Wild-type Mammary Glands Research Articles

Abstract PD1-09: Constitutively active estrogen receptor mutants enhance breast cancer pathogenesis by co-opting progesterone receptor activity, which can be countered by Giredestrant

Abstract ESR1 mutations contribute significantly to resistance against standard-of-care endocrine therapies and are associated with poor outcomes for estrogen receptor-positive (ER+) breast cancer patients. Structural, biochemical and cellular data all support a model in which such mutations trigger estrogen-independent ER activity, consistent with their emergence in patients who have been treated with Aromatase Inhibitors. Engineered cell line models have further suggested that the ESR1 mutations display gain-of-function effects that extend beyond constitutive canonical ER activity by influencing, for example, epithelial to mesenchymal transition and metastatic potential. However, concerns over potential engineering artifacts and limitations of in vitro conditions have challenged interpretation. Importantly, the mechanisms by which ER mutants might broadly impact cell behavior and whether the latest candidate ER therapeutics will inhibit the full spectrum of ESR1 mutant phenotypes are currently unknown. To address these questions, we generated conditional knock-in Esr1-mutant mice, allowing us to study the fundamental properties of the mutations under physiological conditions (equivalent to those experienced by wildtype ESR1) in the mammary gland, as a hormone-sensitive tissue and the origin of breast tumors. We find that ER mutations profoundly re-wire the transcriptional and cellular response of the mammary gland to hormones, including over the normal estrus cycle. In particular, mutant ER cells display hyper-sensitivity to progesterone, which triggers a proliferative program reminiscent of pregnancy (in nulliparous/virgin mice)- a phenotype that is entirely distinct from ESR1 wildtype mammary glands, both under physiological conditions and when stimulated with high levels of exogenous hormones. The ESR1 mutant-specific PR-mediated proliferative program does not reflect elevated Pgr expression but rather correlates with an altered PR cistrome and transcriptome. We found that this PR program also appears important for human tumor biology because progesterone stimulates the growth of ESR1-mutant patient-derived xenograft tumors (PDXs), in contrast to previous work showing progesterone to be generally tumor-suppressive in ESR1 wildtype tumor models. Importantly, Giredestrant (GDC-9545), a latest-generation orally bioavailable ER antagonist and degrader (SERD), counters the PR-mediated proliferative program, both in the mouse mammary model and in human PDXs. Taken together, these data suggest that in addition to promoting estrogen-independence, mutant ER collaborates with PR to drive a pro-tumorigenic proliferative program, perhaps co-opting a relationship between the hormone receptors established to be key for mammary gland development. Citation Format: Jackson Liang, Ciara Metcalfe. Constitutively active estrogen receptor mutants enhance breast cancer pathogenesis by co-opting progesterone receptor activity, which can be countered by Giredestrant [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD1-09.

Production of human tissue-type plasminogen activator (htPA) using in vitro cultured transgenic pig mammary gland cells

Tissue plasminogen activator (tPA) is a protein involved in the breakdown of blood clots. We have previously produced a human tPA (htPA)-overexpressing transgenic pig using a mammary gland-specific promoter. In this study, we have established a transgenic pig mammary gland cell line that produces recombinant htPA. The mammary gland cells grew well and retained their character over long periods of culture. There was no difference in the extent of apoptosis in transgenic cells compared to wild-type mammary gland cells. In addition, the transgenic mammary gland cells expressed and secreted htPA into the conditioned media at a concentration similar to that in milk. This transgenic cell line represents a simple and ethical method for recombinant htPA production.

Gene expression profile and cancer-associated pathways linked to progesterone receptor isoform a (PRA) predominance in transgenic mouse mammary glands

BackgroundProgesterone receptor (PR) is expressed from a single gene as two isoforms, PRA and PRB. In normal breast human tissue, PRA and PRB are expressed in equimolar ratios, but isoform ratio is altered during malignant progression, usually leading to high PRA:PRB ratios. We took advantage of a transgenic mouse model where PRA isoform is predominant (PRA transgenics) and identified the key transcriptional events and associated pathways underlying the preneoplastic phenotype in mammary glands of PRA transgenics as compared with normal wild-type littermates.MethodsThe transcriptomic profiles of PRA transgenics and wild-type mammary glands were generated using microarray technology. We identified differentially expressed genes and analyzed clustering, gene ontology (GO), gene set enrichment analysis (GSEA), and pathway profiles. We also performed comparisons with publicly available gene expression data sets of human breast cancer.ResultsWe identified a large number of differentially expressed genes which were mainly associated with metabolic pathways for the PRA transgenics phenotype while inflammation- related pathways were negatively correlated. Further, we determined a significant overlap of the pathways characterizing PRA transgenics and those in breast cancer subtypes Luminal A and Luminal B and identified novel putative biomarkers, such as PDHB and LAMB3.ConclusionThe transcriptional targets identified in this study should facilitate the formulation or refinement of useful molecular descriptors for diagnosis, prognosis, and therapy of breast cancer.

