Basal Myoepithelial Cells Research Articles

Luminal breast epithelial cells of BRCA1 or BRCA2 mutation carriers and noncarriers harbor common breast cancer copy number alterations.

The prevalence and nature of somatic copy number alterations (CNAs) in breast epithelium and their role in tumor initiation and evolution remain poorly understood. Using single-cell DNA sequencing (49,238 cells) of epithelium from BRCA1 and BRCA2 carriers or wild-type individuals, we identified recurrent CNAs (for example, 1q-gain and 7q, 10q, 16q and 22q-loss) that are present in a rare population of cells across almost all samples (n = 28). In BRCA1/BRCA2 carriers, these occur before loss of heterozygosity (LOH) of wild-type alleles. These CNAs, common in malignant tumors, are enriched in luminal cells but absent in basal myoepithelial cells. Allele-specific analysis of prevalent CNAs reveals that they arose by independent mutational events, consistent with convergent evolution. BRCA1/BRCA2 carriers contained a small percentage of cells with extreme aneuploidy, featuring loss of TP53, BRCA1/BRCA2 LOH and multiple breast cancer-associated CNAs. Our findings suggest that CNAs arising in normal luminal breast epithelium are precursors to clonally expanded tumor genomes.

Molecular complexity of mammary glands development: a review of lactogenic differentiation in epithelial cells

The mammary gland is a dynamic organ with various physiological processes like cellular proliferation, differentiation, and apoptosis during the pregnancy-lactation-involution cycle. It is essential to understand the molecular changes during the lactogenic differentiation of mammary epithelial cells (MECs, the milk-synthesizing cells). The MECs are organized as luminal milk-secreting cells and basal myoepithelial cells (responsible for milk ejection by contraction) that form the alveoli. The branching morphogenesis and lactogenic differentiation of the MECs prepare the gland for lactation. This process is governed by many molecular mediators including hormones, growth factors, cytokines, miRNAs, regulatory proteins, etc. Interestingly, various signalling pathways guide lactation and understanding these molecular transitions from pregnancy to lactation will help researchers design further research. Manipulation of genes responsible for milk synthesis and secretion will promote augmentation of milk yield in dairy animals. Identifying protein signatures of lactation will help develop strategies for persistent lactation and shortening the dry period in farm animals. The present review article discusses in details the physiological and molecular changes occurring during lactogenic differentiation of MECs and the associated hormones, regulatory proteins, miRNAs, and signalling pathways. An in-depth knowledge of the molecular events will aid in developing engineered cellular models for studies related to mammary gland diseases of humans and animals.

Abstract P5-03-09: Cell composition changes in healthy breast tissue is associated with advancing age and epigenetic age acceleration

Abstract Introduction: Risk factors for breast cancer include advancing age, lifetime estrogen exposure, and breast density. DNA methylation-based estimates of age are elevated in breast tissue of healthy women compared with paired blood samples, and the degree of age acceleration is associated with lifetime estrogen exposure. However, no prior work has examined cell compositional changes associated with breast epigenetic aging. In this study we estimated the abundance of different cell types in healthy breast, computed using gene expression data, and investigate cell composition changes that accompany advancing chronologic age and breast epigenetic age acceleration. Methods: DNA/RNA were extracted (AllPrep, Qiagen) from breast tissue specimens from 192 healthy women aged 19-90 years who donated breast tissue to the Susan G. Komen Tissue Bank at the Indiana University Simon Comprehensive Cancer Center. Transcriptome analysis was performed using the QuantSeq 3’mRNA-SeqFWD kit to generate RNA sequencing libraries. DNA methylation age was estimated using beta-values from Illumina EPIC 850K array platform. Age acceleration is defined using the residual of a linear regression of methylation age on chronologic age. Cell deconvolution was performed using CIBERSORTx to estimate the abundance of adipocytes, luminal epithelial cells, basal myoepithelial cells, vascular endothelial cells, lymphatic endothelial cells, immune cells (dendritic cells & macrophages), pericytes & smooth muscle cells, and fibroblasts. We examined cell composition changes with chronologic age, and with age-adjusted measures of acceleration in 6 epigenetic clocks: the Horvath Pan-tissue, Hannum, Phenotypic, Grim, Skin & Blood, and Epigenetic Pacemaker clocks, as well as the DNA methylation-based estimate of telomere length, DNAmTL. Results: Advancing chronologic age was associated with an increase in the imputed proportion of adipocytes (R=0.40, p< 0.0001), vascular endothelial cells (R=0.23, p=0.0033), and immune cells (dendritic cells & macrophages) (R=0.29, p=0.00016), and a decrease in the proportion of luminal epithelial cells (R=-0.43, p< 0.0001), and basal myoepithelial cells (R=-0.27, p=0.00047). Epigenetic age acceleration was significantly associated with increases in proportions of luminal epithelial cells (p< 0.0001 for age-adjusted Hannum, Phenotypic, Grim, and Skin & Blood clocks, p< 0.05 for Pan-tissue) and basal myoepithelial cells (p< 0.0001 for Phenotypic and Skin & Blood clocks), and decreased proportions of adipocytes and vascular endothelial cells (p< 0.0001 for Hannum, Phenotypic, Grim, and Skin & Blood clocks, p< 0.05 for Pan-tissue). Conclusion: Using gene expression data in healthy female breast tissue, we identified significant changes in cell-type-specific abundance that accompany advancing chronologic age, with an increase in adipocytes and immune cells, and a decline in luminal epithelial cells and basal myoepithelial cells. By contrast, epigenetic age acceleration in breast tissue is associated with decreasing proportions of adipocytes and vascular endothelial cells and a rise in basal myoepithelial cells and luminal epithelial cells. Our findings suggest distinct patterns of cellular composition changes that accompany normal aging compared with accelerated aging in breast tissue. Citation Format: Mary E. Sehl, Wenbin Guo, Collin Farrell, Natascia Marino, Jill Henry, Anna Maria Storniolo, Jeanette Papp, Jingyi Li, Steve Horvath, Matteo Pellegrini, Patricia A. Ganz. Cell composition changes in healthy breast tissue is associated with advancing age and epigenetic age acceleration [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-03-09.

