Estrogen mitogenic action. II. Negative regulation of the steroid hormone-responsive growth of cell lines derived from human and rodent target tissue tumors and conceptual implications

Summary In an accompanying report (Moreno-Cuevas, J. E.; Sirbasku, D. A., In Vitro Cell. Dev. Biol.; 2000), we demonstrated 80-fold estrogen mitogenic effects with MTW9/PL2 rat mammary tumor cells in cultures supplemented with charcoaldextran-treated serum. All sera tested contained an estrogen reversible inhibitor(s). The purpose of this report is to extend those observations to additional sex steroid-responsive human and rodent cell lines. Every line tested showed a biphasic response to hormone-depleted serum. Concentrations of ≤10% (v/v) promoted substantive growth. At higher concentrations, serum was progressively inhibitory. With estrogen receptor-positive (ER⁺) human breast cancer cells, rat pituitary tumor cells, and Syrian hamster kidney tumor cells, 50% (v/v) serum caused significant inhibition, which was reversed by very low physiologic concentrations of estrogens. This same pattern was observed with the steroid hormone-responsive LNCaP human prostatic carcinoma cells. Because steroid hormone mitogenic effects are now easily demonstrable using our new methods, the identification of positive results has nullified our original endocrine estromedin hypothesis. We also evaluated autocrine/paracrine growth factor models of estrogen-responsive growth. We asked if insulin-like growth factors I and II, insulin, transforming growth factor alpha, or epidermal growth factor substituted for the positive effects of estrogens. Growth factors did not reverse the serum-caused inhibition. We asked also if transforming growth factor beta (TGFβ) substituted for the serum-borne inhibitor. TGFβ did not substitute. Altogether, our results are most consistent with the concept of a unique serum-borne inhibitor as has been proposed in the estrocolyone model. However, the aspect of the estrocolyone model related to steroid hormone mechanism of action requires more evaluation. The effects of sex steroids at picomolar concentrations may reflect mediation via inhibitor “activated” intracellular signaling pathways.     

Mitogenic regulation of normal and malignant breast epithelium

The multiple roles of both estrogenic and polypeptide regulators of mammary epithelial cell growth are reviewed in this article. Effects of both steroidal and peptide hormones are complex and involve multiple interactions with malignant cells and non-malignant host components. Initial carcinogenesis and progression of mammary epithelium to cancer probably require both proliferative stimuli (estrogen, polypeptide growth factors) and genetic damage. This condition may lead to qualitatively different hormonal responses (hormone-responsive cancer). Estrogens can be shown to induce growth-regulatory polypeptide growth factors and interact with them in hormone-dependent breast cancer. Progression of hormone-dependent (estrogen-responsive) breast cancer to hormone independence probably involves multiple mechanisms, including oncogene activation, loss of the estrogen receptor, or loss of hormone responsivity of other gene products. One direction for further therapies may be blockade of hormonal stimulation and interference with necessary activated or induced components of malignant progression such as oncogenes or polypeptide growth factor-receptor systems.     

Growth factors in breast cancer: mitogenesis to transformation.

While endocrine steroid hormones have been known for many years to regulate normal and malignant mammary epithelium, recent studies have led to an appreciation of polypeptide growth factors as locally-acting autocrine and paracrine effectors. In the current article we summarize what is known about growth factor regulation and action in the normal mammary gland and about perturbations of the steroid-growth factor interplay as cancer progresses. A major theme is that oncogenic activation modulates both regulation of production and function of growth factors in the mammary gland.     

Growth factors and steroid hormone action in endometrial cancer

There is compelling evidence that growth factors are involved in mediating estrogen action in target tissues. The role of growth factors in the development and progression of endometrial cancer is less clear. Steroid hormones can regulate the expression of the transforming growth factors and epidermal growth factor receptors in endometrial cancer cells in culture. It is also possible to demonstrate that these growth factors function in an autocrine fashion to regulate proliferation of endometrial cancer cells in culture. Constitutive expression or overexpression of such autocrine/paracrine factors and/or their receptors may be important in the growth progression of endometrial neoplasia. However, to date the evidence to support the hypothesis is limited.     

