Do Myoepithelial Cells Hold the Key for Breast Tumor Progression?

Mammary myoepithelial cells have been a neglected facet of breast cancer biology, largely ignored since they have been considered to be less important for tumorigenesis than luminal epithelial cells from which most of breast carcinomas are thought to arise. In recent years as our knowledge of stem cell biology and the cellular microenvironment has been increasing, myoepithelial cells are slowly starting to gain more attention. Emerging data raise the hypothesis whether myoepithelial cells play a key role in breast tumor progression by regulating the in situ to invasive carcinoma transition and that myoepithelial cells are part of the mammary stem cell niche. Paracrine interactions between myoepithelial and luminal epithelial cells are known to be important for regulation of cell cycle progression, establishing epithelial cell polarity, and inhibiting cell migration and invasion. Based on these functions, normal mammary myoepithelial cells have been called “natural tumor suppressors.” However, during tumor progression myoepithelial cells seem to loose these properties, and eventually this cell population diminishes as tumors become invasive. Better understanding of myoepithelial cell function and their role in tumor progression may lead to their exploitation for cancer therapeutic and preventative measures.     

Myoepithelial Cells in the Control of Mammary Development and Tumorigenesis: Data From Genetically Modified Mice

Until recently, myoepithelial cells—the second major cell population in the mammary epithelium—were not considered to play an important role in the morphogenetic events during gland development. Mouse mutants with changes in the gene expression pattern characteristic of the basal myoepithelial cell layer have been generated and used to show that these cells influence the proliferation, survival and differentiation of luminal cells, modulate stromal–epithelial interactions and actively participate in mammary morphogenesis. Various cellular and molecular mechanisms may underlie the observed phenotypes. These include an unbalanced expression of matrix degrading metalloproteinases (MMPs) and their inhibitors, leading to changes in the composition and organization of the (extracellular matrix) ECM, the production of soluble growth factors affecting stromal and epithelial cell growth and differentiation and direct signaling through cell–cell contacts between the myoepithelial and luminal cell layers.     

Maintenance of Cell Type Diversification in the Human Breast

Recent genome-wide expression analysis of breast cancer has brought new life to the classical idea of tumors as caricatures of the process of tissue renewal as envisioned by Pierce and Speers (Cancer Res 1988;48:1996–2004) more than a decade ago. The search for a cancer founder cell or different cancer founder cells is only possible if a hierarchy of differentiation has been established for the particular tissue in question. In the human breast, the luminal epithelial and myoepithelial lineages have been characterized extensively in situ by increasingly elaborate panel of markers, and methods to isolate, culture, and clone different subpopulations have improved dramatically. Comparisons have been made with the mouse mammary gland in physiological three-dimensional culture assays of morphogenesis, and the plasticity of breast epithelial cells has been challenged by immortalization and transformation. As a result of these efforts, several candidate progenitor cells have been proposed independently of each other, and some of their features have been compared. This research has all been done to better understand breast tissue homeostasis, cell-type diversification in general and breast cancer evolution in particular. The present review discusses the current approaches to address these issues and the measures taken to unravel and maintain cell type diversification for further investigation.     

Hormone-Responsive Model of Primary Human Breast Epithelium

Retention of hormone responsiveness in primary culture models of human breast is essential for studies aimed at understanding the mechanisms of action of the ovarian hormones in the human breast. In this chapter we describe the development of a culture model of primary human breast that retains critical features of the tissue in vivo. We find that primary normal human breast tissue in embedded culture recapitulates the morphology, cell lineages, functional gene expression characteristics and estrogen and progesterone receptor responsiveness of the breast in vivo. The ratio of luminal to myoepithelial cells after culture recapitulates that observed in the uncultured tissue, highlighting the fact that progenitor cells capable of giving rise to both epithelial cell lineages are retained in this model system. By contrast, primary cells placed into monolayer culture, even for a single passage, lose bipotent progenitors, and the myoepithelial lineage predominates, demonstrating the rapidity with which phenotypic changes and selection occur in normal breast cells, unless cultured under conditions that prevent this outcome. Primary matrix-embedded culture of normal human breast cells provides researchers with a new opportunity to understand ovarian hormone action in the human breast.     

