Mammary Epithelial Cell Populations Research Articles

Sorted stem/progenitor epithelial cells of pubertal bovine mammary gland present limited potential to reconstitute an organised mammary epithelium after transplantation.

The development and maintenance of mammary gland tissue depend on the proliferation and differentiation of mammary stem and progenitor cells. Here, we investigated populations of mammary epithelial cells that are potential candidates for bovine mammary gland development using xenotransplantation into mice cleared mammary fat pad. Transplanted mammary explants from 17-month-old Holstein heifers developed outgrowths exhibiting the archetypal morphology and molecular marker distributions of the bovine gland. Xenotransplantation of sorted mammary epithelial cells (CD49fpos) into bovinised fat pads using inactivated bovine fibroblasts resulted in outgrowth developments with 50% take rate, but these lacked the ductal or alveolar epithelial structures of the normal mammary gland. Similar results were obtained with xenografts of candidate bovine mammary epithelial stem cells (CD49fhighCD24pos) or epithelial cells of the basal lineage (CD49fhighCD24neg) which also developed as clumps of cells surrounded by stromal stretches within the mouse adipose tissue. In conclusion, sorted cells showed compromised regenerative potential for epithelial morphogenesis. Further work is therefore needed to identify mammary stem/progenitor cells with full regenerative capabilities for biogenesis of normal mammary gland structure, with milk-secreting function.

Rank ectopic expression in the presence of Neu and PyMT oncogenes alters mammary epithelial cell populations and their tumorigenic potential

Determination of the mammary epithelial cell that serves as the cell of origin for breast cancer is key to understand tumor heterogeneity and clinical management. In this study, we aimed to decipher whether Rank expression in the presence of PyMT and Neu oncogenes might affect the cell of origin of mammary gland tumors. We observed that Rank expression in PyMT+/− and Neu+/− mammary glands alters the basal and luminal mammary cell populations already in preneoplasic tissue, which may interfere with the tumor cell of origin restricting their tumorigenesis ability upon transplantation assays. In spite of this, Rank expression eventually promotes tumor aggressiveness once tumorigenesis is established.

Dormant Nfatc1 reporter-marked basal stem/progenitor cells contribute to mammary lobuloalveoli formation

SummaryThe Mammary gland undergoes complicated epithelial remodeling to form lobuloalveoli during pregnancy, in which basal epithelial cells remarkably increase to form a basket-like architecture. However, it remains largely unknown how dormant mammary basal stem/progenitor cells involve in lobuloalveolar development. Here, we show that Nfatc1 expression marks a rare population of mammary epithelial cells with the majority being basal epithelial cells. Nfatc1 reporter-marked basal epithelial cells are relatively dormant mammary stem/progenitor cells. Although Nfatc1 reporter-marked basal epithelial cells have limited contribution to the homeostasis of mammary epithelium, they divide rapidly during pregnancy and contribute to lobuloalveolar development. Furthermore, Nfatc1 reporter-marked basal epithelial cells are preferentially used for multiple pregnancies. Using single-cell RNA-seq analysis, we identify multiple functionally distinct clusters within the Nfatc1 reporter-marked cell-derived progeny cells during pregnancy. Taken together, our findings underscore Nfatc1 reporter-marked basal cells as dormant stem/progenitor cells that contribute to mammary lobuloalveolar development during pregnancy.

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.

MiR-424/503 modulates Wnt/β-catenin signaling in the mammary epithelium by targeting LRP6.

During the female lifetime, the expansion of the epithelium dictated by the ovarian cycles is supported by a transient increase in the mammary epithelial stem cell population (MaSCs). Notably, activation of Wnt/β-catenin signaling is an important trigger for MaSC expansion. Here, we report that the miR-424/503 cluster is a modulator of canonical Wnt signaling in the mammary epithelium. We show that mammary tumors of miR-424(322)/503-depleted mice exhibit activated Wnt/β-catenin signaling. Importantly, we show a strong association between miR-424/503 deletion and breast cancers with high levels of Wnt/β-catenin signaling. Moreover, miR-424/503 cluster is required for Wnt-mediated MaSC expansion induced by the ovarian cycles. Lastly, we show that miR-424/503 exerts its function by targeting two binding sites at the 3'UTR of the LRP6 co-receptor and reducing its expression. These results unveil an unknown link between the miR-424/503, regulation of Wnt signaling, MaSC fate, and tumorigenesis.