Laminin provides a better substrate than fibronectin for attachment,growth, and differentiation of 1003 embryonal carcinoma cells

Summary Culture of cells in hormonally defined media has allowed (a) the demonstration of physiological responses from cells usually unable to express them in vitro and (b) the study of the effects on growth and differentiation of diffusible factors and attachment factors. The embryonal carcinoma line 1003 forms multidifferentiated tumors in vivo but is unable to differentiate in vitro when grown in serum-containing medium. In a defined medium containing insulin, transferrin, selenium, and fibronectin as attachment factors, 1003 cells grow for several generations and differentiate into neurons and embryonic mesenchyme (Darmon et al., 1981, Dev. Biol. 85: 463–473). In the present work the effects of fibronectin and laminin were compared. In the presence of laminin the cells attached and spread better, grew faster, and could be plated at lower densities. Neurite extension was also better under these conditions and most importantly, it was found that laminin induced an important formation of muscular tissue when the cells had been seeded at low densities. Multinucleated myotubes could be stained with antibodies directed against embryonic muscular myosin. Coating the dishes with polylysine or adding FGF or serum-spreading factor to the medium allowed growth of low-density cultures with fibronectin instead of laminin but muscular differentiation was not detected under these conditions. Addition of fibronectin to laminin-containing medium did not inhibit muscular differentiation. Presented in the symposium on Plant and Animal Physiology in Vitro at the 33rd Annual Meeting of the Tissue Culture Association, San Diego, California, June 6–10, 1982. This research was supported in part by grants from the “Centre National de la Recherche Scientifique” (LA 269), the “Délégation Générale à la Recherche Scientifique et Technique,” the Fondation pour la Recherche Médicale Fran?aise,” the “Institut National de la Santé et de la Recherche Medicale,” the “Ligue Nationale Fran?aise centre le Cancer,” and the “Fondation André Meyer.” This symposium was supported in part by the following organizations: Bellco Glass, Inc., California Branch of the Tissue Culture Association, Collaborative Research, Hana Media, Hybridtech, K C Biological, Inc., and Millipore Corporation.     

Ceruloplasmin,a copper-transport protein,can act as a growth promoter for some cell lines in serum-free medium

Summary Ceruloplasmin (Cp) is a copper-transport protein with ferric oxidase activity found in high concentrations in the plasma of all vertebrates. Five cell lines (TR-1, TR-M, TR-ST, TM₃, and TM₄) derived from the testis can be grown in hormone-supplemented serum-free medium. Cp stimulates the growth of four of these five cell lines in serum-free medium. The growth stimulation by Cp is not affected by the addition or deletion of free copper, nor does copper itself elicit any significant growth response. Cp can stimulate growth also in the absence of TF suggesting that it is not acting solely to promote Fe(III)-TF binding. A strong interaction is seen between Cp and high density lipoprotein (HDL), with the presence of either decreasing the growth-promoting activity of the other. It is suggested that these cell lines may provide an ideal system for studying the action of Cp at the cellular level. Presented in the symposium on Plant and Animal Physiology in Vitro at the 33rd Annual Meeting of the Tissue Culture Association, San Diego, California, June 6–10, 1982. This work was supported by NIH P50 Grant HO-13541. I am grateful to Ms. Florence Kaczorowski for her expert technical assistance. This symposium was supported in part by the following organizations: Bellco Glass, Inc., California Branch of the Tissue Culture Association, Collaborative Research, Hana Media, Hybridtech, K C Biological, Inc., and Millipore Corporation.     

Hybridoma monoclonal antibodies in the analysis fo human cell surface antigens

Summary In the present paper, we will summarize studies we have performed on two distinct human lymphocyte cell surface antigens defined by monoclonal antibodies: Leu-1 and HLA-DR. Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association, Washington, D.C., June 7–11, 1981. This work was supported by USPHS-NIH Grants CA-21223, AI-11313, and CA-09302. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

Autocrine/Paracrine Human Growth Hormone-stimulated MicroRNA 96-182-183 Cluster Promotes Epithelial-Mesenchymal Transition and Invasion in Breast Cancer

