Mammary Gland Involution Research Articles

Transcription Factor Sox4 as a Potential Player in Mammary Gland Involution.

Mammary gland involution is a regressive process for the gland to return to its prepregnancy state after lactation and comprises an initial reversible and second remodeling stage. Although many genes and the multiple expression profiles of their mRNAs have been found in this process, the mechanisms controlling the profiles are largely unknown. In this study, we identified and analyzed transcription factor Sox4 in mammary gland involution. Elevated expression of Sox4 gene in the first stage (48 h after weaning) was observed at the mRNA and protein levels in the mouse mammary gland. Immunohistochemistry of the involuting gland indicated that Sox4 was located in the nuclei of epithelial cells. Nuclear Sox4 was also detected in the second stage, but unlikely to be involved in cell death, one of the characteristic events of involution. To clarify the functional roles of Sox4 in involution, we introduced a model, including a normal mammary epithelial cell line, for finding candidate target genes of this transcription factor and examined its effect on tenascin C mRNA expression.

Invited review: Accelerating mammary gland involution after drying-off in dairy cattle

Bovine mammary gland involution, as a part of the reproductive cycle in dairy cows, is a very important remodeling transformation of the mammary gland for the subsequent lactation. There is considerable incentive to accelerate mammary gland involution to improve udder health, shorten the dry period, and simplify the management process by reducing dietary changes. The complex process of mammary involution is characterized by morphological changes in the epithelial cells and mammary tissue, changes in the composition of mammary secretions, and changes in the integrity of tight junctions. Involution is facilitated by elements of the immune system and several types of proteases and is coordinated by various types of hormones. This review first describes the involution process and then argues for the need to accelerate it. Last, this review focuses on various intervention methods for accelerating involution. Our aim is to provide a comprehensive overview of bovine mammary gland involution as well as potential techniques and new opinions for dry cow management.

PD-1 Blockade During Post-partum Involution Reactivates the Anti-tumor Response and Reduces Lymphatic Vessel Density.

Post-partum breast cancer patients, or breast cancer patients diagnosed within 10 years of last childbirth, are ~3–5 times more likely to develop metastasis in comparison to non-post-partum, or nulliparous, patients. Additionally, post-partum patients have increased tumor-associated lymphatic vessels and LN involvement, including when controlled for size of the primary tumor. In pre-clinical, immune-competent, mouse mammary tumor models of post-partum breast cancer (PPBC), tumor growth and lymphogenous tumor cell spread occur more rapidly in post-partum hosts. Here we report on PD-L1 expression by lymphatic endothelial cells and CD11b+ cells in the microenvironment of post-partum tumors, which is accompanied by an increase in PD-1 expression by T cells. Additionally, we observed increases in PD-L1 and PD-1 in whole mammary tissues during post-partum mammary gland involution; a known driver of post-partum tumor growth, invasion, and metastasis in pre-clinical models. Importantly, implantation of murine mammary tumor cells during post-partum mammary gland involution elicits a CD8+ T cell population that expresses both the co-inhibitory receptors PD-1 and Lag-3. However, upon anti-PD-1 treatment, during post-partum mammary gland involution, the involution-initiated promotional effects on tumor growth are reversed and the PD-1, Lag-3 double positive population disappears. Consequently, we observed an expansion of poly-functional CD8+ T cells that produced both IFNγ and TNFα. Finally, lymphatic vessel frequency decreased significantly following anti-PD-1 suggesting that anti-PD-1/PD-L1 targeted therapies may have efficacy in reducing tumor growth and dissemination in post-partum breast cancer patients.

NHERF1 Is Required for Localization of PMCA2 and Suppression of Early Involution in the Female Lactating Mammary Gland.

Prior studies have demonstrated that the calcium pump, plasma membrane calcium ATPase 2 (PMCA2), mediates calcium transport into milk and prevents mammary epithelial cell death during lactation. PMCA2 also regulates cell proliferation and cell death in breast cancer cells, in part by maintaining the receptor tyrosine kinase ErbB2/HER2 within specialized plasma membrane domains. Furthermore, the regulation of PMCA2 membrane localization and activity in breast cancer cells requires its interaction with the PDZ domain-containing scaffolding molecule sodium-hydrogen exchanger regulatory factor (NHERF) 1. In this study, we asked whether NHERF1 also interacts with PMCA2 in normal mammary epithelial cells during lactation. Our results demonstrate that NHERF1 expression is upregulated during lactation and that it interacts with PMCA2 at the apical membrane of secretory luminal epithelial cells. Similar to PMCA2, NHERF1 expression is rapidly reduced by milk stasis after weaning. Examining lactating NHERF1 knockout (KO) mice showed that NHERF1 contributes to the proper apical location of PMCA2, for proper apical-basal polarity in luminal epithelial cells, and that it participates in the suppression of Stat3 activation and the prevention of premature mammary gland involution. Additionally, we found that PMCA2 also interacts with the closely related scaffolding molecule, NHERF2, at the apical membrane, which likely maintains PMCA2 at the plasma membrane of mammary epithelial cells in lactating NHERF1KO mice. Based on these data, we conclude that, during lactation, NHERF1 is required for the proper expression and apical localization of PMCA2, which, in turn, contributes to preventing the premature activation of Stat3 and the lysosome-mediated cell death pathway that usually occur only early in mammary involution.

