The Role of the Gut-Breast Axis During Critical Windows of Development and its Effects on Mammary Gland Development Later in Life

Abstract

During critical developmental periods such as pregnancy, early life, and puberty, when the mammary gland is developing, any perturbations in the gut microbiome can lead to long-lasting disturbances in mammary gland homeostasis and may predispose individuals to breast cancer later in life. These perturbations, often triggered by exposure to immune stressors such as low-dose penicillin or lipopolysaccharides (LPS), can disrupt the gut microbiota equilibrium. Notably, prebiotics have emerged as a countermeasure to inflammation and microbiota imbalances caused by antibiotics or LPS exposure, though the mechanisms underlying their effectiveness have not been fully discovered. This thesis hypothesizes that antibiotic exposure during pregnancy and early life, or LPS exposure during puberty, could disrupt mammary gland homeostasis, thereby predisposing individuals to breast cancer in later life. Conversely, prebiotic intake may ameliorate disturbances associated with antibiotic- or LPS-induced inflammation by modulating the immune system epigenetically through microRNAs (miRNAs) and DNA methylation. This research aims to deepen our understanding of the mechanisms by which prebiotic intake, specifically Lentinula edodes cultured extract (AHCC), offers protective effects against the lasting immune deregulation associated with exposure to immune stressors during pivotal developmental phases. The objectives are: 1. To investigate the effectiveness of AHCC supplementation during puberty in mitigating LPS-induced inflammation and its corrective effects on mammary gland homeostasis and breast cancer risk. This will involve a comprehensive assessment of the structure and morphology of mammary glands, cytokines, miRNAs, and DNA methylation. Additionally, the study aims to measure tumor growth, the number of cancer stem cells, and the level of inflammatory cytokines in the tumor and adjacent mammary glands. 2. To explore whether AHCC intake can mitigate the potential impacts of prenatal and early-life antibiotic exposure on the development of mammary glands. This involves assessing the influence of early low-dose penicillin (LDP) on the maternal immune response, and gene expression of lactating mammary glands. The research will also analyze the immune response in offspring and gene expression of mammary glands at genetic and epigenetic levels. This study utilizes Balb/c mice, exposing them to LPS at puberty or low-dose penicillin (LDP) during pregnancy to assess the effects on mammary glands and tumor development. LDP exposure involves feeding dams in the last week of gestation through weaning. The study examines the impact of AHCC intake on mitigating the long-term consequences of LPS or LDP by analyzing immune responses, gene and miRNA expression, and DNA methylation in mammary glands and tumors. These studies highlight the effectiveness of AHCC, a prebiotic, in preventing gene dysregulation in critical genes such as Pdgfc, Ksr1, and Il2rb, especially in LPS-exposed groups. AHCC mitigates disruptions in cytokine levels (IL-1β, IL-10, IL-17a/f, IL-23) and miRNAs (let-7a/c, miR-34a, miR-130a) in mammary glands due to puberty LPS exposure. It also counters LPS-induced cytokine and miRNA alterations in tumor samples, particularly affecting IL-1β, IL-6, and IL-10 levels, along with the TGF-β family. AHCC regulates cytokine expression, reduces the IL-6 upsurge in mammary glands, and increases tumor-suppressing miRNAs (miR-125, miR-140, and miR-200c), contrasting the LPS-induced rise in oncogenic miRNAs (miR-21, miR-92, and miR-155). It also controls immune responses to LDP-induced dysbiosis by altering IL-10, IgG, and IL-6 levels, and affecting a broad range of genes (Fgf1, Fgfr2, Fzd7) and miRNAs (let-7a/c, miR-34b, and miR-146a), showcasing its regulatory impact on mammary gland homeostasis through gene and microRNA expression modulation. This is the first time that the role of the gut-breast axis has been studied. The data will shed light on the importance of preventing gut dysbiosis during critical windows of development in the mammary gland during pregnancy and adolescence.