Abstract 5043: A vimentin binding small molecule (FiVe1) is a novel candidate to target CSC-rich TNBC
Authors: Kuburich Nick A. , Rosolen Matthew , Martinez Francisco , den Hollander Petra , Bollong Michael J. , Soundararajan Rama , Lairson Luke L. , Mani Sendurai A. 
Published: September 2020

Introduction: Epithelial mesenchymal transition (EMT) and Cancer stem cells (CSCs) are associated with metastasis and tumor recurrence. We previously identified that EMT induces stem cell properties and the transcription factor FOXC2 is one of the key mediators. Recently, in a high throughput small molecule screen using FOXC2-overexpressing cells, we identified FiVe1 that binds to vimentin resulting in mitotic collapse and multinucleation. Vimentin is an intermediate filament that is upregulated during EMT and is present in CSCs, where it provides increased motility as well as resistance to mechanical stress in these cells. In this work, we hypothesize that FiVe1 selectively targets mesenchymal-looking, CSC-enriched populations via vimentin.

Experimental procedures: Different subtypes of breast cancer cells and non-transformed cells, as well as CSC-enriched or depleted populations, were assessed for the occurrence of multinucleation (&gt;2 nuclei) after FiVe1 treatment for two population doubling (2PD) through counting by fluorescent microscopy. Cell lines that became multinucleated were then tested for the reversibility of multinucleation after passaging cells with fresh media. Levels of vimentin phosphorylation (S56) and total vimentin were assessed by immunoblot.

Summary of data: Multinucleation was observed only in the mesenchymal-like subtypes of TNBC cell lines relative to other TNBC subtypes and non-transformed cell lines. This multinucleation was irreversible upon passaging with fresh media. The levels of S56 phosphorylation of vimentin increased in these cell lines, but gradually decreased upon passaging with fresh media. To determine if multinucleation was due to the higher number of CSCs in mesenchymal-like TNBC cells, CSC-enriched populations were treated with FiVe1. The CSC-enriched populations had significantly increased levels of multinucleation upon FiVe1 treatment relative to CSC-depleted and control populations.

Conclusions: The induction of multinucleation by Five1 correlates with higher CSC levels. This was observed both in the mesenchymal-like subtypes of TNBCs, which have higher stem-like properties relative to other TNBC subtypes, as well as cell populations enriched for CSCs. The increase in vimentin phosphorylation at S56 after FiVe1 treatment in TNBC cell lines is physiologically significant as misregulated S56 phosphorylation results in multinucleation and dysregulation of vimentin assembly. While the S56 phosphorylation decreased upon withdrawal of FiVe1 in all cells, the kinetics of reversibility is lower in mesenchymal-looking cells, which corroborate with the multinucleation. Further research regarding the fate of multinucleated cells is in progress. Inhibiting CSCs by targeting vimentin is a promising approach for sensitizing these inherently resistant tumors to chemotherapeutic agents.

Citation Format: Nick A. Kuburich, Matthew Rosolen, Francisco Martinez, Petra den Hollander, Michael J. Bollong, Rama Soundararajan, Luke L. Lairson, Sendurai A. Mani. A vimentin binding small molecule (FiVe1) is a novel candidate to target CSC-rich TNBC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5043.