Mouse Mammary Gland Research Articles

Vulnerable periods for the mouse mammary gland: Comparison of the effects of ethinyl estradiol exposures during two early stages of development

The mammary gland is responsive to endogenous hormones and environmental chemicals that are estrogen receptor (ER) agonists. The mouse mammary gland offers the opportunity to dissect the most sensitive windows of exposure. 17α-ethinyl estradiol (EE2) is a pharmaceutical ER agonist that often serves as a positive control for estrogen-active chemicals. Here, adult female mice were exposed to EE2 starting either at pregnancy day 7, or on lactational day 1, and exposures continued until the litters were weaned. The pups were therefore exposed during gestation + the juvenile period, or during the juvenile period alone. The morphology of the mammary gland was evaluated in both male and female offspring at two life stages: weaning (postnatal day [PND]21) and at puberty (PND32). Other hormone-sensitive outcomes evaluated included body weight, anogenital index, frequency of open vagina, and weight of the uterus. We found age- and sex-dependent effects of EE2 on these estrogen-responsive endpoints including the morphology of the mammary gland. Importantly, EE2 altered mammary gland morphology even when exposures were limited to the juvenile period. However, the number of endpoints that were affected in animals from the EE2-Juvenile-Only period were fewer, and typically of a lower magnitude, compared to those observed in the EE2-Gest-Juvenile group. Understanding the effects of environmental estrogen exposures during the juvenile period is critical because humans are exposed to estrogenic pollutants throughout life, including in early childhood.

The chromatin remodeler DEK promotes proliferation of mammary epithelium and is associated with H3K27me3 epigenetic modifications.

The DEK chromatin remodeling protein was previously shown to confer oncogenic phenotypes to human and mouse mammary epithelial cells using in vitro and knockout mouse models. However, its functional role in normal mammary gland epithelium remained unexplored. We developed two novel mouse models to study the role of Dek in normal mammary gland biology in vivo . Mammary gland-specific Dek over-expression in mice resulted in hyperproliferation of cells that visually resembled alveolar cells, and a transcriptional profile that indicated increased expression of cell cycle, mammary stem/progenitor, and lactation-associated genes. Conversely, Dek knockout mice exhibited an alveologenesis or lactation defect, resulting in dramatically reduced pup survival. Analysis of previously published single-cell RNA-sequencing of mouse mammary glands revealed that Dek is most highly expressed in mammary stem cells and alveolar progenitor cells, and to a lesser extent in basal epithelial cells, supporting the observed phenotypes. Mechanistically, we discovered that Dek is a modifier of Ezh2 methyltransferase activity, upregulating the levels of histone H3 trimethylation on lysine 27 (H3K27me3) to control gene transcription. Combined, this work indicates that Dek promotes proliferation of mammary epithelial cells via cell cycle deregulation. Furthermore, we report a novel function for Dek in alveologenesis and histone H3 K27 trimethylation.

Restoration of TFPI2 by LSD1 inhibition suppresses tumor progression and potentiates antitumor immunity in breast cancer

Histone lysine-specific demethylase 1 (LSD1) is frequently overexpressed in triple negative breast cancer (TNBC), which is associated with worse clinical outcome in TNBC patients. However, the underlying mechanisms by which LSD1 promotes TNBC progression remain to be identified. We recently established a genetically engineered murine model by crossing mammary gland conditional LSD1 knockout mice with Brca1-deficient mice to explore the role of LSD1 in TNBC pathogenesis. Cre-mediated Brca1 loss led to higher incidence of tumor formation in mouse mammary glands, which was hindered by concurrent depletion of LSD1, indicating a critical role of LSD1 in promoting Brca1-deficient tumors. We also demonstrated that the silencing of a tumor suppressor gene, Tissue Factor Pathway Inhibitor 2 (TFPI2), is functionally associated with LSD1-mediated TNBC progression. Mouse Brca1-deficient tumors exhibited elevated LSD1 expression and decreased TFPI2 level compared to normal mammary tissues. Analysis of TCGA database revealed that TFPI2 expression is significantly lower in aggressive ER-negative or basal-like BC. Restoration of TFPI2 through LSD1 inhibition increased H3K4me2 enrichment at the TFPI2 promoter, suppressed tumor progression, and enhanced antitumor efficacy of chemotherapeutic agent. Induction of TFPI2 by LSD1 ablation downregulates activity of matrix metalloproteinases (MMPs) that in turn increases the level of cytotoxic T lymphocyte attracting chemokines in tumor environment, leading to enhanced tumor infiltration of CD8+ T cells. Moreover, induction of TFPI2 potentiates antitumor effect of LSD1 inhibitor and immune checkpoint blockade in poorly immunogenic TNBC. Together, our study identifies previously unrecognized roles of TFPI2 in LSD1-mediated TNBC progression, therapeutic response, and immunogenic effects.