The 3-layered ductal epithelium in gynecomastia

Gynecomastia is the most common abnormality in the male breast and has been associated with male breast cancer, but whether there is an etiological role remains unknown. In the present study we conducted an immunohistochemical investigation to further characterize gynecomastia. A total of 46 cases of gynecomastia were immunohistochemically stained on tissue microarrays for estrogen receptor (ER), progesterone receptor, HER2, androgen receptor, cytokeratins (CK5, CK14, CK7, and CK8/18), p63, E-cadherin, BRST2, cyclin D1, Bcl-2, p53, p16, p21, and Ki67. In addition, 8 cases of male ductal carcinoma in situ and normal breast tissue obtained from autopsies (n=10) and adjacent to male breast cancer (n=5) were studied. Normal ductal male breast epithelial cells were very often ER and Bcl-2 positive (>69%), and progesterone receptor and androgen receptor expression was also common (>39%). Gynecomastia showed a consistent 3-layered pattern: 1 myoepithelial and 2 epithelial cell layers with a distinctive immunohistochemical staining pattern. The intermediate luminal layer, consisting of vertically oriented cuboidal-to-columnar cells, is hormone receptor positive and expresses Bcl-2 and cyclin D1. The inner luminal layer is composed of smaller cells expressing CK5 and often CK14 but is usually negative for hormone receptors and Bcl-2. Male ductal carcinoma in situ was consistently ER positive and CK5/CK14 negative. In conclusion, for the first time we describe the 3-layered ductal epithelium in gynecomastia, which has a distinctive immunohistochemical profile. These results indicate that different cellular compartments exist in gynecomastia, and therefore gynecomastia does not seem to be an obligate precursor lesion of male breast cancer.     

Myoepithelial Cells: Pathology,Cell Separation and Markers of Myoepithelial Differentiation

Until recently the myoepithelial cell has been studied relatively little in terms of its role in breast cancer. A number of malignancies showing myoepithelial differentiation have been reported in the literature, although they are still thought to be relatively rare and only limited studies are published. As a result of recent expression profiling experiments, one type of tumor with myoepithelial features, the so-called ‘basal’ breast cancer, has received a renewed interest, although it has been known to pathologists for more than two decades. These tumors, which express markers of both luminal and myoepithelial cells, are now being studied using antibodies against some new molecules that have emerged from studies of sorted normal luminal and myoepithelial cells. These immunohistochemical data, combined with genomic studies, may lead to better identification and management of patients with ‘basal’ tumors.     

Myoepithelial cell cocktail (p63+SMA) for the evaluation of sclerosing breast lesions

Sclerosing breast lesions may sometimes mimic the appearance of infiltrating carcinoma due to the entrapment of ductular structures in a fibrotic core. The immunohistochemical detection of the outer myoepithelial cell layer that is indicative of a non-infiltrating lesion is a valuable clue for the diagnosis of such ambiguous cases. The myoepithelial cell markers smooth muscle actin (SMA) and p63 are most commonly used since their specificity and sensitivity are well established. However, recent studies have indicated that some morphologically distinct myoepithelial cells fail to stain for SMA and that p63 positivity can be rarely expressed by a subset of malignant epithelial cells. Moreover, SMA can also be positive in stromal myofibroblastic cells and normal vessels that can be found close to the entrapped ductules and might be erroneously interpreted as myoepithelial cells. In this study, we used a double-immunolabeling technique combining both SMA and p63 antibodies (myoepithelial cell cocktail), in order to investigate whether this technique is advantageous over either marker used alone, in diagnosing sclerosing breast lesions. Our results indicate that p63 alone is not a useful myoepithelial cell marker if applied in large sclerosing breast lesions, however, in smaller lesions it is still of high value. On the contrary, SMA proved significantly useful in the evaluation of myoepithelial cells in larger but not in smaller complex sclerosing lesions. The myoepithelial cell cocktail has a staining sensitivity identical to that of SMA. Nevertheless, in a certain number of cases the cocktail might be useful in differentiating myoepithelial cells from stromal myofibroblasts or vascular smooth muscle cells due to the false impression of a higher staining intensity of the cocktail resulting from the expression of both nuclear and cytoplasmic/membranous antibodies that occupy a wider area of the cell under control.     

