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 ERα− MCF-10A human breast epithelial cells to investigate ERα-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 ERα-independent mechanism(s) in estrogen carcinogenesis and implicate superiority of aromatase inhibitors to antiestrogens for breast cancer prevention.
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PFOS induces proliferation, cell-cycle progression, and malignant phenotype in human breast epithelial cells
Archives of Toxicology Open Access 23 October 2017
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Key T, Appleby P, Barnes I, Reeves G, Endogenous Hormones and Breast Cancer Collaborative Group. Endogenous sex hormones and breast cancer in postmenopausal women: reanalysis of nine prospective studies. J Natl Cancer Inst 2002; 94: 606–616.
Kotsopoulos J, Chen WY, Gates MA, Tworoger SS, Hankinson SE, Rosner BA . Risk factors for ductal and lobular breast cancer: results from the nurses’ health study. Breast Cancer Res 2010; 12: R106.
Chen WY, Manson JE, Hankinson SE, Rosner B, Holmes MD, Willett WC et al. Unopposed estrogen therapy and the risk of invasive breast cancer. Arch Intern Med 2006; 166: 1027–1032.
Feinleib M . Breast cancer and artificial menopause: a cohort study. J Natl Cancer Inst 1968; 41: 315–329.
Trichopoulos D, MacMahon B, Cole P . Menopause and breast cancer risk. J Natl Cancer Inst 1972; 48: 605–613.
Kaaks R, Rinaldi S, Key TJ, Berrino F, Peeters PHM, Biessy C et al. Postmenopausal serum androgens, oestrogens and breast cancer risk: the European prospective investigation into cancer and nutrition. Endocr Relat Cancer 2005; 12: 1071–1082.
Howell A, Cuzick J, Baum M, Buzdar A, Dowsett M, Forbes JF et al. Results of the ATAC (Arimidex, Tamoxifen, Alone or in Combination) trial after completion of 5 years’ adjuvant treatment for breast cancer. Lancet 2005; 365: 60–62.
Breast International Group (BIG) 1-98 Collaborative Group, Thürlimann B, Keshaviah A, Coates AS, Mouridsen H, Mauriac L et al. A comparison of letrozole and tamoxifen in postmenopausal women with early breast cancer. N Engl J Med 2005; 353: 2747–2757.
Goss PE, Ingle JN, Alés-Martínez JE, Cheung AM, Chlebowski RT, Wactawski-Wende J et al. Exemestane for breast-cancer prevention in postmenopausal women. N Engl J Med 2011; 364: 2381–2391.
Berkovitz GD, Guerami A, Brown TR, MacDonald PC, Migeon CJ . Familial gynecomastia with increased extraglandular aromatization of plasma carbon 19-steroids. J Clin Inv 1985; 75: 1763–1769.
Bulard J, Mowszowicz I, Schaison G . Increased aromatase activity in pubic skin fibroblasts from patients with isolated gynecomastia. J Clin Endocrinol Metab 1987; 64: 618–623.
DeOme KB, Faulkin LJ, Bern HA, Blair PB . Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice. Cancer Res 1959; 19: 515–520.
Tekmal RR, Ramachandra N, Gubba S, Durgam VR, Mantione J, Toda K et al. Overexpression of int-5/aromatase in mammary glands of transgenic mice results in the induction of hyperplasia and nuclear abnormalities. Cancer Res 1996; 56: 3180–3185.
Yue W, Wang J-P, Li Y, Fan P, Liu G, Zhang N et al. Effects of estrogen on breast cancer development: role of estrogen receptor independent mechanisms. Int J Cancer 2010; 127: 1748–1757.
Cavalieri E, Chakravarti D, Guttenplan J, Jankowiak R, Muti P, Rogan E et al. Catechol estrogen quinones as initiators of breast and other human cancers: implications for biomarkers of susceptibility and cancer prevention. Biochim Biophys Acta 2006; 1766: 63–78.
Yager JD, Davidson NE . Estrogen carcinogenesis in breast cancer. N Engl J Med 2006; 354: 270–282.
