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Article

Simulated Galactic Cosmic Radiation Exposure-Induced Mammary Tumorigenesis in ApcMin/+ Mice Coincides with Activation of ERα-ERRα-SPP1 Signaling Axis

by
Kamendra Kumar
1,
Jerry Angdisen
1,
Jinwenrui Ma
1,
Kamal Datta
1,
Albert J. Fornace, Jr.
1,2 and
Shubhankar Suman
1,*
1
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA
2
Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC 20057, USA
*
Author to whom correspondence should be addressed.
Cancers 2024, 16(23), 3954; https://doi.org/10.3390/cancers16233954
Submission received: 5 November 2024 / Revised: 19 November 2024 / Accepted: 20 November 2024 / Published: 26 November 2024
(This article belongs to the Special Issue Radiation Exposure, Inflammation and Cancers)
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Simple Summary

Female astronauts on deep-space missions are at increased risk of breast cancer due to exposure to galactic cosmic radiation (GCR). This study aimed to understand how GCR might lead to breast cancer by focusing on a hormone-related pathway involving estrogen receptor alpha (ERα) and other related molecules (ERRα and SPP1). In mice exposed to simulated GCR, we observed increased levels of estrogen, changes in breast tissue growth, and activation of genes that promote tumor formation. Similar results were found when analyzing human breast cancer tissues, suggesting that this pathway is also important in human breast cancer. These findings highlight the ERα-ERRα-SPP1 pathway as a key player in radiation-induced breast cancer risk, providing a potential target for developing protective therapies to safeguard female astronauts during deep-space missions.

Abstract

Background: Exposure to galactic cosmic radiation (GCR) is a breast cancer risk factor for female astronauts on deep-space missions. However, the specific signaling mechanisms driving GCR-induced breast cancer have not yet been determined. Methods: This study aimed to investigate the role of the estrogen-induced ERα-ERRα-SPP1 signaling axis in relation to mammary tumorigenesis in female ApcMin/+ mice exposed to simulated GCR (GCRsim) at 100–110 days post-exposure. Results: In GCRsim-exposed mice, we observed marked elevations in serum estradiol, increased ductal overgrowth, ERα activation, and upregulation of ERα target genes with pro-tumorigenic functions in mammary tissues that was coupled with a higher mammary tumorigenesis, relative to control. Additionally, the ERα target gene Esrra, which encodes ERRα, was also upregulated along with its oncogenic target gene Spp1, indicating the activation of the ERα-ERRα-SPP1 axis in mouse mammary tissues after GCRsim exposure. Using a human tissue microarray and human breast cancer gene expression analysis, we also highlighted the conserved nature of the ERα-ERRα-SPP1 signaling in human breast cancer development. Conclusions: We identified the ERα-ERRα-SPP1 signaling axis as a potential key mediator in GCR-induced breast cancer with conserved activation in human breast cancer. These findings suggest that targeting this pathway could serve as a potential target for therapeutic intervention to safeguard female astronauts during and after a prolonged outer space mission.

1. Introduction

Female astronauts are considered to be at an increased risk of developing breast cancer due to occupational exposure to ionizing radiation (IR) during space missions [1,2]. This risk is primarily inferred from epidemiological studies of women exposed to low-linear energy transfer (LET) IR, such as γ-rays and X-rays [3,4,5,6]. On Earth, the average radiation background is approximately 2.4 mSv per year, whereas astronauts aboard the International Space Station (ISS) are exposed to radiation doses of around 0.5 mSv per day [7,8]. In deep space, beyond Earth’s magnetosphere, the IR dose rate from galactic cosmic radiation (GCR) is estimated to be around 1 mSv per day, and for longer-duration missions, such as those to Mars, the estimated cumulative dose ranges from 0.30 to 0.45 Gy (equivalent to 0.87 to 1.20 Sv) [9]. Unlike low-LET IR on Earth, GCR consists of highly energetic protons, alpha particles, and heavy ions [10,11]. High-LET heavy ions, due to their ability to penetrate current spacecraft shielding, can induce persistent oxidative stress, genotoxic damage, accelerated senescence, and a senescence-associated secretory phenotype (SASP)-driven inflammatory response, thereby contributing to an elevated cancer risk [12,13,14,15,16].
Since no female astronaut has ventured into deep space, understanding the adverse health risks and associated molecular perturbations after space radiation exposure requires ground-based in vivo studies using simulated GCR (GCRsim) exposures [17]. The female ApcMin/+ mice harboring a germline mutation in one allele of the Apc (adenomatous polyposis coli) gene has been successfully used to study mammary tumorigenesis after IR exposure and is considered as a suitable surrogate model of human breast cancer, specifically for studying IR-induced mammary tumor initiation, progression, and associated molecular signaling events [18,19,20].
In women, activation of estrogen receptor-alpha (ERα), encoded by the Esr1 gene, plays a crucial role in the initiation, development, and progression of breast cancer [21,22,23]. Similarly, studies using IR-exposed animal models have demonstrated a persistent estrogenic response (PER), characterized by elevated systemic estrogen levels and activation of ERα in mammary epithelial cells [19,24,25]. This response coincides with upregulation of oncogenic markers and an increased frequency of hormone receptor-positive tumors [19]. In addition to PER activation, female ApcMin/+ mice exposed to GCRsim have shown increased accumulation of secreted phosphoprotein 1 (SPP1, also known as osteopontin or OPN) in the ductal epithelium, which serves as a preneoplastic marker for breast cancer risk [20]. Notably, SPP1 expression in mammary epithelial cells has been implicated in mammary cancer initiation, and elevated plasma levels of SPP1 protein are associated with increased tumor burden [26,27,28]. However, the molecular links between IR-induced PER signaling and SPP1 are not yet well understood.
In addition to ERα, estrogen-related receptor alpha (ERRα), encoded by the Esrra gene, is another critical factor in both ERα positive and negative in breast cancers [29,30,31]. ERRα is frequently overexpressed in ER-positive human breast cancers and is associated with poor prognosis, contributing to increased tumor aggressiveness [32,33]. Due to the structural similarity in the DNA binding domain of ERRα and ERα, they are likely to compete and modulate the expression of a similar set of genes implicated in breast cancer development [34,35]. Furthermore, activation of both ERα and ERRα has been implicated in the overexpression of SPP1 [36,37,38,39,40]. Therefore, understanding GCRsim-induced alterations in the signaling axis involving ERα, ERRα, and SPP1 is crucial for elucidating the molecular events associated with GCRsim-induced mammary cancer risk.
In this ground-based mouse model study, we exposed female ApcMin/+ mice to GCRsim at NASA’s Space Radiation Laboratory (NSRL) and focused on understanding the roles of PER, ERRα, and SPP1 signaling in GCRsim-exposed ApcMin/+ mice to elucidate their contributions to breast cancer development. GCRsim exposure was associated with a PER characterized by elevated serum estradiol levels and increased activation of ERα, along with enhanced expression of downstream target genes in mammary tissues. Additionally, ERRα expression was elevated, and there was an accumulation of the preneoplasia marker SPP1 in GCRsim-exposed mice, indicating a synergistic effect of both receptors on mammary cancer progression. Notably, the overexpression of ERα, ERRα, and SPP1 was further corroborated by their presence in human breast cancer tissues, suggesting a conserved mechanism across species. This study aims to elucidate the molecular mechanisms underlying GCRsim-induced breast cancer risk, providing insights that could inform future pharmacological investigations to safeguard female astronauts during long-duration space missions

