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Introduction. The focus of this study is on early lifestyle factors and their effect on mammary development during puberty and how they relate to increased breast cancer risk and disparities. At this time we do not understand what biological changes occur during pubertal mammary development which leads to a greater risk of developing cancer in later life. Identifying the molecular mechanisms that cause aberrant pubertal mammary development may lead to defined strategies to reduce breast cancer burden in later life. As our bodies use the sugars that we consume for energy they generate waste chemicals known as advanced glycation end products or AGEs for short. Significantly, low income, obesity and a sedentary lifestyle are established factors driving health disparity that also contribute to increased AGE accumulation levels in our bodies. In particular, AGE content in the Western Diet has consistently increased over the last 50 years due to increased consumption of sugar-laden and cheap processed/manufactured foods which are high in reactive AGE metabolites and can promote obesity. Methods. We use a dietary mouse model to assess impact of AGE on normal mammary development. Wild type FVB/n and RAGE null (RAGE-/-) mice are fed the respective diets from weaning until 7 (pubertal) or 12 (adult) weeks of age. Mammary glands are extracted for whole mounting and paraffin embedded for histology. Fibroblasts were isolated from mammary glands and cultured ex vivo. Transwell migration assays were performed with isolated fibroblasts and HC11 mouse mammary epithelial cells. qPCR was performed on the isolated fibroblasts to assess their activation status. Results. Early life exposures during mammary development influence the breast microenvironment to increase breast cancer risk. We show that due to an innate ability to influence the cellular matrix, dietary AGEs disrupt developmental programs during puberty and promote breast tumor growth. Through receptor for AGE (RAGE) dependent and independent mechanisms, chronic AGE consumption delayed ductal extension, increased ductal branching and caused aberrant terminal end bud (TEB) morphology. Dietary AGE activation of RAGE mediated a program of activated stroma leading to hyperplastic growth and the formation of pre-neoplastic lesions which persisted into adulthood. Importantly, AGE mediated effects remained even after diet intervention after puberty. In dietary-AGE breast tumor models, AGE mediated changes in tissue architecture and cell function were recapitulated and resulted in 3-fold increase in neoplastic growth. Through the perpetual activation of a reactive stroma, AGEs derived from diet represent a common early life exposure which can influence tumor behavior. Conclusions. A greater mechanistic understanding of the link between AGE intake during puberty and increased breast cancer risk may define novel potential strategies for lifestyle and pharmacological intervention aimed at reducing breast cancer risk and cancer disparities. Citation Format: Callan C Frye, Bradley A Krisanits, Reid Schuster, Jaime Randise, Lourdes M Nogueira, Kristi Helke, Amanda C LaRue, David P Turner, Victoria J Findlay. Consumption of dietary AGEs during puberty and increased breast cancer risk: A link between lifestyle and cancer disparity [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr C029.

Evidence supports the notion that critical events during mammary development permanently alter developmentally regulated programs which influence the breast microenvironment to increase breast cancer risk. This is analogous to metabolic memory in diabetic patients where early metabolic events have been found to be remembered and affect disease severity later in life. Advanced glycation end products (AGEs) are highly reactive metabolites that irreversibly accumulate in tissues as we age. AGE accumulation can contribute to pro-inflammatory and -oxidant phenotypes when signaling through the receptor for advanced glycation end products (RAGE). The pathogenic effects of AGE-RAGE signaling include tissue degeneration, protein dysfunction, aberrant cell signaling, and reduced genetic fidelity. AGEs form during normal metabolism but critically, lifestyle factors such as poor diet, a sedentary lifestyle and being obese also contribute to the AGE accumulation pool. The permanence of AGE adducts and their ability to mediate chronic and persistent inflammatory and oxidative stresses is particularly compatible to the concept of metabolic memory. Our dietary studies in pubertal FVB/n mice fed a high AGE diet show a significant dysregulation of mammary gland development and hyperplastic lesion formation. We observe delayed mammary ductal extension, increased ductal branching and aberrant terminal end-bud (TEB) morphology. The basal myoepithelial cell layer surrounding mammary ducts