605 Estrogen receptor regulates insulin-like growth factor-I receptor gene expression in breast tumor cells: involvement of transcription factor Sp1 Sharon Maor, Doris Mayer1, Ronit I Yarden2, Adrian V Lee3, Rive Sarfstein, Haim Werner and Moshe Z Papa4 Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel 1Group on Hormones and Signal Transduction, German Cancer Research Center, Heidelberg D-69120, Germany 2Laboratory of Genomic Applications, Chaim Sheba Medical Center, Tel Hashomer 52621, Israel 3Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, USA 4Department of Oncological Surgery, Chaim Sheba Medical Center, Tel Hashomer 52621, Israel (Requests for offprints should be addressed to H Werner; Email: [email protected]) Abstract The insulin-like growth factors, IGF-I and IGF-II are a familyof along with the Sp1-family-binding inhibitor Mithramycin A. mitogenic polypeptides with important roles in growth and The results obtained indicate that basal IGF-IR promoter differentiation. The biological actions of the IGFs are mediated activity was 5.8-fold higher in MCF-7 than in C4 cells. Estradiol by the IGF-I receptor (IGF-IR), a cell-surface tyrosine kinase, treatment significantly activated the IGF-IR promoter in whose activation by serum IGF-I seems to be a key step in breast MCF-7, but not in C4 cells. Furthermore, the estrogen cancer initiation. Evidence accumulated indicates that estrogens responsive region in the IGF-IR promoter was mapped to a stimulate the expression and activity of IGF axis components. GC-rich sequence located between nucleotides K40 and The aim of our study was to examine the transcriptional K188 in the 50 flanking region. ChIPexperiments revealed that mechanisms involved in regulation of IGF-IR gene expression at least part of the estrogen effect on IGF-IR expression was by the estrogen receptor (ER). For this purpose, transient mediated through activation of the Sp1 transcription factor. In transfections using an IGF-IR promoter-luciferase reporter summary, our studies demonstrate that IGF-IR gene transcrip- plasmid were performed in breast cancer-c derived ER-positive tion in breast cancer cells is controlled by interactions between MCF-7 cells and isogenic ER-negative C4 cells. Toexamine the ERa and Sp1. Dysregulated expression of the IGF-IR gene may potential involvement of zinc-finger nuclear proteins in the have pathologic consequences with relevance in breast cancer transactivating effect of estrogens, chromatin immunoprecipita- etiology. tion(ChIP) experimentswere performed using an Sp1 antibody, Journal of Endocrinology (2006) 191, 605–612 Introduction expressed in breast tumors, including ligands, receptors, and binding proteins (IGFBPs) (Ye e et al. 1989, Schnarr et al. The insulin-like growth factors, IGF-I and IGF-II, are a 2000, Surmacz 2000). The central role of the IGF-IR as a family of mitogenic polypeptides with important roles in mediator of both IGF-I and IGF-II action in breast cancer is growth and differentiation. The biological actions of the IGFs illustrated by the results of several groups showing that are mediated by the IGF-I receptor (IGF-IR), a trans- blockage of the IGF-IR by a variety of methods, including membrane tyrosine kinase structurally and evolutionarily anti-IGF-IR antibodies, antisense oligonucleotides against related to the insulin receptor (LeRoith et al. 1995, Baserga IGF-IR mRNA, and IGFBPs significantly inhibited cellular et al. 1997, Werner & LeRoith 2000). The IGF-IR axis has a proliferation (Arteaga et al. 1989, Van der Burg et al. 1990, central role in cell cycle progression, as demonstrated by the Surmacz et al. 1998). Further evidence in support of an fact that receptor overexpression in fibroblasts abrogates important role for the IGF system in breast carcinogenesis is all requirements for additional growth factors in order to also provided by epidemiological studies, showing a positive promote cellular proliferation (Pietrzkowski et al. 1992). correlation between circulating IGF-I concentrations and In addition, evidence is mounting for a pivotal role for the relative risk of breast cancer (Hankinson et al. 1998). IGF-IR in tumorigenesis (Werner & LeRoith 1996, Baserga Solid evidence accumulated in recent years indicating that 1999). Essentially, all components of the IGF system are the biological activity of the IGF system is strongly associated Journal of Endocrinology (2006) 191, 605–612 DOI: 10.1677/joe.1.07016 0022–0795/06/0191–605 q 2006 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/28/2021 04:38:28AM via free access 606 SMAORand others . Regulation of IGF-IR gene expression by estrogen receptor with estrogen status (Lee et al. 1999, Yee & Lee 2000). Western immunoblots Estrogens were shown to increase IGF-I binding and IGF-IR Cells were serum-starved overnight and then incubated with mRNA levels in MCF-7 cells by sevenfold, suggesting that increasing