Normal Myoepithelial Cells Research Articles

9218 Highly Multiplexed Mapping of the Ductal Carcinoma In Situ Ecosystem to Predict Disease Outcome

Abstract Disclosure: G.M. Wilson: None. T.B. Doan: None. B.J. Guild: None. N. Pathmanathan: None. J.D. Graham: None. Mammographic screening programs have resulted in remarkable improvements in breast cancer survival due to early-stage detection. However, this has resulted in a dramatic increase in the detection of non-invasive ductal carcinoma in situ (DCIS), a non-obligate precursor of invasive ductal carcinoma (IDC), which is confined to the ducts and surrounded by a normal myoepithelial cell layer. Many DCIS cases will not progress to IDC within the lifetime of the patient. However, there are limited tools to accurately predict progression, thus virtually all DCIS is surgically removed, and most patients receive adjuvant radiotherapy as it halves recurrence rates. This likely represents overtreatment for a majority of patients. Emerging evidence has revealed that the adjacent microenvironment becomes progressively altered in DCIS and IDC compared to normal tissue, and this is correlated with disease outcome. The objective of this study was to investigate changes to the DCIS microenvironment during disease progression and identify disease states that predict patient outcome. We used immunohistochemistry and imaging mass cytometry with a well-annotated cohort of DCIS, with or without adjacent IDC, to measure a panel of 40 phenotypic and functional markers that allowed us to distinguish the diverse cell populations present in the tissues. We observed striking changes to almost all cell types within the DCIS tissue when comparing pure DCIS cases to DCIS adjacent to IDC. In particular, there were considerable changes to tissue-resident immune populations, with differences observed in the type, location and population size of a variety of immune cell subsets, in IDC-associated DCIS compared with DCIS-only cases. Computational mapping of cell type communities in pure and IDC-adjacent DCIS tissues revealed specific differences that could be used to predict the emergence of invasive disease, informing clinical management of this good prognosis malignancy and potentially avoiding over-treatment. Presentation: 6/3/2024

9218 Highly Multiplexed Mapping Of The Ductal Carcinoma In Situ Ecosystem To Predict Disease Outcome

Abstract Disclosure: G.M. Wilson: None. T.B. Doan: None. B.J. Guild: None. N. Pathmanathan: None. J.D. Graham: None. Mammographic screening programs have resulted in remarkable improvements in breast cancer survival due to early-stage detection. However, this has resulted in a dramatic increase in the detection of non-invasive ductal carcinoma in situ (DCIS), a non-obligate precursor of invasive ductal carcinoma (IDC), which is confined to the ducts and surrounded by a normal myoepithelial cell layer. Many DCIS cases will not progress to IDC within the lifetime of the patient. However, there are limited tools to accurately predict progression, thus virtually all DCIS is surgically removed, and most patients receive adjuvant radiotherapy as it halves recurrence rates. This likely represents overtreatment for a majority of patients. Emerging evidence has revealed that the adjacent microenvironment becomes progressively altered in DCIS and IDC compared to normal tissue, and this is correlated with disease outcome. The objective of this study was to investigate changes to the DCIS microenvironment during disease progression and identify disease states that predict patient outcome. We used immunohistochemistry and imaging mass cytometry with a well-annotated cohort of DCIS, with or without adjacent IDC, to measure a panel of 40 phenotypic and functional markers that allowed us to distinguish the diverse cell populations present in the tissues. We observed striking changes to almost all cell types within the DCIS tissue when comparing pure DCIS cases to DCIS adjacent to IDC. In particular, there were considerable changes to tissue-resident immune populations, with differences observed in the type, location and population size of a variety of immune cell subsets, in IDC-associated DCIS compared with DCIS-only cases. Computational mapping of cell type communities in pure and IDC-adjacent DCIS tissues revealed specific differences that could be used to predict the emergence of invasive disease, informing clinical management of this good prognosis malignancy and potentially avoiding over-treatment. Presentation: 6/3/2024

