Luminal Mammary Epithelial Cells Research Articles

An ovine model for investigation of the microenvironment of the male mammary gland.

The specific biology of the male breast remains relatively unexplored in spite of the increasing global prevalence of male breast cancer. Delineation of the microenvironment of the male breast is restricted by the low availability of human samples and a lack of characterisation of appropriate animal models. Unlike the mouse, the male ovine gland persists postnatally. We suggest that the male ovine mammary gland constitutes a promising adjunctive model for the male breast. In this study, we evaluate the male ovine mammary gland microenvironment, comparing intact and neutered males. Assessment of the glandular histo-anatomy highlights the resemblance of the male gland to that of neonatal female sheep and confirms the presence of rudimentary terminal duct lobular units. Irrespective of neutered status, cell proliferation in epithelial and stromal compartments is similarly low in males, and cell proliferation in epithelial cells and in the intralobular stroma is significantly lower than in pubertal female sheep. Between 42% and 72% of the luminal mammary epithelial cells in the male gland express the androgen receptor and expression is significantly reduced by neutering. Luminal epithelial cells within the intact and neutered male gland also express oestrogen receptor alpha, but minimal progesterone receptor expression is observed. The distribution of leukocytes within the ducts and stroma is similar to the mammary gland of female sheep and females of other species. Both macrophages and T lymphocytes are intercalated in the epithelial bilayer and are more abundant in the intralobular stroma than the interlobular stroma, suggesting that they may have a protective immunological function within the vestigial glandular tissue of the male sheep. Mast cells are also observed within the stroma. These cells cluster near the glandular tissue and are frequently located adjacent to blood vessels. The abundance of mast cells is significantly higher in intact males compared to neutered males, suggesting that hormone signalling may impact mast cell recruitment. In this study, we demonstrate the utility of the male ovine mammary gland as a model for furthering our knowledge of postnatal male mammary biology.

Targeting NANOG and FAK via Cx26-derived Cell-penetrating Peptides in Triple-negative Breast Cancer.

Triple-negative breast cancer (TNBC) represents the most lethal and treatment-resistant breast cancer subtype with limited treatment options. We previously identified a protein complex unique to TNBC composed of the gap junction protein connexin 26 (Cx26), the pluripotency transcription factor NANOG, and focal adhesion kinase (FAK). We sought to determine whether a peptide mimetic of the interaction region of Cx26 attenuated tumor growth in preclinical models. We designed peptides based on Cx26 juxtamembrane domains and performed binding experiments with NANOG and FAK using surface plasmon resonance. Binding studies revealed that the Cx26 C-terminal tail and intracellular loop bound to NANOG and FAK with submicromolar-to-micromolar affinity and that a 5-amino acid sequence in the C-terminal tail of Cx26 (RYCSG) was sufficient for binding. Peptides with high affinity were engineered with a cell-penetrating antennapedia sequence and assessed in functional assays including cell proliferation, tumorsphere formation, and in vivo tumor growth, and downstream signaling changes were measured. The cell-penetrating Cx26 peptide (aCx26-pep) disrupted self-renewal while reducing nuclear FAK and NANOG and inhibiting NANOG target gene expression in TNBC cells but not luminal mammary epithelial cells. In vivo, aCx26-pep reduced tumor growth and proliferation and induced cell death. Here, we provide proof-of-concept that a Cx26 peptide-based strategy inhibits growth and alters NANOG activity specifically in TNBC, indicating the therapeutic potential of this targeting approach.

Loss of epigenetic suppression of retrotransposons with oncogenic potential in aging mammary luminal epithelial cells.

A primary function of DNA methylation in mammalian genomes is to repress transposable elements (TEs). The widespread methylation loss that is commonly observed in cancer cells results in the loss of epigenetic repression of TEs. The aging process is similarly characterized by changes to the methylome. However, the impact of these epigenomic alterations on TE silencing and the functional consequences of this have remained unclear. To assess the epigenetic regulation of TEs in aging, we profiled DNA methylation in human mammary luminal epithelial cells (LEps)-a key cell lineage implicated in age-related breast cancers-from younger and older women. We report here that several TE subfamilies function as regulatory elements in normal LEps, and a subset of these display consistent methylation changes with age. Methylation changes at these TEs occurred at lineage-specific transcription factor binding sites, consistent with loss of lineage specificity. Whereas TEs mainly showed methylation loss, CpG islands (CGIs) that are targets of the Polycomb repressive complex 2 (PRC2) show a gain of methylation in aging cells. Many TEs with methylation loss in aging LEps have evidence of regulatory activity in breast cancer samples. We furthermore show that methylation changes at TEs impact the regulation of genes associated with luminal breast cancers. These results indicate that aging leads to DNA methylation changes at TEs that undermine the maintenance of lineage specificity, potentially increasing susceptibility to breast cancer.

