Mammary Gland Involution Research Articles

AKT and EZH2 inhibitors kill TNBCs by hijacking mechanisms of involution.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and has the highest rate of recurrence1. The predominant standard of care for advanced TNBC is systemic chemotherapy with or without immunotherapy; however, responses are typically short lived1,2. Thus, there is an urgent need to develop more effective treatments. Components of the PI3K pathway represent plausible therapeutic targets; more than 70% of TNBCs have alterations in PIK3CA, AKT1 or PTEN3-6. However, in contrast to hormone-receptor-positive tumours, it is still unclear whether or how triple-negative disease will respond to PI3K pathway inhibitors7. Here we describe a promising AKT-inhibitor-based therapeutic combination for TNBC. Specifically, we show that AKT inhibitors synergize with agents that suppress the histone methyltransferase EZH2 and promote robust tumour regression in multiple TNBC models in vivo. AKT and EZH2 inhibitors exert these effects by first cooperatively driving basal-like TNBC cells into a more differentiated, luminal-like state, which cannot be effectively induced by either agent alone. OnceTNBCs are differentiated, these agents kill them by hijacking signals that normally drive mammary gland involution. Using a machine learning approach, we developed a classifier that can be used to predict sensitivity. Together, these findings identify a promising therapeutic strategy for this highly aggressive tumour type and illustrate how deregulated epigenetic enzymes can insulate tumours from oncogenic vulnerabilities. These studies also reveal how developmental tissue-specific cell death pathways may be co-opted for therapeutic benefit.

Effects of rumen-protected 5-hydroxytryptophan on circulating serotonin concentration, behaviour, and mammary gland involution in goats

The risk of acquiring new intramammary infections is high at the end of lactation, especially for the high milk-producing dairy animals. Resistance to bacterial infection increases following the completion of mammary gland involution after milking cessation. The serotonin precursor 5-hydroxytryptophan (5-HTP) could accelerate involution by increasing circulating serotonin levels, but ruminal microbes may degrade 5-HTP if orally administered to adult ruminants. It is unclear whether rumen-protected 5-HTP could effectively mediate circulating serotonin (5-hydroxytryptamine, 5-HT) and therefore accelerate mammary gland involution in ruminants. Goats were used as a model in the current study to investigate the effects of rumen-protected 5-HTP on behaviour, 5-HT metabolism, and mammary involution in ruminants. In the first experiment, 16 female Dazu black goats were assigned to one of four groups in a randomised block design. The treatments included a basal diet plus 0, 4, 20, or 100 mg/kg BW of rumen-protected 5-HTP. Serum was collected at 0, 3, 6, 12, and 24 h after offering the rumen-protected 5-HTP in the morning feed, and the behaviours were monitored. In the second experiment, 12 female Dazu black goats (Somatic cell count < 250 000) were randomly assigned to the control (basal diet) or rumen-protected 5-HTP group (basal diet plus 20 mg/kg BW). Milk or mammary secretions were manually collected aseptically on d −1, 1, 2, 3, 4, and 5 around weaning. The results depicted that rumen-protected 5-HTP supplementation elevated circulating 5-HTP and 5-hydroxyindole acetic acid concentrations, while 20 mg/kg BW of rumen-protected 5-HTP supplementation lowered the goats’ locomotive activity. A high concentration of rumen-protected 5-HTP (100 mg/kg BW) increased serum alkaline phosphatase and gamma-glutamyl transpeptidase concentrations. Moreover, oral supplementation with 20 mg/kg BW of rumen-protected 5-HTP accelerated mammary gland involution and reduced feed intake in goats after weaning. These results demonstrate that oral supplementation with rumen-protected 5-HTP influences 5-HT metabolism and accelerates mammary gland involution after milking cessation in ruminants.

Breastfeeding Intensity and Its Association With Mammary Gland Involution Early Post-partum Among Mothers in Low-Income Households

Breastfeeding Intensity and Its Association With Mammary Gland Involution Early Post-partum Among Mothers in Low-Income Households