8770 Highly Multiplexed Mapping of the Ductal Carcinoma In Situ Ecosystem to Predict Disease Outcome

Abstract Disclosure: G.M. Wilson: None. T.B. Doan: None. B.J. Guild: None. N. Pathmanathan: None. J.D. Graham: None. Mammographic screening programs have resulted in remarkable improvements in breast cancer survival due to early-stage detection. However, this has resulted in a dramatic increase in the detection of non-invasive ductal carcinoma in situ (DCIS), a non-obligate precursor of invasive ductal carcinoma (IDC), which is confined to the ducts and surrounded by a normal myoepithelial cell layer. Many DCIS cases will not progress to IDC within the lifetime of the patient. However, there are limited tools to accurately predict progression, thus virtually all DCIS is surgically removed, and most patients receive adjuvant radiotherapy as it halves recurrence rates. This likely represents overtreatment for a majority of patients. Emerging evidence has revealed that the adjacent microenvironment becomes progressively altered in DCIS and IDC compared to normal tissue, and this is correlated with disease outcome. The objective of this study was to investigate changes to the DCIS microenvironment during disease progression and identify disease states that predict patient outcome. We used immunohistochemistry and imaging mass cytometry with a well-annotated cohort of DCIS, with or without adjacent IDC, to measure a panel of 40 phenotypic and functional markers that allowed us to distinguish the diverse cell populations present in the tissues. We observed striking changes to almost all cell types within the DCIS tissue when comparing pure DCIS cases to DCIS adjacent to IDC. In particular, there were considerable changes to tissue-resident immune populations, with differences observed in the type, location and population size of a variety of immune cell subsets, in IDC-associated DCIS compared with DCIS-only cases. Computational mapping of cell type communities in pure and IDC-adjacent DCIS tissues revealed specific differences that could be used to predict the emergence of invasive disease, informing clinical management of this good prognosis malignancy and potentially avoiding over-treatment. Presentation: 6/3/2024

8770 Highly Multiplexed Mapping Of The Ductal Carcinoma In Situ Ecosystem To Predict Disease Outcome

Abstract Disclosure: G.M. Wilson: None. T.B. Doan: None. B.J. Guild: None. N. Pathmanathan: None. J.D. Graham: None. Mammographic screening programs have resulted in remarkable improvements in breast cancer survival due to early-stage detection. However, this has resulted in a dramatic increase in the detection of non-invasive ductal carcinoma in situ (DCIS), a non-obligate precursor of invasive ductal carcinoma (IDC), which is confined to the ducts and surrounded by a normal myoepithelial cell layer. Many DCIS cases will not progress to IDC within the lifetime of the patient. However, there are limited tools to accurately predict progression, thus virtually all DCIS is surgically removed, and most patients receive adjuvant radiotherapy as it halves recurrence rates. This likely represents overtreatment for a majority of patients. Emerging evidence has revealed that the adjacent microenvironment becomes progressively altered in DCIS and IDC compared to normal tissue, and this is correlated with disease outcome. The objective of this study was to investigate changes to the DCIS microenvironment during disease progression and identify disease states that predict patient outcome. We used immunohistochemistry and imaging mass cytometry with a well-annotated cohort of DCIS, with or without adjacent IDC, to measure a panel of 40 phenotypic and functional markers that allowed us to distinguish the diverse cell populations present in the tissues. We observed striking changes to almost all cell types within the DCIS tissue when comparing pure DCIS cases to DCIS adjacent to IDC. In particular, there were considerable changes to tissue-resident immune populations, with differences observed in the type, location and population size of a variety of immune cell subsets, in IDC-associated DCIS compared with DCIS-only cases. Computational mapping of cell type communities in pure and IDC-adjacent DCIS tissues revealed specific differences that could be used to predict the emergence of invasive disease, informing clinical management of this good prognosis malignancy and potentially avoiding over-treatment. Presentation: 6/3/2024

WT1 as a myoepithelial marker: a comparative study of breast, cutaneous, and salivary gland lesions

WT1 immunostain is expressed in various benign and malignant neoplasms, as well as normal myoepithelial cells. WT1 shows differential expression in non-neoplastic, benign, and malignant neoplastic myoepithelial cells of the salivary gland. In this study, WT1 immunostain and other myoepithelial markers were compared to investigate the value of WT1 as a myoepithelial marker, and to delineate the expression profile of WT1 in nonsalivary gland myoepithelial cells. WT1, p63, and calponin immunostains were performed on normal and lesional tissues from the breast (adenosis, sclerosing adenosis, lactating adenoma, nipple adenoma, tubular adenoma, adenomyoepithelioma, and adenoid cystic carcinoma [ACC]), skin (cutaneous mixed tumor, hidradenoma, spiradenoma, and ACC), and salivary gland (pleomorphic adenoma and ACC). The stained slides were digitized and orientated with H&E images and assessed simultaneously using QuPath. A total of 129, 58, and 56 breast, cutaneous, and salivary gland lesions, respectively, were included. There was poor agreement between WT1-p63 and WT1-calponin (κ<0.1) in all organs, with absence of WT1 expression in normal salivary gland myoepithelium and most ACCs. There were no significant differences in WT1 expression in myoepithelial cells in normal breast tissue and benign breast neoplasms. Compared to pleomorphic adenomas, cutaneous mixed tumors showed lower WT1 expression (P<.001). WT1 is a less sensitive myoepithelial marker than calponin and p63. However, its unique pattern of expression in salivary gland primary for pleomorphic adenomas/cutaneous mixed tumor can favor a diagnosis of benign salivary gland tumors, particularly in small biopsy specimens.

