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Progesterone receptor (PR) assay was performed on tumor specimen obtained from 145 women consisting of 82 women with advanced breast cancer and 73 women with early breast cancer (EBC). Advanced breast cancer patients consisted of 50 (61%) of 82 women with advanced primary breast cancer (APBC) and 32 (39%) women with recurrent or secondary breast cancer (SBC). Of 82 advanced breast cancer, 35 (42.7%) were PR+. These consisted of 21 APBC and 14 SBC patients. 43 (59%) of 73 EBC patients had PR+ tumors. Quantitative value of PR correlated with age in overall and in all groups. There was also a significant difference in PR quantitative value between PR+ tumors and PR- tumors. Finally premenopausal APBC patients with PR+ tumors had a 2:1 survival advantage over those with PR- tumors. PR assay is as an invaluable a tool as ER assay in the management of breast cancer.

Estrogen receptor (ER) analyses are widely used in the management of patients with breast cancer. Few data are available concerning variations in ER as a function of time and therapy. We examined patients in whom multiple biopsies and receptor analyses were either performed simultaneously or over time with or without intervening chemotherapy or endocrine therapy. In 27 patients with simultaneous assays from different metastatic sites, concordant results were obtained in 23 (85%) with respect to classifying a tumor as ER+ or ER-. In 34 patients, multiple assays were performed over time. The receptor concentrations were concordant in patients receiving no intervening therapy. In 10 patients receiving hormonal therapy, the median receptor concentration changed from 66 femtomoles per mg cytoplasmic protein pretherapy to 8 femtomoles post therapy (P less than 0.05). This change suggested that hormonal therapy selectively eliminates ER+ cells. In 19 patients receiving intervening chemotherapy, the median receptor concentrations pre and post therapy were similar. We conclude that simultaneous assays from multiple metastatic sites are highly concordant and unlikely to change over time without intervening therapy, but marked changes occur following therapy with a decrease in receptor concentration after endocrine therapy.

Specific binding proteins are thought to modulate the effects of IGF-I. Previous work has demonstrated that media conditioned by human breast cancer cells contains IGF-I binding activity. Radiolabelled IGF-I incubated with serum-free conditioned media from the breast cancer cell line MDA-MB 231 eluted with an apparent M.W. of 35-40 kDa when analyzed by gel filtration chromatography at pH 7.4. The M.W. of this binding activity corresponded to that of BP-25, a binding protein cloned from the hepatocellular carcinoma cell line HepG2. Two breast cancer cell lines, MDA-MB 231 and Hs578T, were found to express BP-25 RNA. Specific BP-25 radioimmunoassay detected BP-25 production in the conditioned media of these two cell lines. Immunoprecipitation confirmed that metabolically labelled MDA-MB 231 released 30 kDa BP-25 into its medium. This study demonstrates that some breast cancer cells express the IGF-I binding protein, BP-25.

In a retrospective study we determined the relation between estrogen receptors and the response rate to cytotoxic chemotherapy in 70 patients with metastatic breast cancer. Thirty-four of 45 patients with low or absent estrogen-receptor values (less than 10 fmol per milligram of cytoplasmic protein) had objective responses to chemotherapy, whereas only three of 25 patients with higher values (greater than 10 fmol per milligram of cytoplasmic protein) responded (P less than 0.0001). There were no statistically significant differences between the two groups in age, menopausal status, disease-free interval, Karnofsky index or prior therapy. Differences in sites of involvement or type of chemotherapy did not account for the increased response rate in receptor-negative patients. We conclude that estrogen-receptor values are an important predictor of response to cytotoxic chemotherapy in metastatic breast cancer.

The influence of steroid hormone receptors on response rate to cytotoxic chemotherapy in 70 patients with metastatic breast cancer was determined in a retrospective study. We have previously reported that 34 of 45 patients with tumors containing low or absent estrogen-receptor values had objective responses to chemotherapy while three of 25 patients with positive estrogen-receptor tumors responded. In the present study, 22 of 34 patients with low or absent progesterone-receptor tumors had an objective response to cytotoxic chemotherapy, while none of eight patients with a positive progesterone-receptor tumor responded (P less than 0.05). Patients having tumors with a negative estrogen receptor and a negative progesterone receptor had a response rate of 88% (21 of 24 patients). There were three patients whose tumors were estrogen-receptor negative but progesterone-receptor positive; none had a response to chemotherapy. Chemotherapy response was not associated with the presence or absence of either androgen or glucocorticoid receptor. We conclude that progesterone-receptor values in addition to estrogen-receptor status may prove to be important correlates of response to cytotoxic chemotherapy in metastatic breast cancer. Androgen- and glucocorticoid-receptor analyses are not helpful in predicting response to chemotherapy.