Effect of milk stasis on mammary gland involution and the microRNA profile

The presence of an autocrine factor in milk that can trigger mammary gland involution was proposed more than 50 yr ago. To provide evidence for the existence of one or more autocrine factors, 10 multiparous cows in late lactation were quarter-milked for 7 d. Following this baseline period, the right front quarter of each cow was left unmilked, and the other quarters were milked for 7 d. Before the last milking of that period, milk (mammary secretions) was collected aseptically from both front quarters. After that milking, 250 mL of the collected samples were infused in the cows' respective rear quarters. No quarters were milked for the following 7 d (milk stasis period), and quarter milking was then resumed in all quarters for the last 7 d of the experiment (remilking period). Quarter milk samples were collected during the baseline period, before the milk stasis period, and during the remilking period. These samples were used for measuring milk components and the concentration of involution markers (SCC, BSA, and lactoferrin). Samples of mammary secretions were collected manually from the quarters during the milk stasis period for involution marker determination. We extracted RNA from samples collected from front quarters before the last milking before the milk stasis period for microRNA (miRNA) determination. As anticipated, the longer milk stasis period implemented for the right front quarter resulted in a more advanced involution than in the left front quarter, based on the concentration of involution markers in the mammary secretions, lower milk production recovery, and changes in milk composition during the remilking period. All 3 involution marker concentrations in the mammary secretions increased in both rear quarters, but were greater in the right quarter secretions than in the left quarter secretions. Resuming milking reinitiated milk production in all quarters, but milk production recovery in the right rear quarters was less robust than that in the left rear quarters (54.3 ± 1.4% vs. 61.6 ± 1.4%, respectively). Milk from the quarters infused with mammary secretions (right rear) had a lower lactose content, but a higher milk protein content and higher SCC than the quarters infused with milk. We detected a total of 359 miRNAs, 76 of which were differentially expressed in milk and mammary secretions. Expression of bta-miR-221 and bta-miR-223 was upregulated in mammary secretions 34- and 40-fold, respectively. The results of the present experiment support the contention that milk stasis leads to the accumulation of one or more factors that trigger involution. The results also indicate that milk stasis leads to changes in the miRNA profile of the milk, but whether such changes are a cause or a consequence of the involution process remains to be established.

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.

Abstract PO4-24-05: Fluid forces and hormone levels during mammary gland development drive changes in breast epithelium that are relevant to the progression of postpartum breast cancer

Abstract Postpartum breast cancer (PPBC), diagnosed in the 5-10 years after childbirth, has an elevated risk of metastasis and death. Poor outcomes are thought to be due to factors involved in mammary gland involution, an important stage of mammary gland development. Involution functions to return the mammary gland to the normal post-lactation state and involves activation of multiple processes such as inflammation, wound healing, and lymphangiogenesis. Previously, these processes have been linked to mechanical forces induced by fluid flowing past cells, known as fluid shear stress (FSS). We and others have shown FSS impacts processes associated with lactation and breast cancer metastasis. Herein, we describe the results of a cell model for mimicking lactation and the cessation of lactation as an investigative tool for probing the mechanisms involved in PPBC development and progression. We identified changing levels of FSS as a potential physiologic biomarker through which pathways associated with the progression of PPBC could be identified. The role of fluid shear stress in the progression of cancer remains relatively unexplored, mostly due to practical research barriers. Since FSS cannot be accurately measured in vivo, most research to date has relied on in vitro modelling. In contrast, the role of the involuting mammary gland on the progression of breast cancer has often been studied using rodent models. As a result, studying the interactions between fluid shear stress and involution poses a novel engineering challenge. To address this problem, we developed a bioreactor cell model to enable cell exposure to fluid flow, mimicking forces experienced during lactation, in presence of lactogenic hormones (dexamethasone, insulin, prolactin). Previous work has induced FSS using parallel plate flow chambers, and induced lactation in mammary epithelial cells grown in vitro using lactogenic hormone treatment. To our knowledge, this is the first time these experimental conditions have been studied in combination. We established three distinct stages of treatment, consistent with the fluid shear stress and hormonal levels expected in (1) lactation, (2) cessation of lactation, and (3) involution. To determine whether the model mimicked expected in vivo conditions in the postpartum mammary gland, we used morphological markers and measured the protein expression of β-casein (CSN2), a milk protein that is an established marker of lactation. Each stage was validated and analyzed using proteomic and genomic sequencing. Generated datasets were further analyzed using unsupervised clustering, combining publicly available datasets with our data. We determined that β-casein levels were highest when protein was collected after the lactation stage, followed by an initial drop-off in β-casein expression when fluid shear stress were lowered during the cessation period. The β-casein levels continued at a constant, low level during the involution period, despite a reintroduction of fluid shear stress. This suggests that breast cancer cells are initially responsive to shear stress stimulation, consistent with what would be expected in the postpartum mammary gland in vivo. Proteomic and genomic datasets generated from cells exposed to FSS demonstrated that multiple pathways associated with metastasis are upregulated, as compared to static controls. When combined with publicly available involution datasets, pathways involved in extracellular matrix remodeling, inflammation, and lymphangiogenesis emerged. Multiple targets identified through this analysis have been previously linked to metastatic activities during breast cancer, suggesting that FSS and involution are relevant physiologic biomarkers in the context of PPBC. Work to validate hormonal and fluid shear stress conditions in combination is ongoing. Future work will use the validated model to test relevant targets identified from analysis of generated datasets. Citation Format: Maya Stibbards-Lyle, Kristina Rinker, Laura Hall, Seleem Badawy, Kathy Zhan. Fluid forces and hormone levels during mammary gland development drive changes in breast epithelium that are relevant to the progression of postpartum 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 PO4-24-05.

