Expression analysis of epithelial cadherin in human breast cancer cell lines IBH-4 and IBH-6

LAPYCKYJ, VAZQUEZ-LEVIN Y COL (VER DETALLE EN RESUMEN)

Ginebra Suiza

Congreso; Biennal International Union Against Cancer (UICC) World Cancer Congress; 2008

Union Against Cancer (UICC)

EXPRESSION ANALYSIS OF EPITHELIAL CADHERIN IN HUMAN BREAST CANCER CELL LINES IBH-4 AND IBH-6  L. Lapyckyj*1, L. F. Castillo1, M. L. Matos1, E. M. Lentz1, I. A. Luthy1, M. H. Vazquez-Levin11Instituto de Biología y Medicina Experimental, CONICET - UBA, Buenos Aires, Argentina ABSTRACT: Background: Epithelial cadherin (Ecad) is a cell-cell adhesion molecule involved in the establishment of epithelial adherens junctions. It is connected to the actin cytoskeleton by accessory proteins, such as beta-catenin. Loss of Ecad expression/function has been related to tumor development and progression, and CDH1 (Ecad gene) has been proposed as a tumor supressor gene (1). Several molecules associated with down-regulation of Ecad have been described, within them Neural cadherin (Ncad) (2), transcription factor Twist (3) and dysadherin (Dys) (4). Human breast cancer cell lines IBH-4 and IBH-6 were developed from primary tumors, are sensitive to estradiol, progesterone and EGF, and show characteristic features of malignant epithelial cells (i.e. cytokeratin expression, growth in soft agar and nude mice) (5). Objective: To perform Ecad expression analysis in IBH-4 and IBH-6 cell lines. Methods: Ecad localization analysis was done by confocal microscopy; in addition, Ecad protein forms were identified by Western immunoblotting of cell extracts with specific antibodies directed towards different protein domains. Ecad mRNA analysis included end point and real-time PCR to search for transcript changes and determine mRNA levels, respectively. Expression analysis of Ncad, Dys and Twist was done using the same experimental approaches. MCF-7 cells served as control. Results: In IBH-4 and IBH-6 cell extracts, an 89 KDa Ecad protein form (Ecad89) was only detected, which was truncated at the C-terminal end. Ecad89 protein levels were significantly lower (less than 30%) than those of 120 KDa Ecad whole protein (Ecad120) present in MCF-7. Accumulation of a 35 kDa Ecad C-terminal fragment (Ecad35) resulting from Ecad120 processing by metalloproteinases, was not observed in IBH-4 and IBH-6 protein extracts, suggesting a novel yet unknown mechanism of Ecad89 production. Ecad localization analysis of IBH-4 and IBH-6 cells revealed a punctuated intracellular staining pattern that contrasted with the characteristic signal in cell-cell junctions observed in MCF-7. Beta-catenin localization accompanied Ecad in both cell types. A lack of F-actin and E-cad co-localization was also found. mRNA Ecad levels were remarkably lower in IBH-4 and IBH-6 compared to MCF-7, reaching up to 1000 times less. The sequence of the Ecad mRNA encoding exons 14-16 could not be amplified by nested RT-PCR in either cell lines. Expression of Ncad and Dys was neither detected in mRNA studies nor in protein analyses. An up-regulation of Twist mRNA expression was found in both cell lines. Conclusions: Ecad expression analysis of human breast cancer cell lines IBH-4 and IBH-6 has shown: 1) presence of a C-terminal truncated 89 kDa Ecad form 2) a significant decrease in both protein and mRNA Ecad levels 3) an intracellular Ecad and beta-catenin localization and a lack of co-localization with F-actin 4) up-regulation of transcription factor Twist mRNA expression. REFERENCES: 1. Berx and van Roy. Breast Cancer Res. 3(2001): 289-93. 2. Hazan et al. Ann.N.Y.Acad.Sci. 1014(2004): 155-63. 3. Nam et al. Cancer Letters 255(2007): 161-69. 4. Vesuna et al. Biochem Biophys Research Commun 367(2008): 235-41. 5. Vazquez, S. M. et al. J.Cell Physiol 199(2004): 460-69. Conflict of Interest: None declared