Abstract 2030: Eribulin disrupts TGF-β-mediated Smad2/3-dependent transcription of EMT promoting proteins in triple negative breast cancer cells

Microtubule targeting agents (MTAs) are of significant utility in the treatment of breast cancer. While decades of research have shown that these drugs cause mitotic arrest in cells by suppressing the dynamic instability of microtubules, recent evidence demonstrates that the ability of MTAs to disrupt the microtubule-dependent transport of key signaling proteins in interphase likely contributes to their anticancer actions. Ligand-mediated activation of TGF-β receptors leads to the activation of downstream pathways that induce the expression of Snail and Slug, key transcriptional-repressors that promote EMT through Smad-dependent and independent pathways. Eribulin was shown by Yoshida and colleagues to reverse epithelial-to-mesenchymal transition (EMT) in preclinical models of triple negative breast cancer (TNBC) within 7 days. We tested the hypothesis that the initial disruption of microtubule dynamics and structure by eribulin could rapidly inhibit TGF-β-dependent signaling related to EMT as compared to other clinically relevant MTAs. A panel of four TGF-β responsive triple negative breast cancer (TNBC) cells, BT-549, MDA-MB-231, HCC1937 and Hs578T, were pre-treated with clinically relevant concentrations of the MTAs eribulin, vinorelbine, paclitaxel, or ixabepilone, which caused maximum disruption of the interphase microtubule network within 2 h. These cells were then stimulated with 2 ng/mL of TGF-β ligand for 30 min - 3 h, followed by evaluation of downstream TGF-β targets. Immunofluorescence experiments showed that the microtubule destabilizers eribulin and vinorelbine inhibited the nuclear accumulation of phosphorylated-Smad 2/3 to an extent greater than the microtubule stabilizers paclitaxel and ixabepilone or the vehicle control. Further, eribulin and vinorelbine caused significant inhibition of TGF-β-induced Snail expression in all four cell lines when compared to vehicle or microtubule stabilizer-treated cells. Knock-down studies confirmed that the effects of MTAs on Snail expression are mediated by Smad 2/3. Consistent with current literature, a 7-day eribulin treatment began to reverse their mesenchymal characteristics as measured by loss of N-cadherin in two TNBC cell lines. These data support the premise that rapid, eribulin-mediated, inhibition of TGF-β signaling contributes to its ability to reverse EMT, through inhibition of Smad-2/3 dependent transcription. Ongoing studies are investigating whether eribulin differentially inhibits Smad 2/3 protein interactions with its upstream scaffold SARA that is integral for optimal signaling as compared to other MTAs. Dissecting how MTAs differentially disrupt the TGF-β-Smad-2/3 signaling pathway might facilitate a better understanding of the molecular contexts of breast cancers that might benefit from treatment with specific MTAs. These studies are supported by Eisai Inc. Citation Format: Roma Kaul, April L. Risinger, Susan L. Mooberry. Eribulin disrupts TGF-β-mediated Smad2/3-dependent transcription of EMT promoting proteins in triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2030.