Abstract P5-15-01: Rediscovering IRF5: A prognostic indicator with novel roles in mammary gland development, ribosome biogenesis, tumor initiation, and metastasis

Abstract Breast cancer is the second leading cause of death for women in the United States. However, only 10% of breast cancers have been linked to inherited genomic mutations. Thus, identifying biomarkers to predict cancer progression and metastasis remains a clear need in the research and medical world. Here, we report a novel role for Interferon Regulatory Factor 5 (IRF5) in mammary gland development, tumorigenesis, and metastasis. Historically, IRF5 has been studied as a transcription factor in the context of genetic risk for autoimmune diseases. However, mining of The Cancer Genome Atlas revealed that loss of IRF5 expression in human breast cancer is significantly linked with progression to high grade carcinoma, increased metastasis, and decreased overall and recurrence-free survival. Supporting these analyses, we demonstrated that female Irf5-/- BALB/c mice have higher incidence of spontaneous atypical ductal hyperplasia (ADH), increased progression to DCIS, and ultimately, increased incidence of IDC. Using qPCR, FISH and IHC, we confirmed that IRF5 is expressed in both luminal and basal myoepithelial cells, but expression is higher in basal cells. Histologic analysis of whole mount preparations of Irf5-/- mammary glands revealed aberrant ductal morphogenesis, characterized by expansion of luminal and basal myoepithelial cells with a loss of organized glandular structure. RNAseq of primary mammary epithelial cells from wild type and Irf5-/- littermate mice showed Irf5-/- mammary epithelial cells to be enriched in ribosome biogenesis pathways, the physiologic consequences of which were demonstrated through increased rates of protein synthesis. Transferring our studies in vivo, we demonstrated that loss of tumor IRF5 expression resulted in decreased tumor-infiltrating lymphocytes and increased pulmonary metastasis in the murine orthotopic 4T1 implantation model. Mechanistically, we found that—as in our studies utilizing primary mammary epithelial cells—IRF5 expression in tumors regulated protein translation and ribosome biogenesis. In light of these findings, we propose IRF5 as a novel prognostic biomarker, loss of which alters mammary gland development, drives tumor initiation and metastasis, and dysregulates mammary epithelial cell protein synthesis. Citation Format: Betsy Barnes, Zarina Brune, Matthew Rice, Carter Somerville. Rediscovering IRF5: A prognostic indicator with novel roles in mammary gland development, ribosome biogenesis, tumor initiation, and metastasis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-15-01.

