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Multiple Imprinted and Stemness Genes Provide a Link Between Normal and Tumor Progenitor Cells of the Developing Human Kidney

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Multiple Imprinted and Stemness Genes Provide a Link Between Normal and Tumor Progenitor Cells of the Developing Human Kidney Research Article Multiple Imprinted and Stemness Genes Provide a Link between Normal and Tumor Progenitor Cells of the Developing Human Kidney Benjamin Dekel,1 Sally Metsuyanim,1 Kai M. Schmidt-Ott,5 Edi Fridman,2 Jasmin Jacob-Hirsch,3 Amos Simon,3 Jehonathan Pinthus,4 Yoram Mor,4 Jonathan Barasch,5 Ninette Amariglio,3 Yair Reisner,6 Naftali Kaminski,7 and Gideon Rechavi3 Departments of 1Pediatrics, 2Pathology, 3Pediatric Hemato-Oncology, and 4Urology, Chaim Sheba Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 5Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York; 6Department of Immunology, Weizmann Institute of Science, Rehovot, Israel; and 7Simmons Center for Interstitial Lung Disease, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania Abstract Introduction Wilms’ tumor (WT), the embryonic kidney malignancy, is Metanephroi are the primordia of adult mammalian kidneys (1). suggested to evolve from a progenitor cell population of The metanephric blastema contains multipotential precursors uninduced metanephric blastema, which typically gives rise that give rise to all cell types of the nephron (2). Errors in to nephrons. However, apart from blastema, WT specimens nephrogenesis that lead to human disease are common (1). They frequently contain cells that have differentiated into renal include congenital abnormalities (e.g., renal dysplasia) and cancer. tubular or stromal phenotypes, complicating their analysis. Wilms’ tumor (WT) or nephroblastoma is a pediatric kidney cancer We aimed to define tumor-progenitor genes that function believed to arise from multipotent embryonic renal precursors of in normal kidney development using WT xenografts (WISH- the metanephric blastema (3), which fail to terminally differentiate WT), in which the blastema accumulates with serial pas- into epithelium and continue to proliferate, thus forming blastemal sages at the expense of differentiated cells. Herein, we did elements in the tumor. Nevertheless, WTs contain structures at transcriptional profiling using oligonucleotide microarrays different stages of maturation that mimic those present in the of WISH-WT, WT source, human fetal and adult kidneys, nephrogenic zone of the growing fetal kidney (tubular epithelia and and primary and metastatic renal cell carcinoma. Among stromal elements in addition to uninduced blastema), and also the most significantly up-regulated genes in WISH-WT, other mesoderm elements (rhabdomyoblats, cartilage), suggesting we identified a surprising number of paternally expressed that blastemal cells have differentiated at least in part (3). Derived genes (PEG1/MEST, PEG3, PEG5/NNAT, PEG10, IGF2,and from primitive embryonic tissue and being strongly associated with DLK1), as well as Meis homeobox genes [myeloid ecotropic various congenital syndromes, WT is an attractive model to study viral integration site 1 homologue 1 (MEIS1) and MEIS2], which developmental pathways leading to cancer. In fact, WT had already suppress cell differentiation and maintain self-renewal. A provided significant information regarding the genetic and comparison between independent WISH-WT and WT sam- epigenetic events leading to the development of cancer in general ples by real-time PCR showed most of these genes to be (4–7). Global gene profiling during kidney development has highly overexpressed in the xenografts. Concomitantly, provided novel insights into the genetic program that control they were significantly induced in human fetal kidneys, murine and human nephrogenesis (8–11). Furthermore, molecular strictly developmentally regulated throughout mouse nephro- signatures of normal human kidney development set a reference genesis and overexpressed in the normal rat metanephric for abnormal one (12). Accordingly, we could also show that the blastema. Furthermore, in vitro differentiation of the unin- overall gene expression profile of a WT specimen was most similar duced blastema leads to rapid down-regulation of PEG3, to that observed for an 8-week human gestation kidney (11). DLK1,andMEIS1. Interestingly, ischemic/reperfusion in- Li et al. (13) have recently done more detailed experiments jury to adult mouse kidneys reinduced the expression of involving gene expression profiling in WT. They identified 357 PEG3, PEG10, DLK1, and MEIS1, hence simulating embryo- genes as differentially expressed between favorable histology WTs genesis. Thus, multiple imprinted and stemness genes that and fetal kidneys (16-22 weeks of gestation). To determine stage- function to expand the renal progenitor cell population