Mammary Gland Involution Research Articles

Abstract B011: The anti-cancer effects of vitamin D are blocked postpartum, due to suppression of vitamin D metabolism in the involuting liver

Abstract Postpartum mammary gland involution is a physiologic window of increased breast cancer risk. It has been proposed that the poor prognosis associated with postpartum breast cancer is due to the involuting mammary microenvironment promoting progression of indolent lesions to invasive disease. As such, the involuting gland has been implicated as a target for preventive strategies. Vitamin D has anti-cancer properties, and there are data demonstrating that vitamin D supplementation protects against breast cancer progression in mouse models. Moreover, vitamin D deficiency is prevalent in postpartum women, suggesting that vitamin D supplementation during the vitamin D-deficient, at-risk window of involution may be an approach for preventing the progression of indolent lesions. Here, we characterized how vitamin D deficiency and supplementation affect tumor growth in a mouse model of postpartum breast cancer. Vitamin D deficiency and sufficiency were established in BALB/c mice through feeding diets containing low or high levels of vitamin D. Quantification of serum 25(OH)D verified that diets resulted in vitamin D deficiency (25.8±4.3nmol/L;mean±stdev) or sufficiency (66.4±11.7nmol/L) at levels comparable to humans. Murine mammary cancer cells (D2A1, 2×104 cells, 20µL) were injected into mammary fatpads of involuting (2 days post wean), or age-matched nulliparous mice (n=5-12 mice/group), and tumor growth tracked for 4 weeks. Independent of vitamin D status, tumors exposed to the involuting mammary gland grew 1.8-fold larger than tumors exposed to the nulliparous gland (p<0.001); consistent with prior data showing that the microenvironment of the involuting gland is tumor promotional. Interestingly, while vitamin D supplementation of nulliparous mice associated with a 3.4-fold reduction in tumor growth (p=0.03), vitamin D supplementation of involution mice did not reduce tumor growth. Dietary vitamin D is metabolized to its active form in the liver. Our group has previously shown that the liver also undergoes weaning-induced involution. Thus, we tested if involution of the liver impairs metabolism of vitamin D to its active form, potentially contributing to the null effects of vitamin D supplementation in involution mice. We collected serum from mice on vitamin D deficient and supplemented diets across reproductive time points (nulliparous, lactation, involution days 2, 4, 6), and found that vitamin D supplementation during involution was insufficient to restore serum vitamin D to levels of sufficiency. Further, gene expression analysis of livers show that expression of Cyp2r1 and Cyp27a1—genes involved in vitamin D activation—were reduced during involution. Together, these findings suggest that impaired metabolism of vitamin D during involution may reduce the availability of active vitamin D, and contribute to the null effect observed in involution mice. Understanding the mechanisms by which mammary gland involution influences the anti-cancer effects of vitamin D is required to optimize cancer prevention strategies that target this window. Citation Format: Sarah M Bernhardt, Pepper Schedin. The anti-cancer effects of vitamin D are blocked postpartum, due to suppression of vitamin D metabolism in the involuting liver [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr B011.

Abstract B002: A facilitating role for semaphorin-7a in regulating cell invasion and matrix remodeling for DCIS progression

Abstract Approximately 25% of newly diagnosed breast cancer cases are non-invasive or pre-invasive breast cancers known as ductal carcinoma in situ (DCIS). DCIS can progress to invasive ductal carcinoma (IDC), conferring a more dangerous prognosis; however, there is a gap in knowledge regarding what ultimately causes this transition. Understanding epithelial invasion in the mammary gland will provide insight on how DCIS ultimately progresses to IDC. Our lab has found that DCIS transforms to IDC under the “wound-healing” pressure of postpartum tissue remodeling – termed postpartum mammary gland involution. This invasive transformation is dependent, in part, on collagen-induced cyclooxygenase (COX2) expression. We identified that a membrane-bound signaling molecule, semaphorin-7a (SEMA7A), promotes both collagen deposition and COX-2 expression and therefore DCIS progression. SEMA7A is also naturally upregulated in mammary epithelial cells during postpartum involution and in DCIS relative to normal epithelium. We have identified that inhibition or silencing of SEMA7A can inhibit DCIS invasion. While this demonstrates that SEMA7A expression is sufficient to promote innate capacity for tumor cell invasiveness, we have yet to investigate how SEMA7A interacts with the tumor microenvironment (TME) to promote stromal changes known to advance this transition, such as collagen remodeling or immune cell recruitment. We have demonstrated a potential role for DCIS-associated macrophages in facilitating this transition. Additionally, we have found that SEMA7A polarizes macrophages to an M2-like state, and this promotes remodeling of the lymphatic vasculature as well as tumor cell entry into lymphatic vessels. Furthermore, we have shown that SEMA7A promotes expression of matrix remodeling enzymes that can, in turn, promote shedding of SEMA7A into the TME where it can affect other cell types. We are utilizing intravital imaging to visualize tumor/lymphatic/macrophage interactions as well as second harmonic imaging to visualize SEMA7A-dependent changes in the collagen structure surrounding DCIS lesions. Our results suggest that SEMA7A mediates DCIS invasion through both intrinsic and extrinsic mechanisms, which could facilitate progression to metastasis. By understanding how SEMA7A regulates DCIS progression we can begin to introduce potential targets for preventative therapies for development of invasive disease – including targeting SEMA7A itself. Since SEMA7A is low in most adult tissues it is expected that such a therapeutic would be less toxic for patients than the current prescribed therapies. Citation Format: Petra Dahms. A facilitating role for semaphorin-7a in regulating cell invasion and matrix remodeling for DCIS progression [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr B002.