Abstract 2657: Preclinical model to evaluate the safety and efficacy of PODO83: A monoclonal antibody against podocalyxin that targets tumor growth and metastasis

Abstract Podocalyxin (PODXL) is a highly glycosylated transmembrane sialomucin normally found on the apical free surface of mesothelial, vascular endothelial, luminal mammary epithelial cells, kidney podocytes and pluripotent and hematopoietic stem cells. We were the first to show that PODXL is upregulated on human cancers and an independent predictor of poor prognosis in breast and high-grade serous ovarian carcinoma (HGSOC). We also demonstrated that PODXL expression is crucial for primary tumor growth, local invasion, and experimental metastasis to distant organs. Subsequently, these results were extended to a wide range of other tumor types. We next developed a large series of high-affinity monoclonal antibodies (mAb) to human PODXL and evaluated these for therapeutic activity against the PODXL+ metastatic breast cancer cell line, MDA-MB-231, in xenografted NOD-SCID IL2Rγ-/- (NSG) mice. One of these, PODO83, potently attenuated primary tumor growth and formation of distal metastases. The PODO83 reactive epitope maps to the juxtamembrane stalk domain (non-glycosylated region) of the PODXL core protein. As a promising therapeutic it is imperative to perform toxicity studies and determine if PODO83 adversely affects the function of PODXL on normal tissues. Accordingly, we generated a transgenic mouse strain (C57Bl/6 congenic) replacing 4 of the 8 mouse PODXL-encoding exons with the orthologous human exons (including those encoding the PODO83 epitope). These mice were healthy and we confirmed by immunohistochemistry that they exhibit appropriate PODO83 immunoreactivity in all expected tissues (podocytes, endothelia, etc). Strikingly, administration of PODO83 in vivo at high doses (4 treatments of 5 mg/kg over 14 days) resulted in no overt signs of toxicity. To now fully assess the safety and efficacy profile of the PODO83 mAb in this preclinical humanized mouse model we have developed mouse metastatic tumor cell line expressing human PODXL and will evaluate the ability of PODO83 to inhibit tumor growth and metastasis while sparing normal tissues in vivo. The results from this study will help us advance PODO83 mAb as a safe and effective approach to treating the wide variety of cancer subtypes that express PODXL. Citation Format: Julyanne Brassard, Michael R. Hughes, Pamela Dean, Diana Canals Hernaez, Janella Schwab, Yasmine Lau, Calvin D. Roskelley, Kelly M. McNagny. Preclinical model to evaluate the safety and efficacy of PODO83: A monoclonal antibody against podocalyxin that targets tumor growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2657.

Inactivation of LATS1/2 drives luminal-basal plasticity to initiate basal-like mammary carcinomas

Basal-like breast cancers, an aggressive breast cancer subtype that has poor treatment options, are thought to arise from luminal mammary epithelial cells that undergo basal plasticity through poorly understood mechanisms. Using genetic mouse models and ex vivo primary organoid cultures, we show that conditional co-deletion of the LATS1 and LATS2 kinases, key effectors of Hippo pathway signaling, in mature mammary luminal epithelial cells promotes the development of Krt14 and Sox9-expressing basal-like carcinomas that metastasize over time. Genetic co-deletion experiments revealed that phenotypes resulting from the loss of LATS1/2 activity are dependent on the transcriptional regulators YAP/TAZ. Gene expression analyses of LATS1/2-deleted mammary epithelial cells notably revealed a transcriptional program that associates with human basal-like breast cancers. Our study demonstrates in vivo roles for the LATS1/2 kinases in mammary epithelial homeostasis and luminal-basal fate control and implicates signaling networks induced upon the loss of LATS1/2 activity in the development of basal-like breast cancer.