Deficiency of X-Linked Protein Kinase Nrk during Pregnancy Triggers Breast Tumor in Mice

The onset and/or growth of breast tumor are controlled, at least in part, by estrogen. Therefore, to prevent the development of breast tumor, estrogen-dependent proliferation of mammary epithelial cells during pregnancy needs to be suppressed once the mammary gland is fully developed to enable lactation. However, the underlying molecular mechanisms remain unknown. Nrk is an X-linked protein serine/threonine kinase in the germinal center kinase family. Herein, we demonstrate a frequent occurrence of breast tumors in homozygous and heterozygous Nrk mutant mice that have experienced pregnancy/parturition. The tumors never developed in the mutant mice without a history of pregnancy/parturition. They exhibited histopathological features of noninvasive tubular adenocarcinoma, and expressed estrogen receptor α. At late gestation when estrogen receptor α expression was significantly reduced in the wild-type mammary gland, grossly normal mammary glands in the pregnant Nrk mutant mice occasionally contained hyperplastic foci continuously expressing the receptor. Consistently, Nrk expression was induced in the wild-type mammary gland at this period of pregnancy. On the other hand, the pregnant Nrk mutant mice also showed elevated blood estrogen levels at late gestation. We suggest that Nrk suppresses the excessive proliferation of mammary epithelial cells during pregnancy, and the impairment of this regulatory system leads to frequent occurrence of breast tumor in Nrk mutant mice.

Abstract P2-04-01: The role of Jmjd1a in mammary gland development and breast tumor growth

Abstract Histone modification alters chromatin architecture and thereby influences gene transcription. Histone methylation status is reversible and counter-regulated by methyltransferases and demethylases. Jmjd1a (also known as KDM3A, TSGA, JMJD1, JHDM2A and JHMD2A) is a histone demethylase. It belongs to JmjC domain-containing protein family and could specifically remove di- and mono- methyl residues from di or mono-methylated histone H3K9 (H3K9me2/me1). Recent studies showed that Jmjd1a plays an important role in embryonic stem cell self-renewal, spermatogenesis, regulation of metabolic gene expression and body weight, sex determination, tumor angiogenesis, and macrophage infiltration. However, its role in mammary gland (MG) development, breast carcinogenesis and breast cancer progression hasn't been systemically investigated. In this study, we found that Jmjd1a is expressed in mouse luminal epithelial cells. Genetic disruption of the Jmjd1a gene significantly slowed down MG development as indicated by retarded MG elongation and decreased ductal density in virgin mice observed at the ages of 4, 6 and 8 weeks. In agreement with the retarded MG development, the expression of Ki67 and cyclinD1 in epithelial cells of MGs from Jmjd1a knockout (KO) mice dramatically reduced compared with that from wild type (WT) mice. H3K9me1 and H3K9me2 levels in the epithelial cells of KO MGs are much higher than that in WT MGs. To assess the role of Jmjd1a in breast cancer progression, we crossbred Tg(Jmjd1a-/-) mice with MMTV-TVA(RCAS-PyMT) mice and obtained Tg(Jmjd1a-/-)×MMTV-TVA(RCAS-PyMT) mice. Infection of the TVA-expressing MG epithelial cells with the RCAS-PyMT virus induced mammary tumors in these mice and MMTV-TVA(RCAS-PyMT) control mice. We found that KO of Jmjd1a slightly accelerated mammary tumor initiation but significantly decreased tumor growth. Ki67 and cyclinD1 expression statistically reduced in KO tumors versus WT tumors. At the molecular level, Jmjd1a expression positively correlated with cyclin D1 expression in mammary epithelial cells and mammary tumors. Knockdown of Jmjd1a in MCF-7 cells significantly reduced cyclin D1 expression, while ectopic expression of Jmjd1a in MCF-7 cells increased cyclin D1 expression. ChIP assay revealed that Jmjd1a is associated with a promoter region of cyclin D1. Co-expression of c-Myc and Jmjd1a boosted the activity of the cyclin D1 reporter. In conclusion, our study indicated that Jmjd1a plays an important role in promoting mammary gland development and breast tumor growth by up-regulating cyclin D1 expression. Targeting Jmjd1a may inhibit breast cancer progression. Citation Format: Qin L, Xu Y, Wu Y, Yu X, Toneff MJ, Liao L, Li Y, Xu J. The role of Jmjd1a in mammary gland development and breast tumor growth. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-04-01.

