Ewing Sarcoma Cells Research Articles

Deciphering the Mechanisms of Action of Altertoxin II in Ewing Sarcoma

Ewing sarcomas (ES) are bone and soft tissue tumors that affect children and adolescents. These cancers are characterized by chromosomal translocations that result in expression of an oncogenic fusion protein (typically EWSR1-FLI1). Despite advances in cure rates for many childhood malignancies, advanced, metastatic or relapsed ES remain a challenge. We identified the fungal metabolite altertoxin II (ATXII) as having highly selective activity against six ES cell lines compared to a panel of pediatric and adult cancer cells. On average, ATXII is 94-fold (range 16- to 400-fold) more potent against ES cells compared to rhabdomyosarcoma (RMS) cells, indicating high selectivity for ES. Mechanism of action studies indicated that ATXII selectively induces DNA damage in ES cells. However, the high degree of selectivity for ES suggests other mechanisms may be involved. To determine if ES sensitivity to ATXII is a dominant or recessive phenotype, we hybridized sensitive EW8 cells (ES) with resistant Rh30 cells (RMS) and determined the sensitivity of these hybrids to ATXII. Five single-cell subclones derived from these hybrids were resistant to ATXII, suggesting that sensitivity to ATXII is a recessive phenotype in ES cells. Based on these data, we further investigated the mechanism of action of ATXII to identify which cellular pathways confer resistance. A genome-wide CRISPR-Cas9 knockout screen was conducted in resistant Rh30 cells using the GeCKOv2 library. Cells were treated for three weeks with ATXII or vehicle and genomic DNA was isolated every 4 days. Next-generation sequencing and bioinformatic analysis identified multiple differentially expressed sgRNAs in ATXII-treated Rh30 cells compared to vehicle-treated cells, suggesting loss of these genes influences growth in the presence of ATXII. Ongoing studies are aimed at confirming the influence of these genes on resistance to ATXII with additional gene knockdown experiments. Additionally, mass spectrometry-based thermal proteome profiling was conducted on ATXII-treated ES cell lysates and identified potential protein binding partners of ATXII. Overall, these studies will identify mechanisms of resistance to ATXII and ultimately identify new molecular targets for the development of ES therapies.

ERG transcription factors have a splicing regulatory function involving RBFOX2 that is altered in the EWS-FLI1 oncogenic fusion

ERG family proteins (ERG, FLI1 and FEV) are a subfamily of ETS transcription factors with key roles in physiology and development. In Ewing sarcoma, the oncogenic fusion protein EWS-FLI1 regulates both transcription and alternative splicing of pre-messenger RNAs. However, whether wild-type ERG family proteins might regulate splicing is unknown. Here, we show that wild-type ERG proteins associate with spliceosomal components, are found on nascent RNAs, and induce alternative splicing when recruited onto a reporter minigene. Transcriptomic analysis revealed that ERG and FLI1 regulate large numbers of alternative spliced exons (ASEs) enriched with RBFOX2 motifs and co-regulated by this splicing factor. ERG and FLI1 are associated with RBFOX2 via their conserved carboxy-terminal domain, which is present in EWS-FLI1. Accordingly, EWS-FLI1 is also associated with RBFOX2 and regulates ASEs enriched in RBFOX2 motifs. However, in contrast to wild-type ERG and FLI1, EWS-FLI1 often antagonizes RBFOX2 effects on exon inclusion. In particular, EWS-FLI1 reduces RBFOX2 binding to the ADD3 pre-mRNA, thus increasing its long isoform, which represses the mesenchymal phenotype of Ewing sarcoma cells. Our findings reveal a RBFOX2-mediated splicing regulatory function of wild-type ERG family proteins, that is altered in EWS-FLI1 and contributes to the Ewing sarcoma cell phenotype.

