Lifetime genistein intake increases the response of mammary tumors to tamoxifen in rats

Abstract

Purpose

Whether it is safe for estrogen receptor positive (ER+) breast cancer patients to consume soy isoflavone genistein (GEN) remains controversial. We compared the effects of GEN intake mimicking either Asian (lifetime) or Caucasian (adulthood) intake patterns to that of starting its intake during tamoxifen (TAM) therapy using a preclinical model.

Experimental Design

Female Sprague-Dawley rats were fed an AIN93G diet supplemented with 0 (control diet) or 500 ppm GEN from postnatal day 15 onwards (lifetime GEN). Mammary tumors were induced with 7,12-dimethylbenz(a)anthracene (DMBA), after which a group of control diet fed rats were switched to GEN diet (adult GEN). When the first tumor in a rat reached 1.4 cm in diameter, TAM was added to the diet, and a subset of previously only control diet fed rats also started GEN intake (post-diagnosis GEN).

Results

Lifetime GEN intake reduced de novo resistance to TAM, compared with post-diagnosis GEN groups. Risk of recurrence was lower both in the lifetime and adult GEN groups than in the post-diagnosis GEN group. We observed downregulation of unfolded protein response (UPR) and autophagy related genes (GRP78, IRE1α, ATF4 and Beclin-1), and genes linked to immunosuppression (TGFβ and Foxp3), and upregulation of cytotoxic T cell marker CD8a in the tumors of the lifetime GEN group, compared with controls, post-diagnosis, and/or adult GEN groups.

Conclusions

GEN intake mimicking Asian consumption patterns improved response of mammary tumors to TAM therapy, and this effect was linked to reduced activity of UPR and pro-survival autophagy signaling, and increased anti-tumor immunity.

INTRODUCTION

High soy food intake among women living in Asian countries is thought to contribute to their low breast cancer risk [1, 2]. Soybeans contain the isoflavone genistein (GEN) that has physicochemical properties similar to 17β-estradiol (E2). GEN activates both estrogen receptors ERα and ERβ in a manner comparable to E2 but with a lower affinity [3]. Studies done using ER+ human MCF-7 breast cancer cells indicate that physiological doses of E2 or GEN stimulate the growth of these cells in vitro and in vivo in athymic nude mice [4]. Since estrogenic compounds, including hormone replacement therapy, are not recommended for breast cancer patients, oncologists often advise their patients not to take isoflavone supplements or to consume soy foods [5]. Also, GEN intake can reduce the efficacy of both tamoxifen (TAM) and aromatase inhibitors in eliminating breast cancer cells in athymic nude mice [6–8].

Studies in women show no evidence of adverse effects of soy intake on breast cancer outcome [9]. Indeed, breast cancer patients consuming more than 10 mg isoflavones daily, corresponding to 1/3 cup of soymilk, have the lowest risk of recurrence, both among Caucasian and Asian populations [10–12]. The protective effect is seen in ER+ and ER− breast cancer patients [11, 12], and in patients treated with endocrine therapy [12]. However, it is unclear whether the protective effect reflects high lifetime soy intake or whether a similar outcome can be reached by starting soy intake for the first time during endocrine therapy.

Resistance to endocrine therapies is a significant problem in treating ER+ breast cancers [13]. While ~50% of ER+ breast cancer patients respond to TAM, tumors can either exhibit de novo resistance (never respond to the treatment) or acquire resistance after initially responding and recur [14, 15]. Factors involved in determining which patients will respond to endocrine therapy and which will recur remain largely unknown [16, 17]. TAM induces endoplasmic reticulum (EnR) stress and the unfolded protein response (UPR); when pro-death signaling dominates cancer cells are eliminated by apoptosis [16, 18]. However, UPR which involves GRP78 as a key activator of the process, and three signaling arms consisting of IRE1α, PERK and ATF6 pathways, can also activate autophagy-related genes to induce cancer cell survival. Earlier in vitro studies indicate that GEN suppresses UPR signaling [19, 20], but as these findings were obtained using 50–200 times higher doses of GEN than can be achieved by consuming soyfoods, their clinical relevance is not known.

The UPR regulates inflammatory responses in antigen presenting macrophages and dendritic cells [21]. Specifically, activation of UPR reduces antigen processing and presentation and consequently suppresses CD8+ cytotoxic T-cells [22], leading to evasion of antitumor immunity [23]. UPR activation is linked to upregulation of FOXP3, a master transcription factor that participates in the development and function of regulatory T-cells (Tregs) [23]. High FOXP3 expression is associated with promotion of immunosuppression. Autophagy also modulates immune responses by increasing Tregs [24] and downregulating CD8+ cells [25]. GEN has several effects on the immune system in vitro and in vivo [26], including increasing CD8+ T-cells and reducing Tregs [27]. Since high FOXP3 levels are predictive of poor survival among ER+ breast cancer patients [28, 29], GEN intake may promote anti-tumor immunity and prevent recurrence.

