Supplementary Figure 1 from Transcriptional Regulation of Estrogen Receptor-α by p53 in Human Breast Cancer Cells

Figure S1 shows representative GTT and ITT results of the parents used to produce study animals.

Figure S3 shows the FACS gating strategy used to isolate MaSCs.

Insulin-like growth factor I (IGF-1) has been implicated in breast cancer due to its mitogenic and anti-apoptotic effects. Despite substantial research on the role of IGF-1 in tumor progression, the relationship of IGF-1 to tissue stem cells, particularly in mammary tissue, and the resulting tumor susceptibility has not been elucidated. Previous studies with the BK5.IGF-1 transgenic (Tg) mouse model reveals that IGF-1 does not act as a classical, post-carcinogen tumor promoter in the mammary gland. Pre-pubertal Tg mammary glands display increased numbers and enlarged sizes of terminal end buds, a niche for mammary stem cells (MaSCs). Here we show that MaSCs from both wild-type (WT) and Tg mice expressed IGF-1R and that overexpression of Tg IGF-1 increased numbers of MaSCs by undergoing symmetric division, resulting in an expansion of the MaSC and luminal progenitor (LP) compartments in pre-pubertal female mice. This expansion was maintained post-pubertally and validated by mammosphere assays in vitro and transplantation assays in vivo. The addition of recombinant IGF-1 promoted, and IGF-1R downstream inhibitors decreased mammosphere formation. Single-cell transcriptomic profiles generated from 2 related platforms reveal that IGF-1 stimulated quiescent MaSCs to enter the cell cycle and increased their expression of genes involved in proliferation, plasticity, tumorigenesis, invasion, and metastasis. This study identifies a novel, pro-tumorigenic mechanism, where IGF-1 increases the number of transformation-susceptible carcinogen targets during the early stages of mammary tissue development, and “primes” their gene expression profiles for transformation.

Diet is a critical environmental factor affecting breast cancer risk, and recent evidence shows that dietary exposures during early development can affect lifetime mammary cancer susceptibility. To elucidate the underlying mechanisms, we used our established crossover feeding mouse model, where exposure to a high-fat and high-sugar (HFHS) diet during defined developmental windows determines mammary tumor incidence and latency in carcinogen-treated mice. Mammary tumor incidence is significantly increased in mice receiving a HFHS post-weaning diet (high-tumor mice, HT) compared to those receiving a HFHS diet during gestation (low-tumor mice, LT). The current study revealed that the mammary stem cell (MaSC) population was significantly increased in mammary glands from HT compared to LT mice. Igf1 expression was increased in mammary stromal cells from HT mice, where it promoted MaSC self-renewal. The increased Igf1 expression was induced by DNA hypomethylation of the Igf1 Pr1 promoter, mediated by a decrease in Dnmt3b levels. Mammary tissues from HT mice also had reduced levels of Igfbp5, leading to increased bioavailability of tissue Igf1. This study provides novel insights into how early dietary exposures program mammary cancer risk, demonstrating that effective dietary intervention can reduce mammary cancer incidence.

Female breast cancer (BrCa) is the most common noncutaneous cancer among women in the United States. Human epidemiological studies reveal that a p53 single-nucleotide polymorphism (SNP) at codon 72, encoding proline (P72) or arginine (R72), is associated with differential risk of several cancers, including BrCa. However, the molecular mechanisms by which these variants affect mammary tumorigenesis remain unresolved. To investigate the effects of this polymorphism on susceptibility to mammary cancer, we used a humanized p53 mouse model, homozygous for either P72 or R72. Our studies revealed that R72 mice had a significantly higher mammary tumor incidence and reduced latency in both DMBAinduced and MMTV-Erbb2/Neu mouse mammary tumor models compared to P72 mice. Analyses showed that susceptible mammary glands from E-R72 (R72 x MMTV-Erbb2/Neu) mice developed a senescence-associated secretory phenotype (SASP) with influx of proinflammatory macrophages, ultimately resulting in chronic, protumorigenic inflammation. Mammary tumors arising in E-R72 mice also had an increased influx of tumor-associated macrophages, contributing to angiogenesis and elevated tumor growth rates. These results demonstrate that the p53 R72 variant increased susceptibility to mammary tumorigenesis through chronic inflammation.