Abstract 2394: Cancer-associated fibroblasts in invasive lobular breast carcinoma

Abstract Cancer-associated fibroblasts (CAFs) can promote breast cancer progression and therapy response through paracrine and juxtacrine signaling pathways. CAFs are abundantly observed in especially the invasive lobular carcinoma (ILC) subtype of breast cancer. Surprisingly, there is nothing known about the function of these CAFs in ILC. Given the fact that human ILC is generally resistant to standard chemotherapy, we believe that the cause of this therapy resistance lies into the active crosstalk between CAFs and tumor cells leading to a more progressive and resistant phenotype. Therefore, we aim to discover the exact function of CAFs in ILC as well as their drugable targets. In order to reliably study the role of CAFs in ILC, we generated a genetically engineered mouse model for ILC based on the tissue-specific loss of E-cadherin and PI3K signaling pathway activation, two hallmarks of human ILC. This clinically relevant mouse model spontaneously developed tumors that grow as typically invasive ‘indian files’ into a rigid extracellular matrix (ECM) that is very rich of CAFs. Uniquely, these tumors closely resemble human classical ILC, including its growth rate. Based on a mammary gland transplantation setting using fluorescent labeled hosts, we have shown that fibroblasts are recruited by ILC, both in vivo and in vitro, suggesting ILC-secreted mediators. Interestingly, in vitro inhibition of PDGFRβ (expressed by fibroblasts) can at least in part inhibit this recruitment. We are currently investigating whether inhibition of PDGFRβ in vivo can prevent the recruitment of fibroblasts and subsequently tumor development or progression. In parallel, we have isolated fibroblasts and epithelial cells from primary mouse ILCs and wild-type mammary glands to screen for other important players in the tumor-stroma crosstalk using RNA-seq and secretome proteomics. We will subsequently assess their functional significance during ILC development, progression and therapy response. Citation Format: Mirjam C. Boelens, Ellen Wientjens, Eva Schut, Sjoerd Klarenbeek, Karin E. de Visser, Jos Jonkers. Cancer-associated fibroblasts in invasive lobular breast carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2394. doi:10.1158/1538-7445.AM2015-2394

Abstract B77: The role of fibroblasts in invasive lobular breast carcinoma

Abstract Cancer-associated fibroblasts (CAFs) can promote breast cancer growth and metastasis, but the underlying mechanisms are still largely unknown. In order to study the role of CAFs in breast cancer development in an in vivo setting, we make use of a spontaneous mammary tumor mouse model for invasive lobular carcinoma (ILC). ILC is the second most common type of breast cancer, and is characterized by loss of E-cadherin, a rich stromal compartment, infiltrative growth, and treatment resistance. Often, the PI3K pathway is observed activated in the ILC subtype. Based on these characteristics, we have generated mouse models with mammary epithelial-specific conditional knockout of E-cadherin and PTEN that develop classical ILCs with a strong resemblance to human ILC. Using PDGFRβ as a fibroblast marker, we have observed an abundant presence of stromal fibroblasts in these ILCs, similar to human ILC. Based on a mammary gland transplantation setting using fluorescent labeled recipient mice, we have shown that fibroblasts get recruited from the host during tumor development. In vitro, we have also shown that tumor cells recruit fibroblasts. Inhibition of PDGFRβ in this in vitro model can at least in part inhibit this recruitment. We are currently investigating whether inhibition of PDGFRβ in vivo can prevent the recruitment of fibroblasts and subsequently tumor development or progression. In parallel, we have isolated fibroblasts from primary mouse ILCs and wild-type mammary glands to screen for other important players in the tumor-stroma crosstalk using RNA-seq and cytokine profiling. We will subsequently assess their functional significance during ILC development and progression. This research is supported by a fellowship of the Dutch Cancer Society Citation Format: Mirjam C. Boelens, Ellen Wientjens, Eva Schut, Sjoerd Klarenbeek, Karin E. de Visser, Jos Jonkers. The role of fibroblasts in invasive lobular breast carcinoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B77. doi:10.1158/1538-7445.CHTME14-B77

Distinct luminal-type mammary carcinomas arise from orthotopic Trp53-null mammary transplantation of juvenile versus adult mice.