Transcriptome Analysis of Goat Mammary Gland Tissue Reveals the Adaptive Strategies and Molecular Mechanisms of Lactation and Involution.

To understand how genes precisely regulate lactation physiological activity and the molecular genetic mechanisms underlying mammary gland involution, this study investigated the transcriptome characteristics of goat mammary gland tissues at the late gestation (LG), early lactation (EL), peak lactation (PL), late lactation (LL), dry period (DP), and involution (IN) stages. A total of 13,083 differentially expressed transcripts were identified by mutual comparison of mammary gland tissues at six developmental stages. Genes related to cell growth, apoptosis, immunity, nutrient transport, synthesis, and metabolism make adaptive transcriptional changes to meet the needs of mammary lactation. Notably, platelet derived growth factor receptor beta (PDGFRB) was screened as a hub gene of the mammary gland developmental network, which is highly expressed during the DP and IN. Overexpression of PDGFRB in vitro could slow down the G1/S phase arrest of goat mammary epithelial cell cycle and promote cell proliferation by regulating the PI3K/Akt signaling pathway. In addition, PDGFRB overexpression can also affect the expression of genes related to apoptosis, matrix metalloproteinase family, and vascular development, which is beneficial to the remodeling of mammary gland tissue during involution. These findings provide new insights into the molecular mechanisms involved in lactation and mammary gland involution.

OR30-5 Androgen-Regulated Secreted Factors Promote Tumorigenesis and Immune Suppression in Breast Cancer

Abstract Background Breast cancer (BC) is the most diagnosed cancer and the second-leading cause of cancer-associated death in women in the United States. BCs that express the estrogen and/or progesterone receptor (ER/PR) are treated with ER-targeting endocrine therapies that effectively reduce disease burden and extend lifespan. However, up to 50% of patients with ER+BCs will experience recurrence, often at a metastatic site, within 10 years of diagnosis. Novel targeted therapies are needed to treat both metastatic ER+BC resistant to current treatment strategies and chemoresistant ER-BCs.We identified chitinase 3–like-1 (CHI3L1/BRP-39/YKL-40) and prolactin inducible protein (PIP/GCDFP-15) as androgen receptor (AR)-regulated in BC. PIP is reported to be detectable in >90% of BCs, but its function in BC is not well-characterized. CHI3L1 is a glycosyl hydrolase family 18 protein member with reported roles in aging and numerous pathologies. During mammary gland involution, CHI3L1 contributes to a pro-tumorigenic wound-healing-like environment and increases macrophage infiltration. Further, CHI3L1 correlates with tumor-suppressive M2 macrophage infiltration in ER-BC. We previously reported that ER+ cells and patient-derived xenografts with activating mutations in the ESR1 gene encoding ER—a known mechanism of endocrine therapy resistance—had elevated AR and CHI3L1 gene and protein expression. Based on the above and our finding that metastatic BCs biopsies harboring ESR1 mutations exhibited increased AR expression, we hypothesized that AR regulation of CHI3L1 and PIP promote progression to metastatic disease in part through immune suppressive effects. Methods A ConTra(V3) transcription factor binding site analysis was performed on CHI3L1 and PIP promoters. CHI3L1 and PIP were measured via immunohistochemistry in xenograft tumors in mice treated with or without the androgen dihydrotestosterone (DHT). Immunoblotting, immunohistochemistry, and immunofluorescence approaches were also utilized to assess CHI3L1 protein expression in DHT-treated, tamoxifen resistant (TAMR), and long term E2 deprived (LTED) ER+BC cells. CIBERSORT analysis was conducted on the Firehouse Legacy TCGA dataset. Macrophage efferocytosis assays were performed on an IncuCyte Live Cell Imaging system. Results We identified highly conserved AR binding sites in the CHI3L1 and PIP promoters. CHI3L1 protein was elevated in TAMR and LTED BC cells, which was further elevated in BC cells with ESR1 mutations, as well as DHT-treated cells. Further, CHI3L1 and PIP were increased in xenograft tumors after DHT treatment. CIBERSORT analysis revealed that CHI3L1 and PIP high tumors had altered immune profiles, including differential macrophage levels. Finally, CHI3L1 and PIP treatment significantly reduced macrophage efferocytosis of BC cells (p ≤ 0.0001). Conclusions Our work suggests that AR-mediated regulation of CHI3L1 and PIP may alter immune cell infiltration/function in BC and may be important in endocrine therapy resistant ER+BCs. Further characterization and manipulation of these AR-regulated proteins is underway to test the potential to target and reverse their immune suppressive functions. Presentation: Tuesday, June 14, 2022 10:45 a.m. - 11:00 a.m.