BACKGROUND: Physical inactivity contributes to incidence of diseases and decreased life expectancy. Previous data has shown that chronic overfeeding via high fat high sugar diet (HFHS) reduces voluntary wheel running (WR) activity in mice. PURPOSE: Determine the effects of a 12% calorie restriction diet (DR) vs. an ad-lib HFHS diet on physical activity (PA) levels (distance, duration, speed) throughout total lifetime in female SENCAR mice. METHODS: SENCAR mice were bred and offspring were weaned at 3 weeks of age onto a HFHS (20% protein, 45% fat, 24% sucrose + 10% fructose water), an ad-lib standard chow (CONT; 20% protein, 10% fat, 57% cornstarch), or a DR (12% kcal restriction, 20% protein, 10% fat, 57% cornstarch). At 4 weeks of age, female mice were housed in pairs and two plastic running wheels were mounted inside each cage. WR distance (km/day) and duration (min/day) were recorded and used to calculate average speed (m/min) via a mounted computer system. Repeated measures ANOVA determined the effect of diet on WR activity relative to varying percentages of total lifetime (15%, 25%, 50%, 75%, and 100%). RESULTS: 116 female mice [HFHS (n=42), DR (n=55), CNTL (n=19)] were analyzed. Both ad lib HFHS and ad lib CONT diets significantly decreased distance, duration, and speed after 25% of the total lifespan. All PA variables remained unchanged for the DR mice with the only significant reduction occurring in duration between 75% (221 ± 98min/day) to 100% (188 ± 79min/day) of total lifetime. Additionally, correlations of determination were observed for body weight vs HFHS (.44), DR (.04), and CONT (.34) diets. CONCLUSIONS: DR mice maintained activity levels across their lifespans as compared to ad lib CONT and ad lib HFHS mice whose activities decreased over their lifespan. These findings substantiate our previous data and propose that minimal calorie restriction may serve a novel intervention to prevent physical inactivity across the lifespan.

PURPOSE: Accuracy of data collection is essential in reducing variability in voluntary wheel running which could potentially hide statistically significant results. When measuring physical activity in rodents, a large portion of each collection period is unobserved and thus, systematic checks to reassure functionality must be a priority. The purpose of this study was to create a procedure that would determine if a running wheel was accurately functioning based on the measured data. METHODS: The TAMU IACUC approved all procedures. SENCAR breeder pairs were mated and at 3 weeks of age, the female pups were co-caged. At 4 weeks of age, two running wheels were mounted in each cage, with an odometer attached to the top of the cage to record daily distance (km/day), duration (mins/day), and to calculate speed (m/min). At 14 weeks of age, the number of manual rotations required to reach a 0.01 km change on the odometer was determined with the number of rotations averaged across three trials. Each wheel’s position in relation to the computer pick-up was then adjusted until the total spins to reach a 0.01 km odometer change were as close to 61 (calculated to be the true distance of 0.01 km) as possible. Resulting average daily distance, duration, and speed were compared to the amount of rotations before and after adjustment using a one-way ANOVA. RESULTS: Wheel rotations needed to reach 0.01 km before adjustments to the wheel were significantly higher (p=0.002) than after adjustments (74.7±15.3 vs. 64.5±6.6 rotations, respectively). Before adjustments, rotations had varying correlations with distance (R2=0.080; p=0.18), duration (R2=0.027; p=0.44), and speed (R2=0.50; p<0.0001). After adjustments, all correlations were lower and insignificant with distance (R2=0.0034; p=0.79), duration (R2=0.0032; p=0.51), and speed (R2=0.0091; p=0.66). CONCLUSIONS: Completing ongoing and regular manual checks on the functionality of running wheels will allow for higher accuracy and lower variance in data, especially running speed, which could otherwise hide significant differences between treatment groups.