Abstract 6852: Immune microenvironment profiling of pregnancy associated breast cancer: A transcriptomic and multiplex immunohistochemical analyses

Abstract Background: Pregnancy-associated breast cancer (PABC) is BC diagnosed during pregnancy or within 2 years after pregnancy. Although PABC is a rare subgroup of BC, the outcome of PABC is worse than BC developed in the same age group unrelated to pregnancy. The proliferation and involution of mammary gland during and after pregnancy create a unique immune microenvironment in the breast. Comprehensive profiling of the tumor immune microenvironment in PABC is important but lacking. Methods: We retrospectively identified PABC patients from electronic medical records and their tumor blocks from pathology department in our institution. Age- and stage-matched BC patients were selected as control group. NanoString Breast Cancer 360 Gene Expression panel and Opal Polaris 7 color immunohistochemical (IHC) were utilized to profile PABC from transcriptome and protein levels. Result: PABC (N=26), BC developed 2 years (BC&amp;gt;2Y, N=54) after pregnancy, and matched non-pregnant BC patients (NPBC, N=27) were included in the final analysis respectively. Samples from BC&amp;gt;2Y (remote influence from pregnancy) and NPBC (pregnancy did not contribute to BC) both served as control groups for PABC patients in our study. PAM50 subtyping categorized patients into luminal A(N=42), luminal B(N=26), basal(N=12), and HER2 (N=25) enriched subgroups. In multiplex IHC analysis, CD4+ cells, CD8+ cells, CD20+ cells, and CD68+/CD163+ (M2) cells were all statistically higher in the tumor samples from PABC and BC&amp;gt;2Y than from NPBC. This suggests a potential contribution of pregnancy in the immune microenvironment. In the luminal A subgroup, CD8+ cells, CD20+ cells, and CD68+/CD163+ cells remain statistically higher in the PABC than in the NPBC samples. In NanoString BC360 transcriptomic profiling, immune cell abundance was evaluated from representative gene signatures. Mast cells were significantly lower in the PABC group than in the NPBC group. Cytotoxic T cells are significantly higher both in the PABC and BC&amp;gt;2Y groups than in the NPBC group. Conclusion: We are the first study to profile immune microenvironment using both transcriptomic and IHC methods. Our study is also unique in using NPBC and BC&amp;gt;2Y patients as control groups for PABC patients. CD8+ T cells are statistically higher in PABC patients than in NPBC patients using either transcriptomic profiling or multiplex IHC analysis. M2 macrophages, which promote tumor growth, are abundant in all the PABC and the luminal A PABC samples. Citation Format: I-Chun Chen, Ching-Hsuan Chen, Chia-Lang Hsu, Tzu-Pin Lu, Yen-Shen Lu, Ching-Hung Lin. Immune microenvironment profiling of pregnancy associated breast cancer: A transcriptomic and multiplex immunohistochemical analyses [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 6852.

Abstract 2138: Understanding the link between breastfeeding and the risk of breast cancer through comparative analysis of the murine-based model of mammary gland involution

Abstract Introduction: A meta-analysis of 47 global epidemiological studies highlights a higher breast cancer risk in women who did not breastfeed or breastfed for a short time. Further studies showed this is especially true for triple-negative breast cancer (TNBC) patients. Premenopausal AA women (AAW) have a lower prevalence of breastfeeding and a higher incidence of TNBC and mortality. Our previous study compares short-term breastfeeding, abrupt involution (AI) with prolonged breastfeeding called gradual involution (GI), revealing that AI alone induces ductal hyperplasia four months postpartum. Our current investigation delves into early events during AI versus GI, employing a comprehensive approach encompassing histology, gene expression, and myeloid cell involvement. Methods: Utilizing FVB female mice, we conducted a comparative analysis of AI and GI. AI involved early pup removal, while GI was achieved through staggered weaning. The evaluation included analysis of histomorphology, gene/protein expression, and myeloid cell infiltration. Sequential mammary gland (MG) changes were monitored through H&amp;E staining, TUNEL assay, and DNA damage analysis. 3D-organoid cultures of luminal progenitors (LPs) were employed to assess the impact of AI versus GI. qRT-PCR, IHC, Western blot, and flow cytometry/multiplex imaging were employed for the differential expression analysis of molecular and cellular factors associated with AI/GI. Results: Our research showed that AI had early adipocyte repopulation, rapid cell death, DNA repair, and myeloid cell infiltration, resulting in a chronically inflamed microenvironment. In contrast, the GI triggers a controlled immune response and prolonged cell death, facilitating comprehensive remodeling of the MG. Our flow cytometric or multiplexing imaging analyses revealed that AI-affected glands exhibit an enrichment of CCL9-producing CD206+ M2-like macrophages and CD11b+Gr1+ myeloid-derived suppressor cells. Moreover, exogenous CCL9 treatment on LPs in 3D-organoid culture results in disorganized acinar-type organoids, mirroring morphological differences observed in LPs from AI mammary glands on day56 PPM. Further analysis of CCL9 treated organoids revealed the expansion of Esr1- LPs population in ex-vivo organoid culture which might indicate an increase in the putative cells of origin of TNBC. Conclusion: Our studies comparing AI and GI demonstrate that AI is producing a pro-tumorigenic environment in the breast. It is important to note that prolonged breastfeeding protects the breast, although this cannot be a singular risk factor for TNBC. Therefore, understanding the mechanism will lead to prevention strategies to improve outcomes for all women but, has the potential to have a significant benefit in AAW. Citation Format: Sanjay Mishra, Neelam Shinde, Maria Cuitino, Morgan Bauer, Dinesh Ahirwar, Vijaya Bharti, Kate Ormiston, Resham Mawalkar, Sara Alsammerai, Gautam Sarathy, Xiaoli Zhang, Anna Vilgelm, Ramesh Ganju, Sarmila Majumder, Bhuvaneswari Ramaswamy. Understanding the link between breastfeeding and the risk of breast cancer through comparative analysis of the murine-based model of mammary gland involution [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 2138.