Abstract P2-05-01: Inactivation of LATS1/2 drives luminal-basal plasticity to initiate basal-like mammary carcinomas

Abstract Basal-like breast cancers, an aggressive breast cancer subtype that has poor treatment options, are thought to arise from luminal mammary epithelial cells but undergo plasticity to a basal-like state upon transformation. Identifying cellular mechanisms underlying this luminal-basal plasticity will allow an improved understanding of the pathogenesis of basal-like breast cancers. The Hippo signaling pathway, an important cell fate regulator across many tissues, has been implicated in breast cancer development. Central to Hippo signaling are the tumor suppressor kinases LATS1 and LATS2 (LATS1/2), which function to inhibit the transcriptional regulators YAP and TAZ (YAP/TAZ). In this study, we investigated the roles of LATS1/2 in the maintenance of mammary epithelial homeostasis and tumorigenesis in vivo. Using genetic mouse models, we found that conditional co-deletion of LATS1/2 in adult luminal cells promotes rapid proliferation and acquisition of basal cell traits, leading to early development of basal-like ductal carcinoma in situ and an eventual transition to a metastatic state. Further co-deletion experiments demonstrated that these phenotypes are dependent on YAP/TAZ. Transcriptional analyses of LATS1/2-null cells sorted from mammary glands demonstrated that these cells adopt gene expression signatures that correlate with mammary basal cells and human basal-like breast cancers, and revealed an association with Sox9, a transcription factor implicated in luminal progenitor fate and basal-like carcinomas. Collectively, this study demonstrates that LATS1/2 are important mediators of luminal cell fate in mammary epithelial homeostasis and that dysregulation of Hippo signaling in vivo promotes luminal-basal cell plasticity leading to basal-like mammary carcinoma development. Citation Format: Joseph Kern, Andrew Tilston-Lunel, Anthony Federico, Boting Ning, Grace Peppler, Eleni Stampouloglou, Marc Lenburg, Jennifer Beane, Stefano Monti, Xaralabos Varelas. Inactivation of LATS1/2 drives luminal-basal plasticity to initiate basal-like mammary carcinomas [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 P2-05-01.

Coordinate control of basal epithelial cell fate and stem cell maintenance by core EMT transcription factor Zeb1.

Maintenance of undifferentiated, long-lived, and often quiescent stem cells in the basal compartment is important for homeostasis and regeneration of multiple epithelial tissues, but the molecular mechanisms that coordinately control basal cell fate and stem cell quiescence are elusive. Here, we report an epithelium-intrinsic requirement for Zeb1, a core transcriptional inducer of epithelial-to-mesenchymal transition, for mammary epithelial ductal side branching and for basal cell regenerative capacity. Our findings uncover an evolutionarily conserved role of Zeb1 in promoting basal cell fate over luminal differentiation. We show that Zeb1 loss results in increased basal cell proliferation at the expense of quiescence and self-renewal. Moreover, Zeb1 cooperates with YAP to activate Axin2 expression, and inhibition of Wnt signaling partially restores stem cell function to Zeb1-deficient basal cells. Thus, Zeb1 is a transcriptional regulator that maintains both basal cell fate and stem cell quiescence, and it functions in part through suppressing Wnt signaling.

Loss of function of GATA3 induces basal-like mammary tumors.

Purpose: GATA3 is a transcription factor essential for mammary luminal epithelial cell differentiation. Expression of GATA3 is absent or significantly reduced in basal-like breast cancers. Gata3 loss-of-function impairs cell proliferation, making it difficult to investigate the role of GATA3 deficiency in vivo. We previously demonstrated that CDK inhibitor p18INK4c (p18) is a downstream target of GATA3 and restrains mammary epithelial cell proliferation and tumorigenesis. Whether and how loss-of-function of GATA3 results in basal-like breast cancers remains elusive.Methods: We generated mutant mouse strains with heterozygous germline deletion of Gata3 in p18 deficient backgrounds and developed a Gata3 depleted mammary tumor model system to determine the role of Gata3 loss in controlling cell proliferation and aberrant differentiation in mammary tumor development and progression.Results: Haploid loss of Gata3 reduced mammary epithelial cell proliferation with induction of p18, impaired luminal differentiation, and promoted basal differentiation in mammary glands. p18 deficiency induced luminal type mammary tumors and rescued the proliferative defect caused by haploid loss of Gata3. Haploid loss of Gata3 accelerated p18 deficient mammary tumor development and changed the properties of these tumors, resulting in their malignant and luminal-to-basal transformation. Expression of Gata3 negatively correlated with basal differentiation markers in MMTV-PyMT mammary tumor cells. Depletion of Gata3 in luminal tumor cells also reduced cell proliferation with induction of p18 and promoted basal differentiation. We confirmed that expression of GATA3 and basal markers are inversely correlated in human basal-like breast cancers.Conclusions: This study provides the first genetic evidence demonstrating that loss-of-function of GATA3 directly induces basal-like breast cancer. Our finding suggests that basal-like breast cancer may also originate from luminal type cancer.

