Pregnancy and Breast Cancer: Pathways to Understand Risk and Prevention  Priscila F. Slepicka, Samantha L. Cyrill, Camila O. dos Santos  Trends in Molecular Medicine  DOI: 10.1016/j.molmed.2019.06.003 Copyright © 2019 The Authors Terms and Conditions

Figure 1 The Clock of Mammary Gland Development. A schematic representation of a murine mammary duct throughout the mammary development cycle: birth, puberty, pregnancy, lactation, involution, and post-pregnancy (clockwise). Ductal branching at each developmental stage is depicted in the outer circle. The ducts comprise luminal cells (inner layer) and basal cells (outer layer), and are surrounded by the basal membrane, fibroblasts, and adipocytes (fat pad). At birth, the duct system is rudimentary. Estrogen and progesterone trigger puberty: cell differentiation and the formation of terminal end buds (TEBs) that invaginate into the fat pad to promote ductal expansion. With conception, prolactin signals differentiation of luminal alveolar cells and tertiary ducts, and lobuloalveolar structures begin to form during early pregnancy. Adipocyte depletion makes way for ductal tissue expansion and mammary epithelial cell (MEC) differentiation. By the end of gestation, luminal alveolar cells increase milk production, whereas oxytocin (Oxt) during lactation triggers myoepithelial cell contraction, luminal constriction, and milk release into the lumen. The initial phase of involution is reversible, whereas during the succeeding irreversible phase MEC apoptosis and adipogenesis take place, macrophages clear dead MECs, and alveolar cells undergo apoptosis. Meanwhile, the extracellular matrix (ECM) is reconstituted with linearized, highly fibrillar collagen, and adipogenesis continues. Immediately post-pregnancy, protumor attributes emerge: heterogeneous cell populations (parity-induced MECs, PI-MECs) trigger tumorigenic pathways, the expansion and self-renewal of luminal progenitors, remodeling of the ECM with high fibrillar collagen, and macrophage infiltration. In the later parous antitumor microenvironment, fibrillar collagen organizes randomly in the ECM and DNA hypomethylation modulates gene expression and the establishment of antitumorigenic pathways in MECs. Trends in Molecular Medicine DOI: (10.1016/j.molmed.2019.06.003) Copyright © 2019 The Authors Terms and Conditions

Figure 2 Signaling Networks in Parity-Induced Tumorigenesis and in Early Parity Protection against Breast Cancer. (A) Parity-Induced mammary tumorigenesis. In the presence of p63, p53, and STAT3 pathways, the gene expression of luminal markers (K18 and CD24) is inhibited whereas STAT5 and NRG1 pathways and gene expression of the basal cell-surface marker, K14, are triggered, further increasing proliferation signals in basal cells. Overexpression of p63 in luminal CD10− cells increases K14 expression while reducing the expression of K18 and CD24, leading to basal cell fate determination. pSTAT5 translocates to the nucleus and initiates the expression of cyclin D1, whose activity increases prosurvival signals in parity-induced mammary epithelial cells (PI-MECs). Notch1 and p63 play antagonistic roles in tumorigenesis. Whereas p63 steers the pathway towards basal transformation, increased Notch1 activation in mammary stem cells (MaSCs) engages luminal lineage commitment and promotes the self-renewal and proliferation of luminal alveolar progenitors. (B) Early parity protection. Early parous glands show an overall decrease in the pool of luminal hormone-sensing cells (including p27+ cells), which reduces the expression and secretion of Wnt4. The ‘X’ represents the lack of Wnt signaling in basal progenitors/MaSCs that downregulates the expression of Wnt-related genes (Lgr5, Axin2, Vcan). Increased Notch pathway signaling leads to the expression of its gene targets and prodifferentiation genes, inducing differentiation but reducing proliferative signaling. Proliferation and cyclin D1/Cdk4 pathways are decreased because p27+ cells (luminal progenitors and CD44+) are less prominent in early-parous glands. Early parity also induces gene hypomethylation, thereby altering the cellular gene expression profile. Trends in Molecular Medicine DOI: (10.1016/j.molmed.2019.06.003) Copyright © 2019 The Authors Terms and Conditions

Figure 3 Advanced Technologies for Studying Breast Cancer Development and Prevention in Humans. Schematic representation of models derived from ex vivo human mammary epithelial tissue for molecular analysis. (A) Human breast biopsy material (i.e., mammary tumor) is extracted and fragmented using mechanical shearing. Controlled treatment with digestive enzymes is then used to either obtain mammary epithelial cell (MEC) clusters (short dissociation duration, low-speed centrifugal separation) or MEC single-cell suspensions (long dissociation duration, high-speed centrifugal separation). (B) Preparation of 3D mammary organoid cultures. MEC clusters are plated in a 3D matrix to form organoids. These organoids can then be stored, in cryoprotective medium, in Biobanks or used to assay drug sensitivity or molecular tumor dynamics. (C) Preparation of patient-derived xenograft (PDX) models of human tumors. MECs, in single-cell suspensions, are labeled with fluorescently tagged antibodies targeting specific cell-surface markers and sorted into specific cell populations using fluorescence-assisted cell sorting. For instance, basal and luminal cells are distinguished from stromal cells as CD24+CD29high and CD24highCD29+, respectively. The sorted tumor cells are then injected into immunocompromised mice as a PDX. After or before tumor development, mice undergo treatment with new compounds and drugs, and the response to treatment is established according to several analyses that include drug sensitivity and resistance, gene signatures and molecular profiling, histopathology, and further reanalysis of MEC composition. Trends in Molecular Medicine DOI: (10.1016/j.molmed.2019.06.003) Copyright © 2019 The Authors Terms and Conditions