Abstract Obesity and alterations in metabolic programming from early diet exposures can affect the propensity to disease in later life. Through dietary manipulation, developing mouse pups were exposed to a hyperinsulinemic, hyperglycemic milieu during three developmental phases: gestation, lactation, and postweaning. Analyses showed that a postweaning high fat/high sugar (HF/HS) diet had the main negative effect on adult body weight, glucose tolerance, and insulin resistance. However, dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis revealed that animals born to a mother fed a HF/HS gestation diet, nursed by a mother on a mildly diet-restricted, low fat/low sugar diet (DR) and weaned onto a HF/HS diet (HF/DR/HF) had the highest mammary tumor incidence, while HF/HF/DR had the lowest tumor incidence. Cox proportional hazards analysis showed that a HF/HS postweaning diet doubled mammary cancer risk, and a HF/HS diet during gestation and postweaning increased risk 5.5 times. Exposure to a HF/HS diet during gestation, when combined with a postweaning DR diet, had a protective effect, reducing mammary tumor risk by 86% (HR = 0.142). Serum adipocytokine analysis revealed significant diet-dependent differences in leptin/adiponectin ratio and IGF-1. Flow cytometry analysis of cells isolated from mammary glands from a high tumor incidence group, DR/HF/HF, showed a significant increase in the size of the mammary stem cell compartment compared with a low tumor group, HF/HF/DR. These results indicate that dietary reprogramming induces an expansion of the mammary stem cell compartment during mammary development, increasing likely carcinogen targets and mammary cancer risk. Cancer Prev Res; 10(10); 553–62. ©2017 AACR. See related editorial by Freedland, p. 551–2.

BACKGROUND: Physical inactivity in combination with poor nutrition promotes obesity and is the second leading cause of death in the US. It is well known that physical activity (PA) mitigates the incidence of obesity related diseases. Yet, most individuals fail to meet daily recommended PA requirements. Available animal literature suggests that one primary factor that regulates PA is sex hormones. PURPOSE: This study examined the effects of a high fat/high sugar (HF/HS) diet on Testosterone (T) and 17β-Estradiol (E2) concentrations in female mice with and without physical activity. METHODS: All procedures were approved by TAMU IACUC. The offspring of SENCAR breeder pairs (Charles River) were weaned and co-caged at three weeks of age and randomly assigned to a HF/HS diet (45% fat/10% fructose drinking water) (n=6) or ad-lib (AL) control diet (10% fat) (n=4). At four weeks of age, mice were provided with running wheel access. Running speed, distance, and duration were recorded daily until termination at 20wks of age. Serum T and E2 concentrations were analyzed via ELISA. A 2-way ANOVA was utilized to assess significant differences. RESULTS: E2 concentrations in the AL fed mice with- and without-PA (0.30±0.03; 0.26±0.03 ng/ml) and in the HF/HS fed mice with- and without-PA (0.28±0.04; 0.27±0.23 ng/ml), were not affected by diet type, but were significantly affected by PA (p=0.03). T concentrations in the AL fed mice with- and without-PA (0.33±0.01; 0.31±0.34 ng/ml) and in the HF/HS fed mice with- and without-PA (0.46±0.22; 0.21±0.14 ng/ml) demonstrated no significant effect of diet or PA. CONCLUSION: In this study neither T nor E2 concentrations were altered by HF/HS diet. However, PA increased the serum concentrations of E2, but not T. Therefore, physical activity can affect estradiol levels independent of diet. ACKNOWLEDGMENTS: This project was funded by the US Army through the Department of Defense projects W81XWH-13-1-0278 (Fuchs-Young) and W81XWH-13-1-0279 (Lightfoot).

