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The WT1 Wilms' Tumor Suppressor Gene Product Interacts with Estrogen Receptor-Α and Regulates IGF-I Receptor Gene Transcripti

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The WT1 Wilms' Tumor Suppressor Gene Product Interacts with Estrogen Receptor-Α and Regulates IGF-I Receptor Gene Transcripti 135 The WT1 Wilms’ tumor suppressor gene product interacts with estrogen receptor- and regulates IGF-I receptor gene transcription in breast cancer cells Naama Reizner, Sharon Maor, Rive Sarfstein, Shirley Abramovitch, Wade V Welshons1, Edward M Curran1, Adrian V Lee2 and Haim Werner Department of Clinical Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel 1Department of Veterinary Biomedical Sciences, University of Missouri, Columbia, Missouri 65211, USA 2Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, USA (Requests for offprints should be addressed to H Werner; Email: [email protected]) Abstract The IGF-I receptor (IGF-IR) has an important role in breast cancer development and progression. Previous studies have suggested that the IGF-IR gene is negatively regulated by a number of transcription factors with tumor suppressor activity, including the Wilms’ tumor protein WT1. The present study was aimed at evaluating the hypothesis that IGF-IR gene transcription in breast cancer cells is under inhibitory control by WT1 and, furthermore, that the mechanism of action of WT1 involves functional and physical interactions with estrogen receptor- (ER). Results of transient coexpression experiments showed that all four predominant isoforms of WT1 (including or lacking alternatively spliced exons 5 and 9) repressed IGF-IR promoter activity by 39–49%. To examine the potential interplay between WT1 and ER in control of IGF-IR gene transcription we employed ER-depleted C4 cells that were generated by clonal selection of ER-positive MCF-7 cells that were maintained in estrogen-free conditions. IGF-IR levels in C4 cells were |43% of the values in MCF-7 cells whereas WT1 levels in C4 cells were 4·25-fold higher than in MCF-7. Triple cotransfection experiments using an ER expression vector in the absence or presence of WT1 expression vectors, along with an IGF-IR promoter reporter plasmid, revealed that ER stimulated IGF-IR promoter activity whereas coexpression of WT1 abrogated the effect of ER. In addition, co-immunoprecipitation experiments demonstrated a specific association between WT1 and ER. Combined, our results suggest that WT1 suppresses IGF-IR gene transcription in breast cancer cells via a mechanism that involves protein–protein association with ER. As a result of this interaction, the ability of ER to transactivate the IGF-IR promoter is abrogated. These findings are consistent with a potential tumor suppressor role for WT1 in breast cancer and suggest that WT1 inactivation in tumoral cells may result in deregulated IGF-IR gene expression and enhanced mitogenic activation by locally produced and/or circulating IGFs. Journal of Molecular Endocrinology (2005) 35, 135–144 Introduction Baserga 1999). Ligand-induced phosphorylation of the IGF-IR tyrosine kinase domain leads to the activation The involvement of the insulin-like growth factor (IGF) of a number of cytoplasmic signal transduction mol- system in the biology of breast cancer has been well ecules, including the ras–raf–MAPK and PI3–PKB/Akt established (Surmacz 2000, Yee & Lee 2000). Evidence cascades. of both an experimental and a clinical nature identified Consistent with its antiapoptotic, cell-survival promot- IGF-I and IGF-II as major players in mammary gland ing role, most available evidence supports the notion growth and development as well as in breast cancer that the IGF-IR gene is highly expressed in many types initiation and progression. In addition, a number of of cancer, including breast tumors (Happerfield et al. epidemiological surveys demonstrated a positive corre- 1997, Mitsiades et al. 2004). Accordingly, IGF-IR lation between circulating IGF-I concentrations and overexpression in breast tissue was linked to tumor relative risk of breast cancer in premenopausal women recurrence at the primary site and radioresistance (Hankinson et al. 1998, Pollak 2000). The biological (Turner et al. 1997). Interestingly, immunohistochemical actions of both endocrine and locally produced IGF-I analyses revealed that progression to advanced meta- and IGF-II on breast cell proliferation, survival and static stages was associated with a significant decrease in transformation are mediated by the IGF-I receptor IGF-IR levels (Schnarr et al. 2000). Combined, these (IGF-IR) (LeRoith et al. 1995, Werner & LeRoith 1996, results suggest that normal breast epithelial cells respond Journal of Molecular Endocrinology (2005) 35, 135–144 DOI: 10.1677/jme.1.01761 0952–5041/05/035–135 © 2005 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 10/01/2021 03:30:54AM via free access 136 N REIZNER and others · Regulation of the IGF-IR gene by WT1 in breast cancer cells to endocrine and autocrine/paracrine IGFs to maintain KTS tripeptide were impaired in their ability to bind to an appropriate balance between proliferation and the