Combination Flavopiridol and SN38 drives toxicity and loss of DNA damage repair in Ewing Sarcoma

EWS-Fli1 is an oncogenic transcription factor that causes Ewing sarcoma. EWS-Fli1 is a fusion protein comprised of an N-terminal low complexity domain from the RNA-binding protein EWSR1 and the C-terminal DNA-binding domain of the ETS transcription factor Fli1. EWS-Fli1 drives expression of oncogenes and decreases expression of tumor suppressor proteins. EWS-Fli1 acts as a transcriptional regulator by recruiting RNA Pol II to promoter regions. Ewing sarcoma is particularly susceptible to DNA damaging chemotherapy compared to other cancers. However, the underlying reason for this sensitivity to DNA damage has not been elucidated. Given the role of EWS-Fli1 in disrupting transcription, we hypothesized that EWS-Fli1 is concurrently disrupting the DNA damage response. During transcription, RNA Pol II is phosphorylated by CDK9 to escape initiation into elongation. We tested the ability of transcription inhibition to affect the DNA damage response by combining treatment a CDK9 inhibitor, flavopiridol, and the DNA damage agent, SN38. This combination drastically diminished cell viability in Ewing cell lines. This sensitivity was not found in non-Ewing cell lines U2OS and HEK293T/17. We also find in Ewing cells that the combination treatment decreases the DNA damage markers gamma-H2A.X and phospho-CHK1 compared to non-Ewing's cells. Together, these results suggest Ewing sarcoma cells are sensitized to DNA damage due in part to transcriptional dysregulation and suggest combination CDK9 inhibition with DNA damage treatment could provide a therapeutic approach for Ewing Sarcoma.

Sarcoma IL-12 overexpression facilitates NK cell immunomodulation

Interleukin-12 (IL-12) is an inflammatory cytokine that has demonstrated efficacy for cancer immunotherapy, but systemic administration has detrimental toxicities. Lentiviral transduction eliciting IL-12-producing human sarcoma for autologous reintroduction provides localized delivery for both innate and adaptive immune response augmentation. Sarcoma cell lines and primary human sarcoma samples were transduced with recombinant lentivirus engineering expression of human IL-12 (hu-IL-12). IL-12 expressing sarcomas were assessed in vitro and in vivo following implantation into humanized NSG and transgenic human IL-15 expressing (NSG.Tg(Hu-IL-15)) murine models. Lentiviral transduction (LV/hu-IL-12) of human osteosarcoma, Ewing sarcoma and rhabdomyosarcoma cell lines, as well as low-passage primary human sarcomas, engendered high-level expression of hu-IL-12. Hu-IL-12 demonstrated functional viability, eliciting specific NK cell-mediated interferon-γ (IFN-γ) release and cytotoxic growth restriction of spheroids in vitro. In orthotopic xenograft murine models, the LV/hu-IL-12 transduced human sarcoma produced detectable IL-12 and elicited an IFN-γ inflammatory immune response specific to mature human NK reconstitution in the NSG.Tg(Hu-IL-15) model while restricting tumor growth. We conclude that LV/hu-IL-12 transduction of sarcoma elicits a specific immune reaction and the humanized NSG.Tg(Hu-IL-15) xenograft, with mature human NK cells, can define in vivo anti-tumor effects and systemic toxicities. IL-12 immunomodulation through autologous tumor transduction and reintroduction merits exploration for sarcoma treatment.

Reverse Engineering of Ewing Sarcoma Regulatory Network Uncovers PAX7 and RUNX3 as Master Regulators Associated with Good Prognosis.

