CIC-DUX4 Induces Small Round Cell Sarcomas Distinct from Ewing Sarcoma
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Author Manuscript Published OnlineFirst on April 12, 2017; DOI: 10.1158/0008-5472.CAN-16-3351 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. CIC-DUX4 induces small round cell sarcomas distinct from Ewing sarcoma Toyoki Yoshimoto 1, 2, Miwa Tanaka1, Mizuki Homme1, Yukari Yamazaki1, Yutaka Takazawa3, Cristina R Antonescu4, and Takuro Nakamura1 1Division of Carcinogenesis, The Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan. 2Department of Pathology, Toranomon Hospital, Tokyo Japan. 3Division of Pathology, The Cancer Institute, Japanese Foundation for Cancer Research, Tokyo Japan. 4Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York. Running title: Mouse model for CIC-DUX4 sarcoma Keywords: CIC-DUX4, sarcoma model, Ccnd2, Muc5ac, biomarker The abbreviations used are: CDS, CIC-DUX4 sarcoma; ES, Ewing sarcoma; eMC, embryonic mesenchymal cell; SS, synovial sarcoma; RS, rhabdomyosarcoma; EMCS, extraskeletal myxoid chondrosarcoma; GSEA, gene set enrichment analysis; ECM, extracellular matrix; MSC, mesenchymal stem cell. Grant Support: The study was supported by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science (no. 26250029 to T. Nakamura), and Cancer Center Support grants (P50 CA140146 and P30-CA008747 to C. R. Antonescu) from the U.S. National Institute of Health. Corresponding Author: Takuro Nakamura, Division of Carcinogenesis, The Cancer Institute, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan. Phone: 81-3-3570-0462; E-mail: [email protected] T. Yoshimoto and M. Tanaka contributed equally to this article. The manuscript includes 3,775 words, five figures and two tables. 1 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 12, 2017; DOI: 10.1158/0008-5472.CAN-16-3351 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract CIC-DUX4 sarcoma (CDS) or CIC-rearranged sarcoma is a subcategory of small round cell sarcoma resembling the morphological phenotypes of Ewing sarcoma (ES). Hoever, recent clinicopathologic and molecular genetic analyses indicate that CDS is an independent disease entity from ES. Few ancillary markers have been used in the differential diagnosis of CDS, and additional CDS-specific biomarkers are needed for more definitive classification. Here we report the generation of an ex vivo mouse model for CDS by transducing embryonic mesenchymal cells (eMC) with human CIC-DUX4 cDNA. Recipient mice transplanted with eMC expressing CIC-DUX4 rapidly developed an aggressive, undifferentiated sarcoma composed of small-round to short-spindle cells. Gene expression profiles of CDS and eMC revealed upregulation of CIC-DUX4 downstream genes such as PEA3 family genes, Ccnd2, Crh and Zic1. Immunohistochemical analyses for both mouse and human tumors showed that CCND2 and MUC5AC are reliable biomarkers to distinguish CDS from ES. Gene silencing of CIC-DUX4 as well as Ccnd2, Ret, and Bcl2 effectively inhibited CDS tumor growth in vitro. The CDK4/6 inhibitor palbociclib and the soft tissue sarcoma drug trabectedin also blocked the growth of mouse CDS. In summary, our mouse model provides important biological information about CDS and provides a useful platform to explore biomarkers and therapeutic agents for CDS. 2 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 12, 2017; DOI: 10.1158/0008-5472.CAN-16-3351 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction CIC-DUX4 sarcomas (CDS) belong to a highly aggressive subgroup of small round cell sarcoma, affecting predominantly children and young adults (1). Although EWS-ETS-negative small round cell sarcomas were previously classified as Ewing-like sarcoma or Ewing sarcoma-like round cell sarcoma, increasing evidence suggests the distinct biology of CDS with CIC-DUX4 gene fusions, secondary to either a t(4;19)(q35;q13) or t(10;19)(q26.3;q13) translocation (1, 2). CDS morphologically show small- to medium-sized, round to ovoid cells, packed in solid sheets, lacking any line of differentiation. Thus the differential diagnosis from Ewing sarcoma (ES) is often difficult without detecting the CIC related fusions (1, 3). CDS show a poor outcome and overall survival of CDS patients was worse than that of ES patients (3). It is therefore important to clarify the biological characteristics responsible for the different