<p>Supplementary Figures S1-6. Supplementary Fig S1: Combined patient characteristics and subjected methods Supplementary Fig. S2 A: ROC curves were constructed by using the somatic scores from the sequenced genotypes. Supplementary Fig. S3: Circos plots of representative Ewing sarcomas. Supplementary Fig. S4: Sanger sequencing results confirmed (A) somatic inactivating mutations of the STAG2 gene (positions according to RefSeq NG 033796.2) and (B) the activating N546 mutation of FGFR1. Supplementary Fig. S5: FGFR-1 expression in Ewing Sarcomas by means of real time RT PCR (A, normalized to FGFR-1 expression in VH-64). (B) Array-based analysis of FGFR1 expression in ES compared to normal tissue (NBA; SI Materials and Methods). (C) Knock-down by means of lentiviral transduced FGFR1.shRNA effectively reduced FGFR1 mRNA expression by 70 % or more in Ewing sarcoma cell lines as compared to scrambled shRNA. (D) Knockdown was confirmed on the protein level by means of western blotting. Supplementary Fig. S6: Amplification of the FGFR-1 gene (A) as well as gene expression (B) are associated with a trend towards inferior survival in patients with ES. (C) Treatment with Ponatinib inhibited growth of ES cell lines significantly. (D) FGFR1 tyrosine kinase inhibitor therapy in a patient with relapsed Ewing sarcoma.</p>
Abstract Introduction: Recent evidence demonstrated that a low mutation rate seems a general feature of pediatric cancers. Ewing sarcoma (ES) is defined by balanced chromosomal EWS/ETS translocations, which give rise to oncogenic chimeric proteins (EWS-ETS). Other contributing somatic mutations involved in disease development have only been observed at low frequency. Thus, cancer in children is not solely a genetic disease and can neither be understood nor cured presumably without epigenetics. Previously, we identified the histone methyl-transferase enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), the enzymatic subunit of the polycomb PRC2 complex, to be over-expressed in ES. RNA interference of EZH2 suppressed tumor development and metastasis in vivo and microarray analysis of EZH2 knock down revealed an EZH2-maintained, undifferentiated, reversible phenotype in ES. Experimental procedures: The role of class I histone deacetylases (HDACs) was determined using different potent inhibitors like Trichostatin A (TSA), Romidepsin (FK22), Entinostat (MS-275) and PCI-34051 as well as a CRISPR/Cas9 approach to knock out specific HDACs. To analyze resulting changes qRT-PCR, Western Blotting, proliferation, apoptosis and invasion assays as well as immunofluorescence experiments were deployed. Results: Interestingly, the effects of gene silencing after RNA interference of EZH2 could be mimicked by treatment of ES with TSA or MS-275. Microarray analysis revealed that treatment with MS-275 or TSA resulted in the induction of a similar pattern of differentiation genes as was previously observed after EZH2 blockade and seems to be dependent on histone deacetylase (HDAC) activity. Furthermore, in proliferation assays ES were more susceptible to treatment with HDAC inhibitors than other small round blue cell tumors such as neuroblastoma or pediatric cALL cells. Treatment of enzymatic inhibitors blocking EZH2 activity could not mimic the results observed after EZH2 RNA interference indicating that EZH2-containing PRC2 complexes may serve as a building block of class I HDAC activity in ES. Conclusion: Class I HDACs seem to be important mediators of the pathognomonic EWS-ETS-mediated transcription program in ES and thus interesting new treatment opportunities for this malignant disease. Citation Format: Kristina von Heyking, Tim Hensel, Julia Singer, Oxana Schmidt, Stefan Burdach, Günther H. Richter. Investigating the role of class I HDACs in Ewing sarcoma pathology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3866. doi:10.1158/1538-7445.AM2017-3866
Microarray analysis revealed genes of the posterior HOXD locus normally involved in bone formation to be over-expressed in primary Ewing sarcoma (ES). The expression of posterior HOXD genes was not influenced via ES pathognomonic EWS/ETS translocations. However, knock down of the dickkopf WNT signaling pathway inhibitor 2 (DKK2) resulted in a significant suppression of HOXD10, HOXD11 and HOXD13 while over-expression of DKK2 and stimulation with factors of the WNT signaling pathway such as WNT3a, WNT5a or WNT11 increased their expression. RNA interference demonstrated that individual HOXD genes promoted chondrogenic differentiation potential, and enhanced expression of the bone-associated gene RUNX2. Furthermore, HOXD genes increased the level of the osteoblast- and osteoclast-specific genes, osteocalcin (BGLAP) and platelet-derived growth factor beta polypeptide (PDGFB), and may further regulate endochondral bone development via induction of parathyroid hormone-like hormone (PTHLH). Additionally, HOXD11 and HOXD13 promoted contact independent growth of ES, while in vitro invasiveness of ES lines was enhanced by all 3 HOXD genes investigated and seemed mediated via matrix metallopeptidase 1 (MMP1). Consequently, knock down of HOXD11 or HOXD13 significantly suppressed lung metastasis in a xeno-transplant model in immune deficient mice, providing overall evidence that posterior HOXD genes promote clonogenicity and metastatic potential of ES.