Abstract B079: Vitamin D metabolism is suppressed postpartum: Implications for targeting the postpartum window for cancer prevention

Abstract Postpartum mammary gland involution is a physiologic inflammatory process that associates with increased risk of postpartum breast cancer (PPBC). Pre-clinical studies provide rationale for targeting this pro-tumor window of involution for PPBC prevention. However, the liver also undergoes physiological remodeling after weaning—consistent with weaning-induced mammary gland involution—which could potentially impact the efficacy of anti-cancer prodrugs administered during the postpartum period. Here, we used mouse models to test the efficacy of vitamin D, a prodrug with known anti-cancer properties, during the postpartum period. Vitamin D supplementation is of particular interest due to its inclusion in dietary guidelines for postpartum women, where vitamin D deficiency is prevalent. Dietary vitamin D is biologically inactive and requires hydroxylation in the liver by Cyp2r1 to produce its circulating form (25(OH)D), which is further hydroxylated in the kidneys to produce the active form (1,25(OH)2D). To understand the impact of liver involution on vitamin D hydroxylation, RNAseq was performed on livers collected from nulliparous or involution mice. Expression of Cyp2r1 was 2-fold lower in involution livers, compared to nulliparous livers (p&amp;lt;0.001). Further, Cyp24a1, which is responsible for degradation of active vitamin D, was increased 5.5-fold in involution livers compared to nulliparous livers (p&amp;lt;0.001). These findings suggest that vitamin D supplementation would be ineffective in the postpartum period. Indeed, nulliparous mice supplemented with vitamin D showed a &amp;gt;2-fold increase in serum 25(OH)D (67.4±8.1nmol/L) compared to nulliparous mice fed a deficient diet (28.7±11.7nmol/L, p&amp;lt;0.01). In contrast, supplementation of involution mice did not increase serum 25(OH)D (supplemented=46.6±8.7nmol/L, deficient=33.7±8.5nmol/L, p&amp;gt;0.05). In the liver, tissue-specific concentrations of 25(OH)D were lower in supplemented involution mice (1.3±0.6ng/g), compared to supplemented nulliparous mice (2.0±0.4ng/g; p=0.02). To assess whether suppression of vitamin D metabolism impacts the anti-cancer activity of dietary vitamin D, mammary cancer cells (D2A1, 2 × 104 cells) were injected into mammary fatpads of mice. In nulliparous mice, vitamin D supplementation associated with a 3-fold reduction in tumor growth (p=0.03); whereas no anti-cancer effects were observed in involution mice. These findings suggest the anti-cancer effects of vitamin D are blocked postpartum due to suppression of vitamin D metabolism in the liver. Understanding how the postpartum liver metabolizes drugs is essential for targeting treatments to this window. A retrospective analysis of our RNAseq data identified 22 additional Cytochrome P450 (CYP450) enzymes—key players in drug metabolism—whose expression was regulated by reproductive stage, suggesting that the effect of liver involution extends beyond suppressed vitamin D metabolism. These findings highlight the importance of considering the postpartum involution period in the design and administration of pharmacological interventions. Citation Format: Sarah M Bernhardt, Pepper Schedin. Vitamin D metabolism is suppressed postpartum: Implications for targeting the postpartum window for cancer prevention [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B079.