BACKGROUND: It is well known that a caloric deficit will cause detrimental bone loss. Physical activity has been shown to improve bone mineral density as long as over training does not occur. However, the effects of excess fat and sugar consumption on bone health are unclear, especially when coupled with physical activity. PURPOSE: The purpose of this study was to investigate the effect of diet and physical activity on bone mineral density (BMD) and body fat percentage (BF) in outbred, female SENCAR mice. METHODS: The TAMU IACUC approved all procedures. SENCAR breeder pairs (Charles River) were mated and at 3 weeks of age, the pups were co-caged and randomly assigned to one of three diet types with wheel running access (WR) or without (no-WR): 1) ad-lib (AL) (10% fat) (n=2 WR; n=3 no-WR); 2) diet restricted (DR) (12% kcal reduction from control) (n=8 WR; n=7 no-WR); or 3) high fat/high sugar (HFHS) (45% fat/10% fructose solution drinking water) (n=5 WR; n=9 no-WR). At 4 weeks of age, the mice were randomly assigned to WR or no-WR groups. After 20 weeks, BF and BMD were determined with a PIXImus DEXA scan at termination. RESULTS: BF percent was reduced by WR across all diet types (p-value<0.0001). Average BF across all groups was 166% higher in no-WR (42.9 ± 9.0 gms) than in WR (25.8 ± 9.5 gms). WR did not reverse decreases in overall BMD associated with the HFHS (0.067 ± 0.005 gms/cm2) or DR (0.066 ± 0.002 gms/cm2) diets compared to the AL (0.080 ± 0.003 gms/cm2) diet (p<0.01). CONCLUSION: Wheel running reduced body fat percentages similarly in HFHS, DR, and AL diet fed mice. However, wheel running did not affect the loss of BMD in animals on HF/HS diets. ACKNOWLEDGMENTS: This project was funded by the US Army through the Department of Defense projects W81XWH-13-1-0278 (Fuchs-Young) and W81XWH-13-1-0279 (Lightfoot).

BACKGROUND: A growing body of literature suggests that dietary intake influences voluntary physical activity in both human and rodent models. It is known that caloric restriction leads to increases in activity; however, it is unknown if excessive caloric intake affects daily activity. Determining whether excessive caloric and decreased physical activity are causally linked would be the first step towards identifying a mechanism through which diet influences activity. PURPOSE: The overall purpose of this project was to determine the effect of diet type on voluntary wheel running in SENCAR female mice. METHODS: All procedures were approved by TAMU IACUC. SENCAR breeder pairs (Charles River) and offspring at 3 weeks of age were group housed and randomly assigned to one of three diet types: 1) control ad-lib (AL; 10% fat) diet (n=2); 2) diet restricted (DR; 12% kcal reduction from AL fed mice) (n=8); or 3) high fat (HFHS; 45% fat/10% fructose drinking water) fed diet (n=7). At 4 weeks of age, these mice were then provided running wheel access and daily speed, distance, and duration of activity were recorded until their termination at 20 weeks of age. Weekly food weights were also calculated to determine average daily caloric (kcal) intake. A two way ANOVA was employed to determine the effect of diet on activity with factors of time and diet. RESULTS: Overall, the HFHS mice consumed 18% more kcals per day than the control AL mice (25.9 ±1.0 and 21.2±1.3 kcals/day; p<0.0001). Over the 17-week period, there was a significant difference in the distance (p=0.01), duration (p=0.01), and speed (p=0.005) ran between the diet groups, with the HFHS fed mice displaying the lowest levels of daily running wheel activity. The HFHS mice ran 58% (±4.2%) and 44.7% (±5.2%) less distance than the AL and DR groups respectively, and this reduction was due to a decreased duration (p<0.001) without a change in activity speed (p>0.05). CONCLUSION: Our data shows that excess caloric intake from HFHS feeding leads to a decrease in daily activity, with the decrease primarily caused by a reduced duration of activity. ACKNOWLEDGMENTS: This project was funded by the US Army through the Department of Defense projects W81XWH-13-1-0278 (Fuchs-Young) and W81XWH-13-1-0279 (Lightfoot).