2. Materials and Methods

2.1. Mice and Radiation Exposure

Male ApcMin/+ mice in C57BL6 background were bred with wild-type female C57BL6 mice at the Georgetown University (GU) animal facility, and all female pups were genotyped to identify ApcMin/+ mice, as described on the Jackson Laboratory website (https://www.jax.org/Protocol?stockNumber=002020&protocolID=529, accessed on 1 October 2015). The female ApcMin/+ mice were randomly assigned to the respective experimental groups and were transported to the Brookhaven National Laboratory (BNL, Upton, NY, USA) animal facility using an approved laboratory animal courier service. Following one week of acclimatization, at the age of 8 to 10 weeks, mice were either sham irradiated or irradiated to 50 cGy of chronic GCRsim beam (Figure 1A). The 4-week irradiation period (at 2.08 cGy/day, 6 days a week, delivering a total dose of 50 cGy) in mice corresponds to 2–3 years of human age, aligning with the estimated duration of a manned Mars mission. This irradiation protocol effectively models the chronic, low-dose exposure astronauts would experience over an extended period. Although condensed into 4 weeks, the dose rate and cumulative dose are biologically relevant, providing an experimental timeline that simulates the long-term, low-dose radiation conditions of space while ensuring feasibility for animal studies. To closely mimic an “actual-GCR”-like scenario and dose rates, we irradiated animals to the full-spectrum GCRsim developed by NSRL at BNL that consists of seven different ion species (1H, 2He, 6C, 16O, 28Si, 44Ti, and 26Fe) and a wide energy spectrum (20–1000 MeV/n) [10,11,41]. Both at GU and BNL facilities, all animals were group-housed (5 per cage) and maintained with standard laboratory conditions including specific pathogen-free (SPF) environment and 12 h:12 h shift of light–dark cycles with easy access to water and food. All experiments were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) approved protocol # 2019-0070 at GU and #515 at BNL.

2.2. Biospecimen Collection, Tumor Counting, and Histological Assessments

The mice were euthanized using carbon dioxide (CO2) asphyxiation at 100 to 110 days post-irradiation and surgically dissected to reveal the mammary fat pads, and the macroscopic mammary tumors were quantified. Other pairs of mammary tissues and serum were flash-frozen and stored at −80 °C for further studies. Samples of normal-appearing mammary tissues and tumors were preserved in 10% buffered formalin for 24 h and then transferred to 70% ethanol prior to paraffin embedding and sectioning. Hematoxylin and eosin (H&E) staining was performed on formalin-fixed, paraffin-embedded (FFPE) mouse mammary gland sections of 5 μm thickness. The sections were deparaffinized in xylene, rehydrated through a graded ethanol series, and stained with hematoxylin for nuclear visualization, followed by eosin for cytoplasmic staining. Finally, digital images of the H&E-stained tissue sections were obtained using a bright-field microscope (Olympus BX63, Olympus America Inc., Center Valley, PA, USA), and the number of ducts in a given microscopic field were visually quantified at 200X magnification.

2.3. Mammary Gland Whole Mount Staining

Freshly isolated normal appearing mammary glands (i.e., mammary fat pad without any visible tumor node under a dissecting scope) were carefully spread and mounted on a microscope slide, and whole-mount staining was performed using the VitroView™ Mammary Gland Whole Mount Stain Kit (VB-3001, VitroVivo Biotech, Rockville, MD, USA), according to the manufacturer’s instructions. Briefly, the mammary glands were fixed in Carnoy’s fixative for 2 h, followed by a wash in 70% ethanol. The tissues were then rehydrated through graded ethanol concentrations, including 50% and 30% ethanol, and subsequently rinsed in distilled water. Staining was performed using carmine alum staining solution, with tissues incubated overnight at room temperature. The next day, the tissues were subjected to a series of dehydration steps using graded ethanol concentrations (70%, 90%, and 100%), followed by clearing in xylene. Finally, the tissues were mounted using Permount mounting medium (SP15-100, Fisher Chemical, Frederick, MD, USA), and digital images were obtained using a bright-field microscope followed by visual assessments of ductal morphology in irradiated and non-irradiated mammary gland tissues.

