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CIC-DUX4 Induces Small Round Cell Sarcomas

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CIC-DUX4 Induces Small Round Cell Sarcomas Published OnlineFirst April 12, 2017; DOI: 10.1158/0008-5472.CAN-16-3351 Cancer Molecular and Cellular Pathobiology Research CIC-DUX4 Induces Small Round Cell Sarcomas Distinct from Ewing Sarcoma Toyoki Yoshimoto1,2, Miwa Tanaka1, Mizuki Homme1, Yukari Yamazaki1, Yutaka Takazawa3, Cristina R. Antonescu4, and Takuro Nakamura1 Abstract CIC-DUX4 sarcoma (CDS) or CIC-rearranged sarcoma is a short spindle cells. Gene-expression profiles of CDS and eMC subcategory of small round cell sarcoma resembling the morpho- revealed upregulation of CIC-DUX4 downstream genes such as logical phenotypes of Ewing sarcoma (ES). However, recent PEA3 family genes, Ccnd2, Crh, and Zic1. IHC analyses for both clinicopathologic and molecular genetic analyses indicate that mouse and human tumors showed that CCND2 and MUC5AC CDS is an independent disease entity from ES. Few ancillary are reliable biomarkers to distinguish CDS from ES. Gene silenc- markers have been used in the differential diagnosis of CDS, and ing of CIC-DUX4 as well as Ccnd2, Ret, and Bcl2 effectively additional CDS-specific biomarkers are needed for more defini- inhibited CDS tumor growth in vitro. The CDK4/6 inhibitor tive classification. Here, we report the generation of an ex vivo palbociclib and the soft tissue sarcoma drug trabectedin also mouse model for CDS by transducing embryonic mesenchymal blocked the growth of mouse CDS. In summary, our mouse cells (eMC) with human CIC-DUX4 cDNA. Recipient mice trans- model provides important biological information about CDS planted with eMC-expressing CIC-DUX4 rapidly developed an and provides a useful platform to explore biomarkers and ther- aggressive, undifferentiated sarcoma composed of small round to apeutic agents for CDS. Cancer Res; 77(11); 1–11. Ó2017 AACR. Introduction CIC–DUX4 and EWS–ETS as well as the potential different cell-of- origin of each tumor type. CIC-DUX4 sarcomas (CDS) belong to a highly aggressive IHC detection of ETV4, a transcriptional target of CIC-DUX4, is subgroup of small round cell sarcoma, affecting predominantly a useful marker for the histologic diagnosis of CDS (2–6). This children and young adults (1). Although EWS–ETS-negative small suggests that further molecular characterization of CDS will round cell sarcomas were previously classified as Ewing-like expand our understanding on their biological behavior, as well sarcoma or Ewing sarcoma (ES)–like round cell sarcoma, increas- as provide novel biomarkers and molecular targets for therapy. To ing evidence suggests the distinct biology of CDS with CIC–DUX4 this end we developed an animal model that recapitulates the gene fusions, secondary to either a t(4;19)(q35;q13) or t(10;19) phenotypes of human CDS. CIC–DUX4 gene fusion is the initi- (q26.3;q13) translocation (1, 2). CDS morphologically show ating and causative event in CDS, encoding a chimeric transcrip- small- to medium-sized, round to ovoid cells, packed in solid tion factor consisting of the large part of CIC including its DNA- sheets, lacking any line of differentiation. Thus, the differential binding HMG box, and the transcriptional activation domain diagnosis from ES is often difficult without detecting the CIC- derived from the DUX4 C-terminus (2). Deregulation of CIC related fusions (1, 3). CDS show a poor outcome and overall target genes such as ETV4 is one of the key events in the devel- survival of CDS patients was worse than that of ES patients (3). It is opment and progression of CDS (2), and chimeric transcription therefore important to clarify the biological characteristics respon- factors act as an oncogene only in proper cellular context (7–9). sible for the different behaviors between CDS and ES. The differ- We have previously successfully generated an ES mouse model ences may be caused by distinct signaling pathways regulated by using an ex vivo–based technology (7). In this model, EWS–FLI1 appropriately upregulates its target genes in the sarcoma cells as well as in the embryonic osteochondrogenic progenitors, consid- 1 Division of Carcinogenesis, The Cancer Institute, Japanese Foundation for ered the likely cell-of-origin of ES, exhibiting morphologic and 2 Cancer Research, Tokyo, Japan. Department of Pathology, Toranomon Hos- molecular features of human ES. In addition, an alveolar soft pital, Tokyo Japan. 3Division of Pathology, The Cancer Institute, Japanese Foundation for Cancer Research, Tokyo Japan. 4Department of Pathology, part sarcoma model mouse that recapitulates histologic features Memorial Sloan-Kettering Cancer Center, New York, New York. and high metastatic potency of human counterpart has been generated (10). Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Taking advantage of this ex vivo gene transduction system, we have developed a similar mouse model for human CDS. Mouse T. Yoshimoto and M. Tanaka contributed equally to this article. embryonic mesenchymal cells transduced with the CIC–DUX4 Corresponding Author: Takuro Nakamura, The Cancer Institute, Japanese develop small blue-round cell tumors, as seen in human CDS, Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan. which showed a highly aggressive growth. Gene microarray anal- Phone: 813-3570-0462; Fax: 813-3570-0463; E-mail: [email protected] yses of CDS revealed distinct gene-expression profiles of CDS doi: 10.1158/0008-5472.CAN-16-3351 from ES, with significant upregulation of the cell-cycle progres- Ó2017 American Association for Cancer Research. sion pathway, such as Ccnd2 gene in CDS. Furthermore, cyclin D2 www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst April 12, 2017; DOI: 10.1158/0008-5472.CAN-16-3351 Yoshimoto et al. and MUC5AC were found as novel biomarkers for diagnosis of In vivo imaging CDS, and a growth inhibitory effect of trabectedin was observed. The mice transplanted CDS tumor cells introduced with the Modeling fusion gene-associated sarcomas thus provides impor- luciferase cDNA were administrated with luciferin. Illumination tant tools for further our understanding in the pathogenesis of from the labeled tumor cells were monitored by the IVIS Lumina these rare types of neoplasms. LT imaging system (PerkinElmer). Materials and Methods RT-PCR and real-time quantitative RT-PCR Total RNA extraction, reverse transcription and RNA quantifi- Generation and characterization of the CDS model mouse cation were performed according to methods described previously N-terminal HA-tagged CIC–DUX4 was introduced into the (2). Conventional RT-PCR and real-time quantitative RT-PCR pMYs–IRES–GFP vector. Limbs of Balb/c mouse embryo (Clea were performed using a Gene Amp 9700 thermal cycler (Applied Japan) were removed aseptically on 18.5 dpc, and embryonic Biosystems) and a 7500 Fast Real-Time PCR System (Applied mesenchymal cells (eMC) were obtained by dissection using Biosystems), respectively. The sequences of the oligonucleotide two mg/mL collagenase (Wako Pure Chemical) at 37Cfor primers used are shown in Supplementary Table S1. 2 hours. eMC were cultured in growth medium composed of Iscove's Modified Dulbecco's Medium (Invitrogen) supplemen- ted with 15% FBS and subjected immediately to retroviral Microarray analysis infection without further purification. The analysis of cell GeneChip analysis was conducted to determine gene expres- fi surface marker expression suggested that eMC might share sion pro les. The murine HT MG-430 PM array (Affymetrix) was overlapping characteristics with mesenchymal stem cells (Sup- hybridized with aRNA probes generated from eMC 48 hours after plementary Fig. S1). Retroviral stock was added into the medi- transduction with pMYs-CIC-DUX4 or empty vector, CDS and ES um containing eMC with 6 mg/mL of Polybrene (Sigma) and tumor tissues, or a mixture of mouse normal tissues according to then spun at 700 Â g for 1 hour. The spin infection was repeated methods described previously (11). The expression data were after 24 hours. Transduced eMC were mixed with growth factor- analyzed using GeneSpring ver 12.6 (Agilent Technologies) reduced Matrigel (Becton Dickinson) and 1 Â 106 cells were and gene set enrichment analysis (GSEA) was performed using transplanted into the subcutaneous regions of Balb/c nude GSEA-P 2.0 software (12). The microarray datasets are acces- mice was performed as described previously (7). Transduction sible through the NCBI Gene Expression Omnibus database efficiency of the CIC-DUX4 retrovirus was confirmed by flow (http://www.ncbi.nlm.nih.gov/geo), with an accession number cytometry using FACSCalibur (Beckton Dickinson; Supplemen- GSE90978. tary Fig. S2). All experiments described in this study were performed in strict accordance with standard ethical guidelines Data comparisons and clustering between murine and human and approved by the animal care committee at the Japanese microarray data Foundation for Cancer Research under licenses 10-05-9 and The microarray data from six mCDS to five hCDS samples (1) 0604-3-13. were compared with human sarcoma microarray data sets. Data from the ONCOMINE data base (https://www.oncomine.org/) Human sarcoma specimens were accessed in June 2011. CEL files from E-MEXP-1142, Paraffin blocks from 10 cases of both CDS and ES specimens E-MEXP-353, GSE21122, GSE7529, mCDS and hCDS samples were obtained from the Memorial Sloan-Kettering Cancer Center. were summarized by MAS5 algorithm with Affymetrix Power The study was approved by the Institutional Review Board at Tool software version 1.12.0 (Affymetrix). Each probe set that Memorial Sloan-Kettering Cancer Center (Protocol 02-060). had same Gene Symbols was collapsed into a single probe set Synovial sarcoma (hSS), rhabdomyosarcoma (hRS), and extra- that showed the highest median value in the raw MAS5 dataset. skeletal myxoid chondrosarcoma (hEMCS) samples were Each mouse gene of collapsed datasets was joined into the same obtained from the Japanese Foundation for Cancer Research. gene symbol of human collapsed datasets that resulted in one dataset with 10,166 probe sets.
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