RESUMEN
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.
Asunto(s)
Exposición Dietética , Neoplasias Mamarias Animales , Animales , Carcinógenos , Dieta , Neoplasias Mamarias Animales/metabolismo , Ratones , Células Madre/metabolismoRESUMEN
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.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina , Glándulas Mamarias Animales , Animales , Proliferación Celular , Femenino , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Transgénicos , Células Madre/metabolismoRESUMEN
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 DMBA-induced 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.
Asunto(s)
Carcinogénesis/genética , Codón/genética , Polimorfismo de Nucleótido Simple/genética , Proteína p53 Supresora de Tumor/genética , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinogénesis/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Inflamación/genética , Inflamación/patología , Macrófagos/patología , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/patología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos , Receptor ErbB-2/genéticaRESUMEN
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 AACRSee related editorial by Freedland, p. 551-2.
Asunto(s)
Carcinogénesis/metabolismo , Dieta Alta en Grasa/efectos adversos , Azúcares de la Dieta/efectos adversos , Glándulas Mamarias Animales/embriología , Neoplasias Mamarias Animales/etiología , Neoplasias Mamarias Experimentales/metabolismo , Células Madre/patología , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Adiponectina/sangre , Animales , Peso Corporal/fisiología , Dieta con Restricción de Grasas , Conducta Alimentaria , Femenino , Resistencia a la Insulina/fisiología , Factor I del Crecimiento Similar a la Insulina/análisis , Lactancia/metabolismo , Leptina/sangre , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Animales/sangre , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/prevención & control , Neoplasias Mamarias Experimentales/sangre , Neoplasias Mamarias Experimentales/inducido químicamente , Neoplasias Mamarias Experimentales/prevención & control , Exposición Materna/efectos adversos , Ratones , Ratones Endogámicos SENCAR , Obesidad/etiología , Obesidad/metabolismo , Factores de Riesgo , Factores de TiempoRESUMEN
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.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Factor I del Crecimiento Similar a la Insulina/fisiología , Glándulas Mamarias Animales/enzimología , Transducción de Señal/fisiología , Animales , Celecoxib , Inhibidores de la Ciclooxigenasa 2/farmacología , Activación Enzimática , Ensayo de Inmunoadsorción Enzimática , Femenino , Glándulas Mamarias Animales/irrigación sanguínea , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Transgénicos , Neovascularización Patológica , Pirazoles/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Prostaglandina E/metabolismo , Sulfonamidas/farmacologíaRESUMEN
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.
Asunto(s)
Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Animales , Neoplasias de la Mama/etiología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Bovinos , Proliferación Celular , Ciclina D1/metabolismo , Estradiol/farmacología , Receptor alfa de Estrógeno/deficiencia , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Sustrato del Receptor de Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/farmacología , Sistema de Señalización de MAP Quinasas , Glándulas Mamarias Animales/crecimiento & desarrollo , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Biológicos , Comunicación Paracrina , Proteínas Proto-Oncogénicas c-raf/metabolismo , Maduración Sexual , Transducción de SeñalRESUMEN
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.
Asunto(s)
Antineoplásicos Hormonales/uso terapéutico , Receptor alfa de Estrógeno/metabolismo , Neoplasias Mamarias Experimentales/genética , Tamoxifeno/uso terapéutico , Proteína p53 Supresora de Tumor/genética , Animales , Transformación Celular Neoplásica/genética , Receptor alfa de Estrógeno/genética , Femenino , Expresión Génica , Genotipo , Heterocigoto , Pérdida de Heterocigocidad , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/prevención & control , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína p53 Supresora de Tumor/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMEN
Estrogen receptor alpha (ER) and p53 are critical prognostic indicators in breast cancer. Loss of functional p53 is correlated with poor prognosis, ER negativity, and resistance to antiestrogen treatment. Previously, we found that p53 genotype was correlated with ER expression and response to tamoxifen in mammary tumors arising in mouse mammary tumor virus-Wnt-1 transgenic mice. These results lead us to hypothesize that p53 may regulate ER expression. To test this, MCF-7 cells were treated with doxorubicin or ionizing radiation, both of which stimulated a 5-fold increase in p53 expression. ER expression was also increased 4-fold over a 24-h time frame. In cells treated with small interfering RNA (siRNA) targeting p53, expression of both p53 and ER was significantly reduced (>60%) by 24 h. Induction of ER by DNA-damaging agents