Simple SummaryEwing Sarcoma is a rare cancer that, when localized, has an overall five-year survival rate of 70%. Patients with metastasis have a five-year survival rate of 15 to 30%. Early analysis of patient prognosis can be crucial to provide adequate treatment and increase chances of survival. Besides, it is a disease with several gaps in our understanding, including regulation of genes and which transcription factors are master regulators. This work addresses these two topics by inferring gene regulatory networks that allow us to identify putative master regulators to predict patient prognosis. We were able to identify two sets of master regulators that can predict good and bad patient outcomes.Ewing Sarcoma (ES) is a rare malignant tumor occurring most frequently in adolescents and young adults. The ES hallmark is a chromosomal translocation between the chromosomes 11 and 22 that results in an aberrant transcription factor (TF) through the fusion of genes from the FET and ETS families, commonly EWSR1 and FLI1. The regulatory mechanisms behind the ES transcriptional alterations remain poorly understood. Here, we reconstruct the ES regulatory network using public available transcriptional data. Seven TFs were identified as potential MRs and clustered into two groups: one composed by PAX7 and RUNX3, and another composed by ARNT2, CREB3L1, GLI3, MEF2C, and PBX3. The MRs within each cluster act as reciprocal agonists regarding the regulation of shared genes, regulon activity, and implications in clinical outcome, while the clusters counteract each other. The regulons of all the seven MRs were differentially methylated. PAX7 and RUNX3 regulon activity were associated with good prognosis while ARNT2, CREB3L1, GLI3, and PBX3 were associated with bad prognosis. PAX7 and RUNX3 appear as highly expressed in ES biopsies and ES cell lines. This work contributes to the understanding of the ES regulome, identifying candidate MRs, analyzing their methilome and pointing to potential prognostic factors.

The miR-146b-5p promotes Ewing's sarcoma cells progression via suppressing the expression of BTG2.

Ewing sarcoma (ES) is a malignant tumor that occurs mostly in children. However, the underlying mechanisms of ES are still unknown. Analyzing the results of two previous miRNA array reports, we found that miR-146b-5p might be an onco-miRNA in ES progression. To test this hypothesis, we detected the expression levels of miR-146b-5p by real-time PCR and observed the effects of miR-146b-5p on the progression of ES cells by CCK8 and transwell assays. Bioinformatics and luciferase assays were used to identify the target genes of miR-146b-5p. It showed that the expression levels of miR-146b-5p were upregulated in ES cell lines compared with human mesenchymal stem cells (MSCs). Up- or downregulation of miR-146b-5p in ES cell lines could effectively promote or block the proliferation, migration, and invasion of ES cells, respectively. Furthermore, we demonstrated that BTG2 was one of the target genes and mediated the effects of miR-146b-5p in ES cells. Interestingly, we also found that miR-146b-5p was partly involved in the anticancer effects of pemetrexed in ES cells. Our study revealed that miR-146b-5p affected the progression of ES by suppressing BTG2, which might shed light on anticancer drug development and ES treatment in the future.

Cooperative treatment effectiveness of ATR and HSP90 inhibition in Ewing\u2019s sarcoma cells

IntroductionEwing's sarcoma is an aggressive childhood malignancy whose outcome has not substantially improved over the last two decades. In this study, combination treatments of the HSP90 inhibitor AUY922 with either the ATR inhibitor VE821 or the ATM inhibitor KU55933 were investigated for their effectiveness in Ewing's sarcoma cells.MethodsEffects were determined in p53 wild-type and p53 null Ewing's sarcoma cell lines by flow cytometric analyses of cell death, mitochondrial depolarization and cell-cycle distribution as well as fluorescence and transmission electron microscopy. They were molecularly characterized by gene and protein expression profiling, and by quantitative whole proteome analysis.ResultsAUY922 alone induced DNA damage, apoptosis and ER stress, while reducing the abundance of DNA repair proteins. The combination of AUY922 with VE821 led to strong apoptosis induction independent of the cellular p53 status, yet based on different molecular mechanisms. p53 wild-type cells activated pro-apoptotic gene transcription and underwent mitochondria-mediated apoptosis, while p53 null cells accumulated higher levels of DNA damage, ER stress and autophagy, eventually leading to apoptosis. Impaired PI3K/AKT/mTOR signaling further contributed to the antineoplastic combination effects of AUY922 and VE821. In contrast, the combination of AUY922 with KU55933 did not produce a cooperative effect.ConclusionOur study reveals that HSP90 and ATR inhibitor combination treatment may be an effective therapeutic approach for Ewing's sarcoma irrespective of the p53 status.