Abstract C025: Dietary-AGE ingestion during puberty modifies the breast microenvironment to alter mammary gland development: Linking lifestyle with cancer disparity

Abstract Advanced glycation end products (AGEs) are highly reactive metabolites that irreversibly accumulate in tissues as we grow older. Accumulation of AGEs in the body can contribute to proinflammatory and pro-oxidant phenotypes when signaling through the receptor for advanced glycation end products (RAGE).The pathogenic effects of AGE-RAGE signaling include tissue degeneration, protein dysfunction, aberrant cell signaling, and reduced genetic fidelity. AGEs are formed during normal metabolism but, critically, lifestyle factors such as poor diet, a sedentary lifestyle and being obese also contribute to the AGE accumulation pool. The permanent nature of AGE adducts and their ability to mediate chronic and persistent inflammatory and oxidative stresses is particularly compatible with the concept of metabolic memory. Our dietary studies in pubertal FVB/n mice after chronic consumption of AGE show a significant dysregulation of mammary gland development and the formation of hyperplastic lesions. We observe delayed mammary ductal extension, increased ductal branching and aberrant terminal end-bud (TEB) morphology. The basal myoepithelial cell layer surrounding mammary ducts and TEBs was irregular and epithelial cell proliferation was increased. Molecular characterization of these hyperplastic lesions was defined by using markers of DCIS progression by histopathologic staining and qRT-PCR. Elevated AGE levels were accompanied by increased expression of RAGE and increased immune (macrophage) and stromal (fibroblast) infiltration around the TEBs. In an attempt to reverse the effects caused by a high-AGE diet, mice were fed a control diet after a pubertal high-AGE diet. Hyperplastic lesions persisted despite diet intervention. Importantly, hyperplastic lesions were not observed in mice fed a control diet during puberty, then switched to a high-AGE diet after. These data not only illustrate the importance of the pubertal window in the mediation of lifestyle factors but also indicate that exposure to AGE-induced changes during puberty may leave a long-lasting imprint analogous to the concept of metabolic memory. Additionally, in our feasibility studies, both pharmacologic (NCT02946996; NCT03092635) and interventional (NCT03459755) strategies were shown to reduce circulating AGE levels in cancer survivors. Based on associations between diet, lifestyle, and race, increases in AGE accumulation may represent a novel biologic mechanism contributing to cancer disparity and may represent a new paradigm contributing to the increased cancer incidence and mortality figures observed within health-disparity populations. Given the potential benefits of lifestyle changes and the potential biologic role of AGEs in promoting cancer, opportunities exist for collaborations impacting basic, translational, epidemiologic and cancer prevention initiatives. Citation Format: Bradley A. Krisanits, Jaime F. Randise, Lourdes M. Nogueira, Kristi Helke, Michael C. Ostrowski, Katie Theis, Maria Cuitino, Gayenel Magwood, Marvella E. Ford, Victoria J. Findlay, David P. Turner. Dietary-AGE ingestion during puberty modifies the breast microenvironment to alter mammary gland development: Linking lifestyle with cancer disparity [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C025.

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 2234: Dietary-AGE ingestion during puberty modifies the breast microenvironment to alter mammary gland development: Linking diet, development and breast cancer risk

Abstract Evidence supports the notion that critical events during mammary development permanently alter developmentally regulated programs which influence the breast microenvironment to increase breast cancer risk. This is analogous to metabolic memory in diabetic patients where early metabolic events have been found to be remembered and affect disease severity later in life. Advanced glycation end products (AGEs) are highly reactive metabolites that irreversibly accumulate in tissues as we age. AGE accumulation can contribute to pro-inflammatory and -oxidant phenotypes when signaling through the receptor for advanced glycation end products (RAGE). The pathogenic effects of AGE-RAGE signaling include tissue degeneration, protein dysfunction, aberrant cell signaling, and reduced genetic fidelity. AGEs form during normal metabolism but critically, lifestyle factors such as poor diet, a sedentary lifestyle and being obese also contribute to the AGE accumulation pool. The permanence of AGE adducts and their ability to mediate chronic and persistent inflammatory and oxidative stresses is particularly compatible to the concept of metabolic memory. Our dietary studies in pubertal FVB/n mice fed a high AGE diet show a significant dysregulation of mammary gland development and hyperplastic lesion formation. We observe delayed mammary ductal extension, increased ductal branching and aberrant terminal end-bud (TEB) morphology. The basal myoepithelial cell layer surrounding mammary ducts and TEBs was irregular and epithelial cell proliferation was increased. Molecular characterization of these hyperplastic lesions were defined using DCIS progression markers by histopathological staining and qRT-PCR. Elevated AGE levels accompanied increased RAGE expression and increased macrophage and fibroblast infiltration around the TEBs. In attempt to reverse the effects caused by a high AGE diet, mice were fed a control diet after a pubertal high AGE diet. Hyperplastic lesions persisted despite diet intervention. Importantly, hyperplastic lesions were not observed in mice fed a control diet during puberty, then switched to a high AGE diet. These data indicate that exposure to AGE induced changes during puberty may leave a long-lasting imprint analogous to metabolic memory. In conclusion, increased AGE consumption during pubertal growth results in significant disruption of normal mammary development and the appearance of hyperplastic lesions by adulthood. Consumption of a high AGE diet despite a control diet intervention, reveals hyperplastic lesions indicative of metabolic memory. We hypothesize that the high AGE diet may leave a metabolic imprint on the mammary gland microenvironment, increasing the risk of future breast cancer development. Citation Format: Jaime F. Randise, Bradley A. Krisanits, Lourdes M. Nogueira, Kristi L. Helke, Taaliah Campbell, Victoria J. Findlay, David P. Turner. Dietary-AGE ingestion during puberty modifies the breast microenvironment to alter mammary gland development: Linking diet, development and breast cancer risk [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 2234.