2.4. Serum Immunoassays

SPP1 and estradiol levels in serum samples were determined using immunoassay kits following the manufacturer’s instructions. SPP1 concentrations in 50-fold diluted serum samples from both control and chronic GCRsim-irradiated mice were quantified by using RayBio® Mouse Osteopontin (SPP1) ELISA Kit (ELM-OPN; RayBiotech, Peachtree Corners, GA, USA). The Mouse SPP1 ELISA had a minimum detectable concentration of 4 pg/mL. Estradiol levels were measured in 2-fold diluted serum samples using the RayBio® Human/Mouse/Rat Estradiol EIA Kit (EIAM-E2; RayBiotech). The estradiol EIA standard curve range was 0.1–1000 ng/mL, with a minimum detectable concentration of 1.5 ng/mL.

2.5. Immunohistochemistry and Image Quantification

Protein expression of hormone receptors (ERα and ERRα) and down downstream targets (Cyclin D1) were analyzed using immunohistochemistry. Formalin-fixed paraffin-embedded (FFPE) mammary tissue sections were deparaffinized and rehydrated, followed by a thermal antigen retrieval step using either citrate buffer (pH 6.0) (SKU:64142-08; Electron Microscopy Sciences, Hatfield, PA, USA) or Tris EDTA buffer (pH 9.0) (10-0037; Torrance, CA, USA). Tissue sections were incubated overnight in a humidified chamber at 4 °C with primary antibodies, ERα (ab32063; dilution 1:200; Abcam, Boston, MA, USA), ERRα (ab137489; dilution 1:100; Abcam, Boston, MA, USA), SPP1 (SC-21742; Santa Cruz, Dallas, TX, USA) and Cyclin D1 (MA5-14512; dilution 1:100; Invitrogen, Waltham, MA, USA). Finally, immunohistochemical signals were detected using a Mouse and Rabbit Specific HRP/DAB IHC Detection Kit (ab236466, Abcam, Boston, MA, USA). Further, sections were counter-stained with hematoxylin (SKU: 26043-06; Electron Microscopy Sciences), dehydrated and mounted using Permount mounting medium (SP15-100, Fisher Chemical, Frederick, MD, USA). Digital images (10–12 per group) were acquired and saved in TIFF format using cellSens Entry v1.15 software (Olympus, Center Valley, PA, USA). Quantification of IHC stained sections was performed using Fiji (ImageJ2) software v2.9.0/1.53t, either by counting the number of positively stained nuclei per high power field (HPF) or by measuring the diaminobenzidine (DAB) chromogen signal intensity within the defined region of interest (ROI) [42].

2.6. mRNA Expression Analysis

Total RNA was isolated from the flash-frozen mammary tissues by using Qiagen RNeasy mini kit (74104; Qiagen, Germantown, MD, USA). RNA purity was measured by the A260 nm/A280 nm ratio using a NanoDrop spectrophotometer (ND-1000, Thermo Scientific, Waltham, MA, USA). Next, 2 μg of RNA was reverse-transcribed into cDNA using iScript cDNA Synthesis Kit (1708891; Bio-rad, Hercules, CA, USA), according to the manufacturer’s instructions. Finally, quantitative real-time RT-PCR (qPCR) was performed to assess mRNA expression of Esr1, Esrra, Ccnd1, cMyc, and Nrip1 genes using SsoAdvanced Universal SYBR Green Supermix (Cat # 1725271, Bio-Rad, Hercules, CA, USA) on a real-time PCR system (CFX96; Bio-rad, Hercules, CA, USA) as per the manufacturer’s instructions. PrimeTime qPCR premixed primer assays for all genes (Supplementary Table S1) were obtained from Integrated DNA Technology (www.idtdna.com, accessed on 21 September 2022). All predesigned primeTime qPCR primer sets are reported to achieve >90% efficiency. Amplification specificity for each PCR reaction was verified using a melting curve analysis and mRNA expression was analyzed by the comparative Cq method, normalized using Polr2a as a reference housekeeping gene.

2.7. Tissue Microarray Analysis of ERRα and SPP1 Protein Expression

TMA slides including normal human breast tissues and carcinoma tissues were purchased from Novus Biologicals (NBP2-78114; Centennial, CO, USA). These slides were processed for immunohistochemistry using anti-SPP1 and anti-ERRα antibodies as described above. Once prepared, the slides were scanned using an Aperio GT450 v1.3 whole slide scanner (Leica Biosystems, Dear Park, IL, USA) at a 40X magnification and high-quality digital images were used to identify individual tissue cores on the TMA slides using the Qpath 0.4.3 software [43].

2.8. Co-Expression Analysis of Esr1, Esrra, and Spp1 Genes in Human Breast Cancer

The co-expression of the Esr1, Esrra, and Spp1 genes in human breast cancer was investigated using RNA sequencing data from The Cancer Genome Atlas (TCGA). Specifically, Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values were utilized to assess gene expression levels across a total of 1075 human breast cancer samples. The FPKM data for each gene were obtained from the Human Protein Atlas (https://www.proteinatlas.org, accessed on 2 December 2023). First, we calculated the average FPKM for each gene across all samples and classified samples based on the expression threshold, i.e., samples exhibiting FPKM values less than 25% of the calculated average were categorized as negative for that gene, while those with FPKM values exceeding 25% of the average were classified as positive. Subsequently, co-expression analyses were performed for each gene pair, specifically Esr1–Esrra, Esr1–Spp1, and Esrra–Spp1. The percentage of positive and negative tumors for each gene combination was computed. These results were expressed as a percentage of the total number of tumors analyzed, facilitating a comprehensive understanding of the relationships between gene expressions in breast cancer samples. Data were presented as proportions of positive and negative classifications to allow for straightforward interpretation of gene co-expression patterns within the context of human breast cancer.

