Full-length cytokeratin-19 is released by human tumor cells: a potential role in metastatic progression of breast cancer

Abstract

Introduction: We evaluated whether CK19, one of the main cytoskeleton proteins of epithelial cells, is released as full-length protein from viable tumor cells and whether this property is relevant for metastatic progression in breast cancer patients.

Methods: EPISPOT (EPithelial ImmunoSPOT) assays were performed to analyze the release of full-length CK19 by carcinoma cells of various origins, and the sequence of CK19 was analyzed with mass spectrometry. Additional functional experiments with cycloheximide, Brefeldin A, or vincristine were done to analyze the biology of the CK19-release. CK19-EPISPOT was used to detect disseminated tumor cells in bone marrow (BM) of 45 breast cancer patients who were then followed up over a median of 6 years.

Results: CK19 was expressed and released by colorectal (HT-29, HCT116, Caco-2) and breast (MCF-7, SKBR3, and MDA-MB-231) cancer cell lines. The CK19-EPISPOT was more sensitive than the CK19-ELISA. Dual fluorescent EPISPOT with antibodies against different CK19 epitopes showed the release of the full-length CK19, which was confirmed by mass spectrometry. Functional experiments indicated that CK19 release was an active process and not simply the consequence of cell death. CK19-releasing cells (RCs) were detectable in BM of 44% to 70% of breast cancer patients. This incidence and the number of CK19-RCs were correlated to the presence of overt metastases, and patients with CK19-RCs had a reduced survival as compared with patients without these cells (P = 0.025, log-rank test; P = 0.0019, hazard ratio, 4.7; multivariate analysis).

Conclusions: Full-length CK19 is released by viable epithelial tumor cells, and CK19-RCs might constitute a biologically active subset of breast cancer cells with high metastatic properties.

Figure 1

Detection of CK19 protein. (a) CK19-EPISPOT assay procedure. Day 1: The membranes of the ELISPOT plates are coated with an anti-human CK19 monoclonal antibody (mAb, Ks19.1). Days 2 to 3: Next, the cells are seeded in each well and cultured for 48 hours. During this incubation period, the released specific proteins are directly immunocaptured by the immobilized mAb on the bottom of the well. Plates are then washed, and cells are removed. The presence of the released CK19 protein is revealed by the addition of an anti-human CK19 mAb (Ks19.2) conjugated with a fluorochrome. Day 4: Fluorescent immunospots are counted with an automated reader. One immunospot corresponds to the fingerprint left only by one viable releasing epithelial tumor cell. (b) Schematic representation of the assumed binding sites of anti-cytokeratin antibodies and cleavage site for caspase 3 on the human CK19 protein [3,40].

Figure 2

EPISPOT assays and immunocytochemistry experiments. (a) Dual-fluorescent CK19-EPISPOT assay with two different couples of mAbs. Red and green immunospots represent protein fingerprints of cells releasing CK19 detected with anti-CK19 AE1^Alexa555 and Ks19.2^Alexa488 mAbs, respectively. Yellow fluorescent immunospots (Merge) are the results of dual staining with both mAbs. (b) Single-fluorescent CK19 Ks19.2^(Alexa488)-EPISPOT assay on MCF-7 cells without and with the addition of cycloheximide (50 μg/ml). (c) Dual-fluorescent CK19 Ks19.2^(Alexa488)/MUC1^(Alexa555) EPISPOT assay on the MCF-7 cancer cell line. Green and red immunospots represent CK19-RCs and MUC1-RCs, respectively. Yellow fluorescent immunospots (Merge) are the results of dual staining with both mAbs. (d) Immunostaining of the nucleus^(dapi), the MUC1^(Alexa555), and the CK19 Ks19.2^(Alexa488) of breast MCF-7 cells (left). Immunostaining of the nucleus^(dapi), the CK19 Ks19.2^(Alexa555), and the cleaved cytokeratin 18 with the M30 CytoDEATH^(FITC)of MCF-7 cells after an incubation with vincristine (20 μmol/l), an inducer of apoptosis (right). The antibody M30 CytoDEATH which recognizes a neo-epitope formed after caspase cleavage of cytokeratin 18 at Asp396 (CK18Asp396-NE M30 neo-epitope) during and after apoptosis, does not bind native CK18 of normal cells and is a very reliable and convenient tool for demonstration of apoptosis in single cells [41]. CK19 vesicles were detectable in the viable but not in the apoptotic MCF-7 cells.

Figure 3

Survival data. Kaplan – Meier estimates of cancer-related survival of breast cancer patients, according to the presence or absence of viable CK19-RCs. (a), MUC1-RCs (b), and subpopulations of cells CK19⁺MUC1^-, CK19⁺MUC1⁺ (c) in the BM. RCs = releasing cells.