Is it ductal carcinoma in situ with microinvasion or “Ductogenesis”? The role of myoepithelial cell markers

Mammary myoepithelial cells have been under‐recognized for many years since they were considered less important in breast cancer tumorigenesis compared to luminal epithelial cells. However, in recent years with advances in genomics, cell biology, and research in breast cancer microenvironment, more emphasis has been placed on better understanding of the role that myoepithelial cells play in breast cancer progression. As the result, it has been recognized that the presence or absence of myoepithelial cells play a critical role in the assessment of tumor invasion in diagnostic breast pathology. In addition, advances in screening mammography and breast imaging has resulted in increased detection of ductal carcinoma in situ and consequently more diagnosis of ductal carcinoma in situ with microinvasion. In the present review, we discuss the characteristics of myoepithelial cells, their genomic markers and their role in the accurate diagnosis of ductal carcinoma in situ with microinvasion. We also share our experience with reporting of various morphologic features of ductal carcinoma in situ that may mimic microinvasion and introduce the term of ductogenesis.     

Myoepithelial Cells: Autocrine and Paracrine Suppressors of Breast Cancer Progression

Host cellular paracrine regulation of tumor progression is an important determinant of tumor biology but one cell that has been ignored in this regulation is the myoepithelial cell. Myoepithelial cells surround normal ducts and precancerous lesions, especially of the breast and form a natural border separating proliferating epithelial cells from proliferating endothelial cells (angiogenesis). Myoepithelial cells may thus negatively regulate tumor invasion and metastasis. Whereas epithelial cells are susceptible targets for transforming events, myoepithelial cells are resistant. Therefore, it can be said that myoepithelial cells function as both autocrine as well as paracrine tumor suppressors. Our laboratory has found that myoepithelial cells secrete a number of suppressor molecules including high amounts of diverse proteinase inhibitors and angiogenic inhibitors but low amounts of proteinases and angiogenic factors compared to common malignant cell lines. This observation has been made in vitro, in mice, and in humans and suggests that myoepithelial cells exert pleiotropic suppressive effects on tumor progression. The gene expression profile of myoepithelial cells may explain the pronounced anti-invasive and anti-angiogenic effects of myoepithelial cells on carcinoma cells and may also account for the reduced malignancy of myoepithelial tumors, which are devoid of appreciable angiogenesis and invasive behavior.     

Characterization of epimyoepithelial islands in benign lymphoepithelial lesions of major salivary gland: an immunohistochemical and ultrastructural study

Knowledge of the processes leading to the development of epimyoepithelial islands bears on histogenetic and morphogentic processes in salivary gland tumors. Immunohistochemical and ultrastructural investigations of the cellular composition of epimyoepithelial islands were carried out on three examples of benign lymphoepithelial lesions with varying histologic features. The monoclonal anti-keratin antibody 312C8-1, which specifically decorates myoepithelial cells of the normal salivary gland, also stains the myoepithelial cells surrounding residual acini and intercalated ducts in benign lymphoepithelial lesions and the cell population of epimyoepithelial islands, with the exception of persisting luminal epithelial cells. Ultrastructurally, the myoepithelial cells of involuting acini and ducts and the modified myoepithelial cells of epimyoepithelial islands, identified in both locations by the monoclonal antibody 312C8-1, show an increasing complement of tonofilament bundles. In addition, persisting lumens (often distended with lymphocytes) and definite luminal epithelial cells can be seen in electron micrographs of some epimyoepithelial islands. The designation for this characteristic epithelial feature of benign lymphoepithelial lesions is therefore appropriate.     

