Estrogen receptor inhibition mediates radiosensitization of ER-positive breast cancer models

Abstract

Endocrine therapy (ET) is an effective first-line therapy for women with estrogen receptor-positive (ER + ) breast cancers. While both ionizing radiation (RT) and ET are used for the treatment of women with ER+ breast cancer, the most effective sequencing of therapy and the effect of ET on tumor radiosensitization remains unclear. Here we sought to understand the effects of inhibiting estrogen receptor (ER) signaling in combination with RT in multiple preclinical ER+ breast cancer models. Clonogenic survival assays were performed using variable pre- and post-treatment conditions to assess radiosensitization with estradiol, estrogen deprivation, tamoxifen, fulvestrant, or AZD9496 in ER+ breast cancer cell lines. Estrogen stimulation was radioprotective (radiation enhancement ratios [rER]: 0.51–0.82). Conversely, when given one hour prior to RT, ER inhibition or estrogen depletion radiosensitized ER+ MCF-7 and T47D cells (tamoxifen rER: 1.50–1.60, fulvestrant rER: 1.76–2.81, AZD9496 rER: 1.33–1.48, estrogen depletion rER: 1.47–1.51). Combination treatment resulted in an increase in double-strand DNA (dsDNA) breaks as a result of inhibition of non-homologous end joining-mediated dsDNA break repair with no effect on homologous recombination. Treatment with tamoxifen or fulvestrant in combination with RT also increased the number of senescent cells but did not affect apoptosis or cell cycle distribution. Using an MCF-7 xenograft model, concurrent treatment with tamoxifen and RT was synergistic and resulted in a significant decrease in tumor volume and a delay in time to tumor doubling without significant toxicity. These findings provide preclinical evidence that concurrent treatment with ET and RT may be an effective radiosensitization strategy.

Androgen and oestrogen receptor co-expression determines the efficacy of hormone receptor-mediated radiosensitisation in breast cancer

“…Further, combination treatment of AR inhibition or knockout with ER inhibition or degradation does not have a synergistic effect on radiosensitisation, suggesting that AR and ER are not directly and exclusively compensating for their role in the radiation response. While our previous data suggest that inhibition or degradation of ER results in radiosensitisation [ 22 ], here we demonstrate that ER inhibition is not uniformly sufficient to radiosensitise AR+/ER+ breast cancer models. Therefore, the prior observed radiosensitisation was associated with AR-low/ER+ cell lines, further promoting the notion of an interaction between AR and ER in response to RT.…”

“…To understand whether there were overlapping roles for AR and ER in AR+/ER+ breast cancer models, clonogenic survival assays were performed with the selective estrogen receptor modulator (SERM), tamoxifen, or the selective estrogen receptor degrader (SERD), fulvestrant. While previous work has demonstrated a role for tamoxifen or fulvestrant in the radiosensitisation of ER+ breast cancer models in vitro and in vivo [ 22 ], the role of AR and ER together and the impact on radiosensitisation has not been assessed. ER inhibition with 0.5–2.0 µM tamoxifen treatment alone in CAMA-1 cells resulted in only slight increases in radiosensitisation with rER of 1.04–1.12 (Fig.…”

“…A Kimtron IC-225 orthovoltage machine was used to provide X-ray radiation at the University of Michigan Experimental Irradiation Core. In keeping with previous work, a dose rate of 2 Gy/min was used [ 22 , 29 – 31 ].…”

“…Additional work has demonstrated that AR inhibition with the second-generation anti-androgen, enzalutamide, AR knockdown by siRNA, or treatment with the experimental dual AR and CYP17 lyase inhibitor, seviteronel, sensitises AR+ TNBC cells to radiation therapy [ 7 , 8 ]. Further, our group and others have shown that inhibition or degradation of ER with tamoxifen or fulvestrant, respectively, is sufficient to radiosensitise ER+ breast cancer models [ 18 – 22 ]. Together these findings suggest a role for nuclear hormone receptors in the radiation response and warrant further investigation of AR and ER inhibition in AR+/ER+ breast cancer models.…”

“…Having previously demonstrated that ER inhibition is an effective radiosensitisation strategy in ER+, AR-low breast cancers [ 22 ] and AR inhibition is a potentially effective radiosensitisation strategy in AR+/ER− breast cancer [ 8 ], the purpose of this study was to assess the effectiveness of AR inhibition as a radiosensitisation strategy in AR+/ER+ breast cancer models. In contrast to findings in other AR + diseases, including AR + TNBC, here we demonstrate that AR inhibition with enzalutamide, apalutamide, darolutamide and seviteronel, degradation of AR with the AR PROTAC degrader, ARD-61, or knockout of AR using CRISPR-Cas9, does not radiosensitise AR+/ER+ breast cancer cells in vitro.…”