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

Oncogene. 2001 Jul 5;20(30):4019-28. doi: 10.1038/sj.onc.1204537.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amphiregulin
  • Breast / cytology*
  • Breast Neoplasms / pathology*
  • Cell Division / drug effects
  • Cell Transformation, Neoplastic / genetics
  • Cells, Cultured / drug effects
  • Clone Cells / cytology
  • Clone Cells / drug effects
  • Culture Media, Conditioned / pharmacology
  • Culture Media, Serum-Free
  • DNA, Complementary / genetics
  • EGF Family of Proteins
  • Epidermal Growth Factor / pharmacology*
  • Epithelial Cells / cytology
  • Epithelial Cells / drug effects
  • ErbB Receptors / genetics
  • ErbB Receptors / physiology
  • Female
  • Gene Library
  • Genes, Wilms Tumor
  • Genetic Complementation Test
  • Genetic Techniques*
  • Genetic Vectors / genetics
  • Glycoproteins / genetics
  • Glycoproteins / physiology*
  • Growth Substances / genetics
  • Growth Substances / physiology*
  • Humans
  • Intercellular Signaling Peptides and Proteins*
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / physiology
  • Neoplastic Stem Cells / cytology
  • Neoplastic Stem Cells / drug effects
  • Phenotype
  • Retroviridae / genetics
  • Transfection
  • Tumor Cells, Cultured / cytology
  • Tumor Cells, Cultured / drug effects


  • AREG protein, human
  • Amphiregulin
  • Culture Media, Conditioned
  • Culture Media, Serum-Free
  • DNA, Complementary
  • EGF Family of Proteins
  • Glycoproteins
  • Growth Substances
  • Intercellular Signaling Peptides and Proteins
  • Neoplasm Proteins
  • Epidermal Growth Factor
  • ErbB Receptors