Combined Radiation and Endocrine Therapies Elicit Benefit in ER+ Breast Cancer

Abstract

Background: Standard treatment for patients with early-stage estrogen receptor-positive (ER+) breast cancer often includes sequential adjuvant radiation and endocrine therapies. Unfortunately, ~1/3 of patients eventually experience disease recurrence, partly due to residual disease in the form of drug-tolerant persister cancer cells. The anti-cancer efficacy of radiation therapy is partly attributable to the production of oxyradicals that damage biomolecules. We previously showed that endocrine therapy increases mitochondrial content in ER+ breast cancer cells; we postulated that this may also increase oxidative stress.

Methods: Herein, we tested the efficacy of concurrent endocrine and radiation therapies, including both conventional (CDR) and ultra-high dose rate (UHDR) radiation.

Results: We found that estrogen deprivation and radiation inhibit cell growth, induce apoptosis, and force cells into an oxidatively stressed state. DNA damage was almost exclusive to cells treated with the combination of endocrine and radiation therapy. Radiation slowed tumor growth in two xenograft models, and combination with estrogen deprivation prolonged the time to regrowth in ZR75-1 tumors.

Conclusions: These findings indicate that simultaneous treatment with endocrine and radiation therapies can be advantageous, warranting further evaluation to identify tumor features predictive of response to individual and combination treatments.

Keywords: antiestrogen; breast cancer; drug-tolerant persisters; endocrine therapy; oxidative stress; radiation.

Figure 1

Endocrine therapy slows growth and induces oxidative stress in ER+ BC cells. (A) Cells were seeded in triplicate and allowed to grow for 21 d. Cells underwent HD for the indicated time frame immediately prior to analysis. (B) Cells underwent HD as indicated, and lysates were analyzed by immunoblot. (C,D) Cells were maintained in growth medium or HD for 14 d prior to being treated with 6 Gy CDR or UHDR radiation. Lysates were harvested 1 h after radiation and analyzed by immunoblot. (E) Orthotopic xenografts were established in ovariectomized NSG mice supplemented with E2 by s.c. pellet. E2 pellets were removed from tumor-bearing mice for 0, 6, or 90 d to induce ED. Tumors and residual tumor beds were harvested and analyzed by IHC. Signal intensity in five microscopic fields was measured per tumor. (F) A gene expression signature of oxidative stress response (OSR) was compared with transcriptional profiles of human tumors sampled before and after presurgical endocrine therapy in 3 patient cohorts. Spearman correlation R values were calculated as correlation coefficients between the OSR signature and tumor gene expression values. In (A,E), data are presented as mean ± SD. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 by Bonferroni-adjusted post hoc test ((A,E), and cohort GSE20181 in (F)) or paired t-test (cohorts GSE71791 and GSE111563 in (F)). Original blot images can be found in Supplementary File S1.

Figure 2

Oxidative state increases upon hormone deprivation and irradiation in ER+ BC cells. (A) Treatment plan overview. (B) Cells underwent HD as indicated and then were treated with ±6 Gy CDR RT or UHDR RT. At 72 h post-RT, relative levels of total and reduced glutathione were measured, which are shown as the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG). (C) Cells were treated as in (B). Relative levels of total and reduced NADP were measured, which are shown as the ratio of reduced NADP (NADPH) to oxidized NADP (NADP+). < LD: below limit of detection. Data are presented as mean of triplicate ± SD. ** p ≤ 0.01, **** p ≤ 0.0001 by Bonferroni-adjusted post hoc test compared to respective Day 0 group unless otherwise indicated with brackets. ns: not significant; FC: fold change represented by the ratio for NRF2 to Actin signal measured via ImageJ.

Figure 3

Radiation inhibits growth and HD apoptosis in ER+ BC cells. (A,B) Cells were treated ±6 Gy CDR RT or UHDR RT and then maintained for 21 d. Representative images are shown in (A). Quantification of relative cell numbers is shown in (B). (C) Cells underwent HD as indicated and then were treated ± RT as above. Medium was changed, and 3 d later, cells were analyzed for apoptosis by annexin V labeling and flow cytometry. Data are presented as mean of triplicate ± SD. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 by Bonferroni-adjusted post hoc test compared to respective “Day 0” group unless otherwise indicated. ns: not significant.

Figure 4

Combination treatment with hormone deprivation and radiation induces DNA damage in ER+ BC cells. Cells underwent HD as indicated and then were treated ±6 Gy CDR RT or UHDR RT. After 16 h, cells were immunostained for γH2AX and counterstained with DAPI. (A) Representative images are shown. (B) Quantification of γH2AX foci per nucleus in ≥100 cells/group. Data are presented as violin plots. * p ≤ 0.05, **** p ≤ 0.0001 by Bonferroni-adjusted post hoc test compared to respective Day 0 group unless otherwise indicated with brackets. ns: not significant.

Figure 5

Radiation and endocrine therapies combine to prevent tumor growth. (A) Treatment plan overview. Ovariectomized mice with E2 supplementation bearing tumors underwent ± ED for 7 d and then ± CDR RT or UHDR RT. (B) Representative images from a CT scan of a mouse bearing an MCF-7/Luc tumor, which was used to help aim the radiation beam. (C) ZR75-1/Luc tumor growth curves displayed as percent change from tumor volume at study enrollment. (D) Allred score of ER IHC staining from tumors (n = 3/group) harvested at the end of the experiment in (C). (E) Quantification of relative flux (p/s) of bioluminescence images from tumors in (C). (F) MCF-7/Luc tumor growth curves displayed as in (C). (G) Quantification of relative flux (p/s) of bioluminescence images from tumors in (F). (H) Percent positivity of Ki67 IHC staining of tumors (n ≥ 3/group) harvested 24 h after RT. (I) Representative images and (J) quantification of ICC staining for γH2AX from tumors (n ≥ 3/group, ≥1200 nuclei/group) harvested 24 h after RT. Data are shown as mean ± SD, except tumor growth curves are shown with SEM (C/F). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 by Bonferroni-adjusted post hoc test compared to vehicle (D), and by mixed-effects model with Bonferroni adjustment for multiple comparisons (C,E–G).