Epithelial-mesenchymal transition increases during the progression of in situ to invasive basal-like breast cancer

Summary

Epithelial-mesenchymal transition (EMT) is known to play an important role in breast cancer invasion and metastatic progression. However, the pattern of expression of EMT markers in the progression from in situ to invasive breast carcinoma is not clear. To investigate this, we performed immunohistochemical analyses of EMT markers (expression of vimentin, smooth muscle actin, osteonectin, and N-cadherin; loss of E-cadherin; alteration of β-catenin), breast cancer stem cell (CSC) markers (CD44⁺/CD24⁻, ALDH1), and CD146, an EMT inducer, in invasive carcinomas and ductal carcinoma in situ (DCIS) of the breast. Expression of EMT markers was closely associated with the basal-like subtype and CSC phenotype in invasive carcinoma but not in pure DCIS, except for vimentin. The expression of smooth muscle actin and N-cadherin, loss of E-cadherin, and alteration of β-catenin were significantly higher in invasive carcinomas than in pure DCIS (P = .015, P = .029, P = .001, and P = .007, respectively). Subgroup analyses revealed greater loss of E-cadherin and alteration of β-catenin in invasive carcinoma than in pure DCIS in basal-like subtype (P = .001) but not in non–basal-like subtypes. Moreover, expression of EMT markers and CD146 was higher in the invasive than in the DCIS component of basal-like cancers. Our study confirmed that EMT is an intrinsic characteristic of basal-like subtype and is associated with CSC phenotype. Furthermore, we showed higher expression of EMT markers in invasive carcinomas than in pure DCIS, especially in basal-like subtype, and in the invasive component of basal-like breast cancers, suggesting that EMT may be involved in the progression from in situ to invasive basal-like breast cancers.

Introduction

Epithelial-mesenchymal transition (EMT) is a developmental process during which epithelial cells lose epithelial characteristics and acquire mesenchymal properties by disassembly of cell-cell junctions, loss of cell polarity, and reorganization of the cytoskeleton, thereby acquiring increased motility [1], [2]. It also occurs during wound healing, tissue regeneration, fibrosis, and cancer progression [1]. Because EMT endows cancer cells with migratory and invasive properties, it is implicated in tumor invasion and metastatic dissemination [1], [3], [4]. Recently, it has been reported that induction of EMT in immortalized human mammary epithelial cells resulted in the acquisition of cancer stem cell (CSC) properties [5], suggesting a direct link between the EMT and CSCs.

In breast cancer, basal-like subtype, which is a highly aggressive type of breast cancer associated with a poor clinical outcome [6], [7], has been linked to EMT. The associations of basal-like breast cancers with increased expression of mesenchymal markers including vimentin, smooth muscle actin (SMA), osteonectin, and N-cadherin, as well as reduced expression of epithelial markers such as E-cadherin, β-catenin, and cytokeratin, has been described in breast cancer cell lines and clinical samples [8], [9], [10], [11]. There is also a relationship between basal-like breast cancers and breast CSCs. Breast cancer cell lines with basal/mesenchymal phenotype were found to be composed of a high proportion of CD44⁺/CD24⁻ cells, putative breast CSCs [12]. We also reported that basal-like breast cancers are enriched with breast CSCs, CD44⁺/CD24⁻ cells, and ALDH1⁺ cells using breast cancer tissue samples [13]. Thus, there seems to be a close relationship between EMT, CSC phenotype, and the basal-like subtype of breast cancer.

Ductal carcinoma in situ (DCIS) is a precursor of invasive breast cancer characterized by proliferation of tumor cells within the ductal-lobular system but not extending through the basement membrane. Although the mechanisms of progression from in situ to invasive carcinoma are not well understood, it is thought to be a complex process, depending on both tumor cell properties and tumor microenvironment [14], [15], [16], [17]. Because EMT promotes tumor invasion by facilitating tumor cells to escape from the rigid constraints of the surrounding tissue architecture, such as basement membrane, it is expected to play an important role in the progression of in situ to invasive carcinoma. Recently, gene expression profiling revealed that the genes associated with EMT were enriched in invasive cancer relative to pure DCIS [18]. However, there are few studies of the expression of EMT markers in pure DCIS and invasive carcinoma.

In this study, we investigated the expression of EMT markers (expression of vimentin, SMA, osteonectin, and N-cadherin; loss of E-cadherin; alteration of β-catenin) and breast CSC markers (ALDH1 and CD44⁺/CD24⁻) in invasive carcinomas and pure DCIS using tissue microarrays to correlate the expression of EMT markers with molecular subtype, CSC phenotype, and histologic stage. We also tried to establish whether the expression of EMT markers is intrinsic characteristics of basal-like breast cancer and, hence, is also found in basal-like DCIS, which is thought to be a precursor of invasive basal-like breast cancer [19], [20], or whether it occurs during the transition from in situ to invasive carcinoma. We analyzed the expression of EMT markers and CD146, an EMT inducer, in the in situ and invasive areas of basal-like invasive breast cancers with adjacent DCIS component to define progression-related changes within individual cases.