Laboratory evidence is presented that estrogens are able to induce the production of numerous growth factors which can act in an autocrine or paracrine fashion in estrogen dependent breast cancer. Estrogen independent tumors can produce these same growth factors constitutively and so escape the need for estrogen stimulation. Growth inhibitory factors such as TGF-beta can also be controlled by estrogens and antiestrogens. It is unclear at present, however, how much of the cytostatic effect of antiestrogens in vivo is explained by the production of growth inhibitors. The overall control of breast cancer growth is mediated by the combined effects of these growth stimulatory and inhibitory factors in both breast stroma and epithelium. Interruption of the action of growth factors and the use of growth inhibitors may provide opportunities for new approaches to the treatment of breast cancer.

Somatomedin activity is required for proliferation of normal cells; recently somatomedin activity in the cellular environment was shown to be necessary for expression of the transformed phenotype. We demonstrate here that authentic serum-derived insulin-like growth factor-I (IGF-I) stimulates the proliferation of four human breast cancer cell lines, MCF-7, MDA-MB-231, ZR-75-1, and Hs578T in serum-free monolayer culture and that each of these lines produces and secretes an IGF-I-related growth factor. The two highly tumorigenic estrogen-independent cell lines, MDA-MB-231 and Hs578T, produced 2- to 10-fold more IGF-I than did two estrogen responsive cell lines, MCF-7 and ZR-75-1, which are not tumorigenic in the absence of estrogen. These breast cancer cells also secrete a Mr 50,000 binding activity which partially obscured detection of IGF-I by radioimmunoassay. Acid-ethanol extraction allowed dissociation of the high molecular weight complex; whereupon, fully immunoreactive IGF-I comigrated on acid gel exclusion chromatography with authentic human serum-derived IGF-I. Radioimmunoassay displacement curves for breast cancer cell line-derived IGF-I were parallel to those for authentic IGF-I. Northern blot analysis of mRNA from four breast cancer cell lines demonstrated specific hybridization with a human IGF-I probe corresponding to one of the two major transcripts seen in human liver mRNA. These data suggest that breast cancer cell line-derived IGF-I is similar to liver-synthesized, serum-derived IGF-I.

After nuclear translocation of estrogen receptors in MCF-7 human breast cancer cells, a processing takes place resulting in a 30-70% decline in the number of estradiol-binding sites measured in nuclear extracts. We have investigated the mechanism of estrogen receptor processing and obtained evidence that multiple events are involved. We confirm, as others have shown previously, that processing involves a decrease in the amount of estradiol binding in MCF-7 cells. In addition, evidence is provided for the generation of a rapidly dissociating population of estradiol-binding sites as an early event in processing. There is a single, slowly dissociating population of estrogen binding sites when MCF-7 cells are exposed to estradiol in the presence of actinomycin D, an inhibitor of receptor processing. One hour after the addition of sufficient estradiol to induce receptor processing, an additional, more rapidly dissociating population of estrogen binding sites is detected. When cells are exposed to estradiol and ethidium bromide, a drug which shares many actions with actinomycin D, but does not inhibit receptor processing, the rapidly dissociating population of estradiol-binding sites is again observed. Significantly, the loss of estradiol-binding sites from MCF-7 cells associated with processing between 1 and 6 h of estradiol exposure, occurs exclusively from the rapidly dissociating population of sites. Whole cell equilibrium-binding assays were performed with MCF-7 cells after 30 min or 5 h of estradiol exposure to determine whether the detected changes in estradiol dissociation reflected affinity changes in a subpopulation of estrogen-binding sites. Although the number of sites detected per cell varied with the assay method employed, binding to a single saturable class of higher affinity sites is always observed. High affinity estradiol-binding sites were reduced by 45% after a 5-h incubation with estradiol in both assay methods. The loss of estradiol binding during processing may therefore be explained by the conversion of certain high affinity estrogen receptors to a rapidly dissociating form which then fails to rebind hormone, or undergoes subsequent reactions that destroy hormone binding activity. Additionally, after 6 h of exposure to estradiol, the remaining receptor-bound estradiol dissociates from intact cells with a rate increased by 50% over that seen from the slow dissociating receptors present at 1 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Human breast cancer cells in long-term tissue culture are responsive to estrogens as assessed by activation of specific gene products. A group of estrogen-regulated, secreted growth factors are responsible for the proliferative responses of these cells both in vitro and in vivo.