2.9. Statistical Analysis

Statistical analysis was conducted using GraphPad Prism software v6.0a for Mac (La Jolla, CA, USA). Non-parametric analysis was used to determine equality of variance for the quantitative analysis of tumor number. The incidence of mammary tumors between the control group and the group exposed to GCRsim was compared using a relative risk analysis. A two-tailed p-value of less than 0.05 was considered statistically significant. The confidence interval (CI) for the relative risk was calculated to estimate the precision of the effect size. Moreover, the IHC and qPCR data from control and irradiated mice were evaluated for statistical significance using a two-tailed paired student t-test. Results are presented as mean ± standard error of the mean (SEM), with a p-value < 0.05 considered statistically significant.

3. Results

3.1. Increased Ductal Overgrowth and Mammary Tumorigenesis After GCRsim

We analyzed the established histological and molecular markers of mammary preneoplastic lesions in normal-appearing mammary gland samples from both sham-treated and GCRsim-exposed ApcMin/+ mice (Figure 1A). To investigate GCRsim-induced changes in the mammary tissue, we performed whole-mount staining and H&E staining. Whole-mount analysis of mammary gland ductal morphology revealed increased ductal outgrowth in GCRsim-exposed ApcMin/+ mice compared to the control group (Figure 1B). In line with this observation, histological analysis of H&E-stained mammary tissue showed a significantly higher number of ducts per microscopic field in GCRsim-exposed ApcMin/+ mice compared to controls (Figure 1B,C). Additionally, the incidence of mammary tumors significantly increased in GCRsim-exposed mice (Figure 1D,E). In the control group (n = 40), the incidence of mammary tumors was 5%, while in the GCRsim group (n = 25), the incidence increased to 24%. The relative risk of developing mammary tumors in the GCRsim group compared to the control group was 4.8, with a 95% confidence interval (CI) ranging from 1.05 to 21.95, indicating a statistically significant increase in mammary tumor development risk (p = 0.043). This suggests that exposure to GCRsim is associated with increased ductal cell proliferation accompanied with a significantly higher risk of mammary tumorigenesis in ApcMin/+ mice.

3.2. GCRsim-Exposure Induces Activation of ERα and Downstream Target Genes

To determine whether GCRsim-exposure can alter estrogen and associated ERα signaling, we first analyzed serum estradiol concentrations in the GCRsim irradiated mice at 100–110 days post radiation, which was ~1.4-fold higher than in the unirradiated control groups (Figure 2A). Concurrent to systemic increase in estradiol, increased protein expression accompanied with enhanced nuclear localization of ERα and Cyclin D1 in mammary tissues was also noted using immunohistochemical staining in the GCRsim group at 100–110 days post radiation (Figure 2B). Quantification of the immunohistochemical results exhibited significantly increased ERα (Figure 2C) and Cyclin D1 (Figure 2D) positive nuclei in GCRsim-exposed mice in comparison with controls, suggesting GCRsim-induced activation of ERα signaling. Further, we looked into the mRNA expression level of ERα downstream genes, i.e., Ccnd1 and cMyc in mammary tissues, and found a significantly higher expression of Ccnd1 (Figure 2E) and cMyc (Figure 2F) in the GCRsim-irradiated group compared to the respective controls. These results indicate that GCRsim exposure leads to a systemic increase in estradiol levels and activation of ERα signaling in mammary tissues, potentially driving the expression of oncogenic targets such as Cyclin D1 and cMyc.

3.3. GCRsim-Exposure Induces Activation of ERRα and Downstream Target Genes

Higher estrogen level is known to activate ERRα directly as well as through ERα-mediated transcriptional activation of Esrra gene [38,44,45,46,47,48,49]. In order to relate ERRα with activation of GCR-induced estrogen signaling, we noted a significant increase in mRNA levels of Esrra in the GCRsim-irradiated group compared to the controls (Figure 3A). Further, immunohistochemical analysis of ERRα in the mammary tissues of GCRsim-irradiated mice also showed increased nuclear staining, relative to the control group (Figure 3B,C). Additionally, mRNA expression of ERRα downstream transcriptional targets, i.e., Spp1 (Figure 3D) and Nrip1 (Figure 3E), was also significantly increased in the GCRsim-irradiated group compared to the control group mice. Next, we looked into the SPP1 protein expression in the serum, and we found a significantly increased level of SPP1 approximately 1.38-fold higher than in the unirradiated control groups (Figure 4A). In addition to overexpression of SPP1 in serum, protein expression of SPP1 was also enhanced in GCRsim-irradiated mice mammary ductal epithelial cells (Figure 4B,C). These findings suggest that GCRsim irradiation enhances ERRα activity and its downstream signaling in mammary tissues, potentially contributing to radiation-induced tumorigenesis through estrogen-related pathways.

3.4. ERα, ERRα and SPP1 Protein Expression in Mouse and Human Mammary Tumors

We investigated the expression pattern of ERα, ERRα and SPP1 in the tumor tissues of both GCRsim exposed and control mice. Notably, a similar pattern in the expression of ERα, ERRα and SPP1 was observed, as they were all overexpressed in the GCRsim-exposed tumors compared to the tumors from control mice (Figure 5A–D). Further, we attempted to ascertain the expression of these protein markers in human breast cancer tissues to test the relevance of these GCRsim-induced proteins in human breast cancer. Interestingly, using serial normal and tumor tissue sections, we found that both ERRα and SPP1 concurrently overexpressed in the different breast cancer tissues including invasive lobular carcinoma, fibroadenoma, and invasive ductal carcinoma compared to the normal breast tissues (Figure 6). Notably, ERRα was localized to the nucleus and SPP1 expression was generally cytoplasmic with a higher likelihood of co-expression in tumor samples than in the normal breast tissue. These findings suggest that the overexpression of ERRα and SPP1 in GCRsim-induced tumors mirrors their elevated expression in human breast cancers, highlighting their potential relevance as biomarkers or therapeutic targets in radiation-induced and spontaneous breast tumorigenesis.

