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Transforming growth factor beta (TGF beta) has potent inhibitory effects upon epithelial proliferation and malignant progression may be associated with breakdown of the autocrine and paracrine inhibitory loops in which TGF beta participates. The therapeutic effecs of anti-estrogens may be partially attributable to boosting of local endogenous levels of TGF beta. This article reviews the evidence in support of TGF beta being a proximate effector in mediation of the anti-neoplastic effects of anti-estrogens. Both the conventional estrogen receptor (ER) dependent and ER independent mechanisms of action are likely to be involved. Evidence for preferential stromal induction of TGF beta by anti-estrogens is emphasized, together with the therapeutic potential of this strategy for improving outcome in early breast cancer irrespective of ER status.

Tamoxifen may mediate its effect in early breast cancer in part via an oestrogen receptor (ER)-independent pathway by directly stimulating fibroblasts to produce the negative paracrine growth factor transforming growth factor (TGF)-beta. We have previously shown that secretion of this factor is induced 3-to 30-fold in human fetal fibroblasts in vitro, and by stromal fibroblasts in vivo following tamoxifen treatment of ER-positive and ER-negative breast cancer patients. Primary cultures of breast tumour fibroblasts have been exposed to tamoxifen for 48 h, and rates of secretion of TGF-beta 1 and TGF-beta 2 measured using a quantitative immunoassay. Fibroblast strains derived from malignant and benign tumours produced and secreted similar amounts of TGF-beta 1, but benign breast tumour fibroblasts secreted significantly higher levels of TGF-beta 2 compared with fibroblasts of malignant origin. Tamoxifen did not induce any consistent increase in TGF-beta secretion into the conditioned medium, but immunofluorescence analysis for the intracellular form of TGF-beta 1 revealed evidence of increased immunoreactive protein in tamoxifen-treated fibroblasts, which is localised to the nucleus. Therefore synthesis of TGF-beta 1 appears to be stimulated by tamoxifen, but increased secretion may be abrogated in vitro. Furthermore, using immunocytochemistry and transient transfection with an ER-responsive reporter construct, no ER was demonstrable in these fibroblasts supporting the proposed ER-independent paracrine pathway.

The molecular mechanism of action of anti-oestrogens such as tamoxifen appears to be a complex mixture of antagonism of the mitogenic action of oestradiol at the level of the oestrogen receptor, plus a range of other activities from enzyme inhibition to growth factor modulation. This article will concentrate on two specific areas: 1) the inhibition of protein kinase C and calmodulin-dependent cAMP phosphodiesterase; and 2) the regulation by tamoxifen of peptide regulators of breast cancer epithelial cell growth such as insulin-like growth factor I (IGF I) and transforming growth factor beta (TGF-beta). The elucidation of these mechanisms is potentially important in the treatment and chemoprevention of breast cancer-the quantitative contribution of each individual mechanism of the overall antineoplastic action of anti-oestrogens is central to developing new and possibly more effective anti-oestrogens and optimizing strategies for their use.

The transforming growth factors beta are a family of peptides which are involved in the regulation of cell growth and differentiation. It has been suggested that the loss of sensitivity to growth inhibition by endogenous TGF-beta may contribute to the process of carcinogenesis in epithelial systems. However, many breast cancer cells remain sensitive to the growth inhibitory effects of these peptides, suggesting that the local induction of TGF-beta could provide a pharmacological approach to chemoprevention. Triphenylethylene anti-oestrogens, synthetic progestins and retinoids all offer potential as chemopreventative agents. A common feature of their mechanism of action is the ability to locally increase the production of the transforming growth factors beta.

Growth of the human endometrial carcinoma Ishikawa cell line was stimulated by transforming growth factor-beta1 when cultured in serum-containing and chemically defined culture medium. This response was not unique to endometrial carcinoma cells, as the breast-cancer cell line, T47-D, was similarly stimulated by TGF-beta 1. TGF-beta 1 stimulated growth of MCF-7 breast-cancer cells in chemically defined medium but inhibited growth of this cell line in serum-containing medium. The data provide a demonstration of a positive growth response to TGF-beta 1 in oestrogen-receptor-positive cells and do not support the hypothesis that this growth factor is simply a negative growth regulator in epithelial-cancer cell lines.