Use of Three-Dimensional Basement Membrane Cultures to Model Oncogene-Induced Changes in Mammary Epithelial Morphogenesis

The development of breast carcinomas involves a complex set of phenotypic alterations in breast epithelial cells and the surrounding microenvironment. While traditional transformation assays provide models for investigating certain aspects of the cellular processes associated with tumor initiation and progression, they do not model alterations in tissue architecture that are critically involved in tumor development. In this review, we provide examples of how three-dimensional (3D) cell culture models can be utilized to dissect the pathways involved in the development of mammary epithelial structures and to elucidate the mechanisms responsible for oncogene-induced phenotypic alterations in epithelial behavior and architecture. Many normal mammary epithelial cell lines undergo a stereotypic morphogenetic process when grown in the presence of exogenous matrix proteins. This 3D morphogenesis culminates in the formation of well-organized, polarized spheroids, and/or tubules that are highly reminiscent of normal glandular architecture. In contrast, transformed cell lines isolated from mammary tumors exhibit significant deviations from normal epithelial behavior in 3D culture. We describe the use of 3D models as a method for both reconstructing and deconstructing the cell biological and biochemical events involved in mammary neoplasia.These authors contributed equally to this review.     

Culture requirements of prostatic epithelial cell lines for acinar morphogenesis and lumen formation in vitro: role of extracellular calcium

BACKGROUND: Three-dimensional (3D) culture of benign prostatic epithelial cell lines can recapitulate acinar morphogenesis in vitro, but the broad applicability of this approach has not been described. The present studies examine the culture conditions important for prostatic acinar morphogenesis in vitro and the role of extracellular calcium in this process. METHODS: With optimized culture conditions, RWPE-1, pRNS-1-1, PZ-HPV-7, PNT1A, BPH-1, and PrEC were analyzed for their ability to undergo acinar morphogenesis in 3D culture and by immunoblotting. RWPE-1 cells were further examined for the effects of calcium on morphology, E-cadherin membrane localization and multicellular layering in 2D culture and for acinar morphogenesis, luminal apoptosis, and luminal filling in 3D. RESULTS: Cell lines grown in low-calcium medium have the ability to form acinar structures with lumens, which correlates with E-cadherin expression, but low calcium is not required for this process. Adding CaCl(2) to the medium strongly inhibits lumen formation, luminal apoptosis and induces luminal filling, and luminal filling is blocked by an interfering antibody. CONCLUSIONS: Optimized medium composition allows nearly all seeded RWPE-1 cells to undergo acinar morphogenesis, forming consistent structures representative of normal adult prostate glands. Low-calcium-containing medium appears selective for cells capable of undergoing acinar morphogenesis in vitro, and branching and luminal space within the acini are strongly influenced by extracellular calcium levels, likely through the actions of E-cadherin. These results provide important information about a relevant in vitro model with which to study prostate development and carcinogenesis and highlight the importance of extracellular calcium in regulating 3D morphology.     

Morphologic and molecular evolutionary pathways of low nuclear grade invasive breast cancers and their putative precursor lesions: further evidence to support the concept of low nuclear grade breast neoplasia family

We have previously provided evidence showing an association between some precursor lesions with low nuclear grade breast carcinomas (LNGBCs). In this study, further immunophenotypic support to our proposed route of pathogenesis of LNGBC and their precursor lesions was provided. Precursor lesions including columnar cell lesions, atypical ductal hyperplasia, ductal carcinoma in situ, usual epithelial hyperplasia, and lobular neoplasia were compared with matching "morphologically normal" terminal lobular duct units and matching invasive carcinoma. The epithelial cells in the putative precursor flat epithelial atypia, atypical ductal hyperplasia, lobular neoplasia, ductal carcinoma in situ lesions, and their coexisting LNGBC were negative for basal and myoepithelial markers, but positive for CK19/18/8, estrogen receptor (ER)-alpha, Bcl-2, and cyclin D1. The ER-alpha/ER-beta expression ratio increased during carcinogenesis, as did expression of cyclin D1 and Bcl-2. p53 immunopositivity was found 3% in LNGBC versus 43% in high nuclear grade breast carcinoma (HNGBC), whereas ataxia telangiectasia mutated expression was absent or reduced in 22% of LNGBC versus 53% of HNGBC cases. In summary, our findings support the concept that flat epithelial atypia is the earliest morphologically identifiable nonobligate precursor lesion of LNGBC. These may represent a family of precursor, in situ and invasive neoplastic lesions belonging to the luminal "A" subclass of breast cancer. The balance between ER-alpha and ER-beta expression may be important in driving cyclin D-1 and Bcl-2 expression. Ataxia telangiectasia mutated may be one of the alternative regulatory mechanisms to TP53 mutation or dysfunction in low-grade and high-grade breast carcinoma. Our findings support the concept that progression of LNGBC to HNGBC (basal-like or HER2+) phenotype is an unlikely biologic phenomenon.     

