Role of p120ctn in Cadherin Mediated Suppression of Breast Cancer

Abstract

SUPPLEMENTARY NOTES 12a. DISTRIBUTION / AVAILABILITY STATEMENT Approved for Public Release; Distribution Unlimited 12b. DISTRIBUTION CODE Abstract (Maximum 200 Words) (abstract should contain no proprietary or confidential information)This research aims to understand the mechanism by which the catenin pi20 regulates the cell adhesion molecule E-cadherin, a protein whose function is critical to inhibiting metastasis of human tumors. My hypothesis is that uncoupling pl20 from E-cadherin by mutation of the juxtamembrane domain will result in an E-cadherin molecule that is defective for the function contributed by pi20. In this reporting period, I have mutated the pi20 binding site on E-cadherin and generated minimally altered E-cadherin proteins that are selectively unable to bind pl20. Expression of the pl20-uncoupled cadherin proteins in the E-cadherin deficient human breast cancer cell line MDA231 eliminates the ability of E-cadherin to induce tightly organized epithelial colonies. The cells form poor junctions and are not able to mediate strong cell-cell adhesion. We are planning to analyze the metastatic potential of these cells in mice. These results significantly clarify the role of pl20 in cadherin function and could ultimately lead to rational therapeutic approaches to inhibit metastasis of breast cancer.14. SUBJECT TERMS cell adhesion, cadherins, catenins, pl20ctn, metastasis invasion, motility Thoreson, Molly A, Thoreson, Molly A. INTRODUCTIONCell-cell adhesion is a fundamental property of epithelial cells which is lost during the transition from solid to metastatic tumors. E-cadherin is the major cell adhesion molecule involved in this process, and evidence indicates that defects not only in the cadherin itself, but also in its cytoplasmic binding partners can affect the transition to metastasis. We aim to better understand this process by studying the interaction of Ecadherin with pl20, a src substrate and cadherin binding partner discovered in our lab.To dissect the influence of pl20 on E-cadherin function, we proposed to uncouple pl20 from E-cadherin in vivo and examine effects on the cadherin related to cell adhesion and invasion. These experiments seek to understand at the mechanistic level, the epithelial to mesynchymal transition which occurs as a loss of cadherin function, and may suggest targets for inhibiting progression to metastasis. Research: The statement of work for this grant is included below for reference. We have completed Tasks 1 and 2, and parts of Task 3, and these results are published in the Journal of Cell Biology 1 (see Appendix ). For Task 1,1 subcloned the E-cadherin mutants 761AAA and 764AAA (which are uncoupled from pi20) into the pLK expression vector. These mutants were then transfected into MDA231 cells to obtain stable cell lines. Simultaneously, A431D cells were also transfected with the same constructs, and experiments were carried out in both cell lines. Cells were screened by immunofluorescence and western blotting for E-cadherin, pi20, and several other catenins (a...