Abstract B18: Identification and characterization of NAC1 as a profilin1/actin-binding protein that is essential for effective cytokinesis in cancer cells

Abstract

Abstract The BTB (bric-a-brac, tramtrack, broad complex) family of proteins share a conserved BTB protein-protein interaction domain that mediates homodimer or heterodimer formation. Nucleus accumbens-associated 1 (NAC1) is a member of the family and forms a functionally active homodimer through the interaction of its N-terminus BTB domains. Our laboratory has demonstrated that homodimerization of the NAC1 protein is essential for ensuring cancer cell survival and growth. NAC1 has also been suggested to participate in maintaining the stemness of mouse embryonic stem cells. We observed the silencing or dominant-negative inactivation of NAC1 in cancer cell lines with high expression levels resulted in polyploidy. This led us to the hypothesis that NAC1 may play an important role during cytokinesis of cancer cells. To test this hypothesis, we silenced NAC1 using RNA-interference and functionally inactivated NAC1 by expressing a dominant-negative truncated protein containing only the BTB domain that prevented homodimerization of full-length NAC1. Live-cell imaging coupled with cell tracking demonstrated the incomplete abscission during late stage cytokinesis is the cause of the polyploid cancer cells. Using traction force microscopy, we investigated our hypothesis that this failure in abcission during cytokinesis is due to a reduction in traction force generated by NAC1 silenced or inactivated cells. This parameter was measured by the reduction in maximal displacement of the fluorescent beads embedded in the polyacrylamide layer immediately below the dividing cells. In noncancer cell systems, cultured mouse embryonic fibroblasts from a homozygous knockout of NAC1 (NAC1 −/−) also exhibited a significant percentage of polyploid cells. This phenotype could be partially reversed by ectopic expression of NAC1. To elucidate the mechanisms of NAC1 function, we demonstrated the co-immunoprecipitation of the actin/profilin1 complex with NAC1. Silencing of profilin1 also led to a similar polyploid phenotype in cancer cells. Direct protein interactions of NAC1 and actin were validated by purified recombinant protein binding assays and we observed a dose dependent binding property of actin to NAC1. Since the complex of profilin1 and actin has been known to be essential for the polymerization of actin filaments, NAC1 could play an important role in the cytokinesis of cancer cells by modulating the function of this complex. Our findings suggest that the NAC1/actin/profilin1 complex is essential for actin remodeling and kinetics to generate sufficient traction forces for complete abscission and ensures effective cytokinesis in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B18.