Signaling pathways regulating aromatase and cyclooxygenases in normal and malignant breast cells

https://doi.org/10.1016/S0960-0760(01)00187-XGet rights and content

Abstract

Aromatase (estrogen synthase) is the cytochrome P450 enzyme complex that converts C19 androgens to C18 estrogens. Aromatase activity has been demonstrated in breast tissue in vitro, and expression of aromatase is highest in or near breast tumor sites. Thus, local regulation of aromatase by both endogenous factors as well as exogenous medicinal agents will influence the levels of estrogen available for breast cancer growth. The prostaglandin PGE2 increases intracellular cAMP levels and stimulates estrogen biosynthesis, and our recent studies have shown a strong linear association between CYP19 expression and the sum of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) expression in breast cancer specimens. Knowledge of the signaling pathways that regulate the expression and enzyme activity of aromatase and cyclooxygenases (COXs) in stromal and epithelial breast cells will aid in understanding the interrelationships of these two enzyme systems and potentially identify novel targets for regulation. The effects of epidermal growth factor (EGF), transforming growth factor-β (TGFβ), and tetradecanoyl phorbol acetate (TPA) on aromatase and COXs were studied in primary cultures of normal human adipose stromal cells and in cell cultures of normal immortalized human breast epithelial cells MCF-10F, estrogen-responsive human breast cancer cells MCF-7, and estrogen-unresponsive human breast cancer cells MDA-MB-231. Levels of the constitutive COX isozyme, COX-1, were not altered by the various treatments in the cell systems studied. In breast adenocarcinoma cells, EGF and TGFβ did not alter COX-2 levels at 24 h, while TPA induced COX-2 levels by 75% in MDA-MB-231 cells. EGF and TPA in MCF-7 cells significantly increased aromatase activity while TGFβ did not. In contrast to MCF-7 cells, TGFβ and TPA significantly increased activity in MDA-MB-231 cells, while only a modest increase with EGF was observed. Untreated normal adipose stromal cells exhibited high basal levels of COX-1 but low to undetectable levels of COX-2. A dramatic induction of COX-2 was observed in the adipose stromal cells by EGF, TGFβ, and TPA. Aromatase enzyme activity in normal adipose stromal cells was significantly increased by EGF, TGFβ and TPA after 24 h of treatment. In summary, the results of this investigation on the effects of several paracrine and/or autocrine signaling pathways in the regulation of expression of aromatase, COX-1, and COX-2 in breast cells has identified more complex relationships. Overall, elevated levels of these factors in the breast cancer tissue microenvironment can result in increased aromatase activity (and subsequent increased estrogen biosynthesis) via autocrine mechanisms in breast epithelial cells and via paracrine mechanisms in breast stromal cells. Furthermore, increased secretion of prostaglandins such as PGE2 from constitutive COX-1 and inducible COX-2 isozymes present in epithelial and stromal cell compartments will result in both autocrine and paracrine actions to increase aromatase expression in the tissues.

Introduction

Cancer is the leading cause of death among women between the ages of 30 and 54 years. An estimated 184,000 new cases of breast cancer will be diagnosed, and 40,800 women in the US will die from breast cancer in 2001. Currently, one out of eight American women will develop breast cancer in her lifetime [1]. The rate of breast cancer incidence has increased 2–3% per year over the past decade in both premenopausal and postmenopausal women. This research focuses on examination of the role of steroid hormones in breast cancer etiology. An estimated 60–70% of human breast cancers are associated with sex hormone exposure. Approximately 60% of all breast cancer patients have hormone-dependent breast cancer, which contains estrogen receptors and requires estrogen for tumor growth. The possible biochemical roles of estrogens in the development of breast cancer remain to be fully elucidated. Estrogens promote growth of estrogen-responsive tumor cells, and production of growth factors by estrogens in established tumor cell culture lines has been demonstrated in the past few years.

The biosynthesis of estradiol (E2) by the enzyme aromatase occurs locally within breast tissue [2], [3], [4], [5], [6], [7], [8], [9]. In addition, increased expression of aromatase cytochrome P450arom is observed in breast cancer tissues, and regulation of aromatase expression is associated with a switch in the major promoter region utilized in gene expression [10], [11]. As a result of the use of the alternate promoter, the regulation of estrogen biosynthesis switches from one controlled primarily by glucocorticoids and cytokines to a promoter regulated through cAMP-mediated pathways. The prostaglandin PGE2 increases intracellular cAMP levels and stimulates estrogen biosynthesis [10], whereas other paracrine or autocrine factors such as IL-1β do not appear to act via PGE2 [12]. Therefore, local production of PGE2 via the cyclooxygenase (COX) isozymes may influence estrogen biosynthesis and estrogen-dependent breast cancer growth.

Prostaglandin G/H endoperoxide synthase, also called COX, is the enzyme which catalyzes the conversion of arachidonic acid to prostaglandins. Two isoforms of this enzyme have been identified, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) [13], [14], [15]. Both isoforms of the COX gene are found at detectable levels in most human tissues, and COX-1, believed to be responsible for synthesis of prostaglandins during homeostasis, is constitutively co-expressed at low levels [16]. In contrast, COX-2 gene expression is an immediate-early response gene that is inducible following stimulation by serum, tumor promoters, mitogens, endotoxin, cytokines, and hormones [17], [18]. In breast cancer, greater amounts of prostaglandins are produced in tumor tissue than normal tissue derived from the same organ [19], [20]. The COX-2 isoform was detected only in breast tumors and was not detectable in normal human breast tissue [21], and COX-2 protein was localized specifically to breast tumor cells.