Effect of experimental stress and cortisol release induced by ACTH administration on expression of key genes related to milk synthesis and apoptosis during mammary involution of Saanen goats.

This research paper addresses the hypothesis that stress, induced by ACTH administration and cortisol release increases somatic cell count (SCC) in mammary secretion, and improves the effectiveness of dry off in goats. We report indicators of milk synthesis and mammary gland involution during dry off. Thirty Saanen goats were subjected to abrupt dry off and treatments: (1) ACTH administration (ACTH) or (2) placebo (Control) on days 1, 3, 6, 9, 12, 15, 30, and 60 of dry off. The expression of target genes in mammary tissue that are related to milk synthesis and cell survival such as insulin-like growth factor 1 receptor (IGF1R), phosphatidylinositol-3-kinase (PIK3CA), protein kinase B (AKT1) and mechanistic target of rapamycin (MTOR), casein (CSN2), lactalbumin (LALBA) and lactoferrin (LF) were evaluated, and plasma cortisol concentration, SCC, leucocyte count, and microbiological analyses in milk and mammary secretions were assessed. ACTH significantly downregulated the expression of IGF1R and upregulated the expression of PIK3CA in mammary tissue, increased lactoferrin concentration and SCC, and changed immune cell levels in mammary secretions compared to Control. Furthermore, ACTH administration increased the percentage of dry goats compared to the Control (73 vs. 46%, respectively). We conclude that the effect of stress via ACTH administration and cortisol release accelerated mammary involution during the early dry-off period.

Characterization of long noncoding RNA in nonlactating goat mammary glands reveals their regulatory role in mammary cell involution and remodeling

Long noncoding RNA (lncRNA) can regulate mammary gland development and lactation physiological activities. However, the molecular genetic mechanisms of lncRNA in mammary gland involution and cell remodeling remain unclear. This work analyzed the expression characteristics and molecular functions of lncRNA in goat mammary gland tissue at the late lactation (LL), dry period (DP), and late gestation (LG) stages. Sequencing results showed that 3074 lncRNAs were identified in non-lactating goat mammary gland tissue. Statistical analysis of lncRNA length characteristics and exon number found that goat lncRNAs were shorter in length, had fewer exons, and significantly lower expression levels than those of protein-coding genes. 331 differentially expressed lncRNAs were identified in the three comparison groups (LLvsDP, DPvsLG, and LLvsLG), which indicated that the lncRNAs expression at the transcriptional level were changed during mammary involution. Interestingly, lncRNAs were more actively expressed during the dry period compared to lactation, suggesting that lncRNAs in mammary glands are developmentally specific. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that lncRNAs could regulate immune function, cell proliferation, apoptosis, hormones, substance metabolism, transport, and intercellular communication in the mammary gland through various action modes. Among them, cis-acting lncRNAs enhanced the protection of mammary gland health during the dry period and late gestation. The above reflects the particular mechanisms of lncRNA to adapt to the developmental needs of mammary involution and remodeling. Furthermore, in the lncRNA-miRNA-mRNA network associated with mammary gland development, the expression of LOC102168552 was higher in late gestation than in the dry period and late lactation. Its expression was positively correlated with PRLR and negatively correlated with chi-miR-324-3p. Overexpression of LOC102168552 in goat mammary epithelial cells cultured in vitro could up-regulate PRLR to activate the prolactin signaling pathway by competitively binding to chi-miR-324-3p, promoting cell proliferation, reducing cell cycle arrest in the G1 / S phase, and inhibiting apoptosis. However, overexpression of LOC102168552 alone did not affect mammary cell growth status and the prolactin signaling pathway. This indicates that LOC102168552 must rely on chi-miR-324-3p to inhibit mammary cell apoptosis. In conclusion, the above analysis revealed that lncRNAs in goat mammary tissue are differentially expressed at different stages of involution. As expected, lncRNAs adaptively regulate various physiological activities during mammary gland involution through multiple modes of action, in preparation for a new round of lactation. These findings provide a reference and help further understand the regulatory role of lncRNAs in mammary cell involution and remodeling.

Breast cancers co-opt normal mechanisms of tolerance to promote immune evasion and metastasis.

Normal developmental processes, such as those seen during embryonic development and postpartum mammary gland involution, can be reactivated by cancer cells to promote immune suppression, tumor growth, and metastatic spread. In mammalian embryos, paternal-derived antigens are at risk of being recognized as foreign by the maternal immune system. Suppression of the maternal immune response toward the fetus, which is mediated in part by the trophoblast, is critical to ensure embryonic survival and development. The postpartum mammary microenvironment also exhibits immunosuppressive mechanisms accompanying the massive cell death and tissue remodeling that occurs during mammary gland involution. These normal immunosuppressive mechanisms are paralleled during malignant transformation, where tumors can develop neoantigens that may be recognized as foreign by the immune system. To circumvent this, tumors can dedifferentiate and co-opt immune-suppressive mechanisms normally utilized during fetal tolerance and postpartum mammary involution. In this review, we discuss those similarities and how they can inform our understanding of cancer progression and metastasis.