Targeting EYA3 in Ewing Sarcoma Retards Tumor Growth and Angiogenesis.

EWSR1/FLI1, the most common fusion gene in Ewing sarcoma, upregulates expression of the Eyes Absent 3 (EYA3) transactivator-phosphatase protein. The purpose of this study was to investigate molecular and cellular mechanisms through which EYA3 might promote Ewing sarcoma tumor growth and to determine whether the EYA3 tyrosine phosphatase activity represents a viable therapeutic target. We used genetic and pharmacologic modulation of EYA3 in cell line-based xenografts to examine how loss of EYA3 tyrosine phosphatase activity affects tumor growth and angiogenesis. Molecular mechanisms were evaluated in vivo and in vitro through analyses of tumor tissue and multicellular tumor spheroids. Our results show that both loss of EYA3 in Ewing sarcoma cells and pharmacologic inhibition of the EYA3 tyrosine phosphatase activity inhibit tumor growth and tumor angiogenesis. EYA3 regulates levels of VEGFA in Ewing tumors, as well as promoting DNA damage repair and survival of Ewing sarcoma tumor cells. Target engagement is demonstrated in tumor tissue through elevated levels of the EYA3 substrate H2AX-pY142 upon loss of EYA3 or with Benzarone treatment. The efficacy of EYA3 tyrosine phosphatase inhibition in attenuating tumor growth and angiogenesis is corroborated in an Ewing sarcoma patient-derived tumor xenograft. Together, the results presented here validate EYA3 as a target for the development of novel Ewing sarcoma therapeutic strategies, and set the stage for evaluating the efficacy of combining the antiangiogenic and anti-cell survival effects of EYA3 inhibition with cytotoxic chemotherapy.

The Clinical Characteristics and Prediction Nomograms for Primary Spine Malignancies.

BackgroundPrimary spine malignancies (PSMs) are relatively rare in bone tumors. Due to their rarity, the clinical characteristics and prognostic factors are still ambiguous. In this study, we aim to identify the clinical features and proposed prediction nomograms for patients with PSMs.MethodsPatients diagnosed with PSMs including chordoma, osteosarcoma, chondrosarcoma, Ewing sarcoma, and malignant giant cell tumor of bone (GCTB) between 1975 and 2016 were selected from the Surveillance, Epidemiology, and End Results (SEER) database. The patient and tumor characteristics were described based on clinical information. The significant prognostic factors of overall survival (OS) and cancer-specific survival (CSS) were identified by the univariate and multivariate Cox analysis. Then, the nomograms for OS and CSS were established based on the selected predictors and their accuracy was explored by the Cox–Snell residual plot, area under the curve (AUC) of receiver operator characteristic (ROC) and calibration curve.ResultsThe clinical information of 1,096 patients with PSMs was selected from the SEER database between 1975 and 2016. A total of 395 patients were identified with full survival and treatment data between 2004 and 2016. Chordoma is the commonest tumor with 400 cases, along 172 cases with osteosarcoma, 240 cases with chondrosarcoma, 262 cases with Ewing sarcoma and 22 cases with malignant GCTB. The univariate and multivariate analyses revealed that older age (Age > 60), distant metastasis, chemotherapy, and Surgery were independent predictors for OS and/or CSS. Based on these results, the nomograms were established with a better applicability (AUC for CSS: 0.784; AUC for OS: 0.780).ConclusionsThis study provides the statistics evidence for the clinical characteristics and predictors for patients with PSMs based on a large size population. Additionally, precise prediction nomograms were also established with a well-applicability.

PDX-Derived Ewing's Sarcoma Cells Retain High Viability and Disease Phenotype in Alginate Encapsulated Spheroid Cultures.