Liriodendrin stimulates proliferation and milk protein synthesis of mammary epithelial cells via the PI3K-DDX18 signaling.

Liriodendrin is a lignan compound that is involved in a wide variety of physiological functions, however it is unknown whether liriodendrin plays an important role in milk production in the mammary glands. In this study, we explored the role and molecular mechanism of Liriodendrin in milk synthesis of mammary epithelial cells (MECs). Bovine MECs were treated with liriodendrin (0, 0.45, 0.9, 1.35, 1.8, and 2.25mM) for 24 h. Liriodendrin dose-dependently increased cell number, cell cycle transition, and milk protein synthesis, as well as Cyclin D1 and mTOR phosphorylation, with the maximal effects observed at a dose of 1.35mM. Liriodendrin increased the expression of DDX18, which mediated liriodendrin stimulation of Cyclin D1 and mTOR mRNA expression. PI3K inhibition and DDX18 knockdown experiments further confirmed that liriodendrin regulates the mRNA expression of Cyclin D1 and mTOR via the PI3K-DDX18 signaling. Mouse feeding experiment showed that liriodendrin dose-dependently promotes β-casein and DDX18 expression in mouse mammary gland. In this study, DDX18 was found to be a novel positive regulator that plays a role in cell proliferation and synthesis of milk protein. These findings reveal that liriodendrin stimulates proliferation and milk protein synthesis of MECs via the PI3K-DDX18 signaling.

Disruption of Circadian Clock Induces Abnormal Mammary Morphology and Aggressive Basal Tumorigenesis by Enhancing LILRB4 Signaling.

Epidemiological studies have shown that circadian rhythm disruption (CRD) is associated with the risk of breast cancer. However, the role of CRD in mammary gland morphology and aggressive basal mammary tumorigenesis and the molecular mechanisms underlying CRD and cancer risk remain unknown. To investigate the effect of CRD on aggressive tumorigenesis, a genetically engineered mouse model that recapitulates the human basal type of breast cancer was used for this study. The effect of CRD on mammary gland morphology was investigated using wild-type mice model. The impact of CRD on the tumor microenvironment was investigated using the tumors from LD12:12 and CRD mice via scRNA seq. ScRNA seq was substantiated by multiplexing immunostaining, flow cytometry, and realtime PCR. The effect of LILRB4 immunotherapy on CRD-induced tumorigenesis was also investigated. Here we identified the impact of CRD on basal tumorigenesis and mammary gland morphology and identified the role of LILRB4 on CRD-induced lung metastasis. We found that chronic CRD disrupted mouse mammary gland morphology and increased tumor burden, and lung metastasis and induced an immunosuppressive tumor microenvironment by enhancing LILRB4a expression. Moreover, CRD increased the M2-macrophage and regulatory T-cell populations but decreased the M1-macrophage, and dendritic cell populations. Furthermore, targeted immunotherapy against LILRB4 reduced CRD-induced immunosuppressive microenvironment and lung metastasis. These findings identify and implicate LILRB4a as a link between CRD and aggressive mammary tumorigenesis. This study also establishes the potential role of the targeted LILRB4a immunotherapy as an inhibitor of CRD-induced lung metastasis.

Bacillus subtilis Feed Supplementation Combined with Oral E. coli Immunization in Sows as a Tool to Reduce Neonatal Diarrhea in Piglets.

To investigate the effects of B. subtilis on the specific immune response of lactating sows to E. coli and the diarrhea rate in suckling piglets, thirty large white sows with similar farrowing dates were randomly divided into two groups: a feedback feeding (i.e., feeding a homogenate of intestinal contents and tissues from E. coli-infected piglets to sows; FB) group and a feedback feeding with B. subtilis (FB + BS) group. Serum, colostrum, and intestinal tissues from sows and piglets were collected to assess the immune response and intestinal barrier function at weaning. T and B cells from Peyer's patches (PPs) and mesenteric lymph nodes (MLNs) in lactating mice (with treatments consistent with the sows') were isolated to explore the underlying mechanism. The results showed that, compared with the FB group, the reproductive performance of sows and the growth performance of their offspring were effectively improved in the FB + BS group. Moreover, the levels of IgG/IgA and those of IgG/IgA against E. coli in the serum and colostrum of sows in the FB+BS group were increased (p < 0.05). Meanwhile, the ratio of CD4+/CD8+, CD4+CXCR5+PD1+, and B220+IgA+ cells in MLNs and PPs, and the IgA levels in the mammary glands of mice, were also increased in the FB + BS group (p < 0.05). Notably, in suckling piglets in the FB + BS group, the diarrhea rate was decreased (p < 0.05), and the intestinal barrier function and intestinal flora composition at weaning were significantly improved. Overall, these results indicated that B. subtilis feed supplementation combined with feedback feeding in pregnant and lactating sows can reduce diarrhea in suckling piglets by enhancing the maternal immune response against E. coli and intestinal barrier function in their offspring, improving survival rates and pre-weaning growth.