BACKGROUND: Regular exercise has been shown to reduce the risk of occurrence for certain cancers. In animal models, DMBA is a synthetic carcinogen that has been established as the gold standard for inducing cancerous tumors in rodents. However, it has yet to be established whether DMBA has an effect on voluntary wheel running in mice. If there is an effect, it would confound any experiment which investigates exercise effects on tumor growth. PURPOSE: The overall purpose of this project was to determine if DMBA altered voluntary wheel running in mice. METHODS: All procedures were approved by TAMU IACUC. SENCAR mice breeder pairs (Charles River) and offspring at 3 weeks of age were group housed and randomly assigned to a group receiving the DMBA (n=69) or not receiving the DMBA treatment (n=22). At 4 weeks of age, two running wheels were placed inside the cages and connected to a computer that measured distance and time. The running wheels were mounted to the cage tops of standard rat cages and equipped with a cycling computer (BC8.12, Sigma Sport) to record running distance and duration. The running wheels were plastic and had a 410mm circumference with a solid running surface. From 8 to 14 weeks of age, mice in the DMBA group were gavaged daily with a DMBA dose (20 μg/mouse) dissolved in corn oil. A two way ANOVA was employed to determine the effect of DMBA on activity with factors of time and treatment. RESULTS: DMBA had no effect on the distance (p=0.51) or duration ran (p=0.12), but significantly decreased the speed at which the mice ran (p=0.02). A post-hoc analysis indicated that significant decreases in speed occurred at weeks 12 (35.2 ±9.2 vs. 46.4 ± 14.6; p=0.0002) and 20 (35.4 ±10.3 vs. 46.2 ± 14.1; p<0.0001) of age. CONCLUSION: Our data suggest that DMBA does not affect the distance or time spent running on a wheel, but does affect the speed at which the mice run. While DMBA decreased speed, the significant effects on speed are minor given that neither distance nor duration were different between the groups. Therefore, we can conclude that DMBA does not prevent voluntary wheel running in mice. ACKNOWLEDGMENTS: This project was funded by the US Army through the Department of Defense projects W81XWH-13-1-0278 (Fuchs-Young) and W81XWH-13-1-0279 (Lightfoot).

BACKGROUND: Regular exercise has been shown to diminish the risk of certain cancers. DMBA, (7, 12-dimethylbenz(a)-anthracene) is a complete carcinogen that is used to induce tumors in mice. It has yet to be established whether DMBA has an effect on voluntary wheel running (WR) in mice, and whether these effects may be exacerbated via consumption of a high fat high sugar (HFHS) diet. PURPOSE: Determine if DMBA treatment altered voluntary WR in female SENCAR mice, and whether a HFHS diet exacerbated treatment effects on voluntary WR. METHODS: Offspring of SENCAR breeder pairs were weaned at 3 weeks (wks) of age onto either an ad lib fed HFHS (20% protein, 45% fat/24% sucrose + 10% fructose water) or a diet restricted (DR) (12% kcal restriction, 20% protein, 10% fat, 57% cornstarch) diet. Animals were double-housed and randomly assigned to either a DMBA (n=40) treatment with HFHS (n=20) and DR (n=20) diets; or a control (CNTL) (n=18) treatment with HFHS (n=10) and DR (n=8) diets. At 4 wks of age, two plastic running wheels were mounted inside standard rat cages, and connected to a computer to record WR duration and distance. At 7-9 wks of age, mice were gavaged with DMBA dissolved in corn oil (20 μg/mouse/day) or with corn oil vehicle only (CNTL) for 5 days/wk for 6 weeks. A two-way ANOVA was employed to determine the effect of DMBA on activity with factors of treatment and diet for wks 9-20. RESULTS: Compared to CNTL, DMBA significantly decreased distance (7.41 ± 0.45 vs. 11.08 ± 0.68 km/day; p=0.0002), and duration (175.19 ± 8.24 vs. 261.23 ± 12.36 min; p<0.0001). No significant difference in speed was noted (40.31 ± 1.37 vs. 40.34 ± 2.05 m/min; p=0.77). HFHS diet compared to DR diet significantly decreased distance (5.84 ± 0.60 vs. 11.08 ± 0.55 km/day; p<0.0001), duration (168.41 ± 10.9 vs. 233.14 ± 10.1 min; p<0.0001), and speed (33.04 ± 1.81 vs. 47.12 ± 1.67 m/min; p<0.0001). No significant interactions were observed between treatment and diet groups. CONCLUSIONS: DMBA and HFHS diet decrease WR distance and duration, while only the HFHS diet decreased WR speed. Although DMBA and HFHS independently decreased WR, a lack of interaction suggest that they are not additive or synergistic. ACKNOWLEDGMENTS: Project was funded by the US Army through the Department of Defense projects W81XWH-13-1-0278 (Fuchs-Young) and W81XWH-13-1-0279 (Lightfoot).

