Volume 10 Number 1 January 2008 pp. 69–78 69 www.neoplasia.com RESEARCH ARTICLE † Marianne K.-H. Kim*, Jacqueline M. Mason , A Pathologic Link between Wilms ‡ § Chi-Ming Li , Windy Berkofsky-Fessler , ∥ WT1 Le Jiang , Divaker Choubey¶, Paul E. Grundy#, Tumor Suppressor Gene, , ∥ and IFI161,2 Benjamin Tycko and Jonathan D. Licht* *Division of Hematology/Oncology, Feinberg School of Medicine, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL, USA; †The Campbell Family Institute for Breast Cancer Research at the Ontario, Cancer Institute, Ontario, Canada; ‡Translational Medicine, Amgen, Thousand Oaks, CA, USA; §Section of Bioinformatics, Genetics and Genomics, Hoffmann-La Roche Inc, Nutley, NJ, USA; ∥Institute for Cancer Genetics and Department of Pathology, Columbia University College of Physicians and Surgeons, New York, NY, USA; ¶University of Cincinnati, Cincinnati, OH, USA; #University of Alberta, Alberta, Canada Abstract The Wilms tumor gene (WT1) is mutated or deleted in patients with heredofamilial syndromes associated with the development of Wilms tumors, but is infrequently mutated in sporadic Wilms tumors. By comparing the micro- array profiles of syndromic versus sporadic Wilms tumors and WT1-inducible Saos-2 osteosarcoma cells, we iden- tified interferon-inducible protein 16 (IFI16), a transcriptional modulator, as a differentially expressed gene and a candidate WT1 target gene. WT1 induction in Saos-2 osteosarcoma cells led to strong induction of IFI16 expression and its promoter activity was responsive to the WT1 protein. Immunohistochemical analysis showed that IFI16 and WT1 colocalized in WT1-replete Wilms tumors, but not in normal human midgestation fetal kidneys, suggesting that the ability of WT1 to regulate IFI16 in tumors represented an aberrant pathologic relationship. In addition, en- dogenous IFI16 and WT1 interacted in vivo in two Wilms tumor cell lines. Furthermore, IFI16 augmented the transcriptional activity of WT1 on both synthetic and physiological promoters. Strikingly, short hairpin RNA (shRNA)–mediated knockdown of either IFI16 or WT1 led to decreased growth of Wilms tumor cells. These data suggest that IFI16 and WT1, in certain cellular context including sporadic Wilms tumors, may support cell survival. Neoplasia (2008) 10, 69–78 Introduction the notion that WT1 functions as both a tumor suppressor and an Loss of functional mutations and deletion of WT1 occurs in ap- oncogene is widely supported (reviewed in Yang et al. [7]). proximately 10% of Wilms tumor cases. Most cases of Wilms tumor are unilateral, sporadic, not associated with a malformation syndrome, Abbreviations: GST, glutathione-S-Transferase; IFI16, interferon-inducible protein 16; and express abundant levels of wild-type WT1 protein. This suggests shRNA, short hairpin RNA; siRNA, small interfering RNA; STAT3, signal transducer that WT1 is a tumor suppressor only in certain contexts. When re- and activators of transcription protein 3; WT1, Wilms tumor gene Address all correspondence to: Jonathan D. Licht, MD, Division of Hematology/ constituted into Wilms tumor cell lines devoid of wild-type WT1 as Oncology, Northwestern University, Feinberg School of Medicine, Lurie 5-123, 303 well as a number of other cell lines including Saos cells, WT1 can slow E. Superior Street, Chicago, IL 60611. E-mail: [email protected] cell growth, induce cell cycle arrest, apoptosis, and initiate features of 1Supported by NIH grant R01CA102270 to B. T. and J. D. L. and by a Postdoctoral – Fellowship (#PF-05-252-01-MGO) from the American Cancer Society to M. K.-H. K. epithelial differentiation [1 5]. However, the high level of expression 2This article refers to supplementary materials, which are designated by Figures W1, of WT1 in leukemia, as well as in a number of solid tumors, such as W2, W3, and W4 and are available online at www.neoplasia.com. breast cancer, lung cancer, primary thyroid cancer, and colorectal car- Received 19 September 2007; Revised 7 November 2007; Accepted 8 November 2007 cinoma, suggests that, in certain contexts, the protein is at least per- Copyright © 2008 Neoplasia Press, Inc. All rights reserved 1522-8002/08/$25.00 missive for, if not stimulating, neoplastic cell growth [6]. Therefore, DOI 10.1593/neo.07869 70 Novel WT1-Associated Protein Kim et al. Neoplasia Vol. 10, No. 1, 2008 IFI16 is a human member of hematopoietic interferon-inducible Coimmunoprecipitation and Glutathione-S-Transferase nuclear proteins, can act as a transcriptional repressor, and affects the Fusion Protein Binding Assays transcriptional activity of other transcription factors [8–10]. Over- Cells were washed once in 1× PBS and lysed in ice-cold lysis buffer expression of IFI16 in prostate and thyroid cancer cells inhibited