Over 40 years ago, Jordan et al. [30] used 7,12-dimethylbenz[a]anthracene (DMBA) to induce ER+ mammary tumors in rats and showed that these tumors stopped growing and many disappeared when animals were treated with tamoxifen. Using this same model, with some modifications [31], we compared the effects of lifetime GEN exposure that mimics the soy food intake of Asian women, with GEN intake starting during adult life (mimicking soy food intake among some Western women), or intake that began during TAM treatment. Most breast cancers in the U.S. likely fall into the latter category, if patients start using soy supplements or soy foods to alleviate menopausal symptoms induced by antiestrogens. Our results show that either lifetime or adult GEN intake inhibits TAM resistance and reduces local mammary cancer recurrence. Starting to consume GEN during TAM treatment has the opposite effect. We also found changes in tumor UPR and immune markers that are indicative of lower levels of EnR stress and improved anti-tumor immune responses in the lifetime GEN exposed group.

MATERIALS AND METHODS

Animals

Sixteen female Sprague-Dawley rats, each nursing ten 10-day-old female pups, were obtained from Charles River Laboratory (Frederick, MD) and housed in the Department of Comparative Medicine at Georgetown University. Rats were kept in a temperature- and humidity- controlled room with free access to water and food under a 12:12-hour light:dark cycle. All experimental procedures were approved by Georgetown University Animal Care and Use Committee.

Dietary exposures and DMBA administration

When pups were 15 days of age, 16 litters were divided into two groups: (i) 7 dams with a total of 70 female pups were fed AIN93G laboratory diet supplemented with 500 ppm GEN (GEN diet), and (ii) 9 dams with a total of 90 female pups were fed an AIN93G diet (control diet). Pups were weaned on postnatal day (PND) 21 and kept on the GEN or control diet until PND 30. Between PND 31 and 55, all rats were fed the control diet to avoid GEN’s potential effects on the metabolism and activation of 7,12-dimethylbenz(a)anthracene (DMBA).

On PND 48, mammary tumors were initiated by administering 1 ml of peanut oil containing 10 mg DMBA by oral gavage. One week later, rats were divided into five groups: (1) those previously fed GEN diet received 500 ppm GEN from PND 55 onwards until the end of the study (lifetime GEN group mimicking Asian women) (n=35); (2) those fed GEN between PNDs 15 and 30 received control diet until they developed mammary tumors and started antiestrogen treatment. Once antiestrogen treatment started, these rats were again fed 500 ppm GEN (prepubertal GEN group) (n=35). This group does not represent either Caucasian or Asian soy intake patterns; this group was included as a control group for the lifelong GEN intake group. (3) Those animals previously fed control diet received 500 ppm GEN from PND 55 onwards until the end of the study (adult GEN group mimicking Caucasian women) (n=35). (4) Rats previously fed control diet remained in the control diet until they developed mammary tumors and started antiestrogen treatment at which point they were fed for the first time 500 ppm GEN (antiestrogen treatment only GEN group) (n=20). Finally, (5) a control group composed of rats kept on the control diet throughout the study (control) (n=15). This control group was not included to assessing tumor responses to TAM treatment. Instead, a historical control group [31] was utilized to compare tumor responses to TAM. All the diets were provided by Harlan Laboratories (Madison, WI) and the ingredients are listed in Supplementary Table 1. Outline of dietary exposures are provided in Fig. 1.

Figure 1. Study design.

Rats were fed 0 or 500 ppm genistein (GEN) containing diet either before puberty, adult life or both, or rats started consuming it for the first time during tamoxifen (TAM) treatment. Mammary tumors were initiated with DMBA at postnatal day (PND) 48, and when a first tumor per rat reached a size of 1.4 cm in diameter, 337 ppm tamoxifen citrate (TAM) was added to the diet. Response of tumors to TAM was monitored for up to 38 weeks.

To determine the effect of GEN intake on UPR, autophagy and immune parameters in the mammary tumors prior to antiestrogen treatment, we euthanized 15 rats per group when their tumor reach a size of 14±1 mm in diameter.

Mammary tumor latency, incidence and burden

Tumor latency was the length of the tumor-free period (in weeks) from the date of DMBA administration until a rat developed the first measurable mammary tumor. Incidence was assessed weekly and was defined as the percentage of animals per group that had developed at least one tumor. Tumor burden was the overall tumor area, calculated by measuring the width × length of each tumor per animal, and was assessed when rats started receiving antiestrogen treatment.

Antiestrogen Treatment

Mammary tumor growth was monitored weekly by palpation, and tumor locations and sizes (measured by a caliper) were recorded. When a tumor reached a size of 14±1 mm in diameter, a rat was switched to a diet containing 337 ppm TAM citrate. This dose results in a daily exposure of 15 mg/kg TAM. The response of the tumors to TAM was assessed by the criteria outlined in Supplementary Table 2.