Altered expression of epidermal growth factor receptor in non-involved tissue of cancer-containing breasts

Previous studies have identified functional differences in non-involved breast tissue from cancer-containing breasts. This study has examined the expression of epidermal growth factor (EGFR) protein and mRNA in the non-involved breast of over 100 cancer-containing breasts and compared these with the same number of normal breast tissues from age-matched women with no history of breast cancer. Immunohistochemistry with EGFR1 antibody applied to frozen sections was used for the detection of protein, and in-situ hybridization using a digoxigenin-labelled oligonucleotide probe was the method for detecting mRNA. EGFR protein was detected in myoepithelial cells and to a lesser extent in epithelial cells, where it was predominantly basal or baso-lateral. There was a significant difference in the extent of staining in ducts and lobules between non-involved tissue from cancer-containing breasts and age-matched normal breasts, it being significantly greater in the latter (P<0.001). Labelling for EGFR mRNA was greater and more consistent in myoepithelial cells than epithelial cells overall. Differences were found for intensity of labeling, with it being significantly greater for normal breast tissue (P<0.001) than non-involved tissue from cancer-containing breasts. There is reduced EGFR expression in normal breast tissue from cancer-containing breasts when compared to age-matched breast tissue from women with no history of breast cancer. The mechanisms underlying this are unclear but in previous studies we have identified alterations in myoepithelial cells in cancer-containing breasts and the present findings may represent altered myoepithelial cell function.     

Epithelial Progenitors in the Normal Human Mammary Gland

The human mammary gland is organized developmentally as a hierarchy of progenitor cells that become progressively restricted in their proliferative abilities and lineage options. Three types of human mammary epithelial cell progenitors are now identified. The first is thought to be a luminal-restricted progenitor; in vitro under conditions that support both luminal and myoepithelial cell differentiation, this cell produces clones of differentiating daughter cells that are exclusively positive for markers characteristic of luminal cells produced in vivo (i.e., keratins 8/18 and 19, epithelial cell adhesion molecule [EpCAM] and MUC1). The second type is a bipotent progenitor. It is identified by its ability to produce mixed colonies in single cell assays. These colonies contain a central core of cells expressing luminal markers surrounded by cells with a morphology and markers (e.g., keratin 14⁺) characteristic of myoepithelial cells. Serial passage in vitro of an enriched population of bipotent progenitors promotes the expansion of a third type of progenitor that is thought to be myoepithelial-restricted because it only produces cells with myoepithelial features. Luminal-restricted and bipotent progenitors can prospectively be isolated as distinct subpopulations from freshly dissociated suspensions of normal human mammary cells. Both are distinguished from many other cell types in mammary tissue by their expression of EpCAM and CD49f (6 integrin). They are distinguished from each other by their differential expression of MUC1, which is expressed at much higher levels on the luminal progenitors. To relate the role of these progenitors to the generation of the three-dimensional tubuloalveolar structure of the mammary tree produced in vivo, we propose a model in which the commitment to the luminal versus the myoepithelial lineage may play a determining role in the generation of alveoli and ducts.     