Studies show that elevated insulin‐like growth factor‐1 (IGF‐1) levels are associated with an increased risk of breast cancer; however, mechanisms through which IGF‐1 promotes mammary tumorigenesis in vivo have not been fully elucidated. To assess the possible involvement of COX‐2 signaling in the pro‐tumorigenic effects of IGF‐1 in mammary glands, we used the unique BK5.IGF‐1 mouse model in which transgenic (Tg) mice have significantly increased incidence of spontaneous and DMBA‐induced mammary cancer compared to wild type (WT) littermates. Studies revealed that COX‐2 expression was significantly increased in Tg mammary glands and tumors, compared to age‐matched WTs. Consistent with this, PGE 2 levels were also increased in Tg mammary glands. Analysis of expression of the EP receptors that mediate the effects of PGE 2 showed that among the four G‐protein‐coupled receptors, EP3 expression was elevated in Tg glands. Up‐regulation of the COX‐2/PGE 2 /EP3 pathway was accompanied by increased expression of VEGF and a striking enhancement of angiogenesis in IGF‐1 Tg mammary glands. Treatment with celecoxib, a selective COX‐2 inhibitor, caused a 45% reduction in mammary PGE 2 levels, attenuated the influx of mast cells and reduced vascularization in Tg glands. These findings indicate that the COX‐2/PGE 2 /EP3 signaling pathway is involved in IGF‐1‐stimulated mammary tumorigenesis and that COX‐2‐selective inhibitors may be useful in the prevention or treatment of breast cancer associated with elevated IGF‐1 levels in humans. © 2011 Wiley Periodicals, Inc.

Abstract Background The obesity epidemic in the U.S. has substantially increased the number of people with altered glucose metabolism. Alterations in metabolic programming, resulting from early exposures can affect development, metabolism as well as propensity to later diseases, including cancer. The Warburg effect describes a mechanism by which diet-induced hyperglycemia and hyperinsulinemia can contribute to cellular transformation. To investigate the effect of a hyperinsulinemia-, hyperglycemia-inducing (HI/HG) diet on metabolic programming and susceptibility to mammary cancer, we exposed developing mouse pups during three developmental stages: gestation, lactation and post-weaning. Materials and Methods: Female SENCAR mice were fed either a mildly restricted, defined, “chow like” control diet (DR) or a HI/HG, high sucrose/high fat (HS/HF) diet. At 14 weeks, both DR and HS/HF fed female mice were bred and the resulting offspring were randomized into 8 groups to model all combinations of gestational, lactational and post-weaning dietary exposures. Body weights (BW) were recorded weekly and Glucose Tolerance Tests (GTTs) were conducted on the female offspring at 10–12 weeks of age. Starting at 7–9 weeks of age, mice received 20 mg/day of DMBA or vehicle by daily gavage to induce mammary carcinogenesis. Results: Animals in the DR/DR/DR (gestational diet/nursing diet/post-weaning diet) and HS/DR/DR groups had the lowest average BW and retained normal response to glucose, while mice in the DR/HS/HS and HS/HS/HS groups had the highest average BW. Interestingly, animals born to DR-fed and nursed by HS/HF mothers and weaned onto a HS/HF diet (DR/HS/HS) were the most glucose intolerant. In response to DMBA, animals in the different dietary regimens partitioned into high, moderate or low mammary tumor incidence groups. Mice fed consistent diets throughout gestation, lactation and post-weaning (DR/DR/DR or HS/HS/HS) had intermediate tumor incidence. However, mice exposed to DR during gestation and/or lactation and then switched to a HS/HF diet at weaning had the highest tumor incidence and shortest latency. Mice exposed to a HS/HF diet during gestation and/or lactation and then switched to a DR diet at weaning had the fewest mammary tumors and longest latency. Conclusion: Our data indicate that substantial changes in the type and abundance of calories during gestation and/or lactation had long-lasting impacts on BW, glucose metabolism, and mammary tumorigenesis. Since BW did not consistently correlate with GTT results, the effects of dietary manipulation on obesity and glucose metabolism appear to be distinguishable. Results also indicate that diet-induced changes in the glucose metabolism of the mother profoundly affected tumor susceptibility in the exposed female offspring. These findings support the contention that rising levels of obesity, metabolic syndrome, and diabetes, especially in young individuals, may contribute to the increasing incidence of early onset breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-05-02.