Location, Isolation, and Identification of Mesenchymal Stem Cells from Adult Human Sweat Glands.

Sweat glands (SGs) are spread over almost the entire surface of the human body and are essential for thermoregulation. Theoretically, tissue-specific stem cells (TSSCs) are excellent candidate cells for the regeneration of SGs due to their genetic stability and differentiation ability. Herein, we attempted to isolate TSSCs derived from adult human sweat glands (ahSGs). ahSGs were localized and identified by H&E staining, double immunofluorescence staining, transmission electron microscope (TEM), and immuno-TEM. We found a population of cells with stem cell characteristics (SGSCs), located in basal myoepithelial cells of the secretory portion of the solenoid bulb. The SGSCs expressed alpha-smooth muscle actin (α-SMA) and showed the typical characteristics of mesenchymal stem cells (MSCs), with a positive antigen profile for CD44, CD73, CD90, and CD105, and had the multilineage differentiation potential to osteoblasts and adipocytes. In addition, the isolated α-SMA positive cells remained stably phenotypic and proliferative cycles at passage 12. This is the first report of successful isolation of MSC-like cells from ahSGs, which may contribute to wound repair and SG regeneration.

Podoplanin regulates mammary stem cell function and tumorigenesis by potentiating Wnt/β-catenin signaling.

Stem cells (SCs) drive mammary development, giving rise postnatally to an epithelial bilayer composed of luminal and basal myoepithelial cells. Dysregulation of SCs is thought to be at the origin of certain breast cancers; however, the molecular identity of SCs and the factors regulating their function remain poorly defined. We identified the transmembrane protein podoplanin (Pdpn) as a specific marker of the basal compartment, including multipotent SCs, and found Pdpn localized at the basal-luminal interface. Embryonic deletion of Pdpn targeted to basal cells diminished basal and luminal SC activity and affected the expression of several Wnt/β-catenin signaling components in basal cells. Moreover, Pdpn loss attenuated mammary tumor formation in a mouse model of β-catenin-induced breast cancer, limiting tumor-initiating cell expansion and promoting molecular features associated with mesenchymal-to-epithelial cell transition. In line with the loss-of-function data, we demonstrated that mechanistically Pdpn enhances Wnt/β-catenin signaling in mammary basal cells. Overall, this study uncovers a role for Pdpn in mammary SC function and, importantly, identifies Pdpn as a new regulator of Wnt/β-catenin signaling, a key pathway in mammary development and tumorigenesis.

Abstract P3-05-05: PTHrP affects breast cancer initiation and progression through Stat5 signaling pathway