3.5. Co-Expression of Spp1 in ERα and ERRα Positive Human Breast Cancer

The protein ERα is a known transcriptional regulator of Esrra and Spp1 genes [36,37,38,39,40]. Additionally, ERRα is also a transcriptional regulator of the Spp1 gene [38,44,45,46,47,48,49] (Table S2). To understand the crosstalk between expression pattens of Spp1 upstream factors, i.e., ERα and ERRα in breast cancer, analysis of RNAseq data from a total of 1075 human breast cancer samples revealed a higher co-expression of Esrra and Spp1 (70.4% of total tumors) compared to relatively lower co-expression of Esr1 and Spp1 (48.4% of total tumors). This indicates that upregulation/activation of both ERα and ERRα is important for SPP1 positive breast cancer cells (Figure 7). These results suggest that the co-activation of both ERα and ERRα plays a crucial role in regulating SPP1 expression in breast cancer, with ERRα potentially being a more dominant regulator in SPP1-positive tumors in both mouse and human mammary tumors (Figure 5, Figure 6 and Figure 7).

4. Discussion

Our findings demonstrate increased ductal outgrowth and duct density, along with a higher incidence of mammary tumors following GCRsim exposure. Further investigation into estrogenic responses revealed that GCRsim exposure led to a sustained elevation in serum estradiol levels, coupled with enhanced activation of ERα and its downstream targets, which are known to promote cell proliferation in mammary ductal epithelial cells. In parallel with increased ERα activation, we observed significant overexpression and nuclear localization of ERRα in GCRsim-exposed mouse mammary tissues. Moreover, elevated expression of its downstream target, Spp1 gene and protein, supports the involvement of ERRα in GCRsim-induced mammary tumorigenesis. Notably, the concurrent upregulation of ERα, ERRα, and SPP1 suggests a crosstalk between estrogen signaling and inflammatory pathways mediated by SPP1 in the context of GCRsim exposure [28,31,38,47,48,49]. Collectively, these results suggest that GCRsim exposure induces a sustained estrogenic response, promoting both preneoplastic and neoplastic changes involving ERα, ERRα, and SPP1 signaling axis (Figure 8).
A high level of bioavailable estradiol is a risk factor for breast cancer development [50,51]. It has been reported that sublethal IR exposure enhances the aberrant proliferation of ductal epithelial cells, and risk of ER-positive mammary tumor development by activating PER signaling in both animal models and atomic bomb survivors [19,50,52,53]. Our results show a significant increase in serum estradiol concentrations and ERα protein expression, and nuclear localization in GCRsim-irradiated mice compared to controls. The concurrent increase in downstream target genes, like Ccnd1 and cMyc, further underscores the activation of ERα signaling. This observation is consistent with prior findings that radiation exposure enhances ER-positive tumor formation [19]. These findings align with the role of estrogen signaling in breast cancer progression by activating genes such as Ccnd1 and cMyc [54,55,56].
The DNA-binding domains of ERRα and ERα display ~70% homology but only 36% similarity in ligand-binding domain, and therefore, direct activation of ERRα by estradiol is not expected, and estrogen response is likely to be primarily through ERα activation [57,58]. Notably, using chromatin immunoprecipitation assay, the interaction between ERα and multi-hormone response elements (MHREs) present in the ERRα gene promoter region has been demonstrated earlier, and estrogen is also known to augment the association of ERα and MHREs in vivo [44]. Therefore, binding of ERα on ERRα MHRE in response to higher estradiol bioavailability is likely to co-upregulate both ERα and ERRα in response to GCRsim exposure. ERRα is known to regulate SPP1 expression via a non-canonical ERRα response element, and this regulation is dependent on the specific cellular context [39]. SPP1 overexpression has been implicated in mammary cancer development [27], and our study corroborates these findings by showing enhanced SPP1 protein levels in both serum and mammary ductal epithelial cells of GCRsim-exposed mice. Interestingly, SPP1 has been associated with tumor burden and reduced survival in breast cancer patients [59].
In concurrence to activation of ERα, ERRα, and SPP1 signaling axis in GCRsim-induced tumors, using TMA, we noted elevated expression of ERRα and SPP1 in human breast cancer tissues, including invasive lobular carcinoma and invasive ductal carcinoma. This co-expression of SPP1 and ERRα, particularly in ERα and ERRα-positive breast cancer cells, suggests that upregulation of ERRα is critical for SPP1-positive preneoplastic and neoplastic cells [27,60]. Furthermore, the higher co-expression of Esr1, Esrra and Spp1 mRNA in human breast cancers emphasizes the importance of ERRα in conjunction with ERα in driving SPP1 expression and, potentially, breast cancer progression. Notably, ERRα inhibitors have also shown potential in breast cancer risk prevention and mitigation through disruption of cancer cell metabolism, proliferation, and tumor growth by interfering with genes associated with oncogenic processes [39,61,62]. Therefore, further studies are required to test and validate the efficacy of ERRα inhibitors in breast cancer prevention after GCRsim exposure to safeguard female astronauts.
Overall, our findings provide novel insights into the effects of GCRsim on mammary gland biology, emphasizing the role of estrogenic and inflammatory signaling in mediating GCRsim-induced preneoplastic and neoplastic changes. The increased ductal outgrowth and tumor incidence observed in the ApcMin/+ mice suggest that GCRsim exposure leads to alterations in mammary gland architecture and microenvironment, promoting neoplastic transformation. The persistent estrogenic response, activation of ERα and ERRα, and overexpression of SPP1 following GCRsim exposure indicate a complex interplay between hormonal signaling, inflammation, and cancer development. Future studies should aim to dissect the exact molecular mechanisms linking radiation exposure to hormonal dysregulation and inflammatory responses. Additionally, it would be valuable to evaluate the potential of pharmacological interventions, such as selective estrogen receptor modulators (SERMs), estrogen receptor degraders, and aromatase inhibitors, to mitigate estrogen-related mammary tumorigenesis after GCRsim exposure [63,64]. These insights have important implications for understanding breast cancer risk in individuals exposed to space radiation and may help in developing strategies to mitigate these risks.

