Windows of Susceptibility

Studies in humans and animals indicate that the origins of breast cancer likely occur early in development, especially during puberty, a time of rapid breast development. Animal studies show that pubertal diet and environmental exposures can increase mammary cancer susceptibility. Human and animal studies of breast cancer show inflammatory processes (IP) contribute to tumor proliferation and metastasis. Preliminary results in BALB/c mice show pubertal exposure to a high fat diet (HFD) increases pubertal mammary gland proliferation and DMBA-induced mammary tumorigenesis. The investigators will also show that pubertal exposure to HFD induces IP. Importantly, these effects occur without obesity. They hypothesize that HFD increases mammary gland proliferation and mammary tumorigenesis through induction of IP. IP are a necessary and key component of normal mammary gland development. The investigators recently identified a novel pathway linking progesterone (P) to regulation of IP during mammary gland development. They further hypothesize that P-induced RANKL expression in the mammary epithelium is critical in promoting normal IP by activating NFkB, a transcriptional regulator of proinflammatory gene products, and that RANKL is the central regulator of HFD-induced IP in the mammary gland. The investigators propose that the observed HFD-induced RANKL expression, subsequent IP activation through NFkB, and peri-epithelial infiltration of macrophages and eosinophils promote both mammary gland proliferation and tumorigenesis. They will test these hypotheses during normal pubertal mammary gland development, and during DMBA and p53-/- carcinogenesis in BALB/c mice fed control and HFD. The investigators will also test several experimental intervention strategies to overcome the negative effects of diet on inflammation, and on mammary cancer development. Understanding the regulation of IP in normal pubertal mammary gland development, as well as in carcinogenesis, may provide relevant information about how other environmental factors (i.e., endocrine disrupters, toxicants) may increase susceptibility to mammary cancer. With their community breast cancer advocacy partner, Michigan Breast Cancer Coalition, the investigators will translate and communicate their research findings to the concerned public.

Breast cancer (BC) is a hormone-dependent malignancy. Endocrine disrupters (EDs) are exogenous substances with hormonal activity in the endocrine system that disrupts the physiologic function of endogenous hormones. Animal studies unequivocally demonstrated that EDs disrupt development of mammary tissues and increase the risk of mammary tumors depending upon the dose and timing of exposure. However, evidence directly linking EDs and BC in human populations is far from conclusive. Two major issues have seriously limited translation of laboratory experiments into population studies. First, the window of susceptibility has not been clearly elucidated in humans. Second, most animal studies have tested one ED at a time often with doses orders of magnitude higher than common human exposure. Thus dose-extrapolations are usually performed neglecting possible synergistic or antagonist effects in a complex mixture of EDs that humans are more likely to encounter. Population studies of ED-BC associations are urgently needed that incorporate realistic exposure schemes and the concept of susceptibility windows. This proposed study uses an innovative approach of combining both an animal and a population study. First, animals will be exposed to three commonly-used EDs (individually and in combination) in physiological relevant doses at 6 different windows of development. Whole-genome expression and pathological features of rat mammary tissues will be profiled to provide both quantitative and qualitative biological effects of EDs. The investigators will then validate the laboratory findings in the population-based Long Island Breast Cancer Study Project (LIBCSP) through the use of its extensive database and biorepository. The overarching goal is to explore whether environmental EDs are acting in specific developmental windows and whether they exert their biological effects independently or synergistically/antagonistically in breast tissue leading to breast cancer development. Results from this study would provide invaluable information on effects of environmental exposure during women’s life course, which may be used to direct future molecular epidemiological studies on ED exposure and BC. Should this study approach provide the information as expected, it will lead to the development of an animal model that can be used to study a wide range of EDs and other exogenous factors with significant implications for future investigations on the timing of environmental exposures to human diseases.

