A functional screen for genes inducing epidermal growth factor autonomy of human mammary epithelial cells confirms the role of amphiregulin.
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.
Berquin
I M
IM
Department of Radiation Oncology, Division of Radiation and Cancer Biology, University of Michigan, Ann Arbor, Michigan, MI 48109, USA.
Dziubinski
M L
ML
Nolan
G P
GP
Ethier
S P
SP
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
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