Age and physiologic status, such as menopause, are risk factors for breast cancer. Less clear is what factors influence the diversity of breast cancer. In this study, we investigated the effect of host age on the distribution of tumor subtypes in mouse mammary chimera consisting of wild-type hosts and Trp53 nullizygous epithelium, which undergoes a high rate of neoplastic transformation. Wild-type mammary glands cleared of endogenous epithelium at 3 weeks of age were subsequently transplanted during puberty (5 weeks) or at maturation (10 weeks) with syngeneic Trp53-null mammary tissue fragments and monitored for one year. Tumors arose sooner from adult hosts (AH) compared with juvenile hosts (JH). However, compared with AH tumors, JH tumors grew several times faster, were more perfused, exhibited a two-fold higher mitotic index, and were more highly positive for insulin-like growth factor receptor phosphorylation. Most tumors in each setting were estrogen receptor (ER)-positive (80% JH vs. 70% AH), but JH tumors were significantly more ER-immunoreactive (P = 0.0001) than AH tumors. A differential expression signature (JvA) of juvenile versus adult tumors revealed a luminal transcriptional program. Centroids of the human homologs of JvA genes showed that JH tumors were more like luminal A tumors and AH tumors were more like luminal B tumors. Hierarchical clustering with the JvA human ortholog gene list segregated luminal A and luminal B breast cancers across datasets. These data support the notion that age-associated host physiology greatly influences the intrinsic subtype of breast cancer.

Peroxisomal membrane channel Pxmp2 in the mammary fat pad is essential for stromal lipid homeostasis and for development of mammary gland epithelium in mice

To understand the functional role of the peroxisomal membrane channel Pxmp2, mice with a targeted disruption of the Pxmp2 gene were generated. These mice were viable, grew and bred normally. However, Pxmp2−/− female mice were unable to nurse their pups. Lactating mammary gland epithelium displayed secretory lipid droplets and milk proteins, but the size of the ductal system was greatly reduced. Examination of mammary gland development revealed that retarded mammary ductal outgrowth was due to reduced proliferation of epithelial cells during puberty. Transplantation experiments established the Pxmp2−/− mammary stroma as a tissue responsible for suppression of epithelial growth. Morphological and biochemical examination confirmed the presence of peroxisomes in the mammary fat pad adipocytes, and functional Pxmp2 was detected in the stroma of wild-type mammary glands. Deletion of Pxmp2 led to an elevation in the expression of peroxisomal proteins in the mammary fat pad but not in liver or kidney of transgenic mice. Lipidomics of Pxmp2−/−mammary fat pad showed a decrease in the content of myristic acid (C14), a principal substrate for protein myristoylation and a potential peroxisomal β-oxidation product. Analysis of complex lipids revealed a reduced concentration of a variety of diacylglycerols and phospholipids containing mostly polyunsaturated fatty acids that may be caused by activation of lipid peroxidation. However, an antioxidant-containing diet did not stimulate mammary epithelial proliferation in Pxmp2−/− mice.The results point to disturbances of lipid metabolism in the mammary fat pad that in turn may result in abnormal epithelial growth. The work reveals impaired mammary gland development as a new category of peroxisomal disorders.

Abstract A083: Neutrophils promote metastasis of invasive lobular carcinoma

Abstract The multistep process of metastasis is highly dependent on the intrinsic mutations that accumulate in cancer cells. However, recent experimental evidence has shown that these mutations are not sufficient to facilitate metastatic disease. Cues from the tumor microenvironment are also required. In order to understand how systemic changes might affect metastasis formation, we profiled the cellular and molecular alterations in various organs of K14cre;EcdF/F;p53F/F mammary tumor-bearing mice – a model of invasive lobular breast cancer. Among all immune cells, increased neutrophil accumulation was the most pronounced. To test the functional importance of neutrophils in the metastatic process, we utilized our recently described model of spontaneous metastasis. This model is based on the transplantation of K14cre;EcdF/F;p53F/F tumor pieces into wild-type recipient mice, and subsequent surgical removal of the primary tumor, permitting us to investigate neutrophil function in both the neoadjuvant and adjuvant settings. Interestingly, mice treated with neutrophil-depleting antibodies in the neoadjuvant setting displayed a profound reduction in pulmonary metastasis, whereas metastases in the adjuvant group were equivalent to controls. These data indicate that neutrophils play a prominent role during the very early stages of the metastatic cascade. Our preliminary data suggest that these tumor-educated neutrophils can suppress the activation of T cells, as one mechanism by which they may promote metastasis. In addition, cytokine analysis of K14cre;EcdF/F;p53F/F mammary tumors demonstrated an increase in the IL17 pathway when compared to wild-type mammary glands. Subsets of T cells are known producers of IL17 and this pathway regulates neutrophil expansion indirectly. In accordance with this, K14cre;EcdF/F;p53F/F mice lacking the adaptive immune system displayed reduced IL17 levels and reduced circulating neutrophils. Current efforts are underway to determine whether T cells play a role in the pro-metastatic function of neutrophils. Together, these findings indicate that neutrophils are crucial for the establishment of breast cancer metastasis. Citation Format: Seth B. Coffelt, Chris W. Doornebal, Kelly Kersten, Metamia Ciampricotti, Cheei-Sing Hau, Kim Vrijland, Jorieke Weiden, Jos Jonkers, Karin E. de Visser. Neutrophils promote metastasis of invasive lobular carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A083.

