Transforming growth factor a (TGFalpha), amphiregulin (AR) and cripto-1 (CR-1) are proteins that are structurally and in some cases functionally related to epidermal growth factor (EGF) in that TGFalpha and AR can bind to the EGF receptor (c-erb B). TGFalpha has been circumstantially implicated in the autocrine growth of a number of different human carcinoma cells such as breast and colon tumors. However, the regulation of expression of this growth factor and interference with its biological activity have not been thoroughly examined. Moreover, the relative levels of expression and biological function of AR and CR-1 in these malignancies are unknown. The present studies have demonstrated that MCF-10A human mammary epithelial cells are mitogenically responsive to exogenous EGF, TGFalpha or AR and that transformation of these cells with a point-mutated c-Ha-ras protooncogene but not with a c-erb B-2 protooncogene results in an increase in the expression of endogenous TGFalpha. Furthermore, overexpression of a human TGFalpha cDNA in these cells leads to their in vitro transformation. Addition of an anti-EGF receptor blocking antibody or an anti-TGFalpha neutralizing antibody can partially or completely inhibit the growth of the Ha-ras or TGFalpha transformed mammary cells suggesting that an external autocrine loop is operative in these cells. In contrast, AR expression is increased in both Ha-ras and c-erb B-2 transformed MCF-10A cells and the growth of these transformants can be inhibited by AR antisense phosphorothioate oligonucleotides demonstrating that AR is functioning as an autocrine intermediary in the transformation pathway that is utilized by both Ha-ras and erb B-2. Estrogens can increase the expression of TGFalpha mRNA and protein in estrogen- responsive human breast cancer cell lines such as MCF-7 or ZR-75-1 cells. Transient transfection assays in MCF-7 or ZR-75-1 cells using a plasmid containing the TGFalpha promoter ligated to either the chloramphenicol acetyltransferase (CAT) or luciferase genes have demonstrated that physiological concentrations of estrogens can induce a 5-to 50-fold increase in the activity of these reporter genes, suggesting that the TGFalpha promoter contains a cis-acting estrogen-responsive element(s) (ERE). MCF-7 or ZR-75-1 cells were infected with a recombinant amphotropic TGFalpha antisense mRNA expression vector. Expression of this antisense mRNA lead to a reduction in estrogen-induced TGFalpha protein production and to an equivalent degree of inhibition of estrogen-induced proliferation in these cells. Specific mRNA and immunoreactivity for AR and CR-1 have been detected in approximately 50% to 80% of primary and metastatic human colorectal tumors, whereas only 5% of normal adjacent colon or liver tissue express these genes. Likewise, immunoreactive AR and CR-1 was detected in approximately 70% of primary human breast tumors at a level that exceeded the level found in adjacent normal mammary epithelium.