Identification and profiling of microRNAs involved in the regenerative involution of mammary gland

Regenerative involution is important for the subsequent lactation, but molecular mechanism has not been revealed. The crucial miRNA in tissue development indicates that miRNAs might participate in regenerative involution. In the present study, the mammary tissues of the dairy goats (n = 3) were collected via biopsy at wk-8 (time to dry off), –6, –4, –1, and + 1 relative to lambing for the Hematoxylin and Eosin staining and miRNA sequencing. Alveolar structures collapsed during regenerative involution, but the structures remained intact and distended. Among the 50 miRNA expression trajectories categorized by short time-series expression miner, two significant patterns were clustered. The differentially expressed miRNAs in the two patterns were mainly related to the self-renewal of tissue and enriched in pathways containing vesical-mediated transport, tissue development, tube development, vasculature development and epithelial development. The identification of the miRNAs will help in elucidating their regulatory roles in mammary gland development.

Abstract 983: RNA-seq analysis of murine liver to identify breast cancer metastatic potential during liver involution

Abstract Background: Postpartum breast cancer (PPBC), variably defined as breast cancer diagnosed within 5-10 years after a patient’s last pregnancy, has overall increased risk of metastasis even after controlling for patient age, year of diagnosis, and tumor subtype and stage. Metastasis to the liver is specifically increased in PPBC compared to non-PPBC cases and likely contributes to the overall poor prognosis of PPBC. Our lab has previously shown that the rodent liver undergoes physiological remodeling after pregnancy, in a process reminiscent of weaning-induced mammary gland involution. Further, rodent models confirm that breast cancer cells delivered to the involuting liver grow more robustly than tumor cells delivered to nulliparous liver. Our rodent data suggest that weaning induced liver involution may be responsible for increased tumor cell seeding and ultimately higher rates of liver metastasis observed in women diagnosed with PPBC. Since weaning-induced liver involution is a recently recognized physiology, little is known of its molecular underpinnings. Here we employ RNA-seq of murine livers to better understand how reproductive status influences liver physiology with a focus on exploring the pro-metastatic niche formed after weaning. Study design: Livers were isolated from age-matched mice at the following reproductive time points: nulliparous (never pregnant), lactation, involution days 2, 4, 6, and 8, and fully regressed (4 weeks post-wean) and flash frozen for RNA isolation (n=3-6 mice per group). RNA-seq was utilized to identify gene expression differences between reproductive groups. We employed single-sample gene set enrichment analysis (ssGESA) to identify key pathways modulated by reproductive state. Results: We identify clear reproductive control of liver physiology. We find increases in proliferation, MTORC1, and anabolic metabolism pathways during lactation when compared to any other reproductive state. In the transition from lactation to involution, we find increases in apoptotic pathways, and a switch to catabolic metabolism. Correspondingly, during involution we see enrichment in pro-cancer pathways including increased TGFβ signaling, Epithelial to Mesenchymal Transition, and Inflammatory Responses Hallmark Pathways. Within the immune signatures changes of involution, we observe myeloid-derived cell signatures including enrichment in M2 associated genes, and increases in cytotoxic and helper T cell signatures that parallels a signature for disease-specific T cell exhaustion. Conclusion: This work highlights novel regulation of liver physiology by reproductive state, and finds pathways regulated during the natural physiological process of involution that have been previously identified as contributing to metastatic niches. This study has the potential to identify new targets for the prevention and treatment of breast cancer liver metastasis. Citation Format: Michelle K. Ozaki, Alexandra Q. Bartlett, Zheng Xia, Pepper Schedin. RNA-seq analysis of murine liver to identify breast cancer metastatic potential during liver involution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 983.

Transcriptome profiling of the nonlactating mammary glands of dairy goats reveals the molecular genetic mechanism of mammary cell remodeling

The mammary gland redevelops to the prepregnancy state during involution, which shows the mammary cells have the characteristics of remodeling. The rapidity and degree of mammary gland involution vary across species (e.g., between model organism mice and dairy livestock). However, the molecular genetic mechanism of involution and remodeling of goat mammary gland has not yet been clarified. This work investigated the structural changes and transcriptome characteristics of the mammary gland tissue of nonlactating dairy goats during the late lactation (LL), the dry period (DP), and late gestation (LG). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining revealed significant changes in the structure of the nonlactating goat mammary gland, and obvious cell apoptosis occurred at LL and DP. Sequencing identified 1,381 genes that are differentially expressed in mammary gland tissue at the 3 developmental stages. Genes related to cell growth, apoptosis, immunity, nutrient transport, synthesis, and metabolism exhibited adaptive transcriptional changes to meet the needs of a new set of mammary gland lactation functions. The significant enrichment of Gene Ontology terms such as humoral immune response, complement activation, and neutrophil-mediated immunity indicates that the innate immune system plays an important role in maintaining the health of degenerative mammary glands and eliminating apoptotic cells. The peroxisome proliferator-activated receptor signaling pathway plays an important regulatory role in lipid metabolism, especially the adaptive changes in expression of genes encoded lipid transport and enzymes, which promote the formation of milk fat during the lactation. The mammary gland development gene module revealed that pregnancy hormone receptors, cell growth factors and their receptors, and genes encoding insulin-like growth factor binding proteins regulate the physiological process of mammary gland involution through adaptive transcriptional changes. Interestingly, ERBB4 was identified as the hub gene of the network that regulates mammary gland growth and development. Overexpression of ERBB4 in mammary epithelial cells cultured in vitro can reduce cell cycle arrest in G1/S phase and apoptosis by regulating the PI3K/Akt signaling pathway and promote the proliferation of mammary epithelial cells. The gene ERBB4 also affects the expression of genes that initiate mammary gland involution and promote mammary gland remodeling. These findings contribute to an in-depth understanding of the molecular mechanisms involved in mammary gland involution and remodeling.

