The Stimulation of HSD17B7 Expression by Estradiol Provides a Powerful Feed-Forward Mechanism for Estradiol Biosynthesis in Breast Cancer Cells

ORIGINAL RESEARCH The Stimulation of HSD17B7 Expression by Estradiol Provides a Powerful Feed-Forward Mechanism for Estradiol Biosynthesis in Breast Cancer Cells Aurora Shehu, Constance Albarracin, Y. Sangeeta Devi, Kristin Luther, Julia Halperin, Jamie Le, Jifang Mao, Rachel W. Duan, Jonna Frasor, and Geula Gibori Department of Physiology and Biophysics (A.S., Y.S.D., K.L., J.H., J.L., J.M., R.W.D., J.F., G.G.), University of Illinois at Chicago, Chicago, Illinois 60612; and Department of Pathology (C.A.), University of Texas, M.D. Anderson Cancer Center, Houston, Texas 77030 Our laboratory has previously cloned and purified an ovarian protein found to be a novel 17␤hydroxysteroid dehydrogenase type 7 enzyme (HSD17B7) (formerly prolactin receptor-associated protein) that converts the weak estrogen, estrone, to the highly potent estradiol. The regulation of this enzyme has not yet been explored. In this report, we show high expression of HSD17B7 in human ductal carcinoma and breast cancer cell lines and present evidence for a strong up-regulation of this enzyme by estradiol at the level of mRNA, protein expression, and promoter activity in MCF-7 cells. The effect of estradiol is mediated by estrogen receptor (ER)␣, whereas ER␤ prevents this stimulation. ER antagonists, ICI 182,780 and 4-hydroxytamoxifen, prevent estradiolinduced stimulation of the endogenously expressed HSD17B7, suggesting that these inhibitors not only block estradiol action but also its production. We have identified a ⫺185-bp region of the hsd17b7 promoter that is highly conserved among rat, mouse, and human and confers regulation by estradiol in MCF-7 cells. This region is devoid of a classical estradiol-response element but contains a nuclear factor 1 (NF1) site that is essential for estradiol action. We found that estradiol stimulates the recruitment and DNA binding of NF1 to this region of the hsd17b7 promoter. Furthermore, knockdown of NF1 family members, NF1B, NF1A, and NF1X, completely prevents induction of this gene by estradiol. In summary, our findings demonstrate that estradiol stimulates HSD17B7 transcriptional activity in breast cancer cells through a novel mechanism requiring NF1 and strongly suggest a positive feedback mechanism to increase local estradiol synthesis causing growth of estrogen-dependent breast cancers. (Molecular Endocrinology 25: 754 –766, 2011) NURSA Molecule Pages: Nuclear Receptors: ER-␣ 兩 ER-␤; Ligands: 17␤-estradiol 兩 Fulvestrant 兩 4-Hydroxytamoxifen. 7␤-Hydroxysteroid dehydrogenase type 7 (HSD17B7) is a 32-kDa microsomal protein involved in estradiol production. This enzyme was first discovered in our laboratory and named prolactin (PRL) receptor-associated protein, because it associates specifically with the cytoplasmic domain of the short form of the PRL receptor (1). Prolactin Receptor Associated Protein (known as PRAP) 1 has been found since to be a novel isoform of 17␤-hydroxysteroid dehydrogenase that is responsible for the conversion of estrone, a weak estrogen, to the more potent estradiol (2, 3). To date, 15 different isozymes of 17␤-hydroxysteroid dehydrogenase have been cloned (4 – 8). They belong to a family of enzymes responsible for the activation/inactivation of hormones. All require nicotin- ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2011 by The Endocrine Society doi: 10.1210/me.2010-0261 Received June 30, 2010. Accepted February 11, 2011. First Published Online March 3, 2011 Abbreviations: CDT, Charcoal-dextran treated; ChIP, chromatin immunoprecipitation; Egr-1, early growth response-1; Elk-1, Ets like gene 1; ER, estrogen receptor; ERE, estradiol-response element; FBS, fetal bovine serum; HSD17B7, 17␤-hydroxysteroid dehydrogenase type 7; ICI, ICI 182,780; IPTG, isopropyl-␤-D-thiogalactopyranoside; NADPH, nicotinamide adenine dinucleotide phosphate; NF1, nuclear factor 1; PRL, prolactin; Q-PCR, quantitative PCR; si, small interfering; Sp1, specificity protein 1; Tam, 4-hydroxytamoxifen. 754 mend.endojournals.org Mol Endocrinol, May 2011, 25(5):754 –766 Mol Endocrinol, May 2011, 25(5):754 –766 mend.endojournals.org 755 FIG. 1. Generation of HSD17B7 polyclonal antibody. A, Expression of His-tagged HSD17B7 in bacteria. Cells were grown in culture and induced to express His-HSD17B7 with increasing doses of IPTG. His-HSD17B7 proteins were purified on Talon column. The different fractions obtained were run on SDS-PAGE and stained with Coomassie blue dye. Fraction number denotes individual eluates obtained via sequential elution from the same set of Talon column as described in Materials and Methods. These eluted proteins were used to generate antibodies in two rabbits. B and C, Western blot analysis was performed with serially diluted antibodies from first (B) and fourth bleed (C) using proteins from corpora lutea known to highly express HSD17B7. D, Immunoprecipitation of luteal HSD17B7 with the two antibodies generated to native HSD17B7 protein. amide adenine dinucleotide phosphate (NADPH) for activity and are short chain dehydrogenases/reductases, with the exception of HSD17B5. All of these enzymes, beside types 6 and 9, have been found in humans. The majority of these isoenzymes use steroids as their substrates (4, 7), and most, including HSD17B7, recognize specific substrates (2). HSD17B7 is highly expressed in the ovarian corpus luteum of every mammalian species examined and is responsible for luteal estradiol biosynthesis in the ovary (1, 9, 10). Several HSD17B isoforms have also been found to be of importance in hormone-dependent tumors (11–13). HSD17B7 was detected by RT-PCR and immunohistochemistry in normal and pathological human breast tissue (14). The local production of estradiol in breast cancer cells is presently a subject of great interest, because it is becoming clear that locally produced estradiol can exacerbate growth of hormone-dependent breast tumors. The local mechanisms responsible for high estradiol concentrations observed in the breast are not completely understood (15) but most probably involve increased expression of enzymes involved in estradiol biosynthesis. Both P450aromatase, which converts androstenedione to estrone, and HSD17B7, which converts estrone to estradiol, are expressed in the breast (16). Although extensive efforts have been invested in defining regulatory mechanisms for P450aromatase in breast cancer (17, 18), no information is available to date as to what regulates HSD17B7 expression. Because it is estradiol, not estrone, that plays a critical role in the progression of breast cancer (15, 19 –22), the control of HSD17B7 gene expression in cancer cells can be of great significance (23). In this investigation, we show that although HSD17B7 is expressed at low levels in normal epithelial cells of breast ductal tissue, it becomes highly expressed in neighboring cancerous cells. Using breast cancer cells and a 1.16-kb HSD17B7 promoter isolated in our laboratory (...truncated)