In vivo and in vitro anti-inflammation of Rhapontici Radix extract on mastitis via TMEM59 and GPR161

Ethnopharmacological relevanceRhapontici Radix ethanol extract (RRE) is derived from the dried root of Rhaponticum uniflorum (L.) DC belonging to the Asteraceae family. RRE exhibits significant anti-inflammatory and antioxidant properties; however, the potential of RRE in mastitis treatment requires further investigation. Aim of this studyThis research was performed to examine the protective properties of RRE against mastitis and the mechanisms underlying the effects of RRE. Material and methodsRRE components were analyzed by HPLC-MS/MS and DPPH methods. Isochlorogenic acid B (ICAB) was obtained commercially. MTT assay was utilized to assess RRE or ICAB cytotoxicity in bovine mammary alveolar (MAC-T) cells. Immunohistochemistry were used to investigate the pathological alterations in mammary tissue. The protein levels of inflammatory cytokines and mediators were analyzed using ELISA, and the expression of MAPK and NF-κB signaling pathways, as well as p65 nuclear translocation, were analyzed through Western blotting and immunofluorescence techniques, respectively. Target proteins of RRE were screened by RNA-seq and tandem mass tag analyses. Protein interaction was revealed and confirmed using co-immunoprecipitation and CRISPR/Cas9-based knockdown and overexpression of target genes. ResultsICAB was revealed as one of the main components in RRE, and it was responsible for 84.33% of RRE radical scavenging activity. Both RRE and ICAB mitigated the infiltration of T lymphocytes in the mammary glands of mice, leading to decreased levels of inflammatory mediators (COX-2 and iNOS) and cytokines (TNF-α, IL-6, and IL-1β) in lipopolysaccharide (LPS)-induced MAC-T cells. Furthermore, RRE and ICAB suppressed the LPS-induced phosphorylation of NF-κB inhibitor and p65, thereby impeding p65 nuclear translocation in mouse mammary glands and MAC-T cells. In addition, RRE and ICAB attenuated the LPS-triggered activation of c-Jun N-terminal kinase 1/2, p38, and extracellular regulated protein kinase 1/2. Importantly, co-treated with LPS and ICAB in MAC-T cells, an upregulation of G-protein coupled receptor 161 (GPR161) and transmembrane protein 59 (TMEM59) was observed; the interact between TMEM59 and was found, leading to inhibition of NF-κB activity and inflammatory cytokine production. ConclusionICAB is a prominent antioxidant in RRE. RRE and ICAB reduce mammary inflammation via MAPK and NF-κB pathways and the interaction between TMEM59 and GPR161 mediates the control of ICAB in NF-κB signaling.

Transcriptomic Analysis of Pubertal and Adult Virgin Mouse Mammary Epithelial and Stromal Cell Populations

Conflicting data exist as to how mammary epithelial cell proliferation changes during the reproductive cycle. To study the effect of endogenous hormone fluctuations on gene expression in the mouse mammary gland, we performed bulk RNAseq analyses of epithelial and stromal cell populations that were isolated either during puberty or at different stages of the adult virgin estrous cycle. Our data confirm prior findings that proliferative changes do not occur in every mouse in every cycle. We also show that during the estrous cycle the main gene expression changes occur in adipocytes and fibroblasts. Finally, we present a comprehensive overview of the Wnt gene expression landscape in different mammary gland cell types in pubertal and adult mice. This work contributes to understanding the effects of physiological hormone fluctuations and locally produced signaling molecules on gene expression changes in the mammary gland during the reproductive cycle and should be a useful resource for future studies investigating gene expression patterns in different cell types across different developmental timepoints.

Hormonal regulation of miRNA during mammary gland development.

The mammary gland is a unique organ as most of its development occurs after birth through stages of proliferation, differentiation and apoptosis that are tightly regulated by circulating hormones and growth factors. Throughout development, hormonal cues induce the regulation of different pathways, ultimately leading to differential transcription and expression of genes involved in this process, but also in the activation or inhibition of post-transcriptional mechanisms of regulation. However, the role of microRNAs (miRNAs) in the different phases of mammary gland remodeling is still poorly understood. The objectives of this study were to analyze the expression of miRNA in key stages of mammary gland development in mice and to determine whether it could be associated with hormonal variation between stages. To do so, miRNAs were isolated from mouse mammary glands at stages of adulthood, pregnancy, lactation and involution, and sequenced. Results showed that 490, 473, 419, and 460 miRNAs are detected in adult, pregnant, lactating and involuting mice, respectively, most of them being common to all four groups, and 58 unique to one stage. Most genes could be divided into six clusters of expression, including two encompassing the highest number of miRNA (clusters 1 and 3) and showing opposite profiles of expression, reaching a peak at adulthood and valley at lactation, or showing the lowest expression at adulthood and peaking at lactation. GO and KEGG analyses suggest that the miRNAs differentially expressed between stages influence the expression of targets associated with mammary gland homeostasis and hormone regulation. To further understand the links between miRNA expression and hormones involved in mammary gland development, miRNAs were then sequenced in breast cells exposed to estradiol, progesterone, prolactin and oxytocin. Four, 38, 24 and 66 miRNAs were associated with progesterone, estradiol, prolactin, and oxytocin exposure, respectively. Finally, when looking at miRNAs modulated by the hormones, differentially expressed during mammary gland development, and having a pattern of expression that could be correlated with the relative levels of hormones known to be found in vivo, 16 miRNAs were identified as likely regulated by circulating hormones. Overall, our study brings a better understanding of the regulation of miRNAs throughout mammary gland development and suggests that there is a relationship between their expression and the main hormones involved in mammary gland development. Future studies will examine this role more in detail.

Expression Patterns of Grainyhead-Like 2 and Ovo-Like 2 in Mouse Mammary Gland Development During Pregnancy, Lactation, and Weaning.

Grainyhead-like 2 (Grhl2) is a transcription factor that regulates cell adhesion genes in mammary ductal development and serves as a repressor of the epithelial-mesenchymal transition. Conversely, Ovo-like2 (Ovol2) is a target gene of Grhl2 but functions as a substitute in Grhl2-deficient mice, facilitating successful epithelial barrier formation and lumen expansion in kidney-collecting ductal epithelial cells. Our objective was to examine the expression patterns of Grhl2, Ovol2, and their associated genes during the intricate phases of mouse mammary gland development. The mRNA expression of Grhl2 and Ovol2 increased after pregnancy. We observed Grhl2 protein presence in the epithelial cell's region, coinciding with acini formation, and its signal significantly correlated with E-cadherin (Cdh1) expression. However, Ovol2 was present in the epithelial region without a correlation with Cdh1. Similarly, Zeb1, a mesenchymal transcription factor, showed Cdh1-independent expression. Subsequently, we explored the interaction between Rab25, a small G protein, and Grhl2/Ovol2. The expressions of Grhl2 and Ovol2 exhibited a strong correlation with Rab25 and claudin-4, a tight junction protein. These findings suggest that Grhl2 and Ovol2 may collaborate to regulate genes associated with cell adhesion and are crucial for maintaining epithelial integrity during the different phases of mammary gland development.

The basement membrane regulates the cellular localization and the cytoplasmic interactome of Yes-Associated Protein (YAP) in mammary epithelial cells.

The Hippo pathway, a signaling cascade involved in the regulation of organ size and several other processes, acts as a conduit between extracellular matrix (ECM) cues and cellular responses. We asked whether the basement membrane (BM),a specialized ECM component known to induce quiescence and differentiation in mammary epithelial cells, would regulate the localization, activity, and interactome of YAP, a Hippo pathway effector. To address this question, we used a broad range of experimental approaches, including 2D and 3D cultures of both mouse and human mammary epithelial cells, as well as the developing mouse mammary gland. In contrast to malignant cells, nontumoral cells cultured with a reconstituted BM (rBM)displayed higher concentrations of YAP in the cytoplasm. Incidentally, when in the nucleus of rBM-treated cells, YAP resided preferentially at the nuclear periphery. In agreement with our cell culture experiments, YAP exhibited cytoplasmic predominance in ductal cells of developing mammary epithelia, where a denser BM is found. Conversely, terminal end bud (TEB)cells with a thinner BM displayed higher nucleus-to-cytoplasm ratios of YAP. Bioinformatic analysis revealed that genes regulated by YAP were overrepresented in the transcriptomes of microdissected TEBs. Consistently, mouse epithelial cells exposed to the rBM expressed lower levels of YAP-regulated genes, although the protein level of YAP and Hippo components were slightly altered by the treatment. Mass spectrometry analysis identified a differential set of proteins interacting with YAP in cytoplasmic fractions of mouse epithelial cells in the absence or presence of rBM. In untreated cells, YAP interactants were enriched in processes related to ubiquitin-mediated proteolysis, whereas in cells exposed to rBM YAP interactants were mainly key proteins related to amino acid, amino sugar, and carbohydrate metabolism. Collectively, we unraveled that the BM induces YAP translocation or retention in the cytoplasm of nontumoral epithelial cells and that in the cytoplasm YAP seems to undertake novel functions in metabolic pathways.

ABCD4 is associated with mammary gland development in mammals.

Mammary gland development is a critical process in mammals, crucial for their reproductive success and offspring nourishment. However, the functional roles of key candidate genes associated with teat number, including ABCD4, VRTN, PROX2, and DLST, in this developmental process remain elusive. To address this gap in knowledge, we conducted an in-depth investigation into the dynamic expression patterns, functional implications, and regulatory networks of these candidate genes during mouse mammary gland development. In this study, the spatial and temporal patterns of key genes were characterized in mammary gland development. Using time-series single-cell data, we uncovered differences in the expression of A bcd4, Vrtn, Prox2, and Dlst in cell population of the mammary gland during embryonic and adult stages, while Vrtn was not detected in any cells. We found that only overexpression and knockdown of Abcd4 could inhibit proliferation and promote apoptosis of HC11 mammary epithelial cells, whereas Prox2 and Dlst had no significant effect on these cells. Using RNA-seq and qPCR, further analysis revealed that Abcd4 can induce widespread changes in the expression levels of genes involved in mammary gland development, such as Igfbp3, Ccl5, Tlr2, and Prlr, which were primarily associated with the MAPK, JAK-STAT, and PI3K-AKT pathways by functional enrichment. These findings revealed ABCD4 as a candidate gene pivotal for regulating mammary gland development and lactation during pregnancy by influencing PRLR expression.

Sympathetic nerve signals: orchestrators of mammary development and stem cell vitality.

The mammary gland is a dynamic organ that undergoes significant changes at multiple stages of postnatal development. Although the roles of systemic hormones and microenvironmental cues in mammary homeostasis have been extensively studied, the influence of neural signals, particularly those from the sympathetic nervous system, remains poorly understood. Here, using a mouse mammary gland model, we delved into the regulatory role of sympathetic nervous signaling in the context of mammary stem cells and mammary development. Our findings revealed that depletion of sympathetic nerve signals results in defective mammary development during puberty, adulthood, and pregnancy, accompanied by a reduction in mammary stem cell number. Through in vitro three-dimensional culture and in vivo transplantation analyses, we demonstrated that the absence of sympathetic nerve signals hinders mammary stem cell self-renewal and regeneration, while activation of sympathetic nervous signaling promotes these capacities. Mechanistically, sympathetic nerve signals orchestrate mammary stem cell activity and mammary development through the ERK signaling pathway. Collectively, our study unveils the crucial roles of sympathetic nerve signals in sustaining mammary development and regulating mammary stem cell activity, offering a novel perspective on the involvement of the nervous system in modulating adult stem cell function and organ development.

Methionine and Leucine Promote mTOR Gene Transcription and Milk Synthesis in Mammary Epithelial Cells through the eEF1Bα-UBR5-ARID1A Signaling.

Amino acids are essential for the activation of the mechanistic target of rapamycin (mTOR), but the corresponding molecular mechanism is not yet fully understood. We previously found that Met stimulated eukaryotic elongation factor α (eEF1Bα) nuclear localization in bovine mammary epithelial cells (MECs). Herein, we explored the role and molecular mechanism of eEF1Bα in methionine (Met)- and leucine (Leu)-stimulated mTOR gene transcription and milk synthesis in MECs. eEF1Bα knockdown decreased milk protein and fat synthesis, cell proliferation, and mTOR mRNA expression and phosphorylation, whereas eEF1Bα overexpression had the opposite effects. QE-MS analysis detected that eEF1Bα was phosphorylated at Ser106 in the nucleus and Met and Leu stimulated p-eEF1Bα nuclear localization. eEF1Bα knockdown abrogated the stimulation of Met and Leu by mTOR mRNA expression and phosphorylation, and this regulatory role was dependent on its phosphorylation. Akt knockdown blocked the stimulation of Met and Leu by eEF1Bα and p-eEF1Bα expression. ChIP-PCR detected that p-eEF1Bα bound only to the -548 to -793 nt site in the mTOR promoter, and ChIP-qPCR further detected that Met and Leu stimulated this binding. eEF1Bα mediated Met and Leu' stimulation on mTOR mRNA expression and phosphorylation through inducing AT-rich interaction domain 1A (ARID1A) ubiquitination degradation, and this process depended on eEF1Bα phosphorylation. p-eEF1Bα interacted with ARID1A and ubiquitin protein ligase E3 module N-recognition 5 (UBR5), and UBR5 knockdown rescued the decrease of the ARID1A protein level by eEF1Bα overexpression. Both eEF1Bα and p-eEF1Bα were highly expressed in mouse mammary gland tissues during the lactating period. In summary, we reveal that Met and Leu stimulate mTOR transcriptional activation and milk protein and fat synthesis in MECs through eEF1Bα-UBR5-ARID1A signaling.

Abstract PO5-09-11: Abrupt involution of mouse mammary gland leads to inflammatory systemic changes along with mammary specific metabolic shifts that may enhance risk of breast cancer

Abstract Epidemiological data links higher parity and lack of breastfeeding with increased risk of breast cancer, specifically aggressive triple negative breast cancer (TNBC), and higher mortality rate. Following pregnancy and lactation, breast remodels to near pre-pregnancy stage through apoptotic cell death and adipocyte repopulation process. Long-term breastfeeding and gradual weaning of an infant leads to gradual involution (GI) of the breast, while lack of or abrupt discontinuation of breastfeeding after birth leads to abrupt involution (AI), when rapid and massive cell death takes place. Our studies have shown several precancerous changes, such as increased collagen deposition, inflammation, and hyperplasia in the mammary gland of mice after AI. However, the systemic impact of AI and how this increases risk of breast cancer is yet to be elucidated. Objectives: Our objective is to evaluate the systemic effects that are prompted by the AI mammary gland. We hypothesize that AI leads to marked alteration in lipid metabolism and systemic inflammation that enhances risk for breast cancer. Methods: FVB/n mice (8week old) were paired for breeding. At partum (day 0), dams were randomized to AI or GI cohort and standardized to 6 pups. AI mice had pups removed on day 7 postpartum (ppm) to mimic short-term breastfeeding. For GI mice 3 pups each were removed on day 28 and 31ppm to mimic gradual weaning. Tissues harvested on day 28, 56, and 120 postpartum. Body composition was measured by echo MRI. Glucose tolerance test (GTT) was performed after a 6 hour fast using a 2g/kg glucose intraperitoneal injection. Blood glucose was measured by glucometer. Serum insulin was analyzed by ELISA. HOMA-IR was calculated using blood glucose and serum insulin results. Serum was analyzed using multiplex ELISA by MesoScale Diagnostics. Mammary glands were subjected to untargeted lipidomics. Results: There were no significant differences in body weight, percent body fat or lean mass between AI and GI groups at any time point. However, at day 120 ppm (4 months after partum), we have observed significantly larger amount (1.29-fold increase) of perigonadal adipose tissue (visceral adipose) in AI mice than GI mice (p=0.0112; n=24-38/group). There were no significant differences in blood glucose, serum insulin, HOMA-IR, or GTT results between AI and GI groups at day 120. AI mice had significantly higher levels of cytokines IL-1β (3.1-fold increase, p=0.0417) and KC/GRO (1.5-fold increase, p=0.0196) than GI mice at day 120. At day 28, AI mammary glands had significantly higher amounts of level 3 identified oxidized ceramide containing sphingolipids that were linked to insulin resistance and diabetes. At day 56, GI mammary glands had significantly higher amounts of level 2 and 3 identified metabolites linked to cellular signaling and lipid metabolism. On day 120, there were no significant differences in lipid metabolites between groups. Conclusion: Although histologically GI and AI mammary glands return to near pre-pregnancy state within a month after partum, our data shows specific lipid changes in the AI mammary gland similar to what has been shown in women with TNBC. Furthermore, AI of the mammary gland leads to systemic effects on adiposity and inflammation that could be key to increased breast cancer risk. Further studies along these lines are in progress to understand the whole-body effects of AI and stratify preventive measures for women who cannot breast feed. Significance: Lack of breastfeeding is more prevalent in African American (AA) women and linked to higher risk of developing aggressive TNBC1. Our novel animal models of AI and GI help to link the impact of AI and systemic changes that may enhance breast cancer risk. In particular, we see an increase in visceral adiposity with AI. Understanding this mechanism will help identify strategies to reduce risk in women who are unable or choose not to breastfeed and ultimately help to reduce TNBC and TNBC-related mortality in AA women. Citation Format: Kate Ormiston, Kirti Kaul, Neelam Shinde, Djawed Bennouna, Rachel Kopec, Ramesh Ganju, Sarmila Majumder, Bhuvaneswari Ramaswamy. Abrupt involution of mouse mammary gland leads to inflammatory systemic changes along with mammary specific metabolic shifts that may enhance risk of breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-09-11.

Puerarin Delays Mammary Gland Aging by Regulating Gut Microbiota and Inhibiting the p38MAPK Signaling Pathway.

Mammary gland aging is one of the most important problems faced by humans and animals. How to delay mammary gland aging is particularly important. Puerarin is a kind of isoflavone substance extracted from Pueraria lobata, which has anti-inflammatory, antioxidant, and other pharmacological effects. However, the role of puerarin in delaying lipopolysaccharide (LPS)-induced mammary gland aging and its underlying mechanism remains unclear. On the one hand, we found that puerarin could significantly downregulate the expression of senescence-associated secretory phenotype (SASP) and age-related indicators (SA-β-gal, p53, p21, p16) in mammary glands of mice. In addition, puerarin mainly inhibited the p38MAPK signaling pathway to repair mitochondrial damage and delay mammary gland aging. On the other hand, puerarin could also delay the cellular senescence of mice mammary epithelial cells (mMECs) by targeting gut microbiota and promoting the secretion of gut microbiota metabolites. In conclusion, puerarin could not only directly act on the mMECs but also regulate the gut microbiota, thus, playing a role in delaying the aging of the mammary gland. Based on the above findings, we have discovered a new pathway for puerarin to delay mammary gland aging.

