INDUCTION OF ESTROGEN INDEPENDENCE OF ZR-75-1 HUMAN BREAST-CANCER CELLS BY EPIGENETIC ALTERATIONS

Antagonists of steroid hormones are clinically important in the manage ment of breast cancer. However, the duration of response!s limited due to the development of hormone-independent tumors in virtually all cas es. In an attempt to obtain insight into the mechanisms underlying ant iestrogen resistance, the consequences of epigenetic changes in gene e xpression were studied in vitro. Estrogen-dependent ZR-75-1 human brea st cancer cells were treated with 5-azacytidine, an inhibitor of DNA m ethylation, and cultured in the absence of estradiol or in the presenc e of antiestrogens. Estrogen-independent cell colonies developed withi n 3 weeks at high frequency in 5-azacytidine-treated, cultures (0.7 x 10(-3)), in contrast to control Cultures (less than or equal to 10(-8) ). The derived cells (ZR/AZA) were resistant to 4-hydroxy-tamoxifen an d ICI 164,384, independent of the selection protocol, but had lost the ability to grow anchorage-independent. Whereas expression of estrogen receptor, progesterone receptor, and pS2 were down-regulated, express ion of epidermal growth factor (EGF) receptor and HER2/neu were increa sed in ZR/AZA cells. In contrast to the stable altered expression patt erns of estrogen receptor and EGF receptor, transient keratin 7 expres sion was observed. Transforming growth factor-alpha mRNA was identifie d in ZR-75-1 cells and ZR/AZA cells and EGF-like peptides were secrete d in the culture medium. Proliferation of ZR/AZA cells could be partia lly inhibited with an EGF receptor-blocking antibody. Presence of both growth factor receptors and possible ligands suggests the development of an autocrine growth mechanism. Our data show that epigenetic alter ations of gene expression result in rapid progression of breast cancer cells to hormone independence.