Invasiveness of breast carcinoma cells and transcript profile: Eph receptors and ephrin ligands as molecular markers of potential diagnostic and prognostic application

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Abstract

The Eph family of receptors, with 14 members in humans, makes up the largest group of receptor tyrosine kinases. These Eph receptors, along with their ligands, the 8 members of the ephrin family of ligands are involved in diverse developmental functions, including hindbrain development in vertebrates, tissue patterning, and angiogenesis. These Eph receptors and ephrin ligands have also been identified as important regulators in the development and progression of cancer. We have presented here a systematic and comprehensive investigation of the Eph/ephrin expression profiles of MCF-10A, MCF-7, and MDA-MB-231 cells representing normal breast, non-invasive breast tumor, and invasive tumor, respectively, based on their characteristic phenotypes in Matrigel matrix. The data have allowed us to correlate the gene expression profile with the cell phenotype that has potential application in tumor diagnostics. We demonstrate here that upregulation of EphA2, A7, A10, and ephrinA2 and B3 is likely involved in tumorigenesis and/or invasiveness, while downregulation of EphA1, A3, A4, A8, B3, B4, B6, and ephrinA1 and B1 may be particularly important in invasiveness. Based on these results we discuss the role of EphA2 and ephrinA1 combination in malignancy. The data have provided clues as to the importance of these molecules in the progression of breast cancer and specifically identified EphB6, a kinase-deficient receptor, which is downregulated in the most aggressive cell line, as reported for several other cancer types including neuroblastoma and melanoma suggesting its potential as a prognostic indicator in breast cancer as well.

Section snippets

Materials and methods

Cell cultures. All cells were cultured at 37 °C/7% CO2. MCF-10A cells were grown in 1:1 DMEM:F12 media (Gibco) with 5% Horse Serum (Gibco), 20 mM Hepes, 10 ng/ml EGF (Invitrogen), 10 ml/L PenStrep-Glutamine 10,000 U/ml penicillin, 10,000 μg/ml streptomycin, and 29.2 mg/ml l-glutamine (Gibco), 10 g/ml insulin (Invitrogen), 0.1 μg/ml Cholera Toxin (Sigma), and 500 ng/ml Hydrocortisone (Sigma). MCF-7 and MDA-MB-231 cells were grown in DMEM (Gibco) supplemented with 2 mM l-glutamine (Gibco), 1 mM sodium

Results

We have first confirmed the invasive phenotype of the breast carcinoma cell lines used in this study. We have then analyzed the abundance of 13 members of the Eph receptor family and 8 members of the ephrin ligand family in three cell lines, namely, MCF-10A, MCF-7, and MDA-MB-231. The results are presented in the following manner: (1) The relative abundance of each Eph receptor and ephrin ligand in individual cell lines to obtain a cell-type profile of these transcripts. (2) A comparative

Discussion

The evaluation of cell lines established from normal and cancer tissue has often served as a primer for biological characterization of a number of genes involved in tumorigenesis. While global gene profiling via gene microarrays is currently being used to arrive at a molecular signature of tumor tissues [42], [43], the analysis of such large-scale expression profiles is a major challenge. We reasoned that a systematic analysis of gene families involved in specific signaling pathways may allow

Acknowledgements

We wish to acknowledge the Department of Defense for supporting the P.I’s (R.P.K.) research. Our thanks to Dr. Berish Rubin and Dr. Sylvia Anderson for use of real time PCR machine, Jinsong Qiu for his help with designing and running real time experiments, and Nagaraja Ganachari for technical assistance.

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