Simple SummaryEwing’s Sarcoma (ES) is the second most frequent bone tumour in children and young adults, with very aggressive behaviour and significant disease recurrence. To better study the disease and find new therapies, experimental models are needed. Recently, patient-derived xenografts (PDX), obtained by implanting patient tumour samples in immunodeficient mice, have been developed. However, when ES cells are extracted from the patient’s tumour or from PDX and placed on plasticware surfaces, they lose their original 3D configuration, cell identity and function. To overcome these issues, we implemented cultures of PDX-derived ES cells, by making them aggregate to form ES cell spheroids and then encapsulating these 3D spheroids into a hydrogel, alginate, to stabilize the culture. We show that this methodology maintained ES cell viability and intrinsic characteristics of the original ES tumour cells for at least one month and that it is suitable for study the effect of anticancer drugs.Ewing’s Sarcoma (ES) is the second most frequent malignant bone tumour in children and young adults and currently only untargeted chemotherapeutic approaches and surgery are available as treatment, although clinical trials are on-going for recently developed ES-targeted therapies. To study ES pathobiology and develop novel drugs, established cell lines and patient-derived xenografts (PDX) are the most employed experimental models. Nevertheless, the establishment of ES cell lines is difficult and the extensive use of PDX raises economic/ethical concerns. There is a growing consensus regarding the use of 3D cell culture to recapitulate physiological and pathophysiological features of human tissues, including drug sensitivity. Herein, we implemented a 3D cell culture methodology based on encapsulation of PDX-derived ES cell spheroids in alginate and maintenance in agitation-based culture systems. Under these conditions, ES cells displayed high proliferative and metabolic activity, while retaining the typical EWSR1-FLI1 chromosomal translocation. Importantly, 3D cultures presented reduced mouse PDX cell contamination compared to 2D cultures. Finally, we show that these 3D cultures can be employed in drug sensitivity assays, with results similar to those reported for the PDX of origin. In conclusion, this novel 3D cell culture method involving ES-PDX-derived cells is a suitable model to study ES pathobiology and can assist in the development of novel drugs against this disease, complementing PDX studies.

Ubiquitin-Specific Protease 6 Functions as a Tumor Suppressor in Ewing Sarcoma through Immune Activation.

Ewing sarcoma is the second most common pediatric bone cancer, with a 5-year survival rate for metastatic disease of only 20%. Recent work indicates that survival is strongly correlated with high levels of tumor-infiltrating lymphocytes (TIL), whose abundance is associated with IFN-inducible chemokines CXCL10 and CCL5. However, the tumor-intrinsic factors that drive chemokine production and TIL recruitment have not been fully elucidated. We previously showed that ubiquitin-specific protease 6 (USP6) directly deubiquitinates and stabilizes Jak1, thereby inducing an IFN signature in Ewing sarcoma cells. Here, we show that this gene set comprises chemokines associated with immunostimulatory, antitumorigenic functions, including CXCL10 and CCL5. USP6 synergistically enhanced chemokine production in response to exogenous IFN by inducing surface upregulation of IFNAR1 and IFNGR1. USP6-expressing Ewing sarcoma cells stimulated migration of primary human monocytes and T lymphocytes and triggered activation of natural killer (NK) cells in vitro. USP6 inhibited Ewing sarcoma xenograft growth in nude but not NSG mice and was accompanied by increased intratumoral chemokine production and infiltration and activation of NK cells, dendritic cells, and macrophages, consistent with a requirement for innate immune cells in mediating the antitumorigenic effects of USP6. High USP6 expression in patients with Ewing sarcoma was associated with chemokine production, immune infiltration, and improved survival. This work reveals a previously unrecognized tumor-suppressive function for USP6, which engenders an immunostimulatory microenvironment through pleiotropic effects on multiple immune lineages. This further raises the possibility that USP6 activity may be harnessed to create a "hot" tumor microenvironment in immunotherapy. SIGNIFICANCE: This study reveals a novel tumor-suppressive function for USP6 by inducing an immunostimulatory microenvironment, suggesting that USP6 activity may be exploited to enhance immunotherapy regimens.

Augmented Antitumor Activity for Novel Dual PI3K/BDR4 Inhibitors, SF2523 and SF1126 in Ewing Sarcoma.