Laminin alpha 5 regulates mammary gland remodeling through luminal cell differentiation and Wnt4-mediated epithelial crosstalk.

ABSTRACTEpithelial attachment to the basement membrane (BM) is essential for mammary gland development, yet the exact roles of specific BM components remain unclear. Here, we show that Laminin α5 (Lama5) expression specifically in the luminal epithelial cells is necessary for normal mammary gland growth during puberty, and for alveologenesis during pregnancy. Lama5 loss in the keratin 8-expressing cells results in reduced frequency and differentiation of hormone receptor expressing (HR+) luminal cells. Consequently, Wnt4-mediated crosstalk between HR+ luminal cells and basal epithelial cells is compromised during gland remodeling, and results in defective epithelial growth. The effects of Lama5 deletion on gland growth and branching can be rescued by Wnt4 supplementation in the in vitro model of branching morphogenesis. Our results reveal a surprising role for BM-protein expression in the luminal mammary epithelial cells, and highlight the function of Lama5 in mammary gland remodeling and luminal differentiation.

Bench Research, Human Milk, and SARS-CoV-2.

* Abbreviations: ACE2 — : angiotensin-converting enzyme 2 COVID-19 — : coronavirus disease 2019 SARS-CoV-2 — : severe acute respiratory syndrome coronavirus 2 In this issue of Pediatrics , Conzelmann et al1 examined whether Holder pasteurization of human milk, that is, heating to 62.5°C for 30 minutes, can inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In the laboratory, the authors “spiked” 5 individual women’s expressed milk samples with 5 different SARS-CoV-2 isolates, conducted Holder pasteurization (to 63°C), and then assessed tissue culture infectious dose 50 by infecting susceptible cells and monitoring the cytopathic effect. Holder pasteurization effectively inactivated SARS-CoV-2, and additionally, they noted a 40.9% to 92.8% viral titer decrease in human milk as compared with the control medium, confirming human milk’s unique antiviral properties. This important study adds to the limited evidence that pasteurized donor human milk is safe,2 but placing it within the clinical context is key.3 Providers and parents should not leap to any of several possible unfounded conclusions: (1) that the milk of a mother who has SARS-CoV-2 infection will be infected, (2) that her milk must be expressed to be fed to her infant, or (3) that her milk should be Holder pasteurized before feeding. Although much remains to … Address correspondence to Lydia Furman, MD, Division of General Academic Pediatrics, Department of Pediatrics, University Hospitals Rainbow Babies and Children’s Hospital, 11100 Euclid Ave, Cleveland, OH 44106. E-mail: lydia.furman{at}uhhospitals.org

The effect of radiation on the ability of rat mammary cells to form mammospheres.

As classical transplantation repopulation assays for studying the radiobiology of rat mammary stem/progenitor cells are extremely time-consuming, this study aimed to characterize the radiobiological properties of mammospheres, spherical clumps of mammary cells formed under non-adherent culture conditions, which are a simple and widely used technique for assessing progenitor cell activity. Rat mammary cells were dissociated and used in transplantation repopulation assays and for the formation of mammospheres. Immunofluorescence for cytokeratin 14 and 18 was used to identify basal and luminal mammary epithelial cells, respectively. Incorporation of 5-bromo-2'-deoxyuridine was used to evaluate cell proliferation. The repopulating activity of the transplanted primary rat mammary cells demonstrated their radiosensitivity, reproducing previous data, with a significant reduction in repopulating activity at ≥ 2Gy. Cells constituting rat mammospheres were positive for either cytokeratin 14 or 18, with occasional double-positive cells. Both proliferation and aggregation contributed to sphere formation. Cells obtained from the spheres showed lower repopulating activity after transplantation than primary cells. When primary cells were irradiated and then used for sphere formation, the efficiency of sphere formation was significantly decreased at 8Gy but not at ≤ 6Gy, indicating radioresistance of the formation process. Irradiation at 8Gy reduced the proliferation of cells during sphere formation, whereas the cellular composition of the resulting spheres was unaffectes. Thus, mammosphere formation assays may measure a property of putative mammary progenitors that is different from what is measured in the classic transplantation repopulation assay in radiobiology.