Identification of novel polymorphism in buffalo stanniocalcin-1 gene and its expression analysis in mammary gland under different stages of lactation

Stanniocalcin-1 (STC1) is secreted by the variety of tissues having a major role in the regulation of calcium ions in the involuting mammary gland. The present work aims to sequence and structural characterization as well as expression profiling of STC1 gene in buffalo. Polymorphism identified in the 3-untranslated region (UTR) was analysed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) genotyping in riverine and swamp buffaloes. Expression profiling of STC1 was performed in different lactation stages of mammary gland and peripheral blood mononuclear cells to study the impact of 3'-UTR polymorphism on its expression. Different polymorphic sites were detected in the entire coding and noncoding regions of riverine and swamp buffaloes, including two INDELs. An identified polymorphic nucleotide locus A324G, having target sites for two miRNAs, namely bta-miR-2382 and bta-miR-1343, reported in cattle, was genotyped by PCR-RFLP to reveal variable allelic distribution among swamp and riverine buffaloes. Gene expression profiling across buffalo mammary tissues representing different lactation stages showed maximum expression of the STC1 gene in the involuting mammary gland. Ruminants' specific genetic variation has been observed in STC1 and its implication in buffalo mammary gland involution as well as coregulation of gene expression through miRNA binding in the 3'-UTR is suggested.

Abstract ES2-2: Breast cancer's metastatic moment: How postpartum tissue involution facilitates progression

Abstract Virginia F. Borges* and Pepper J. Schedin*# Young Women's Breast Cancer Translational Program, University of Colorado, Anschutz Medical Campus, Aurora, Colorado*, and Knight Cancer Institute, Oregon Health & Sciences University, Portland, Oregon#. Young women diagnosed with breast cancer (YWBC) have worse outcomes than older women, however the reasons remain poorly understood. The two dominant hypotheses, centered on tumor cell intrinsic and extrinsic biology respectively, appear in conflict. One hypothesis emphasizes the role of poor prognostic factors increased in young patients, such as ER-, PR- and Her2- disease. A second theory posits that host biology related to reproductive history drives poor outcomes, independent of tumor biologic subtype or size. However, studies addressing the roles of pregnancy in breast cancer risk and outcomes can appear discordant. Consequently, metrics of reproductive history are not always included in standard risk assessment tools, nor utilized to optimize treatment strategies. This education session is focused on bridging the gap between these two theories. Specifically, our data shows that tumor intrinsic risk factors for breast cancer progression are significantly modulated by reproductive biology and support the incorporation of reproductive history into clinical care decisions. There is a strong connection between reproductive history and risk of metastasis in breast cancer. Epidemiologic studies find a recent, but not concurrent, pregnancy to be an independent risk factor for breast cancer metastasis. Our own studies find a ∼3 fold increase in metastases in patients diagnosed postpartum (PPBC). Further, we and others find the postpartum risk-window persists for ≥10 years after child birth. Finally, interactions between breast cancer subtype and pregnancy history are observed, supporting roles for both tumor cell intrinsic and extrinsic biology in driving metastasis in young women. To investigate the mechanism of postpartum host biology on tumor progression, we developed rodent models of PPBC. We find the physiologic process of weaning-induced mammary gland involution to support immune suppression, tumor cell dissemination, and tumor cell access to vasculature — concerted processes that lead to breast cancer metastasis. In a prospective clinical trial, we developed a cohort of healthy, recently nursing women, and find similar tumor-promotional hallmarks in the human breast. Newly identified postpartum liver biology may also shed insight into metastasis in young women. We recently reported weaning-induced involution in the rodent liver. Further, the rodent involuting liver is highly permissive to breast cancer outgrowth. Potential relevance to women is suggested by a first-site-of-metastasis study in YWBC patients. Postpartum patients were at increased risk for liver metastasis, but not lung, brain or bone. Finally, our data showing normal mammary gland involution to be a COX-2 mediated process has led to preclinical modeling of NSAID based PPBC prevention and treatment strategies. Translating NSAID interventions to young women is potentially high-impact, as ∼50% of all YWBC patients are diagnosed within 10 years of their last pregnancy, putting them into an under-recognized, high risk, poor prognostic group. Citation Format: Schedin P. Breast cancer's metastatic moment: How postpartum tissue involution facilitates progression [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr ES2-2.

