No abstract

Withaferin A (WFA) is purified from the plant Withania somnifera and inhibits the vimentin cytoskeleton. Vimentin overexpression in cancer correlates with metastatic disease, induction of epithelial to mesenchymal transition and reduced patient survival. As vimentin functions in cell motility, we wanted to test the hypothesis that WFA inhibits cancer metastasis by disrupting vimentin function. These data showed that WFA had weak cytotoxic and apoptotic activity at concentrations less than or equal to 500 nM, but retained potent anti-invasive activity at these low doses. Imaging of breast cancer cell lines revealed that WFA induces perinuclear vimentin accumulation followed by rapid vimentin depolymerization. A concomitant induction of vimentin ser56 phosphorylation was observed, which is consistent with vimentin disassembly. Structure activity relationships were established using a set of chemically modified WFA analogs and showed that the predicted vimentin-binding region of WFA is necessary to induce vimentin ser56 phosphorylation and for its anti-invasive activity. Pharmacokinetic studies in mice revealed that WFA reaches peak concentrations up to 2 lM in plasma with a half-life of 1.36 hr following a single 4 mg/kg dose. In a breast cancer metastasis mouse model, WFA showed dose-dependent inhibition of metastatic lung nodules and induced vimentin ser56 phosphorylation, with minimal toxicity to lung tissue. Based upon these studies, we conclude that WFA is a potent breast cancer anti-metastatic agent and the anti-metastatic activity of WFA is, at least in part, mediated through its effects on vimentin and vimentin ser56 phosphorylation.Metastatic disease is the major cause of death in almost all cancer types; however, most treatments are developed to target the primary tumor and not the metastases. This is due to metastatic cells being difficult to detect, highly aggressive, chemoresistant and experimentally challenging to model. 1 At present, there is no agent in clinical use that effectively prevents metastasis and most patients ultimately succumb to metastatic disease.The metastatic process can be broadly categorized into three stages-tumor cell invasion into surrounding tissue, intravasation into blood or lymphatic vessels and extravasation into a new host environment. These events are triggered by genetic and epigenetic alterations that transform stationary epithelial cells into migratory cells, a process termed epithelialmesenchymal transition (EMT). 2,3 Recent data suggests that cancer cells can re-trigger EMT to migrate into surrounding tissue. 3,4 A classical EMT protein is vimentin; numerous reports show that vimentin is overexpressed in invasive human tumors but is nearly undetectable in non-invasive, stationary tumors. 3,[5][6][7] Vimentin is a Type III intermediate filament 8 and its overexpression correlates with metastatic disease, EMT induction, poor prognosis and reduced patient survival. 7,[9][10][11] Similar correlations between vimentin overexpression and invasion are observed in cancer ce...

Vimentin is an intermediate filament protein whose expression correlates with increased metastatic disease, reduced patient survival, and poor prognosis across multiple tumor types. Despite these well-characterized correlations, the molecular role of vimentin in cancer cell motility remains undefined. To approach this, we used an unbiased phosphoproteomics screen in lung cancer cell lines to discover cell motility proteins that show significant changes in phosphorylation upon vimentin depletion. We identified the guanine nucleotide exchange factor (GEF) VAV2 as having the greatest loss of phosphorylation due to vimentin depletion. Since VAV2 serves as a GEF for the small Rho GTPase Rac1, a key player in cell motility and adhesion, we explored the vimentin-VAV2 pathway as a potential novel regulator of lung cancer cell motility. We show that VAV2 localizes to vimentin positive focal adhesions (FAs) in lung cancer cells and complexes with vimentin and focal adhesion kinase (FAK). Vimentin loss impairs both pY142-VAV2 and downstream pY397-FAK activity showing that vimentin is critical for maintaining VAV2 and FAK activity. Importantly, vimentin depletion reduces the activity of the VAV2 target, Rac1, and a constitutively active Rac1 rescues defects in FAK and cell adhesion when vimentin or VAV2 is compromised. Based upon this data, we propose a model whereby vimentin promotes FAK stabilization through VAV2-mediated Rac1 activation. This model may explain why vimentin expressing metastatic lung cancer cells are more motile and invasive.

We have recently identified a mechanistic link between disruption of the microtubule cytoskeleton and inhibition of tumor angiogenesis via the hypoxia-inducible factor-1 (HIF-1) pathway. Based on this model, we hypothesized that other microtubule-targeting drugs may have a similar effect on HIF-1A. To test that hypothesis, we studied the effects of different clinically relevant microtubule-disrupting agents, including taxotere, epothilone B, discodermolide, vincristine, 2-methoxyestradiol, and colchicine. In all cases, HIF-1A protein, but not mRNA, was down-regulated in a drug dose-dependent manner. In addition, HIF-1A transcriptional activity was also inhibited by all drugs tested. To further examine whether these effects were dependent on microtubule network disruption, we tested the ability of epothilone B to inhibit HIF-1A protein in the human ovarian cancer cell line 1A9 and its B-tubulin mutant epothilone-resistant subclone 1A9/A8. Our data showed that epothilone B treatment down-regulated HIF-1A protein in the parental 1A9 cells but had no effect in the resistant 1A9/A8 cells. These observations were confirmed by confocal microscopy, which showed impaired nuclear accumulation of HIF-1A in parental 1A9 cells at epothilone B concentrations that induced extensive microtubule stabilization. In contrast, epothilone B treatment had no effect on either microtubules or HIF-1A nuclear accumulation in the resistant 1A9/A8 cells. Furthermore, epothilone B inhibited HIF-1 transcriptional activity in 1A9 cells, as evidenced by a hypoxia response element-luciferase reporter assay, but had no effect on HIF-1 activity in the resistant 1A9/A8 cells. These data directly link B-tubulin drug binding with HIF-1A protein inhibition. Our results further provide a strong rationale for testing taxanes and epothilones in clinical trials targeting HIF-1 in cancer patients. (Cancer Res 2005; 65(19): 9021-8)