Mechanostimulation of breast myoepithelial cells induces functional changes associated with DCIS progression to invasion

Women with ductal carcinoma in situ (DCIS) have an increased risk of progression to invasive breast cancer. Although not all women with DCIS will progress to invasion, all are treated as such, emphasising the need to identify prognostic biomarkers. We have previously shown that altered myoepithelial cells in DCIS predict disease progression and recurrence. By analysing DCIS duct size in sections of human breast tumour samples, we identified an associated upregulation of integrin β6 and an increase in periductal fibronectin deposition with increased DCIS duct size that associated with the progression of DCIS to invasion. Our modelling of the mechanical stretching myoepithelial cells undergo during DCIS progression confirmed the upregulation of integrin β6 and fibronectin expression in isolated primary and cell line models of normal myoepithelial cells. Our studies reveal that this mechanostimulated DCIS myoepithelial cell phenotype enhances invasion in a TGFβ-mediated upregulation of MMP13. Immunohistochemical analysis identified that MMP13 was specifically upregulated in DCIS, and it was associated with progression to invasion. These findings implicate tissue mechanics in altering the myoepithelial cell phenotype in DCIS, and that these alterations may be used to stratify DCIS patients into low and high risk for invasive progression.

Roles of Proteoglycans in the Tumourigenesis and Development of Adenoid Cystic Carcinoma and Pleomorphic Adenoma of the Salivary Gland: A Systematic Review.

Salivary adenoid cystic carcinoma (ACC) and pleomorphic adenoma (PA) are the most common types of salivary gland tumours; the former is malignant and the latter is benign but with features of a border tumour. Proteoglycans (PGs) produced by neoplastic myoepithelial cells are ubiquitous in both types of tumours. However, normal myoepithelial cells of salivary glands do not have the ability to secrete PGs. When the synthesis of PGs is blocked, the pulmonary metastasis and perineural growth of salivary ACC as well as the implanting growth of salivary PA are inhibited, highlighting the important functions of PGs in the tumourigenesis and development of these two tumours. In this review, we summarise literature from the past 40 years, including more recent findings from our laboratory, to clarify the pivotal roles of PGs produced by neoplastic myoepithelial cells in both the histogenesis and biological behaviours of ACC and PA.

TGF-\u03b21 stimulates epithelial\u2013mesenchymal transition and cancer-associated myoepithelial cell during the progression from in situ to invasive breast cancer

BackgroundThe progression of ductal carcinoma in situ (DCIS) into invasive ductal carcinoma (IDC) is prevented by normal breast myoepithelial cells. Studies have suggested that EMT-associated genes were enriched in IDC in contrast to DCIS. This paper explored the relationship and potential mechanism between myoepithelial cells and EMT-associated genes in facilitating the transformation from DCIS to breast cancer.MethodsEMT markers and myoepithelial phenotypic markers in IDC, DCIS, and healthy breast tissue were characterized using immunohistochemical assay. Both in vivo and in vitro models were created to mimic the various cell–cell interactions in the development of invasive breast cancer.ResultsWe found that EMT markers were more abundant in invasive carcinomas than DCIS and adjacent normal breast tissue. Meanwhile, TGF-β1 regulated the morphology of MCF-7 (epithelial cells substitute) migration and EMT markers during the transformation from DCIS to invasive breast cancer. Additionally, TGF-β1 also regulated invasion, migration and cytokines secretion of MDA-MB-231 (myoepithelial cells substitute) and epithelial cells when co-cultured with MCF-7 both in vitro and in vivo.ConclusionsIn conclusion, these findings demonstrated that both EMT phenotypes and cancer-associated myoepithelial cells may have an impact on the development of invasive breast cancer.