Abstract A88: RUNX1-deficiency in luminal mammary epithelial cells leads to development of a unique type of immune “hot” ER+ mammary tumor

Abstract Genetic mutations in cancer cells play key roles in shaping the tumor immune microenvironment. RUNX1 is a transcription factor (TF) essential for estrogen receptor (ER)+ luminal mammary epithelial cells (MECs) and is mutated in a significant portion of human ER+ luminal breast cancers. It is also an important regulator of immune cells, raising an intriguing question as to whether its loss-of-function mutations found in ER+ breast cancers affect both MECs and immune cells. To address this, we established an in vivo mouse model by inducing loss of both RUNX1 and p53 in luminal MECs, upon intraductal injection of a Cre-expressing adenovirus under the control of the Keratin 8 promoter (Ad-K8-Cre) to mammary glands of female mice carrying their conditional knockout alleles. Compared to similarly injected wild-type (WT) control mice or mice with induced loss of p53 alone, induced loss of both led to hyperproliferation of Runx1/Trp53-null MECs and eventually development of mammary tumors after a long latency (a tumor-free period of ~9-13 months after Ad-K8-Cre injection). Characterization of these tumors revealed that they are ER+ mammary tumors with extensive leukocyte infiltration. CD45+ leukocytes in them constituted ~32-84% of total live tumor cells, which is unusual for a mouse cancer model. Among CD45+ cells, ~45% were macrophages and ~15% were CD3+ T cells. Among tumor-infiltrating myeloid cells (CD45+CD11b+), many were myeloid-derived suppressor cells (MDSCs). In addition, significant differential expression of PD-1 on CD3+ T cells (38.5% vs. 7.3%) and PD-L1 on CD45- cells (2.7% vs. 0.3%) was observed in these Runx1/Trp53-null tumors, when compared to WT controls. This immune profile suggests an immunosuppressive microenvironment in these tumors. The tumor immune phenotype induced by RUNX1-loss in our model is supported by genomic data from human breast cancers analyzed by the TIMER online tool (Li et al., Cancer Res 77(21):e108-e110). The TIMER analysis showed that in human breast cancers, levels of most infiltrating immune cell subtypes (e.g., macrophages, T cells) in RUNX1-mutated cases are higher than those of RUNX1-WT cases; in contrast, a similar analysis for GATA3-mutated cases exhibited the opposite. As both RUNX1 and GATA3 are key TFs for luminal MECs and their mutations are both found in luminal breast cancers, the data from both human cases and our mouse model suggest that RUNX1-mutated breast cancers may represent a unique, immune “hot” subtype of ER+ luminal breast tumors. ER+ breast cancers are typically immune “cold” and do not respond to immune checkpoint blockade (ICB) well. Our data suggest that RUNX1-mutated breast cancers may represent a subset of ER+ breast cancers that may respond to ICB-based therapy. Collectively, our data suggest that loss of RUNX1 promotes development of ER+ breast cancer and modulates the immune microenvironment; a better understanding of how these two processes are interconnected may provide novel strategies to treat breast cancer with RUNX1-mutation. Citation Format: Dongxi Xiang, Zhe Li. RUNX1-deficiency in luminal mammary epithelial cells leads to development of a unique type of immune “hot” ER+ mammary tumor [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A88.

Downregulation of miR-223 Expression Is an Early Event during Mammary Transformation and Confers Resistance to CDK4/6 Inhibitors in Luminal Breast Cancer.

miR-223 is an anti-inflammatory miRNA that in cancer acts either as an oncosuppressor or oncopromoter, in a context-dependent manner. In breast cancer, we demonstrated that it dampens the activation of the EGF pathway. However, little is known on the role of miR-223 during breast cancer onset and progression. miR-223 expression was decreased in breast cancer of luminal and HER2 subtypes and inversely correlated with patients' prognosis. In normal luminal mammary epithelial cells, miR-223 acted cell autonomously in the control of their growth and morphology in three-dimensional context. In the MMTV-Δ16HER2 transgenic mouse model, oncogene transformation resulted in a timely abrogation of miR-223 expression, likely due to activation of E2F1, a known repressor of miR-223 transcription. Accordingly, treatment with CDK4/6 inhibitors, which eventually results in restraining E2F1 activity, restored miR-223 expression and miR-223 ablation induced luminal breast cancer resistance to CDK4/6 inhibition, both in vitro and in vivo. Notably, miR-223 expression was lost in microdissected ductal carcinoma in situ (DCIS) from patients with luminal and HER2-positive breast cancer. Altogether, these results identify downmodulation of miR-223 as an early step in luminal breast cancer onset and suggest that it could be used to identify aggressive DCIS and predict the response to targeted therapy. SIGNIFICANCE: miR-223 may represent a predictive biomarker of response to CDK4/6 inhibitors and its loss could identify DCIS lesions that are likely to progress into invasive breast cancer.

