Abstract 737: Estrogen receptor inhibition with tamoxifen mediates radiosensitization of ER+ breast cancer models
Authors: Michmerhuizen Anna R. , Pesch Andrea M. , Schwartz Rachel , Wilder-Romans Kari , Liu Meilan , Azaria Ruth , Jelley Alexa , Pierce Lori J. , Speers Corey W. 
Published: July 2021

Abstract
Purpose: Estrogen receptor (ER) expression is present in over 80% of breast tumors and has been shown to be a significant driver of tumor initiation and progression. Therefore, patients with ER-positive (ER+) breast cancers are given first-line therapies which target the ER and downstream ER signaling. Ionizing radiation (RT) has been shown to significantly improve locoregional control and increase overall survival in patients with ER+ breast cancer. Similarly, endocrine therapy (ET) has also been shown to improve metastasis-free and overall survival in women with ER+ breast cancer. While both radiation and ET are used in women with ER+ breast cancer, the effect of endocrine therapies on tumor radiosensitization remains unclear. Here we assessed the efficacy and mechanism of ER inhibition in ER+ breast cancers in combination with radiation therapy.
Methods: Clonogenic survival assays were performed to assess radiosensitization and calculate radiation enhancement ratios (enhR) with the selective estrogen receptor modulator (SERM), tamoxifen, in ER+ MCF-7 and T47D cells or ER-negative (ER-) SUM-159 cells. DNA damage was assessed by yH2AX foci. Efficiency of homologous recombination (HR) or non-homologous end joining (NHEJ) was measured by RAD51 foci or using a pYFP reporter, respectively. Cell cycle effects were measured using flow cytometry with propidium iodide (PI) staining. Apoptosis was assessed by annexin V/PI via flow cytometry. Western blotting was used to quantify expression of proteins and phospho-proteins involved in DNA repair, cell cycle, and apoptosis. An MCF-7 xenograft model was used to assess the efficacy of tamoxifen with RT in vivo.
Results: ER inhibition with tamoxifen radiosensitized ER+ MCF-7 (enhR: 1.14-1.50) and T47D (enhR: 1.33-1.60) cells but not ER- SUM-159 cells (enhR: 0.99-1.02). MCF-7 and T47D cells treated with tamoxifen did not have changes in the kinetics of dsDNA break repair as measured by yH2AX foci (p&gt;0.05) but demonstrated a decrease in NHEJ-mediated repair (p&lt;0.05). No changes were observed in HR-mediated repair by Rad51 foci (p&gt;0.05). While cell cycle arrest was induced at 24 hours after RT, no changes were observed with tamoxifen treatment in combination with RT. In addition, there were no significant changes in apoptosis in MCF-7 or T47D cells with treatment of tamoxifen, radiation, or the combination (p&gt;0.05). In vivo xenograft studies demonstrate a significant delay in time to tumor doubling and a significant difference in tumor growth between mice treated with tamoxifen or RT alone compared to mice treated with tamoxifen and RT.
Conclusion: Our data suggest that tamoxifen may be effectively used to radiosensitize ER+ breast tumors. This work also supports further clinical investigation of the timing of radiation for patients receiving endocrine therapy as concurrent use may be more effective than sequential.
Citation Format: Anna R. Michmerhuizen, Andrea M. Pesch, Rachel Schwartz, Kari Wilder-Romans, Meilan Liu, Ruth Azaria, Alexa Jelley, Lori J. Pierce, Corey W. Speers. Estrogen receptor inhibition with tamoxifen mediates radiosensitization of ER+ breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 737.