Yager JD, Santen RJ . Mechanisms relating estrogens to breast cancer. Transl Endocrinol Metab 2012; 3: 1–19.
Zhou D, Pompon D, Chen S . Stable expression of human aromatase complementary DNA in mammalian cells: a useful system for aromatase inhibitor screening. Cancer Res 1990; 50: 6949–6954.
Maurice KT . Diagnosis and surgical removal of a granulosa–theca cell tumor in a mare. Can Vet J 2005; 46: 644–646.
Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF . Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 2003; 100: 3983–3988.
Yue W, Wang J-P, Hamilton CJ, Demers LM, Santen RJ . In situ aromatization enhances breast tumor estradiol levels and cellular proliferation. Cancer Res 1998; 58: 927–932.
O’Neill JS, Elton RA, Miller WR . Aromatase activity in adipose tissue from breast quadrants: a link with tumor site. BMJ 1988; 296: 741–743.
Bulun SE, Price TM, Aitken J, Mahendroo MS, Simpson ER . A link between breast cancer and local estrogen biosynthesis suggested by quantification of breast adipose tissue aromatase cytochrome P450 transcripts using competitive polymerase chain reaction after reverse transcription. J Clin Endocrinol Metab 1993; 77: 1622–1628.
Hu Y, Ghosh S, Amleh A, Yue W, Lu Y, Katz A et al. Modulation of aromatase expression by BRCA1: a possible link to tissue-specific tumor suppression. Oncogene 2005; 24: 8343–8348.
Lu M, Chen D, Lin Z, Reierstad S, Trauernicht AM, Boyer TG et al. BRCA1 negatively regulates the cancer-associated aromatase promoters I.3 and II in breast adipose fibroblasts and malignant epithelial cells. J Clin Endocrinol Metab 2006; 91: 4514–4519.
Agarwal VR, Bulun SE, Leitch M, Rohrich R, Simpson ER . Use of alternative promoters to express the aromatase cytochrome P450 (CYP19) gene in breast adipose tissues of cancer-free and breast cancer patients. J Clin Endocrinol Metab 1996; 81: 3843–3849.
Chand AL, kConFab, Simpson ER, Clyne CD . Aromatase expression is increased in BRCA1 mutation carriers. BMC Cancer 2009; 9: 148.
Diaz-Cruz ES, Sugimoto Y, Gallicano GI, Brueggemeier RW, Furth PA . Comparison of increased aromatase versus ERα in the generation of mammary hyperplasia and cancer. Cancer Res 2011; 71: 5477–5487.
Rebbeck TR, Kauff ND, Domchek SM . Meta-analysis of risk reduction estimates associated with risk-reducing salpingo-oophorectomy in BRCA1 or BRCA2 mutation carriers. J Natl Cancer Inst 2009; 101: 80–87.
Lakhani SR, Van De Vijver MJ, Jacquemier J, Anderson TJ, Osin PP, McGuffog L et al. The pathology of familial breast cancer: predictive value of immunohistochemical markers estrogen receptor, progesterone receptor, HER-2, and p53 in patients with mutations in BRCA1 and BRCA2. J Clin Oncol 2002; 20: 2310–2318.
Foulkes WD, Metcalfe K, Sun P, Hanna WM, Lynch HT, Ghadirian P et al. Estrogen receptor status in BRCA1- and BRCA2-related breast cancer: the influence of age, grade, and histological type. Clin Cancer Res 2004; 10: 2029–2034.
Russo J, Lareef HM, Tahin Q, Hu Y-F, Slater C, Ao X et al. 17β-Estradiol is carcinogenic in human breast epithelial cells. J Steroid Biochem Mol Biol 2002; 80: 149–162.
Russo J, Fernandez SV, Russo PA, Fernbaugh R, Sheriff FS, Lareef HM et al. 17-Beta-estradiol induces transformation and tumorigenesis in human breast epithelial cells. FASEB J 2006; 20: 1622–1634.