Selenium Counteracts Tight Junction Disruption and Attenuates the NF-κB-Mediated Inflammatory Response in Staphylococcus aureus-Infected Mouse Mammary Glands.

Tight junctions (TJs) are the key determinant of barrier function in the mammary gland, with their disruption being associated with the pathogenesis and progression of mastitis, especially in the case of Staphylococcus aureus (S. aureus) infection. This study investigated whether selenium (Se) could attenuate S. aureus-induced mastitis by inhibiting inflammation and protecting mammary gland TJs in mice. The expression profiles of S. aureus-infected gland tissues derived from the gene expression omnibus dataset were analyzed. We found cytokine production, cell junctions, the nuclear transcription factor-κB (NF-κB) signalling pathway, and inflammatory responses associated with the differentially expressed genes, as revealed by Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses. Se reduced the mRNA expression and production of inflammatory cytokines, including tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and decreased phosphorylation levels of the NF-κB complex. Moreover, Se alleviated structural damage and microvillus injury in mammary glands. Immunohistochemical staining revealed that Se increased the expression of Claudin-3; Western blot analysis revealed increased protein levels of Occludin and Tricellulin in the group supplemented with dietary Se. In summary, Se counteracted TJ disruption and attenuated NF-κB-mediated inflammatory responses in S. aureus-infected mouse mammary glands.

Adipocytes Are the Only Site of Glutamine Synthetase Expression Within the Lactating Mouse Mammary Gland

BackgroundGlutamine in milk is believed to play an important role in neonatal intestinal maturation and immune function. For lactating mothers, glutamine utilization is increased to meet the demands of the enlarged intestine and milk production. However, the source of such glutamine during lactation has not been studied. ObjectivesWe aimed to assess the effects of lactation on the expression of glutamine synthetase (GS) in the mammary gland and other tissues of lactating mice. MethodsMouse tissues were sampled at 4 time points: 8-wk-old (virgin, control), post-delivery day 5 (PD5, early lactation), PD15 (peak lactation), and involution (4 days after weaning at PD21). We examined the gene expression and protein concentrations of GS and the first 2 enzymes of branched-chain amino acid catabolism: branched-chain aminotransferase 2 (BCAT2) and branched-chain ketoacid dehydrogenase subunit E1α (BCKDHA). ResultsThe messenger RNA (mRNA) expression and protein concentrations of GS in mammary glands were significantly lower at PD5 and PD15 compared with the control but were restored at involution. Within the mammary gland, GS protein was only detected in adipocytes with no evidence of presence in mammary epithelial cells. Compared with the control, mRNA and protein concentrations of BCAT2 and BCKDHA in mammary glands significantly decreased during lactation and involution. No changes in GS protein concentrations during lactation were found in the liver, skeletal muscle, and lung. In non-mammary adipose tissue, GS protein abundance was higher during lactation compared with the virgin. ConclusionsThis work shows that, within the mouse mammary gland, GS is only expressed in adipocytes and that the relative GS abundance in mammary gland sections is lower during lactation. This suggests that mammary adipocytes may be a site of glutamine synthesis in the lactating mouse. Identifying the sources of glutamine production during lactation is important for optimizing milk glutamine concentration to enhance neonatal and maternal health.

Abstract 2181: Endocrine-targeting therapies modify the breast microbiome to shift metabolism and reduce cancer risk

