Changes in Cytoskeletal Protein Composition Indicative of an Epithelial-Mesenchymal Transition in Human Micrometastatic and Primary Breast Carcinoma Cells

Abstract

Purpose:The bone marrow is a frequent and clinically important homing site for early disseminated breast cancer cells. Here, we aimed to profile the protein expression of these cells using unique cell line models and to evaluate the prognostic relevance of candidate gene expression for breast cancer patients. Experimental Design: To identify expression patterns characteristic for micrometastatic cells, three different cell lines (BC-K1, BC-P1, and BC-S1) established by SV40 immortalization of cancer cells isolated from the bone marrow of patients with breast cancer were compared with MCF-7 breast cancer and SV40 immortalized normal breast ductal cells (MTSV-1.7) using twodimensional gel electrophoresis followed by MALDI-ToF analysis.The prognostic significance and clinicopathologic associations of selected differentially expressed proteins were evaluated using high-density breast cancer tissue microarrays. Results: In contrast to MCF-7 and MTSV1-7 reference cell lines, all micrometastatic cancer cell lines displayed loss of epithelial cytokeratins (CK8, CK18, and CK19) and ectopic expression of vimentin commonly present in mesenchymal cells. Immunohistochemical analysis of 2,517 samples of breast cancer further showed that loss of cytokeratin and ectopic vimentin expression were significantly associated with a higher tumor grade, high mitotic index, and negative estrogen/ progesterone-receptor status. Although in univariate analyses significantly related to clinical outcome, none of the cytokeratins analyzed were independently associated with either overall or cancer-specific survival. Conclusions: Micrometastatic cancer cells exhibit marked changes in the expression pattern of cytoskeletal proteins indicative of an epithelial-mesenchymal transition. This phenotypical change could already be detected in primary tumors and is associated with the aggressive behavior of breast cancer cells in vivo.Breast cancer, the most common malignancy in females, kills f300,000 women worldwide every year and is the main reason for cancer-related death in the postoperative development of distant metastasis in secondary organs (1). Many of the patients who are primarily diagnosed as having apparently localized or regional breast cancer eventually develop distant metastases. Clinical experience showed that in early clinical stages, cells could already dissociate from the tumor's parenchyma via the hematogenous route and colonize the bone marrow, the clinically most relevant site of metastatic disease in patients with solid epithelial tumors (2). Often, many years after resection of the primary tumor and treatment of cancer patients, micrometastatic cancer cells can grow out to form overt metastases. Indeed, evidence is accumulating that the presence of occult carcinoma cells of epithelial origin in the bone marrow is an independent risk factor in breast cancer (3 -5). Understanding the biology of these dormant tumor cells in the bone marrow is therefore pivotal for the development of novel therapeutic approaches for the t...

Acantholytic Squamous Cell Carcinoma in Upper Aerodigestive Tract: Histopathology, Immunohistochemical Profile and Epithelial Mesenchymal Transition Phenotype Change

“…During tumor growth and progression, the local microenvironment and growth factors may induce epithelial tumor cells to dedifferentiate and acquire an epithelial mesenchymal transition like phenotype characterized by loss of cell to cell adhesion, down regulation of E-cadherin and a shift of intermediate filaments to vimentin [25]. Suppression of CK19 expression in carcinomas during tumor differentiation has been reported in both human tissues, such as breast cancers, and cultured malignant cell lines [26,27]. Down-regulation of CK19 expression and overexpression of vimentin were found in highly aggressive breast cancer cells with strong migratory and invasive abilities [28].…”

Keratin down-regulation in vimentin-positive cancer cells is reversible by vimentin RNA interference, which inhibits growth and motility

“…For example, in breast epithelia, K8 and K18 are expressed in the differentiated compartment and are down-regulated in a high percentage of breast carcinomas, a feature that correlates with a worse prognosis (13,26,27). Similarly, micrometastatic cell lines derived from the bone marrow of breast cancer patients showed loss of cytokeratin expression with concomitant up-regulation of vimentin (28). Furthermore, these authors were able to correlate changes in keratin and vimentin expression with more aggressive lesions, indicating clinical significance.…”

Epithelial—mesenchymal and mesenchymal—epithelial transitions in carcinoma progression

“…It has long been recognized that CTCs show reduced expression of specific cytokeratins (Pantel et al, 1994). More recently, Pantel and coworkers have shown that cell lines derived from breast cancer micrometastases stably express the mesenchymal marker vimentin (Willipinski-Stapelfeldt et al, 2005). The possible roles of EMT in escape and survival in the blood stream as CTCs, and establishment as micrometastases, are summarized in Figure 3.…”