Abstract A045: Elevated PGC1α during abrupt mammary gland involution leads to long-term metabolic reprogramming and genomic instability; hallmarks 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, and associated higher mortality rate. 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 show several precancerous changes, such as increased collagen deposition, inflammation, and hyperplasia in the mammary gland (MG) of mice after AI1. Recent studies indicate peroxisome proliferator-activated receptor-gamma coactivator 1-apha (PGC1α) as a regulator of involution and energy metabolism. As metabolic reprogramming is a hallmark of cancer, metabolic changes in the MG related to involution warrant investigation. Objective: Our objective was to evaluate the metabolic effects of AI in MG. We hypothesized that AI leads to marked alteration in mammary lipid metabolism, mitochondrial biogenesis, and oxidative stress through elevations of PGC1α, which cause long-term metabolic reprogramming and genomic instability. Methods: FVB/n mice were paired for breeding. At partum, dams were randomized to AI or GI cohort and standardized to 6 pups per dam. AI mice had pups removed on day 7 postpartum (PPM). For GI mice 3 pups each were removed on day 28 and 31ppm. Tissues were harvested on day 28, 56, and 120 PPM. MG were subjected to Affymetrix, Gene Set Enrichment Analysis (GSEA), Seahorse Analysis, and lipidomics. Results were validated by qPCR and Western Blot. Superoxide species were detected by flow cytometry. DNA damage was analyzed via 8-hydroxy-2’-deoxygnuanosine (8-OHdG) ELISA. Results: Day 28 AI glands had significantly higher PGC1α expression than GI glands (p=0.006). Affymetrix and GSEA data showed day 28 AI glands to have enriched pathways related to fatty acid metabolism (p=0.004) and oxidative phosphorylation (p&amp;lt;0.001). Lipidomics showed elevated levels of oxidized sphingolipids and production of prostaglandin J2 (PGJ2) in day 28 AI glands (all p&amp;lt;0.02). Day 56 AI glands had higher levels of mitochondria superoxide species (p&amp;lt;0.0001) and higher PGJ2 synthesis (p=0.01). Day 120 AI MG had upregulation of an oxidized lipid (p=0.0476). Day 120 AI glands had significantly higher 8-OHdG levels (p=0.02), higher reliance of fatty acid substrates for energy (p=0.0185), and elevated extracellular acidification rates (p=0.0194). Conclusion: Although histologically both GI and AI MG return to near pre-pregnancy state within a month, our data shows long-term metabolic reprogramming in the AI MG similar to what is shown in breast cancer cells. These metabolic changes link to early elevations in PGC1α. Addressing the metabolic changes by targeting PGC1α provide a potential option to reduce the risk of developing breast cancer if a woman is unable to breastfeed. PMID6637535 *Fellowship T32CA229114 Citation Format: Kate S Ormiston, Kirti Kaul, Neelam Shinde, Gautam Sarathy, Morgan Bauer, Djawed Bennouna, Rachel Kopec, Ramesh Ganju, Sarmila Majumder, Bhuvaneswari Ramaswamy. Elevated PGC1α during abrupt mammary gland involution leads to long-term metabolic reprogramming and genomic instability; hallmarks of breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A045.

A randomized control trial to test the effect of pegbovigrastim treatment at dry-off on plasma and milk oxylipid profiles during early mammary gland involution and the postparturient period

The early period of mammary gland involution is a critical juncture in the lactation cycle that can have significant effects on milk production and mammary gland health. Pegbovigrastim (PEG) administered 1 wk prior and on the day of parturition can enhance immune function and reduce the incidence of mastitis in the early postpartum period. Oxylipids are potent metabolites of polyunsaturated fatty acids (PUFA) and are important mediators of inflammation. The objective of this study was to evaluate effects of PEG given 1 wk before and at the day of dry-off (D0) on concentrations of oxylipids in plasma and milk from 7 d before D0 to 14 d after, as well as the effects during the first 14 d of the subsequent lactation. We hypothesized that both pro- and anti-inflammatory oxylipids would vary based on initiation of mammary gland involution and that pegbovigrastim would affect oxylipid concentrations, particularly those related to leukocytes. A complete randomized blocked design was used to enroll cows into either a PEG treatment group (n = 10) or control group (n = 10; CON). Blood samples were collected -7, -2, -1, 0, 1, 2, 4, 7, and 14 d relative to dry-off and 5, 10, and 14 d postcalving. Samples were analyzed for PUFA and oxylipids in milk and plasma by ultra-performance mass spectrometry and liquid chromatography tandem quadrupole mass spectrometry, respectively. Overall, 30 lipid mediators were measured in both milk and plasma. Repeated measures analyses revealed a significant interaction of treatment by time for milk 8-iso-keto-15-prostaglandin E2, prostaglandin F2α, plasma 8,12-iso-prostaglandin Fα-VI, 11-hydroxyeicosatetraenoic acid, and 12-hydroxyheptadecatienoic acid. The majority of milk PUFA and oxylipids differed significantly during early mammary gland involution and into the early postpartum period. This study demonstrated changes in oxylipids in milk secretions and plasma during early involution, and further investigation may illuminate multiple complex processes and reveal targets for optimization of mammary gland involution.