The investigators propose a unique study of human exposure to organchlorines during two critical windows of exposure for the breast: 1) pregnancy when preparation for lactation places the breast at risk for carcinogenesis in the mother and 2) the prenatal period when breast differentiation places the breast at risk for carcinogenesis in the daughter. There are few human studies where exposure to endocrine active compounds during these critical periods can be measured directly in relation to subsequent breast cancer risk. The proposed research addresses this gap and represents a novel and unique opportunity, efficiently using an existing cohort that spans two generations. The investigators will also develop the capacity for cohort members to participate as full research partners, enhancing the relevance and success of continuing breast cancer research in this unique study population. This study is made possible by a 50-year follow-up of the Child Health and Development Studies Pregnancy Cohort (CHDS). Maternal pregnancy serum samples were collected during peak exposure to organochlorine pesticides and polychlorinated biphenyls in the 1960’s, prior to the ban on these chemicals. The proposed research funds organochlorine serum assays for recently diagnosed cases of breast cancer in mothers to extend the sample size for a prior published study of maternal breast cancer, collects 200 mammograms for daughters of mothers who have been diagnosed with breast cancer, assays 400 adult daughter specimens for DNA methylation, supports new data analysis to 1) test a novel hypothesis that maternal exposure during a critical window (prior to age 14) increases susceptibility of the fetus to the effects of environmental exposures in the womb; 2) test a well accepted hypothesis where data have not heretofore been available; that daughters of women who developed breast cancer are more susceptible to environmental exposures in the womb; and 3) examine the role of pregnancy hormone exposures in explaining observed organochlorine associations with breast cancer in mothers and age at menarche, mammographic density and DNA methylation of their daughters.

Humans are exposed to a complex mixture of environmental chemicals. This is evident from the internal concentrations of these chemicals in humans, such as those from girls recruited via the Breast Cancer and the Environment Research Program. It is the investigators’ goal to determine the potential of combinational, low dose exposures to three of these common environmental chemicals, administered orally in rats during the prepubertal period, to alter lifetime mammary cancer susceptibility. The investigators hypothesize that combinational exposure to Bisphenol A (BPA) and di-2-ethylhexyl phthalate (DEHP) will predispose for an additive or synergistic increase in mammary cancer development in a rodent model of mammary cancer. Furthermore, they will investigate the potential of genistein, when administered concurrently, to suppress the ability of BPA and DEHP to increase mammary cancer susceptibility. The specific aims are: 1) to determine if oral, prepubertal exposure to the combination of BPA and DEHP will result in an additive or synergistic increase in chemically-induced mammary cancer in rats, and if dietary genistein will result in negating the effects of BPA and DEHP; 2) to identify genomic signatures, via gene microarrays, from mammary glands of rats exposed to these chemicals to elucidate affected signaling pathways; 3) using the information from the gene array data and MIRA-seq (methylated CpG islands recovery assay coupled with massively parallel sequencing), the investigators will identify candidate DNA methylated genes and determine if prepubertal BPA, DEHP and genistein will effect epigenetic DNA methylation; 4) to investigate in vivo mechanisms of action at the cellular and protein levels (mammary gland morphology, cell proliferation, apoptosis and signaling pathways determined to be differentially regulated via gene array and eipigenetic assays); and 5) to measure concentrations of chemicals in blood and urine of rats exposed, alone or in combination, to BPA, DEHP and/or genistein during the prepubertal period. The identification of early life combinational chemical exposures commonly reported to occur in humans to alter mammary cancer susceptibility will facilitate our understanding of the complex role environmental chemicals play in breast cancer causation and prevention.

It is thought that endocrine-disrupting chemicals cause the most harm to developing bodies. They may change the timing of when a girl develops breasts or gets her first period. Girls who get their first periods earlier than expected seem to be at greater risk of developing breast cancer as an adult. This is why researchers are exploring the effect of certain endocrine-disrupting chemicals on girls, and whether or not reducing a girl’s exposure might help lower her later risk of developing breast cancer.

Two types of endocrine-disrupting chemicals that researchers are studying particularly closely are phthalates (THA-lates) and bisphenol A (BPA). Both are found in certain types of plastic food containers. BPA is also sometimes found in the lining of canned foods. Phthalates are also found in many personal care products (like cosmetics, fragrance, nail polish, deodorant, hair care, and body lotion).