APKCi triggers basal extrusion of luminal mammary epithelial cells by tuning contractility and vinculin localization at cell junctions

Metastasis is the main cause of cancer-related deaths. How a single oncogenic cell evolves within highly organized epithelium is still unknown. Here, we found that the overexpression of the protein kinase atypical protein kinase C ι (aPKCi), an oncogene, triggers basally oriented epithelial cell extrusion in vivo as a potential mechanism for early breast tumor cell invasion. We found that cell segregation is the first step required for basal extrusion of luminal cells and identify aPKCi and vinculin as regulators of cell segregation. We propose that asymmetric vinculin levels at the junction between normal and aPKCi+ cells trigger an increase in tension at these cell junctions. Moreover, we show that aPKCi+ cells acquire promigratory features, including increased vinculin levels and vinculin dynamics at the cell-substratum contacts. Overall, this study shows that a balance between cell contractility and cell-cell adhesion is crucial for promoting basally oriented cell extrusion, a mechanism for early breast cancer cell invasion.

MicroRNA-455-3p mediates GATA3 tumor suppression in mammary epithelial cells by inhibiting TGF-β signaling

GATA3 is a basic and essential transcription factor that regulates many pathophysiological processes and is required for the development of mammary luminal epithelial cells. Loss-of-function GATA3 alterations in breast cancer are associated with poor prognosis. Here, we sought to understand the tumor-suppressive functions GATA3 normally performs. We discovered a role for GATA3 in suppressing epithelial-to-mesenchymal transition (EMT) in breast cancer by activating miR-455-3p expression. Enforced expression of miR-455-3p alone partially prevented EMT induced by transforming growth factor β (TGF-β) both in cells and tumor xenografts by directly inhibiting key components of TGF-β signaling. Pathway and biochemical analyses showed that one miRNA-455-3p target, the TGF-β-induced protein ZEB1, recruits the Mi-2/nucleosome remodeling and deacetylase (NuRD) complex to the promotor region of miR-455 to strictly repress the GATA3-induced transcription of this microRNA. Considering that ZEB1 enhances TGF-β signaling, we delineated a double-feedback interaction between ZEB1 and miR-455-3p, in addition to the repressive effect of miR-455-3p on TGF-β signaling. Our study revealed that a feedback loop between these two axes, specifically GATA3-induced miR-455-3p expression, could repress ZEB1 and its recruitment of NuRD (MTA1) to suppress miR-455, which ultimately regulates TGF-β signaling. In conclusion, we identified that miR-455-3p plays a pivotal role in inhibiting the EMT and TGF-β signaling pathway and maintaining cell differentiation. This forms the basis of that miR-455-3p might be a promising therapeutic intervention for breast cancer.

Establishment and Characterization of a Lactating Caprine Mammary Gland Luminal Epithelial Cell Line

Mammary gland is a defining characteristic of mammalian species which produces nutritious milk and plays a major role in the development of newborns. The gland contains a series of ducts and crevices leading back to alveoli, which contain milk producing cells called luminal epithelial cells. These cells, if cultured in-vitro, can be utilized to explore the metabolic processes occurring during milk production. The knowledge thus gained can be used to manipulate the system to enhance milk production and/or modify its composition. The main objective of this study was to establish a luminal epithelial cell-line from a lactating goat. Explant culture technique was used to produce primary cells from the mammary tissue of a 4-year-old lactating Saanen goat. The outgrowing cells were purified by selective trypsinization to remove fibroblast cells in 3-4 serial passages. The purified cell cultures exhibited cobblestone morphology, typical of the mammary epithelial cells, formed clear islands when plated in low density, and exhibited dome-shaped structures, if cultured for extended time. The cells stained positive with anti-human cytokeratin 18 antibodies, confirming their epithelial nature. Cell cultures also stained positive with rabbit anti-bovine β-lactoglobulin antibodies, indicating milk production in these cells. The cell-line has potential as an in-vitro cell model to understand signaling during milk synthesis, mammary gland development, and testing DNA constructs for therapeutic protein secretion in milk, prior to production of transgenic goats.