In vitro effects of 5-Hydroxy-L-tryptophan supplementation on primary bovine mammary epithelial cell gene expression under thermoneutral or heat shock conditions

Serotonin (5-HT) is an autocrine-paracrine molecule within the mammary gland regulating homeostasis during lactation and triggering involution after milk stasis. Exposure of dairy cows to hyperthermia during the dry period alters mammary gland involution processes leading to reduced subsequent yields. Herein, primary bovine mammary epithelial cells (pBMEC) under thermoneutral (TN, 37 °C) or heat shock (HS, 41.5 °C) conditions were cultured with either 0, 50, 200, or 500 μM 5-Hydroxy-L-tryptophan (5-HTP; 5-HT precursor) for 8-, 12- or 24-h. Expression of 95 genes involved in 5-HT signaling, involution and tight junction regulation were evaluated using a Multiplex RT-qPCR BioMark Dynamic Array Circuit. Different sets of genes were impacted by 5-HTP or temperature, or by their interaction. All 5-HT signaling genes were downregulated after 8-h of HS and then upregulated after 12-h, relative to TN. After 24-h, apoptosis related gene, FASLG, was upregulated by all doses except TN-200 μM 5-HTP, and cell survival gene, FOXO3, was upregulated by HS-50, 200 and 500 μM 5-HTP, suggesting 5-HTP involvement in cell turnover under HS. Supplementing 5-HTP at various concentrations in vitro to pBMEC modulates the expression of genes that might aid in promoting epithelial cell turn-over during involution in dairy cattle under hyperthermia.

Peripartal treatment with low-dose sertraline accelerates mammary gland involution and has minimal effects on maternal and offspring bone.

Women mobilize up to 10% of their bone mass during lactation to provide milk calcium. About 8%–13% of mothers use selective serotonin reuptake inhibitors (SSRI) to treat peripartum depression, but SSRIs independently decrease bone mass. Previously, peripartal use of the SSRI fluoxetine reduced maternal bone mass sustained post‐weaning and reduced offspring bone length. To determine whether these effects were fluoxetine‐specific or consistent across SSRI compounds, we examined maternal and offspring bone health using the most prescribed SSRI, sertraline. C57BL/6 mice were given 10 mg/kg/day sertraline, from the beginning of pregnancy through the end of lactation. Simultaneously, we treated nulliparous females on the same days as the primiparous groups, resulting in age‐matched nulliparous groups. Dams were euthanized at lactation day 10 (peak lactation, n = 7 vehicle; n = 9 sertraline), lactation day 21 (weaning, n = 9 vehicle; n = 9 sertraline), or 3m post‐weaning (n = 10 vehicle; n = 10 sertraline) for analysis. Offspring were euthanized at peak lactation or weaning for analysis. We determined that peripartum sertraline treatment decreased maternal circulating calcium concentrations across the treatment period, which was also seen in nulliparous treated females. Sertraline reduced the bone formation marker, procollagen 1 intact N‐terminal propeptide, and tended to reduce maternal BV/TV at 3m post‐weaning but did not impact maternal or offspring bone health otherwise. Similarly, sertraline did not reduce nulliparous female bone mass. However, sertraline reduced immunofluorescence staining of the tight junction protein, zona occludens in the mammary gland, and altered alveoli morphology, suggesting sertraline may accelerate mammary gland involution. These findings indicate that peripartum sertraline treatment may be a safer SSRI for maternal and offspring bone rather than fluoxetine.

Abstract P5-01-08: Mechanistic differences between abrupt and gradual involution of mouse mammary gland