and TEBs was irregular and epithelial cell proliferation was increased. Molecular characterization of these hyperplastic lesions were defined using DCIS progression markers by histopathological staining and qRT-PCR. Elevated AGE levels accompanied increased RAGE expression and increased macrophage and fibroblast infiltration around the TEBs. In attempt to reverse the effects caused by a high AGE diet, mice were fed a control diet after a pubertal high AGE diet. Hyperplastic lesions persisted despite diet intervention. Importantly, hyperplastic lesions were not observed in mice fed a control diet during puberty, then switched to a high AGE diet. These data indicate that exposure to AGE induced changes during puberty may leave a long-lasting imprint analogous to metabolic memory. In conclusion, increased AGE consumption during pubertal growth results in significant disruption of normal mammary development and the appearance of hyperplastic lesions by adulthood. Consumption of a high AGE diet despite a control diet intervention, reveals hyperplastic lesions indicative of metabolic memory. We hypothesize that the high AGE diet may leave a metabolic imprint on the mammary gland microenvironment, increasing the risk of future breast cancer development. Citation Format: Jaime F. Randise, Bradley A. Krisanits, Lourdes M. Nogueira, Kristi L. Helke, Taaliah Campbell, Victoria J. Findlay, David P. Turner. Dietary-AGE ingestion during puberty modifies the breast microenvironment to alter mammary gland development: Linking diet, development and breast cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2234.

Advanced glycation end products (AGEs) are highly reactive metabolites that irreversibly accumulate in tissues as we grow older. Accumulation of AGEs in the body can contribute to proinflammatory and pro-oxidant phenotypes when signaling through the receptor for advanced glycation end products (RAGE).The pathogenic effects of AGE-RAGE signaling include tissue degeneration, protein dysfunction, aberrant cell signaling, and reduced genetic fidelity. AGEs are formed during normal metabolism but, critically, lifestyle factors such as poor diet, a sedentary lifestyle and being obese also contribute to the AGE accumulation pool. The permanent nature of AGE adducts and their ability to mediate chronic and persistent inflammatory and oxidative stresses is particularly compatible with the concept of metabolic memory. Our dietary studies in pubertal FVB/n mice after chronic consumption of AGE show a significant dysregulation of mammary gland development and the formation of hyperplastic lesions. We observe delayed mammary ductal extension, increased ductal branching and aberrant terminal end-bud (TEB) morphology. The basal myoepithelial cell layer surrounding mammary ducts and TEBs was irregular and epithelial cell proliferation was increased. Molecular characterization of these hyperplastic lesions was defined by using markers of DCIS progression by histopathologic staining and qRT-PCR. Elevated AGE levels were accompanied by increased expression of RAGE and increased immune (macrophage) and stromal (fibroblast) infiltration around the TEBs. In an attempt to reverse the effects caused by a high-AGE diet, mice were fed a control diet after a pubertal high-AGE diet. Hyperplastic lesions persisted despite diet intervention. Importantly, hyperplastic lesions were not observed in mice fed a control diet during puberty, then switched to a high-AGE diet after. These data not only illustrate the importance of the pubertal window in the mediation of lifestyle factors but also indicate that exposure to AGE-induced changes during puberty may leave a long-lasting imprint analogous to the concept of metabolic memory. Additionally, in our feasibility studies, both pharmacologic (NCT02946996; NCT03092635) and interventional (NCT03459755) strategies were shown to reduce circulating AGE levels in cancer survivors. Based on associations between diet, lifestyle, and race, increases in AGE accumulation may represent a novel biologic mechanism contributing to cancer disparity and may represent a new paradigm contributing to the increased cancer incidence and mortality figures observed within health-disparity populations. Given the potential benefits of lifestyle changes and the potential biologic role of AGEs in promoting cancer, opportunities exist for collaborations impacting basic, translational, epidemiologic and cancer prevention initiatives. Citation Format: Bradley A. Krisanits, Jaime F. Randise, Lourdes M. Nogueira, Kristi Helke, Michael C. Ostrowski, Katie Theis, Maria Cuitino, Gayenel Magwood, Marvella E. Ford, Victoria J. Findlay, David P. Turner. Dietary-AGE ingestion during puberty modifies the breast microenvironment to alter mammary gland development: Linking lifestyle with cancer disparity [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C025.