In our recent study, the levels of mRNA expression of CYP19, COX-1, and COX-2 genes were examined in human breast cancer specimens and normal breast tissue samples using semi-quantitative RT-polymerase chain reaction (PCR) methods [22]. Positive correlations were observed between CYP19 and COX-2 and the greater extent of breast cancer cellularity. Linear regression analysis using a bivariate model shows a strong linear association between CYP19 expression and the sum of COX-1 and COX-2 expression. In addition, we have recently reported that aromatase activity was significantly induced by PGE2 and by agonists for the EP1 and EP2 receptor subtypes in primary human breast stromal cell cultures [23]. This significant relationship between the aromatase and COX enzyme systems suggests that autocrine and paracrine mechanisms may be involved in hormone-dependent breast cancer development via growth stimulation from local estrogen biosynthesis.

The thrust of the research described in this manuscript evaluates the effects of several paracrine and/or autocrine signaling pathways in the regulation of expression of aromatase, COX-1, and COX-2 in breast cells (Fig. 1). Specifically, the effects of epidermal growth factor (EGF, stimulates tyrosine kinase activity), transforming growth factor-β (TGFβ, suppresses growth involving SMAD proteins), and tetradecanoyl phorbol acetate (TPA, stimulates protein kinase C activity) were studied in primary cultures of normal human adipose stromal cells and in cell cultures of normal immortalized human breast epithelial cells (MCF-10F), an estrogen-responsive human breast cancer cell line (MCF-7), and an estrogen-unresponsive human breast cancer cell line (MDA-MB-231). EGF, TGFβ, and TPA acting via their respective signaling pathways have been identified as key components and implicated as underlying factors in regulation of cellular events in breast tumorigenesis. Investigations on the potential roles of these pathways in expression of aromatase and COXs in stromal and epithelial breast cells may aid in elucidation of their importance in breast cancer development.

Section snippets

Chemicals, biochemicals, and reagents

Radiolabeled [1β-3H]-androst-4-ene-3,17-dione was obtained from NEN Life Science Products (Boston, MA). Recombinant human EGF and TPA (also known as phorbol myristate acetate, PMA) were obtained from Gibco BRL (Grand Island, NY). Recombinant human TGFβ was obtained from R&D Systems (Minneapolis, MN). Steroids were purchased from Steraloids (Wilton, NH), and all other chemicals were obtained from Aldrich (Milwaukee, WI). Enhanced chemiluminescent kit (ECL) was obtained from Roche Molecular

Regulation of COX-1 and COX-2 in mammary adenocarcinoma cancer cell lines

Selected agonists of signaling pathways associated with tumorogenesis were used to assess COX isozyme inducibility in the estrogen-responsive MCF-7 and estrogen-independent MDA-MB-231 cell lines. A 24 h dose with 50 pM TGFβ or 4 nM EGF did not alter COX-1 or COX-2 levels relative to untreated controls. However, the phorbol ester, TPA, which stimulates PKC pathways, induced COX-2 levels by 75% in MDA-MB-231 cells (Table 1). The estrogen-responsive MCF-7 cells showed no significant change in COX-1

Discussion

The effects of EGF, TGFβ, and TPA on the regulation of expression of aromatase, COX-1, and COX-2 in breast cells were examined in primary cultures of normal human adipose stromal cells and in cell cultures of normal immortalized human breast epithelial cells MCF-10F, estrogen-responsive human breast cancer cells MCF-7, and estrogen-unresponsive human breast cancer cells MDA-MB-231. This research strategy with EGF, TGFβ, and TPA enables comparisons of mitogenic versus

Acknowledgements

This research is supported by NCI Grant R01 CA73698 and DOD Grant DAMD 17-00-1-0388. The authors would like to thank Dr. Gilles-Eric Seralini of the University of Caen, Caen, France for the equine aromatase polyclonal antibody and Dr. Yasuro Sugimoto, The Ohio State University, for contributions of primers and competitors for RT-PCR.

References (34)

  • R.W. Brueggemeier et al.

    Correlation of aromatase and cyclooxygenase gene expression in human breast cancer specimens

    Cancer Lett.

    (1999)
  • R.W. Brueggemeier et al.

    Molecular pharmacology of aromatase and its regulation by endogenous and exogenous agents

    J. Steroid Biochem. Mol. Biol.

    (2001)
  • A.L. Quinn et al.

    Effects of matrix components on aromatase activity in breast stromal cells in culture

    J. Steroid Biochem. Mol. Biol.

    (1999)
  • M. Dowsett et al.

    Control of aromatase in breast cancer cells and its importance for tumor growth

    J. Steroid Biochem. Mol. Biol.

    (1993)
  • M.J. Reed et al.

    Control of aromatase activity in breast cancer cells: the role of cytokines and growth factors

    J. Steroid Biochem. Mol. Biol.

    (1993)
  • American Cancer Society, Facts and Figures,...
  • E.R. Simpson et al.

    Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis

    Endocrinol. Rev.

    (1994)
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