IGF-IR promoter region and, therefore, displayed a differentiation. Excess IGF may increase the probability reduced repressing activity. that cells become premalignant while transition to Recent studies have suggested a potential role for advanced-stage disease appears to be associated with a WT1 in various aspects of mammary gland biology. reduction in IGF-IR concentrations and IGF responsive- Thus, the involvement of WT1 in mammary gland ness (Pennisi et al. 2002). The molecular mechanisms morphogenesis was inferred from immunohistochemical that are responsible for regulation of IGF-IR gene analyses showing that WT1 is differentially regulated expression in breast cancer cells remain to be identified. between developmentally divergent ducts and lobules as The regulatory region of the IGF-IR gene comprises a well as within the mature duct (Silberstein et al. 1997). unique ‘initiator’ motif, from which transcription is With regard to breast tumorigenesis, a number of initiated in vivo, and which acts in concert with upstream observations suggested that WT1 may function as a Sp1 sites (Werner et al. 1992, Beitner-Johnson et al. candidate tumor suppressor. Specifically: (i) a high 1995). The region flanking the transcription start site is percentage of breast cancer cells lacked immunodetected extremely G-C rich, with no obvious TATA or CAAT WT1; (ii) the cytoplasmic localization of WT1 sug- elements. In addition, the IGF-IR promoter contains gested that functional inactivation of WT1 occurs in a numerous binding sites for members of the early growth subset of tumors; (iii) changes in the intracellular response (EGR) family of transcriptional activators localization of WT1 accompanied tumor progression; (Werner et al. 1993, 1994). The EGR family includes, and (iv) common tumor-associated perturbations of among other members, EGR1 (NGFI-A, Zif 268, Krox WT1 mRNA splicing were demonstrated (Silberstein 24), EGR2, EGR3 and the Wilms’ tumor suppressor et al. 1997). gene, WT1 (Call et al. 1990, Gessler et al. 1990, Rauscher In view of the important role of IGF-IR in breast 1993). The WT1 gene is located on chromosome 11p13 cancer etiology and to extend our previous observations and encodes a DNA-binding protein with a proline- and on regulation of IGF-IR gene expression by WT1, we glutamine-rich N-terminal end and four zinc-finger investigated the potential involvement of WT1 in domains in its C-terminus (Morris et al. 1991). The WT1 transcriptional control of the IGF-IR gene in breast gene undergoes alternative splicing at two different cancer cells. Specifically, our aim was to elucidate the positions, a 51 bp sequence encoded by exon 5 and a mechanisms that are responsible for regulation of the 9 bp sequence that results from the use of an alternative IGF-IR gene, a bona fide WT1 downstream target, in 5 splice junction in exon 9. Use of exon 5 results in mammary gland-derived cells, and to evaluate the isoforms that include a 17 amino acid insert while use of potential molecular interactions between WT1 and exon 9 leads to the inclusion of a tripeptide breast cell-specific transactivators. The results obtained (Lys-Thr-Ser, KTS) between zinc-fingers 3 and 4 (Haber indicate that WT1 suppresses IGF-IR gene transcription et al. 1991) (Fig. 1A). The consensus binding sequence via a mechanism that involves protein–protein interac- for the EGR/WT1 gene products is 5-GCGGGG tion with estrogen receptor- (ER). These results are GCG-3 (Rauscher et al. 1990). Unlike other members of consistent with a tumor suppressor role for WT1 in the EGR family, WT1 has been shown to usually repress breast cancer cells and suggest that WT1 inactivation in the activity of target promoters containing this tumoral cells may lead to deregulated IGF-IR expression cis-element, including the IGF-II, platelet-derived and enhanced proliferative capacity. growth factor A-chain, transforming growth factor-1 and others (Madden et al. 1991, Drummond et al. 1992, Gashler et al. 1992, Dey et al. 1994). Inactivation of the Materials and methods WT1 gene has been postulated to be a key event in the etiology of Wilms’ tumor, a pediatric kidney cancer (Call et al. 1990, Rose et al. 1990). Cell cultures In the specific context of the IGF-IR gene, we have Breast carcinoma-derived MCF-7 cells were obtained previously demonstrated that WT1 isoforms lacking the from the American Type Culture Collection (Mannassas, KTS insert suppressed IGF-IR promoter activity in a VA, USA). C4 cells were generated by clonal selection number of cell types, including osteosarcoma- and of MCF-7 cells cultured in estrogen-free medium for colorectal cancer-derived cell lines (Werner et al. 1993, 9 months (Oesterreich et al. 2001). C4 cells were grown 1994, 1995, Idelman et al. 2003). Furthermore, the in phenol red-free Dulbecco’s modified Eagle’s medium biological activity of WT1 isoforms lacking KTS was (DMEM) supplemented with 10% charcoal-stripped associated with specific binding to cis-elements in the fetal bovine serum (FBS), 2 mM glutamine, 50 µg/ml IGF-IR promoter region, as demonstrated using gentamicin sulfate and 2·5 µg/ml Fungizone.
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