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Antisense expression for amphiregulin suppresses tumorigenicity of a transformed human breast epithelial cell line

Oncogene volume 18, pages 6513–6520 (1999)Cite this article

Abstract

The epidermal growth factor (EGF) family of receptors and their respective ligands play a major role in breast cancer progression and are the targets of new therapeutic approaches. Following immortalization with SV40 T antigen of normal human breast epithelial cells, a transformed variant cell line (NS2T2A1) was selected for its increased tumorigenicity in nude mice. This cell line was shown to have a higher expression of EGF receptors (EGFR) and amphiregulin (AR) when compared to their normal counterparts or less aggressive transformed cells. Dual staining of EGFR and AR was observed in 50 – 60% of NS2T2A1 cells, while 30 – 40% cells expressed AR only. To explore the potential tumorigenic role of AR, a 1.1 kb AR cDNA in an antisense orientation was transfected in NS2T2A1 cells. Three clones, selected by hygromycin B, expressed AR antisense RNA (AR AS1, AR AS2 and AR AS3 cell lines) in which AR protein expression was reduced (ranging from about 50 to <5%). The anchorage-independent growth of AR AS cell lines was reduced to levels ranging from 32.4 – 6.8% relative to the control cell line transfected with the vector alone. The clones expressing AR antisense RNA showed a reversion of the malignant phenotype when injected in nude mice, since a significant reduction of tumor intake was observed coincident with a significant tumor mass reduction (>96%). Moreover, intra-tumoral vascularization decreased significantly in tumors derived from AR AS cells (26.7, 70.7 and 50.4% of control). These in vitro and in vivo data reveal the oncogenic nature of AR in transformed breast epithelial cells and imply a role for AR in tumor angiogenesis.

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Acknowledgements

We are grateful to Dr A Lilienbaum, ML Lacombe, G Linares-Cruz, T Le Bricon and A Faille for helpful discussions. We wish to thank Drs WJ Gullick, DS Salomon and H Lu for providing antibodies, Dr M Pla for the use of animal facilities, and B Boursin for photografic support. This work was supported by a grant from CNAMTS-INSERM to F Calvo.

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Authors and Affiliations

  1. Laboratoire de Pharmacologie Expérimentale et Clinique, INSERM EP-9932, Institut de Génétique Moléculaire, 27, rue Juliette Dodu, 75010, Paris, France

    Lin Ma, Christiane Gauvillé, Guy Millot & Fabien Calvo

  2. Faculté de Médecine Nord, Bd P. Dramar, Marseille, 13916, France

    Yolande Berthois

  3. Division of Cytokine Biology, FDA, CBER, HFM-511, Building 29A, Room 3B-16, 8800 Rockville Pike, Bethesda, Maryland, 20892, MD, USA

    Gibbes R Johnson

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  1. Lin Ma
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Ma, L., Gauvillé, C., Berthois, Y. et al. Antisense expression for amphiregulin suppresses tumorigenicity of a transformed human breast epithelial cell line. Oncogene 18, 6513–6520 (1999). https://doi.org/10.1038/sj.onc.1203042

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