Abstract LB-276: CLCA2 interactor EVA1 maintains epithelial differentiation of HMEC.

Abstract

CLCA2 is a p53-inducible transmembrane protein that is frequently downregulated in breast cancer. It is induced during MET in immortalized HMEC, and its knockdown causes EMT. To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with the cell junctional protein EVA1 (Epithelial V-like Antigen 1), confirmed by co-immunoprecipitation and immunolocalization experiments. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53. It mediates homophilic cell-cell adhesion in diverse epithelial tissues. We found that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype. Like CLCA2, knockdown of EVA1 resulted in rapid EMT in immortalized HMEC and increased invasiveness. The interacting domains were delimited by deletion analysis, revealing that the CLCA2 transmembrane segment (TMS) alone was sufficient for the interaction with EVA1. Surprisingly, EVA1 bound to the TMS only in the context of full length CLCA2 precursor, but not in the context of the C-terminal cleavage product, suggesting that cleavage of CLCA2 causes a conformational shift and release of EVA1. The interaction was specific, as other transmembrane proteins did not interact with CLCA2 or EVA1. To determine whether CLCA2 regulates EVA1 localization to tight junctions or hemophilic ligation, we fused the CLCA2 or EVA1 TMS to a secreted heterologous protein to create competitive inhibitors. These results identify the CLCA2-EVA1 complex as pivotal in maintaining epithelial differentiation and explain the downregulation of both genes during tumor progression. Citation Format: Randolph C. Elble, Grace Ramena, Vijay Walia. CLCA2 interactor EVA1 maintains epithelial differentiation of HMEC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-276. doi:10.1158/1538-7445.AM2013-LB-276