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

To gain better understanding of the molecular alterations responsible for t he aggressive growth potential of epidermal growth factor receptor (EGFR)-p ositive breast cancers, we utilized an expression cloning strategy to seek gene products that mediate the EGF-independent growth of human breast cance r 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 sel ected for their ability to proliferate in serum-free, EGF-free medium. Libr ary 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,vas able to confer EGF independence when tra nsduced into HME cells. SUM-149PT and H55a#1 cells overexpressed amphiregul in transcripts, and secreted moderate EGF-like activity in conditioned medi a, indicating a possible autocrine loop. EGFR membrane levels and constitut ive phosphorylation were consistent with this hypothesis, as well as the se nsitivity of the cells to an ErbB-specific kinase inhibitor. Expression of the WT1 Wilms' tumor suppressor gene, a transcriptional activator of amphir egulin, did not parallel amphiregulin transcript levels, suggesting that an other factor regulates amphiregulin in SUM-149PT, Our data confirm the impo rtance of amphiregulin in the etiology of breast cancer.