Ewing sarcoma (ES) is the second most common pediatric bone cancer. Despite recent advances in the treatment, patients with metastatic tumors have dismal prognosis and hence novel therapies are urgently needed to combat this cancer. A recent study has shown that phosphoinositide-3 kinase (PI3K) inhibitors can synergistically increase sensitivity to bromodomain and extraterminal domain inhibitors in ES cells and therefore combined inhibition of PI3K and bromodomain and extraterminal domain bromodomain proteins might provide benefit in this cancer. Herein, we have investigated the efficacy of dual PI3K/BRD4 inhibitors, SF2523 and SF1126, for their antitumor activity in ES cell lines. The effect of SF1126 and SF2523 on cell viability and PI3K signaling was assessed on a panel of human ES cell lines. To evaluate the antitumor activity of SF1126, A673 cells were injected intrafemorally into RAG-2-/- mice and treated with 50 mg/kg SF1126 6 days per week, for 30 days. Both SF1126 and SF2523 decreased cell survival and inhibited phosphorylation of AKT in human ES cell lines. In vivo, SF1126 showed a significant reduction in tumor volume. These results suggest that dual PI3K/BRD4 inhibitor, SF1126, has antitumor activity in ES models.

P21-activated kinases as viable therapeutic targets for the treatment of high-risk Ewing sarcoma.

Ewing sarcoma (ES) is the second most common bone tumor in children and young adults. Unfortunately, there have been minimal recent advancements in improving patient outcomes, especially in metastatic and recurrent diseases. In this study, we investigated the biological role of p21-activated kinases (PAKs) in ES, and the ability to therapeutically target them in high-risk disease. Via informatics analysis, we established the inverse association of PAK1 and PAK4 expression with clinical stage and outcome in ES patients. Through expression knockdown and small-molecule inhibition of PAKs, utilizing FRAX-597, KPT-9274, and PF-3758309 in multiple ES cell lines and patient-derived xenograft models, we further explored the role of PAKs in ES tumor growth and metastatic capabilities. In vitro studies in several ES cell lines indicated that diminishing PAK1 and PAK4 expression reduces tumor cell viability, migratory, and invasive properties. In vivo studies using PAK4 inhibitors, KPT-9274 and PF-3758309 demonstrated significant inhibition of primary and metastatic tumor formation, while transcriptomic analysis of PAK4-inhibitor-treated tumors identified concomitant suppression of Notch, β-catenin, and hypoxia-mediated signatures. In addition, the analysis showed enrichment of anti-tumor immune regulatory mechanisms, including interferon (IFN)-ɣ and IFN-α responses. Altogether, our molecular and pre-clinical studies are the first to establish a critical role for PAKs in ES development and progression, and consequently as viable therapeutic targets for the treatment of high-risk ES in the near future.

LncRNA TUG1 promotes Ewing's sarcoma cell proliferation, migration, and invasion via the miR-199a-3p-MSI2 signaling pathway.

The aim of this study was to investigate the roles and potential mechanisms of long non-coding RNA (lncRNA) taurine upregulated gene 1 (TUG1) in the proliferation, migration, and invasion of Ewing's sarcoma cells. RT-qPCR was used to detect the expression of TUG1, microRNA-199a-3p (miR-199a-3p), and musashi2 (MSI2) in Ewing's sarcoma tissues and cell lines. Kaplan-Meier overall survival curves showed the survival rates of Ewing's sarcoma patients with high and low expression of TUG1. The association between the expressions of TUG1/MSI2 and miR-199a-3p in Ewing's sarcoma tissues was assessed by Pearson's correlation analysis. Cell proliferation, migration, and invasion were detected by CCK-8 assay and Transwell assay, respectively. The protein level of MSI2 was determined using western blotting. The interaction between TUG1/MSI2 and miR-199a-3p was validated by the dual-luciferase reporter assay. The levels of TUG1 and MSI2 were increased, while the level of miR-199a-3p was decreased in Ewing's sarcoma tissues and cells. High expression of TUG1 or MSI2 indicated a decreased overall survival rate of Ewing's sarcoma patients. TUG1/MSI2 level was negatively correlated with miR-199a-3p level. While TUG1 level was positively correlated with MSI2 level. In Ewing's sarcoma cells, knockdown of TUG1/MSI2 or overexpression of miR-199a-3p inhibited cell proliferation, migration, and invasion, whereas the overexpression of TUG1/MSI2 presented the opposite results. TUG1 functioned as a competing endogenous RNA to regulate MSI2 expression by sponging miR-199a-3p. Finally, miR-199a-3p inhibitor or MSI2 overexpression counteracted the TUG1 knockdown-mediated inhibitory effect on Ewing's sarcoma cell proliferation, migration, and invasion. TUG1 promotes proliferation, migration, and invasion of Ewing's sarcoma cells via sequestering miR-199a-3p to enhance the MSI2 expression, suggesting that TUG1 might be a potential target for treating Ewing's sarcoma.