Additional file 2: Fig. S1 a, Expression levels of different class I HDAC genes in different pediatric small-round-blue-cell tumors, carcinomas and normal tissues by box plot presentation using a comparative study of the amc onco-genomics software tool ( https://hgserver1.amc.nl/cgi-bin/r2/main.cgi ). The number of samples in each cohort is given in brackets. b, Differential expression levels of class I HDAC genes in primary EwS at different tumor sites by box plot presentation using the GSE63157 study set and the amc onco-genomics software tool. The number of samples in each cohort is given in brackets, ND: not determined. p-value < 0.05. c, Retroviral gene transfer of EWS-FLI1 cDNA into MSC lines L87 and V54.2 [4] results in HDAC3 and HDAC8 induction as measured via qRT-PCR, while no change of HDAC1 and HDAC2 expression was observed. Induction of EWS-FLI1-dependent EZH2 expression served as control. Fig. S2 a, Tube formation assay with the EwS cell lines CHLA-10 and SK-N-MC after incubation with 3µM MS275 or 4nM FK228 over-night compared to WT control. Both HDACi clearly enhanced endothelial differentiation potential (scale bar 0.5mm). b, Analysis of neurogenic differentiation potential of the EwS cell lines CHLA-10, EW7 and SK-N-MC treated for six days with 0.5µM MS-275 or 0.2nM FK228. The neurogenic differentiation marker GFAP (glial fibrillary acidic protein) and GAP43 (growth associated protein 43) were significantly upregulated after incubation with both HDACi as demonstrated by qRT-PCR. Fig. S3 a, Cell cycle analysis of CRISPR/Cas9 HDAC1 or HDAC2 knock outs compared to their controls (Cas9) in three different EwS cell lines are shown. Distributional analysis of cell cycle phases of HDAC1 or HDAC2 knock outs compared to their control were performed by propidium iodine staining and flow cytometry measurement, respectively. b, To analyze apoptosis in HDAC1 and HDAC2 CRISPR/Cas9 knock outs, DNA double strand breaks were measured with anti-phospho-histone H2AX-FITC conjugated mAbs and counterstained with DAPI. Left, the frequency of g-H2AX positive foci per cell was summarized in bar graphs. Right, fluorescence images show a representative experiment with HDAC1 (top) and HDAC2 (bottom) in two different EwS cell lines each, compared to their controls. Fig. S4 a, Western blot analysis of class I HDAC protein levels and their compensation in CRISPR/Cas9 HDAC1 and HDAC2 knock outs compared to their controls (Cas9). Protein levels were detected by antibodies against HDAC1, HDAC2, HDAC3 and HDAC8. b-actin or GAPDH antibodies were used as loading control. b, Heat map of 229 genes differentially expressed in three different EwS lines CHLA-10, EW7 and SK-N-MC after CRISPR/Cas9 HDAC1 knock out, are shown. Each column represents one individual array. Microarray data with their normalized fluorescent signal intensities were used (robust multichip average (RMA); GSE162786). c, Circos plots of downregulated genes (left column) and heatmaps of pathways and ontology terms the downregulated genes are enriched for (right column). The plots are based on gene lists for three EwS cell lines (CHLA-10, EW7, SK-N-MC), containing the 300 strongest downregulated genes after HDAC1 or HDAC2 knock out, respectively. The lists of downregulated genes for HDAC knock out effects in the top row is based on averaged expression data from HDAC1 and HDAC2 knock outs. The circos plots show overlaps in the gene sets, where each gene is a spot on the inner arc. Purple lines indicate genes shared by the gene lists, and blue lines indicate functional overlaps in the lists. A blue line connects two different genes belonging to the same enriched ontology term. The strongest enriched ontology terms are depicted in the heatmaps. The cells are colored by p-value. Grey cells indicate that a term is not significantly enriched in a gene list. Hence, the heatmap shows common and unique enrichments for the three cell lines. Fig. S5 a, HDAC3 or HDAC8 expression after transient shRNA transfection measured by qRT-PCR in EwS cell lines CHLA-10, SK-N-MC or EW7, respectively. Induction of three different shRNAs was done with Doxycycline for 72 hours. b, Proliferation of EwS cells after transfection with HDAC3 (top) or HDAC8 (bottom, left) specific shRNA. Further