PRIMARY INTRAOSSEOUS SOFT TISSUE MYOEPITHELIOMA OF THE MANDIBLE: CASE REPORT AND LITERATURE REVIEW

Introduction Soft tissue myoepithelioma (STM) is a benign tumor composed of spindled and epithelioid cells arranged in various patterns within chondromyxoid stroma. While salivary gland myoepithelioma has been well-recognized as a variant of pleomorphic adenoma, the absence of normal myoepithelial cells within STM may be why STM was only recently-recognized. The occurrence of STM within the bone is rare with only 16 cases reported in the literature, none of which have been reported in the mandible. Case Report A 14-year-old female presented with a lobulated gingival nodule measuring 1.2 × 0.8 cm between teeth #20 and 21. Radiographically, the lesion was a well-circumscribed, non-corticated multilocular radiolucency between the roots of #20 and #21 measuring 1.4 × 1.0. cm, extending from the alveolar crest to close to the root apices. An incisional biopsy was performed. Microscopically, the lesion consisted of a non-encapsulated, multilobular tumor composed of a proliferation of spindle and epithelioid cells within a delicate myxochondroid stroma. Tumor cells were positive for S-100, vimentin, neuron-specific enolase (NSE) and epithelial membrane antigen (EMA) and negative for CAM5.2, AE1/3, SMA, SOX-10, CD57, glial fibrillary acidic protein (GFAP), and p63. Ki-67 labeled less than 5% of the cells. These findings are suggestive of STM, and the bone and soft tissue consultant pathologist concurred with this diagnosis. Discussion The putative cell of origin for this tumor is a stem cell within the soft tissues that differentiates towards a cell with myoepithelial phenotype. Unlike salivary gland myoepithelioma which harbors PLAG1 and HMGA2rearrangements, STM harbors the EWSR1 rearrangement in up to 44% of the cases. Ectomesenchymal chondromyxoid tumor may represent the same entity since it has similar histomorphology and immunohistochemical profile but exhibits EWSR1 rearrangement in only 25% of cases. To our knowledge, this is the first report of a STM occurring in the mandible.

Abstract P5-18-08: Defining molecular signatures to personalise management of patients with early breast cancer

Abstract Background A review of breast screening highlighted the need to reduce overdiagnosis. Ductal Carcinoma In-Situ (DCIS) contributes significantly to this overdiagnosis. Epithelial cells in DCIS are as genetically advanced as those in invasive disease, focusing attention on the tumour microenvironment (ME). A key components of the ME in DCIS is the myoepithelial cell(MEC). These cells lie at the interface of the epithelial and stromal compartments, regulating cell function. We previously have identified changes in the MEC that contribute to tumour progression. Here we investigate the functional and clinical significance of a novel change in MEC phenotype: loss of Galectin-7 (Gal-7) expression. Gal-7 is proposed to play a role in apoptosis. We hypothesise that changes in MEC phenotype in DCIS alter the ME towards a pro-invasive phenotype, and hypothesise that loss of Gal-7 modifies the ME, destabilizes the MEC interface and ultimately may lead to loss of the MEC population through apoptosis. Methods Gal-7 expression and function was investigated in clinical samples and in-vitro model systems, respectively. Gal-7 expression was assessed in a series of pure DCIS samples (low risk model) and DCIS with co-existant invasion (high risk model). Tissue sections were stained for Gal-7 and MEC expression scored on a duct-by-duct basis as positive, heterogeneous or negative. An in-vitro model of normal primary myoepithelial cells isolated from reduction mammoplasty was used to investigate the functional impact of loss of Gal-7. These cells have high endogenous levels of Gal-7. Gal-7 was knocked down using siRNA and apoptosis assessed using cleaved caspase-3. The effect of Gal-7 on MEC layer integrity was assessed using immunofluorescence and adhesion assays. The global impact of loss of Gal-7 was investigated using RNA sequencing. Results In the tissue analysis 1926 DCIS ducts were scored for MEC expression of Gal-7. Significantly more ducts showed loss of Gal-7 in DCIS with co-existant invasion, with pure DCIS showing 388 ducts positive and DCIS with invasion 144 DCIS ducts positive (p=0.0014). Pure DCIS and DCIS with invasion had 99 and 646 negative DCIS ducts respectively (p=0.0002). In model systems of primary MEC, knockdown of Gal-7 resulted in increased expression of cleaved caspase-3, suggesting lower levels of Gal-7 increases apoptosis. In functional assays silencing Gal-7 reduces adhesion to both fibronectin and laminin extracellular matrices (p-value 0.005 and 0.001 respectively) RNA sequencing indicates silencing Gal-7 increases LOX expression - a key regulator of the collagen matrix of the microenvironment. Conclusion Normal MEC strongly express Gal-7. Expression is lost in DCIS, with significantly more frequent loss in DCIS with co-existant invasion, suggesting that loss is associated with a more advanced phenotype. Functional assays indicate that loss of MEC Gal-7 enhances MEC apoptosis, which may be one mechanism by which this interface is lost during progression. Gal-7 negative MEC also show impaired adhesion to matrix proteins and lead to up-regulation of LOX, an enzyme key in promoting tumourigenesis. The incorporation of Gal-7 expression into a risk stratification algorithm has functional evidence and is currently being investigated. Citation Format: Allen N, Allen M, Ahmed K, Gomm J, Nelan R, Nagano A, Chelala C, Gadaleta E, Thorat M, Cuzick J, Jones LJ. Defining molecular signatures to personalise management of patients with early breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-18-08.

