a1-Antitrypsin Blocks the Release of Transforming Growth Factor-a from MCF-7 Human Breast Cancer Cells*

Human breast cancer cells synthesize and release a variety of growth-modulating substances in response to estrogen stimulation, and it is generally accepted that the growth-promoting effects of estrogens are due at least in part to this autocrine/paracrine mechanism. Several of these growth-modulating substances, including transforming growth factor-a (TGFa) and its analogs, have been shown to require pericellular proteolysis for activation or release. Recently, we reported that MCF-7 human breast cancer cells are able to synthesize a1-antitrypsin (a1-AT), the major elastase inhibitor in human serum, and that there is a negative correlation between anchorage-independent growth ofMCF-7 cells in soft agar and synthesis of a1-AT. The studies we present here were undertaken to gain an understanding of the mechanisms responsible for this observation. We show that release of TGFa from itsmembrane-bound precursor on MCF-7 cells is blocked by a1-AT whether the cells were maintained in the presence or absence of estradiol and that there is a clear dose-response relationship between the a1-AT concentration and both the release of TGFa and growth in soft agar. Consistent with this, TGFa release was increased in the presence of antibody to a1-AT. In contrast, TGFa release and growth in soft agar were not blocked by peptide inhibitors specific for trypsinor chymotrypsin-like enzymes. The a1-AT concentration required for a half-maximal effect is lower for inhibition of TGFa release than it is for inhibition of colony formation (0.4 vs. 1.5 mmol/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 a1-AT than its parent cell line was constructed by stable transfection of MCF-7 ML cells (a subline producing low levels of a1-AT) with an a1-AT complementary DNA. Growth in soft agar and release of TGFa were significantly decreased in cells transfected with the a1-AT complementary DNA compared to those in cells transfected with vector alone, although, TGFa expression was the same. The above observations support amodel for growth regulation in human breast ductal epithelial cells in which growth factor activation and release are dependent on the coordinate action of proteases and protease inhibitors. This model would predict that a1-AT can act as a tumor suppressor in inhibiting the growth of breast cancer cells. (J Clin Endocrinol Metab 82: 745–752, 1997) T growth factor-a (TGFa), a peptide structurally and functionally related to epidermal growth factor (EGF), interacts with the EGF receptor and elicits a mitogenic response in a variety of cell types (1). Proteolytic cleavage is required to split out the soluble, 50amino acid form of TGFa from its membrane-bound precursor (pro-TGFa) on the cell surface (2), and this process may constitute an important regulatory step in the release of soluble TGFa. The observation that cleavage occurs on cells growing in serum-free medium indicates that the protease(s) is synthesized by the cell itself. Pro-TGFa has been shown to interact with EGF/TGFa receptors on the surface of adjacent cells (3). This juxtacrine action may play a role in cell-cell adhesion and mitogenesis, and cleavage may be an obligate step in a process generating two active forms of TGFa, each with different properties. The amino acid residues at the two cleavage sites in pro-TGFa suggest that both reactions should be accomplished by a yet to be identified elastase-like enzyme(s) (4). Because of this elastase-like specificity, the TGFa proteasewould not appear to be a Kex2or furin-like enzyme (5, 6) or hepsin (7). Onewould also expect it to be neutralized by a1-antitrypsin (a1-AT also known as a1-proteinase inhibitor), the major human extracellular elastase inhibitor (8), or other extracellular elastase inhibitors, such as the secretory leukocyte protease inhibitor (SLPI) (9). Chinese hamster ovary (CHO) cells, which normally do not express pro-TGFa, can release soluble TGFa after transfection with a TGFa complementary DNA (cDNA) (10); however, the CHO cell protease responsible for this hydrolysis does not appear to be sensitive to a1-AT. This does not rule out such a role for an a1-AT-sensitive protease in other cell types. For example, cell surface elastase-like enzymes sensitive to a1-AT have been isolated from transformed rat liver epithelial cells and Schwann cells, both of which express TGFa (11). The expression of TGFa occurs in normal breast tissue, breast tumors, and breast cancer cells in culture, and TGFa Received September 11, 1996. Revision received November 12, 1996. Accepted November 20, 1996. Address all correspondence and requests for reprints to: Thomas H. Finlay, Ph.D., New York University Medical Center, 550 First Avenue, New York, New York 10016. * This work was supported in part by grants from the Elena Calas Memorial Fund, the Children’s Brain Tumor Foundation, the NIH (Grant CA-67348), and the U.S. Army (Grant DAMD17–96-1–6238). The National Science Foundation is thanked for its support of the computing resources through Grant BIR-9318128. † Current address: Department of Biochemistry, University of Medicine and Dentistry, Johnson Medical School, Piscataway, New Jersey 08854. 0021-972X/97/$03.00/0 Vol. 82, No. 3 Journal of Clinical Endocrinology and Metabolism Printed in U.S.A. Copyright © 1997 by The Endocrine Society