Shen Y-M, Troxel AB, Vedantam S, Penning TM, Field J . Comparison of p53 mutations induced by PAH o-quinones with those caused by anti-benzo[α]pyrene diol epoxide in vitro: role of reactive oxygen and biological selection. Chem Res Toxicol 2006; 19: 1441–1450.
Gaikwad NW, Yang L, Muti P, Meza JL, Pruthi S, Ingle JN et al. The molecular etiology of breast cancer: evidence from biomarkers of risk. Int J Cancer 2008; 122: 1949–1957.
Gaikwad NW, Yang L, Pruthi S, Ingle JN, Sandhu N, Rogan EG et al. Urine biomarkers of risk in the molecular etiology of breast cancer. Breast Cancer: Basic Clin Res 2009; 3: 1–8.
Sheridan C, Kishimoto H, Fuchs RK, Mehrotra S, Bhat-Nakshatri P, Turner CH et al. CD44+/CD24− breast cancer cells exhibit enhanced invasive properties: an early step necessary for metastasis. Breast Cancer Res 2006; 8: R59.
Asselin-Labat M-L, Vaillant F, Sheridan JM, Pal B, Wu D, Simpson ER et al. Control of mammary stem cell function by steroid hormone signalling. Nature 2010; 465: 798–802.
Joshi PA, Jackson HW, Beristain AG, Di Grappa MA, Mote PA, Clarke CL et al. Progesterone induces adult mammary stem cell expansion. Nature 2010; 465: 803–807.
Fillmore CM, Gupta PB, Rudnick JA, Caballero S, Keller PJ, Lander ES et al. Estrogen expands breast cancer stem-like cells through paracrine FGF/Tbx3 signaling. Proc Natl Acad Sci USA 2010; 107: 21737–21742.
Yue W, Wang J-P, Conaway MR, Li Y, Santen RJ . Adaptive hypersensitivity following long-term estrogen deprivation: involvement of multiple signal pathways. J Steroid Biochem Mol Biol 2003; 86: 265–274.
Yue W, Wang J-P, Conaway M, Masamura S, Li Y, Santen RJ . Activation of the MAPK pathway enhances sensitivity of MCF-7 breast cancer cells to the mitogenic effect of estradiol. Endocrinology 2002; 143: 3221–3229.
Russo J, Hu YF, Yang X, Russo IH . Developmental, cellular, and molecular basis of human breast cancer. J Natl Cancer Inst Monogr 2000; 27: 17–37.
Lu F, Zahid M, Saeed M, Cavalieri EL, Rogan EG . Estrogen metabolism and formation of estrogen–DNA adducts in estradiol-treated MCF-10F cells: The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin induction and catechol-O-methyltransferase inhibition. J Steroid Biochem Mol Biol 2007; 105: 150–158.
Rogan EG, Badawi AF, Devanesan PD, Meza JL, Edney JA, West WW et al. Relative imbalances in estrogen metabolism and conjugation in breast tissue of women with carcinoma: potential biomarkers of susceptibility to cancer. Carcinogenesis 2003; 24: 697–702.
Fan P, Wang J-P, Santen RJ, Yue W . Long-term treatment with tamoxifen facilitates translocation of estrogen receptor α out of the nucleus and enhances its interaction with EGFR in MCF-7 breast cancer cells. Cancer Res 2007; 67: 1352–1360.
This study is supported by DOD Grants: DOD DAMD17-01-1-0444 and DAMD17-01-1-0445. Human aromatase cDNA plasmid was kindly provided by Dr Shiuan Chen (Beckman Institute at City of Hope, Duarte, CA, USA). Measurement of estrogen metabolites was performed by Dr Cavalieri at the Laboratory of Eppley Institute for Research in Cancer and Allied Diseases (University of Nebraska Medical Center Omaha, NE, USA).
The authors declare no conflict of interest.
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Wang, J., Gildea, J. & Yue, W. Aromatase overexpression induces malignant changes in estrogen receptor α negative MCF-10A cells. Oncogene 32, 5233–5240 (2013). https://doi.org/10.1038/onc.2012.558
- breast cancer
- stem cell
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