Abstract 1168: Characterization of tumor initiation cells in mammary tissues of C3-1-TAg transgenic mice

Abstract Animal models are powerful tools for the understanding of tumor development and cancer stem cell (CSC) origin. C3-1-TAg transgenic mice are a model for human triple negative breast cancer. Although general patterns of tumor development in this model have been well characterized, the tumor initiation cells of this model have not been defined. The objective of this study was to characterize the stemness and growth properties of mammary tumor cells in context with mammary epithelial cell (MEC) subpopulations of C3-1-TAg transgenic mice to identify the origin of tumor initiation cells. We first isolated MECs from mammary tissues of C3-1-TAg mice at various ages (8, 17, and 23 weeks old) and primary tumor cells from tumors of this model. Flow cytometry analysis with CD24 and CD49f as cell markers showed that cells from mammary tissues were separated into three subpopulations, including luminal, basal, and stromal populations. We found that the percentage of luminal subpopulation increased with age. Analysis of tumor derived cells using the same markers indicated that the majority of the cells in the plots overlapped with the position of luminal epithelial cells, suggesting their mutual connections. Next, we performed FACS sorting of mammary epithelial cells from the glands based on CD24 and CD49f and obtained four subpopulations of cells, including P1(stromal), P2(luminal), P3(CD24high/CD49fhigh, top of basal population on the plot), and P4(CD24mid/CD49fhigh, lower part of basal population), followed by the characterization of each subpopulations. Colony formation cell (CFC) assay and mammosphere assay were used for evaluation of luminal progenitor cells and self-renewal potential, respectively. Data from CFC assays showed that CFC colony numbers were the highest from the P2 population, followed by a few colonies from P3 population. Mammosphere assay showed that cells from P1 population formed typical mammospheres, while as cells from P2 and P3 formed cellular clusters different from typical mammospheres. FACS sorting with cells from C3-1-TAg mammary tumors obtained P2 and P3 subpopulations. Cells from P2, not P3, subpopulation were efficient in mammosphere formation. Because cells in P2 population are luminal mammary epithelial cells and are enriched with luminal progenitor cells, integration of data above suggest that C3-1-TAg mammary tumor cells are mainly derived from luminal progenitor cells. Using CD61, which is a luminal progenitor cell marker, we found that the percentage of luminal progenitor cells (CD61+/CD49fhigh) in the mammary glands was increased with age, and most tumor derived cells were CD61+/CD49fhigh. Taken together, our data demonstrate that the tumor initiation cells in the C3-1-TAg mammary tumors were derived from luminal progenitor cells. These findings lay a foundation for further characterization of cellular basis and molecular analysis of tumorigenesis in C3-1-TAg transgenic models. Citation Format: Qiong Cheng, Amanda B. Parris, Zhikun Ma, Yujie Shi, Lingfei Kong, Xiaohe Yang. Characterization of tumor initiation cells in mammary tissues of C3-1-TAg transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1168.

Abstract 1118: FOXQ1 stimulates WNT-signaling through transcriptional activation of WNT5B to promote the epithelial to mesenchymal transition

Abstract Aberrant induction of the epithelial to mesenchymal transition (EMT) is an acknowledged mechanism of acquired metastatic competence and drug resistance in many epithelial cancer types. Both physiological and pathological forms of EMT are thought to occur through a network of transcription factors (TFs) that drive cell plasticity and the acquisition of stem-like traits. The forkhead box transcription factor, FOXQ1, is a potent driver of EMT. FOXQ1 has been shown to be specifically upregulated in the triple negative subtype of breast cancer (TNBC) where is it associated with worse clinical prognosis. However, the role of FOXQ1 within the EMT transcriptional network has not been extensively characterized. In this study, we employed chromatin immunoprecipitation and RNA sequencing in a human mammary luminal epithelial cell line with ectopic FOXQ1 (HMLE/FOXQ1), as a model of breast EMT. We identified FOXQ1 localization within the promoter regions of prominent EMT-TFs, including TWIST1, ZEB1, FOXC2 and SIX2. Further, FOXQ1 direct gene targets are enriched in signaling pathways previously associated with EMT, including the WNT-signaling pathway. Within the WNT pathway, the WNT5B signaling ligand was a direct target for transcriptional activation by FOXQ1. Gene expression analysis in a panel of breast cancer cell lines revealed both FOXQ1 and WNT5B are enriched in the basal B subtype of TNBC. FOXQ1 and WNT5B expression were also found to be upregulated and correlated downstream of TGFβ induced EMT. Disruption of WNT5B expression attenuated EMT-associated stem-like traits, including decreases in mammosphere formation and in the CD44+/CD24- population, and significantly reduced cell invasion. Analysis of breast cancer gene expression from the TCGA revealed that FOXQ1 and WNT5B expression are correlated across all breast samples (r=0.46) and differentially upregulated in ER- breast cancer. Additionally, both FOXQ1 and WNT5B expression were associated with worse recurrence-free survival. Our data supports that WNT5B is a critical FOXQ1 target for mediating EMT features and suggests that disrupting the FOXQ1-WNT5B axis could be a potential strategy to target the EMT cell population in TNBC patients. Citation Format: Allison V. Mitchell, Guojun Wu. FOXQ1 stimulates WNT-signaling through transcriptional activation of WNT5B to promote the epithelial to mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1118.