proliferation of SK-N-MC HDAC1 knock out cells with transient HDAC3 or HDAC8 knock down (bottom, right). Control cells were transfected with irrelevant shRNA. Proliferation and cell impendence was measured by the xCELLigence assay every 4 hours. Data are shown as mean ± SEM (hexaplicates/group; p-value < 0.001, respectively < 0.0001). c, Analysis of the invasive potential of EwS cell line CHLA-10 after transient shRNA transfection with HDAC3 (top) or SK-N-MC HDAC1 knock out with HDAC8 (bottom) specific shRNA 48 hours after seeding. d, Evaluation of tumorigenicity of CRISPR/Cas9 knock outs of HDAC1 and their controls (Cas9) in EwS cell line CHLA-10. Immune deficient Rag2-/-γC-/-mice were injected s.c. with 4x106 EwS cells. Mice with an average tumor size >10 mm in diameter were considered positive and sacrificed. Kaplan-Meier plots of individual experiments with six mice per group are shown. Log-rank test was used to test for differences in survival. Fig. S6 a, Proliferation of SK-N-MC and CHLA-10 after treatment with Doxorubicin and/or HDACi (MS-275 or FK228) was analyzed with the xCELLigence system. Cell impedance was measured every 4 hours. Data are shown as mean ± SEM (hexaplicates/group; p-value < 0.0001). Fig. S7 a, Proliferation of EwS CRISPR/Cas9 HDAC 1 knock outs and their controls (Cas9) in CHLA-10 or SK-N-MC cells after treatment with Vincristine (top 2 panels) or combined treatment of SK-N-MC with MS-275 and Vincristine (bottom panel). Proliferation and cell impendence were measured by the xCELLigence assay every 4 hours. Data are shown as mean ± SEM (hexaplicates/group; p-value > 0.0001). b, Heatmaps of pathways and ontology terms that are enriched among up- and downregulated genes. The plots are based on gene lists for two EwS cell lines (EW7, SK-N-MC), containing the 300 strongest differentially expressed genes after FK228, Vincristine or combined treatment, compared to solvent controls, respectively. The strongest enriched ontology terms are depicted in the heatmaps. The cells are colored by p-value. Grey cells indicate that a term is not significantly enriched in a gene list. Hence, the heatmap shows common and unique enrichments for the two cell lines. c, Spheroid growth was monitored in Greiner bio-one CELLSTAR® Cell-Repellent Surface 96-well round bottom plates. Left, CHLA-10 or EW7 cells were plated in Matrigel-containing medium and cells were treated for 48 hours with the inhibitors as indicated. Results were compared to solvent controls. Right, primary EwS tumor cells derived from PDX mice. Cell viability was measured with CellTiter Glo® Luminescent assay (quadruplets/group). Fig. S8 a, Western blot analysis of apoptosis susceptibility after FK228 or MS-275 and/or A-395 treatment, respectively. Protein levels measured by antibodies against, PARP, CASP3, and GAPDH as loading control. CHLA-10, EW7 or SK-N-MC cells were treated for 48 hours with inhibitors. b, Left, heat map of 824 genes, 3-fold differentially expressed in different EwS tumor samples (CHLA-10 and SK-N-MC) at the end of treatment, are shown. Right, zoomed in heat map with 132 genes contains only those genes with a p-value < 0.05. Each column represents one individual array. Microarray data with their normalized fluorescent signal intensities were used (robust multichip average (RMA); GSE162788). Cells were treated for 27 hours with solvent control or EEDi (A-395), HDACi (FK228) or with both inhibitors. c, Volcano plot of differentially expressed genes of EwS cells at the end of treatment (CHLA-10, SK-N-MC). The plot shows fold changes of log2 expression values (log FC) and p-values obtained from differential expression analysis comparing tumors treated with A-395 + FK228 to solvent controls. Depicted in red are genes obtaining p-value < 0.05 and absolute log FC > 1; in blue, genes with p-value < 0.05 and absolute log FC ≤ 1; in green, genes with p-value ≥ 0.05 and absolute log FC > 1; and in black, genes with p-value ≥ 0.05 and absolute log FC ≤ 1. Positive log FCs indicate higher expression of the gene in the treated cell lines. D, GSEA enrichment plots of up- and downregulated gene sets after combined A-395 and FK228 treatment. NES: normalized enrichment score. GSEA: http://www.broadinstitute.org/gsea/index.jsp .