Tissue collection

At the end of the tumor monitoring period, rats were euthanized by CO₂; earlier if burden reached 10% of the body weight, or if animals lost weight or were sick, as required by the Georgetown University Animal Care and Use Committee. At necropsy, blood was collected by cardiac puncture, and mammary glands and tumors were removed and flash frozen in liquid N₂ for future analysis or were fixed in 10% formalin for histopathology purpose.

Tumor Pathologic Evaluation

Formalin fixed mammary tumors were embedded in paraffin and cut into 5 µm sections. Hematoxylin and eosin (H&E) stained tumor sections were used for the evaluation. Tumors were classified according to their histopathology as evaluated by an experienced pathologist (ARUP Laboratory, Salt Lake City, Utah). In all the molecular biology assays done using mammary tumors, only tumors that were malignant adenocarcinomas were included to the analysis.

RNA Extraction and cDNA Synthesis

One hundred grams of frozen mammary tissue was ground using a mortar and pestle in liquid nitrogen. Total RNA was isolated from the ground mammary glands by RNeasy Lipid Tissue Mini Kit (Qiagen, MD), following the manufacturer’s instructions. Total RNA from malignant mammary tumors was extracted using TRIzol reagent (Life technologies, CA) followed by one step of DNase I treatment to prevent genomic DNA contamination (Roche, Germany), as the manufacturer instructed. Quantity and quality of RNA was determined according to the optical density ratio (OD260:OD280) using a ND1000 Nanodrop spectrophotometer (Thermo scientific, MA). A total of 2 µg RNA per sample was used to generate cDNA via reverse transcription in a PTC-100 thermal cycler (Bio-Rad, CA) using the following steps: initiation at 25°C for 10 min, reverse transcribing at 37°C for 2 hours and deactivation at 85°C for 5 min.

Quantitative Real-Time Polymerase Chain Reaction

To measure the relative mRNA abundance of UPR and immune related genes, quantitative real-time PCR was conducted. Briefly, 12.5 µg cDNA was used as template with primers specific for PgR, Ki67, Tgfβ1, Foxp3, Cd8a, spliced Xbp1 (Xbp1s), unspliced Xbp1 (Xbp1us), Hspa5 (GRP78), and Hprt using 5 µL Absolute QPCR SYBR Green ROX Mix in a 10 µL reaction (Thermo Scientific, Foster City, CA). Serially diluted cDNA samples (20 ng/µL -to 0.625 ng/µL) were included with each primer. To determine the relative quantity of the gene, the expression was normalized to the level of the house-keeping gene Hprt. Real-time PCR reactions were carried out in an ABI Prism 7900 Sequence Detection System (Life Technologies, Carlsbad, CA) with the following thermo cycler setting: activation of the enzyme at 95°C for 15 min, 40 cycles of denaturing at 95°C for 15 sec, annealing at 60°C for 30 sec, and elongation at 72°C for 30 sec, followed by one step of dissociation to ensure the purity of the product. Primers used in the real-time PCR were designed using Vector NTI software (Life Technologies) and are listed in Supplementary Table 3. The result of the reaction was checked and exported using SDS 2.3 software (Life Technologies). The highest efficiency of the machine was confirmed by ensuring that the R² of the standard curve was >0.98 and that the slope was within 3.3 ± 0.3.

Protein Isolation and Immunoblotting

Protein isolated from mammary glands and malignant tumors was used to determine whether GEN intake and TAM treatment affected the expression of estrogen receptor alpha (ERα), ERβ, progesterone receptor (PgR), erb2/HER2, GRP78, IRE1α, XPB-1, sXBP-1, PERK, ATF4, ChOP, ATF6, Beclin-1, ATG7, LC3I, LC3II, and p62. Detailed description of the procedure and antibodies used can be found in Supplementary Text.

Serum Isoflavone quantification

Blood was drawn by cardio-puncture at necropsy and serum was separated and kept at −80°C until analysis. Serum levels of total isoflavone (aglycones and conjugated forms) were determined by LC-ES/MS/MS. Briefly, complete enzymatic hydrolysis was performed by incubating the serum samples overnight with H. pomatia preparation containing glucuronidase, sulfatase, and glucosidase, followed by isotope dilution quantification of GEN, daidzein, and equol. Inter- and intra-day analysis was performed and the precisions of measurements were ensured with 1–6% relative standard deviation. For each sample, the method detection limit for GEN was approximately 0.005 µM per aliquot of 10 µL. Quality control samples were included for the analysis of every sample test including the analysis of blank and spiked serum samples (glucuronidase/sulfatase), blank injections, and injections of authentic standards.