Co-expression of transcription factor AP-2beta (TFAP2B) and GATA3 in human mammary epithelial cells with intense, apicobasal immunoreactivity for CK8/18

AP-2β is a new mammary epithelial differentiation marker and its expression is preferentially retained and enhanced in lobular carcinoma in situ and invasive lobular breast cancer. In normal breast epithelium AP-2β is expressed in a scattered subpopulation of luminal cells. So far, these cells have not been further characterized. Co-expression of AP-2β protein and luminal epithelium markers (GATA3, CK8/18), hormone receptors [estrogen receptor (ER), androgen receptor (AR)] and candidate stem cells markers (CK5/14, CD44) were assessed by double-immunofluorescence staining in normal mammary gland epithelium. The subpopulation of AP-2β-positive mammary epithelial cells showed an almost complete, superimposable co-expression with GATA3 and a peculiar intense, ring-like appearing immunoreactivity for CK8/18. Confocal immunofluorescence microscopy revealed an apicobasal staining for CK8/18 in AP-2β-positive cells, which was not seen in in AP-2β-negative cells. Furthermore, AP-2β-positive displayed a partial co-expression with ER and AR, but lacked expression of candidate stem cell markers CK5/14 and CD44. In summary, AP-2β is a new luminal mammary epithelial differentiation marker, which is expressed in the GATA3-positive subpopulation of luminal epithelial cells. These AP-2β-positive/GATA3-positive cells also show a peculiar CK8/18-expression which may indicate a previously unknown functionally specialized mammary epithelial cell population.

Weighing the Risk: effects of Obesity on the Mammary Gland and Breast Cancer Risk.

Obesity is a preventable risk factor for breast cancer following menopause. Regardless of menopausal status, obese women who develop breast cancer have a worsened prognosis. Breast tissue is comprised of mammary epithelial cells organized into ducts and lobules and surrounded by adipose-rich connective tissue. Studies utilizing multiple in vivo models of obesity as well as human breast tissue have contributed to our understanding of how obesity alters mammary tissue. Localized changes in mammary epithelial cell populations, elevated secretion of adipokines and angiogenic mediators, inflammation within mammary adipose tissue, and remodeling of the extracellular matrix may result in an environment conducive to breast cancer growth. Despite these significant alterations caused by obesity within breast tissue, studies have suggested that some, but not all, obesity-induced changes may be mitigated with weight loss. Here, we review our current understanding regarding the impact of obesity on the breast microenvironment, how obesity-induced changes may contribute to breast tumor progression, and the impact of weight loss on the breast microenvironment.

Flow Cytometry Definition of Rat Mammary Epithelial Cell Populations and Their Distinct Radiation Responses.

Breast tissue is very susceptible to radiation-induced carcinogenesis, and mammary stem/progenitor cells are potentially important targets of this. The mammary epithelium is maintained as two mostly independent lineages of luminal and basal cells. To elucidate their immediate radiation responses, we analyzed the mammary glands of female Sprague-Dawley rats, a radiation carcinogenesis model, using colony formation, flow cytometry and immunofluorescence. The results revealed that flow cytometry successfully fractionates rat mammary cells into CD49fhi CD24lo basal, CD49fmed CD24hi luminal progenitor, and CD49flo CD24hi mature luminal populations, resembling human breast, rather than mouse tissues. The colony-forming ability of the basal cells was more radiosensitive than the luminal progenitor cells. Flow cytometry and immunofluorescence showed more efficient cell cycle arrest, γ-H2AX responses, and apoptosis in the irradiated luminal progenitor cells, than in the basal cells. These results provide important insights into the early phase of radiation-induced breast cancer.