Abstract LB-42: In vivo screen for novel breast cancer promoting genes.

Abstract Mis-regulation of oncogenes and tumor suppressor genes causes cancer. Although the evolution of cancer is not conclusive, most evidence supports that tumors arise from collaboration of multiple genes. To better understand the process of breast tumor initiation and cancer progression we carried out an in vivo screen, for genes that promote tumor formation. Our screen was performed using lentiviral transduction of primary murine mammary epithelial cells, and orthotopic implantation into syngeneic recipient mice. Wild type mammary stem cells can regenerate an entire mammary ductal tree in the recipients. We chose a well-characterized ErbB2/Her2 over-expression mouse model as a source of donor cells because HER2 over-expression occurs in >20% of human breast cancer patients. A cDNA library consisting of a “druggable” set of ~1000 genes was screened in 12 subsets segregated by ORF length. Ten months after transplantation the recipient mice were euthanized and DNA from mammary gland tumors was harvested. No tumors formed in mice transplanted with cells into which a control virus had been transduced. Genes from the library that were expressed in the tumors were identified by sequencing. The ability of these genes to drive tumor formation was confirmed by transduction and transplantation into a second cohort of mice. One of the genes identified in this screen is GTF2I repeat domain containing 1 (GTF2IRD1). Multi-focal atypical hyperplasia formed in mammary glands over-expressing GTF2IRD1 while no hyperplasia was seen in the control mice. This effect depends on the expression of ErbB2 because expression of GTF2IRD1 itself in wild type mammary glands did not cause abnormal growth before the end point. Analysis of the cancer genome atlas database demonstrates that GTF2IRD1 expression is significantly increased in invasive breast cancer compared to normal controls. Therefore, our screen has identified a novel tumor-promoting gene that is of potential clinical importance. A major advantage of our method is the use of normal cells of origin and immunocompetent mice. Moreover, it could be expanded to genome-wide searches for new tumor- and metastasis-promoting genes. Citation Format: Yongliang Huo, Ian Macara. In vivo screen for novel breast cancer promoting genes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-42. doi:10.1158/1538-7445.AM2013-LB-42

Abstract P2-04-04: Prolactin cooperates with loss of p53 to promote mammary tumorigenesis.