Intracellular calcium links milk stasis to lysosome-dependent cell death during early mammary gland involution

Involution of the mammary gland after lactation is a dramatic example of coordinated cell death. Weaning causes distension of the alveolar structures due to the accumulation of milk, which, in turn, activates STAT3 and initiates a caspase-independent but lysosome-dependent cell death (LDCD) pathway. Although the importance of STAT3 and LDCD in early mammary involution is well established, it has not been entirely clear how milk stasis activates STAT3. In this report, we demonstrate that protein levels of the PMCA2 calcium pump are significantly downregulated within 2–4 h of experimental milk stasis. Reductions in PMCA2 expression correlate with an increase in cytoplasmic calcium in vivo as measured by multiphoton intravital imaging of GCaMP6f fluorescence. These events occur concomitant with the appearance of nuclear pSTAT3 expression but prior to significant activation of LDCD or its previously implicated mediators such as LIF, IL6, and TGFβ3, all of which appear to be upregulated by increased intracellular calcium. We further demonstrate that increased intracellular calcium activates STAT3 by inducing degradation of its negative regulator, SOCS3. We also observed that milk stasis, loss of PMCA2 expression and increased intracellular calcium levels activate TFEB, an important regulator of lysosome biogenesis through a process involving inhibition of CDK4/6 and cell cycle progression. In summary, these data suggest that intracellular calcium serves as an important proximal biochemical signal linking milk stasis to STAT3 activation, increased lysosomal biogenesis, and lysosome-mediated cell death.

Effect of different inflammation states on the antimicrobial components in milk of goat udders after milking cessation.

The aim of this study was to examine the effects of milking cessation under different inflammatory conditions on the changes in antimicrobial components in milk and the process of mammary gland involution. Twenty udder halves were divided into two groups: those with (LPS) and without (control) lipopolysaccharide infusion, followed by cessation of milking for 8weeks. Milk samples were collected weekly. Udder tissue was collected 4weeks after milking cessation to measure the area of the lobule and connective tissue. After milking cessation, the somatic cell count (SCC) in the control group increased, whereas that in the LPS group did not. Lactoferrin (LF) and cathelicidin (Cath)-2 concentrations increased in both groups, whereas only LF was significantly lower in the LPS group than in the control group at week 4. The Cath-7 and S100A8 concentrations were significantly lower in the LPS group than in the control group. The lobule area was higher, and the connective tissue area was lower in the LPS group than in the control group. These results indicate that inflammation at milking cessation decreased the concentrations of some antimicrobial components and interfered with mammary gland involution. Therefore, animals with mastitis should recover prior to the onset of the dry period.

MiR-30a-3p Regulates Autophagy in the Involution of Mice Mammary Glands.

The mammary gland undergoes intensive remodeling during the lactation cycle, and the involution process of mammary gland contains extensive epithelial cells involved in the process of autophagy. Our studies of mice mammary glands suggest that miR-30a-3p expression was low during involution compared with its high expression in the mammary glands of lactating mice. Then, we revealed that miR-30a-3p negatively regulated autophagy by autophagy related 12 (Atg12) in mouse mammary gland epithelial cells (MMECs). Restoring ATG12, knocking down autophagy related 5 (Atg5), starvation, and Rapamycin were used to further confirm this conclusion. Overexpression of miR-30a-3p inhibited autophagy and altered mammary structure in the involution of the mammary glands of mice, which was indicative of alteration in mammary remodeling. Taken together, these results elucidated the molecular mechanisms of miR-30a-3p as a key induction mediator of autophagy by targeting Atg12 within the transition period between lactation and involution in mammary glands.

Poor Outcome in Postpartum Breast Cancer Patients Is Associated with Distinct Molecular and Immunologic Features.

Patients with postpartum breast cancer diagnosed after cessation of breastfeeding (postweaning, PP-BCPW) have a particularly poor prognosis compared with patients diagnosed during lactation (PP-BCDL), or to pregnant (Pr-BC) and nulliparous (NP-BC) patients, regardless of standard prognostic characteristics. Animal studies point to a role of the involution process in stimulation of tumor growth in the mammary gland. However, in women, the molecular mechanisms that underlie this poor prognosis of patients with PP-BCPW remain vastly underexplored, due to of lack of adequate patient numbers and outcome data. We explored whether distinct prognostic features, common to all breast cancer molecular subtypes, exist in postpartum tumor tissue. Using detailed breastfeeding data, we delineated the postweaning period in PP-BC as a surrogate for mammary gland involution and performed whole transcriptome sequencing, immunohistochemical, and (multiplex) immunofluorescent analyses on tumor tissue of patients with PP-BCPW, PP-BCDL, Pr-BC, and NP-BC. We found that patients with PP-BCPW having a low expression level of an immunoglobulin gene signature, but high infiltration of plasma B cells, have an increased risk for metastasis and death. Although PP-BCPW tumor tissue was also characterized by an increase in CD8+ cytotoxic T cells and reduced distance among these cell types, these parameters were not associated with differential clinical outcomes among groups. These data point to the importance of plasma B cells in the postweaning mammary tumor microenvironment regarding the poor prognosis of PP-BCPW patients. Future prospective and in-depth research needs to further explore the role of B-cell immunobiology in this specific group of young patients with breast cancer.

Effects of Serotonin on Cell Viability, Permeability of Bovine Mammary Gland Epithelial Cells and Their Transcriptome Analysis.