cell containing 1% Triton X-100, 50 mM Tris–HCl (pH 8.0), 150 mM proliferation [11,12], whereas immortalized fibroblasts displayed de- NaCl, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, creased expression and promoter hypermethylation of IFI16, impli- 10 mM EDTA, 1 mM sodium orthovanadate, 1 mM PMSF, and cating a role in cellular senescence and growth control as a tumor one tablet of protease inhibitor (Complete; Roche) per 50 ml. After suppressor [13]. In addition, decreased expression of IFI16 was ob- 5 minutes on ice, the lysates were centrifuged at 10,000g for 5 min- served in breast cancer cell lines, suggesting that the loss of its expres- utes at 4°C. Agarose-conjugated antibodies (Santa Cruz Biotech- sion contributes to cancer development [14]. nologyInc.,SantaCruz,CA)wereaddedto2mgoflysatesfor In this study, we find that IFI16 is upregulated in Saos-2 cells on WT1 overnight incubation at 4°C. For glutathione-S-transferase (GST) induction and is overexpressed in WT1-replete tumors. IFI16 forms a pull-down experiments, cell lysates (2 mg) were incubated for 3 hours complex with WT1 and augments its transcriptional activity. Most im- at 4°C with gentle rocking with the indicated GST–WT1 fusion pro- portantly, loss of expression of IFI16 and WT1 inhibited the growth of teins immobilized on glutathione–Sepharose 4B beads (GE Health- Wilms tumors, suggesting that both genes support cell survival. care, Piscataway, NJ). The beads were collected and washed three times. The proteins were resolved by SDS-PAGE and transferred to Immobilon-P PVDF membranes (Millipore, San Francisco, CA) for immunoblot analysis. Materials and Methods Tumor and Control Tissues, and Northern Blot Immunoblot Analysis Tumor and control tissue samples, and Northern blot analysis The following primary antibodies were used (all from Santa Cruz were described previously [15]. Biotechnology): WT1 clone C19 rabbit polyclonal (1/1000); IFI16 clone 1G7 mouse monoclonal (1/1000); and GST clone Z5 rabbit poly- clonal (1/1000). The GAPDH mouse monoclonal (1/20,000) was from Plasmids Chemicon International (Temecula, CA). For the detection, one of the Plasmids RSV-WT1A (−exon5/−KTS), RSV-WT1-F112Y, ZIF- following conjugated antisera was used (GE Healthcare): peroxidase– tk-Luc, and Sprouty1-Luc were described previously [16–18]. donkey anti-rabbit IgG (1/5000); peroxidase–sheep anti-mouse igG pCMV/SPORT6-IFI16B isoform (Clone ID: CS0DF005YG06) (1/5000). The proteins were visualized using enhanced chemiluminis- was purchased from Invitrogen (Carlsbad, CA) and subcloned into cence (GE Healthcare). the pSP7, pCDNA3.1(+), or pRc/RSV vectors (Invitrogen). A WT1A isoform cut out with Sau3A from pSP64-WT1A [2] was cloned into BamHI-digested pBIG2i (gift from Dr. C. Strathdee). Reporter Gene Assays The IFI16 promoter–reporter construct, containing DNA se- quences between nt 231031 and 232710 of GenBank accession Cell Culture and Transfection Methods AP002534, was cloned upstream of the firefly luciferase gene in Saos-2–WT1A cells [3] were maintained in DMEM containing the pGL3-basic vector (Promega, Madison, WI). An artificial 10% FBS, 10 mM penicillin–streptomycin, 0.5 mg/ml G418, and WT1-responsive and Spry1 promoter reporter constructs were pre- 1 μg/ml of tetracycline. WT1A or a mutant WT1A-F112Y expres- viously described [18]. Transient transfection was performed using sion was induced by removing the tetracycline. 293 and 293T cells FuGENE 6 (Roche) in six-well plates. The Renilla luciferase plasmid were transfected using a transfection reagent (FuGENE 6; Roche (Dual-Luciferase Reporter Assay System; Promega) was cotransfected Diagnostics Corp., Indianapolis, IN). CCG99-11 cells isolated as an internal control and dual luciferase activities were measured af- from a sporadic Wilms tumor were cultured in DMEM, 10% ter 48 hours. heat-inactivated FBS, 1 mM sodium pyruvate, and transfected using a reagent (Lipofectamine Plus; Invitrogen). SK-NEP-1 cells (Ameri- Immunohistochemistry can Type Culture Collection, Manassas, VA) were maintained in Antigen retrieval for detection of IFI16 and WT1 was done ’ McCoy s 5a medium (Invitrogen) with 10% FBS. Plasmid pBIG2i- by boiling the deparaffinized sections on slides for 10 minutes in WT1A was transfected into CCG99-11 cells and stable clones were 1 mM EDTA (pH 8.0) in a microwave oven [15]. IFI16 clone μ selected with 400 g/ml hygromycin (Invitrogen). Stable clones were 1G7 mouse monoclonal antibody, the WT1 clone 180 rabbit poly- maintained in 50% of the drug concentration used for selection. clonal antibody (Santa Cruz Biotechnology) and the secondary anti- body (biotinylated horse anti-mouse and goat anti-rabbit; Vector Laboratories, Burlingame, CA) were used at a dilution of 1:400.