5. Conclusions

In conclusion, our study demonstrates that GCRsim exposure induces ductal overgrowth, increased duct density, and a heightened incidence of mammary tumors in ApcMin/+ mice. Chronic increase in estradiol levels and the concurrent activation of ERα–ERRα signaling in GCRsim-irradiated mice highlight the influence of estrogenic responses in promoting mammary ductal epithelial cell proliferation. Additionally, ERRα expression, particularly through its regulatory effects on SPP1, underscores the potential importance of estrogen and inflammatory crosstalk in IR-induced breast cancer. Our analysis of human breast cancer samples revealed elevated expression of ERRα and SPP1, mirroring the findings in GCRsim-exposed mammary tissues and suggesting the relevance of these markers in both IR-induced and spontaneous breast cancers. The co-expression of ERα and ERRα, alongside SPP1 upregulation, points to a complex interplay of hormonal and inflammatory signaling that could underlie mammary carcinogenesis following radiation exposure. Overall, this study provides insights into the effects of GCRsim on mammary tissue biology, with implications for breast cancer risk after space radiation. Future research should focus on the detailed mechanisms linking radiation to hormonal dysregulation and inflammatory responses and evaluate the efficacy of pharmacological interventions, such as anti-estrogens or ERRα inhibitors, in mitigating GCRsim-induced carcinogenesis. These findings could contribute to protective strategies for astronauts and others at risk of radiation exposure, ultimately helping to reduce breast cancer incidence.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cancers16233954/s1. Table S1: ERα and ERRα downstream target genes identified using TFLink database; and Table S2: Sequence details of qPCR primers.

Author Contributions

Conceptualization, S.S. and K.D.; methodology, K.K. and S.S.; formal analysis, S.S. and K.K.; investigation, K.K., J.A. and J.M.; resources, S.S. and A.J.F.J.; data curation, S.S. and K.K.; writing—original draft preparation, S.S.; writing—review and editing, S.S., K.K., A.J.F.J. and K.D.; visualization, S.S.; supervision, S.S.; project administration, S.S. and A.J.F.J.; funding acquisition, S.S., K.D. and A.J.F.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Aeronautics and Space Administration (NASA), grant number 80NSSC19K1649 and 80NSSC24K0287.

Institutional Review Board Statement

This study was conducted in accordance with approval from the Institutional Animal Care and Use Committee (IACUC) at Georgetown University (protocol code 2019-0070 and date of approval: 11 March 2020) and Brookhaven National Lab (protocol code 515 and date of approval: 7 January 2020).

Informed Consent Statement

No human studies were conducted. The TMA used in this study is commercially available.

Data Availability Statement

All relevant data have been included in this manuscript and supplementary information.

Acknowledgments

We extend our sincere gratitude to Pelagie Ake for her invaluable support in animal care. We also greatly appreciate the assistance provided by Peter Guida, as well as all the scientists, physicists, and support staff at the NASA Space Radiation Laboratory (NSRL).