Abstract BL1: Deciphering stem and progenitor cells to understand breast cancer

Abstract Breast cancer is a highly heterogeneous disease at the molecular and pathological levels. To understand heterogeneity and potential ‘cells of origin’ of breast cancer, it is important to dissect the normal mammary epithelial differentiation hierarchy as well as the cellular composition of breast tumors. Discrete populations of mouse and human mammary epithelial cells have been isolated on the basis of cell surface marker expression, yielding three primary populations including a stem cell-enriched population and two luminal populations. Their responsiveness to female steroid hormones was investigated, revealing the importance of paracrine regulation of ER/PR– stem/progenitor cells by ER/PR+ luminal ‘hormone sensor’ cells. RANKL was found to be a key paracrine effector of progesterone signaling, underscoring the central role that female hormones play in normal mammary gland homeostasis and cancer. Consistent with this observation, a RANK+ luminal progenitor cell was found to be the likely target cell that gives rise to breast cancer in BRCA1 mutation carriers. Pre-clinical studies further indicate that it may be possible to target this ‘culprit’ cell with the RANKL inhibitor denosumab. This finding is now being tested in a randomized phase III prevention study, ‘BRCA-P’. In further studies, a quiescent stem cell that remains hormone responsive has been identified. Lineage tracing, combined with high resolution 3D imaging (visualizing large regions of intact tissue), was carried out to track stem and progenitor cells in situ. These studies have revealed the presence of long-lived stem cells that replenish mammary epithelium, while unipotent progenitors appear to play an essential role in sustaining the independent lineages on a day-to-day basis. In order to generate a comprehensive single cell expression resource and explore molecular heterogeneity in the mouse mammary gland, single cell profiling at key developmental stages was performed. This has revealed novel intermediates and unexpected changes in the transcriptional landscape that could inform a role for distinct cell types during mammary oncogenesis. To gain insight into cancer heterogeneity and clonal dynamics at cellular resolution, 3D imaging was combined with lineage tracing to track specific cells during cancer development. Through the application of a novel pipeline, the epithelial-to-mesenchymal transition (EMT) was found to be a frequent event, thus highlighting the inherent plasticity of mammary tumors. These findings are currently being extended to human tumors to further understand inter- and intra-tumoral heterogeneity. In parallel, our group has established a bank of patient-derived xenograft (PDX) and patient derived organoid (PDO) models to facilitate the transfer of laboratory findings to the clinic. These serve as useful pre-clinical models to test promising agents. Initial efforts have focused on BH3 mimetics, a new class of drug that targets the BCL-2 family of pro-survival proteins. This work is leading to early phase clinical trials, currently focused on patients with metastatic ER+ breast cancer. Citation Format: JE Visvader, GJ Lindeman. Deciphering stem and progenitor cells to understand breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr BL1.

Lifelong n-3 Polyunsaturated Fatty Acid Exposure Modulates Size of Mammary Epithelial Cell Populations and Expression of Caveolae Resident Proteins in Fat-1 Mice.

Omega-3 polyunsaturated fatty acids (n-3 PUFA) have been associated with reduced breast cancer risk; however, the exact mechanism remains elusive. Female wildtype (WT) and fat-1 mice were fed a 10% safflower diet until 6 weeks of age. Mammary gland epithelial cells (EC) were isolated and EC populations were determined by CD24 surface expression. Fat-1 mice expressed 65%, 20%, and 15% while WT mice expressed 65%, 26% and 9% for non-, myo- and luminal ECs, respectively. The luminal EC population was significantly greater in fat-1 mice (p ≤ 0.05), while the total number of mammary ECs were similar between groups (p = 0.79). Caveolae was isolated from ECs and Her-2/neu, ER-α and cav-1 protein expression was determined by Western blotting. Fat-1 mice had a two-fold greater ER-α (p ≤ 0.05) and a 1.5-fold greater cav-1 (p ≤ 0.05) expression than WT with a similar amount of Her-2/neu protein (p = 0.990) between groups. Overall, this study provides novel mechanistic evidence by which n-3 PUFA modifies early mammary gland development that may potentially reduce breast cancer risk later in life.

A new view of the mammary epithelial hierarchy and its implications for breast cancer initiation and metastasis.

The existence of mammary epithelial stem cell (MaSC) populations capable of mediating mammary gland development and homeostasis has been established for over a decade. A combination of lineage tracing and mammary gland transplantation studies has affirmed that MaSCs and their downstream progenitors are organized in a hierarchal manner; however, these techniques have failed to illuminate the complete spectrum of epithelial intermediate populations or their spatial and temporal relationships. The advent of single cell sequencing technology has allowed for characterization of highly heterogeneous tissues at high resolution. In the last two years, the remarkable advances in single cell RNA sequencing (scRNA-seq) technologies have been leveraged to address the heterogeneity of the mammary epithelium. These studies have afforded fresh insights into the transcriptional differentiation hierarchy and its chronology. Importantly, these data have led to a major conceptual shift in which the rigid boundaries separating stem, progenitor, and differentiated epithelial populations have been deconstructed, resulting in a new more fluid and flexible model of epithelial differentiation. The emerging view of the mammary epithelial hierarchy has important implications for mammary development, carcinogenesis, and metastasis, providing novel insights into the underlying cellular states that may promote malignant phenotypes.