Matrix Metalloproteinase-Induced Epithelial-Mesenchymal Transition in Breast Cancer

Matrix metalloproteinases (MMPs) degrade and modify the extracellular matrix (ECM) as well as cell-ECM and cell-cell contacts, facilitating detachment of epithelial cells from the surrounding tissue. MMPs play key functions in embryonic development and mammary gland branching morphogenesis, but they are also upregulated in breast cancer, where they stimulate tumorigenesis, cancer cell invasion and metastasis. MMPs have been investigated as potential targets for cancer therapy, but clinical trials using broad-spectrum MMP inhibitors yielded disappointing results, due in part to lack of specificity toward individual MMPs and specific stages of tumor development. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells take on the characteristics of invasive mesenchymal cells, and activation of EMT has been implicated in tumor progression. Recent findings have implicated MMPs as promoters and mediators of developmental and pathogenic EMT processes in the breast. In this review, we will summarize recent studies showing how MMPs activate EMT in mammary gland development and in breast cancer, and how MMPs mediate breast cancer cell motility, invasion, and EMT-driven breast cancer progression. We also suggest approaches to inhibit these MMP-mediated malignant processes for therapeutic benefit.     

A Double-Clear Variant of Epithelial-Myoepithelial Carcinoma of the Parotid Gland

The hallmark of the histology of epithelial-myoepithelial carcinoma (EMC) is the presence of a regular repetitive mixture of bilayered duct-like structures with an outer layer of myoepithelial cells and inner ductal epithelial cells. Clear cell change in the myoepithelial component is common, but clearing of both cell types, giving an impression of a monocellular neoplasm, is rare. A parotid biopsy was received from an 83-year-old male and subject to routine histologic processing for conventional staining and immunohistochemistry. The encapsulated tumour was composed of sheets of PAS/diastase negative clear cells, separated by fibrous septae. The clear myoepithelial cells were positive for S-100 protein, SMA, and p63 and negative for CK19 and surrounded CK19-positive luminal cells. It is important to utilise immunohistochemistry to differentiate this tumour from others with a similar histologic pattern. Information about the behaviour of the double-clear EMC is limited since there are few cases reported.     

Immunohistochemical distinction of invasive from noninvasive breast lesions: a comparative study of p63 versus calponin and smooth muscle myosin heavy chain

Identification of myoepithelial cells using antibodies to cytoskeletal proteins, such as smooth muscle myosin heavy chain (SMM-HC) and calponin, can play an important role in distinguishing invasive carcinoma from its histologic mimics. However, antibodies to these proteins may also cross-react with stromal myofibroblasts and vascular smooth muscle cells. It has recently been demonstrated that myoepithelial cells express the nuclear protein, p63, a member of the p53 gene family. We compared the patterns of reactivity of antibodies with p63, calponin, and SMM-HC on 85 breast lesions, including 11 cases of sclerosing adenosis, 33 cases of ductal carcinoma in situ, including 10 that showed microinvasion, 6 cases of lobular carcinoma in situ, and 35 cases of infiltrating ductal carcinoma. All three antibodies were positive on the vast majority of myoepithelial cells in all cases. A small minority of cases showed focal gaps in the revealed myoepithelial cell layer, reflected in discontinuous positive immunostaining around noninvasive epithelial nests (including ductal carcinoma in situ). No case showed p63 expression by myofibroblasts or vascular smooth muscle cells, whereas myofibroblasts expressed, in 8% and 76% of cases, SMM-HC and calponin, respectively. Although no tumor cell reactivity was noted with antibodies to calponin or SMM-HC, tumor cells in 11% of cases showed at least focal p63 expression. And although antibodies to p63 offer excellent sensitivity and increased specificity for myoepithelial detection relative to antibodies to calponin and SMM-HC, they have the following diagnostic limitations: 1) they occasionally demonstrate an apparently discontinuous myoepithelial layer, particularly around ductal carcinoma in situ, and 2) they react with a small but significant subset of breast carcinoma tumor cells. p63 may represent a myoepithelial marker that can complement or replace SMM-HC and/or calponin in the analysis of difficult breast lesions.