New insights into the catalytic inactivity of mammary gland protein-40, a chitinase-like protein expressed during mammary gland involution

MGP-40 is a mammary gland-specific glycoprotein which is expressed during involution and is an important marker for mammary gland apoptosis. It is an inactive chitinase-like protein belonging to Glycosyl Hydrolase family 18. The present study reports sequence characterization, tissue-specific expression analysis, production of recombinant MGP-40 and its mutant (A117D and L119E) in both E. coli and COS1 cells for their chitin-binding and chitinase activity analysis. The cDNA of buffalo MGP-40 was cloned and sequenced which corresponded to 1803bp with an open reading frame of 1152bp (361 aa), signal sequence of 63bp (21 aa), 5' and 3' UTR of 144bp and 507bp, respectively. The 3' UTR analysis revealed potential sites for high level expression and stability during involution. The half-life of buffalo MGP-40 was found to be 11.7h. MGP-40 was highly expressed in mammary gland followed by small intestine, spleen and mammary epithelial cells. The purified recombinant MGP-40 and its mutant expressed in E.coli were observed to bind chitin efficiently, however, no chitinase activity was observed. Further, chitinase activity was also not observed by expressing mutant recombinant MGP-40 in COS1 cells ruling out the possible role of post-translational modifications. Structure-based in-silico mutagenesis by FoldX algorithm showed a drastic decrease in overall fold stability which might be a possible reason for inability to recover its activity. Therefore, chitinase activity could not be restored in MGP-40 even after reverting back two critical residues in active site which may be due to detrimental effect of mutations on structural stability.

Oxylipid profiles of dairy cattle vary throughout the transition into early mammary gland involution

Successful lactation in multiparous dairy cattle relies on a well-managed dry period that allows the mammary gland to remodel and regenerate between lactations. Oxylipids are potent inflammatory mediators that are capable of regulating all aspects of inflammation. Although an oxylipid profile has been documented for periparturient and lactating cattle, little work has been done to define the profile of cows in the early dry period. Therefore, our group aimed to characterize the oxylipid profile in healthy cows during the transition into early mammary gland involution. Plasma samples were collected from 10 healthy Holstein dairy cows via coccygeal venipuncture 6 d before dry-off (d -6), at dry-off (d 0), and 1 (d +1), 2 (d +2), 6 (d +6), and 12 (d +12) d after the dry-off date. Liquid chromatography-mass spectrometry was used to quantify select monounsaturated fatty acids, polyunsaturated fatty acids, and saturated fatty acids, whereas oxylipids were quantified using liquid chromatography-tandem mass spectrometry. The results of this study revealed a unique profile of pro- and anti-inflammatory oxylipids throughout the transition from late lactation into the dry period. Many compounds reached the highest concentrations of the study at d +1, d +2, or d +12, whereas others reached the lowest concentrations at d +12. The characterization of this profile allows for further understanding of the physiology of early mammary involution. Future studies should investigate how the oxylipid profile of early mammary involution may affect the health and productivity of dairy cows.

TGF-beta signalling in bovine mammary gland involution and a comparative assessment of MAC-T and BME-UV1 cells as in vitro models for its study.