Deletion of Cdkn1b in ACI rats leads to increased proliferation and pregnancy-associated changes in the mammary gland due to perturbed systemic endocrine environment.

Mammary epithelial progenitors are the normal cell-of-origin of breast cancer. We previously defined a population of p27+ quiescent hormone-responsive progenitor cells in the normal human breast whose frequency associates with breast cancer risk. Here, we describe that deletion of the Cdkn1b gene encoding the p27 cyclin-dependent kinase inhibitor in the estrogen-induced mammary tumor-susceptible ACI rat strain leads to a decrease in the relative frequencies of Cd49b+ mammary luminal epithelial progenitors and pregnancy-related differentiation. We show by comprehensive gene expression profiling of purified progenitor and differentiated mammary epithelial cell populations that p27 deletion has the most pronounced effects on luminal progenitors. Cdkn1b-/- females have decreased fertility, but rats that are able to get pregnant had normal litter size and were able to nurse their pups implying that loss of p27 in ACI rats does not completely abrogate ovarian function and lactation. Reciprocal mammary gland transplantation experiments indicate that the p27-loss-induced changes in mammary epithelial cells are not only caused by alterations in their intrinsic properties, but are likely due to altered hormonal signaling triggered by the perturbed systemic endocrine environment observed in Cdkn1b-/- females. We also observed a decrease in the frequency of mammary epithelial cells positive for progesterone receptor (Pr) and FoxA1, known direct transcriptional targets of the estrogen receptor (Erα), and an increase in phospho-Stat5 positive cells commonly induced by prolactin (Prl). Characterization of genome-wide Pr chromatin binding revealed distinct binding patterns in mammary epithelial cells of Cdkn1b+/+ and Cdkn1b-/- females and enrichment in genes with known roles in Notch, ErbB, leptin, and Erα signaling and regulation of G1-S transition. Our data support a role for p27 in regulating the pool size of hormone-responsive luminal progenitors that could impact breast cancer risk.

Overexpression of miR-489 derails mammary hierarchy structure and inhibits HER2/neu-induced tumorigenesis.

Although it has been demonstrated that transformed progenitor cell population can contribute to tumor initiation, factors contributing to this malignant transformation are poorly known. Using in vitro and xenograft based models, previous studies demonstrated that miR-489 acts as a tumor suppressor miRNA by targeting various oncogenic pathways. It has been demonstrated that miR-489 directly targets HER2 and inhibits the HER2 signaling pathway; however, its role in mammary gland development and HER2 induced tumor initiation hasn’t been studied. To dissect the role of miR-489, we sorted different populations of mammary epithelial cells and determined that miR-489 was highly expressed in mammary stem cells. MMTV-miR-489 mice that overexpressed miR-489 in mammary epithelial cells were developed and these mice exhibited an inhibition of mammary gland development in early ages with a specific impact on highly proliferative cells. Double transgenic MMTV-Her2-miR489 mice were then generated to observe how miR-489 overexpression affects HER2 induced tumorigenesis. miR-489 overexpression delayed HER2 induced tumor initiation significantly. Moreover, miR-489 overexpression inhibited tumor growth and lung metastasis. miR-489 overexpression reduced mammary progenitor cell population significantly in preneoplastic mammary glands of MMTV-Her2 mice which showed a putative transformed population in HER2 induced tumorigenesis. The miR-489 overexpression reduced CD49fhiCD61hi populations in tumors that have stem- like properties, and miR-489 overexpression altered the HER2 signaling pathway in mammary tumors. Altogether, these data indicate that the inhibition of HER2 induced tumorigenesis by miR-489 overexpression was due to altering progenitor cell populations while decreasing tumor growth and metastasis via influencing tumor promoting genes DEK and SHP2.

