CIC-DUX4 Induces Small Round Cell Sarcomas Distinct from Ewing Sarcoma

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). However, 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 IHC 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. Cancer Res; 77(11); 2927-37. ©2017 AACR.

Figure 1

The CDS ex vivo model. A, Cumulative incidence of CDS tumors arising in the transplanted mice induced by eMC expressing CIC-DUX4 (blue line) or containing an empty vector (black line). The survival curve of mouse ES is shown (red line) and survival was compared by the log-rank test. *p < 0.01. B, The CDS tumors could be observed as subcutaneous masses in recipient nude mice (arrows). C-F, Histology of murine CDS. H&E staining for primary sites (C-E) and metastasis in the lung (F). Mouse CDS are composed of predominantly small round cells (C) or short spindle cells (D). Abundant ECM accumulation was indicated as dense fibrous septa (E). Scale bars indicate 50 μm (C and D) and 200 μm (E and F). Insets show high-magnification images. G, Systemic metastases by injecting CDS cells via tail vein. H, Expression of the CIC-DUX4 chimeric protein was detected by immunoprecipitation with an anti-DUX4 antibody followed by western blotting by an anti-CIC antibody. Immunoprecipitated samples with rabbit IgG were loaded as negative controls.

Figure 2

Gene expression profiles of eMC expressing CIC-DUX4 and mCDS tumors. A, Venn diagram showing upregulated genes in eMC expressing CIC-DUX4 (n = 4) or mCDS tumors (n = 6) vs those containing an empty vector (n = 4). B, GSEA of eMC expressing CIC-DUX4 versus those with an empty vector shows enrichment of the gene set involved in ECM organization (left), and mCDS tumors versus eMC with an empty vector shows the Cyclin D1 pathway (right). C, Principal component analysis for gene expression profiles of mCDS and mES. D, Hierarchical clustering with 1,661 differentially upregulated genes in mCDS or mES. E, Real-time quantitative RT-PCR for Ccnd1, Ccnd2, Bcl2, Ret, Muc5ac and Etv4 in 6 independent mCDS and mES tumors, eMC with an empty vector and the CIC-DUX4 retrovirus. F, GSEA shows enrichment of the VEGF signaling pathway and the Cyclin D1 signature in mCDS. G, Hierarchical clustering of gene expression profiles of 6 samples of mCDS, 5 cases of hCDS, 20 myxoid liposarcomas (MLS), 21 malignant fibrous histiocytomas (MFH), 16 synovial sarcomas (SS), 11 osteosarcomas (OS), 7 chondrosarcomas (CS), 32 Ewing sarcomas (ES) and 10 neuroblastomas (NB).

Figure 3

Immunostaining of CCND2 and MUC5AC in mouse and human CDS and ES. A, mCDShows positive staining for CCND2, ETV4 and MUC5AC (left). No expression for CCND2 and MUC5AC and little expression for ETV4 in mES (center). Scale bars indicate 40 μm. The frequencies of positive cells for CCND2, MUC5AC or ETV4 were quantitated (right). B, Human CIC-rearranged sarcoma (hCIC) shows CCND2 and MUC5AC expression (left). No expression in hES (center). Scale bars indicate 40 μm. The frequencies of positive cells for CCND2 or MUC5AC in hCIC, hES, rhabdomyosarcoma (hRS), extraskeletal myxoid chondrosarcoma (hEMCS) and synovial sarcoma (hSS) were quantitated (right). The numbers of cases are indicated in parentheses.

Figure 4

Growth suppression of tumor cells by gene silencing. A, Inhibition of cell proliferation by knockdown of CIC-DUX4 and upregulated genes in mCDS. Relative growth of tumor cells 48 hours after siRNA treatment was calculated by comparing each cell number to cells treated with control siRNA (NC). Two oligonucleotide sequences were used for each gene and the symbols of siRNA used are indicated. B, Gene knockdown was confirmed by RT-PCR. Knockdown of CIC-DUX4 was achieved using human CIC-specific siRNAs. CIC-DUX4 of human origin was extensively downregulated (bottom left), whereas endogenous mouse Cic expression was not reduced significantly (bottom right).

Figure 5

Effects of small molecule inhibitors and anti-cancer agents on mCDS. A, In vitro growth inhibition of mCDS (mCDS 4, 15 and 16) cell lines by palbociclib, vandetanib and ABT199. Human clear cell sarcoma (KAS) and human osteosarcoma (U2OS) cell lines were also tested. Cells were treated with each reagent at the indicated concentration for 48 hours. The experiment was performed in triplicate, and average suppression rates with SEM, and IC50 are indicated. B, In vitro growth inhibition of mCDS and ES cell lines by bortezomib and trabectedin as in A. C, Growth inhibitory effects of bortezomib and palbociclib for mCDS in vivo. mCDS 16 cells were transplanted subcutaneously into nude mice, and tumor volume was measured every other day. Mean tumor volumes ± SEM for 5 mice of each group are plotted. The treatment times of trabectedin and palbociclib are indicated as arrows. *p < 0.01, **p < 0.001.