Abstract 1447: An unbiased screen for Nm23-H1 interacting proteins identifies the cytoskeletal crosslinking protein Ezrin

We hypothesized that Nm23-H1 may suppress metastasis through 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 model system. Flag tagged Nm23-H1 and its mouse variant, -M1, were transfected into 4T1 mouse mammary cancer cells. Clonal lines were inoculated into the number four mammary fat pad of mice and allowed to grow for 10 days prior to excision due to size of the primary tumor. Mice were then imaged weekly for the remainder of the experiment, 8 weeks to follow the metastatic spread of the 4T1 cells from each group. 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 < 0.001). To characterize Nm23-H1/M1 binding proteins, Flag tagged Nm23 from both H1 and M1 clones were immunoprecipitated (IP) from in vitro cell lines as well as isolated primary tumors, lymph nodes, spleen and liver tissue after verification of metastasis by histological examination of the tissue. Mass spectrometry analysis of IP samples from cell lines and primary tumors showed 99 proteins of interest including Ezrin. Co-IP of Ezrin and Nm23-H1/M1 has been confirmed using both Nm23 and Ezrin as the IP antibodies, implicating Ezrin as a potential binding partner of NM23-H1 and M1. Ezrin belongs to a family of widely expressed proteins known as ERMs which have been described to link the actin cytoskeleton to membrane signaling pathways. To rule out potential interaction due to F-actin, cells were exposed to latrunculin B and co-IP of Nm23-H1 and Ezrin was again verified, indicating that the Ezrin and Nm23-H1/M1 interaction is not due to F-actin binding. To further elucidate this interaction plasmid constructs expressing either the N terminal FERM domain or C terminal F-actin binding domain of Ezrin coupled to GFP were constructed. Co-IP showed a binding of Nm23-H1 to the N terminal domain of Ezrin. In addition the expression of the N terminal overcame the motility and invasive inhibitory phenotype of Nm23-H1 expression in both 4T1 and MDA-MB-231 cells. The data identify a novel interaction of the metastasis suppressor Nm23-H1 and the cytoskeletal linker Ezrin. Suppression of metastasis by Nm23-H1 may be due, in part, to the abrogation of a pro-invasive function of the N-terminus of Ezrin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1447. doi:10.1158/1538-7445.AM2011-1447