MiR-145 suppresses epithelial-mesenchymal transition by targeting stem cells in Ewing sarcoma cells.

We aimed to examine the performance of stem cell markers and epithelial-mesenchymal transition (EMT) process in miR-145 transfected EWS cells (TC71, TC106). EWS cells were utilized for functional analysis of mir-145. Proliferation, migration, invasion and soft agar colony assay were performed to observe the alterations in migration behavior of transfected cells. Caspase assay was used to investigate the underlying reasons of proliferative inhibition in cells in whichmiR-145 is overexpressed. QRT-PCR was used to determine the role of miR-145 in EMT transcription markers and mir-145 targeted genes, KLF4, SOX2 and OCT4 expression levels. The miR-145 expression has been shown to be down-regulated in EWS. The miR-145 overexpression caused inhibition of proliferation and reduced migration in EWS cells through induction of apoptosis. Mir-145 suppresses EMT capacity and SOX2, KLF4 and OCT4 expression levels. This is the first time in the literature we have shown deregulation of miR-145 inhibits EMT process by targeting stem cell properties leading to the inhibition of tumor growth and metastasis in TC71 and TC106 cells. Based on these results, we propose that miR-145, as an important regulator of SOX2, KLF4 and OCT4 carries crucial roles in EWS tumorigenesis and EMT (Tab. 1, Fig. 4, Ref. 26).

High Expression of TIM 3 and Galectin 9 on Immunohistochemistry Staining of Tumor Specimen at Diagnosis in Pediatric Patients with Ewing Sarcoma.

Significant progress has been made in the advancement of immune system modulation for cancer treatment in recent years. In particular, immune checkpoint inhibitors and chimeric antigen receptor (CAR) T-cell therapy have demonstrated remarkable clinical benefit in relapsed/refractory cancers. However, our understanding of the immuno-oncologic landscape in pediatric solid tumors remains limited and is a barrier to continued progress. We examined the immunohistochemical expression of checkpoint receptors PD-1, TIM-3, LAG-3 and their respective ligands in various pediatric cancers at diagnosis and found high expression of TIM-3/Galectin-9 in the infiltrating cells of Ewing sarcoma. Location of checkpoint receptor/ligand expressions is important, as some staining patterns were only seen along tumor borders. Finally, peripheral T cell function varied significantly among different tumors supporting a complex relationship between the tumor microenvironment and the global immune system.

Establishment and Characterisation of Metastatic Extraskeletal Ewing Sarcoma Mouse Models.

Ewing sarcomas most commonly arise in the bones, but can also manifest as extraskeletal tumours in soft tissues. Metastases from extraskeletal Ewing sarcomas occur in more diverse anatomical sites than skeletal tumours, and have poorer survival rates. Few animal models replicate the extraskeletal form of Ewing sarcoma, and those that have been developed do not reflect the widespread metastatic spread of these cancers. Luciferase-expressing Ewing sarcoma cells derived from a muscle tumour were intramuscularly or intravenously injected into nude mice. Both models achieved metastatic spread to numerous sites including the lungs, liver, kidneys, and brain. We characterized the cellular composition of primary and metastatic tumours, observing a greater level of immune cell infiltration in metastases compared to primary intramuscular tumours. These pre-clinical models will hopefully facilitate the evaluation of novel therapies and contribute to better understanding the disease progression of metastatic extraskeletal Ewing sarcoma.

