ALPHA(1)-ANTITRYPSIN BLOCKS THE RELEASE OF TRANSFORMING GROWTH-FACTOR-ALPHA FROM MCF-7 HUMAN BREAST-CANCER CELLS

Human breast cancer cells synthesize and release a variety of growth-m odulating substances in response to estrogen stimulation, and it is ge nerally accepted that the growth-promoting effects of estrogens are du e at least in part to this autocrine/paracrine mechanism. Several of t hese growth-modulating substances, including transforming growth facto r-alpha (TGF alpha) and its analogs, have been shown to require perice llular proteolysis for activation or release. Recently, we reported th at MCF-7 human breast cancer cells are able to synthesize alpha(1)-ant itrypsin (alpha(1)-AT), the major elastase inhibitor in human serum, a nd that there is a negative correlation between anchorage-independent growth of MCF-7 cells in soft agar and synthesis of alpha(1)-AT. The s tudies we present here were undertaken to gain an understanding of the mechanisms responsible for this observation. We show that release of TGF alpha from its membrane-bound precursor on MCF-7 cells is blocked by alpha(1)-AT whether the cells were maintained in the presence or ab sence of estradiol and that there is a clear dose-response relationshi p between the alpha(1)-AT concentration and both the release of TGF al pha and growth in soft agar. Consistent with this, TGF alpha release w as increased in the presence of antibody to alpha(1)-AT. In contrast, TGF alpha release and growth in soft agar were not blocked by peptide inhibitors specific for trypsin- or chymotrypsin-like enzymes. The alp ha(1)-AT concentration required for a half-maximal effect is lower for inhibition of TGF alpha release than-it is for inhibition of colony f ormation (0.4 vs. 1.5 mu mol/L). However, both values are in the range of concentrations one might expect at the cell surface in vivo. A new MCF-7 cell subline producing 10-fold higher levels of alpha(1)-AT tha n its parent cell line was constructed by stable transfection of MCF-7 ML cells (a subline producing low levels of alpha(1)-AT) with an alph a(1)-AT complementary DNA. Growth in soft agar and release of TGF alph a were significantly decreased in cells transfected with the alpha(1)- AT complementary DNA compared to those in cells transfected with vecto r alone, although, TGF alpha expression was the same. The above observ ations support a model for growth regulation in human breast ductal ep ithelial cells in which growth factor activation and release are depen dent on the coordinate action of proteases and protease inhibitors. Th is model would predict that alpha(1)-AT can act as a tumor suppressor in inhibiting the growth of breast cancer cells.