Abstract B62: Perturbed myoepithelial cell differentiation in BRCA mutation carriers and in DCIS (ductal carcinoma in situ)

Abstract The normal mammary gland is composed of multiple cell types, including luminal and myoepithelial cells. Myoepithelial cells have also been referred to as natural tumor suppressors due to their inhibitory effect on various neoplastic phenotypes, including tumor cell growth, invasion, and angiogenesis. We have previously determined that DCIS-associated myoepithelial cells lack clonal genetic aberrations, but their DNA methylation and gene expression patterns are distinct from that of normal myoepithelial cells, implying perturbed differentiation. We also showed that myoepithelial cells play key roles in preventing tumor growth and invasive progression in experimental models of DCIS, and in immune escape in breast cancer. These data support the hypothesis that the progressive loss of normal myoepithelial cell function in DCIS is a key step in the transition to invasive breast carcinomas, and assessing myoepithelial markers may identify patients with high risk of progression. However, the mechanisms underlying myoepithelial cell aberrations and eventual loss in DCIS are not well understood. In the present study, we used a combination of genomic profiling of human tissue samples and functional assays in a DCIS model to investigate regulators of normal myoepithelial cells and perturbations of these in BRCA mutation carriers and in DCIS. We first explored cellular heterogeneity of the CD10+ cell population in normal human breast tissues of nulliparous and parous women, from reduction mammoplasties and BRCA1 and BRCA2 mutation carriers undergoing prophylactic mastectomy. We identified two distinct CD10+ cell populations distinguished by the expression of CD44 and characterized their gene expression profiles. We also profiled CD10+ cells from BRCA1 and BRCA2 mutation carriers compared to normal noncarriers and found a clear separation of three distinct groups reflecting germline mutation status. Next we defined the p63 cistrome and the enhancer landscape of normal myoepithelial cells and identified alterations of these due to germline BRCA mutation status. Based on our initial results, we focused in p63 and TCF7 as these two transcription factors appear to be central for establishing myoepithelial cell fate. To address the functional relevance of p63 and TCF7 in myoepithelial cell differentiation, we used the MCFDCIS xenograft model of DCIS. These results support the hypothesis that myoepithelial cells play key roles in invasive progression and have significant impact on our understanding of DCIS progression and the increased breast cancer risk of BRCA mutation carriers. Citation Format: Lina Ding, Ying Su, Muhammad Ekram, Sung Jin Huh, Noga Bloushtain-Qimron, Sibgat Choudhury, William Hines, Jun Yao, Mina Bissell, Kornelia Polyak. Perturbed myoepithelial cell differentiation in BRCA mutation carriers and in DCIS (ductal carcinoma in situ) [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 B62.

Loss of MMP-8 in ductal carcinoma in situ (DCIS)-associated myoepithelial cells contributes to tumour promotion through altered adhesive and proteolytic function

BackgroundNormal myoepithelial cells (MECs) play an important tumour-suppressor role in the breast but display an altered phenotype in ductal carcinoma in situ (DCIS), gaining tumour-promoter functions. Matrix metalloproteinase-8 (MMP-8) is expressed by normal MECs but is lost in DCIS. This study investigated the function of MMP-8 in MECs and the impact of its loss in DCIS.MethodsPrimary normal and DCIS-associated MECs, and normal (N-1089) and DCIS-modified myoepithelial (β6-1089) cell lines, were used to assess MMP-8 expression and function. β6-1089 lacking MMP-8 were transfected with MMP-8 WT and catalytically inactive MMP-8 EA, and MMP-8 in N-1089 MEC was knocked down with siRNA. The effect on adhesion and migration to extracellular matrix (ECM), localisation of α6β4 integrin to hemidesmosomes (HD), TGF-β signalling and gelatinase activity was measured. The effect of altering MEC MMP-8 expression on tumour cell invasion was investigated in 2D and 3D organotypic models.ResultsAssessment of primary cells and MEC lines confirmed expression of MMP-8 in normal MEC and its loss in DCIS-MEC. Over-expression of MMP-8 WT but not MMP-8 EA in β6-1089 cells increased adhesion to ECM proteins and reduced migration. Conversely, knock-down of MMP-8 in N-1089 reduced adhesion and increased migration. Expression of MMP-8 WT in β6-1089 led to greater localisation of α6β4 to HD and reduced retraction fibre formation, this being reversed by MMP-8 knock-down in N-1089. Over-expression of MMP-8 WT reduced TGF-β signalling and gelatinolytic activity. MMP-8 knock-down enhanced TGF-β signalling and gelatinolytic activity, which was reversed by blocking MMP-9 by knock-down or an inhibitor. MMP-8 WT but not MMP-8 EA over-expression in β6-1089 reduced breast cancer cell invasion in 2D and 3D invasion assays, while MMP-8 knock-down in N-1089 enhanced cancer cell invasion. Staining of breast cancer cases for MMP-8 revealed a statistically significant loss of MMP-8 expression in DCIS with invasion versus pure DCIS (p = 0.001).ConclusionsThese data indicate MMP-8 is a vital component of the myoepithelial tumour-suppressor function. It restores MEC interaction with the matrix, opposes TGF-β signalling and MMP-9 proteolysis, which contributes to inhibition of tumour cell invasion. Assessment of MMP-8 expression may help to determine risk of DCIS progression.

