Background In utero endocrine disruption is linked to increased risk of breast cancer later in life. Despite numerous studies establishing this linkage, the long-term molecular changes that predispose mammary cells to carcinogenic transformation are unknown. Herein, we investigated how endocrine disrupting compounds (EDCs) drive changes within the stroma that can contribute to breast cancer susceptibility. Methods We utilized bisphenol A (BPA) as a model of estrogenic endocrine disruption to analyze the long-term consequences in the stroma. Deregulated genes were identified by RNA-seq transcriptional profiling of adult primary fibroblasts, isolated from female mice exposed to in utero BPA. Collagen staining, collagen imaging techniques, and permeability assays were used to characterize changes to the extracellular matrix. Finally, gland stiffness tests were performed on exposed and control mammary glands. Results We identified significant transcriptional deregulation of adult fibroblasts exposed to in utero BPA. Deregulated genes were associated with cancer pathways and specifically extracellular matrix composition. Multiple collagen genes were more highly expressed in the BPA-exposed fibroblasts resulting in increased collagen deposition in the adult mammary gland. This transcriptional reprogramming of BPA-exposed fibroblasts generates a less permeable extracellular matrix and a stiffer mammary gland. These phenotypes were only observed in adult 12-week-old, but not 4-week-old, mice. Additionally, diethylstilbestrol, known to increase breast cancer risk in humans, also increases gland stiffness similar to BPA, while bisphenol S does not. Conclusions As breast stiffness, extracellular matrix density, and collagen deposition have been directly linked to breast cancer risk, these data mechanistically connect EDC exposures to molecular alterations associated with increased disease susceptibility. These alterations develop over time and thus contribute to cancer risk in adulthood.

The pervasive nature of estrogenic industrial and dietary compounds is a growing health concern linked to cancer, obesity, and neurological disorders. Prior analyses of endocrine disruptor action have focused primarily on the short-term consequences of exposure. However, these studies are unlikely to reflect the consequences of constant exposures common to industrialized countries. Here we examined the global effects of long-term endocrine disruption on gene transcription and estrogen signaling. Estrogen-dependent breast cancer cell lines were chronically treated with physiologically relevant levels of bisphenol A or genistein for more than 70 passages. Microarray analysis demonstrated global reprogramming of the transcriptome when compared with a similarly cultured control cell line. Estrogen-responsive targets showed diminished expression in both the presence and absence of estrogen. Estrogen receptor recruitment, H3K4 monomethylation, and deoxyribonuclease accessibility were reduced at nearby response elements. Based on these observations, we investigated the potential of long-term endocrine disruptor exposure to initiate persistent transcriptional reprogramming. Culture of chronically exposed cell lines in the absence of the endocrine disruptors did not reverse many of the signaling defects that accumulated during treatment. Taken together, these data demonstrate that chronic exposure to endocrine disrupting compounds can permanently alter physiological hormone signaling.

AbstractIn utero endocrine disruption is linked to increased risk of breast cancer later in life. Despite numerous studies establishing this linkage, the long-term molecular changes that predispose mammary cells to carcinogenic transformation are unknown. Several lines of evidence indicate the stroma mediates endocrine disruption following early-life (or in utero) exposure. Herein, we utilized BPA as a model of estrogenic endocrine disruption to analyze the long-term consequences in the stroma. Using RNA-seq transcriptional profiling of adult primary fibroblasts isolated from female mice exposed to BPA in utero, we identified deregulated genes associated with the extracellular matrix. Specifically, multiple collagen genes had increased expression in exposed mice. In line with the transcriptional data, collagen deposition is increased in adult BPA-exposed mice. We further demonstrate in vitro that fibroblasts exposed to BPA in utero remodel a collagen matrix, thereby decreasing permeability of the collagen matrix. These alterations to the mammary gland resulted in increased gland stiffness in the adult mice. Our data connects early life endocrine disruption to breast density. Interestingly, increased collagen deposition and gland stiffness were not observed in the developing glands of younger mice, suggesting risk factors for breast cancer continue to develop throughout life following these exposures. Finally, we assessed whether in utero exposure to two other endocrine disruptors, BPS and DES, also increase breast stiffness in adult mice. While DES increased breast stiffness, BPS did not, suggesting this BPA alternative may in fact pose less breast cancer risk than its predecessor. As breast stiffness, extracellular matrix density, and collagen deposition have been directly linked to breast cancer risk, these data mechanistically link endocrine disruptor exposures and molecular alterations to increased disease susceptibility in the gland.