Abstract Prolactin (PRL) is critical for mammary development and lactation. Epidemiological studies also support a role for PRL in breast cancer. In order to study the contributions of PRL to tumor development and progression, we developed a transgenic mouse model that expresses PRL in the mammary epithelial cells (NRL-PRL), mimicking the mammary PRL production in women. Loss of function of the tumor suppressor p53 is a common occurrence in many human cancers including breast tumors. Interactions between PRL and p53 were examined by crossing the NRL-PRL mouse to p53 knockout mice which were made congenic on the FVB/N background. To circumvent the problem that p53−/− mice are prone to multiple nonmammary tumors, mammary cells from 10–12 week old wild-type, NRL-PRL, p53−/−, and NRL-PRL/ p53−/− were transplanted to wild type mammary glands. Histological analysis of the donor glands showed that NRL-PRL glands displayed marked epithelial proliferation and focally dilated ducts, whereas p53−/− glands exhibited irregular ductal epithelium with increased stromal density compared to wild type mice. In contrast, glands of donor PRL/ p53−/− females showed widespread epithelial hyperplasias and highly irregular ductal epithelium often surrounded by dense stroma. By one year of age, no tumors had developed from either wild-type or NRL-PRL transplanted epithelium. However, recipients of either p53−/− or NRL-PRL/ p53−/− mammary epithelium developed histologically similar anaplastic carcinomas. The presence of transgenic PRL decreased tumor latency (205 vs 244 days) and significantly increased tumor cell proliferation. In addition, these carcinomas appeared to be more aggressive: 5/16 (32%) of the NRL-PRL/ p53−/− tumors invaded into the peritoneal cavity, compared to 0/10 p53−/− tumors. Expression of matrix metalloproteinase 9 (MMP-9), but not MMP-2, was found to be significantly higher in the NRL-PRL/ p53−/−, compared to p53−/− tumors. MMP-9 is regulated by the transcription factor, AP-1, and PRL can signal through AP-1. NRL-PRL/ p53−/− tumors displayed significantly increased expression of the AP-1 family members, c-Jun and FosL, but not JunD or c-Fos compared to tumors from p53−/− mice. In addition, levels of c-Jun and FosL proteins increased in a similar pattern. In summary, interactions between PRL and loss of p53 promote breast cancer by increasing proliferation and invasiveness, potentially via AP-1 target genes. Supported by CDMRP BC053412. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-04-04.

Epithelial cell-directed efferocytosis in the post-partum mammary gland is necessary for tissue homeostasis and future lactation

BackgroundMammary glands harbor a profound burden of apoptotic cells (ACs) during post-lactational involution, but little is known regarding mechanisms by which ACs are cleared from the mammary gland, or consequences if this process is interrupted. We investigated AC clearance, also termed efferocytosis, during post-lactational remodeling, using mice deficient for MerTK, Axl, and Tyro3, three related receptor tyrosine kinases (RTKs) regulating macrophage-mediated efferocytosis in monocytes. MerTK expression, apoptosis and the accumulation of apoptotic debris were examined in histological sections of MerTK-deficient, Axl/Tyro3-deficient, and wild-type mammary glands harvested at specific time points during lactation and synchronized involution. The ability of primary mammary epithelial cells (MECs) to engulf ACs was assessed in culture. Transplant of MerTK-deficient mammary epithelium into cleared WT mammary fat pads was used to assess the contribution of WT mammary macrophages to post-lactational efferocytosis.ResultsACs induced MerTK expression in MECs, resulting in elevated MerTK levels at the earliest stages of involution. Loss of MerTK resulted in AC accumulation in post-lactational MerTK-deficient mammary glands, but not in Axl and Tyro3-deficient mammary glands. Increased vascularization, fibrosis, and epithelial hyperproliferation were observed in MerTK-deficient mammary glands through at least 60 days post-weaning, due to failed efferocytosis after lactation, but did not manifest in nulliparous mice. WT host-derived macrophages failed to rescue efferocytosis in transplanted MerTK-deficient mammary epithelium.ConclusionEfferocytosis by MECs through MerTK is crucial for mammary gland homeostasis and function during the post-lactational period. Efferocytosis by MECs thus limits pathologic consequences associated with the apoptotic load following lactation.

Microdialysis Combined with Proteomics for Protein Identification in Breast Tumor Microenvironment In Vivo

Tumor microenvironment constitutes a reservoir for proteins released from tumor cells and the host, which can contribute significantly to tumor growth and invasion. This study aims to apply a method of combining in vivo microdialysis and proteomics to identify proteins in mammary tumor interstitial fluids, a major component of tumor microenvironment. In vivo microdialysis was performed in polyomavirus middle T antigen (PyVmT) transgenic mouse mammary tumors and age-matched control wild-type mammary glands. Over four hundred proteins were identified from the microdialysis perfusates, using the Multidimensional Protein Identification Technology. Osteopontin (OPN) is one of the proteins overexpressed in breast tumor perfusates, as confirmed with immunoassays. OPN was also found to be present in tumor-associated stroma in both PyVmT and human breast tumors, using immunohistochemistry. Specifically, fibroblasts were further shown to express OPN at both mRNA and protein levels. In vitro assays showed that OPN can stimulate PyVmT breast carcinoma cell proliferation and migration. Finally, the expression of OPN was significantly higher in the peripheral blood of mice bearing breast tumors, compared to wild-type mice. Overall, microdialysis combined with proteomics is a unique technique for identifying proteins in a tumor microenvironment in vivo. Mammary fibroblasts can secrete OPN, and its overexpression in mammary tumor microenvironment may contribute significantly to mammary tumor progression. The online version of this article (doi:10.1007/s12307-010-0046-3) contains supplementary material, which is available to authorized users.

