Karen Huff

Autocrine and paracrine growth regulation of human breast cancer

SummaryWe consider the hypothesis that estrogen control of hormone dependent breast cancer is mediated by autocrine and paracrine growth factors secreted by the breast cancer cells themselves. Though we show direct, unmediated effects of estrogen on specific cell functions, we also provide evidence that human breast cancer cells secrete a collection of growth factors (IGF-I, TGFα, TGFβ, a PDGF-like competency factor, and at least one new epithelial colony stimulating factor). Some of these are estrogen-regulated in hormone dependent cells, and are constitutively increased in cells which acquire independence either spontaneously or byras transfection. Collectively, the secreted growth factors are capable of promoting tumor formation by MCF-7 cells in nude mice, though not to the same extent as estrogens. There would seem to be potential for clinical intervention in the autocrine and paracrine control of breast cancer cells, including some cells which are no longer dependent on estrogens.

Estrogen receptor status: an important variable in predicting response to endocrine therapy in metastatic breast cancer

Abstract The influence of estrogen receptor status on response rate to endocrine therapy in 85 patients with metastatic breast cancer was determined in a retrospective study. The specific purpose of this study was to assess the role of estrogen receptor determinations in the light of a host of clinical variables known or suspected to influence response rates to endocrine therapy. Thirty-four of 52 patients whose tumors contained significant amounts of estrogen receptor (> 10 fmole/mg cytoplasmic protein) had objective responses to endocrine therapy while only 333 patients whose tumors did not possess estrogen receptor ( 10 fmole/mg cytoplasmic protein) responded ( P 0.0001 ). A quantitative relationship was found between the amount of estrogen receptor and response rate. The quantity of estrogen receptor was not associated with the duration of response. The predictive value of the estrogen receptor assay was not associated with the receptor dissociation constant. Prior treatment with endocrine or chemotherapy did not diminish the ability of estrogen receptor determinations to predict response to subsequent endocrine therapy. Response rate in estrogen receptor positive tumors was not affected by extent of disease, site of involvement with metastatic tumor, or prior therapy. We conclude that these negative prognostic factors are less important in predicting response to endocrine therapy than estrogen receptor values.

Autocrine and paracrine growth regulation of human breast cancer.

Previous work from our laboratory has demonstrated that human breast cancer (BC) cells in culture can be stimulated by physiologic concentrations of estrogen. In an effort to further understand this process, we have examined the biochemical and biological properties of proteins secreted by human BC cells in vitro. We have developed a defined medium system which simultaneously allows the collection of factors secreted by the BC cells, facilitates their purification and allows for an unequivocal assay of their effect on other BC cells. By both biochemical and radioimmunoassay procedures, MCF-7 cells secrete large quantities of IGF-I-like activity. The cells contain receptors for IGF-I and are stimulated by physiologic concentrations of IGF-I. Multiple additional peaks of growth stimulatory activity can be obtained by partial purification of conditioned media from human BC cells by sequential dialysis, acid extraction and Biogel P60 chromatography. These peaks are induced up to 200-fold by physiologic concentrations of estrogen. Several of these peaks cross-react in a radioreceptor assay with EGF and are thus candidates for transforming growth factors. Monoclonal antibodies (MCA) have been prepared which react with secreted proteins from the MCF-7 cells. One of these MCAs binds to material from MCF-7 and ZR-75-1 hormone-dependent BC cells only when these two lines are treated with estrogen but reacts with conditioned medium from several other hormone-independent cell lines in the absence of estrogen stimulation. This MCA is currently undergoing further characterization and evaluation of its biological potency. We conclude that with estrogen stimulation, hormone-dependent human BC cells secrete peptides which when partially purified can replace estrogen as a mitogen. Their role as autocrine or paracrine growth factors and their effects on surrounding nonneoplastic stroma may suggest a means of interfering with tumor proliferation.

Changes in multiple or sequential estrogen receptor determinations in 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 < 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. Cancer 45:792‐794, 1980.

Growth regulatory peptide production by human breast carcinoma cells

The mechanisms by which human breast cancers regulate their own growth have been studied by us in an in vitro model system. We showed that specific growth factors (IGF-I, TGF alpha, PDGF) are secreted by human breast cancer cells. A variety of experiments suggest that they are involved in tumor growth and progression. These activities are induced by estradiol in hormone-dependent breast cancer cells and secreted constitutively by estrogen-independent cells. Concentrates of conditioned medium derived from breast cancer cells can induce the growth of hormone-dependent cells in vivo in athymic nude mice. Hormone-dependent breast cancer cells also secrete TGF beta. TGF beta is growth inhibitory. Growth inhibitors such as antiestrogens or glucocorticoids increase TGF beta secretion. An antiestrogen-resistant mutant of MCF-7 cells does not secrete TGF beta when treated with antiestrogen, but is growth inhibited when treated with exogenous TGF beta. Thus, TGF beta functions as a negative autocrine growth regulator and is probably responsible for some of the growth inhibitory effects of antiestrogens.

