Abstract P1-18-21: Neratinib reduces HER2+/luminal B breast cancer stem cell activity and inhibits resistance by blocking efflux mechanisms
Authors: Hull Nathan , Clarke Robert , Bundred Nigel 
Published: March 2020

Abstract
Background: Breast cancer stem cells (BCSCs) have been attributed to drug resistance and recurrence of disease following treatment. BCSCs employ various mechanisms to resist chemotherapy. One such mechanism is the increase in expression of the ATPase drug efflux pumps ABCB1 and ABCG2, reducing intracellular Paclitaxel and Mitoxantrone drug concentration, respectively. Neratinib a pan-HER tyrosine kinase inhibitor was reported to block both ABCB1 and ABCG2 pump activity. We aimed to determine the effect of Neratinib on CSC activity (primary and self-renewal) and determine if the mechanism included pump inhibition.
Methods: The effects of 10nM Neratinib alone and in combination with either 1µM Tamoxifen, 0.1µM Paclitaxel or 0.1µM Mitoxantrone on anoikis resistance culture was evaluated in multiple breast cancer cell lines. The mammosphere forming efficiency (MFE) and generation of secondary mammospheres (self-renewal) were calculated. Blockage of the ABCG2 by Neratinib was assessed via FAC analysis. Statistical significance was determined using unpaired t-test and one-way ANOVA.
Results: Neratinib reduced CSC activity assessed by MFE in BT474 cells by 50% (P &lt; 0.01). Combining Neratinib with Tamoxifen further reduced CSC (by 75%) than either treatment alone (P &lt; 0.05 and P &lt; 0.01, respectively). The combination of Neratinib and Paclitaxel also reduced the MFE by a greater extent (90%, P &lt; 0.0001) than either treatment alone (P &lt; 0.05 and P &lt; 0.0001, respectively). Neratinib alone had no effect on MFE in the triple negative breast cancer cell line MDA-MB-453, combining Neratinib with Paclitaxel reduced MFE by 85%, a greater reduction than paclitaxel alone (75%), P &lt; 0.0001. Our data also indicates that Neratinib in combination with Paclitaxel reduces CSC self-renewal in MCF7, MCF7HER2 and MDA-MB-453 cell lines. Combining Neratinib with Mitoxantrone further decreases CSC activity than either treatment alone in MCF7, MCF7HER2, SKBR3 and MDA-MB-453 cells and completely inhibited CSC self-renewal in the triple negative cell line. Neratinib also reduced the side population in BT474 and SKBR3 cells in a dose response manner, demonstrating inhibition of ABCG2 activity.
Conclusions: These findings support the idea that Neratinib has the ability to overcome drug resistance caused by the ABCB1 and ABCG2 pumps. Therefore, we are currently investigating the potential of Neratinib to block the activity of the ABCB1 and ABCG2 pumps using flow cytometry to analyse the side-population and drug accumulation. They also provide insight into a role for Neratinib to enhance paclitaxel chemotherapy in triple negative CSC.
Citation Format: Nathan Hull, Robert Clarke, Nigel Bundred. Neratinib reduces HER2+/luminal B breast cancer stem cell activity and inhibits resistance by blocking efflux mechanisms [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-21.