For more information:

• Fact sheet on endocrine disruptors – National Institute of Environmental Health Sciences (NIEHS)
• Fact Sheet: The Chemical Connection (color) – PDF
• Endocrine Disruptors – NIEHS
• What are Endocrine Disruptors – U.S. Environmental Protection Agency

View a glossary of terms and links to additional information

Breast cancer is the second leading cause of cancer deaths in women and is the most common cancer among women. Exposures to environmental agents can have an effect on mammary gland development and may affect breast cancer risk. This project explores the mechanisms underlying windows of susceptibility during across the lifespan. By defining the molecular architecture of the developing and changing mammary gland over the lifespan the investigators will be able to develop new and improved animal models and biomarkers to study the Impact of environmental stressors on breast cancer, elucidate the effects of timing of exposure during critical windows of vulnerability in both normal and tumor prone animals. This project will use state-of-the-art imaging, proteomic and genetic tools to determining when, which and how environmental insults regulate cell behavior in the mouse mammary gland during development in vivo, and in conversion of mammary epithelial cells to premalignant phenotypes. Experiments will be conducted cooperatively, using organotypic cultures and mouse models to characterize pathways related to breast development during early life, puberty, pregnancy, and other time points, and to determine how they are affected by exposures to environmental stressors occurring at different windows of vulnerability. These studies in mice will be complemented using human breast epithelium in culture to develop new assays that can assess how exogenous agents alter their ability to overcome finite lifespan and constraints imposed by intercellular interactions. By defining critical windows of vulnerability, and creating improved animal and human cell culture models that will lead to identification of biomarkers, these studies have the potential for translation to women to determine the impact of environmental stressors on breast cancer. The investigators propose to determine the alterations in the mechanical properties of mammary microenvironment in mice in vivo and the mammary epithelial cells during the life cycle. The investigators will determine the effects of exposure to prototypical environmental stressors during the life cycle on the mammary gland in normal and cancer prone mice in vivo and in human breast specimens. They will use in vitro mechanism-based assays to detect agents that possess signatures indicative of possible mammary gland carcinogenicity. This project will participate in the BCERP Network activities by exchange of information, interactions and collaborations. An essential feature of this proposal is regular bidirectional communication with the community-based advocacy group to focus on appropriate environmental stressors and to develop biomarkers and to translate the findings into lay terms.

Breast cancer (BCa) is the leading cause of death in women; incidence rates continue to rise globally. Genetic predisposition accounts for less than 15% of BCa risk while main etiological factors are those related to environmental exposure and lifestyle choices. Immigrants to the US from low-risk countries develop a higher BCa risk within one generation. Moreover, risk in the second generation is dependent on whether one is born to mother from homeland or US. The latter finding suggests BCa is of fetal origin. A diet high in fat is likely a major risk factor of BCa in the U.S. Rodent studies showed that the type of fat and the particular critical window of exposure are key determinants of this linkage. The environmental estrogen, Bisphenol A (BPA), found ubiquitously in US populations, is also a suspect. Rats exposed to BPA pre-/peri-natally are more susceptible to DMBA-induced BCa than their unexposed counterparts. Although both BPA and high-fat diets have been separately studied, whether the two, given together, have synergistic action has not been investigated. More importantly, epigenetics, a known mechanism underlying gene by environment interaction, has not been studied in the context of developmental origin of BCa. The investigators recently showed that rats prenatally exposed to high-fat diets (39% of kcal) exhibited a higher DMBA-induced BCa susceptibility than controls exposed to a reference diet (16% of kcal). The high-fat diets also induced marked increases in epithelial cell proliferation along with a unique proliferation gene signature identified by global transcriptome profiling. Using an unbiased methylome profiling technique, the investigators also generated preliminary data in support of epigenetic reprogramming of gene expression in adult mammary glands after prenatal exposure to high-fat diets. Here, they propose the gestational period is a critical developmental window for dietary fatty acids-BPA interaction, that reprograms the mammary methylome, resulting in aberrant gene expression and increased BCa risk in adulthood. Three specific alms are proposed to test this hypothesis and translate findings Into public health action through community outreach: Aim 1: To characterize the dose-response of dietary BPA on the developmental effects of high-fat diets on BCa risk in later-life; Aim 2: To delineate (a) if the type of fat matters in synergizing with an effective developmental dose of BPA in elevating adult BCa risk, and (b) if a methylome, along with an aberrant mammary epithelial cell proliferative gene signature, is reflective of the combined exposure; and Aim 3: To translate research findings into public awareness and action to reduce the burden of adverse lifestyle choices and environmental pollutants on BCa risk through an outreach program that partners with the Pink Ribbon Girls. This study will be the first to examine epigenetics as a mechanism underlying lifestyle choices by environmental toxicant Interaction.