Abstract PTHrP affects mammary development and breast cancer. PTHrP is expressed in basal myoepithelial cells until late pregnancy, when it also becomes expressed in alveolar epithelial cells. During lactation, PTHrP is secreted into milk and into the circulation, and regulates systemic calcium and bone metabolism. Secretion of PTHrP by breast cancers stimulates bone resorption, promoting the skeletal metastases and/or development of hypercalcemia. Less is known about the contribution of PTHrP to development and/or progression of primary breast cancers. Recent GWAS reports have identified the PTHrP gene as a breast cancer susceptibility locus. Using data from the Cancer Genome Atlas (TCGA) Project and a Yale tissue microarray (YTMA49) we have found that increased breast tumor PTHrP expression predicts a more aggressive phenotype and increased mortality, signifying the clinical relevance of high tumor PTHrP levels. In an attempt to define how PTHrP affects breast cancer, we assessed the effects of PTHrP overexpression on the development of mammary tumors in mice. We developed a tetracyline-regulated, MMTV-driven transgenic model of PTHrP overexpression targeted to mammary epithelial cells (MMTV-rtTA;tetO-hPTHrP). Surprisingly, we found that PTHrP overexpression in luminal epithelial cells causes alveolar hyperplasia, secretory differentiation and milk production in virgin mice, associated with a lower number of luminal progenitor cells and basal stem cells upon FACS analysis. Additionally, Signal Transducer and Activator of Transcription 5 (Stat5) is highly activated in the setting of PTHrP-induced alveolar hyperplasisa. This activation is functionally relevant as evidenced by higher levels of β-Casein mRNA and protein, the transcription of which is regulated by activated Stat5 (pStat5). This effect of PTHrP was reversible upon withdrawal of doxycycline (Dox). Addition of Dox to primary mammary epithelial cell cultures from these mice recapitulated the findings. Knocking out PTHrP receptor (PTH1R) in these mice did not affect this phenotype, suggesting that PTHrP acts in a receptor-independent manner. Overexpression of PTHrP in MMTV-PyMT mice dramatically accelerated formation of mammary tumors reducing both latency of tumor formation and survival of the mice. It caused a higher rate of proliferation and a lower rate of apoptosis in vivo. All MMTV-rtTA;tetO-hPTHrP;MMTV-PyMT mice developed palpable tumors in all mammary glands within 3 weeks of age, became hypercalcemic and died before 4.5 weeks of age. These mice had higher levels of pStat5 and βCasein. Tumor cells cultured ex vivo and cell lines established from these tumors overexpressed PTHrP when stimulated with Dox. PTHrP overexpression, and not exogenous PTHrP, caused higher levels of proliferation, pStat5, and β-Casein. This PTHrP-induced activity of Stat5 was blocked by treatment of these cells with a pharmacological inhibitor of Jak2, a tyrosine kinase upstream of Stat5. Additionally, T47D cells transfected with PTHrP showed higher levels of pStat5, suggesting that PTHrP could activate Stat5 signaling regardless of the cell type. We show that PTHrP overexpression results in Stat5 activation and increased proliferation in breast cancer cells in a cell-autonomous fashion and independent of its receptor. Citation Format: Takyar FM, Boras-Granic K, Kim W, Dann P, Wysolmerski JJ. PTHrP affects breast cancer initiation and progression through Stat5 signaling pathway [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-05.

The Hippo kinases LATS1 and 2 control human breast cell fate via crosstalk with ERα.

Cell fate perturbations underlie many human diseases, including breast cancer. Unfortunately, the mechanisms by which breast cell fate are regulated are largely unknown. The mammary gland epithelium consists of differentiated luminal epithelial and basal myoepithelial cells, as well as undifferentiated stem cells and more restricted progenitors. Breast cancer originates from this epithelium, but the molecular mechanisms that underlie breast epithelial hierarchy remain ill-defined. Here, we use a high-content confocal image-based short hairpin RNA screen to identify tumour suppressors that regulate breast cell fate in primary human breast epithelial cells. We show that ablation of the large tumour suppressor kinases (LATS) 1 and 2 (refs 5, 6), which are part of the Hippo pathway, promotes the luminal phenotype and increases the number of bipotent and luminal progenitors, the proposed cells-of-origin of most human breast cancers. Mechanistically, we have identified a direct interaction between Hippo and oestrogen receptor-α (ERα) signalling. In the presence of LATS, ERα was targeted for ubiquitination and Ddb1-cullin4-associated-factor 1 (DCAF1)-dependent proteasomal degradation. Absence of LATS stabilized ERα and the Hippo effectors YAP and TAZ (hereafter YAP/TAZ), which together control breast cell fate through intrinsic and paracrine mechanisms. Our findings reveal a non-canonical (that is, YAP/TAZ-independent) effect of LATS in the regulation of human breast cell fate.

The Potential Role of Hedgehog Signaling in the Luminal/Basal Phenotype of Breast Epithelia and in Breast Cancer Invasion and Metastasis.

The epithelium of the lactiferous ducts in the breast is comprised of luminal epithelial cells and underlying basal myoepithelial cells. The regulation of cell fate and transit of cells between these two cell types remains poorly understood. This relationship becomes of greater importance when studying the subtypes of epithelial breast carcinoma, which are categorized according to their expression of luminal or basal markers. The epithelial mesenchymal transition (EMT) is a pivotal event in tumor invasion. It is important to understand mechanisms that regulate this process, which bears relation to the normal dynamic of epithelial/basal phenotype regulation in the mammary gland. Understanding this process could provide answers for the regulation of EMT in breast cancer, and thereby identify potential targets for therapy. Evidence points towards a role for hedgehog signaling in breast tissue homeostasis and also in mammary neoplasia. This review examines our current understanding of role of the hedgehog-signaling (Hh) pathway in breast epithelial cells both during breast development and homeostasis and to assess the potential misappropriation of Hh signals in breast neoplasia, cancer stem cells and tumor metastasis via EMT.