Serum cytokine levels

To investigate possible changes in serum cytokine levels in rats fed GEN at different times of their life, a rat cytokine multiplex array (#110449RT) was carried out by Quansys Biosciences (Logan, UT). This analysis includes the following nine cytokines: IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, IFN-γ, and TNF-α. Serum was obtained from rats that were euthanized before TAM treatment. Samples were run in triplicates using ELISA-based Chemiluminescent assay and the mean value was calculated per animal to determine the cytokine levels.

Statistical Analysis

Statistical analysis of the tumor latency, serum cytokine levels, mRNA expression, and protein levels were performed using one-way ANOVA followed by post-hoc analysis using Fisher’s least significant difference (LSD) test. If not otherwise specified, p-values given in the Results section represent those obtained from the LSD test. Kaplan-Meier survival analysis and the log-rank test were used to compare the differences in tumor incidence. Chi-square analysis was applied to determine the statistical significance in tumor response to TAM treatment, and in the rate of recurrence among the five groups. Repeated-one-way ANOVA over each tumor-monitoring week was applied to the tumor burden data. All statistical analyses were carried out using SPSS SigmaStat software, and differences were considered significant if p was less than 0.05. Data are expressed as mean ± SEM (standard error of mean).

RESULTS

We measured the serum GEN concentration by LC-ES/MS/MS: they were 4.14 µM ± 0.35 in all of the GEN exposed groups and did not differ by the duration of GEN intake. These levels are comparable to those seen in humans consuming 2–4 servings of soyfoods daily [32]. We did not measure TAM or its metabolite levels here. However, based on previously published study in Sprague Dawley rats receiving either 13.3 or 22.5 mg/kg/day of TAM and having blood TAM levels of ~120 or ~180 ng/ml, respectively [33], we estimate that the levels in our study in rats consuming 15 m/kg/day TAM were about 120–130 ng/ml. These levels are comparable to those reported in breast cancer patients (~84 ng/ml) who took 20 mg of tamoxifen per day for 28 days [34].

Tumor latency and incidence

Lifelong GEN intake (10.9 ± 0.8 weeks) significantly delayed mammary tumorigenesis (p<0.05), when compared with control rats that never consumed GEN (7.9 ± 0.3 weeks). No significant differences were observed among the other groups (Fig. 2A). Survival analysis revealed that the cumulative tumor incidence in the lifelong GEN consumption group was significantly lower than in the control rats (p=0.0071). Since most animals in each group developed mammary tumors, tumor incidence at the end of the study did not differ among the groups (Fig. 2B).

Figure 2. Mammary tumor latency and incidence.

(A) The time between DMBA administration and appearance of first measurable mammary tumor (tumor latency) per rat consuming control diet (blue), fed genistein (GEN) through adulthood (red), fed GEN during prepuberty (green), and fed GEN both before puberty and adulthood (lifelong, purple). Lifelong GEN intake lengthened tumor latency. Means ± SEM are shown. Bars with different letters are significantly different from each other, p<0.05. (B) Mammary tumor incidence, shown as percentage of rats between weeks 5 and 20 after DMBA administration that developed at least one measurable mammary tumor. It was significantly lower in the lifelong GEN group than in the controls (p=0.0071). n=20–35 rats per group.

Tumor responses to TAM treatment and the risk of recurrence

Responses to TAM treatment were monitored for up to 30 weeks from DMBA administration; i.e., until rats were 37 weeks of age. No difference in the length of the monitoring period was seen among the groups (data not shown). Responses (R) to TAM in a historical non-GEN reference group were seen among 54% of the tumors treated with TAM [31] (Fig. 3A). Similar response rates were seen in rats that consumed GEN during prepuberty (56%) or lifetime (52%). These two groups exhibited a higher percentage of partially responding tumors (PR); tumors that stopped growing upon TAM exposure, 21% and 24% respectively, compared with the control group (8%; p=0.007) (Fig. 3A). Consequently, de novo resistance to TAM was significantly lower in the prepubertal (23%) and lifetime GEN (24%) groups than in the control group (38%; p=0.03).

Figure 3. Responses of mammary tumors to tamoxifen (TAM) therapy.

(A) Percentage of responses (green), partial responses (blue) and de novo resistance (red) in rats that consumed genistein (GEN) during different time periods of their life. Numbers of tumors per group were 30 in no GEN controls (historical reference group described in reference [31]), 47 in post-diagnosis GEN, 44 in adult GEN, 40 in prepubertal GEN and 42 in lifelong GEN. Starting GEN intake during TAM treatment (p=0.004) and adult GEN intake (p=0.033) reduced responses, whilst prepubertal (p=0.032) and lifelong GEN intake (p=0.047) reduced de novo resistance, compared with no GEN controls. (B) Percentage of completely responding tumors that recurred locally. Red portion of each bar represents recurrent tumors. Compared with no GEN controls, adult (p=0.003) and prepubertal GEN intakes (p=0.038) reduced recurrence, and recurrence was also significantly lower in the lifelong GEN group than in the post-diagnosis GEN group (p=0.023).