Abstract PR04: Differentiation dynamics of the developing mammary gland revealed by single-cell RNA-sequencing

Abstract The mammary gland consists of epithelial ducts that stretch out in a tree-like structure through a stromal fat pad. The ducts are formed by two layers, the inner layer composed of luminal cells and an outer layer of basal (myoepithelial) cells. Despite this relatively simple anatomy, the precise cellular composition and hierarchies are still ill-defined. Recent efforts to enrich, isolate, and characterize the different mammary epithelial cell compartments only used a handful of markers to define and trace cell populations. Therefore, there is a need for an unbiased and comprehensive description of mammary epithelial cells within the gland at different developmental stages. To this end we herein use single-cell RNA sequencing to determine the gene expression profile of individual cells across four adult developmental stages; nulliparous, mid-gestation, lactation, and post-weaning (full natural involution). Our data from over 23,000 individual cells identify distinct mammary epithelial cell populations and allow their hierarchical structure across development to be charted. The luminal compartment showed a differentiation structure with a common progenitor that can give rise to intermediate progenitors of both alveolar and hormone-sensing luminal cells. In contrast, we captured only few cells that were transitioning between basal and luminal cells, suggesting that the basal compartment is less involved in maintaining luminal cells in adult tissue homeostasis. Interestingly, the transcriptional profile of some cell types appeared to be more affected by gestation and lactation. For example, our analysis revealed a cluster of luminal progenitor cells in post-involution glands, which is distinct from progenitors found in nulliparous glands. The post-involution progenitor cells share all the luminal progenitor characteristics with their nulliparous counterparts but were marked by higher expression of genes involved in milk synthesis and the immune response, suggesting that these cells maintain a memory of having undergone a full pregnancy cycle. This is especially interesting in the light of the protective effect of early pregnancies on breast cancers. The data also showed that only few clusters could be fully characterized by a single marker gene. We argue instead that the epithelial cells—especially in the luminal compartment—should rather be conceptualized as being part of a continuous spectrum of differentiation. This view highlights the plasticity of the tissue and might help to explain some of the conflicting results from lineage tracing studies. Our work provides a foundation for understanding the cellular aspects of the developmental biology of the mammary gland. This is vital to understand the early steps of tumor development. We hope that our dataset can also be mined to help to gain insights into the cells-of-origin for various breast cancers. This abstract is also being presented as Poster A66. Citation Format: Karsten Bach, Sara Pensa, David J. Adams, John C. Marioni, Walid T. Khaled. Differentiation dynamics of the developing mammary gland revealed by single-cell RNA-sequencing [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr PR04.

Abstract 1982: Obesity reversibly enhances mammary epithelial estrogen receptor alpha expression and progenitor activity

Abstract Obesity has been identified as an important risk factor for breast cancer in post-menopausal women and is significantly correlated with poor prognosis in breast cancer patients. Women with an increasing body mass index (BMI) are more likely to develop estrogen receptor alpha (ERα) positive tumors that are larger at the time of diagnosis and demonstrate an increased risk for metastasis. Given that obesity is becoming a global epidemic, understanding how obesity alters the biology of the normal breast is of significant clinical relevance. To examine the effects of obesity on mammary tissue, mice were fed a high fat diet to induce obesity and compared with mice fed a control diet. We also examined reduction mammoplasty tissue from women with a known BMI. In both obese female mice and women, mammary epithelial cell populations demonstrated significant decreases in basal/myoepithelial cells, when examined using either flow cytometry or cell type-specific markers (SMA and p63) in tissue. ERα expression was significantly increased in luminal cells in obese mammary tissue, compared with control mice or breast tissue from lean women. Functional assays demonstrated significantly enhanced mammary epithelial progenitor activity in obese mammary epithelial cells as well as elevated numbers of ERα positive epithelial cells that were co-labeled with markers of proliferation. Weight loss in a group of obese mice reversed increases in progenitor activity and ERα expression observed in obese mammary tissue. Obesity enhances the percentage of ERα positive epithelial cells and reduces the number of basal/myoepithelial cells within normal mammary tissue in both women and female mice. Both aspects of this phenotype have been linked to breast tumor susceptibility. These changes may contribute to lifetime risk for the development of ERα positive tumors in postmenopausal women. However, the changes in epithelial cell populations induced by obesity are reversible with weight loss, suggesting that weight loss interventions in obese women may effectively reduce breast cancer risk by decreasing the number of potential tumor progenitor cells in the breast epithelium. Citation Format: Tamara Chamberlin, Joseph V. D'Amato, Lisa M. Arendt. Obesity reversibly enhances mammary epithelial estrogen receptor alpha expression and progenitor activity [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 1982.