The goal of the dairy industry is ultimately to increase lactation persistency, which is the length of time during which peak milk yield is sustained. Lactation persistency is determined by the balance of cell apoptosis and cell proliferation; when the balance is skewed toward the latter, this results in greater persistency. Thus, we can potentially increase milk production in dairy cows through manipulating apoptogenic and antiproliferative cellular signaling that occurs in the bovine mammary gland. Transforming growth factor beta 1 (TGFβ1) is an antiproliferative and apoptogenic cytokine that is upregulated during bovine mammary gland involution. Here, we discuss possible applications of TGFβ1 signaling for the purposes of increasing lactation persistency. We also compare the features of mammary alveolar cells expressing SV-40 large T antigen (MAC-T) and bovine mammary epithelial cells-clone UV1 (BME-UV1) cells, two extensively used bovine mammary epithelial cell lines, to assess their appropriateness for the study of TGFβ1 signaling. TGFβ1 induces apoptosis and arrests cell growth in BME-UV1 cells, and this was reported to involve suppression of the somatotropic axis. Conversely, there is no proof that exogenous TGFβ1 induces apoptosis of MAC-T cells. In addition to TGFβ1’s different effects on apoptosis in these cell lines, hormones and growth factors have distinct effects on TGFβ1 secretion and synthesis in MAC-T and BME-UV1 cells as well. MAC-T and BME-UV1 cells may behave differently in response to TGFβ1 due to their contrasting phenotypes; MAC-T cells have a profile indicative of both myoepithelial and luminal populations, while the BME-UV1 cells exclusively contain a luminal-like profile. Depending on the nature of the research question, the use of these cell lines as models to study TGFβ1 signaling should be carefully tailored to the questions asked.

Studies of postpartum mammary gland involution reveal novel pro-metastatic mechanisms.

Postpartum involution is the process by which the lactating mammary gland returns to the pre-pregnant state after weaning. Expression of tumor-promotional collagen, upregulation of matrix metalloproteinases, infiltration of M2 macrophages, and remodeling of blood and lymphatic vasculature are all characteristics shared by the involuting mammary gland and breast tumor microenvironment. The tumor promotional nature of the involuting mammary gland is perhaps best evidenced by cases of postpartum breast cancer (PPBC), or those cases diagnosed within 10 years of most recent childbirth. Women with PPBC experience more aggressive disease and higher risk of metastasis than nulliparous patients and those diagnosed outside the postpartum window. Semaphorin 7a (SEMA7A), cyclooxygenase-2 (COX-2), and collagen are all expressed in the involuting mammary gland and, together, predict for decreased metastasis free survival in breast cancer. Studies investigating the role of these proteins in involution have been important for understanding their contributions to PPBC. Postpartum involution thus represents a valuable model for the identification of novel molecular drivers of PPBC and classical cancer hallmarks. In this review, we will highlight the similarities between involution and cancer in the mammary gland, and further define the contribution of SEMA7A/COX-2/collagen interplay to postpartum involution and breast tumor progression and metastasis.

Semaphorin 7A Promotes Macrophage-Mediated Lymphatic Remodeling during Postpartum Mammary Gland Involution and in Breast Cancer.

Postpartum mammary gland involution is a tissue remodeling event that occurs in all mammals in the absence of nursing or after weaning to return the gland to the pre-pregnant state. The tissue microenvironment created by involution has proven to be tumor promotional. Here we report that the GPI-linked protein semaphorin 7A (SEMA7A) is expressed on mammary epithelial cells during involution and use preclinical models to demonstrate that tumors induced during involution express high levels of SEMA7A. Overexpression of SEMA7A promoted the presence of myeloid-derived podoplanin (PDPN)-expressing cells in the tumor microenvironment and during involution. SEMA7A drove the expression of PDPN in macrophages, which led to integrin- and PDPN-dependent motility and adherence to lymphatic endothelial cells to promote lymphangiogenesis. In support of this mechanism, mammary tissue from SEMA7A-knockout mice exhibited decreased myeloid-derived PDPN-expressing cells, PDPN-expressing endothelial cells, and lymphatic vessel density. Furthermore, coexpression of SEMA7A, PDPN, and macrophage marker CD68 predicted for decreased distant metastasis-free survival in a cohort of over 600 cases of breast cancer as well as in ovarian, lung, and gastric cancers. Together, our results indicate that SEMA7A may orchestrate macrophage-mediated lymphatic vessel remodeling, which in turn drives metastasis in breast cancer.Signficance: SEMA7A, which is expressed on mammary cells during glandular involution, alters macrophage biology and lymphangiogenesis to drive breast cancer metastasis. Cancer Res; 78(22); 6473-85. ©2018 AACR.