Abstract Objective: Epidemiological studies indicate a direct relation between length of breast feeding and protection against risk of triple negative breast cancer (TNBC), an aggressive subtype. While prolonged breastfeeding allows gradual involution (GI) of the breast, short-term or no breast feeding leads to abrupt involution (AI). We modelled AI and GI of breast in mice, and showed that mice subjected to GI have better protection from tissue remodeling associated injuries in the mammary gland. Our data revealed late development of ductal hyperplasia aided by pro-tumorigenic microenvironment in the AI glands1. Detailed mechanism of mammary gland involution immediately following cessation of lactation has been studied in the past, but only in the AI setting, that is after abrupt removal of pups at the peak of lactation2,3. The goal of this study is to conduct stepwise comparison of the mechanism of GI vs. AI early on to understand the protective effect of GI against breast cancer risk.Methods: Wild-type FVB mice were used in all our studies. Females were mated at 8 weeks of age and uniparous mice were allowed to nurse (6 pups/dam) for 7 days. Females were then assigned randomly to AI or GI cohort. All pups were removed from the AI dams on postnatal day7 (PND7). Three pups each were removed from GI dams on day28 and 31. Mammary glands were harvested intermittently between PND7 and PND35. H&E stained sections were used for histological studies. Unstained FFPE sections were used for immunohistochemistry and TUNEL assay. Total RNA and protein from whole mammary gland was used for qPCR and western blot respectively. Results: GI glands transitioned from fully active lactating to near involuted glands over a period of 8 days (PND17- PND25), while for AI glands it took < 4 days (PND8.5-PND12). The shrinkage and flattening of tall epithelia and loss of acini was gradual in GI glands as opposed to rapid breakdown of acini and adipocyte repopulation in AI gland. Apoptotic cell count peaked on PND11 (5%) in AI vs. PND25 (3%) in GI glands. The pStat3Y705+ cells were highest on PND8.5 (25%) in AI vs. on PND25 (11%) in GI glands. Macrophage infiltration (F4/80+) peaked on PND11 (35%) and remained elevated at ~24% till PND25, while in GI glands increase was gradual from PND17 through PND25 (27%). Expression of key genes identified in AI mice2,3 have markedly different expression pattern in GI mice (Table). While some peaked at a later time point in GI vs. AI coinciding with maximum cell death, expression of some are significantly low or undetectable in GI glands at both RNA and protein level. Conclusions: We show for the first time that kinetics of cell death, adipocyte repopulation, immune cell infiltration and inflammatory state of glands undergoing abrupt vs. gradual involution are markedly different. Several genes known to play a key role during AI are either not expressed or barely detectable in the GI glands at any time point during involution. These data suggests that not only the kinetics, but mechanism of GI and AI are not identical. We conclude that orchestrated cell death in GI protects from drastic lysosomal, and immune cell activities that predisposes mammary glands to higher risk of neoplastic changes.Significance: Epidemiological data highlights the benefits of prolonged breastfeeding in protecting against breast cancer, particularly, TNBC, an aggressive subtype prevalent in the African American women. Our study highlights the mechanism underlying the benefits of gradual involution of breast. GeneFold Change compared to PND7 (GI vs. AI) Peaked on (GI vs. AI)Stat34.0 vs. 5.9PND25 vs. PND8.5Ctsb2.8 vs. 3.4PND25 vs. PND8.5CD143.4 vs. 20PND25 vs. PND8.5Orm11.7 vs. 10PND25 vs. PND12Lrg130 vs. 216PND25 vs. PND12MMP215.8 vs. 27PND25 vs. PND12Chi3L11350 vs. 572, 859PND28 vs. PND11, 28Cebpδnone vs. 4.4None vs. PND8.5CtsLnone vs. 5.7None vs. PND8.5Orm2none vs. 370None vs. PND12Slpinone vs. 92None vs. PND8.5 Citation Format: Bhuvaneswari Ramaswamy, Neelam Shinde, Morgan Bauer, Maria Cuitino, Saba Mehra, Resham Mawalkar, Mustafa Basree, Allen Zhang, Kirti Kaul, Xiaoli Zhang, Ramesh Ganju, Sarmila Majumder. Mechanistic differences between abrupt and gradual involution of mouse mammary gland [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-08.

Abstract P1-04-06: Semaphorin7a expression in breast cancers promotes susceptibility to immune checkpoint blockade