Paracrine Met signaling triggers epithelial-mesenchymal transition in mammary luminal progenitors, affecting their fate.

HGF/Met signaling has recently been associated with basal-type breast cancers, which are thought to originate from progenitor cells residing in the luminal compartment of the mammary epithelium. We found that ICAM-1 efficiently marks mammary luminal progenitors comprising hormone receptor-positive and receptor-negative cells, presumably ductal and alveolar progenitors. Both cell populations strongly express Met, while HGF is produced by stromal and basal myoepithelial cells. We show that persistent HGF treatment stimulates the clonogenic activity of ICAM1-positive luminal progenitors, controlling their survival and proliferation, and leads to the expression of basal cell characteristics, including stem cell potential. This is accompanied by the induction of Snai1 and Snai2, two major transcription factors triggering epithelial-mesenchymal transition, the repression of the luminal-regulatory genes Elf5 and Hey1, and claudin down-regulation. Our data strongly indicate that paracrine Met signaling can control the function of luminal progenitors and modulate their fate during mammary development and tumorigenesis.

Abstract P2-06-09: Inflammation promotes stem-like characteristics and increases stemness of breast epithelial cells

Abstract Triple negative breast cancer (TNBC) accounts for approximately 15% of all breast cancers and is more likely to affect younger women, women of African American descent, and BRCA1 mutation carriers. TNBC is typically an aggressive cancer and is defined as the absence of positive staining for estrogen (ER) and progesterone (PR) receptor, and a lack of amplification of HER2. Due to its receptor status, TNBC is insensitive to the conventional targeted treatment utilized in ER/PR/HER2 positive breast cancer leading to poor prognosis and early visceral metastasis with survival rates for women who relapse within 5 years of treatment being significantly lower than hormone receptor positive breast cancer. Therefore, understanding the mechanisms that drive the formation of TNBC can aid in determining targets to develop better treatment options. We investigated differences in the tumor microenvironment between TNBC and ER positive breast cancer patient tissue samples. Hematoxylin & eosin stain of invasive ductal carcinomas revealed that TNBC patients had higher amounts of tumor infiltrating leukocytes compared to ER positive breast cancers. In premalignant breast cancer patients, ER negative ductal carcinoma in situ (DCIS) patients had significantly higher infiltrating leukocytes compared to ER positive DCIS indicating that inflammation may be a contributing factor in TNBC. Research on molecular profiling of TNBC has revealed that a subset of gene expression patterns are associated with the basal-myoepithelial cells rather than the luminal cells, however, previous evidence indicates that TNBC arises from luminal progenitors which undergo an abnormal conversion to basal-like progenitors with enhanced growth and survival promoting characteristics. We propose this conversion occurs due to the presence of inflammatory signals early in tumor development which trigger a cascade of events that leads to acquisition of a basal-like phenotype in luminal progenitor cells and upregulation of stem-like properties, resulting in the formation of basal-like breast cancer/TNBC. Treatment of non-malignant breast epithelial cells with IL-6 increased sphere-forming efficiency under stem cell growth promoting conditions and an inhibitor of the IL-6/STAT3 pathway decreased ALDH enzymatic activity as measured by Aldefluor assay. Also, treatment with IL-6 downregulated the luminal marker, epithelial specific antigen (ESA), and increased the expression of the myoepithelial marker, CD49f, on the cell surface of non-malignant breast epithelial cells, indicating an increase in the basal-like cell population and a loss of the luminal cell population. Therefore, inflammation appears to be a common feature in TNBC compared to ER positive breast cancer and the presence of an inflammatory stimulus in the breast may promote the conversion of luminal to basal-like cells, promoting the upregulation of stem-like properties and development of TNBC. These data suggest that anti-inflammatory drugs could potentially be used to inhibit these events from occurring in the development of TNBC, leading to decreased tumor growth and better treatment options. Citation Format: Jennifer Sims-Mourtada, Kimberly M Arnold, Lynn Opdenaker, Daniel Flynn. Inflammation promotes stem-like characteristics and increases stemness of breast epithelial cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-06-09.

Up-regulation of integrin α6β4 expression by mitogens involved in dairy cow mammary development.