A demonstration of androgen and estrogen receptors in a human breast cancer using a new protamine sulfate assay.

Separate receptors for estrogen and androgen are demonstrated in cells from metastic human breast cancer. By criteria of binding affinity, number of binding sites, and specificity of the receptor for different steroids, the receptors are shown to be distinguishable. The protamine sulfate receptor assay technique employed allows both kinds of receptor to be quantified conveniently and reproducibly without interference by plasma steroid‐binding components.

Estrogen receptor values in patients with benign breast disease.

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 (>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 < 0.01). Twenty of 46 (43%) premenopausal patients had benign tumors which were estrogen receptor positive compared to zero of 8 postmenopausal patients (p < 0.05).

Human breast cancer responsive to androgen in long term tissue culture

ALTHOUGH a significant role has been established for oestrogen in the growth of some animal1,2 and human3,4 mammary carcinoma, evidence for androgen dependency of human breast cancer has remained equivocal. Although the existence of a mouse mammary carcinoma which shows modest androgen responsiveness5 has supported the notion that some human breast cancer might also be dependent on androgens, no direct proof has been available. We have described our work characterising the oestrogen responsiveness of MCF-7, a human breast cancer cell line maintained in tissue culture for over 3 yr (ref. 6). This cell line was shown to contain oestrogen receptor, to be killed by antioestrogens, and stimulated by physiological concentrations of oestradiol. We now describe further investigations of this cell line which reveal that in addition to oestrogen responsiveness, this cell line shows threefold enhancement of precursor incorporation into macromolecules by androgens using serum-free conditions which preclude stimulatory effects of other trophic hormones. Furthermore, this stimulation by androgens seems to be mediated by interaction with a cytoplasmic androgen receptor protein which is clearly differentiable from the oestrogen receptor also found in these cells. Aside from defining an interesting new system in which the action of androgen can be studied in a human cell line in tissue culture, the present study provides unequivocal evidence for the androgen responsiveness of some human breast cancer at least in vitro.

Progesterone and glucocorticoid interactions with receptor in breast cancer cells in long-term tissue culture.

I t has long been appreciated that the clinical course of breast cancer can be altered by a variety of hormonal manipulations.’ Despite an awareness that a variety of ablative and additive hormonal therapies may be accompanied by objective tumor regressions in some cases, the exact mechanism whereby these therapeutic interventions cause tumor regression is unclear. While clinical studies of breast cancer and investigation of hormone-dependent breast cancer in animals have contributed much to our understanding, the inherent complexities of studying hormonal regulation of tumor growth in vivo led us to attempt to investigate this process in long-term tissue culture lines of human breast cancer. By using defined media in which single hormones could be added to well-characterized uniform populations of cells, we hoped to avoid some of the difficulties intrinsic in more complexly organized systems. Thus, effects of hormone mediated by alterations in levels or activity of other trophic hormones, or effects of hdrmone on supporting stromal elements or the immune system, could be eliminated from consideration. I n addition, some recent work has suggested that determination of progesterone receptor activity may be helpful in predicting which patients with breast cancer might benefit from hormonal therapy,2 much as the knowledge of estrogen receptor levels has also been shown to be of value.) We have previously reported that some lines of human breast cancer in long-term tissue culture contain glucocorticoid and progesterone receptor^,^.^ and this finding has been confirmed by others.6 Because of the biologic interactions of glucocorticoids and progesterone, the availability of new compounds, such as R 5020, for receptor quantification (see below), and the need to establish assays that would unambiguously measure each of these receptors independently, we undertook a series of experiments aimed at studying progesterone-glucocorticoid interactions with human breast cancer in long-term culture. To substantiate the likelihood that observations made on such tissue culture systems bear any relevance to breast cancer in vivo. it is important to review the criteria that suggest that these cells resemble those found in the tumor in vivo. Four lines of evidence suggest that these cells,* which were initially derived from breast cancer specimens, continue to manifest properties characteristic of the primary tumor. First, morphologically, all of the cell lines employed have epithelial characteristics. In some cell lines, such as the MCF-7,’ the tendency of

Estrogens Regulate Production of Specific Growth Factors in Hormone‐Dependent Human Breast Cancer

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.