Abstract Five-year survival rates for women diagnosed with stage II breast cancer are >85% on average; however, in women diagnosed with stage II breast cancer (BC) within 10 years of last childbirth, we observe a 5-fold increase in risk for developing distant metastasis when compared with nulliparous women1. Major driving factors for stage II diagnosis include increased tumor-associated lymphatic vessel density (LVD), lymphovascular invasion (LVI), and lymph node positivity (LN+) and are all associated with poor prognosis for breast cancer patients. Nonetheless, the mechanisms underlying development of LN metastasis, and how LN metastasis seed distant metastases, has remained elusive. LVD, LVI, and LN+ can be driven by tumor-associated macrophages (TAMs), which have been implicated in creating a pro-tumor tumor microenvironment (TME) in many types of cancer. Additionally, podoplanin (PDPN)-expressing macrophages (PoEMs), are a newly characterized type of TAM that specifically contribute to tumor-associated lymphatic vessel formation2,3. Our lab identified that semaphorin7A (SEMA7A)—a neuroimmune molecule—is significantly associated with increased metastasis in LN+, but not LN-, BC patients, as well as increased recurrence in cases of BCs diagnosed within 10 years of childbirth, designated postpartum breast cancers (PPBCs). In mouse models, SEMA7A expression is associated with increased LVD and TAM presence, including PoEMs3. Furthermore, we and others have shown that PoEMs intercalate into lymphatic vessels to form PoEM-LEC chimeric vessels, where tumor cells can often be found at the PoEM-LEC junctions2. We have also observed that breast cancer cells, lymphatics, macrophages, and PoEMs exhibit SEMA7A-dependent expression of PD-L1 in vitro, which may make SEMA7A+ tumors more sensitive to anti-PD-1/PD-L1 immunotherapies. Therefore, we utilized multiple orthotopic, immunocompetent mouse models to reveal that growth of SEMA7A overexpressing (OE) tumors, but not controls, was significantly slowed with both anti-PD-1 and anti-PD-L1 treatments. Flow cytometric analysis of cells from the TME revealed increased LECs, TAMs, and PoEMs in SEMA7A+ tumors, compared to controls—all populations had higher PD-L1 expression, which was decreased with both anti-PD-1 or anti-PD-L1. We also observed a decrease in PD-L1 expression on the tumor cells with treatment. Furthermore, LEC, TAM, and PoEM presence was decreased within the TME alongside increased presence of activated CD4 and CD8 T cells. Collectively, our results suggest that SEMA7A expression in breast cancers activates a major pathway associated with immune evasion and helps to establish a protumor microenvironment, which can be targeted with readily available FDA approved drugs such as immune checkpoint-based therapies. Since SEMA7A+ and PPBCs have high rates of metastasis, more specific treatments for these patient populations should be explored. 1.Goddard, E.T., et al. Association Between Postpartum Breast Cancer Diagnosis and Metastasis and the Clinical Features Underlying Risk. JAMA Netw Open 2, e186997 (2019). 2.Bieniasz-Krzywiec, P., et al. Podoplanin-Expressing Macrophages Promote Lymphangiogenesis and Lymphoinvasion in Breast Cancer. Cell Metab (2019). 3.Elder, A.M., et al. Semaphorin 7A Promotes Macrophage-Mediated Lymphatic Remodeling during Postpartum Mammary Gland Involution and in Breast Cancer. Cancer research 78, 6473-6485 (2018). Citation Format: Alan Elder, Alexander Stoller, Traci Lyons. Semaphorin7a expression in breast cancers promotes susceptibility to immune checkpoint blockade [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-06.

Longitudinal Intravital Imaging Through Clear Silicone Windows.

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging windows is particularly powerful because it allows longitudinal observation of the same tissue over days to weeks. Typical imaging windows comprise a glass coverslip in a biocompatible metal frame sutured to the mouse's skin. These windows can interfere with the free movement of the mice, elicit a strong inflammatory response, and fail due to broken glass or torn sutures, any of which may necessitate euthanasia. To address these issues, windows for long-term abdominal organ and mammary gland imagingwere developed from a thin film of polydimethylsiloxane (PDMS), an optically clear silicone polymer previously used for cranial imaging windows. These windows can be glued directly to the tissues, reducing the time needed for insertion. PDMS is flexible, contributing to its durability in mice over time-up to 35 days have been tested. Longitudinal imaging is imaging of the same tissue region during separate sessions. A stainless-steel grid was embedded within the windows to localize the same region, allowing the visualization of dynamic processes (like mammary gland involution) at the same locations, days apart. This silicone window also allowed monitoring of single disseminated cancer cells developing into micro-metastases over time. The silicone windows used in this study are simpler to insert than metal-framed glass windows and cause limited inflammation of the imaged tissues. Moreover, embedded grids allow for straightforward tracking of the same tissue region in repeated imaging sessions.

Gestational and lactational xenoestrogen exposure disrupts morphology and inflammatory aspects in mammary gland of gerbil mothers during involution

In the mammary gland (MG), the developmental window for gestational/lactational differentiation and growth is highly vulnerable to hormonal disruption. Here we describe that the MG involution process in female gerbil mothers is delayed by bisphenol A (BPA) exposure during gestation and lactation. The process is directly influenced by changes in expression of extracellular matrix proteases MMP-2, MMP-9, and FAP, and the incidence of collagen and elastin is reduced after 7 and 14 days of weaning. A pro-inflammatory environment in the late involution process was confirmed by higher expression of TNF-α, COX-2 and phospho-STAT3 n the MG stroma, allied to increases in the incidence of macrophages and mast cells. These aspects impacted the proliferative pattern of epithelial cells, which decreased on the 14th post-weaning day. These data confirm that the milk production window of susceptibility is vulnerable to the impact of BPA, which promotes a suggestive pro-tumoral microenvironment during mammary involution.

The Potential of Metalloproteinase-9 Administration to Accelerate Mammary Involution and Boost the Immune System at Dry-Off.