Abstract Studies indicate that breast tissue has a distinct modifiable microbiome population. However, whether endocrine-targeting therapies modify the non-cancerous breast microbiome to impact tumorigenesis is unknown. To examine this question, we performed 16S bacterial sequencing on DNA isolated from female C57BL/6 mouse mammary glands (MG) administered tamoxifen citrate for 16 weeks and breast tissue from ovariectomized non-human primates administered tamoxifen citrate (TAM) for 2.5 years. In both models, we show that TAM significantly shifted β-diversity and increased Firmicutes proportional abundance. TAM elevated the proportional abundance of Lactobacillus spp., Streptococcus luticea, and Staphylococcus sciuri. Female BALB/c mice received intra-nipple injections of Lactobacillus and MG were harvested at 1-, 3-, 7-, and 14-days post-injection to show bacteria colonization and modulation of metabolism-associated gene and protein expression. B6.MMTV-PyMT mice were injected with Lactobacillus bacteria into the MG. Elevating MG Lactobacillus reduced tumorigenesis with an associated decrease in tumor proliferation and shifts in non-cancerous MG tissue metabolism. To elucidate the effects of combining TAM and probiotic bacteria on breast cancer outcomes, 4T1.2ER+ tumor-bearing female BALB/c mice, fed either a control or Western diet were treated with TAM, Lactobacillus probiotic, or a combination of TAM + Lactobacillus. Results indicate that oral probiotic administration in combination with TAM reduced ER+ tumor growth. Plasma metabolomics revealed combination treatment elevated L-carnitine regardless of diet, suggesting drug-bug interactions modifying metabolite profiles. Cell viability assays on human MCF7 BC cells and non-cancerous mammary HMT-3522S1 cells treated with 1% probiotic-conditioned media (Pro-CM) revealed a significant decrease in cell viability in BC cells but not non-cancerous epithelial cells. Mitochondrial fuel oxidation Seahorse assay on murine 4T1.2ER+ cells treated with 1% Pro-CM, 1μm 4-hydroxytamoxifen (4-OHT) or combination revealed changes in metabolic substrate dependency, supporting role of microbial metabolites impacting metabolism. Tumor sections from patients with ER+ BC treated with aromatase inhibitors and/or Faslodex in the neoadjuvant setting were stained for Gram-positive bacteria, Gram-negative bacteria, and Ki67. Gram-positive bacteria negatively correlated with Ki67 immunoreactivity, suggesting endocrine-targeting therapies promote tumor Gram-positive bacteria associated with a decrease in proliferation. Taken together, our data indicates that endocrine-targeting treatments modify the breast microbiome associated with a shift in tissue metabolism to decrease proliferation and reduce ER+ BC risk. Citation Format: Alana Arnone, Yu-Ting Tsai, J Mark Cline, Adam S. Wilson, Brian Westwood, Meghan E. Seger, Akiko Chiba, Marissa Howard-McNatt, Edward A. Levine, Alexandra Thomas, David R. Soto-Pantoja, Katherine L. Cook. Endocrine-targeting therapies modify the breast microbiome to shift metabolism and reduce cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2181.

Abstract 6877: Progesterone promotes immunosuppression in the murine mammary gland by causing T cell death

Abstract Breast Cancer is the most common cancer diagnosed in women in the United States. Second only to lung cancer, breast cancer results in the greatest number of cancer deaths for women. Hormone receptor positive (HR+) breast cancers - cancers expressing the nuclear receptors for steroid hormones estrogen and progesterone, termed the estrogen receptor (ER) and progesterone receptor (PR) - make up the largest proportion of diagnosed breast cancers by a significant margin. So, although the HR+ subtype is less aggressive compared to others, it continues to be the largest contributor to breast cancer mortality. Treatment consists of anti-endocrine therapies, largely targeting ER/estrogen, the established drivers of breast cancer growth. Most women respond well to these therapies, but for women who do not, there are few alternative options. With the advent of immunotherapy and its successes in treating and controlling certain tumor types, many were hopeful that it could be employed for breast cancer as well. However, breast cancers, especially HR+ breast cancers, are considered “immune cold.” They lack high proportions of tumor-infiltrating lymphocytes (TILs), have low neoantigen production, and high percentages of immunosuppressive cells. A contributing factor could be the hormones present in the tumor microenvironment. The accepted paradigm is that lifetime exposure to estrogens drives the development of HR+ breast cancer, but both estrogen and progesterone are part of the normal menstrual cycle. Prior work from our lab has uncovered a novel immune-modulatory role for progesterone, pivotal to promoting the growth of PR-positive mammary tumors. Recent preliminary data from our lab show that progesterone treatment conveys immune protection against T cell killing in a co-culture with E0771 mammary gland tumor cells. However, when activated in monoculture, T cells exposed to increasing concentrations of progesterone have no differing expression of cell surface activation or exhaustion markers, implying that progesterone is not eliciting this effect by interfering with the T cell activation cascade nor is it inducing premature exhaustion. Interestingly, progesterone treatment instead leads to a concentration-dependent increase in T cell death compared to the untreated control. In vivo, these results are supported as progesterone treatment decreases the amount of total CD3+ T cells, and specifically the cytotoxic CD8+ subset, in the mammary glands of mice. These results suggest that progesterone could modulate the breast and tumor microenvironment, providing tumor cells with protection from immunosurveillance and contributing to HR+ breast cancer development. These data also argue for the use of anti-progestins to be added to the HR+ breast cancer treatment regimen to boost anti-tumor immune activity. Citation Format: Amanda Heard, Lauryn R. Werner, Eilidh I. Chowanec, Harmony Saunders, Julio Tinoco, Zachary C. Hartman, Christy R. Hagan. Progesterone promotes immunosuppression in the murine mammary gland by causing T cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6877.