Epidemiologic and experimental data have shown that a full term pregnancy reduces breast cancer risk. However, recent studies have suggested that while full term pregnancy does reduce risk for estrogen receptor and luminal breast cancers, pregnancy may actually increase risk of more aggressive basal-like breast cancers. There are complex relationships between age, race, parity, and obesity in observational human datasets making it difficult to translate these findings into public health messages – behavioral variables such as obesity and pregnancy are often correlated. Experimental studies using rodents have examined parity and obesity individually, but to date, the independent and joint effects of parity and obesity have not been dissected. This proposal will address this gap and will do so within the context of tumor heterogeneity, focusing specifically on the basal-like breast cancer subtype and the microenvironment changes that promote this breast cancer subtype during a vital window of susceptibility, the post partum period. In aim 1, mouse models of basal-like and heterogeneous breast cancer will be used to study the tumor promoting effects of pregnancy and high fat diet. Endpoints will include tumor latency, tumor mass, gene expression and microRNA changes induced by pregnancy and/or obesity. Macrophage infiltration, an important variable in cancer progression and obesity pathogenesis will be characterized in the microenvironment of the tumors that form. In aim 2, a co-culture system will be used to model the effects of obesity- and pregnancy-associated macrophage infiltration on basal-like and luminal breast cancers. In aim 3, the investigators will conduct ancillary histology and expression studies on normal breast tissue from an ongoing study of breast microenvironment and utilize available gene expression and demographic information from that parent study in their analyses. Comparison of results across in vitro and in vivo systems and across species will help to identify the most important pathways and/or biomarkers that are differentially regulated by parity and obesity in the basal-like microenvironment. In aim 4, the investigators will identify and address the knowledge needs of their target population and disseminate outreach tools through a network of national advocates.

An individual woman’s susceptibility to breast cancer is determined by her inherited genetic susceptibility and her personal environment as well as interactions between and within genes and environmental factors. The environment is also complex, consisting of both endogenous and exogenous factors. Importantly, in breast cancer, the susceptibility to environmental agents is highly dependent on the physiological and developmental status of the breast at the time of exposure, i.e., windows of susceptibility. Several major hormonal landmarks in a woman’s life impact on the breast’s developmental status and modulate susceptibility. This proposal will focus on three post-natal windows of breast cancer susceptibility using both rat and human studies. In rats, these windows include the “immature” window (3 week old rats) corresponding to childhood in humans, the “adolescent” window (7 week old rats), and the novel “older adult” window (65 week old rats) corresponding to peri- menopause in women. It is hypothesized that susceptibility to environmental induced breast cancer is modulated by window-specific risk alleles/genes. Results obtained from these rat models will be extended to women using a comparative genomics approach focusing on genetic epidemiological investigations. Aim 1 will determine if seven previously identified mammary carcinoma susceptibility (Mcs) alleles that have been shown to modulate mammary cancer risk at the adolescent window are unique for this window or will also function at the immature or older adult windows. Aim 2a will phenotype mammary gland at all three windows by integrating gland morphology with global gene expression. Aims 2b and 2c will ask if mammary carcinomas induced by exposure at different windows show window-specific patterns of gene expression and pathway signaling. These expression patterns will be compared to human breast cancer gene expression patterns. Aim 3 will extend work on previous aims by genetically identifying unique quantitative risk loci (QTL) that are not active at the adolescent window but modulate risk at the other two windows. Aim 4 will focus on a comparative genomics/molecular epidemiologic case-control study of over 7,000 women to identify established and suspected GWAS SNPs in human regions homologous to our loci (Aim 1) and QTL (Aim 3). Importantly, all major study planning will include input from partners affiliated with several advocacy and community groups within Wisconsin (Aim 5). Advocates and scientists will jointly communicate important results from these studies as well as studies from other BCERP collaborators.