Vimentin: A Novel Chemopreventive Target for Breast Cancer Metastasis.

Abstract

Abstract Background: Cancer metastasis is the major cause of death in nearly all tumor types; however, most treatments target the primary tumor and not the highly invasive metastatic cells. We propose to prevent cancer metastasis by precisely targeting the cancer invasion machinery. To do this we disrupt a novel target-the vimentin cytoskeleton. Vimentin is overexpressed in nearly all invasive solid tumors and is critical for cell migration. Here we show that Withania sominifera root extracts (WRE) and one of its primary constituent, Withaferin A (WFA), targets vimentin to inhibit cancer cell motility and migration while having negligible effects on viability. WFA is a steroidal natural product with 4-rings (A-D) connected to a lactone ring. We test the hypothesis that WFA is an anti-invasive compound that disrupts vimentin function with limited toxicity.Material and Methods: We employ cutting-edge live cell confocal imaging studies in combination with traditional molecular biology techniques to dissect the precise mechanism of how WRE or WFA inhibits cancer cell migration and invasion. We have employed a wounding model and Matrigel invasion assay to determine the anti-migratory and anti-invasive properties of WRE and WFA using MDAMB-231 cells. We have used GFP-vimentin to visualize the changes in vimentin dynamics. Effect of WRE and WFA on cell cycle was analyzed by Flow cytometry.Results: Our results show WFA has weak anti-proliferative activity at low concentrations but potently inhibits breast cancer migration and invasion in a dose-dependent manner. High resolution confocal images reveal that in WFA-treated cells, vimentin fails to invade the lamellipodia resulting in decreased cell migration. This is supported by live cell confocal imaging showing that at high doses WFA treatment depolymerizes GFP: vimentin. Furthermore, treatment with WFA results in aberrant vimentin phosphorylation, likely resulting in a net depolymerized phenotype. In order to determine if the vimentin-binding A ring of WFA is critical for its anti-invasive efficacy, we synthesized and tested A-ring modified analogs of WFA. All three analogs showed severely reduced potency in inhibiting cancer cell migration and invasion, suggesting that the A ring is critical for its anti-invasive activity.Discussion: The present study shows that WRE or WFA inhibits breast cancer cell migration at nanomolar concentrations by disrupting vimentin at concentrations that have minimal effect on proliferation. We believe its strong anti-migratory activity make this an appropriate compound as an anti-metastatic chemopreventative. Ultimately, we envision WFA can be used as a vimentin-targeting chemopreventative in high-risk metastatic patients and has the potential to be used with traditional cytotoxics. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5063.