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Aromatase Overexpression Induces Malignant Changes in Estrogen Receptor a Negative MCF-10A Cells

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Aromatase Overexpression Induces Malignant Changes in Estrogen Receptor a Negative MCF-10A Cells Oncogene (2013) 32, 5233–5240 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE Aromatase overexpression induces malignant changes in estrogen receptor a negative MCF-10A cells J Wang1, JJ Gildea2 and W Yue1 Estrogen is a risk factor of breast cancer. Elevated expression of aromatase (estrogen synthase) in breast tissues increases local estradiol concentrations and is associated with breast cancer development, but the causal relationship between aromatase and breast cancer has not been identified. Accumulating data suggest that both estrogen receptor (ER)-dependent and -independent effects are involved in estrogen carcinogenesis. We established a model by expressing aromatase in ERa À MCF-10A human breast epithelial cells to investigate ERa-independent effects of estrogen in the process of malignant transformation. Overexpression of aromatase significantly increased anchorage-independent growth. Parental- or vector-expressing MCF-10A cells did not form colonies under the same conditions. The anchorage-independent growth of MCF-10Aarom cells can be completely abolished by pre- treatment with the aromatase inhibitor, letrozole. Neither MCF-10Aarom nor MCF-10Avector cells grown in monolayer were affected by short-term exposure to estradiol. Enhanced motility is another characteristic of cellular transformation. Motility of MCF-10Aarom cells was increased, which could be inhibited by letrozole. Increases in stem cell population in breast cancer tissues are associated with tumor recurrence and metastasis. CD44high/CD24low is a stem cell marker. We found that CD24 mRNA levels were reduced in MCF-10Aarom cells compared with those in parental- and vector-transfected cells. By examining individual clones of MCF-10Aarom with various aromatase activities, we found that the CD24 mRNA levels were inversely correlated with aromatase activity. The ability of MCF-10Aarom cells to form mammospheres in the absence of serum was increased. Our results suggest that overexpression of aromatase in MCF-10A cells causes malignant transformation. Estrogen metabolite-mediated genotoxicity and induction of a stem cell/progenitor cell population are possible mechanisms. These studies provide additional evidence for ERa-independent mechanism(s) in estrogen carcinogenesis and implicate superiority of aromatase inhibitors to antiestrogens for breast cancer prevention. Oncogene (2013) 32, 5233–5240; doi:10.1038/onc.2012.558; published online 26 November 2012 Keywords: aromatase; MCF-10A; estrogen; breast cancer; stem cell INTRODUCTION are characteristic of those in women, which predispose to later Long-term exposure to endogenous and exogenous estradiol development of breast cancer. increases the risk of breast cancer in women.1–3 A key piece of Overexpression of aromatase increases tissue estrogen levels. evidence is that bilateral oophorectomy before the age of 35 years The precise mechanism by which estrogen causes breast cancer is 4,5 reduces the lifetime incidence of breast cancer by 75%. Post- not fully understood. A widely accepted concept is that E2, acting menopausal women in the highest quintile of plasma free through estrogen receptor a (ERa), stimulates cell proliferation estradiol (E2) experienced at least a 2.58-fold higher rate of and initiates mutations that occur as a function of errors during breast cancer over the ensuing 10 years compared with those in DNA replication. The promotional effect of E2 then supports the 1,6 the lowest quintile. Inhibition of E2 synthesis with aromatase growth of cells harboring mutations, which accumulate until inhibitors or abrogation of its action with antiestrogens prevents cancer ultimately results. Our studies and those of others the development of contralateral breast cancer during adjuvant suggested that ERa-independent, metabolite-mediated mechan- therapy.7–9 isms are also involved in the process of estrogen carcinogen- 14–17 The aromatase enzyme catalyzes the rate-limiting step in the esis. Increased local E2 concentrations due to aromatase estrogen synthesis pathway. Overexpression of aromatase is expression would increase the levels of oxidative metabolites of associated with cell proliferation of both normal and malignant E2, which cause DNA damage and eventually initiate breast cancer. breast tissues. Increased aromatase expression has been reported Stem cells are defined by their capacity for self-renewal and in breast epithelium, skin fibroblasts and peripheral lymphocytes differentiation into cell lineages present in a specific tissue. The in patients with familial or non-familial gynecomastia.10,11 common characteristics of stem cells and tumor cells imply that Aromatase overexpression occurs in three independent mouse cancer may be originated and sustained by a small population of hyperplastic alveolar nodule (HAN) models of breast cancer,12 as a stem-like cells. The long lifespan of a stem cell would allow the result of an integration