Human growth hormone (hGH) plays critical roles in pubertal mammary gland growth, development, and sexual maturation. Accumulated studies have reported that autocrine/paracrine hGH is an orthotopically expressed oncoprotein that promotes normal mammary epithelial cell oncogenic transformation. Autocrine/paracrine hGH has also been reported to promote mammary epithelial cell epithelial-mesenchymal transition (EMT) and invasion. However, the underlying mechanism remains largely obscure. MicroRNAs (miRNAs) are reported to be involved in regulation of multiple cellular functions of cancer. To determine whether autocrine/paracrine hGH promotes EMT and invasion through modulation of miRNA expression, we performed microarray profiling using MCF-7 cells stably expressing wild type or a translation-deficient hGH gene and identified miR-96-182-183 as an autocrine/paracrine hGH-regulated miRNA cluster. Forced expression of miR-96-182-183 conferred on epithelioid MCF-7 cells a mesenchymal phenotype and promoted invasive behavior in vitro and dissemination in vivo. Moreover, we observed that miR-96-182-183 promoted EMT and invasion by directly and simultaneously suppressing BRMS1L (breast cancer metastasis suppressor 1-like) gene expression. miR-96 and miR-182 also targeted GHR, providing a potential negative feedback loop in the hGH-GHR signaling pathway. We further demonstrated that autocrine/paracrine hGH stimulated miR-96-182-183 expression and facilitated EMT and invasion via STAT3 and STAT5 signaling. Consistent with elevated expression of autocrine/paracrine hGH in metastatic breast cancer tissue, miR-96-182-183 expression was also remarkably enhanced. Hence, we delineate the roles of the miRNA-96-182-183 cluster and elucidate a novel hGH-GHR-STAT3/STAT5-miR-96-182-183-BRMS1L-ZEB1/E47-EMT/invasion axis, which provides further understanding of the mechanism of autocrine/paracrine hGH-stimulated EMT and invasion in breast cancer.     

Coexpression of PTHrP and PTH/PTHrP receptor in a myoepithelial cell line derived from normal human breast

Parathyroid hormone-related peptide (PTHrP) is the cause of humoral hipercalcaemia of malignancy syndrome (HHM). It is known that the peptide as well as its receptors are widely distributed in many normal organs and tissues, where it influences an array of diverse functions which are realized through paracrine or autocrine pathway. PTHrP is present in large amounts in lactating mammary gland but its function is not fully elucidated. In this study, production of parathyroid hormone-related peptide (PTHrP) by the Hs578Bst cell line corresponding to mammary myoepithelial cells was examined by immunocytochemistry. Using RNA extracted from these cells we analyzed expression of mRNA for PTHrP and for the PTH/PTHrP receptor by RT-PCR. The obtained results demonstrated that Hs578Bst cells produced PTHrP and synthesized mRNA for PTHrP and PTH/PTHrP type I receptor. It provides evidence that myoepithelial cells are target cells for PTHrP. The data support that PTHrP may be an important autocrine/paracrine factor, involved in the regulation of myoepithelial cell function as well as in growth and differentiation of the mammary gland.     

IGFBP-2 and ?5: important regulators of normal and neoplastic mammary gland physiology

The insulin-like growth factor (IGF) axis plays an important role in mammary gland physiology. In addition, dysregulation of this molecular axis may have a causal role in the aetiology and development of breast cancer (BC). This report discusses the IGF axis in normal and neoplastic mammary gland with special reference to IGF binding proteins (IGFBPs) -2 and −5. We describe how these high affinity binders of IGF-1 and IGF-2 may regulate local actions of growth factors in an autocrine and/or paracrine manner and how they also have IGF-independent effects in mammary gland. We discuss clinical studies which investigate both the prognostic value of IGFBP-2 and −5 expression in BC and possible involvement of these genes in the development of resistance to adjuvant endocrine therapies.     

The role of junctional communication in animal tissues

Summary Permeable intercellular junctions are a common feature of most animal tissues. These junctions allow the free exchange of small ions and molecules between all the cells in coupled populations. Such limited syncytial interaction contributes to the integration of individual cells into organized tissues. Presented in the symposium on Molecular and Morphological Aspects of Cell-Cell Communication at the 31st Annual Meeting of the Tissue Culture Association, St. Louis, Missouri, June 1–5, 1980. This symposium was supported in part by Contract 263-MD-025754 from the National Cancer Institute and the Fogarty International Center.     