The role of Galectin-7 in normal and DCIS-associated myoepithelial cells: predicting progression of DCIS

The role of Galectin-7 in normal and DCIS-associated myoepithelial cells: predicting progression of DCIS

Immunohistochemical expression of myoepithelial markers in adenomyoepithelioma of the breast: a unique paradoxical staining pattern of high-molecular weight cytokeratins.

To determine which immunohistochemical markers are useful for the identification of neoplastic myoepithelial cells in adenomyoepithelioma of the breast, the expression of seven myoepithelial markers (α-smooth muscle actin (α-SMA), calponin, p63, CD10, cytokeratin 5/6, cytokeratin 14, and S-100) was examined in 19 lesions from 16 patients. The lesion consisted of seven spindle and 12 clear cell lesions. For normal myoepithelial cells, α-SMA, calponin, and p63 were significantly more sensitive than cytokeratin 5/6, cytokeratin 14, and S-100. There was no significant difference in the expression of α-SMA, calponin, p63, and CD10 in neoplastic myoepithelial cells of adenomyoepithelioma regardless of spindle or clear cell types. In spindle cell lesions, high-molecular weight cytokeratins (HMWCK; cytokeratin 5/6 and cytokeratin 14) tended to show higher staining scores and S-100 showed lower staining scores than other markers. In clear cell lesions, HMWCK showed significantly lower staining scores than the other five markers. There was no significant difference in staining scores among the other five markers. HMWCK showed a unique paradoxical staining pattern in clear cell lesions, with diffusely positive inner epithelial cells and completely negative outer myoepithelial cells. Although the sensitivity of HMWCK in clear cell lesions is low, with this unique paradoxical staining pattern and relatively high sensitivity in spindle cell lesions, HMWCK could be useful in diagnosing adenomyoepithelioma. In choosing immunohistochemical markers, any of the seven markers are useful, but combining HMWCK and any one of α-SMA, calponin, and p63 would be a good panel for the diagnosis of adenomyoepithelioma.

αvβ6 Expression in myoepithelial cells: a novel marker for predicting DCIS progression with therapeutic potential.

The tumor microenvironment dynamically regulates the progression of cancer. In the breast, a unique component of the microenvironment is the myoepithelial cell. Normal myoepithelial cells act as "natural tumor suppressors"; however, more recent evidence suggests that these cells develop phenotypic changes, which may contribute to loss of tumor suppressor activity. We have shown that myoepithelial cells in a subset of preinvasive ductal carcinoma in situ (DCIS) upregulate expression of the integrin αvβ6, switching on tumor promoter activity through activation of TGFβ and MMP9. This makes the tumor microenvironment more permissive to invasion, seen both in vitro and in vivo. In human tissue samples, increased myoepithelial αvβ6 expression correlated with increased risk of disease progression and recurrence. Current estimates suggest that as many as 50% of DCIS cases will never progress in the patient's lifetime, but there are no markers to predict the outcome of individual cases. The identification of αvβ6 in a subset of DCIS presents a unique way to stratify patients with DCIS into those who may or may not progress to more serious disease. As αvβ6 is not expressed on most normal adult tissues, this finding may also provide novel targets for therapy in this high-risk group.

Abstract C39: Building a novel molecular model for Twist1-induced epithelial dissemination