By comparison, tumors were least likely to exhibit a response in rats that started consuming GEN either during TAM treatment (R=33%; p=0.004) or as adults (R=38%; p=0.03) when compared with the reference control animals (p=0.004 or p=0.03, respectively), lifetime GEN intake group (p=0.01 or p=0.065) or group that consumed GEN during prepuberty and TAM treatement (p=0.002 and p=0.016). In the adult GEN group, 21% of the tumors exhibited a PR, and thus de novo resistance was not increased in this group (Fig. 3A). De novo resistance in rats that started consuming GEN during TAM treatment was significantly higher compared with the prepubertal (p=0.032) and lifetime (p=0.047) GEN groups.

The rats that started consuming GEN as adults had a significantly higher tumor burden, and the rats that consumed GEN during prepubertal period or lifetime had a significantly lower tumor burden, compared with the reference group (repeated measures ANOVA: p<0.001) (Fig. S1).

Tumors that exhibited a response and were undetectable for at least 6 weeks, but then regrew at the same location to reach ≥1.4 cm in diameter, were characterized as recurring tumors with acquired TAM resistance. Prepubertal (11% recurrence rate) and lifetime GEN intake (18% recurrence) decreased the risk of tumor recurrence, when compared with rats that started consuming GEN with TAM treatment (33% recurrence) (p<0.001) (Fig. 3B). The risk of recurrence was lowest in the rats that started GEN intake as adults (7% recurrence) (p=0.003, compared with the reference group).

Hormone receptor expression in the TAM treated mammary glands and tumors

Western blotting was performed to determine the protein expression of ERα, ERβ, and HER2 in the mammary glands and tumors. Quantitative real-time PCR (qRT-PCR) was applied to measure PgR mRNA expression. In the mammary gland, protein levels of ERα (Fig. 4A), ERβ (Fig. 4B) and HER2 (Fig. 4D) and mRNA level of PgR (Fig. 4C) did not differ among groups. Tumors in TAM treated rats were all ER-positive, including acquired resistant tumors. ERα (Fig. 4F) or PgR (Fig. 4H) protein levels were not affected by the different timings of GEN exposure. Protein levels of ERβ (p =0.02, Fig. 4G) and HER2 (p= 0.007, Fig. 4I) were upregulated in the tumors of prepubertally GEN exposed rats, when compared with the group that started GEN intake during TAM therapy.

Figure 4. Expression of ERα, ERβ, PgR and Erb2/HER2 in the mammary glands and tumors of genistein (GEN) fed and tamoxifen (TAM) treated rats.

Western blot analysis of (A, E) ERα, (B, E) ERβ and (C, E) Erb2/HER2 protein levels, and (D) RT-qPCR analysis of PgR mRNA levels in the mammary glands and tumors of rats fed GEN during different periods of their life. Quantitated data and western blots are shown. Lifelong GEN intake increased ER-β levels. For the adenocarcinomas (either partially responding or de novo resistant), protein levels of (F, J) ERα (G, J), ERβ and (H, J) Erb2/HER2 levels and (I, J) PgR are shown. Data are presented as means ± SEM. Bars with different letters are significantly different from each other, p<0.05. Prepubertal GEN intake increased ER-β levels. n=5–10 mammary glands and 5–10 tumors per group.

Tumor pathologic type

Histopathology of the mammary tumors were assessed before and after TAM treatment by examining H&E stained tumor sections. Results are shown in Fig. S2. No statistical difference in tumor histopathology was seen in animals that were euthanized prior to TAM treatment. 0–27% of the tumors were benign in the control and all GEN groups, and the remaining tumors were either papillary or tubular adenocarcinomas.

Tumor histopathology was assessed after TAM treatment in partially responding, de novo resistant, or recurring mammary tumors. Benign tumors constituted 23% of tumors in the controls, 27% in the rats that started to consume GEN during TAM therapy and 33% in the rats that consumed GEN during adulthood. Prepubertal and lifetime GEN intake significantly increased the rate of benign tumors in TAM treated rats to 53% (p<0.001 compared with control group). Thus, because of TAM therapy, more than half of the partially responding or growing tumors in these groups were no longer malignant.

UPR and autophagy signaling in the mammary glands and tumors before TAM treatment

Mammary glands

Protein levels, determined using Western blots, of UPR and autophagy markers did not change among GEN groups in rats before TAM treatment (Fig. S3).

Mammary tumors

We assessed possible changes in UPR and autophagy using Western blots in the mammary glands and tumors obtained from rats before TAM treatment. All the tumors used here were malignant adenocarcinomas and were approximately 1.4 cm in diameter when harvested. None of the UPR or autophagy genes studied were significantly different among the controls or GEN fed groups (Fig. S3).