Recent experimental work has identified a novel intracellular binding site for the synthetic progestin, Gestodene, that appears to be uniquely expressed in human breast cancer cells. Gestodene is shown here to inhibit the growth of human breast cancer cells in a dose-dependent fashion, but has no effect on endocrine-responsive human endometrial cancer cells. Gestodene induced a 90-fold increase in the secretion of transforming growth factor-beta (TGF-beta) by T47D human breast cancer cells. Other synthetic progestins had no effect, indicating that this induction is mediated by the novel Gestodene binding site and not by the conventional progesterone receptor. Furthermore, in four breast cancer cell lines, the extent of induction of TGF-beta correlated with intracellular levels of Gestodene binding site. No induction of TGF-beta was observed with the endometrial cancer line, HECl-B, which lacks the Gestodene binding site, but which expresses high levels of progesterone receptor. The inhibition of growth of T47D cells by Gestodene is partly reversible by a polyclonal antiserum to TGF-beta. These data indicate that the growth-inhibitory action of Gestodene may be mediated in part by an autocrine induction of TGF-beta.

Gestodene, a new synthetic progestogen, binds to progesterone receptor (PR) derived from normal breast, endometrium and both normal and diseased liver with an affinity 8-10 times higher than that of the natural ligand. PR in malignant breast could not be quantified on account of the binding of gestodene to oestrogen receptor (ER) in that tissue. Sucrose density ultracentrifugation using 3H-oestradiol and 3H-gestodene in normal and malignant breast demonstrated that the gestodene-ER complex in addition to exhibiting the 4S and 5S peaks showed an additional peak which sedimented at 2.9-3.15. Dissociation kinetics of gestodene from ER which was either heat-activated or molybdate-stabilised were comparable to the triphenylethylene class of antioestrogens in that the rate of dissociation, unlike that of oestradiol from ER, was unaffected by these treatments. The binding of ER-gestodene to DNA-cellulose was also investigated and was found to be approximately 30% less than that of ER-oestradiol.

Oestrogen receptor protein (ER) was detected in 9 of 11 samples of malignant breast tissue and 8 of 9 samples of normal breast tissue. Levels of cytosolic ER (ERc) in malignant breast were 21-1102 fmol mg-1 soluble protein (Kd 1.8 X 10(-9)-3.1 X 10(-8) mol l-1) and those of nucleosolic ER (ERn), 13-526 fmol mg-1 soluble protein (Kd 2.1 X 10(-9)-1.4 X 10(-8) mol l-1). In normal breast tissue ERc levels were 33-640 fmol mg-1 soluble protein (Kd 1.3 X 10(-10)-3.2 X 10(-9) mol l-1), ERn was detected in only 2 samples, 8 and 87 fmol mg-1 soluble protein with Kd 3.2 X 10(-9) and 1.4 X 10(-9) l mol-1 respectively. 17 alpha-ethinyl-13 beta-ethyl-17 beta-hydroxy-4,15-gonadiene-3-one (gestodene), a new synthetic progestogen displaced 3H-oestradiol (3H-E2) from both ERc and ERn in malignant tissue but not in normal breast, or these receptors from endometrial tissue. In competition studies gestodene was approximately 3 times more effective in displacing 3H-E2 from ERc and ERn in malignant breast tissue than the natural ligand. Quantitation of ER by gestodene were ERc, 12-1134 fmol gestodene bound mg-1 soluble protein (Kd 1 X 10(-9)-8.1 X 10(-9) mol l-1); ERn, 17-531 fmol gestodene bound mg-1 soluble protein (Kd 1.6 X 10(-9)-1.1 X 10(-8) mol l-1). L-13-ethyl-17 alpha-ethinyl, 17 beta-hydroxy-gonen-3-one (levonorgestrel) showed no binding to ER in malignant breast, normal breast or endometrial tissue. In circulation both gestodene and levonorgestrel displaced E2 from sex hormone binding globulin more than any of the androgens tested. These results suggest that gestodene is a progestogen with oestrogenic and/or antioestrogenic properties and provide strong evidence for differences in ER from malignant and normal breast tissue.

Over-expression of oestrogen and progesterone receptor (ER and PR respectively) has been reported in malignant breast disease but the techniques employed have been relatively insensitive and prone to underestimations. Using a highly sensitive microassay method, we have estimated ER (n = 39), PR (n = 32) and androgen receptor (AR) (n = 37) in cytosolic and nucleosolic fractions obtained from the same tissue samples. Induction of PR by oestrogen is a widely recognized phenomenon, and a relationship between PR and ER is to be expected. There is evidence that sex-steroids and their receptors act in concert and it is therefore surprising that AR has been poorly studied in malignant breast disease, despite indications that its presence increases hormone responsiveness. Our hypothesis is that, unlike the normal breast, in malignancy a possible deregulation of transcriptional control may lead to an over expression of all three classes of sex-steroid receptor. Using the microassay, we have tested this hypothesis and found not only a greater incidence of ER, AR and PR than previously reported in malignant breast disease, but considerably higher levels of these receptors. In addition, our findings demonstrate that these receptors tend to be expressed simultaneously rather than discretely, supporting the view that a deregulation of transcriptional control may account for the clinical and biological heterogeneity of the disease.