Estrogen receptor signaling is reprogrammed during breast tumorigenesis

Limited knowledge of the changes in estrogen receptor (ER) signaling during the transformation of the normal mammary gland to breast cancer hinders the development of effective prevention and treatment strategies. Differences in estrogen signaling between normal human primary breast epithelial cells and primary breast tumors obtained immediately following surgical excision were explored. Transcriptional profiling of normal ER+ mature luminal mammary epithelial cells and ER+ breast tumors revealed significant difference in the response to estrogen stimulation. Consistent with these differences in gene expression, the normal and tumor ER cistromes were distinct and sufficient to segregate normal breast tissues from breast tumors. The selective enrichment of the DNA binding motif GRHL2 in the breast cancer-specific ER cistrome suggests that it may play a role in the differential function of ER in breast cancer. Depletion of GRHL2 resulted in altered ER binding and differential transcriptional responses to estrogen stimulation. Furthermore, GRHL2 was demonstrated to be essential for estrogen-stimulated proliferation of ER+ breast cancer cells. DLC1 was also identified as an estrogen-induced tumor suppressor in the normal mammary gland with decreased expression in breast cancer. In clinical cohorts, loss of DLC1 and gain of GRHL2 expression are associated with ER+ breast cancer and are independently predictive for worse survival. This study suggests that normal ER signaling is lost and tumor-specific ER signaling is gained during breast tumorigenesis. Unraveling these changes in ER signaling during breast cancer progression should aid the development of more effective prevention strategies and targeted therapeutics.

Germline Genetic Variants in GATA3 and Breast Cancer Treatment Outcomes in SWOG S8897 Trial and the Pathways Study

IntroductionGATA3 is a critical transcription factor in maintaining the differentiated state of luminal mammary epithelial cells. We sought to determine the prognostic and predictive roles of GATA3 genotypes for breast cancer. Patients and MethodsTwelve single nucleotide polymorphisms (SNPs) were genotyped in 2 breast cancer cohorts, including the SWOG S8897 trial where patients were treated with adjuvant chemotherapy (CAF [cyclophosphamide, doxorubicin, 5-fluorouracil] vs. CMF [cyclophosphamide, methotrexate, 5-fluorouracil]) or untreated, and the observational Pathways Study. ResultsIn the S8897 trial, rs3802604 and rs568727 were associated with disease-free survival and overall survival in the treated group, regardless of chemotherapy regimen. The GG genotype of rs3802604 conferred poorer overall survival (adjusted hazard ratio, 2.45; 95% confidence interval, 1.48-4.05) and disease-free survival (adjusted hazard ratio, 1.95; 95% confidence interval, 1.27-2.99) compared with the AA genotype. Similar associations were found for rs568727. In contrast, no association with either SNP was found in the untreated group. Subgroup analyses indicated that these 2 SNPs more strongly influenced outcomes in the patients who also received tamoxifen. However, the associations in the subgroup with tamoxifen treatment were not replicated in the Pathways Study, possibly owing to substantial differences between the 2 patient cohorts, such as chemotherapy regimen and length of follow-up. Results from joint analyses across these 2 cohorts were marginally significant, driven by the results in S8897. Bioinformatic analyses support potential functional disruption of the GATA3 SNPs in breast tissue. ConclusionsThe present study provides some evidence for the predictive value of GATA3 genotypes for breast cancer adjuvant therapies. Future replication studies in appropriate patient populations are warranted.