Proteomics of Cell Invasion
Tumor cell invasion and metastasis are strongly associated with the loss of E-cadherin, the main adhesion molecule in epithelial cells. In most tumors, the loss of E-cadherin expression is thought to occur epigenetically, and is usually followed by increased expression of mesenchymal markers, including mesenchymal cadherins. Recent evidence argues strongly that the misexpression of mesenchymal cadherins by epithelial tumor cells promotes cell invasiveness and metastasis. The mechanism by which E-cadherin promotes suppression of invasiveness is still unclear, but is thought to require its intracellular domain, which interacts directly with β-catenin and p120. However, the same proteins also associate with mesenchymal cadherins, which instead of suppressing promote invasiveness. Furthermore, in some cases, despite seemingly normal expression of E-cadherin, cells fail to adhere strongly, and instead they invade surrounding tissues. We postulate that the difference between epithelial and mesenchymal cadherins lies in specific protein-protein interactions that allow E-cadherin to suppress and mesenchymal cadherins to promote motility. To this end, we are undergoing an extensive investigation of cadherin (both epithelial and mesenchymal), catenin and kaiso binding partners in metastatic and non-metastatic cells that express E-cadherin, lack E-cadherin expression, and/or inappropriately express mesenchymal cadherins.
To identify these protein complexes we use two complimentary approaches. One involves the determination of endogenous protein complexes using immunoprecipitation followed by Mass Spectroscopy, while the other involves ectopic retroviral expression of epitope tagged proteins followed by tandem tag affinity purification and Mass Spectroscopy. A number of novel and exciting putative binding partners of E-cadherin have already been identified using these techniques.
In addition to investigating changes during tumor progression, we are also looking at cadherin/catenin binding partners in human epithelial tissues. Most of the current information on the cadherin/catenin complex comes from studies in cultured cells (2D). However, as these adhesion proteins have important roles in cell polarization and tissue morphogenesis, it is likely that protein interactions that promote these processes will not be evident in 2D cultures. To address this concern, we have developed techniques that can identify the interaction partners of the cadherin/catenin complex in small samples of normal epithelial tissue (3D).
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