Abstract 2792: Novel anti-microtubule agents for breast cancer

Abstract

Abstract Background and Objectives: Stathmin is a founding member of a family of microtubule-destabilizing proteins and is one of the key regulators of the microtubule cytoskeleton. Interestingly, stathmin is expressed at high levels in breast cancer and its overexpression is linked with disease progression. Recent studies identified numerous short peptides from the N-terminal region of stathmin family that were shown to impede tubulin polymerization. Here, we tested the hypothesis whether intracellular delivery of stathmin-like peptide(s) would inhibit the malignant proliferation of breast cancer cells by disrupting microtubule assembly in in vitro models of human breast cancer. Methods and Results: The peptide(s) that we designed consisted of a short stathmin-like domain that was fused to a TAT transduction domain, which served as a carrier to facilitate entry into breast cancer cells. We also included a hemagglutinin (HA) epitope tag to track the peptide intracellularly. Since stathmin could be phosphorylated & inactivated intracellularly by p34cdc2 kinase, we also designed another peptide in which Ser-16 was mutated to an alanine to prevent its inactivation by phosphorylation. Previous studies had shown that this substitution results in a more potent form of stathmin that cannot be inactivated by phosphorylation. As a control, we had generated a similar peptide in which the stathmin-like domain was scrambled. We tested the effects of these peptides in different breast cancer cell lines (MDA-MB-231, T47D, & SKBR-3). Immunostaining of breast cancer cells exposed to different peptides showed a vast majority of cells (>95%) positive for anti-HA staining suggesting that the designed peptides can be efficiently taken up by the cells. Proliferation assays in different breast cancer cell lines showed growth inhibitory effects in cells exposed to both, wild type or mutant peptides compared to cells exposed to a control peptide. However, the mutant peptide mediated a more profound anti-proliferative effect as expected. Immunofluorescence analysis of the microtubule organization of breast cancer cells exposed to different peptides using an anti-tubulin antibody showed a marked reduction in the density of microtubules. We also quantified the levels of polymerized and unpolymerized tubulin in breast cancer cells exposed to the different peptides using a sensitive biochemical assay. Immunoblot analysis showed a 1.8 fold decrease in the ratio of polymerized to soluble tubulin in cells exposed to wild-type peptide relative to control cells. The ratio of polymerized to soluble tubulin was further decreased by 3.7 fold in cells exposed to the mutant peptide. This suggests that the observed anti-proliferative effect is likely a result of inhibition of microtubule assembly. Thus, peptide(s) could be used to modulate stathmin function and inhibit malignant proliferation of breast cancer cells by interfering with the microtubule equilibrium. 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 2792. doi:1538-7445.AM2012-2792