Abstract Background: Ewing sarcoma (ES), an osteogenic malignancy that mainly affects children and young adults, is characterized by early metastasis to lung and bone. In the clinical setting, prognosis for patients with metastatic ES at diagnosis is clearly worse than for those without metastases (5-year survival > 30%). Hence, there is an urgent need to understand the fundamental molecular mechanisms of ES differentiation, invasion, and metastasis to possibly identify novel therapeutic strategies to prevent metastasis. The purpose of this study was to shed further light into the function of Chondromodulin 1 (CHM1) on ES pathogenesis, especially on metastasis, and at best to establish new therapeutic targets. Material and Methods: Expression of CHM1 was analyzed using microarrays and its function was examined by RNA interference (RNAi). To analyze resulting changes qRT-PCR, ELISA, FACS, IHC, proliferation and invasion assays, as well as a xeno-transplant model in immune deficient mice were applied. Results: In this study, we investigated the role of the BRICHOS chaperon domain containing endochondral bone protein chondromodulin I (CHM1) in ES pathogenesis. CHM1 is significantly overexpressed in ES and ChIP data demonstrate CHM1 to be directly bound by EWS-FLI1. Using RNA interference we demonstrate that CHM1 enhanced contact-dependent as well as independent proliferation and the invasive potential of ES cells in vitro. This invasiveness was in part mediated via CHM1-regulated MMP9 expression. In a xenograft mouse model CHM1 was essential for the establishment of lung metastases, which is in line with the observed increased CHM1 expression in patient specimens with ES lung metastases. Mechanistically, CHM1 promoted chondrogenic differentiation capacity of ES cells but suppressed endothelial differentiation. Further, CHM1 suppressed the number of TRAP+ osteoclasts in an orthotopic model of tumor growth in line with suppression of osteolytic genes such as HIF1A, IL6, JAG1, and VEGF, indicating that CHM1-blocked osteomimicry might play a role in homing, colonization, and invasion into bone tissues. Conclusions: Our results suggest that CHM1 is an important player suppressing endothelial differentiation capacity and seems essential for the invasive and metastatic capacities of ES. Citation Format: Kristina von Heyking, Julia Calzada-Wack, Stefanie Göllner, Oxana Schmidt, Tim Hensel, David Schirmer, Annette Fasan, Carsten Müller-Tidow, Poul Sorensen, Stefan Burdach, Günther H.S. Richter. The endochondral bone protein CHM1 sustains an undifferentiated, invasive phenotype promoting lung metastasis in Ewing sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B03.
Abstract Background Histone acetylation and deacetylation seem processes involved in the pathogenesis of Ewing sarcoma (EwS). Here histone deacetylases (HDAC) class I were investigated. Methods Their role was determined using different inhibitors including TSA, Romidepsin, Entinostat and PCI-34051 as well as CRISPR/Cas9 class I HDAC knockouts and HDAC RNAi. To analyze resulting changes microarray analysis, qRT-PCR, western blotting, Co-IP, proliferation, apoptosis, differentiation, invasion assays and xenograft-mouse models were used. Results Class I HDACs are constitutively expressed in EwS. Patients with high levels of individual class I HDAC expression show decreased overall survival. CRISPR/Cas9 class I HDAC knockout of individual HDACs such as HDAC1 and HDAC2 inhibited invasiveness, and blocked local tumor growth in xenograft mice. Microarray analysis demonstrated that treatment with individual HDAC inhibitors (HDACi) blocked an EWS-FLI1 specific expression profile, while Entinostat in addition suppressed metastasis relevant genes. EwS cells demonstrated increased susceptibility to treatment with chemotherapeutics including Doxorubicin in the presence of HDACi. Furthermore, HDACi treatment mimicked RNAi of EZH2 in EwS. Treated cells showed diminished growth capacity, but an increased endothelial as well as neuronal differentiation ability. HDACi synergizes with EED inhibitor (EEDi) in vitro and together inhibited tumor growth in xenograft mice. Co-IP experiments identified HDAC class I family members as part of a regulatory complex together with PRC2. Conclusions Class I HDAC proteins seem to be important mediators of the pathognomonic EWS-ETS-mediated transcription program in EwS and in combination therapy, co-treatment with HDACi is an interesting new treatment opportunity for this malignant disease.