mutation in the aromatase gene. accumulation of mutations and epigenetic changes required for Overexpression of aromatase in the mammary glands of neoplastic transformation. Their self-renewal properties would transgenic mice induces pre-malignant lesions.13 These lesions allow expansion of the cells carrying mutant genes. It has been 1Department of Medicine, University of Virginia Health System, Charlottesville, VA, USA and 2Department of Pathology, University of Virginia Health System, Charlottesville, VA, USA. Correspondence: Dr W Yue, Department of Medicine, University of Virginia Health System, Fountaine Research Park, PO Box 801416, Charlottesville, VA 22908, USA. E-mail: [email protected] Received 29 June 2012; revised 2 October 2012; accepted 4 October 2012; published online 26 November 2012 Aromatase and breast epithelial transformation J Wang et al 5234 12 7 0.8 10 0.7 6 8 0.6 5 mg/h) 0.5 4 6 -1 0.4 3 4 0.3 (pmol/mg/h) (pmol/mg/h) 0.2 2 2 1/v (pmol Aromatase activity Aromatase activity 0.1 1 0 0.0 0 0 5 10 15 20 25 30 35 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 10-1010-910-810-710-6 Δ4A (nM) 1/s (nM-1) Letrozole (M) 10 70 70 E1+E2 H2O T+A 60 60 8 50 50 6 40 40 4 30 30 20 20 2 10 10 % radioactivity recovered % radioactivity recovered % radioactivity recovered 0 0 0 Figure 1. Aromatase activity in MCF-10Aarom cells determined by tritiated water release assay (a–c) and product isolation (d–f). (a) Substrate saturation curve of aromatase activity. (b) Lineweave–Burk reciprocal plot. (c) Dose-dependent inhibition of aromatase activity by letrozole. (d) Conversion of testosterone to E1 and E2 (E1 þ E2). (e) Percent radioactivity recovered from water phase. (f) Percentage of aromatase substrate testosterone and androstenedione (T þA) left in the cultures after 2-h incubation in the presence or absence of letrozole. reported that ovarian steroid hormones stimulate the stem cell individual spots representing testosterone, androstenedione, population in MCF-10F cells as well as in mouse mammary glands. estrone (E1) and E2 were measured separately. As MCF-10A cells To investigate the role of aromatase in estrogen carcinogenesis, express 17b-steroid dehydrogenase that catalyzes the conversions we established a model by overexpressing aromatase in MCF-10A of testosterone to androstenedione and E1 to E2, radioactivity human breast epithelial cells lacking ERa. The unique design of presented in Figure 1d is the sum of E1 and E2 and in Figure 1f the the studies was to culture aromatase expressing MCF-10A cells for sum of testosterone and androstenedione. After a 1-h incubation months with or without the aromatase inhibitor, letrozole. of MCF-10Aarom cells with [1,2,6,7-3H]testosterone, the production Aromatization of androgens from the horse serum in the medium of E1 and E2 (Figure 1d) was observed, as well as the by-product, 3 was the only source of estrogens. This model provides a setting of H2O (Figure 1e). Inclusion of letrozole in the culture reduced the long-term exposure of breast epithelial cells to physiological levels of E1 and E2 (Figure 1d). In contrast to estrogen products, concentrations of estrogens, which allows us to investigate ERa- the levels of the androgen substrates, testosterone and andros- independent estrogen carcinogenesis. In this study, we demon- tenedione, were reduced in MCF-10Aarom cells treated with the strated that enhanced aromatase expression can induce transfor- vehicle due to aromatization to estrogens. This reduction was mation of benign breast epithelial cells. An increase in the blocked by letrozole (Figure 1f). population of stem-like cells might have a significant role in the overall carcinogenic process. Aromatase expression promotes anchorage-independent growth of MCF-10A cells RESULTS MCF-10A cells do not express ERa. Any ERa-mediated acute effects Aromatase activity in MCF-10A cells with stable expression of of E2 on proliferation and other biological effects are not expected. aromatase cDNA Therefore, the experiments described in this and the following More than 20 clones of MCF-10A cells were selected after trans- sections were carried out in MCF-10Aarom and MCF-10Avector cells fection with plasmid pHbArm and 2-week treatment with G418. that had been cultured in the medium containing 5% horse serum Aromatase activities measured by tritiated water release assay for at least 3 months. This procedure ascertains that the cells were ranged between 0.37 and 10 pmol/mg protein/h (Supplementary exposed to E2 long enough for E2 to exert mutagenic effects. No Figure 1). androgen was added to the culture of MCF-10Aarom cells. Horse In-depth analysis of aromatase activity was carried out in two serum contains testosterone at nanomolar levels.19 As a clones selected because of high aromatase activity in the initial conservative estimate, 2 nmol/l in horse serum would provide À 10 screening. As expected from the known kinetic properties of 10 M testosterone in the culture. aromatase, estrogen production increased as a function of We first examined the rate of anchorage-dependent growth of substrate concentration and times (data not shown) in the MCF- MCF-10Aarom cells.
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