Autophagy and unfolded protein response (UPR) regulate mammary gland involution by restraining apoptosis-driven irreversible changes

The postnatal mammary gland undergoes repeated cycles of proliferation and cell death, most notably when the fully differentiated (lactating) gland dedifferentiates to a prelactation state. Accumulation of milk proteins in the secretory epithelium creates the stress signal that triggers this process (involution). How this stress is perceived, and the cellular processes that are subsequently activated, remain unclear. We now report that Unfolded Protein Response (UPR), autophagy, and apoptosis related genes cluster separately during lactation and involution in the mouse mammary gland. Time-course experiments in rodents show that autophagy and UPR signaling are tightly co-regulated at the transition from reversible to irreversible involution. Inhibition of autophagy by chloroquine or genetic deletion of one ATG7 allele enhanced progression of mammary involution into the irreversible phase, as characterized by an early/precocious induction of apoptosis. These are the first preclinical in vivo data in support of a clinical trial testing an autophagy inhibitor for prevention of intraductal breast malignancy progression to invasive breast cancer. In marked contrast, stimulation of autophagy by low dose tunicamycin treatment reduced apoptosis and extended the reversible phase of involution by sustaining the secretory epithelium. Autophagy stimulators could be used short-term to promote lactation in women experiencing difficulties or irregularities in nursing. Taken together, these data indicate that UPR and autophagy play a key role in regulating the balance between cell survival and apoptosis during normal mammary gland regression.

Ibuprofen supports macrophage differentiation, T cell recruitment, and tumor suppression in a model of postpartum breast cancer

BackgroundWomen diagnosed with breast cancer within 5 years postpartum (PPBC) have poorer prognosis than age matched nulliparous women, even after controlling for clinical variables known to impact disease outcomes. Through rodent modeling, the poor prognosis of PPBC has been attributed to physiologic mammary gland involution, which shapes a tumor promotional microenvironment through induction of wound-healing-like programs including myeloid cell recruitment. Previous studies utilizing immune compromised mice have shown that blocking prostaglandin synthesis reduces PPBC tumor progression in a tumor cell extrinsic manner. Given the reported roles of prostaglandins in myeloid and T cell biology, and the established importance of these immune cell populations in dictating tumor growth, we investigate the impact of involution on shaping the tumor immune milieu and its mitigation by ibuprofen in immune competent hosts.MethodsIn a syngeneic (D2A1) orthotopic Balb/c mouse model of PPBC, we characterized the impact of mammary gland involution and ibuprofen treatment on the immune milieu in tumors and draining lymph nodes utilizing flow cytometry, multiplex IHC, lipid mass spectroscopy and cytokine arrays. To further investigate the impact of ibuprofen on programming myeloid cell populations, we performed RNA-Seq on in vivo derived mammary myeloid cells from ibuprofen treated and untreated involution group mice. Further, we examined direct effects of ibuprofen through in vitro bone marrow derived myeloid cell cultures.ResultsTumors implanted into the mammary involution microenvironment grow more rapidly and display a distinct immune milieu compared to tumors implanted into glands of nulliparous mice. This milieu is characterized by increased presence of immature monocytes and reduced numbers of T cells and is reversed upon ibuprofen treatment. Further, ibuprofen treatment enhances Th1 associated cytokines as well as promotes tumor border accumulation of T cells. Safety studies demonstrate ibuprofen does not impede gland involution, impact subsequent reproductive success, nor promote auto-reactivity as detected through auto-antibody and naïve T cell priming assays.ConclusionsIbuprofen administration during the tumor promotional microenvironment of the involuting mammary gland reduces overall tumor growth and enhances anti-tumor immune characteristics while avoiding adverse autoimmune reactions. In sum, these studies implicate beneficial prophylactic use of ibuprofen during the pro-tumorigenic window of mammary gland involution.

OXTR overexpression leads to abnormal mammary gland development in mice.

Oxytocin receptor (OXTR) is a G-protein coupled receptor and known for regulation of maternal and social behaviors. Null mutation (Oxtr-/-) leads to defects in lactation due to impaired milk ejection and maternal nurturing. Overexpression of OXTR has never been studied. To define the functions of OXTR overexpression, a transgenic mouse model that overexpresses mouse Oxtr under β-actin promoter was developed (++Oxtr). ++Oxtr mice displayed advanced development and maturation of mammary gland, including ductal distention, enhanced secretory differentiation and early milk production at non-pregnancy and early pregnancy. However, ++Oxtr dams failed to produce adequate amount of milk and led to lethality of newborns due to early involution of mammary gland in lactation. Mammary gland transplantation results indicated the abnormal mammary gland development was mainly from hormonal changes in ++Oxtr mice but not from OXTR overexpression in mammary gland. Elevated OXTR expression increased prolactin-induced phosphorylation and nuclear localization of STAT5 (p-STAT5), decreased progesterone level, leading to early milk production in non-pregnant and early pregnant females, whereas low prolactin and STAT5 activation in lactation led to insufficient milk production. Progesterone treatment reversed the OXTR-induced accelerated mammary gland development by inhibition of prolactin/p-STAT5 pathway. Prolactin administration rescued lactation deficiency through STAT5 activation. Progesterone plays a negative role in OXTR regulated prolactin/p-STAT5 pathways. The study provides evidence that OXTR overexpression induces abnormal mammary gland development through progesterone and prolactin-regulated p-STAT5 pathway.

