Abstract 5125: The identification of Gelsolin as a potential binding partner of Nm23

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Abstract Nm23 was the first metastasis suppressor gene to be identified. To better understand the biological mechanism leading to its anti-metastatic properties, we have investigated new potential binding-proteins of Nm23. Using Mass-spectrometry, we analyzed proteins obtained with co-immunoprecipitation of Nm23 in a mouse mammary cancer cell line overexpressing the human and murine forms of Nm23 (4T1-Nm23H1-flag and 4T1-Nm23M1-flag). Co-immunoprecipitations were also performed from primary tumors and liver metastases obtained from injection of these cell lines into Balb/C mice. Several proteins were found to bind Nm23, both in vitro and in vivo specimens. In addition to previously published Nm23 interacting proteins, we identified new binding partners including the actin-binding protein Gelsolin. Using immunoprecipitation assays, the interaction between Gelsolin and Nm23 was validated in a murine cell line (4T1- Nm23H1/M1 clones) and then in human breast carcinoma cell lines with low (MCF-7) and high metastatic potentials (MDA-MB-435, MDA-MB-231). In the highly metastatic MDA-MB-231 cell line, the interaction was observed only after the overexpression of Nm23 in the system. These results suggest that a Nm23-gelsolin interaction may only take place when relatively high levels of Nm23 are expressed. Moreover, we observed that this interaction involves not only the native form of Gelsolin but also Gelsolin-fragments, NH2-terminal and COOH-terminal, which are generated by the action of caspase-3 cleavage. Full-length Gelsolin binds and severs actin polymers in a Ca2+-dependent manner, whereas caspase-3-cleaved Gelsolin severs actin polymers independent of Ca2+. The overexpression of the NH2-terminal fragment has been shown to induce apoptosis, whereas the COOH Terminal fragment has been shown to have an anti-apoptotic effect. We are therefore interested in exploring which Gelsolin-fragment may be specifically binding to Nm23, in order to determine the biological function regulated by this interaction. Our results show a novel Nm23 binding partner, Gelsolin, which is involved in the actin-cytoskeleton regulation and may, in part, explain the motility inhibitory activity of Nm23. The exact interactions between Nm23 and Gelsolin are currently being explored. In particular we are evaluating the role of this interaction in the anti-metastatic effects of Nm23, both human and murine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5125.