Abstract Most solid tumors originate from epithelial cells, and their metastasis represents the major cause of death among cancer patients. Because each tumor contains many mutations, it has been difficult to isolate how individual genetic events contribute to a tumor cell's ability to disseminate. Two posited mechanisms for metastasis are loss of cell-cell adhesion and activation of an epithelial-mesenchymal transition (EMT). These molecular models converge on the cell adhesion gene E-cadherin (Cdh1). E-cadherin is frequently mutated in human cancers, and it can also be transcriptionally repressed during EMT. In turn, expression of EMT transcription factors in multiple epithelial cancers has been correlated with increased invasion and metastasis. However, the acute consequences of E-cadherin loss or EMT activator expression in otherwise normal epithelial tissues remain incompletely understood. We therefore utilized a combination of organotypic culture, Cre-lox based gene deletion, inducible expression systems, and time-lapse imaging to test the sufficiency of single genetic perturbations to induce dissemination in primary murine mammary epithelium. E-cadherin deletion induced loss of simple epithelial architecture and inhibited branching morphogenesis in 3D culture and in vivo. Furthermore, E-cadherin loss acutely induced collective invasion past basement membrane. However, E-cadherin- cells remained non-protrusive, adhered to one another, and rarely disseminated. Conversely, expression of the EMT transcription factor Twist1 induced rapid dissemination of individual epithelial cells into the extracellular matrix (ECM), and disseminated cells proliferated to form secondary sites. Surprisingly, Twist1+ cells were observed to have membrane-localized E-cadherin during dissemination and during migration in the ECM. Our data thus provide distinct roles for E-cadherin and Twist1 in the early cellular processes of metastasis. We are currently testing whether shRNA knockdown of E-cadherin synergizes with Twist1 to further enhance dissemination. To date, we have demonstrated the sufficiency of constitutive Twist1 expression to induce dissemination of both major mammary cell types: inner luminal epithelial cells and outer myoepithelial cells. Moreover, we observe abnormal localization of myoepithelial cells and basement membrane to the inside of the tissue. However, it remains unclear why some Twist1-expressing cells disseminate while many others remain in the epithelium. We hypothesize that intrinsic, cell type-specific factors influence disseminative capacity. We next seek to test the sufficiency of Twist1 to induce cell dissemination and secondary site formation in distinct mammary epithelial subpopulations. We have developed mouse models that use cell-type specific promoters to drive Twist1 expression in myoepithelial, luminal progenitor, and differentiated luminal lineages. We anticipate that differences across cell types may help identify the subpopulations within a heterogeneous tumor with the highest metastatic potential. Furthermore, we expect that extrinsic factors, such as normal myoepithelial cells, may restrict dissemination of Twist1-expressing luminal cells. Understanding such modulation of Twist1's phenotypic outcomes could suggest new approaches to suppressing metastasis without necessitating direct inhibition of Twist1, itself an “undruggable” target. Citation Format: Eliah R. Shamir, Phuoc T. Tran, Andrew J. Ewald. Building a novel molecular model for Twist1-induced epithelial dissemination. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C39.

Deregulated Expression of ANXA1 in Human High-Grade Breast Cancers

Background: Breast cancer is a heterogeneous disease comprising a diversity of tumor subtypes. All of them differ from each other resulting in a wide array of clinical pictures, risks of recurrence and response to treatment. In recent years, tumor biomarkers have changed the way breast cancers are diagnosed and treated. In this paper we have sought to investigate the differential expression of ANXA1, a multifunctional calcium binding protein, among various molecular subtypes of breast cancers and in particular triple negative tumors. Methods: ANXA1 was first studied using in-silico analysis on available DNA microarray and RNA sequencing data of human breast tissues. Next we ascertained ANXA1 expression on cell lines and breast carcinoma tissue microarrays along with cognate normal breast tissue. Results: Whereas ANXA1 expression is normally restricted to the normal myoepithelial cell layer it becomes ectopically and aberrantly expressed in tumor cells of a significant minority of aggressive breast cancers. Specifically, we found that ANXA1 expression is severely deregulated in high-grade breast cancers that comprise clinically aggressive tumors such as triple-negative and, to some extent, HER2-positive breast cancers. Conclusion: Our results indicate that ANXA1 is a valuable breast cancer biomarker that can help to segregate and dissect out subsets of high histological grade breast cancers paving the way to a better understanding of breast cancer progression and metastasis.

Abstract P1-05-02: Epithelial-to-epithelial transition precedes collective breast cancer invasion

Abstract BACKGROUND: Invasion represents a fundamental step in tumor progression and is a driving force for metastasis and cancer-related mortality. Invasion has been studied largely in the context of single cells, but in vivo, most tumor cells are not found in isolation, but rather are found as ensembles of cells with substantial molecular heterogeneity. It remains poorly understood in this context which cells are invasive, how these cells arise or how these cells interact collectively to accomplish invasion. RESULTS: We developed an ex-vivo 3D culture assay enabling us to distinguish invasive from non-invasive cells and to interrogate their molecular phenotype. In two mouse models of luminal breast cancer, we find that invasive cells express markers of basal epithelium including cytokeratin K14, p63 and P-cadherin. Invasive K14+ cells were also molecularly and functionally distinct from normal myoepithelial cells and co-expressed luminal K8 and E-cadherin. In vivo, K14+ cells were localized to tumor-stromal interfaces in the primary tumor and in lung micro and macro-metastases. Using a molecular biosensor and time-lapse microscopy, we observed that K14+ invasive cells were derived from K14-negative luminal tumor cells by direct phenotypic conversion. Interestingly, the capacity of luminal tumor cells to acquire basal markers varied by model, with few K14+ cells in MMTV-Her2 and many K14+ cells in MMTV-PyMT mice, correlating with the known in vivo kinetics of tumor progression in these models. To block this epithelial to epithelial transition, we tested the activity of an inhibitor of ribosome S6 kinase (RSK), which has been shown previously to inhibit myoepithelial differentiation in normal mammary epithelium. RSK inhibition caused relative depletion of K14+ tumor cells in 3D culture and abrogation of invasion. Finally, our assay revealed that in fresh primary tissue from human ER+ luminal breast cancers, invasive cells also expressed basal epithelial markers. CONCLUSIONS: Taken together, these data suggest that an epithelial-to-epithelial transition precedes collective invasion in luminal breast cancers. Because we observe K14+ leader cells independent of tumor estrogen receptor status, these data may be generally applicable to breast cancer invasion across subtypes. Because many epithelial tissues have basal populations, K14+ tumor cells may also lead invasion in other cancers. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-02.