In dairy cows, the extracellular microenvironment varies significantly from the virgin state to lactation. The function of integrin α6β4 is dependent on cell type and extracellular microenvironment, and the precise expression profile of α6β4 and its effects on mammary development remain to be determined. In the present study, real-time PCR and immunohistochemistry were used to analyze the expression and localization of integrin α6β4 in Holstein dairy cow mammary glands. The effects of integrin α6β4 on the proliferation induced by mammogenic mitogens were identified by blocking integrin function in purified dairy cow mammary epithelial cells (DCMECs). The results showed that the localization of β4 subunit and its exclusive partner the α6 subunit were not consistent but were co-localized in basal luminal cells and myoepithelial cells, appearing to prefer the basal surface of the plasma membrane. Moreover, α6 and β4 subunit messenger RNA (mRNA) levels changed throughout the stages of dairy cow mammary development, reflected well by protein levels, and remained higher in the virgin and pregnancy states, with duct/alveolus morphogenesis and active cell proliferation, than during lactation, when growth arrest is essential for mammary epithelial cell differentiation. Finally, the upregulation of integrin expression by both mammogenic growth hormone and insulin-like growth factor-1 and the inhibited growth of DCMECs by function-blocking integrin antibodies confirmed that integrin α6β4 was indeed involved in dairy cow mammary development.

Mammary stem cells have myoepithelial cell properties.

Contractile myoepithelial cells dominate the basal layer of the mammary epithelium and are considered to be differentiated cells. However, we observe that up to 54% of single basal cells can form colonies when seeded into adherent culture in the presence of agents that disrupt acin-myosin interactions, and on average, 65% of the single-cell-derived basal colonies can repopulate a mammary gland when transplanted in vivo. This indicates that a high proportion of basal myoepithelial cells can give rise to a mammary repopulating unit (MRU). We demonstrate that myoepithelial cells, flow-sorted using 2 independent myoepithelial-specific reporter strategies, have MRU capacity. Using an inducible lineage tracing approach we follow the progeny of α-smooth muscle actin-expressing myoepithelial cells and show that they function as long-lived lineage-restricted stem cells in the virgin state and during pregnancy.

Ceruminous Adenoma

A 58-year-old Filipino man with a two-year history of a left external auditory canal mass associated with ipsilateral hearing loss underwent polypectomy for a clinical impression of aural polyp.
 
 We received several cream tan, irregular tissue fragments with an aggregate diameter of 1.4 cm. Histopathologic examination shows clusters of tumor cells forming variably sized ducts and glands, some of which are cystically dilated; many of these structures have irregular lumina. (Figure 1) Higher magnification shows a dual cell population: an outer layer of round to ovoid cells with clear cytoplasm, corresponding to basal myoepithelial cells; and an inner layer of cuboidal to columnar cells, that have eosinophilic and granular cytoplasm with decapitating apical ends, corresponding to luminal epithelial cells with apocrine morphology. (Figure 2) Nuclear pleomorphism is mild to moderate, nucleoli are not prominent, and mitoses, perineural invasion and necrosis are not seen. In some glands a yellow to golden brown, coarse pigment is seen at the cytoplasm of the luminal cells. (Figure 3) The tumor does not involve the epidermis, and there is a variable amount of chronic inflammation. (Figure 4) Based on these features we diagnosed it as ceruminous adenoma.
 
 Ceruminous neoplasms are uncommon tumors found in the external auditory canal. Benign ceruminous tumors include ceruminous adenoma, ceruminous pleomorphic adenoma and ceruminous syringocystadenoma papilliferum.1 Most common among these is the ceruminous adenoma; making up 88% in one thirty-year review of benign ceruminous neoplasms.1 These tumors occur in a wide age range, most commonly in the sixth decade, and have no sex predilection.2 They present as masses in the outer half of the external auditory canal with hearing changes and pain.2 They can be mistaken for aural polyps, especially when there is associated chronic suppurative otitis media.3
 
 Ceruminous adenomas arise from ceruminous glands,4 which are modified sweat glands of the outer one half of the external auditory canal.1 These are well-circumscribed tumors with glandular architecture composed of two populations of cells with apocrine features, as described above. Two microscopic features reinforce the ceruminous nature of the glands: the apocrine morphology and presence of “ceroid” material, which are lipofuscin-like pigment granules seen in the cytoplasm of ceruminous gland luminal cells.1,2 Both are evident in our case. The main differential diagnosis of ceruminous adenoma is a ceruminous adenocarcinoma.1,2,5,6 Marked pleomorphism, brisk mitoses, necrosis, invasion (e.g. perineural), and loss of the two-cell population favor a diagnosis of ceruminous adenocarcinoma, but some well-differentiated cases can be confused with an adenoma. In these cases, sometimes the only clue of malignancy is invasion, especially at the surgical margins.6 Lassaletta et al.5 stressed the importance of adequate tumor excision for a more accurate diagnosis. The presence of a dual cell population and the absence of malignant features led us to a diagnosis of ceruminous adenoma. Immunohistochemical staining for cytokeratin (specifically CK7), which highlight the luminal cells, and for basal/myoepithelial cell markers like CK 5/6, S-100 and p63 may be done to further demonstrate the dual cell population.2 Complete or adequate local excision is the treatment of choice; however, residual tumor often remains because of the difficulty of surgery at this location, leading to recurrence. Subsequent repeat surgery to completely remove the tumor leads to cure.1,2,6
 
