ABSTRACT
Purpose: The estrogen receptor (ER) is expressed in over 80% of breast tumors and has been shown to be a significant driver of breast cancer (BC) pathogenesis and therefore a target of effective first-line therapies. While both ionizing radiation (RT) and endocrine therapies (ET) are used for the treatment of ER+ BC, the effect of ET on tumor radiosensitization remains unclear, with concerns it may be radioprotective based on G1 cell arrest with ET treatment. Here we assessed the efficacy and mechanism of ER-mediated radiosensitization using various pharmacologic approaches in ER+ BC. Methods: Radiosensitization with ER inhibitors (tamoxifen [TAM], fulvestrant [FULV], AZD9496) was assessed using clonogenic survival assays. DNA damage was assessed by the neutral comet assay. Efficiency of homologous recombination (HR) or non-homologous end joining (NHEJ) as well as changes in cell cycle, apoptosis, and senescence were assessed. The efficacy of TAM with RT in vivo was assessed with an MCF-7 xenograft model. Results: The selective estrogen receptor modulator TAM radiosensitized ER+ MCF-7 (enhancement ratio [enhR]: 1.14-1.50) and T47D (enhR: 1.33-1.60) cells but not ER-negative SUM-159 cells (enhR: 0.99-1.02). The selective estrogen receptor degrader (SERD) FULV had similar radiosensitization effects in MCF-7 (enhR: 1.33-1.76) and T47D cells (enhR: 0.97-2.81) with no radiosensitization observed in SUM-159 cells (enhR: 1.01-1.03). The novel oral SERD AZD9496 radiosensitized MCF-7 cells (enhR: 1.36-1.56). MCF-7 cells treated with TAM and RT had an increase in dsDNA breaks compared to RT alone as measured by the comet assay (p<0.05) and a decrease in NHEJ-mediated repair with TAM (p<0.05). No changes were observed in HR-mediated repair by Rad51 foci or a reporter (p=NS). RT alone and in combination with TAM or FULV induced similar levels of cell cycle arrest, suggesting that radiosensitization with the combination therapy is cell-cycle independent. There were no significant changes in apoptosis with TAM, FULV, RT, or the combination (p=NS). Although TAM or FULV did induce senescence, ET with RT increased senescence induction (p<0.05). In vivo, combination RT and TAM led to a significant delay in days to tumor doubling (control: 17, TAM: 40, RT: 32, TAM+RT: undefined;p<0.0001), and a significant difference in tumor growth between mice treated with TAM or RT alone compared combination treatment, with no increased toxicities or skin lesions from the combination treatment. Conclusion: Our data suggest that TAM, FULV, or AZD9496 can radiosensitize ER+ breast tumors, and these agents with RT may be more effective for radiosensitization. This work also supports further clinical investigation of the timing of RT for patients receiving ET, including using ET during RT, especially as initiating ET prior to RT has been increasingly utilized as a bridging therapy followed by concurrent ET+RT during the COVID-19 pandemic.
ABSTRACT
Purpose: Estrogen receptor (ER) expression is present in over 80% of breast tumors and has been shown to be a significant driver of breast cancer (BC) pathogenesis and therefore a target of first-line therapies for ER-positive (ER+) BC patients. While both ionizing radiation (RT) and endocrine therapies (ET) are used for the treatment of ER+ BC, the sequencing of therapy and the effect of ET on tumor radiosensitization remain unclear. Recently, this question has become much more clinically relevant when many physicians started offering ET as a bridging strategy to surgery and RT during the COVID-19 pandemic. Here we assessed the efficacy and mechanism of ER inhibition in ER+ BC in combination with RT in preclinical models. Methods: Clonogenic survival assays were used to assess radiosensitization. Inhibition of ER signaling was accomplished by treating ER+ MCF-7 and T47D cells with the selective ER modulator (SERM), tamoxifen, or the selective ER degrader (SERD), fulvestrant. The ER-negative SUM-159 cells were used as a negative control. DNA damage was assessed by the neutral comet assay. Efficiency of homologous recombination (HR) was measured by Rad51 foci or a GFP reporter system. Non-homologous end joining (NHEJ) efficiency was assessed with a pEYFP reporter. Cell cycle effects were measured using flow cytometry with propidium iodide (PI) staining. Apoptosis was assessed by annexin V/PI via flow Scytometry. Senescence was measured using β-galactosidase staining. Western blotting was used to quantify expression of proteins and phospho-proteins involved in cell cycle and apoptosis. An MCF-7 xenograft model was used to assess the efficacy of tamoxifen with RT in vivo. Synergy was determined using the fractional tumor volume (FTV) method. Results: ER inhibition with tamoxifen radiosensitized ER+ MCF-7 (10-250 nM, enhR: 1.14-1.50) and T47D (500 nM-2.0 μ M, enhR: 1.33-1.60) cells but not ER-negative SUM-159 cells (500 nM-2.0 μ M, enhR: 0.99-1.02). ER degradation with fulvestrant had similar radiosensitization effects in MCF-7 (1-25 nM, enhR: 1.33-1.76) and T47D cells (0.5-5 nM, enhR: 0.97-2.81) with no radiosensitization observed in SUM-159 cells (1-25 nM, enhR: 1.01-1.03). MCF-7 cells treated with 500 nM tamoxifen and 4 Gy RT had an increase in dsDNA breaks compared to RT alone as measured by the comet assay (p<0.05), and there was a decrease in NHEJ-mediated repair with tamoxifen treatment (p<0.05). No changes were observed in HR-mediated repair by Rad51 foci or an HR reporter (p=NS). RT alone and in combination with tamoxifen and fulvestrant induced similar levels of cell cycle arrest, suggesting that radiosensitization with the combination therapy is a cell-cycle independent effect. In addition, there were no significant changes in apoptosis in MCF-7 or T47D cells with endocrine therapy, RT, or the combination (p=NS). Although treatment with ET did induce senescence in ER+ MCF-7 and T47D cells, the combination treatment of ET with RT induced senescence to a much greater level suggesting this mechanism may contribute to radiosensitization (p<0.05). In vivo, combination RT and tamoxifen led to a significant delay in time to tumor doubling (17 days in control, 40 days with tamoxifen alone, 32 days with RT alone, and undefined with combination;p<0.0001) and a significant difference in tumor growth between mice treated with tamoxifen or RT alone compared to mice treated with tamoxifen and RT with synergy noted with combination treatment (FTV 1.297). Conclusion: Our data suggest that ET can radiosensitize ER+ breast tumors, and ET with RT may be more effective for radiosensitization. Ongoing studies will address concurrent versus sequential ET with RT. This work also supports further clinical investigation of the timing of RT for patients receiving ET, especially as ET prior to RT is increasingly used as a bridging therapy during the COVID-19 pandemic.