Abstract SY38-02: Postpartum tissue remodeling drives breast cancer metastasis

Abstract Women diagnosed with breast cancer within 10 years of a completed pregnancy are at ~2-3 fold increased risk for developing metastatic disease (1,2). This increased risk for metastasis is independent of tumor stage and biologic subtype, implicating a tumor-extrinsic biology unique to the postpartum woman (3,4). Further, 30-50% of all young women diagnosed with breast cancer have had a completed pregnancy within the last 10 years (4), making a postpartum diagnosis an independent poor prognostic indicator for a significant number of patients. One postpartum event that may drive tumor progression in rodents (5-8) and women (9) is weaning-induced mammary gland involution, a physiologic tissue remodeling process that shares numerous attributes with protumorigenic wound healing. We have developed rodent models designed to mimic this highly metastatic subset of young women's breast cancer and find that weaning-induced mammary gland involution supports breast cancer growth, dissemination, and escape to secondary organs in a COX-2 dependent manner (6,10,11). Here we extend these studies by investigating the mammary gland from a mucosal organ perspective, as identifying links with mucosal biology and its associated immune suppression may provide insight into the poor prognosis of postpartum breast cancers. While the mammary gland is not classically considered a mucosal organ, bacteria routinely interface with luminal epithelium during lactation, necessitating barrier function. Similar barrier defenses are anticipated during weaning-induced mammary gland involution, as milk stasis increases risk for mastitis. One key immunologic hallmark of a mucosal organ is the presence of a distinct CD4+ helper T-cell subset, Th17 cells. Th17 cells stimulate epithelial cell junction integrity and epithelial secretion of mucins and defensins, mechanisms that barricade against bacterial activation of antitumor Th1 immune cells. Within the mammary gland we find a baseline mucosal program of Th17 T cells, which is elevated within lactating and involuting mammary glands. Mucosal features are further expanded during involution to include tolerogenic dendritic cell phenotypes, barrier-supportive antimicrobials, alternatively activated Th2 CD4+ cells, and immunosuppressive Treg CD4+T cells. Further, during involution, we find suppression of mammary-derived antigen-dependent CD4+ T-cell activation, data consistent with immune tolerance. We also find antigen-independent accumulation of memory Th17- Treg CD4+ T cells, data consistent with weaning-induced mammary inflammation. Overall, these data elucidate strong mucosal immune programs within the lactating gland and immune-suppressive programs within the involuting gland. Tumor cells within the involution immunologic microenvironment are anticipated to avoid immune detection and readily escape the mammary gland, a mechanism that may contribute to increased metastasis observed in postpartum breast cancer patients. Importantly, to successfully metastasize, a tumor cell must not only escape from the primary tumor, but also seed and grow within the secondary site. Thus, an additional potential mechanism by which a postpartum diagnosis may confer increased risk of metastasis is through involution-specific changes at metastatic sites. We report evidence for such a metastatic advantage within the liver of postpartum hosts. Using both intracardiac and portal vein liver metastasis models, we find that the post-weaning liver supports increased seeding of murine mammary tumor cells compared to livers of nulliparous control mice (12). Relevance to women is suggested by data obtained from a cohort of young women's breast cancer patients, where liver metastatic tropism is observed specifically in postpartum patients. In rodents, the biology driving this metastatic advantage is weaning-induced liver involution, a previously unrecognized tissue remodeling process. Post weaning, we find a 50% reduction in liver volume, hepatocyte cell death, deposition of fibrillar collagen and tenascin-c, increased matrix metalloproteinase activity, and influxes of immune cell populations with immune-suppressive phenotypes (12,13). These tissue-level changes are consistent with the establishment of a prometastatic niche during liver involution. Combined, our studies are supportive of the mammary gland being a hormone-responsive, immune-suppressed mucosal organ, and suggest the liver as a hormone-responsive organ that is functionally coordinated with the mammary gland during the cycle of pregnancy, lactation, and weaning. Further, we identify stromal remodeling in the mammary gland and liver as a mediator of breast cancer metastasis in the postpartum window. These findings shed light on how normal reproductive physiology, specifically mucosal biology interfaced with tissue remodeling, alters site-specific metastasis. These findings provide the framework necessary to investigate postpartum involution as a target for the prevention of breast cancer metastasis in young women.