 

Epithelial‐mesenchymal cross talk in cancer behavior (1047.1)

Epithelial‐mesenchymal interaction is important in the morphogenesis, development, wound healing and also in cancer behavior. It is dependent on the range of cytokines, chemokines, growth, proliferating and transforming factors produced by both the counterpartners. Tumor stroma contains, except the mesenchyme, also blood vessels, and the inflammatory cells. We focused our research on most abundant cell component of cancer stroma on cancer‐associated fibroblasts (CAF). They are producers of extracellular components, which are necessary to formation bioactive cancer microenvironment and are able to influence the biology of tumor predominantly the differentiation status of tumor cells and their migratory potential. We have isolated CAF from malignant tumors (squamous cell carcinoma, basal cell carcinoma (BCCF), melanoma, and skin metastasis of breast cancer) and have shown that these CAF are able to influence the differentiation status of co‐cultured cells from normal squamous or breast cancer epithelium. The results were compared with control experiments using normal human dermal fibroblasts, 3T3 mouse fibroblasts, and 3T3 fibroblasts influenced by the fibroblasts prepared from the basal cell carcinoma. Our results demonstrated that expression of luminal marker keratin 8 was influenced only by CAFs prepared from any tested tumors. In contrast, all tested types of fibroblasts showed a strong stimulatory effect on the expression of basal/myoepithelial marker keratin 14. Since keratin 14 is a marker of basal myoepithelial cells and keratin 8 is a marker of luminal cells, these double‐positive cells can be considered for precursor cells with properties close to stem cells. Their presence in clinical samples indeed signals very poor prognosis in cancer‐suffering patiens. In conclusion, our data indicate that CAFs are able to influence the phenotype of a breast cancer cell line and this effect is based on a tumor type‐unspecific mechanism.

Fully Interlocking: A Story of Teamwork among Breast Epithelial Cells

In this issue of Developmental Cell, Forster etal. (2014) show that the basal myoepithelial cell layer directs the final maturation of the adjacent luminal cell sheet during pregnancy. Do all mammary epithelial cells both give and take instructions from others to create the milk production machinery?

Myc is required for β-catenin-mediated mammary stem cell amplification and tumorigenesis

BackgroundBasal-like breast cancer is a heterogeneous disease characterized by the expression of basal cell markers, no estrogen or progesterone receptor expression and a lack of HER2 ov erexpression. Recent studies have linked activation of the Wnt/β-catenin pathway, and its downstream target, Myc, to basal-like breast cancer. Transgenic mice K5ΔNβcat previously generated by our team present a constitutive activation of Wnt/β-catenin signaling in the basal myoepithelial cell layer, resulting in focal mammary hyperplasias that progress to invasive carcinomas. Mammary lesions developed by K5ΔNβcat mice consist essentially of basal epithelial cells that, in contrast to mammary myoepithelium, do not express smooth muscle markers.MethodsMicroarray analysis was used to compare K5ΔNβcat mouse tumors to human breast tumors, mammary cancer cell lines and the tumors developed in other mouse models. Cre-Lox approach was employed to delete Myc from the mammary basal cell layer of K5ΔNβcat mice. Stem cell amplification in K5ΔNβcat mouse mammary epithelium was assessed with 3D-culture and transplantation assays.ResultsHistological and microarray analyses of the mammary lesions of K5ΔNβcat females revealed their high similarity to a subset of basal-like human breast tumors with squamous differentiation. As in human basal-like carcinomas, the Myc pathway appeared to be activated in the mammary lesions of K5ΔNβcat mice. We found that a basal cell population with stem/progenitor characteristics was amplified in K5ΔNβcat mouse preneoplastic glands. Finally, the deletion of Myc from the mammary basal layer of K5ΔNβcat mice not only abolished the regenerative capacity of basal epithelial cells, but, in addition, completely prevented the tumorigenesis.ConclusionsThese results strongly indicate that β-catenin-induced stem cell amplification and tumorigenesis rely ultimately on the Myc pathway activation and reinforce the hypothesis that basal stem/progenitor cells may be at the origin of a subset of basal-like breast tumors.