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A functional screen for genes inducing epidermal growth factor autonomy of human mammary epithelial cells confirms the role of amphiregulin

Oncogene volume 20, pages 4019–4028 (2001)Cite this article

Abstract

To gain better understanding of the molecular alterations responsible for the aggressive growth potential of epidermal growth factor receptor (EGFR)-positive breast cancers, we utilized an expression cloning strategy to seek gene products that mediate the EGF-independent growth of human breast cancer cells. A retroviral cDNA expression library was constructed from the EGFR-positive SUM-149PT cell line, and transduced into growth factor-dependent human mammary epithelial (HME) cells. Recipient cells were functionally selected for their ability to proliferate in serum-free, EGF-free medium. Library cDNAs were recovered from EGF-independent colonies by PCR amplification or by biological rescue. Clone H55a#1 contained a library insert encoding amphiregulin. This EGFR ligand was able to confer EGF independence when transduced into HME cells. SUM-149PT and H55a#1 cells overexpressed amphiregulin transcripts, and secreted moderate EGF-like activity in conditioned media, indicating a possible autocrine loop. EGFR membrane levels and constitutive phosphorylation were consistent with this hypothesis, as well as the sensitivity of the cells to an ErbB-specific kinase inhibitor. Expression of the WT1 Wilms' tumor suppressor gene, a transcriptional activator of amphiregulin, did not parallel amphiregulin transcript levels, suggesting that another factor regulates amphiregulin in SUM-149PT. Our data confirm the importance of amphiregulin in the etiology of breast cancer.

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Acknowledgements

We thank Dr Alice Telesnitsky for the pMLV Ψ-plasmid, Drs Stephan Stoll and JT Elder for the amphiregulin probe fragment, and Drs Wilber Leopold and David Fry for the CI-1033 EGFR kinase inhibitor. We also express our appreciation to Drs Michael Imperiale, Eric Fearon and the members of the Ethier laboratory for helpful discussions and Mrs Amy Pace for preparing the figures. This work was supported in part by Grant RO1 CA77416 from the NIH (to SP Ethier) and by a postdoctoral fellowship from the Pardee Foundation (to IM Berquin).

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  1. Department of Radiation Oncology, Division of Radiation and Cancer Biology, University of Michigan, Ann Arbor, 48109, Michigan, MI, USA

    Isabelle M Berquin, Michele L Dziubinski & Stephen P Ethier

  2. Department of Molecular Pharmacology, Stanford University, Stanford, 94305, California, CA, USA

    Garry P Nolan

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  1. Isabelle M Berquin
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  2. Michele L Dziubinski
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Correspondence to Stephen P Ethier.

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Berquin, I., Dziubinski, M., Nolan, G. et al. A functional screen for genes inducing epidermal growth factor autonomy of human mammary epithelial cells confirms the role of amphiregulin. Oncogene 20, 4019–4028 (2001). https://doi.org/10.1038/sj.onc.1204537

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