Obesity reversibly depletes the basal cell population and enhances mammary epithelial cell estrogen receptor alpha expression and progenitor activity

BackgroundObesity is correlated with an increased risk for developing postmenopausal breast cancer. Since obesity rates continue to rise worldwide, it is important to understand how the obese microenvironment influences normal mammary tissue to increase breast cancer risk. We hypothesized that obesity increases the proportion of luminal progenitor cells, which are thought to be the cells of origin for the most common types of breast cancer, potentially leading to an increased risk for breast cancer.MethodsTo study the obese microenvironment within the mammary gland, we used a high-fat diet mouse model of obesity and human breast tissue from reduction mammoplasty surgery. We identified changes in breast epithelial cell populations using flow cytometry for cell surface markers, in vitro functional assays and expression of markers on breast tissue sections.ResultsIn both obese female mice and women, mammary epithelial cell populations demonstrated significant decreases in basal/myoepithelial cells, using either flow cytometry or cell-type-specific markers (SMA and p63). Estrogen receptor alpha (ERα) expression was significantly increased in luminal cells in obese mammary tissue, compared with control mice or breast tissue from lean women. Functional assays demonstrated significantly enhanced mammary epithelial progenitor activity in obese mammary epithelial cells and elevated numbers of ERα-positive epithelial cells that were co-labeled with markers of proliferation. Weight loss in a group of obese mice reversed increases in progenitor activity and ERα expression observed in obese mammary tissue.ConclusionsObesity enhances ERα-positive epithelial cells, reduces the number of basal/myoepithelial cells, and increases stem/progenitor activity within normal mammary tissue in both women and female mice. These changes in epithelial cell populations induced by obesity are reversible with weight loss. Our findings support further studies to examine how obesity-induced changes in stem/progenitor cells impact breast tumor incidence and histologic tumor types.

FGFR inhibitor, AZD4547, impedes the stemness of mammary epithelial cells in the premalignant tissues of MMTV-ErbB2 transgenic mice

The fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases (RTKs) regulates signaling pathways involved in cell proliferation and differentiation. Currently, the anti-tumor properties of FGFR inhibitors are being tested in preclinical and clinical studies. Nevertheless, reports on FGFR inhibitor-mediated breast cancer prevention are sparse. In this study, we investigated the anti-cancer benefits of AZD4547, an FGFR1-3 inhibitor, in ErbB2-overexpressing breast cancer models. AZD4547 (1–5 µM) demonstrated potent anti-proliferative effects, inhibition of stemness, and suppression of FGFR/RTK signaling in ErbB2-overexpressing human breast cancer cells. To study the in vivo effects of AZD4547 on mammary development, mammary epithelial cell (MEC) populations, and oncogenic signaling, MMTV-ErbB2 transgenic mice were administered AZD4547 (2–6 mg/kg/day) for 10 weeks during the ‘risk window’ for mammary tumor development. AZD4547 significantly inhibited ductal branching and MEC proliferation in vivo, which corroborated the in vitro anti-proliferative properties. AZD4547 also depleted CD24/CD49f-sorted MEC populations, as well as the CD61highCD49fhigh tumor-initiating cell-enriched population. Importantly, AZD4547 impaired stem cell-like characteristics in primary MECs and spontaneous tumor cells. Moreover, AZD4547 downregulated RTK, mTOR, and Wnt/β-catenin signaling pathways in premalignant mammary tissues. Collectively, our data provide critical preclinical evidence for AZD4547 as a potential breast cancer preventative and therapeutic agent.

Buformin inhibits the stemness of erbB-2-overexpressing breast cancer cells and premalignant mammary tissues of MMTV-erbB-2 transgenic mice

