Abstract 3421: Nm23, a metastasis suppressor gene, binds the cytoskeletal linker Ezrin to regulate motility
Authors: Marshall Jean-Claude A. , Collins Joshua , Zhou Ming , Veenstra Timothy , Khanna Chand , Steeg Patricia 
Published: August 2012

Abstract
Nm23 was the first metastasis suppressor gene characterized. One of the best correlates of the metastasis suppressive effect of Nm23 is its inhibition of tumor cell motility, but the mechanistic basis for this phenotype have not been elucidated. Our data in this abstract describe a novel interaction of Nm23-H1 with the FERM domain of Ezrin, which competitively inhibits the ability of FAK to bind Ezrin and induce motility. We hypothesized that Nm23-H1 may suppress motility and metastasis through the binding and inactivation of proteins that promote aggressive behavior. An unbiased survey of Nm23-H1 binding proteins was conducted in a 4T1 murine mammary carcinoma system. Flag tagged Nm23-H1 and its mouse variant, M1, were transfected into 4T1 cancer cells. Primary tumor size was unaffected by over-expression of Nm23-H1 or M1. Histological examination revealed a 75% decrease in liver metastasis by Nm23-H1 or M1 over-expression (P &lt; 0.001). To characterize Nm23-H1/M1 binding proteins, Flag tagged Nm23 from both H1 and M1 clones were immunoprecipitated (IP) from cell lines in vitro as well as isolated primary tumors and metastases. Mass spectrometry analysis of the IP samples identified Ezrin as a potential binding partner. Ezrin is a member of the ERM family of proteins, which have been described to link the actin cytoskeleton to membrane associated proteins. Using co-IP, Ezrin was validated as a binding partner of Nm23-H1 and M1 in both the 4T1 and human MDA-MB-231 cell lines. Nm23-H1 and M1 were found to bind to the N terminal of Ezrin, which contains the highly conserved FERM domain. Deletion mutants established the interaction of Nm23 in both cell lines with the FERM-C portion, corresponding to Ezrin amino acids 205 to 300. Ezrin exists as a folded circular protein that, when activated by phosphorylation at amino acid 567 unfurls, exposing the FERM domain. Nm23-H1 co-immunoprecipitated constitutively active (T567D) but not inactive (T567A) mutants of Ezrin, suggesting that the active “open” structure permitted access to the FERM domain. As focal adhesion kinase (FAK) binds to Ezrin also through the FERM domain, it was hypothesized that Nm23-H1 would competitively prevent this association, confirmed in triple co-IP experiments. Expression of the T567D mutant of Ezrin increased cell motility of the 4T1 cells by an average of 148% (P&lt;0.01). The expression of the T567D mutant did not overcome the motility inhibiting phenotype of Nm23 when co-expressed in 4T1 cells. We therefore hypothesize that Nm23 functions as a secondary regulator for Ezrin, binding to the N terminus and preventing the FERM domain from interacting with cell membrane associated proteins such as FAK that control tumor cell motility.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3421. doi:1538-7445.AM2012-3421