Use of CD10 as a marker of canine mammary myoepithelial cells

CD10 is an important cell marker in the diagnosis of acute lymphoblastic leukaemia and of breast myoepithelial (ME) cells in humans. The objective of this study was to assess the value of CD10 as a marker of canine ME cells using immunohistochemistry on routinely processed normal, dysplastic and neoplastic mammary tissue. Five different CD10 positive cell types were identified on the basis of cell morphology, pattern of immunoreactivity, and on the co-expression of additional cell lineage-specific markers. Type 1 cells were typical fusiform cells with a ME cell phenotype (calponin- and cytokeratin [CK] 14-positive, CK8/18-negative). Type 2 cells were typical or atypical polyhedral cells with a luminal epithelial (LE) cell phenotype (calponin- and CK14-negative, CK8/18-positive). Type 3 cells had a type 1 phenotype with variable morphology, and type 4 were atypical neoplastic cells with a mixed ME/LE phenotype. Type 5 cells were typical fusiform cells with a stromal phenotype. Type 1 cells were considered normal ME cells and were found in all sample types; type 2 cells were considered normal or neoplastic LE cells and were also found in all sample types; types 3 and 4 cells were restricted to tumour samples and to malignant tumours, respectively, and type 5 cells were found in all sample types, although predominantly in neoplastic tissue. The findings indicate that the CD10 antigen is a sensitive (although not specific) marker of canine ME cells in normal, dysplastic and neoplastic mammary tissue. Differences in the distribution and staining intensity of CD10-positive cells suggest a number of potential roles for this protein in the pathogenesis of canine mammary neoplasia.

Abstract 1060: A functional and proteomics analysis of AKT1(E17K) in human mammary epithelial cells of luminal and myoepithelial cells of origin

Abstract The normal breast epithelium, from which most breast cancers arise, is composed of an inner layer of luminal cells and an outer layer of myoepithelial (basal) cells. A cell culture method recently described allows direct comparison of two genetically matched cells derived from normal myoepithelial and luminal epithelial cell types termed HME and BPE cells respectively (Ince et al, Cancer Cell 2007). Transformation of these cells (subsequently termed HMLER and BPLER) formed tumor xenografts exhibiting major differences in histopathology, tumorigenicity, and metastatic behavior. The non-receptor serine/threonine kinase AKT/PKB is one of the most commonly deregulated pathways in human breast cancer. In this regard we performed functional and proteomics assays to compare the role of wild-type (wt) AKT1 versus the naturally occurring AKT1 mutant (E17K), in human breast cancer. We demonstrated that BPE and HME cells expressing AKT1(E17K) have constitutive plasma membrane localization and hyper-phosphorylation at serine-473 and threonine-308, demonstrating that activation of E17K is growth-factor independent. However, additional studies suggest that HME and BPE cells expressing wt and mutant AKT1 have differing activation kinetics, the reasons of which are unknown. Using a sulforhodamine B and transwell migration assays we demonstrated that E17K inhibits cell growth and migration respectively in BPE and HME cells. However, E17K enhances chemoresistance compared with wt AKT1 function. Interestingly, while E17K inhibits migration in HMLER cells it leads to increased cellular migration in BPLER cells. In order to investigate the molecular basis of AKT1(E17K) function in the different epithelial cell lineages, we performed differential proteomics analysis using liquid chromatography (LC) mass spectrometry (MS) analysis. Although several LCMS methods allowing the characterization and quantification of proteins have been developed we chose two unique styles using 2-dimensional reverse phase reverse phase LC for both. The first method is label-free quantitation utilizing spectral counting. This technique counts the number of spectra identified for a given peptide in each sample and results integrated for all peptides detected for a given protein. The second being iTRAQ, an isobaric labeling technique that uses isotope coded covalent tags for relative quantitation. Using these approaches we identified several hundred differentially expressed proteins between wt and mutant AKT1 and between HME and BPE cells. The proteins from our analysis converged on various cellular functions including pathways underlying cellular migration. Overall the data leads us towards understanding the molecular mechanisms that discriminate between mutant and wt AKT1 function and demonstrates the importance of cellular context in determining genotypic effects of oncogenic mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1060. doi:10.1158/1538-7445.AM2011-1060