behaviors between CDS and ES. The differences may be caused by distinct signaling pathways regulated by CIC-DUX4 and EWS-ETS as well as the potential different cell-of-origin of each tumor type. Immunohistolochemical detection of ETV4, a transcriptional target of CIC-DUX4, is a useful marker for the histologic diagnosis of CDS (2-6). This suggests that further molecular characterization of CDS will expand our understanding on their biological behavior, as well as provide novel biomarkers and molecular targets for therapy. To this end we developed an animal model that recapitulates the phenotypes of human CDS. CIC-DUX4 gene fusion is the initiating and causative event in CDS, encoding a chimeric transcription factor consisting of the large part of CIC including its DNA-binding HMG box, and the transcriptional activation domain derived from the DUX4 C-terminus (2). Deregulation of CIC target genes such as ETV4 is one of the key events in the development and progression of CDS (2), and chimeric transcription factors act as an oncogene only in proper cellular context (7-9). We have previously successfully generated an Ewing sarcoma mouse model utilizing 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, considered the likely 3 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 12, 2017; DOI: 10.1158/0008-5472.CAN-16-3351 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. cell-of-origin of ES, exhibiting morphologic and molecular features of human ES. In addition, an alveolar soft part sarcoma model mouse that recapitulates histologic features and high metastatic potency of human counterpart has been generated (10). Taking advantage of this ex vivo gene transduction system, we have developed a similar mouse model for human CDS. Mouse embryonic mesenchymal cells transduced with the CIC-DUX4 develop small blue round cell tumors, as seen in human CDS, which showed a highly aggressive growth. Gene microarray analyses of CDS revealed distinct gene expression profiles of CDS from ES, with significant upregulation of the cell cycle progression pathway, such as Ccnd2 gene in CDS. Furthermore, Cyclin D2 and MUC5AC were found as novel biomarkers for diagnosis of CDS, and a growth inhibitory effect of trabectedin was observed. Modeling fusion gene-associated sarcomas thus provides important tools for further our understanding in the pathogenesis of these rare types of neoplasms. 4 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 12, 2017; DOI: 10.1158/0008-5472.CAN-16-3351 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Materials and Methods Generation and characterization of the CDS model mouse N-terminal HA-tagged CIC-DUX4 was introduced into the pMYs-IRES-GFP vector. Limbs of Balb/c mouse embryo (Clea Japan, Tokyo, Japan) were removed aseptically on 18.5 dpc, and embryonic mesenchymal cells (eMC) were obtained by dissection using two mg/mL collagenase (Wako Pure Chemical, Tokyo, Japan) at 37˚C for 2 h. eMC were cultured in growth medium composed of Iscove’s Modified Dulbecco’s Medium (Invitrogen) supplemented with 15% fetal bovine serum and subjected immediately to retroviral infection without further purification. The analysis of cell surface marker expression suggested that eMC might share overlapping characteristics with mesenchymal stem cells (Supplemental Fig. S1). Retroviral stock was added into the medium containing eMC with six μg/mL of Polybrene (Sigma) and then spun at 700 g for 1 h. The spin infection was repeated after 24 hours. Transduced eMC were mixed with growth factor-reduced Matrigel (Becton Dickinson) and 1 x 106 cells were transplanted into the subcutaneous regions of Balb/c nude mice was performed as described previously (7). Transduction efficiency of the CIC-DUX4 retrovirus was confirmed by flow cytometry using FacsCalibur (Beckton Dickinson) (Supplemental Fig. S2). All experiments described in this study were performed in strict accordance with standard ethical guidelines and approved by the animal care committee at the Japanese Foundation for Cancer Research under licenses 10-05-9 and 0604-3-13. Human sarcoma specimens Paraffin blocks from ten cases of both CDS and ES specimens were obtained from the Memorial Sloan-Kettering Cancer Center. The study was approved by the Institutional Review Board at Memorial Sloan-Kettering Cancer Center (Protocol 02-060). Synovial sarcoma (hSS), rhabdomyosarcoma (hRS)