hypothesis that Src regulates RhoGDI2 in the bladder cancer metastasis suppression pathway. The first aim will establish that Src is able to influence the formation of bladder cancer metastases in vivo and growth and migration of bladder cancer cells in vitro in relation to cellular RhoGDI2 levels. This will be accomplished by using both activating and inactivating mutations of Src kinase function in order to observe how these mutations affect both in vitro and in vivo activity of bladder cancer cells with varying levels of RhoGDI2, The second aim will establish that Src and RhoGDI2 bind and interact through distinct domains and that these interactions are pivotal in the bladder cancer metastasis suppression pathway. This will be accomplished by mutating domains/sites on both Src and RhoGDI2 and evaluating binding by co-immunoprecipitation and yeast two-hybrid assays, then studying their effects on in vitro and in vivo bladder cancer phenotypes. In summary, the specific aims are as follows: (1) Evaluate the role of Src on motility and migration of bladder cancer cells in vitro and formation of bladder cancer metastases in vivo and the relationship of cellular RhoGDI2 levels and phosphorylation on this effect, and (2) Determine the nature and functional consequence of Src and RhoGDI2 interaction domains.

描述（由申请人提供）：最初患有肌肉侵入性疾病的大量膀胱癌患者通常患有高转移率，这通常是致命的。了解和预防膀胱癌中转移的形成对于降低其死亡率和发病率至关重要。 Theodorescu实验室表明，Rho GDP分解抑制剂2（RHOGDI2）是膀胱癌中的转移抑制基因。尽管已经确定了在RhoGDI2转移抑制途径中很重要的靶分子，但RHOGDI2转移抑制途径的调节尚不清楚。通过使用质谱和共免疫沉淀方法，Theodorescu实验室表明，细胞肉瘤酪氨酸激酶（SRC）能够磷酸化并结合RhogDI2。该实验室还表明，与WT Rhogd12相比，SRC磷酸化RHOGDI2（在RHOGDI2上的Src磷酸化位点的酪氨酸153的谷氨酸取代153）在体内转移抑制了10倍以上的实验性转移。此外，已经表明，转染到侵袭性膀胱癌细胞系UMUC3中的组成性活性SRC降低了细胞运动测定中的迁移。考虑到大多数癌症中SRC的经典教条，后两个结果令人惊讶。该项目的目的是阐明SRC和RHOGDI2在膀胱癌的转移抑制途径方面的相互作用。已经提出了两个目标来评估SRC调节膀胱癌转移抑制途径中RHOGDI2的假设。第一个目的将确定SRC能够影响体内膀胱癌转移的形成以及与细胞RhoGDI2水平有关的膀胱癌细胞的生长和迁移。 This will be accomplished by using both activating and inactivating mutations of Src kinase function in order to observe how these mutations affect both in vitro and in vivo activity of bladder cancer cells with varying levels of RhoGDI2, The second aim will establish that Src and RhoGDI2 bind and interact through distinct domains and that these interactions are pivotal in the bladder cancer metastasis suppression pathway.这将通过在SRC和RHOGDI2上突变域/位点来实现，并通过共免疫沉淀和酵母两种杂交测定法评估结合，然后研究其对体外和体内膀胱癌表型的影响。总而言之，具体目的如下：（1）评估SRC在体外的运动和膀胱癌细胞在体外的运动和迁移的作用，并形成体内膀胱癌转移以及细胞RHOGDI2水平以及磷酸化的关系，以及（2）确定SRC和RhogogDi2互动的性质和功能。