Tissue specimens from 55 female patients with benign breast disease were assayed for estrogen receptor. Twenty-one of 55 patients (38%) had tumors which contained significant amounts of estrogen receptor (greater than 10 femtomoles/mg protein). Fibroadenomas possessed estrogen receptor more frequently than fibrocystic disease or other benign breast tumors. Estrogen receptor positivity did not correlate with laterality of the tumor; location or size of the largest nodule. Patients with estrogen receptor positive tumors had a mean age of 26.9 years compared to 36.4 years for patients with estrogen receptor negative tumors (p less than 0.01). Twenty of 46 (43%) premenopausal patients had benign tumors which were estrogen receptor positive compared to zero of 8 postmenopausal patients (p less than 0.05).

Activity of secreted plasminogen activator (PA) by ZR-75-1 human breast cancer cells in culture is shown to be altered by the addition of physiologically relevant concentrations of the hormones 17 beta-estradiol (E2), insulin, and dexamethasone. After 48 h, E2 stimulated PA activity 6-fold at concentrations as low as 10(-12) M. This stimulation was prevented by the addition of actinomycin D and cycloheximide. The antiestrogen tamoxifen reduced estrogen stimulation of PA, but had slight stimulatory effects on PA secretion by itself. Insulin (5 X 10(-10) M) induced a 2-fold increase in PA activity. Effects of insulin and E2 were additive, suggesting independent sites of control of PA production. Dexamethasone (10(-8) M) decreased PA activity by 20%, but did not inhibit cell growth at the concentration tested. These data suggest that secreted PA activity is differentially regulated by hormones and that effects of PA and growth do not occur in parallel.

LY 117018 is a nonsteroidal compound which has been shown to antagonize more effectively than tamoxifen the effects of estradiol in rats and mice. We have further defined the properties of this compound on cell growth and progesterone receptor (PR) induction and have compared inhibitory effects to relative binding affinity in a human breast cancer cell line. In MCF-7 cells, LY 117018 [( 6-hydroxy-2-(p-hydroxyphenyl)-benzo(b) thien-3-yl-p-(2-(pyrrolidinyl)ethoxy phenyl ketone] is devoid of estrogenic effects, has a greater affinity for the estrogen receptor (ER) than tamoxifen at 4 C, and is 100-1000 times more potent than tamoxifen at inhibiting cell growth. While tamoxifen has been reported to induce PR, a purported estrogen effect, the LY 117018 compound failed to induce PR using a broad range of concentrations. The direct antagonistic effect of LY 117018 was demonstrated by the use of varying concentrations of LY 117018 and estradiol concomitantly. We conclude that LY 117018 is a very effective antiestrogen devoid of any stimulatory effects among these parameters tested.

The possibility of an association between steroid hormone receptor status and disease-free interval was examined in 292 patients with breast cancer. Estrogen receptor positivity was associated with a prolonged disease-free interval. This association was independent of age, menopausal status, tumor size, or nodal status. There was no association between the presence or absence of progesterone, androgen, or glucocorticoid receptor and disease-free interval.

