title: A functional screen for genes inducing epidermal growth factor autonomy of human mammary epithelial cells confirms the role of amphiregulin.
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.
authors.affiliation: Department of Radiation Oncology, Division of Radiation and Cancer Biology, University of Michigan, Ann Arbor, Michigan, MI 48109, USA.
authors.forename: I M
authors.initials: IM
authors.lastname: Berquin
authors.forename: M L
authors.initials: ML
authors.lastname: Dziubinski
authors.forename: G P
authors.initials: GP
authors.lastname: Nolan
authors.forename: S P
authors.initials: SP
authors.lastname: Ethier
mesh_terms: D066258:Amphiregulin; D001940:Breast / Q000166:cytology*; D001943:Breast Neoplasms / Q000473:pathology*; D002455:Cell Division / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D002478:Cells, Cultured / Q000187:drug effects; D002999:Clone Cells / Q000166:cytology / Q000187:drug effects; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary / Q000235:genetics; D066255:EGF Family of Proteins; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects; D066246:ErbB Receptors / Q000235:genetics / Q000502:physiology; D005260:Female; D015723:Gene Library; D016162:Genes, Wilms Tumor; D005816:Genetic Complementation Test; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics / Q000502:physiology*; D006133:Growth Substances / Q000235:genetics / Q000502:physiology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins / Q000235:genetics / Q000502:physiology; D014411:Neoplastic Stem Cells / Q000166:cytology / Q000187:drug effects; D010641:Phenotype; D012190:Retroviridae / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects
chemical_list: C587848:AREG protein, human; D066258:Amphiregulin; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D066255:EGF Family of Proteins; D006023:Glycoproteins; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors