Protons Show Greater Relative Biological Effectiveness for Mammary Tumorigenesis with Higher ERα- and HER2-Positive Tumors Relative to γ-rays in APCMin/+ Mice

Abstract

Purpose: Exposure to ionizing radiation increases risk of breast cancer. Although proton radiation is encountered in outer space and in medicine, we do not fully understand breast cancer risks from protons owing to limited in vivo data. The purpose of this study was to comparatively assess the effects of γ-rays and protons on mammary tumorigenesis in APC^Min/+ mice.

Methods and materials: Female APC^Min/+ mice were exposed to 1 GeV protons (1.88 or 4.71 Gy) and ¹³⁷Cs γ-rays (2 or 5 Gy). Mice were euthanized 100 to 110 days after irradiation, at which point mammary tumors were scored, tumor grades were assessed, and relative biological effectiveness was calculated. Molecular phenotypes were determined by assessing estrogen receptor α (ERα) and human epidermal growth factor receptor 2 (HER2) status. ERα downstream signaling was assessed by immunohistochemistry.

Results: Exposure to proton radiation led to increased mammary tumor frequency at both proton radiation doses compared with γ-rays. The calculated relative biological effectiveness for proton radiation-induced mammary tumorigenesis was 3.11 for all tumors and >5 for malignant tumors relative to γ-rays. Tumor frequency per unit of radiation was higher at the lower dose, suggesting a saturation effect at the higher dose. Protons induced more adenocarcinomas relative to γ-rays, and proton-induced tumors show greater ERα and HER2 positivity and higher activation of the ERα downstream PI3K/Akt and cyclin D1 pathways relative to γ-rays.

Conclusions: Our data demonstrate that protons pose a higher risk of mammary tumorigenesis relative to γ-rays. We also show that proton radiation-induced tumors in APC^Min/+ mice are ERα- and HER2-positive, which is consistent with our previous data on radiation-induced estrogenic response in wild-type mice. Although this study establishes APC^Min/+ as a model with adequate signal-to-noise ratio for space radiation-induced mammary tumorigenesis, further studies will be required to address the uncertainties in space radiation-induced breast cancer risk estimation.

Figure 1.. Higher mammary tumorigenesis in APC^Min/+ mice after proton radiation relative to γ-rays.

A) Mammary tumor frequency was higher after 1.88 Gy of protons relative to control and equitoxic 2 Gy γ-rays (p<0.001 compared to control; p<0.03 compared to γ-rays). Higher mammary tumor frequency was also noted after 2 Gy γ radiation relative to control (p<0.02). B) Higher mammary tumor frequency was observed after 4.71 Gy of protons relative to control and equitoxic 5 Gy of γ-rays (p<0.0001 compared to control and γ-rays). Mammary tumor frequency was also increased after 5 Gy γ radiation relative to control (p<001). C) Percent of tumor-bearing mice was higher after 1.88 Gy protons relative to equitoxic 2 Gy γ-rays. D) Percent of tumor bearing mice was higher after 4.71 Gy of protons relative to 5 Gy of γ-rays. E) Average number of mammary tumors per unit of proton radiation was higher at the lower dose relative to the higher dose (p<0.03 when compared between 1.88 and 4.71 Gy of protons). Comparison between low and high dose γ-rays was not statistically significant. Statistical significance for mammary tumorigenesis data is set at p ≤ 0.05 and the error bars represent mean ± standard error of the mean (SEM). *Significant compared to control. **Significant compared to γ-rays.

Figure 2.. Proton exposure was associated with higher frequency of carcinomas relative to γ-rays.

A) Malignant tumor frequency was higher after 1.88 Gy of proton radiation relative to control and 2 Gy γ-rays (p<0.0001 compared to control and γ-rays). Higher malignant mammary tumor frequency was also scored after 2 Gy γ radiation relative to control (p<0.03). B) Higher malignant mammary tumor frequency was observed after 4.71 Gy of proton radiation relative to control and γ-rays. (p<0.0001 compared to control and γ-rays). Exposure to 5 Gy γ radiation also increased malignant mammary tumor frequency (p<0.04). C) Higher percent of carcinomas was noted after 1.88 Gy protons relative to 2 Gy γ-rays. D) Higher percent of carcinomas was noted after 4.71 Gy protons relative to 5 Gy γ-rays. E) Representative image of an epidermoid cystadenoma and an acinar adenocarcinoma.

Figure 3.. Proton radiation-induced mammary tumors showed higher ERα and HER2 positivity relative to γ-rays.

A) Increased ERα positive nuclei were observed in proton-induced mammary tumors. B) Quantification shows higher percent of ERα positive nuclei after protons relative to control and γ-rays (p<0.0001compared to control and γ-rays). Increased ERα was also observed after γ radiation relative to control (p<0.0001). C) Increased HER2 staining was observed in proton-induced mammary tumors. D) Quantification shows higher staining of HER2 after protons relative to control and γ-rays (p<0.0002 compared to control; p<0.0007 compared to γ-rays). Increased HER2 was also observed after γ radiation relative to control (p<0.0004). Statistical significance for immunohistochemistry quantification data is set at p ≤ 0.05 and the error bars represent mean ± standard error of the mean (SEM). *Significant compared to control. **Significant compared to γ-rays.

Figure 4.. Increased expression of ERα downstream molecules after proton radiation.

A) Higher p85 expression in proton radiation-induced mammary tumors relative to γ-rays. B) Quantification shows increased p85 expression in proton radiation-induced tumors relative to control and γ-rays (p<0.001 compared to control; p<0.002 compared to γ-rays). Exposure to γ-rays also increased p85 level (p<0.002). C) Akt expression was higher in proton radiation-induced tumors. D) Quantification shows increased Akt expression in proton radiation-induced tumors relative to control and γ-rays (p<0.0009 compared to control; p<0.006 compared to γ-rays). Increased Akt was also observed after γ radiation (p<0001). E) Higher number of cyclin D1 positive nuclei in proton radiation-induced mammary tumors. F) Quantification shows increased cyclin D1 expression in proton radiation-induced tumors relative to control and γ-rays (p<0.0001 compared to control and γ-rays). Cyclin D1 was also increased after γ radiation (p<0.0001). Statistical significance for immunohistochemistry quantification data is set at p ≤ 0.05 and the error bars represent mean ± standard error of the mean (SEM). *Significant compared to control. **Significant compared to γ-rays.