Endogenous human prolactin and not exogenous human prolactin induces estrogen receptor alpha and prolactin receptor expression and increases estrogen responsiveness in breast cancer cells

Prolactin (PRL) and estrogen act synergistically to increase mammary gland growth, development, and differentiation. Based on their roles in the normal gland, these hormones have been studied to determine their interactions in the development and progression of breast cancer. However, most studies have evaluated only endocrine PRL and did not take into account the recent discovery that PRL is synthesized by human mammary cells, permitting autocrine/paracrine activity. To examine the effects of this endogenous PRL, we engineered MCF7 cells to inducibly overexpress human prolactin (hPRL). Using this Tet-On MCF7hPRL cell line, we studied effects on cell growth, PRLR, ER alpha, and PgR levels, and estrogen target genes. Induced endogenous hPRL, but not exogenous hPRL, increased ER alpha levels as well as estrogen responsiveness in these cells, suggesting that effects on breast cancer development and progression by estrogen may be amplified by cross-regulation of ER alpha levels by endogenous hPRL. The long PRLR isoform was also upregulated by endogenous, but not exogenous PRL. This model will allow investigation of endogenous hPRL in mammary epithelial cells and will enable further dissection of PRL effects on other hormone signaling pathways to determine the role of PRL in breast cancer.     

Growth of MTW9/PL2 estrogen-responsive rat mammary tumor cells in hormonally defined serum-free media

Summary Growth of the estrogen-responsive MTW9/PL2 rat mammary tumor cells was demonstrated in serum-free defined medium (designated DDM-1) formulated with F12-DME (1:1 vol/vol) supplemented with 15 mM HEPES pH 7.4 insulin 10 μg/ml, transferrin 10 μg/ml, sodium selenite 10 ng/ml, triiodo-l-thyronine 0.3 nM, phosphoethanolamine 5 μM, epidermal growth factor (20 ng/ml), 17 β-estradiol 2 nM, and bovine serum albumin 20 μg/ml. In DDM-1, the growth rate was about one-half that seen in serum-containing medium. When ethanolamine (50 μM), glutathione (20 μg/ml), and linoleic acid/bovine serum albumin (150 μg/ml) were added (formulation DDM-2), the growth rate was 80% of serum-containing medium and not seed-density dependent. Deletion of estradiol from DDM-1 or DDM-2 had no effect on growth rate. Also, cells grown in steroid hormone deficient medium for 4 mo. continued to form estrogen-responsive tumors in rats as did cells cultured for the same period in 2 nM estradiol. To investigate autocrine growth factor secretion, a third medium (DDM-3) was prepared by deleting insulin, epidermal growth factor, phosphoethanolamine, estradiol, and both forms of bovine serum albumin from DDM-2. Growth in mitogen-free medium equaled 86% of the serum-stimulated rate and was seed-density dependent; phenol red deletion from DDM-3 had no effect on growth rate. Evidence presented suggests that autocrine factors stimulate growth of the MTW9/PL2 cells in DDM-3, and that this secretion may support the growth of estrogen-responsive cells in culture in the absence of steroid hormone. This work was supported by National Cancer Institute grants CA-38024 and CA-26617 and American Cancer Society grant BC255.     

BG-1 ovarian cell line: An alternative model for examining estrogen-dependent growth in vitro

Summary Examination of estrogen-responsive processes in cell culture is used to investigate hormonal influence on cancer cell growth and gene expression. Most experimental studies have used breast cancer cell lines, in particular MCF7 cells, to investigate estrogen responsiveness. In this study we examined an ovarian cancer cell line, BG-1, which is highly estrogen-responsive in vitro. This observation, plus the fact that the cells are of ovarian rather than mammary gland origin, makes it an attractive alternative model. 17β-Estradiol, epidermal growth factor, and insulin-like growth factor induced proliferation of BG-1 and MCF7 cells. Viability was dependent on these growth factors in BG-1 cells, but not in MCF7 cells. Therefore, we examined the differences between these two cell lines with respect to estrogen and growth factor receptors. BG-1 cells have twice as many estrogen receptors as MCF7 cells, and BG-1 cells have higher insulin-like growth factor-1 and epidermal growth factor receptor levels than MCF7 cells. This may also explain why BG-1 cells proliferate 56% more robustly in serum and show more serum dependence in culture. In both BG-1 and MCF7 cells, epidermal growth factor receptor number is low (<20 000/cell), while insulin-like growth factor-1 receptor level was highest in estrogen receptor positive cell lines. For example, insulin-like growth factor-1 receptor was higher in BG-1 and MCF7 cells than in estrogen receptor negative cells (HeLa>MDA-MB-435>HBL100). In conclusion, BG-1 cells are an excellent model for understanding hormone responsiveness in ovarian tissue and an alternative for examining estrogen receptor-mediated and insulin-like growth factor-1/epidermal growth factor/estrogen cross-talk processes because of their sensitivity to these factors.     