Simple SummaryThe cow dry period is a critical period presenting a high risk of contracting intramammary infections. Active molecules to boost the innate immunity of the mammary gland and increase infection resilience could be decisive for the milking performance of dairy cows in the next lactation. Metalloproteinase-9 is a protein with a relevant role in facilitating the immune function and activating the regeneration of the mammary gland. The focus of this study was to test the role of the infusion of a recombinant version of metalloproteinase 9 at cow dry off, showing, contrary to expectations, that it is not able to enhance the innate immunity nor to improve the involution and regeneration of the mammary gland.The dry period is decisive for the milking performance of dairy cows. The promptness of mammary gland involution at dry-off affects not only the productivity in the next lactation, but also the risk of new intra-mammary infections since it is closely related with the activity of the immune system. Matrix metalloproteinase-9 (MMP-9) is an enzyme present in the mammary gland and has an active role during involution by disrupting the extracellular matrix, mediating cell survival and the recruitment of immune cells. The objective of this study was to determine the potential of exogenous administration of a soluble and recombinant version of a truncated MMP-9 (rtMMP-9) to accelerate mammary involution and boost the immune system at dry-off, avoiding the use of antibiotics. Twelve Holstein cows were dried abruptly, and two quarters of each cow received an intra-mammary infusion of either soluble rtMMP-9 or a positive control based on immunostimulant inclusion bodies (IBs). The contralateral quarters were infused with saline solution as negative control. Samples of mammary secretion were collected during the week following dry-off to determine SCC, metalloproteinase activity, bovine serum albumin, lactoferrin, sodium, and potassium concentrations. The soluble form of rtMMP-9 increased endogenous metalloproteinase activity in the mammary gland compared with saline quarters but did not accelerate either the immune response or involution in comparison with control quarters. The results demonstrated that the strategy to increase the mammary gland immunocompetence by recombinant infusion of rtMMP-9 was unsuccessful.

Mammary collagen is under reproductive control with implications for breast cancer

Mammographically-detected breast density impacts breast cancer risk and progression, and fibrillar collagen is a key component of breast density. However, physiologic factors influencing collagen production in the breast are poorly understood. In female rats, we analyzed gene expression of the most abundantly expressed mammary collagens and collagen-associated proteins across a pregnancy, lactation, and weaning cycle. We identified a triphasic pattern of collagen gene regulation and evidence for reproductive state-dependent composition. An initial phase of collagen deposition occurred during pregnancy, followed by an active phase of collagen suppression during lactation. The third phase of collagen regulation occurred during weaning-induced mammary gland involution, which was characterized by increased collagen deposition. Concomitant changes in collagen protein abundance were confirmed by Masson's trichrome staining, second harmonic generation (SHG) imaging, and mass spectrometry. We observed similar reproductive-state dependent collagen patterns in human breast tissue obtained from premenopausal women. SHG analysis also revealed structural variation in collagen across a reproductive cycle, with higher packing density and more collagen fibers arranged perpendicular to the mammary epithelium in the involuting rat mammary gland compared to nulliparous and lactating glands. Involution was also characterized by high expression of the collagen cross-linking enzyme lysyl oxidase, which was associated with increased levels of cross-linked collagen. Breast cancer relevance is suggested, as we found that breast cancer diagnosed in recently postpartum women displayed gene expression signatures consistent with increased collagen deposition and crosslinking compared to breast cancers diagnosed in age-matched nulliparous women. Using publicly available data sets, we found this involution-like, collagen gene signature correlated with poor progression-free survival in breast cancer patients overall and in younger women. In sum, these findings of physiologic collagen regulation in the normal mammary gland may provide insight into normal breast function, the etiology of breast density, and inform breast cancer risk and outcomes.

Control of hormone-driven organ disassembly by ECM remodeling and Yorkie-dependent apoptosis

Epithelia grow and shape into functional structures during organogenesis. Although most of the focus on organogenesis has been drawn to the building of biological structures, the disassembly of pre-existing structures is also an important event to reach a functional adult organ. Examples of disassembly processes include the regression of the Müllerian or Wolffian ducts during gonad development and mammary gland involution during the post-lactational period in adult females. To date, it is unclear how organ disassembly is controlled at the cellular level. Here, we follow the Drosophila larval trachea through metamorphosis and show that its disassembly is a hormone-driven and precisely orchestrated process. It occurs in two phases: first, remodeling of the apical extracellular matrix (aECM), mediated by matrix metalloproteases and independent of the actomyosin cytoskeleton, results in a progressive shortening of the entire trachea and a nuclear-to-cytoplasmic relocalization of the Hippo effector Yorkie (Yki). Second, a decreased transcription of the Yki target, Diap1, in the posterior metameres and the activation of caspases result in the apoptotic loss of the posterior half of the trachea while the anterior half escapes cell death. Thus, our work unravels a mechanism by which hormone-driven ECM remodeling controls sequential tissue shortening and apoptotic cell removal through the transcriptional activity of Yki, leading to organ disassembly during animal development.

Anoikis resistance in mammary epithelial cells is mediated by semaphorin 7a

Semaphorin-7a (SEMA7A), best known as a neuroimmune molecule, plays a diverse role in many cellular processes and pathologies. Here, we show that SEMA7A promotes anoikis resistance in cultured mammary epithelial cells through integrins and activation of pro-survival kinase AKT, which led us to investigate a role for SEMA7A during postpartum mammary gland involution—a normal developmental process where cells die by anoikis. Our results reveal that SEMA7A is expressed on live mammary epithelial cells during involution, that SEMA7A expression is primarily observed in α6-integrin expressing cells, and that luminal progenitor cells, specifically, are decreased in mammary glands of SEMA7A−/− mice during involution. We further identify a SEMA7A-α6/β1-integrin dependent mechanism of mammosphere formation and chemoresistance in mammary epithelial cells and suggest that this mechanism is relevant for recurrence in breast cancer patients.