Insulin like growth factor-1 (IGF-1) stimulates increased proliferation and survival of mammary epithelial cells and also promotes mammary tumorigenesis. To study the effects of IGF-1 on the mammary gland in vivo, we used BK5.IGF-1 transgenic (Tg) mice. In these mice, IGF-1 overexpression is controlled by the bovine keratin 5 promoter and recapitulates the paracrine exposure of breast epithelium to stromal IGF-1 that is seen in women. Studies have shown that BK5.IGF-1 Tg mice are more susceptible to mammary tumorigenesis than wild-type littermates. Investigation of the mechanisms underlying increased mammary cancer risk, reported here, revealed that IGF-1 preferentially activated the PI3K/Akt pathway in glands from prepubertal Tg mice, resulting in increased cyclin D1 expression and hyperplasia. However, in glands from postpubertal Tg mice, a pathway switch occurred and activation of the Ras/Raf/MAPK pathway predominated, without increased cyclin D1 expression or proliferation. We further showed that in prepubertal Tg glands, signaling was mediated by formation of an ERα/IRS-1 complex, which activated IRS-1 and directed signaling via the PI3K/Akt pathway. Conversely, in postpubertal Tg glands, reduced ERα expression failed to stimulate formation of the ERα/IRS-1 complex, allowing signaling to proceed via the alternate Ras/Raf/MAPK pathway. These in vivo data demonstrate that changes in ERα expression at different stages of development direct IGF-1 signaling and the resulting tissue responses. As ERα levels are elevated during the prepubertal and postmenopausal stages, these may represent windows of susceptibility during which increased IGF-1 exposure maximally enhances breast cancer risk.

Clinical studies show that estrogen receptor-α (ER) expressing tumors tend to have better prognosis, respond to antiestrogen therapy and have wild-type p53. Conversely, tumors with inactivating mutations in p53 tend to have worse outcomes and to be ER-negative and unresponsive to antihormone treatment. Previous studies from our laboratory have shown that p53 regulates ER expression transcriptionally, by binding the ER promoter and forming a complex with CARM1, CBP, c-Jun, RNA polymerase II and Sp1. In this study, the MMTV-Wnt-1 transgenic mouse model was used to demonstrate that p53 regulation of ER expression and function is not solely an in vitro phenomenon, but it is also operational in mammary tumorigenesis in vivo. The expression of ER and the ability to respond to tamoxifen were determined in mammary tumors arising in p53 wild type (WT) or p53 heterozygous (HT) animals carrying the Wnt-1 transgene. In p53 WT mice, development of ER-positive tumors was delayed by tamoxifen treatment, while tumors arising in p53 HT mice had significantly reduced levels of ER and were not affected by tamoxifen. P53 null tumors were also found in the p53 HT mice and these tumors were ER-negative. ER expression was upregulated in mouse mammary tumor cell lines following transfection with WT p53 or treatment with doxorubicin. These data demonstrate that p53 regulates ER expression in vivo, and affects response to tamoxifen. Results also provide an explanation for the concordant relationship between these prognostic proteins in human breast tumors.