specific expression of these genes, they compared their data set may lead to evolution and maintenance of WT. (Cancer Res to that previously obtained for normal rat nephrogenesis (8). One 2006; 66(12): 6040-9) hundred twenty-four matches to genes on the microarray used by Stuart et al. (8) were found. Mapping between the two data sets showed that WTs systematically overexpressed genes corres- ponding to the earliest stage of metanephric development. Never- theless, profiling gene expression in whole heterogeneous tissues, Note: Supplementary data for this article are available at Cancer Research Online such as WT, is complicated by mixed populations of cells and is (http://cancerres.aacrjournals.org/). Requests for reprints: Benjamin Dekel, Laboratory for Developmental and therefore less powerful for discovering genes involved in specific Regenerative Nephrology, Department of Pediatrics, Safra Children’s Hospital, Sheba developmental processes. In addition, it is becoming clear that Medical Center, 52621 Tel Hashomer, Israel. Phone: 972-3-5302517; Fax: 972-3-5305787; many, if not most, malignancies arise from a rare population of E-mail: [email protected]. I2006 American Association for Cancer Research. cells that exclusively maintain the ability to self-renew and sustain doi:10.1158/0008-5472.CAN-05-4528 the tumor via the expression of tumor-progenitor genes (14, 15). Cancer Res 2006; 66: (12). June 15, 2006 6040 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2006 American Association for Cancer Research. Imprinted Genes in Wilms’ Tumor Xenografts Moreover, these ‘‘cancer stem cells’’ are often biologically distinct Ischemia/reflow experiments. For unilateral ischemia/reflow, a flank from the bulk of differentiated cancer cells that characterize the incision was made and the left renal pedicle was clamped for 40 minutes disease. In that regard, the analysis of WT xenografts established using a vascular clamp (Fine Science Tools, Inc., Foster City, CA). The and propagated in immunodeficient mice, which pressure for meta- abdomen was covered with gauze moistened in PBS, and the mice were j nephric blastemal maintenance/proliferation and the disappearance maintained at 37 C using a warming pad. After 40 minutes, the clamp was removed and reperfusion was confirmed visually. To determine the extent of of differentiated tubular and stromal structures, is advantageous. acute injury, control mice were sacrificed 24 hours after ischemia/reflow, The selection of a more homogenous subset of early metanephric and kidneys were collected and processed for histology using H&E and cancer cells is of paramount importance as these are the cellular sirius red staining. characteristics that might be clinically linked to morbidity. RNA isolation. Total RNA from human and mouse samples was isolated Here, we present molecular analysis of these authentic WT xeno- from each sample using TRIZOL (Life Technologies, Invitrogen, Carlsbad, grafts, concomitant with fetal and adult kidneys, and renal cell carcino- CA).8 Total RNA from rat metanephric mesenchymes was extracted using ma. By comparing the gene expression profiles and analyzing models RNeasy mini kit (Qiagen, Valencia, CA) with on-column DNase digestion of murine kidney development and regeneration, we have identified according to the instructions of the manufacturer. An Agilent Bioanalyzer multiple imprinted and stemness-related genes that participate in early was used to confirm RNA integrity. nephrogenesis and also signify maintenance and expansion of WT Microarray analysis. Malignant adult and fetal kidney specimens were obtained from kidneys removed for stage I clear cell carcinoma or WT. blastema. Thus, epigenetic changes, which regulate imprinted genes, Adult kidney specimens were obtained from the normal kidneys removed may serve as a driving force for the human renal progenitor cell for stage I clear cell carcinoma. Metastatic renal cell carcinoma was population affecting both organogenesis and tumorigenesis. obtained from surgical remnants of biopsies. Fetal kidney tissue was obtained following curettage. Studies with human embryonic kidney tissue were approved by the Helsinki Ethical Committee. Total RNA was extracted Materials and Methods and used as a template to generate double-stranded cDNA and biotin- Animals and surgical procedures. Severe combined immunodeficient labeled cRNA, as recommended by the manufacturer of the arrays and as (SCID) (c.b-17/Icr beige or nonobese diabetic), nude (BALB/c nu/nu), and previously described (17). Hybridization to a Genechip Human Genome C57BL/6 mouse strains were obtained from the Weizmann Institute Animal HU95A oligonucleotide arrays containing 9,632 probe sets was done Breeding Center (Rehovot, Israel). Animals used were 6- to 10-week-old. as described in the Affymetrix human_datasheet.pdf 9 (Affymetrix,
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