Gene expression profiling of cancer progression reveals intrinsic regulation of transforming growth factor-β signaling in ErbB2/Neu-induced tumors from transgenic mice

Upregulation of HER2/ErbB2/Neu occurs in 15-30% of human breast cancers and correlates with poor prognosis. Identification of ErbB2/Neu transcriptional targets should facilitate development of novel therapeutic approaches. Development of breast cancer is a multistep process; thus, to identify the transcriptomes associated with different stages of progression of tumorigenesis, we compared expression profiles of mammary tumors and preneoplastic mammary tissue from MMTV-Neu transgenic mice to expression profiles of wild-type mammary glands using Affymetrix microarrays. We identified 324 candidate genes that were unique to ErbB2/Neu-induced tumors relative to normal mammary gland tissue from wild-type controls. Expression of a subset of these genes (82) was also changed in the preneoplastic mammary glands compared to wild-type controls, indicating that they may play a pivotal role during early events of ErbB2/Neu-initiated mammary tumorigenesis. Further analysis of the microarray data revealed that expression of several known transforming growth factor (TGF)-beta target genes was altered, suggesting that the TGF-beta signaling cascade is downregulated in ErbB2/Neu-induced tumors. Western blot analysis for TGF-beta-Receptor-I/ALK5 and immunohistochemistry for TGF-beta-Receptor-I/ALK5 and phosphorylated/activated Smad2 confirmed that the Smad-dependent TGF-beta signaling cascade was inactive in these tumors. Although absent in most of the tumor, phosphorylated Smad2 was present in the periphery of tumors. Interestingly, presence of phosphorylated/activated Smad2 correlated with expression of Activin-Receptor-IB/ALK4, suggesting that although Smad-dependent TGF-beta signaling is absent in ErbB2/Neu-induced tumors, Activin signaling may be active at the leading edge of these tumors. Cumulatively, these data indicate that the TGF-beta pathway is intrinsically suppressed in ErbB2/Neu tumors via a mechanism involving loss of TGF-beta-Receptor-I/ALK5.

Regulation of Id2 expression by CCAAT/enhancer binding protein

Mice deficient for Id2, a negative regulator of basic helix–loop–helix (bHLH) transcription factors, exhibit a defect in lactation due to impaired lobuloalveolar development during pregnancy, similar to the mice lacking the CCAAT enhancer binding protein (C/EBP) β. Here, we show that Id2 is a direct target of C/EBPβ. Translocation of C/EBPβ into the nucleus, which was achieved by using a system utilizing the fusion protein between C/EBPβ and the ligand-binding domain of the human estrogen receptor (C/EBPβ-ERT), demonstrated the rapid induction of endogenous Id2 expression. In reporter assays, transactivation of the Id2 promoter by C/EBPβ was observed and, among three potential C/EBPβ binding sites found in the 2.3 kb Id2 promoter region, the most proximal element was responsible for the transactivation. Electrophoretic mobility shift assay (EMSA) identified this element as a core sequence to which C/EBPβ binds. Chromatin immunoprecipitation (ChIP) furthermore confirmed the presence of C/EBPβ in the Id2 promoter region. Northern blotting showed that Id2 expression in C/EBPβ-deficient mammary glands was reduced at 10 days post coitus (d.p.c.), compared with that in wild-type mammary glands. Thus, our data demonstrate that Id2 is a direct target of C/EBPβ and provide insight into molecular mechanisms underlying mammary gland development during pregnancy.

Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation

Elf5 is an epithelial-specific ETS factor. Embryos with a null mutation in the Elf5 gene died before embryonic day 7.5, indicating that Elf5 is essential during mouse embryogenesis. Elf5 is also required for proliferation and differentiation of mouse mammary alveolar epithelial cells during pregnancy and lactation. The loss of one functional allele led to complete developmental arrest of the mammary gland in pregnant Elf5 heterozygous mice. A quantitative mRNA expression study and Western blot analysis revealed that decreased expression of Elf5 correlated with the downregulation of milk proteins in Elf5(+/-) mammary glands. Mammary gland transplants into Rag(-/-) mice demonstrated that Elf5(+/-) mammary alveolar buds failed to develop in an Elf5(+/+) mammary fat pad during pregnancy, demonstrating an epithelial cell autonomous defect. Elf5 expression was reduced in Prolactin receptor (Prlr) heterozygous mammary glands, which phenocopy Elf5(+/-) glands, suggesting that Elf5 and Prlr are in the same pathway. Our data demonstrate that Elf5 is essential for developmental processes in the embryo and in the mammary gland during pregnancy.