Serotonin (5-HT) has been reported to play an important role in mammary gland involution that is defined as the process through which the gland returns to a nonlactating state. However, the overall picture of the regulatory mechanisms of 5-HT and the effects of serotonylation on mammary gland involution still need to be further investigated. The current study aimed to investigate the effects of 5-HT on global gene expression profiles of bovine mammary epithelial cells (MAC-T) and to preliminarily examine whether the serotonylation involved in the mammary gland involution by using Monodansylcadaverine (MDC), a competitive inhibitor of transglutaminase 2. Results showed that a high concentration of 5-HT decreased viability and transepithelial electrical resistance (TEER) in MAC-T cells. Transcriptome analysis indicated that 2477 genes were differentially expressed in MAC-T cells treated with 200 μg/mL of 5-HT compared with the control group, and the Notch, p53, and PI3K-Akt signaling pathways were enriched. MDC influenced 5-HT-induced MAC-T cell death, fatty acid synthesis, and the formation and disruption of tight junctions. Overall, a high concentration of 5-HT is able to accelerate mammary gland involution, which may be regulated through the Notch, p53, and PI3K-Akt signaling pathways. Serotonylation is involved in bovine mammary gland involution.

Amplifying local serotonin signaling prior to dry-off hastens mammary gland involution and redevelopment in dairy cows

The monoamine serotonin (5-hydroxytryptamine, 5-HT) has been reported to inhibit milk protein gene expression and increase mammary epithelial cell (MEC) tight junction permeability after milk stasis. We hypothesized that increasing serotonin synthesis and signaling within the mammary epithelium before milk stasis would increase systemic and local involution markers, and downregulate the expression of milk protein and tight junction during involution, leading to more efficient tissue growth during the redevelopment phase. Herein, we examined the outcomes of increasing local mammary 5-HT synthesis before milk stasis on involution biomarkers, mammary gland microstructure, and gene and protein expression during the dry period. Multiparous Holstein cows were administered intramammary infusions (via the teat canal) of sterile water (CON, 4 mL/teat, n = 7) or 5-hydroxy-l-tryptophan (5-HTP, serotonin precursor, 20 mg/teat, n = 7) once daily for 5 d before dry-off (d 0). Blood, milk, and mammary secretions were collected and analyzed for components and metabolites. Mammary secretions were collected 12 h after the last milking and on d 1 to 4 during the dry period at 1200 h. Mammary gland biopsies were performed on d 4 (i.e., involution phase) and d 36 (i.e., redevelopment phase) of the dry period for histological and molecular evaluation. Milk protein and tight junction gene expression was quantified via real-time PCR. Hematoxylin and eosin staining, immunohistochemistry (Ki67), and immunofluorescence (serotonin, cleaved caspase 3) were performed to visualize tissue microstructure and to quantify serotonin intensity and cell turnover. Data were analyzed in SAS (SAS Institute Inc.) using 2-way ANOVA. After d 0, mammary secretions of 5-HTP cows had increased concentrations of 5-HT, lactoferrin, and bovine serum albumin. On d 1, 5-HTP cows had greater α-lactalbumin concentrations in plasma relative to CON. Serotonin intensity was increased in the mammary tissue of 5-HTP cows on d 4, relative to CON. On d 4, milk protein and tight junction gene expression was downregulated, MEC number was reduced, and cleaved caspase 3 protein was greater in mammary tissue of 5-HTP cows, relative to CON. On d 36, milk protein genes were upregulated, and the lumen:outer alveolar area and Ki67-positive cells were increased in the mammary tissue of 5-HTP cows, relative to CON. Amplifying serotonin signaling in the mammary epithelium before milk stasis at dry-off achieves greater apoptosis, leading to a reduction in MEC, allowing for greater cell proliferation, which results in more MEC during the redevelopment phase preceding the onset of lactation.

