Abstract 1779: Inability to regulate ROS content confers sensitivity to ferroptosis in breast cancer cells resistant to T-DM1
Authors: Conilh Louise , Sauveur Juliette , Beaumel Sabine , Scheffges Claire , Nunez Estelle , Cheillan David , Dumontet Charles 
Published: June 2022

Abstract
Clinical success of Trastuzumab-emtansine (T-DM1, Kadcyla) lead its extended approval as a post-surgical adjuvant treatment in early stage HER-2-positive breast cancer. However, intrinsic and acquired resistance remains a major limitation to its clinical efficacy. Adipocytes were shown to exhibit a pro-survival cross talk with breast cancer cells, limiting their sensitivity to T-DM1 and raising the question of metabolic alterations involved in this resistance phenotype. To explore the role of tumor cell metabolic plasticity in T-DM1 resistance, xenografts of the human HER-2 positive breast cancer model BT-474 were exposed in vivo to increasing doses of T-DM1. BT-474 WT and R tumors were characterized by RNAseq and primary explants of in vivo tumors were cultured and analyzed in vitro. Although HER-2 levels were found to be strongly reduced in resistant cells, no increase of its pro-survival downstream partners were observed at the transcriptomic level. Conversely, PIK3CA and KRAS were respectively 2.5- and 2-times downregulated in BT-474 R according to RNAseq data, suggesting the implementation of compensatory pathways for cell survival and proliferation. Tumors resistant to T-DM1 exhibited alterations in lipid metabolism, including an increase in lipid droplet content observed by ORO staining, upregulation of fatty acid metabolism pathways, and lipid transporter FABP4 overexpression. Cells resistant to T-DM1 survived to 2-deoxyglucose treatment (100 mM) as well as glucose deprivation in vitro while these conditions were cytotoxic to BT-474 WT cells. In addition, BT-474 R cells were resistant to palmitate exposure, suggesting energy production is monitored by an increased lipid import and metabolism. Ferroptotic cell death and associated lipid peroxidation have recently been associated with alterations in lipid metabolism. H2D-CFDA staining revealed a 2-fold increase in ROS content in R cells at the basal state compared to WT cells. In vitro, BT-474 R cells were highly sensitive to ROS inducers such as H2O2 and bleomycin when compared to WT cells (IC50s were 7- and 30-fold higher in WT compared to R cells for H202 and bleomycin, respectively). This unusually strong cytotoxicity of H2O2 was found to be correlated with a significant increase of ROS content within the resistant cells compared to wild type cells after H2O2 treatment. Additionally, inhibitors of ferroptosis such as GPX4, and the antiporter system xct were found to be downregulated in resistant cells, while the GPX4 inhibitor RSL3 was 5-fold more cytotoxic in cells resistant to T-DM1 than in WT cells. T-DM1 -resistant cells thus demonstrate increased fatty acid uptake, decreased ability to eliminate ROS and enhanced sensitivity to ferroptosis inducers. Altogether, these results suggest that ferroptosis could constitute a promising target for T-DM1-resistant tumors.
Citation Format: Louise Conilh, Juliette Sauveur, Sabine Beaumel, Claire Scheffges, Estelle Nunez, David Cheillan, Charles Dumontet. Inability to regulate ROS content confers sensitivity to ferroptosis in breast cancer cells resistant to T-DM1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1779.