was p53 dependent as either ionizing radiation or doxorubicin failed to up-regulate ER after treatment with p53-targeting siRNA. To further investigate whether p53 directly regulates transcription of the ER gene promoter, MCF-7 cells were transiently transfected with a wild-type (WT) p53 expression vector along with a luciferase reporter containing the proximal promoter of ER. In cells transfected with WT p53, transcription from the ER promoter was increased 8-fold. Chromatin immunoprecipitation assays showed that p53 was recruited to the ER promoter along with CARM1, CBP, c-Jun, and Sp1 and that this multifactor complex was formed in a p53-dependent manner. These data show that p53 regulates ER expression through transcriptional control of the ER promoter, accounting for their concordant expression in human breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica , Proteína p53 Supresora de Tumor/genética , Neoplasias de la Mama/metabolismo , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/metabolismo , Factor de Unión a CCAAT/genética , Factor de Unión a CCAAT/metabolismo , Línea Celular Tumoral , Doxorrubicina/farmacología , Receptor alfa de Estrógeno/biosíntesis , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Transcripción Genética , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Insulin-like growth factor-1 (IGF-1) stimulates proliferation, regulates tissue development, protects against apoptosis, and promotes the malignant phenotype in the breast and other organs. Some epidemiological studies have linked high circulating levels of IGF-1 with an increased risk of breast cancer. To study the role of IGF-1 in mammary tumorigenesis in vivo, we used transgenic mice in which overexpression of IGF-1 is under the control of the bovine keratin 5 (BK5) promoter and is directed to either the myoepithelial or basal cells in a variety of organs, including the mammary gland. This model closely recapitulates the paracrine exposure of breast epithelium to stromal IGF-1 seen in women. Histologically, mammary glands from transgenic mice were hyperplastic and highly vascularized. Mammary glands from prepubertal transgenic mice had significantly increased ductal proliferation compared with wild-type tissues, although this difference was not maintained after puberty. Transgenic mice also had increased susceptibility to mammary carcinogenesis, and 74% of the BK5.IGF-1 mice treated with 7,12-dimethylbenz[a]anthracene (20 microg/day) developed mammary tumors compared with 29% of the wild-type mice. Interestingly, 31% of the vehicle-treated BK5.IGF-1 animals, but none of the wild-type animals, spontaneously developed mammary cancer. The mammary tumors were moderately differentiated adenocarcinomas that expressed functional, nuclear estrogen receptor at both the protein and mRNA levels. These data support the hypothesis that tissue overexpression of IGF-1 stimulates mammary tumorigenesis.
Asunto(s)
Adenocarcinoma/metabolismo , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Neoplasias Mamarias Experimentales/metabolismo , Comunicación Paracrina/fisiología , Adenocarcinoma/patología , Animales , Western Blotting , Bovinos , Ciclina D1/biosíntesis , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Queratina-5/biosíntesis , Queratina-8/biosíntesis , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos , Microscopía Confocal , Regiones Promotoras Genéticas , Receptores de Estrógenos/biosíntesis , Receptores de Progesterona/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In the past 20 yr, several inbred strains have been derived from SENCAR outbred mice. These strains display different susceptibility to the induction of papillomas and progression to squamous cell carcinomas (SCC) in the skin after chemical carcinogenesis. In the present study, we showed that one of these strains SENCARB/Pt was highly susceptible to the development of N-methyl-N-nitrosourea (MNU)- and 7,12-dimethylbenz[a]anthracene (DMBA)-induced lymphomas. In contrast, the SSIN/Sprd inbred strain is completely resistant to T-cell lymphomagenesis by both carcinogens. Within 175 d after a single injection of 75 mg/kilogram body weight (kbw) of MNU, SENCARB/Pt mice exhibited a 91.6% incidence of lymphoma. In addition, during an independent tumorigenesis study with repeated doses of intragastric DMBA, SENCARB/Pt mice showed an incidence of 75% lymphoma development 300 d after the last treatment. Histopathological and flow cytometric parameters indicated that the lymphomas were of the T-cell lineage. In order to study the genetics of MNU-induced tumorigenesis, we generated F1 hybrid mice between SSIN/Sprd and SENCARB/Pt mice. Tumor incidence in MNU-injected F1 mice suggested that the high tumor incidence is a dominant trait. Loss of heterozygosity (LOH) analysis in these tumor samples revealed allelic imbalances on chromosomes 15 and 19. Given that these inbred strains are closely related, it is likely that a relatively small number of loci are responsible for the observed differences in susceptibility. Therefore, these SENCAR inbred strains constitute important new tools to study the genetic basis of resistance and susceptibility to chemically induced thymic lymphoma formation.