Alterations in UPR and autophagy signaling in the mammary glands and tumors in TAM treated rats

Mammary glands

TAM did not affect UPR or autophagy signaling in the DMBA exposed mammary glands (Fig. S4A and S4C–H). Consumption of GEN throughout the lifetime increased GRP78 mRNA expression, compared with the control rats (p<0.001) or rats that started consuming GEN during TAM treatment (p=0.007). GRP78 mRNA also was elevated in prepubertally GEN fed rats, compared with the same two groups (p<0.001 or p=0.02). No other changes in mRNA levels in UPR or autophagy genes were seen. At the protein level, ATF6 was higher in the lifetime GEN group than in the control (p=0.02) or adult GEN (p=0.01) rats, and CHOP protein was higher in the lifetime group than in the adult GEN rats (p=0.05) (Fig. 5A, 5B and 5D).

Figure 5. Protein levels of genes in unfolded protein response (UPR) pathways in the mammary glands and tumors of genistein (GEN) fed and tamoxifen (TAM) treated rats.

(A) UPR chaperone GRP78, (B) ATF6 and its down-stream targets (C) Xbp1 (ratio of spliced to unspliced version) and (D) ChOP were elevated in the mammary glands of rats fed GEN though the lifetime. In contrast, (F) GRP78, (G) IRE1α, (H) ATF4 and (I) Beclin-1 were downregulated in the mammary tumors of rats fed GEN through the lifetime. (E) Shows western blots of the genes in the tumors, and (B) and (D) show the blots in the mammary tissues. Data are presented as means ± SEM; bars with different letters are significantly different from each other, p<0.05. n=6–10 mammary tissues and n=3–6 tumors per group.

Since ATF6 acts as a transcription factor, we assessed the mRNA levels of several ATF6 target genes. mRNA levels of total Xbp1 were significantly higher in the lifetime (p=0.01) and prepubertally GEN fed rats (p=0.02) than in the control rats. In addition, the ratio of spliced Xbp1 levels to total Xbp1 levels was higher in the lifetime (p=0.02) and prepubertally GEN fed rats (p=0.01) than in the controls (Fig. 5C). These findings show that in the mammary glands with no tumors, UPR was more activated in those groups that received GEN already during pubertal development, suggesting that upregulation of UPR may be involved in protecting against mammary cancer development.

Mammary tumors

Consistent with the earlier studies showing that TAM activates UPR in breast cancer cell lines [18], we found elevated protein levels of GRP78 (p=0.01) and IRE1α (p=0.05), when compared with tumors obtained from rats not treated with TAM (Fig. S4B and S4I–N). We also noted that Beclin-1 (p=0.05) protein levels were increased and p62 protein levels were reduced (p=0.02) in the TAM treated tumors, indicative of higher level of autophagy. These findings are in agreement with the reported effects of TAM on human breast cancer cell lines [18].

In contrast to the mammary glands, several UPR components were significantly down-regulated in the mammary tumors in rats that consumed GEN throughout their life-time. The down-regulated genes included GRP78 (p<0.001, compared with adult GEN group), ATF4 (p=0.01, compared with control group) and Beclin-1 (p=0.05 and p=0.001, compared with control and adult GEN group, respectively) (Fig. 5E–I). Since ATF4 and Beclin-1 both induce autophagy [35], lifetime GEN intake may promote higher responsiveness to TAM therapy by reducing autophagy.

Alterations in tumor immune system markers before and after TAM treatment

The mRNA level of tumor immune markers Tgfβ1, Foxp3, and Cd8a were determined in the mammary tumors from rats before and after treatment with TAM. Foxp3 is a member of the forkhead-box transcription factor family and induces differentiation of immature CD4+ T-cells to CD4+CD25+ Tregs [36]. Before TAM treatment, GEN consumption during childhood (p=0.04), adulthood (p=0.005), or throughout lifetime (p=0.02) reduced Foxp3 mRNA level, when compared with the control rats (Fig. 6E). Tgfβ1 increases differentiation of immature T-cells into Treg lineage, and Foxp3 induces its expression [37]. Tgfβ1 mRNA levels in the mammary tumors were significantly lower in all GEN groups, compared with the control group (p=0.05). GEN exposure tended to reduce Tgfβ1 mRNA level in the tumors of rats exposed during childhood and adulthood, but the difference did not reach statistical significance (Fig. 6D). CD8 is expressed in cytotoxic T-cells. Lifelong GEN exposure increased mRNA level of Cd8a in tumors before TAM treatment, compared with control rats (p=0.03) (Fig. 6F).

Figure 6. Cytokine levels and expression of Foxp3, TFGβ1 and CD8a in the mammary tumors of genistein (GEN) fed and tamoxifen (TAM) treated rats.

