Abstract 3427: Interaction of two metastasis suppressors, Nm23-H1 and Gelsolin, in the proliferation and motility of MDA-MB-231 breast carcinoma cells

Abstract

Abstract Recent studies have highlighted the fact that the metastatic process can be separate from tumorigenesis. Nm23 was the first metastasis suppressor gene to be identified, although the biochemical basis for Nm23's anti-metastatic properties remains to be fully elucidated. Therefore we undertook an mechanistic study of Nm23 binding partners hypothesizing that Nm23's interaction with other proteins may play a role in regulating metastasis. Our data identify a new interaction with the metastasis suppressor Gelsolin, which demonstrates synergistic phenotypic effects. Gelsolin was identified as a binding partner of Nm23-H1 using co-immunoprecipitation assays and mass spectrometry analysis. Gelsolin is an actin binding protein involved in actin-cytoskeleton remodeling through its actin-severing, -capping and newly discovered -nucleating activities. Two-way co-immunoprecipitations were performed in 4T1 cells and several human breast carcinoma cell lines (MCF7, MDA-MB-231, MDA-MB-435) to confirm the association. The potential interaction of Nm23-H1 and Gelsolin was investigated by overexpressing each protein, or the combination, in human MDA-MB-231 breast carcinoma cells. With regard to in vitro motility, overexpression of Nm23-H1 reduced motility in Boyden chambers by 63.1% as compared to vector clones, while Gelsolin overexpression reduced motility by 29.6% and the combined overexpression of both genes reduced motility by 82.3% (p<0.001). Invasion assays showed similar trends with Nm23-H1 overexpression reducing invasion by 42.7% as compared to vector, Gelsolin overexpression reducing invasion by 33%, and the combination reducing invasion by 85.4% (p<0.001). With regard to proliferation, neither single gene had a prominent effect on cell proliferation, but overexpression of both Nm23-H1 and Gelsolin resulted in an anti-proliferative effect, 28.8% of vector or single gene transfectants, p =0.028. The data demonstrate an additive interaction of two metastasis suppressors in motility and invasion, and synergy in proliferation. Since Gelsolin is involved in the actin-cytoskeleton organization, regulating actin polymerization and depolimerization, we investigated the role of Nm23 in these processes. In both MDA-MB-231 and 4T1 cell lines, Gelsolin overexpression showed an increase in F-actin depolimerization (3-fold and 6.2-fold increase respectively) compared the vector clones. Moreover we observed that in both the MDA-MB-231- and 4T1- Nm23-overexpressing cells, F-actin depolimerization increased 8-fold and 5-fold respectively as compared to vector. Studies analyzing the in vivo effect of the co-overexpression of the two proteins are ongoing. The data provide an insight into the interaction of metastasis suppressors and may represent a step for the understanding of their biological mechanisms. 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 3427. doi:1538-7445.AM2012-3427