The distribution and frequency of steroid hormone receptors are described 329 patients with breast cancer. The distribution of each of the steroid hormone receptors is unimodal with a progressive increase in the proportion of patients positive at lower receptor values. Receptor values expressed as fmol/mg cytoplasmic protein are well correlated with values expressed as fmol/mg breast tumor. Estrogen receptor was positive in 53% of the patients; progesterone receptor was positive in 38% of the patients; glucocorticoid receptor was positive in 52% of the patients; and androgen receptor was positive in 31% of the patients. The type of tissue assayed did not affect steroid hormone receptor positivity. For primary tumors, there was no correlation between steroid hormone receptor positivity and location of the tumor in the breast, size of the tumor, or extent of the disease. Each of the steroid hormone receptors was positively associated with each of the other steroid hormone receptors. Estrogen receptor was correlated with menopausal status and axillary nodal status, but these correlations did not exist for the other steroid hormone receptors. Estrogen receptor was not correlated with age after adjustment for menopausal status. The other steroid hormone receptors were not correlated with age.

MCF-7 human breast cancer cells have been studied for hormonal regulation of secretion of an insulin growth factor-I (IGF-I)-related growth factor. 17 beta-Estradiol, which is required for tumorigenesis of the cell line in the nude mouse and which stimulates proliferation in vitro, was able to significantly induce IGF-I secretion at 10(-13) M, with maximal induction at 10(-11) M. Under optimal conditions IGF-I could be induced 4-fold after 4 days. Demonstration of estrogenic stimulations required removal of phenol red, a weak estrogen, from the cell culture medium. In addition to estrogen, insulin, epidermal growth factor, and transforming growth factor alpha induce both cellular proliferation and IGF-I secretion, while growth inhibitory antiestrogens, transforming growth factor beta, and glucocorticoids have the opposite effect. In each case, modulations in IGF-I secretion preceeded effects on cellular proliferation. IGF-I was not regulated by human GH, basic fibroblast growth factor, platelet-derived growth factor, or PRL, none of which affected proliferation rate. Thus, regulation of IGF-I secretion in human breast cancer is controlled by different hormones from those previously reported in human fibroblasts. Regulation of IGF-I by neither estrogen nor antiestrogen was associated with changes in steady-state mRNA levels; thus regulation may occur at a step beyond mRNA. We conclude that IGF-I production is tightly coupled to growth regulation by estrogens, antiestrogens, and other hormones and may contribute to autocrine and/or paracrine growth regulation by these agents in breast cancer.

The MCF-7 human breast cancer cell line responds to estrogen stimulation in vitro by increased secretion of growth factors and proliferation and in vivo by tumor formation in the nude mouse. To test a possible role of growth factor secretion in expression of the tumorigenic phenotype, we stably transfected MCF-7 cells with the v-Ha-ras oncogene to produce the MCF-7ras cell line. The MCF-7ras cell line was tumorigenic in the absence of estrogens and secreted 3- to 5-fold elevated levels of a high molecular weight form of a type alpha transforming growth factor-like growth factor, type beta transforming growth factor, and insulin-like growth factor I. MCF-7ras cells, in contrast to MCF-7, were less sensitive to further growth stimulation by estrogen, type alpha transforming growth factor, and insulin-like growth factor I and showed little change in receptor levels for these hormones. Conditioned medium from MCF-7ras cells as well as two of its component growth factors (insulin-like growth factor I and type alpha transforming growth factor) replaced estrogen in stimulating MCF-7 colony formation in vitro. A coordinate increase in growth factor secretion by human breast cancer may contribute to its escape from estrogen dependence.

MCF-7 human breast cancer cells release polypeptides related to insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) into serum-free culture medium (CM). Treatment of MCF-7 cells with 17 beta-estradiol, which is required in vivo for MCF-7 tumor growth in the nude mouse and stimulates the MCF-7 growth rate in vitro, resulted in selectively enhanced growth factor activities in CM. Autostimulatory growth-promoting activity was elevated at least 2-fold, and EGF-like polypeptides were elevated 5-fold but IGF-I immunoreactivity was not elevated. Several species of estrogen-induced receptor-reactive EGF-like polypeptides, suggestive of high molecular weight transforming growth factor alpha, were detected after gel exclusion chromatography of CM extracted with 1 M acetic acid. A 30,000 mol wt peak of EGF receptor competing activity comigrated with a peak of autostimulatory and fibroblast-transforming activity. It is possible that estradiol stimulation of MCF-7 growth and/or tumor formation may depend on induction of EGF-related polypeptide growth factors. EGF-I- and EGF-related polypeptides may act together as autocrine or paracrine growth factors in breast cancer.