Serum cytokine levels of (A) IL-1α, (B) IL-6 and (C) IL-12 and tumor mRNA levels of (D) Treg cell marker Foxp3 and (E) its down-stream target TGFβ1, and (F) cytotoxic T cell marker CD8a in rats fed GEN before puberty, adulthood or both and not yet treated with tamoxifen (TAM) (n=9 adenocarcinomas per group). GEN intake, either past or current, reduced Foxp3 levels and increased CD8a levels. Same end-points were measured in tumors during TAM treatment (G, H and I) and this assessment included tumors from rats that started GEN intake for the first time during TAM treatment (n=4–6 partially responding or de novo resistant adenocarcinomas per group). Lifelong GEN intake reduced Foxp3 and increased CD8a levels in TAM treated rats. Data are presented as means ± SEM; bars with different letters are significantly different from each other, p<0.05. *significantly different from no GEN control, #statistically different from post-diagnosis GEN exposed rats, p<0.05.

In TAM-treated tumors, mRNA level of Tgfβ1 did not differ among the five groups (Fig. 6G). Lifelong GEN fed rats exhibited the lowest mRNA level of Foxp3 (p=0.05 compared with rats starting to consume GEN with TAM therapy) and highest Cd8a (p=0.02, compared with controls) (Fig. 6H and 6I). Together, these findings suggest that lifetime GEN intake prevents pathways leading to tumor immunosuppression and promotes anti-tumor immune responses.

Alterations in circulating cytokine levels

Cytokine levels in serum were assessed by multiplex rat cytokine arrays only before TAM treatment. Thus, rats fed a GEN containing diet during adulthood and throughout their lifetime were consuming this isoflavone at the time cytokines were measured, unlike the control rats or rats that received GEN before puberty. Of the 9 cytokines tested, only IL-1α, IL-6, and IL-12 had detectable levels in the rat serum. Rats that consumed GEN prepubertally (but not at the time the cytokines were assayed) had higher level of serum IL-1α than the control rats (p<0.001) (Fig. 6A). Serum IL-6 levels also were higher (p<0.001) in this group than in the controls, whilst the levels were significantly reduced in the group consuming GEN during adulthood (p=0.04) (Fig. 6B). IL-12 levels tended to be elevated in rats consuming GEN at the time of measurements but the difference did not reach statistical significance (Fig. 6C).

Proliferation and apoptosis in the TAM treated mammary glands and tumors

To determine the level of cell proliferation, qRT-PCR was carried out to test the relative mRNA level of Ki67 in the mammary glands and tumors. This is a less time consuming measure of cell proliferation than assessing Ki67 though immunohistochemistry, but equally accurate [38]. In the mammary glands, mRNA levels of Ki67 were higher in the rats consuming GEN prepubertally than in the post-diagnosis GEN fed rats (p=0.01) and control rats (p=0.003; p for ANOVA = 0.02) (Fig. S5A). However, Ki67 levels in the adenocarcinomas did not differ among the groups (Fig. S5C).

Apoptosis was assessed by determining the ratio between the protein levels of pro-apoptotic marker Bax and anti-apoptotic marker Bcl2 in the mammary glands and TAM treated tumors. Neither the Bax nor Bcl-2 levels were different among the groups (data not shown). The ratio between the two also was similar in the glands (Fig. S5B) and tumors (Fig. S5D) across all groups.

DISCUSSION

In 2012, over 1.67 million women were diagnosed with breast cancer worldwide [39]; ~70% of these patients had an ER+ tumor. Although endocrine therapies are highly effective in preventing and treating breast cancer [14, 15], approximately half of the treated patients exhibit resistance and recur [13]. In the present study, by using a preclinical model of ER+ breast cancer, we investigated whether GEN intake modifies responsiveness to TAM. Findings obtained in vitro and in immunodeficient mice indicate that GEN impairs response to TAM [6, 8], but observational studies in breast cancer patients show that soy food intake is linked to reduced risk of breast cancer recurrence [10–12]. It is unclear how to explain these conflicting findings. Since GEN does not alter the expression of TAM metabolizing enzymes [40], nor is there any evidence that increased estrogenicity impairs antiestrogen action, as TAM elevates circulating estrogen levels [41], the opposing effects of GEN on human breast cancer cells in vitro or in vivo and on breast cancer patients are unlikely to reflect estrogenicity of this isoflavone.

We found that lifelong GEN intake reduced the risk of de novo and acquired TAM resistance and local recurrence. Similar results were seen in rats that consumed GEN before puberty and again during TAM therapy, suggesting that GEN intake around puberty is critical in preventing TAM resistance. These finding are consistent with childhood soy intake in humans and prepubertal genistein exposure in animal models reducing later breast cancer risk (as reviewed in Ref. [42]). Starting GEN intake during adulthood did not interfere with the ability of TAM to inhibit the growth of rat mammary tumors and was highly effective in preventing recurrence. In contrast to lifetime GEN intake, rats that started consuming GEN only when they were treated with TAM exhibited an increased risk of recurrence. Thus, GEN has a preventative effect in TAM treated animals only if it is consumed before tumors start to develop.

