AMPHIREGULIN AS AN AUTOCRINE GROWTH-FACTOR FOR C-HA-RAS-TRANSFORMED AND C-ERBB-2-TRANSFORMED HUMAN MAMMARY EPITHELIAL-CELLS

Amphiregulin (AR), a member of the epidermal growth factor (EGF) famil y, was found to be as potent as EGF in stimulating the anchorage-depen dent growth (ADG) of immortalized, nontransformed human mammary epithe lial MCF-10A cells. MCF-10A cells transformed by either an activated h uman c-Ha-ras protooncogene (MCF-10A ras) or by overexpression of a no nactivated rat c-neu gene (MCF-10A neu) exhibited a 35% reduction in t he response to AR in ADG when compared to MCF-10A cells, but AR was st ill as potent as EGF in these transformants. Exogenous AR exhibited on ly 15-20% of the activity of EGF in stimulating the anchorage-independ ent growth, a response that is normally dependent upon exogenous EGF, of the oncogene-transformed MCF-10A cells. MCF-10A cells express low l evels of a 1.4-kb AR mRNA transcript, while MCF-10A ras and MCF-10A ne u cells display a 15- to 30-fold increase in the levels of AR mRNA and endogenous AR protein as determined by Western blot analysis. Exogeno us EGF was found to induce both AR mRNA and protein in the MCF-10A par ental and transformed cells. A 20-mer phosphorothioate antisense deoxy oligonucleotide complementary to the 5' sequence of AR mRNA was able t o significantly reduce the levels of endogenous AR protein and to inhi bit the EGF-stimulated ADG and anchorage-independent growth of MCF-10A ras and MCF-10A neu cells. These data suggest that AR may function as an EGF-dependent autocrine growth factor in mammary epithelial cells that have been transformed by either a point-mutated c-Ha-ras or c-neu .