Hormonal defect in maspin heterozygous mice reveals a role of progesterone in pubertal ductal development.

Progesterone and PR are mainly thought to affect tertiary ductal side branching and alveologenesis in late stage of mammary gland development. Here, we present evidence that they also play a role in early ductal development. This conclusion derived from our analysis of maspin heterozygous (Mp+/-) mice that showed defective ductal development at puberty. The defect was due to a reduced systemic level of progesterone. We show that treatment of Mp+/- mice with progesterone rescued the defect of ductal development. When both wild-type and Mp+/- mice were ovariectomized at 4 wk of age, treatment with progesterone alone can stimulate their ductal growth. In addition, treatment of wild-type mice with the progesterone inhibitor RU486 slowed ductal development in a dose-dependent manner. To confirm that progesterone receptor (PR) was required for progesterone action in ductal development at pubertal stage, we treated ovariectomized PR-deficient (PRKO) and wild-type mice with progesterone and examined ductal development at 7 wk of age. Whereas wild-type mammary glands displayed abundant ductal growth after progesterone treatment, there was a significant retardation of ductal growth in PRKO mice. Furthermore, we observed reduced ductal development in intact PRKO mice at 7 wk of age compared with that of wild-type mice. However, the defect was rescued at late stage of mammary development in PRKO mice. These data demonstrate that progesterone signaling, which is mediated by PR, plays an important role in early ductal development. In PRKO mice, a compensatory mechanism occurs that rescues the ductal defect at a late stage of mammary development.

Epithelial Development and Differentiation in the Mammary Gland Is Not Dependent on α3 or α6 Integrin Subunits

In the mammary gland, both laminin and integrins have been shown to be required for normal ductal morphogenesis during development in vivo, and for functional differentiation in culture models. Major integrin receptors for laminins in the mammary gland are α3β1, α6β1, and α6β4. However, the specific subunits that contribute to laminin-mediated mammary cell function and development have not been identified. In this study, we use a genetic approach to test the hypothesis that laminin-binding integrins are required for the function of the mammary gland in vivo. Rudiments of embryonic mammary gland were shown to develop in the absence of these integrin subunits. Postnatal development of the mammary gland was studied in integrin null tissue that had been transplanted into the mammary fat pads of syngeneic hosts. In mammary epithelium lacking α6 integrin, the β4 subunit was not apparent and hemidesmosome formation was only rudimentary. However, despite this deficiency, normal ductal morphogenesis and branching of the mammary gland occurred and myoepithelial cells were distributed normally with respect to luminal cells. Mammary alveoli devoid of α3 or α6 integrin formed in pregnancy and were histologically and functionally identical to those in wild-type mammary gland. The tissue underwent full morphological differentiation, and the epithelial cells retained the ability to synthesize β-casein. This work demonstrates that mammary tissue genetically lacking major laminin-binding integrin receptors is still able to develop and function.

Visualization of X-chromosome inactivation mosaicism of Tfm gene in XTfm/X+ heterozygous female mice.

The testicular feminization (Tfm) locus, which produces a deficiency in androgen receptors, is located on the X-chromosome. Steroid autoradiographic techniques were used to demonstrate the mosaicism of the X-chromosome inactivation in two androgen target tissues of XTfm/X+ heterozygous female mice. In the mesenchyme of urogenital sinuses of wild-type female fetuses (X+/X+), more than 95% of the cells were androgen-receptor positive (labelled with [3H]testosterone) while in that of heterozygous fetuses (XTfm/X+), about half of the cells were receptor positive (Tfm gene inactive). Statistical analysis of coherent clone size was applied to the heterozygous mesenchyme of the urogenital sinus and the coherent clone size of receptor-positive cells was estimated to be two or three cells per clone. This small clone size suggests that considerable cell mixing occurred in the tissue during embryonic development. Androgen binding in the mammary gland rudiments was restricted to the mesenchymal cells only in close vicinity to the epithelial mammary bud. In the wild-type rudiments most of the mesenchymal cells beneath the epithelium were receptor positive, while in heterozygous rudiments, receptor-positive and -negative cells intermingled. This observation suggests that in the wild-type mammary gland rudiments the epithelial bud may induce the formation of androgen receptors in adjacent mesenchymal cells rather than attract pre-existing receptor-rich mesenchymal cells.