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Figure 1. GCRsim exposure promotes mammary tissue overgrowth and increased duct density. (A) Schematic representation of the experimental setup depicting chronic exposure to GCRsim, using a 33-ion mixed beam to simulate the deep-space environment. Experimental animals were exposed to 2.08 cGy per day, 6 days per week, for 4 weeks, resulting in a cumulative dose of 50 cGy. (B) Representative images of mammary tissues: whole-mount micrographs (scale bar = 500 µm) show the extent of ductal branching, while H&E-stained sections (scale bar = 100 µm) highlight histological features. (C) Quantification of ductal density, expressed as the number of ducts per high-power microscopic field (HPF). (D) Representative H&E-stained normal mammary gland and tumor samples. (E) Comparison of mammary tumor incidence between control and GCRsim-exposed animals, showing increased tumor formation in the 50 cGy irradiated GCRsim group. Statistically significant difference (p < 0.05) relative to the control group is denoted by an asterisk (*).
Figure 1. GCRsim exposure promotes mammary tissue overgrowth and increased duct density. (A) Schematic representation of the experimental setup depicting chronic exposure to GCRsim, using a 33-ion mixed beam to simulate the deep-space environment. Experimental animals were exposed to 2.08 cGy per day, 6 days per week, for 4 weeks, resulting in a cumulative dose of 50 cGy. (B) Representative images of mammary tissues: whole-mount micrographs (scale bar = 500 µm) show the extent of ductal branching, while H&E-stained sections (scale bar = 100 µm) highlight histological features. (C) Quantification of ductal density, expressed as the number of ducts per high-power microscopic field (HPF). (D) Representative H&E-stained normal mammary gland and tumor samples. (E) Comparison of mammary tumor incidence between control and GCRsim-exposed animals, showing increased tumor formation in the 50 cGy irradiated GCRsim group. Statistically significant difference (p < 0.05) relative to the control group is denoted by an asterisk (*).
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Figure 2. Chronic GCRsim exposure increases serum estradiol levels and enhances estrogen receptor signaling and proliferation markers in mammary tissue. (A) Serum estradiol levels expressed as fold change (FC) relative to control animals, demonstrating increased estradiol in GCRsim-exposed mice. (B) Representative photomicrographs illustrating increased immunohistochemical staining for estrogen receptor alpha (ERα) and Cyclin D1 in mammary tissues of GCRsim-exposed animals (scale bar = 50 µm). (C) Quantification of ERα-positive nuclei in mammary tissue, showing increased receptor expression in the GCRsim group. (D) Quantification of Cyclin D1-positive nuclei, indicating increased cellular proliferation in response to GCRsim exposure. (E) Fold change in mRNA expression of ERα downstream target gene Cyclin D1 (or Ccnd1), demonstrating activation of estrogen signaling. (F) Fold change in mRNA expression of ERα downstream target gene cMyc, indicating enhanced proliferative signaling. Statistically significant difference (p < 0.05) relative to the control group is indicated by an asterisk (*).
Figure 2. Chronic GCRsim exposure increases serum estradiol levels and enhances estrogen receptor signaling and proliferation markers in mammary tissue. (A) Serum estradiol levels expressed as fold change (FC) relative to control animals, demonstrating increased estradiol in GCRsim-exposed mice. (B) Representative photomicrographs illustrating increased immunohistochemical staining for estrogen receptor alpha (ERα) and Cyclin D1 in mammary tissues of GCRsim-exposed animals (scale bar = 50 µm). (C) Quantification of ERα-positive nuclei in mammary tissue, showing increased receptor expression in the GCRsim group. (D) Quantification of Cyclin D1-positive nuclei, indicating increased cellular proliferation in response to GCRsim exposure. (E) Fold change in mRNA expression of ERα downstream target gene Cyclin D1 (or Ccnd1), demonstrating activation of estrogen signaling. (F) Fold change in mRNA expression of ERα downstream target gene cMyc, indicating enhanced proliferative signaling. Statistically significant difference (p < 0.05) relative to the control group is indicated by an asterisk (*).
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Figure 3. Chronic GCRsim exposure elevates mRNA and protein levels of estrogen-related receptor alpha (ERRα). (A) Fold change in mRNA expression of ERRα (or Esrra) in mammary tissue of control and GCRsim-exposed mice, showing elevated expression in response to GCRsim exposure. (B) Representative photomicrographs of ERRα protein expression in the mammary gland, demonstrating increased levels in GCRsim-exposed mice (scale bar = 50 µm). (C) Quantification of ERRα-positive cells per high-power microscopic field (HPF), indicating increased receptor expression in GCRsim-exposed animals. (D) Gene expression analysis showing activation of ERRα downstream target Spp1, expressed as fold change relative to control. (E) Gene expression analysis showing activation of ERRα downstream target Nrip1, expressed as fold change relative to control. Statistically significant change (p < 0.05) relative to the control group is denoted by an asterisk (*).
Figure 3. Chronic GCRsim exposure elevates mRNA and protein levels of estrogen-related receptor alpha (ERRα). (A) Fold change in mRNA expression of ERRα (or Esrra) in mammary tissue of control and GCRsim-exposed mice, showing elevated expression in response to GCRsim exposure. (B) Representative photomicrographs of ERRα protein expression in the mammary gland, demonstrating increased levels in GCRsim-exposed mice (scale bar = 50 µm). (C) Quantification of ERRα-positive cells per high-power microscopic field (HPF), indicating increased receptor expression in GCRsim-exposed animals. (D) Gene expression analysis showing activation of ERRα downstream target Spp1, expressed as fold change relative to control. (E) Gene expression analysis showing activation of ERRα downstream target Nrip1, expressed as fold change relative to control. Statistically significant change (p < 0.05) relative to the control group is denoted by an asterisk (*).
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Figure 4. GCRsim exposure elevates SPP1 expression at both systemic and tissue levels. (A) Serum levels of SPP1 expressed as fold change relative to the control group, indicating increased levels in GCRsim-exposed mice. (B) Representative photomicrographs of mammary gland tissue showing increased SPP1 expression in GCRsim-exposed animals compared to controls (scale bar = 50 µm). (C) Quantification of SPP1 signal intensity in mammary tissue, demonstrating enhanced expression in response to GCRsim exposure. Statistically significant change (p < 0.05) relative to the control group is denoted by an asterisk (*).
Figure 4. GCRsim exposure elevates SPP1 expression at both systemic and tissue levels. (A) Serum levels of SPP1 expressed as fold change relative to the control group, indicating increased levels in GCRsim-exposed mice. (B) Representative photomicrographs of mammary gland tissue showing increased SPP1 expression in GCRsim-exposed animals compared to controls (scale bar = 50 µm). (C) Quantification of SPP1 signal intensity in mammary tissue, demonstrating enhanced expression in response to GCRsim exposure. Statistically significant change (p < 0.05) relative to the control group is denoted by an asterisk (*).
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Figure 5. Increased ERα, ERRα, and SPP1 protein expression in GCRsim-induced tumors from ApcMin/+ mice compared to controls. (A) Representative immunohistochemically (IHC) stained images of mammary tissue showing the expression of ERα, ERRα, and SPP1 in both control and GCRsim-induced tumor samples (scale bar = 50 µm). (B) Increased tissue expression of ERα in GCRsim-exposed ApcMin/+ tumors compared to controls. (C) Elevated ERRα protein expression in GCRsim-induced tumors compared to controls. (D) Enhanced SPP1 expression in GCRsim-exposed tumors relative to control tumors. Statistically significant changes (p < 0.05) are denoted by an asterisk (*). AU: Arbitrary unit; ROI: Region of interest.
Figure 5. Increased ERα, ERRα, and SPP1 protein expression in GCRsim-induced tumors from ApcMin/+ mice compared to controls. (A) Representative immunohistochemically (IHC) stained images of mammary tissue showing the expression of ERα, ERRα, and SPP1 in both control and GCRsim-induced tumor samples (scale bar = 50 µm). (B) Increased tissue expression of ERα in GCRsim-exposed ApcMin/+ tumors compared to controls. (C) Elevated ERRα protein expression in GCRsim-induced tumors compared to controls. (D) Enhanced SPP1 expression in GCRsim-exposed tumors relative to control tumors. Statistically significant changes (p < 0.05) are denoted by an asterisk (*). AU: Arbitrary unit; ROI: Region of interest.
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Figure 6. Tissue Microarray (TMA) analysis of ERRα and SPP1 protein expression in normal and malignant human breast tissues. Expression of ERRα and SPP1 in different types of breast tissue, including normal tissue, invasive lobular carcinoma (ILC), fibroadenoma (FA), and invasive ductal carcinoma (IDC). Upper panels show representative images of tissue microarray (TMA) cores captured using an Aperio whole-slide digital scanner while lower panels show magnified views (marked by blue color box in the respective upper panel) of selected regions with increased expression (scale bar = 100 µm). Comparisons are provided for normal breast tissue and various tumor types to demonstrate a correlation in ERRα and SPP1 expressions.
Figure 6. Tissue Microarray (TMA) analysis of ERRα and SPP1 protein expression in normal and malignant human breast tissues. Expression of ERRα and SPP1 in different types of breast tissue, including normal tissue, invasive lobular carcinoma (ILC), fibroadenoma (FA), and invasive ductal carcinoma (IDC). Upper panels show representative images of tissue microarray (TMA) cores captured using an Aperio whole-slide digital scanner while lower panels show magnified views (marked by blue color box in the respective upper panel) of selected regions with increased expression (scale bar = 100 µm). Comparisons are provided for normal breast tissue and various tumor types to demonstrate a correlation in ERRα and SPP1 expressions.
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Figure 7. Co-expression analysis of Esr1, Esrra, and Spp1 genes in human breast cancer using RNA-Seq data from The Cancer Genome Atlas (TCGA). Presented bar graph illustrates the co-expression patterns of the genes Esr1, Esrra, and Spp1 in human breast cancer samples, analyzed using RNA sequencing (RNA-Seq) data from TCGA. Samples were categorized based on Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values as negative (i.e., samples with FPKM values in the lowest quartile, specifically Esr1 FPKM ≤ 10.1, Esrra FPKM ≤ 2.45, and Spp1 FPKM ≤ 30.22) and positive (i.e., samples with FPKM values above the 25th percentile cutoff). This analysis provides insight into how these genes are co-expressed and their relationship in the context of human breast cancer progression.
Figure 7. Co-expression analysis of Esr1, Esrra, and Spp1 genes in human breast cancer using RNA-Seq data from The Cancer Genome Atlas (TCGA). Presented bar graph illustrates the co-expression patterns of the genes Esr1, Esrra, and Spp1 in human breast cancer samples, analyzed using RNA sequencing (RNA-Seq) data from TCGA. Samples were categorized based on Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values as negative (i.e., samples with FPKM values in the lowest quartile, specifically Esr1 FPKM ≤ 10.1, Esrra FPKM ≤ 2.45, and Spp1 FPKM ≤ 30.22) and positive (i.e., samples with FPKM values above the 25th percentile cutoff). This analysis provides insight into how these genes are co-expressed and their relationship in the context of human breast cancer progression.
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Figure 8. Schematic representation of putative molecular pathways linking GCRsim-induced estrogen response to increased breast cancer risk. GCRsim exposure results in: elevated estrogen levels, enhancing estrogen signaling in breast tissues involving activation of ERα, ERRα, and proliferation markers such as Cyclin D1 and cMyc. Both ERα and ERRα could induce overexpression of SPP1 at both systemic and tissue levels, ultimately contributing to enhanced breast cancer risk. Arrows in the schematic indicate the putative molecular connections between GCRsim-induced changes, receptor upregulation, proliferative signaling, and tumor development.
Figure 8. Schematic representation of putative molecular pathways linking GCRsim-induced estrogen response to increased breast cancer risk. GCRsim exposure results in: elevated estrogen levels, enhancing estrogen signaling in breast tissues involving activation of ERα, ERRα, and proliferation markers such as Cyclin D1 and cMyc. Both ERα and ERRα could induce overexpression of SPP1 at both systemic and tissue levels, ultimately contributing to enhanced breast cancer risk. Arrows in the schematic indicate the putative molecular connections between GCRsim-induced changes, receptor upregulation, proliferative signaling, and tumor development.
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MDPI and ACS Style