Abstract 2242: Breastfeeding protects against pro-tumorigenic changes in the mammary gland by limiting epithelial luminal progenitor cell expansion

Abstract Introduction: Multiple epidemiological studies have shown that prolonged breastfeeding is associated with a reduced risk of developing triple-negative/basal-like breast cancers (TN/BLBC). However, no preclinical models that delineate the mechanism of this link exist. This understanding is critical not only to prevent TN/BLBC, but also to address disparity in breast cancer outcomes, as African-American women have a higher incidence of TN/BLBC and lower prevalence of breastfeeding. We present our data using mouse models that provides new insights into this link. Experimental procedure: We modeled gradual involution (GI) and abrupt involution (AI) of mammary glands (MGs) in wild type FVB/N mice. Uniparous mice were assigned to AI or GI cohorts either by removal of all pups on day7 postpartum (AI) or allowing the pups to naturally wean (GI). MGs were harvested for analysis on postpartum day28, day56 and day120. We assessed MG morphology/histology using whole mounts and H&E stained sections, collagen deposition using Trichrome and PicroSirus red staining, inflammatory markers and immune cell infiltration using immunohistochemistry. Mammary epithelial cell hierarchy was analyzed by Fluorescence-Activated Cell Sorting of a single cell suspension prepared from the MGs. Gene expression was analyzed in mouse mammary epithelial cells using Affymatrix Gene ChIP Mouse Transcriptome array 1.0 and Gene Set Enrichment Analysis (GSEA) was used to analyze gene expression data. Summary: Abruptly involuted MGs exhibited altered morphology including denser stroma, increased collagen deposition with higher levels of Type I collagen, increased inflammation (pStat3-Y705), increased immune cell infiltration and proliferation compared to GI glands. The mammary epithelial cell hierarchy was disrupted with marked expansion of the luminal progenitor (LP) population in the AI glands but not in the GI glands, a trend frequently observed in women heterozygous for BRCA1 mutation at higher risk of developing BLBC. Enrichment of an LP gene signature and Notch pathway was observed in mouse LP cells isolated from the AI glands. Most strikingly, the AI glands developed alveolar hyperplasia, and squamous metaplasia within 4 months of removal of the pups. Conclusion: Involution leading to a pro-inflammatory milieu in the MG is well established. Whether involution that happens gradually after prolonged breastfeeding differs in its effect on MGs has not been well studied. Using novel animal modeling to study the differences in MGs following abrupt vs gradual involution, we report a distinct morphology in the mammary tissue favoring pro-carcinogenic changes following AI. Furthermore, we show for the first time, expansion of the LP population in AI glands, which is known to be cell of origin for TN/BLBC potentially linking the risk of TN/BLBC with lack of breastfeeding. Further studies are ongoing. Citation Format: Mustafa M. Basree, Neelam Shinde, Christopher Koivisto, Maria Cuitino, Raleigh Kladney, Allen Zhang, Hee Kyung Kim, Anthony Trimboli, Jianying Zhang, Gustavo W. Leone, Gina M. Sizemore, Sarmila Majumder, Bhuvaneswari Ramaswamy. Breastfeeding protects against pro-tumorigenic changes in the mammary gland by limiting epithelial luminal progenitor cell expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2242.

The Multifaceted Role of STAT3 in Mammary Gland Involution and Breast Cancer.

Since seminal descriptions of signal transducer and activator of transcription 3 (STAT3) as a signal transducer and transcriptional regulator, which is most usually activated by phosphorylation of a specific tyrosine residue, a staggering wealth of research has delineated the key role of this transcription factor as a mediator of mammary gland postlactational regression (involution), and paradoxically, a pro-survival factor in breast cancer and some breast cancer cell lines. STAT3 is a critical regulator of lysosomal-mediated programmed cell death (LM-PCD) during mammary gland involution, where uptake of milk fat globules, and consequent high levels of free fatty acids, cause permeabilisation of lysosomal vesicle membranes, in turn leading to cathepsin protease leakage and cell death. A recent proteomic screen of STAT3-induced changes in lysosomal membrane protein components has highlighted wide-ranging effects of STAT3, which may coordinate LM-PCD via the stimulation of endocytosis, intracellular trafficking, and lysosome biogenesis. In parallel, STAT3 regulates the acute phase response during the first phase of involution, and it contributes to shaping the pro-tumourigenic ‘wound healing’ signature of the gland during the second phase of this process. STAT3 activation during involution is important across species, although some differences exist in the progression of involution in dairy cows. In breast cancer, a number of upstream regulators can lead to STAT3 activation and the effects of phosphorylation of STAT3 are equally wide-ranging. Recent studies have implicated microRNAs in some regulatory pathways. In this review, we will examine the multifaceted role of STAT3 in mammary gland involution and tumourigenesis, incorporating a review of these fundamental processes in tandem with a discussion of recent developments in this field.

ZnT2 is critical for lysosome acidification and biogenesis during mammary gland involution.

Mammary gland involution, a tightly regulated process of tissue remodeling by which a lactating mammary gland reverts to the prepregnant state, is characterized by the most profound example of regulated epithelial cell death in normal tissue. Defects in the execution of involution are associated with lactation failure and breast cancer. Initiation of mammary gland involution requires upregulation of lysosome biogenesis and acidification to activate lysosome-mediated cell death; however, specific mediators of this initial phase of involution are not well described. Zinc transporter 2 [ZnT2 ( SLC30A2)] has been implicated in lysosome biogenesis and lysosome-mediated cell death during involution; however, the direct role of ZnT2 in this process has not been elucidated. Here we showed that ZnT2-null mice had impaired alveolar regression and reduced activation of the involution marker phosphorylated Stat3, indicating insufficient initiation of mammary gland remodeling during involution. Moreover, we found that the loss of ZnT2 inhibited assembly of the proton transporter vacuolar ATPase on lysosomes, thereby decreasing lysosome abundance and size. Studies in cultured mammary epithelial cells revealed that while the involution signal TNFα promoted lysosome biogenesis and acidification, attenuation of ZnT2 impaired the lysosome response to this involution signal, which was not a consequence of cytoplasmic Zn accumulation. Our findings establish ZnT2 as a novel regulator of vacuolar ATPase assembly, driving lysosome biogenesis, acidification, and tissue remodeling during the initiation of mammary gland involution.

Gene set analysis of post-lactational mammary gland involution gene signatures in inflammatory and triple-negative breast cancer.

BackgroundEpidemiological studies have found that triple-negative breast cancer (TNBC) and TN inflammatory breast cancer (IBC) are associated with lower frequency and duration of breast-feeding compared to non-TNBC and non-TN IBC, respectively. Limited breast-feeding could reflect abrupt or premature involution and contribute to a “primed” stroma that is permissive to the migration of cancer cells typical of IBC. We hypothesized that gene expression related to abrupt mammary gland involution after forced weaning may be enriched in the tissues of IBC patients and, if so, provide a potential correlation between limited breast-feeding and the development of aggressive breast cancer.MethodsWe utilized the Short Time-series Expression Miner (STEM) program to cluster significant signatures from two independent studies that analyzed gene expression at multiple time-points of mouse mammary gland involution. Using 10 significant signatures, we performed gene ontology analysis and gene set enrichment analysis (GSEA) on training and validation sets from human breast cancer gene expression data to identify specific genes that are enriched in IBC compared to non-IBC and in TN compared to non-TN in IBC and non-IBC groups.ResultsExamining the combined data, we identified 10 involution gene clusters (Inv1-10) that share time-dependent regulation after forced weaning. Inv5 was the only cluster significantly enriched in IBC in the training and validation set (nominal p-values <0.05) and only by unadjusted p-values (FDR q-values 0.26 and 0.46 respectively). Eight genes in Inv5 are upregulated in both the training and validation sets in IBC. Combining the training and validation sets, both Inv5 and Inv6 have nominal p-values <0.05 and q-values 0.39 and 0.20, respectively. The time course for both clusters includes genes that change within 12 hours after forced weaning.ConclusionsResults from this in silico study suggest correlation between molecular events during abrupt involution and aggressive breast cancer. Specifically, candidate genes from Inv5 merit functional investigation regarding the role of limited breast-feeding in IBC development.

The cJUN NH2-terminal kinase (JNK) pathway contributes to mouse mammary gland remodeling during involution

Involution returns the lactating mammary gland to a quiescent state after weaning. The mechanism of involution involves collapse of the mammary epithelial cell compartment. To test whether the cJUN NH2-terminal kinase (JNK) signal transduction pathway contributes to involution, we established mice with JNK deficiency in the mammary epithelium. We found that JNK is required for efficient involution. JNK deficiency did not alter the STAT3/5 or SMAD2/3 signaling pathways that have been previously implicated in this process. Nevertheless, JNK promotes the expression of genes that drive involution, including matrix metalloproteases, cathepsins, and BH3-only proteins. These data demonstrate that JNK has a key role in mammary gland involution post lactation.