BackgroundMetformin, an FDA-approved drug for the treatment of Type II diabetes, has emerged as a promising anti-cancer agent. Other biguanide analogs, including buformin and phenformin, are suggested to have similar properties. Although buformin was shown to reduce mammary tumor burden in carcinogen models, the anti-cancer effects of buformin on different breast cancer subtypes and the underlying mechanisms remain unclear. Therefore, we aimed to investigate the effects of buformin on erbB-2-overexpressing breast cancer with in vitro and in vivo models.MethodsMTT, cell cycle, clonogenic/CFC, ALDEFLUOR, tumorsphere, and Western blot analyses were used to determine the effects of buformin on cell growth, stem cell populations, stem cell-like properties, and signaling pathways in SKBR3 and BT474 erbB-2-overexpressing breast cancer cell lines. A syngeneic tumor cell transplantation model inoculating MMTV-erbB-2 mice with 78617 mouse mammary tumor cells was used to study the effects of buformin (1.2 g buformin/kg chow) on tumor growth in vivo. MMTV-erbB-2 mice were also fed buformin for 10 weeks, followed by analysis of premalignant mammary tissues for changes in morphological development, mammary epithelial cell (MEC) populations, and signaling pathways.ResultsBuformin significantly inhibited SKBR3 and BT474 cell growth, and in vivo activity was demonstrated by considerable growth inhibition of syngeneic tumors derived from MMTV-erbB-2 mice. In particular, buformin suppressed stem cell populations and self-renewal in vitro, which was associated with inhibited receptor tyrosine kinase (RTK) and mTOR signaling. Consistent with in vitro data, buformin suppressed mammary morphogenesis and reduced cell proliferation in MMTV-erbB-2 mice. Importantly, buformin decreased MEC populations enriched with mammary reconstitution units (MRUs) and tumor-initiating cells (TICs) from MMTV-erbB-2 mice, as supported by impaired clonogenic and mammosphere formation in primary MECs. We further demonstrated that buformin-mediated in vivo inhibition of MEC stemness is associated with suppressed activation of mTOR, RTK, ER, and β-catenin signaling pathways.ConclusionsOverall, our results provide evidence for buformin as an effective anti-cancer drug that selectively targets TICs, and present a novel prevention and/or treatment strategy for patients who are genetically predisposed to erbB-2-overexpressing breast cancer.

A Quiescent Bcl11b High Stem Cell Population Is Required for Maintenance of the Mammary Gland

Stem cells in many tissues sustain themselves by entering a quiescent state to avoid genomic insults and to prevent exhaustion caused by excessive proliferation. In the mammary gland, the identity and characteristics of quiescent epithelial stem cells are not clear. Here, we identify a quiescent mammary epithelial cell population expressing high levels of Bcl11b and located at the interface between luminal and basal cells. Bcl11bhigh cells are enriched for cells that can regenerate mammary glands in secondary transplants. Loss of Bcl11b leads to a Cdkn2a-dependent exhaustion of ductal epithelium and loss of epithelial cell regenerative capacity. Gain- and loss-of-function studies show that Bcl11b induces cells to enter the G0 phase of the cell cycle and become quiescent. Taken together, these results suggest that Bcl11b acts as acentral intrinsic regulator of mammary epithelial stem cell quiescence and exhaustion and is necessary for long-term maintenance of the mammary gland.

Mammary Epithelial Cell Hierarchy in the Dairy Cow Throughout Lactation.

The plasticity of the mammary gland relies on adult mammary stem cells (MaSCs) and their progenitors, which give rise to various populations of mammary epithelial cells (MECs). To face global challenges, an in-depth characterization of milk-producing animal mammary gland plasticity is required, to select more sustainable and robust dairy cows. The identification and characterization of MaSC and their progenitors will also provide innovative tools in veterinary/human medicine regarding mammary tissue damage (carcinogenesis, bacterial infections). This study aimed to determine the dynamics of mammary cell populations throughout a lactation cycle. Using mammary biopsies from primiparous lactating dairy cows at 30, 90, 150, and 250 days of lactation, we phenotyped cell populations by flow cytometry. To investigate cell lineages, we used specific cell-surface markers, including CD49f, CD24, EpCAM (epithelial cell adhesion molecule), and CD10. Two cell populations linked to milk production were identified: CD49f(+)/EpCAM(-) (y = 0.88x + 4.42, R(2) = 0.36, P < 0.05) and CD49f(-)/EpCAM(-) (y = -1.15x + 92.44, R(2) = 0.51, P < 0.05) cells. Combining immunostaining analysis, flow cytometry, daily milk production data, and statistical approaches, we defined a stem cell population (CD24(+)/CD49f(+)) and four progenitor cell populations that include bipotent luminal progenitors (CD24(-)/CD49f(+)), lumino-alveolar progenitors (CD24(-)/EpCAM(+)), myoepithelial progenitors (CD24(+)/CD10(-)), and lumino-ductal progenitors (CD49f(-)/EpCAM(+)). Interestingly, we found that the bipotent luminal progenitors (CD24(-)/CD49f(+)) decreased significantly (P < 0.05) during lactation. This study provides the first results of mammary cell lineage, allowing insight into mammary cell plasticity during lactation.