Abstract P1-05-14: ctDNA IN BREAST MILK FOR EARLY DETECTION OF PREGNANCY ASSOCIATED BREAST CANCER

Abstract The potential of cell-free tumor DNA (ctDNA) for early tumor detection in asymptomatic patients is yet to be established. In the case of pregnancy associated breast cancer (BC), early detection is of special interest, since it is an entity of special aggressiveness due to a delay in diagnosis, along with the negative effect of mammary gland involution when BC is diagnosed during the postpartum period (PPBC). Indeed, PPBC has double metastatic risk and worst prognosis. With a potential applicability for cancer screening during breastfeeding, here we explored the presence of ctDNA in breast milk (BM) from women with BC associated to pregnancy. Matched samples from breast tumor, plasma and BM from a cohort of 14 women diagnosed during pregnancy or breastfeeding were analysed by droplet digital PCR and a targeted next generation sequencing panel (NGS). Thirteen patients had early-stage disease (11% Stage I, 61% Stage II and 28% Stage III) whilst one was diagnosed at advanced stage. BM harboured ctDNA, since mutations present in the tumor tissue were detected in 86% of the cases by ddPCR and in 71,4% by NGS (difference owing to technique sensitivity). Matched plasma samples had detectable ctDNA levels in only 8% of the cases. In one of the patients, a BM sample collected 18 months prior to BC diagnosis revealed the presence of a pathogenic PIK3CA mutation later detected in the surgically removed tumor. With the ultimate goal of applying the NGS in BM as a technique for early detection of BC in the postpartum period, we have collected samples from healthy volunteers and patients at high risk of developing BC (defined as women becoming pregnant at &amp;gt;40 years or carriers of germ-line pathogenic variants associated with BC -ie: BRCA1, BRCA2, PALB2, RAD51C/D). The application of NGS in BM as a technique for early detection of BC in the postpartum period, identified in a high-risk woman (criteria of enrolment was the age, 46yo) an AKT1 pathogenic mutation in the right-sided BM anticipating by 6 months the radiological diagnosis of a Luminal A tumor, stage pT1bN0M0. In summary, our data provides evidence that ctDNA in BM is highly prevalent even at initial tumor stages, and could be exploited for early breast cancer screening during breastfeeding. Citation Format: Cristina Saura, Carolina Ortiz, Enrique Javier Arenas, Judit Matito, Anna Suñol-Camas, Octavi Cordoba, Alex Martinez-Sabadell, Itziar Garcia-Ruiz, Ignacio Miranda, Clara Morales-Comas, Estela Carrasco Lopez, Cristina Viaplana, Vicente Peg, Paolo Nuciforo, Neus Bayo, Josep Maria Miquel, Marina Gomez-Rey, Guillermo Villacampa, Silvia Arevalo, Javier Carmona, Martín Espinosa-Bravo, Judith Balmaña, Rodrigo Dienstmann, Joaquin Arribas, Josep Tabernero, miriam sanso, Ana Vivancos. ctDNA IN BREAST MILK FOR EARLY DETECTION OF PREGNANCY ASSOCIATED BREAST CANCER [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-05-14.

Abstract P4-08-11: AKT and EZH2 inhibitors kill TNBCs by hijacking mechanisms of involution

Abstract Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype and has the highest rate of recurrence. The predominant standard of care for advanced TNBC is systemic chemotherapy with or without immunotherapy, however responses are typically short-lived. Thus, there is an urgent need to develop more effective treatments. PI3K pathway components represent plausible therapeutic targets, as approximately 40% of TNBCs have PIK3CA/AKT1/PTEN alterations. However, unlike hormone receptor-positive tumors, it is still unclear if or how PI3K pathway inhibitors will be effective in triple-negative disease. Here we identify a promising AKT inhibitor-based therapeutic combination for TNBC. Specifically, we show that AKT inhibitors potently synergize with agents that suppress the histone methyltransferase, EZH2, and promote robust tumor regression in multiple TNBC models in vivo. AKT and EZH2 inhibitors exert these effects by first cooperatively driving basal-like TNBC cells into a more differentiated, luminal-like state, which cannot be effectively induced by either agent alone. More importantly, once differentiated, these agents kill TNBCs by hijacking signals that normally drive mammary gland involution. Together these findings identify a promising therapeutic strategy for this highly aggressive tumor type and illustrate how deregulated epigenetic enzymes can insulate tumors from oncogenic vulnerabilities. These studies also reveal how developmental tissue-specific cell death pathways may be co-opted for therapeutic benefit. Citation Format: Amy Schade, Naiara Perurena, Marina Watanabe, Carrie L. Rodriguez, Patrick Loi, Natalie Pilla, Rachel A. Davis, Kaia Mattioli, Dongxi Xiang, Jason J. Zoeller, Zhe Li, Ana C. Garrido-Castro, Sara Tolaney, Karen Cichowski. AKT and EZH2 inhibitors kill TNBCs by hijacking mechanisms of involution [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-11.

Fur removal promotes an earlier expression of involution-related genes in mammary gland of lactating mice

Peak lactation occurs when milk production is at its highest. The factors limiting peak lactation performance have been subject of intense debate. Milk production at peak lactation appears limited by the capacity of lactating females to dissipate body heat generated as a by-product of processing food and producing milk. As a result, manipulations that enhance capacity to dissipate body heat (such as fur removal) increase peak milk production. We investigated the potential correlates of shaving-induced increases in peak milk production in laboratory mice. By transcriptomic profiling of the mammary gland, we searched for the mechanisms underlying experimentally increased milk production and its consequences for mother–young conflict over weaning, manifested by advanced or delayed involution of mammary gland. We demonstrated that shaving-induced increases in milk production were paradoxically linked to reduced expression of some milk synthesis-related genes. Moreover, the mammary glands of shaved mice had a gene expression profile indicative of earlier involution relative to unshaved mice. Once provided with enhanced capacity to dissipate body heat, shaved mice were likely to rear their young to independence faster than unshaved mothers.