Abstract P6-02-04: Eribulin mesylate promotes a mesenchymal-epithelial transition (MET) and effectively radio-sensitizes triple negative breast cancer cells

Abstract

Abstract Purpose: Eribulin mesylate is a novel non-taxane microtubule dynamics inhibitor that irreversibly induces mitotic arrest, and has been shown to promote a shift from mesenchymal to epithelial phenotypes in breast cancer. It has demonstrated therapeutic activity in heavily pretreated patients with metastatic breast cancer, with a trend toward greater efficacy in patients with triple negative breast cancer (TNBC). TNBC is an aggressive tumor with a mesenchymal phenotype and higher locoregional recurrence (LRR) compared to estrogen receptor (ER) positive breast cancer. One postulated cause for higher LRR in TNBC is that these tumors have a great proportion of cancer stem cells (CSC). CSCs are radio- and chemo-resistant when compared to the bulk of the tumor, and are thought to be the source of both LRR and distant metastasis after treatment, thus an important target for therapy. Microtubule inhibitors are known radiation sensitizers given that they arrest cells at G2/M, the most radiation sensitive phase of the cell cycle. We hypothesize that because eribulin induces an irreversible mitotic arrest and promotes a mesenchymal-to-epithelial transition (MET) it will enhance the radiation sensitivity of TNBC through reduction in CSCs and inhibition of DNA damage repair (DDR) pathways. Methods: Experiments were conducted in 3 subtypes of TNBC cells, all with p53 mutations and PTEN loss: SUM149 (basal B), BT-549 (mesenchymal-like), and MDA-MB-468 (basal A). Cells were treated with 0.5nM-3nM Eribulin, alone or in combination with increasing doses of radiation from 0-8 Gy. In vitro studies preformed included; cell survival assays, immunoblot analysis for markers of MET, FACS analysis for cell cycle, cancer stem cell population, and apoptosis, and finally P-γH2AX staining to measure DDR. Results: We found that combining ionizing radiation (IR) and eribulin is synergistic. The IC50 of eribulin combined with 6Gy IR, was 0.5-1 nM, 10 fold less than drug alone, and a 40-60% decrease in cell viability was observed with the combination compared to IR alone. A significant decrease in clonogenic survival was seen after 24 hours pretreatment with 1nM eribulin, with the greatest efficacy seen in BT-549 cells. No significant increase in apoptosis was seen with the combination treatment by FACS analysis though a significant delay in DDR was observed. Cell cycle analysis demonstrated eribulin increases the proportion of cells in G2/M arrest following IR, as well as suppressed cell proliferation. 24-hour eribulin treatment also caused morphological changes in culture consistent with transition from a mesenchymal to an epithelial phenotype, and IHC showed a decrease in mesenchymal markers and increase in epithelial markers. Finally eribulin treatment decreased the proportion of CSCs by FACS analysis by 80% in SUM149 cells and prevented an increase in CSC population following IR. Studies are on going to further characterize the effect of combined eribulin and IR on DDR and the CSC population in TNBC cells. Conclusions: These studies demonstrate that eribulin effectively radiosensitizes TNBC cells. We believe this occurs through inhibition of DDR as well as promotion of MET which reduces the radiation resistant CSC population. Citation Format: Eileen P Connolly, Youping Sun, Tom K Hei. Eribulin mesylate promotes a mesenchymal-epithelial transition (MET) and effectively radio-sensitizes triple negative breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-02-04.