Kumar, K.; Angdisen, J.; Ma, J.; Datta, K.; Fornace, A.J., Jr.; Suman, S. Simulated Galactic Cosmic Radiation Exposure-Induced Mammary Tumorigenesis in ApcMin/+ Mice Coincides with Activation of ERα-ERRα-SPP1 Signaling Axis. Cancers 2024, 16, 3954. https://doi.org/10.3390/cancers16233954

AMA Style

Kumar K, Angdisen J, Ma J, Datta K, Fornace AJ Jr., Suman S. Simulated Galactic Cosmic Radiation Exposure-Induced Mammary Tumorigenesis in ApcMin/+ Mice Coincides with Activation of ERα-ERRα-SPP1 Signaling Axis. Cancers. 2024; 16(23):3954. https://doi.org/10.3390/cancers16233954

Chicago/Turabian Style

Kumar, Kamendra, Jerry Angdisen, Jinwenrui Ma, Kamal Datta, Albert J. Fornace, Jr., and Shubhankar Suman. 2024. "Simulated Galactic Cosmic Radiation Exposure-Induced Mammary Tumorigenesis in ApcMin/+ Mice Coincides with Activation of ERα-ERRα-SPP1 Signaling Axis" Cancers 16, no. 23: 3954. https://doi.org/10.3390/cancers16233954

APA Style

Kumar, K., Angdisen, J., Ma, J., Datta, K., Fornace, A. J., Jr., & Suman, S. (2024). Simulated Galactic Cosmic Radiation Exposure-Induced Mammary Tumorigenesis in ApcMin/+ Mice Coincides with Activation of ERα-ERRα-SPP1 Signaling Axis. Cancers, 16(23), 3954. https://doi.org/10.3390/cancers16233954

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