Preclinical study Physiologic estrogen receptor alpha signaling in non-tumorigenic human mammary epithelial cells* Abde M. Abukhdeir 1 , Brian G. Blair 1 , Keith Brenner 1 , Bedri Karakas 1 , Hiroyuki Konishi 1 , Joselin Lim 1 , Vanita Sahasranaman 1 , Yi Huang 1 , Judith Keen 1 , Nancy Davidson 1 , Michele I. Vitolo 2 , Kurtis E. Bachman 2 , and Ben Ho Park 1 1 Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; 2 The Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD, USA Key words: 17-beta-estradiol, breast cancer, ERa, MCF-10A, pre-neoplastic Summary Currently, a number of breast cancer cell lines exist that serve as models for both estrogen receptor alpha (ERa) positive and ERa negative disease. Models are also available for pre-neoplastic breast epithelial cells that do not express ERa; however, there are no ideal systems for studying pre-neoplastic cells that are ERa positive. This has been largely due to the inability to establish an estrogen growth stimulated, non-tumorigenic breast epithelial cell line, as most human breast epithelial cells engineered to overexpress ERa have been found to be growth inhibited by estrogens. We have developed independently derived clones from the non-cancerous MCF-10A human breast cell line that express ERa and are growth stimulated by 17-beta-estradiol (E2) in the absence of epidermal growth factor (EGF), a cytokine normally required for MCF-10A cell proliferation. This effect is blocked by the selective estrogen receptor modulator (SERM), Tamoxifen and the selective estrogen receptor downregulator, ICI 182,780 (Faslodex, Fulvestrant). Exposure of these cells to EGF and E2 results in a growth inhibitory phenotype similar to previous reports. These data present a reconciling explanation for the previously described paradoxical effects of ERa overexpression, and provide a model for examining the carcinogenic effects of estrogens in non-tumorigenic human breast epithelial cells. Introduction ERa is unequivocally involved with human breast can- cer initiation and progression, as evidenced by effective therapies that target the presence of this receptor. However, models for examining ERa signaling are lim- ited to breast cancer cell lines that natively express ERa such as MCF-7, while non-tumorigenic human mam- mary epithelial cell (HMEC) lines expressing endoge- nous ERa have not been successfully cultured. This makes elucidation of the carcinogenic effects of estrogens difficult to analyze, since ERa positive breast cancer cell lines have already acquired a transformed state. It is of interest that despite the fact that most human breast cancers express ERa, the majority of breast cancer cell lines in culture are ERa negative. Additionally, it is estimated that in non-tumorigenic and/or normal breast epithelia, only 10–15% of cells express ERa [1,2]. Thus, it is likely that selective pressures against culturing ERa positive breast cell lines along with the low percentage of non-tumorigenic ERa positive breast cells have ham- pered the ability to successfully establish an ERa positive non-tumorigenic HMEC line. The MCF-10A human breast epithelial cell line and its derivatives have been well characterized and extensively used as models for studying non-tumori- genic/pre-neoplastic disease [3–6]. This cell line has been demonstrated to be genetically stable [7] and contains a known homozygous deletion at chromosome 9p result- ing in the loss of the tumor suppressors p16INK4A, p14ARF and p15INK4B likely accounting for its spon- taneous immortalization [8]. Previous attempts to develop an ERa expressing MCF-10A cell line using stable transfection of an ERa cDNA have yielded conflicting results, as ERa overexpression usually resulted in a growth inhibitory phenotype or no phenotype upon E2 exposure [9,10]. Similarly, human breast cancer cell lines that are ERa negative, also are growth inhibited upon reconstitution of ERa and E2 stimulation [11]. Others have been able to derive * This work was supported by The Flight Attendant’s Medical Re- search Institute (FAMRI), The American Cancer Society (#IRG-58- 005-41), NIH Breast SPORE Grant P50 CA88843, the Maryland Cigarette Restitution Fund, The Entertainment Industry Foundation, The Department of Defense Breast Cancer Research Program (DAMD17-03-1-0241), and the Avon Foundation. B.H.P. is an Avon Scholar for Breast Cancer Research and also receives generous support from The V Foundation for Cancer Research. Breast Cancer Research and Treatment (2006) 99: 23–33 Ó Springer 2006 DOI 10.1007/s10549-006-9177-0