To identify pathways that may explain the differing effects of lifetime GEN intake versus starting GEN consumption during TAM treatment, we focused on three interconnected biological systems. First, the effects on UPR and autophagy were investigated, since these are causally linked to the development of antiestrogen resistance [13, 16, 18]. Earlier studies have reported downregulation of GRP78 by GEN in prostate and liver cancer cells [19, 20], but using pharmacological doses that are not achievable by isoflavone or soy intake in humans. In the TAM treated mammary tumors, GRP78 expression was downregulated in rats consuming GEN through their lifetime or during prepuberty. ATF4 and Beclin-1 protein levels also were significantly reduced in the tumors of lifetime GEN group, compared with controls, indicating that autophagy may be reduced, as these transcription factors both induce autophagy [35]. Previous results obtained in cancer cell lines in vitro indicated reduced autophagy by physiological doses of GEN [43]. The reduced UPR and autophagy in the mammary tumors of rats consuming GEN through their lifetime may be linked to their increased sensitivity to TAM therapy.

In the non-tumor bearing mammary glands, GRP78 levels were increased of rats consuming GEN through their lifetime, as were the levels of CHOP and ATF6, and ATF6’s downstream target Xbp1. Thus, upregulation of UPR may be a successful defense against malignant transformation. Upregulation of Xbp1 in C. elegans was recently found to confer a stress-resistant phenotype and increased longevity [44], raising the possibility that in mammary glands elevated Xbp1 expression also is linked to increased resistance to EnR stress.

Next, we studied if GEN affects markers of cytotoxic T-cells (CD8a) that drive antitumor immune responses, and Tregs (Foxp3 and TGFβ) that induce immunosuppression and allow cancer cells to escape elimination by the immune system [45, 46]. ER+ mammary tumors in our model are sensitive to immunomodulation.

A previous study found that GEN did not prevent DMBA-induced skin carcinogenesis in immunodeficient mice lacking T lymphocytes, while in immunocompetent mice GEN reduced cancer growth; however, this protective effect was seen only if GEN was given prior to tumor induction [27]. These mice, but not those receiving GEN after tumors were detected, exhibited increased activity of cytotoxic T-cells and natural killer (NK) cells, and decreased presence of Tregs in the spleen [27].

Pro- and anti-inflammatory cytokines are produced by immune cells and they have a profound influence on the development and function of T-lymphocytes. Serum IL-12 levels were non-significantly increased in rats consuming GEN at the time of measurement (life-time and adult GEN intake groups), compared with control or prepubertal GEN groups. However, tumor bearing rats fed GEN during prepuberty, but not at the time the cytokine panel was assessed, had significantly higher serum levels of IL-1α and IL-6 than control rats. Since IL-1α or IL-6 both can attenuate Treg function [51], these changes may explain the reduced expression of Foxp3 in the mammary tumors of prepubertally GEN fed rats. Lifetime GEN intake did not modify the levels of either IL-1α or IL-6, suggesting that the presence of GEN reversed the increase in cytokine levels caused by prepubertal GEN intake. This is supported by the finding that adult GEN intake significantly reduced IL-6 levels, in accordance with findings reported by others [52].

In summary, we used a validated preclinical model of ER+ breast cancer to study the response of tumors to TAM in rats that were fed GEN. Our results show that prepubertal and lifetime GEN consumption improved responsiveness to TAM, indicating that improved response to endocrine therapy was pre-programmed early in life. Further, adult GEN intake almost completely eliminated local recurrences. However, animals that received GEN only during TAM treatment were at an increased risk of recurrence. Since no changes in tumor ERα or PgR levels were seen in any GEN group, the differences cannot be caused by alterations in hormone receptor expression. Rather, we found reductions in tumor UPR and autophagy signaling and markers of tumor immune evasion in the lifetime GEN intake group. Although translation of findings from animal models to humans should always be done with caution, our results suggest that it is beneficial to continue to consume soy foods after diagnosis to reduce TAM resistance and breast cancer recurrence.

TRANSLATIONAL RELEVANCE.

Due to its apparent estrogenicity, the safety of genistein in soy foods for ER+ breast cancer patients who are treated with anti-estrogen therapy remains controversial. We show here using a preclinical rat model that lifetime or adult genistein intake improved tamoxifen responsiveness and reduced the risk of recurrence, compared with starting genistein intake during tamoxifen therapy. The positive effects of lifetime genistein intake were linked to reduced unfolded protein response and autophagy, and improved anti-tumor immune responses. If true for women, our results suggest that breast cancer patients should continue consuming soy foods after diagnosis, but not to start if they have not consumed genistein previously.