Mir-129-2-3p Has Tumor Suppressor Role in Ewing Sarcoma Cell Lines and Cancer Tissue Samples

Mir-129-2-3p Has Tumor Suppressor Role in Ewing Sarcoma Cell Lines and Cancer Tissue Samples

YAP/TAZ inhibition reduces metastatic potential of Ewing sarcoma cells

Ewing sarcoma (EwS) is a highly metastatic bone cancer characterized by the ETS fusion oncoprotein EWS-FLI1. EwS cells are phenotypically highly plastic and switch between functionally distinct cell states dependent on EWS-FLI1 fluctuations. Whereas EWS-FLI1high cells proliferate, EWS-FLI1low cells are migratory and invasive. Recently, we reported activation of MRTFB and TEAD, effectors of RhoA and Hippo signalling, upon low EWS-FLI1, orchestrating key steps of the EwS migratory gene expression program. TEAD and its co-activators YAP and TAZ are commonly overexpressed in cancer, providing attractive therapeutic targets. We find TAZ levels to increase in the migratory EWS-FLI1low state and to associate with adverse prognosis in EwS patients. We tested the effects of the potent YAP/TAZ/TEAD complex inhibitor verteporfin on EwS cell migration in vitro and on metastasis in vivo. Verteporfin suppressed expression of EWS-FLI1 regulated cytoskeletal genes involved in actin signalling to the extracellular matrix, effectively blocked F-actin and focal-adhesion assembly and inhibited EwS cell migration at submicromolar concentrations. In a mouse EwS xenograft model, verteporfin treatment reduced relapses at the surgical site and delayed lung metastasis. These data suggest that YAP/TAZ pathway inhibition may prevent EwS cell dissemination and metastasis, justifying further preclinical development of YAP/TAZ inhibitors for EwS treatment.

Mithramycin A Radiosensitizes EWS:Fli1+ Ewing Sarcoma Cells by Inhibiting Double Strand Break Repair

The oncogenic EWS:Fli1 fusion protein is a key transcriptional mediator of Ewing sarcoma initiation, progression, and therapeutic resistance. Mithramycin A (MithA) is a potent and specific inhibitor of transcription mediated by the EWS:Fli1. We tested the hypothesis that pretreatment with MithA could selectively radiosensitize EWS:Fli1+ tumor cells by altering the transcriptional response to radiation injury. A panel of 4 EWS:Fli1+ and 3 EWS:Fli1- Ewing sarcoma cell lines and 1 nontumor cell line were subjected to MithA dose-response viability assays to determine the relative potency of MithA in cells possessing or lacking the EWS:Fli1 fusion. Radiosensitization by MithA was evaluated by clonogenic survival assays invitro and in a murine xenograft model. DNA damage was evaluated by comet assay and γ-H2Ax flow cytometry. Immunoblotting, flow cytometry, and reverse-transcription, polymerase chain reaction were used to evaluate DNA damage-induced signaling and repair processes and apoptosis. We found that MithA alone could potently and selectively inhibit the growth of EWS:Fli1+ tumor cells, but not cells lacking this fusion. Pretreatment with MithA for 24 hours before irradiation significantly reduced clonogenic survival invitro and delayed tumor regrowth invivo, prolonging survival of EWS:Fli1+ tumor-bearing mice. Although MithA did not increase the level of DNA double-strand breaks, mechanistic studies revealed that MithA pretreatment selectively inhibited DNA double-strand break repair through downregulation of EWS:Fli1-mediated transcription, leading to tumor cell death by apoptosis. Our data indicate that MithA is an effective radiosensitizer of EWS:Fli1+ tumors and may achieve better local control at lower doses of radiation.