concentrations of b-estradiol (Sigma-Aldrich Corp.) the potential mechanism by which estrogens stimulate breast for 24 h. Following incubation, cells were harvested and lysates cancer cell proliferation involves sensitization to the mitogenic effects of IGFs by enhancing IGF-IR concen- were prepared as described previously (Idelman et al. 2002). m tration (Stewart et al. 1990). In addition, estrogens can Samples (50 g) were subjected to 8% SDS-PAGE, followed by modulate IGF signaling by regulating the expression of other electrophoretic transfer of the proteins to nitrocellulose members of the IGF family, including ligands, IGFBPs, and membranes. After blocking with 3% bovine serum albumin or milk in T-TBS (20 mM Tris–HCl, pH 7.5, 135 mM NaCl, and insulin receptor substrate (IRS)-1 (Osborne et al. 1989, . McGuire et al. 1992, Salerno et al. 1999). Using MCF-7- 0 1% Tween-20), blots were incubated with polyclonal derived sublines that have been selected for loss of estrogen antibodies against IGF-IR b subunit, Sp1 and ERa (C-20, receptor a (ERa) by long-term estrogen withdrawal, it was PEP-2, and MC-20 respectively; Santa Cruz Biotechnology, previously demonstrated that the loss of ERa caused reduced Santa Cruz, CA, USA), and against actin or tubulin, followed by expression of IGF-signaling molecules and failure to incubation with a horseradish peroxidase-conjugated secondary proliferate in response to IGF-I or estrogen. Re-expression antibody. Proteins were detected using the SuperSignal West of ERa restored the IGF-responsive phenotype, suggesting Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA). that ERa is a crucial regulator of the IGF mitogenic loop In selected experiments, the Sp1-family inhibitor Mithramycin (Oesterreich et al. 2001). A (Sigma-Aldrich Corp.) was added at a concentration of The IGF-IR promoter is a TATA-less, CAAT-less, and 500 nM 1 h before the estradiol treatment. initiator type of promoter. The region flanking the transcription start site is extremely GC-rich, and contains IGF-IR promoter activity measurements numerous potential binding sites for transcription factor Sp1, a zinc-finger-containing nuclear protein that has been shown For transient transfection experiments, luciferase reporter K C K C to strongly transactivate the IGF-IR promoter (Beitner- constructs p( 2350/ 640)LUC, p( 476/ 640)LUC, K C K C Johnson et al. 1995). Given the involvement of ERa in p( 188/ 640)LUC, and p( 40/ 640)LUC (nucleotide 1 control of IGF-IR gene expression and IGF-I action, and in corresponds to the transcription start site) were employed. view of the fact that the transcriptional mechanisms involved These plasmids include 2350, 476, 188, or 40 nucleotides of the 0 0 in regulation of the IGF-IR gene by ERa have not yet been 5 -flanking region, linked to 640 bp of the 5 -untranslated characterized, the present study was designed to analyze region of the rat IGF-IR gene. The basal promoter activities of the molecular mechanisms responsible for regulation of the these constructs were previously reported (Maor et al. 2000). IGF-IR gene by ERa. Specifically, we hypothesized that ERa The ERa expression vector was generated by subcloning the regulates IGF-IR gene expression through interaction with human ERa cDNA into the pSG5 vector using EcoRI sites. transcription factor Sp1. The results obtained prove that ERa The ERa vector was provided by Dr Yitzhak Koch, Weizmann is a potent transactivator of the IGF-IR gene and that GC-rich Institute of Science, Rehovot, Israel. An Sp1 expression vector sequences in the proximal IGF-IR promoter region are (pPacSp1), under the control of an actin promoter in the pPac0 required for this effect. Impaired interactions between ERa vector, was provided by Dr Robert Tjian (University of and zinc-finger proteins may lead to aberrant IGF-IR California, Berkeley, CA, USA). Cells were seeded in six-well expression in breast cancer cells. plates 48 h before transfection, and transfected with 0.5 mgof the indicated IGF-IR promoter reporter plasmids, along with 1.3 mgERa expression vector (or empty pSG5 vector), 1 mg pPacSp1 (or empty pPac0 vector), and 0.2 mg b-galactosidase Materials and Methods expression plasmid (pCMVb,Clontech,PaloAlto,CA,USA), using the jetPEI reagent (Polyplus Transfection, Illkirch, Cell cultures France). Twenty-four hours after transfection, cells were treated The MCF-7 human breast cancer cell line was maintained in with estradiol (1, 10 and 100 nM) or left untreated. Cells were Dulbecco’s modified Eagle’s medium (DMEM) supplemented harvested after an additional 24 h, and luciferase and with 10% fetal bovine serum (FBS), 2 mM glutamine, and b-galactosidase activities were measured as previously described 50 mg/ml gentamicin sulfate. C4 cells were generated by clonal (Maor et al. 2000). In addition, some experiments included selection of MCF-7 cells, cultured in estrogen-free medium Mithramycin A.