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Tamoxifen is a Selective Estrogen Receptor (ER) Modulator extensively used in the adjuvant and first line treatment of ER positive breast cancer. Recently, we reported that tamoxifen resistant breast cancer cells are cross-resistant to irradiation, possibly hampering optimal treatment of breast cancer patients. Tamoxifen has been shown to also exhibit ER-independent effects such as inhibition of mitochondrial oxidative phosphorylation, which might explain subsequent radioresistance. Here, we aim to investigate the mechanisms associated with tamoxifen induced radioresistance, and whether this depends on ER status.

To identify mechanisms underlying this cross-resistance, we induced tamoxifen resistance in ER positive MCF7 and ER negative MDA-MB-231 breast cancer cells by chronic treatment with increasing doses. Several metabolic characteristics were assessed in wild type (WT) cells and resistant (TAM) cells such as oxygen consumption and glycolysis, using the Seahorse metabolic analyzer. Additionally, we measured real-time ROS production in response to tamoxifen and H₂O₂ as well as toxicity of these compounds to WT and TAM cells and assessed total antioxidant capacity. Attempting to increase the sensitivity of TAM cells to ROS, NRF2, the main activator for the antioxidant response element, was inhibited and the cells sensitivity to several ROS as well as irradiation was examined.

Clonogenic survival reveals that also MDA-MB-231 cells lacking an ER can become resistant to irradiation after chronic tamoxifen treatment. We show that tamoxifen resistant cells exhibit a decreased oxygen consumption rate and have developed a more glycolytic phenotype compared to untreated wild type cells (Fig.1A), indicating mitochondrial dysfunction. Real time measurement of ROS demonstrates significantly lower overall ROS levels in resistant cells in response to tamoxifen (Fig.1), but also after H₂O₂ treatment. Additionally, we found treatment with H₂O₂ to be less toxic on tamoxifen resistant cells, suggesting ROS protective mechanisms as antioxidants to be more active in these cells. Indeed, we find higher antioxidant levels in tamoxifen resistant cells likely protecting cells from ROS induced DNA damage. By inhibiting NRF2, the activator of the antioxidant response element, tamoxifen resistant cells reestablished their sensitivity to ROS (Fig.1C).

These data indicate two mechanisms of ER independent cellular adaptations underlying tamoxifen induced radioresistance. We report an increased glycolytic capacity in tamoxifen resistant cells, which is generally associated with radioresistance. Additionally, increased metabolism of ROS and higher expression of antioxidants serve to protect cells from DNA damage and death related to irradiation induced ROS as we show that tamoxifen resistant cells regain their sensitivity to ROS when NRF2 is inhibited which expectedly increases sensitivity to irradiation.