Ovarian substance induces steroid production in cultured granulosa cells

Summary The rat ovary produces an apparently low molecular weight substance that mimics the action of follitropin (FSH) on ovarian granulosa cells in culture. Similar to FSH action, the ovarian substance (OS) induces temporal cell rounding and, later on, intensive progestin production. However, unlike FSH, OS does not induce accumulation of cyclic AMP (cAMP) in the granulosa cells. The ovarian factor cannot be cAMP as its action is not abolished by phosphodiesterase (PDE) treatment. Neither is it a possible PDE inhibitor, as it does not augment cAMP accumulation in granulosa cells or Friend erythroleukemic cells induced by FSH or PGE₁, respectively. The factor is still active after heating for 20 min at 90° C but is rapidly inactivated by alkali treatment. In addition, treatment with various proteases did not abolish the steroidogenic activity. These findings suggest a possible novel intraovarian regulator of the granulosa cell function. Presented in the symposium on Plant and Animal Physiology in Vitro at the 33rd Annual Meeting of the Tissue Culture Association, San Diego, California, June 6–10, 1982. This work was supported by the United States-Israel Binational Science Foundation, Grant 2656/81. This symposium was supported in part by the following organizations: Bellco Glass, Inc., California Branch of the Tissue Culture Association, Collaborative Research, Hana Media, Hybridtech, K C Biological, Inc., and Millipore Corporation.     

Verhulstian analysis of the growth of transplantable mammary tumours in sialoadenectomized mice

In this report we have analysed data published in 1989 by Inui et al. (Incidence of precancerous foci of mammary glands and growth rate of transplantable mammary cancers in sialoadenectomized mice. J. Natl Cancer Inst. 81, 1660) involving the effects of perturbation of the epidermal growth factor (EGF) status of mammary tumour-bearing mice on subsequent volumetric responses. Removal of an endogenous EGF stimulus by surgical ablation of the submaxillary glands, the major EGF-producing organ in mice, produced significantly slower growth of rodent mammary neoplasms, decreased success rate of transplantation, and an increase in the latent period before growth occurred. Administration of i.p. EGF (5 micrograms/mouse/day) to sialadectomized tumour-bearing mice would however, increase tumour growth rate. Data were analysed using the Verhulst equation which indicated that the observed effects on tumour volumetrics by either sialoadenectomy or EGF administration could be interpreted as being produced through paracrine pathways. The use of the Verhulstian analysis indicates that it is possible to analyse neoplastic responses and infer whether paracrine or autocrine pathways are involved.     

GAS6 is an estrogen-inducible gene in mammary epithelial cells

To identify estrogen-responsive genes in mammary glands, microarray assays were performed. Twenty genes were found to be up-regulated while 16 genes were repressed in the 9h estrogen treated glands. The induction of GAS6, one of the genes up-regulated by estrogen, was confirmed by RNase protection assay. Furthermore, GAS6 was also demonstrated to be induced by estrogen in ER positive breast cancer cells. Analysis of GAS6 promoter revealed that GAS6 promoter was regulated by estrogen. An estrogen response element (ERE) was identified in the GAS6 promoter. Electrophoretic mobility shift assay revealed that ERalpha interacted with the ERE in the GAS6 promoter. Chromatin immunoprecipitation demonstrated that ERalpha was recruited to the GAS6 promoter upon estrogen stimulation. These results suggested that GAS6 is an estrogen target gene in mammary epithelial cells.     

MCF-7 Human Breast Cancer Cells Form Differentiated Microtissues in Scaffold-Free Hydrogels

Three-dimensional (3D) cultures are increasing in use because of their ability to represent in vivo human physiology when compared to monolayer two-dimensional (2D) cultures. When grown in 3D using scaffold-free agarose hydrogels, MCF-7 human breast cancer cells self-organize to form directionally-oriented microtissues that contain a luminal space, reminiscent of the in vivo structure of the mammary gland. When compared to MCF-7 cells cultured in 2D monolayer culture, MCF-7 microtissues exhibit increased mRNA expression of luminal epithelial markers keratin 8 and keratin 19 and decreased expression of basal marker keratin 14 and the mesenchymal marker vimentin. These 3D MCF-7 microtissues remain responsive to estrogens, as demonstrated by induction of known estrogen target mRNAs following exposure to 17β-estradiol. Culture of MCF-7 cells in scaffold-free conditions allows for the formation of more differentiated, estrogen-responsive structures that are a more relevant system for evaluation of estrogenic compounds than traditional 2D models.