Ewing Sarcoma Family Of Tumors Cell Research Articles

MicroRNA Content of Ewing Sarcoma Derived Extracellular Vesicles Leads to Biomarker Potential and Identification of a Previously Undocumented EWS-FLI1 Translocation.

Objective: Ewing Sarcoma Family of Tumors (ESFT) are a highly aggressive pediatric bone and soft tissue malignancy with poor outcomes in the refractory and recurrent setting. Over 90% of Ewing Sarcoma (ES) tumors are driven by the pathognomonic EWS-ETS chimeric transcripts and their corresponding oncoproteins. It has been suggested that the EWS-ETS oncogenic action can mediate microRNA (miRNA) processing. Importantly, small extracellular vesicles (sEVs), including those frequently referred to as exosomes have been shown to be highly enriched with tumor-derived small RNAs such as miRNAs. We hypothesized that ESFT-specific sEVs are enriched with certain miRNAs which could be utilized toward an exo-miRNA biomarker signature specific to this disease. Methods: We performed miRNAseq to compare both the exo-derived and cell-derived miRNA content from 8 ESFT, 2 osteosarcoma, 2 non-cancerous cell lines, and pediatric plasma samples. Results: We found that sEVs derived from ESFT cells contained nearly 2-fold more number of unique individual miRNAs as compared to non-ESFT samples. Quantitative analysis of the differential enrichment of sEV miRNAs resulted in the identification of 62 sEV-miRNAs (exo-miRNAs) with significant (P < .05) enrichment variation between ESFT and non-ESFT sEV samples. To determine if we could utilize this miRNA signature to diagnose ESFT patients via a liquid biopsy, we analyzed the RNA content of total circulating sEVs isolated from 500 µL plasma from 5 pediatric ESFT patients, 2 pediatric osteosarcoma patients, 2 pediatric rhabdomyosarcoma patients, and 4 non-cancer pediatric controls. Pearson's clustering of 60 of the 62 candidate exo-miRNAs correctly identified 80% (4 of 5) of pathology confirmed ESFT patients. Importantly, RNAseq analysis of tumor tissue from the 1 outlier, revealed a previously uncharacterized EWS-FLI1 translocation.Conclusions: Taken together, these findings support the development and validation of an exo-miRNA-based liquid biopsy to aid in the diagnosis and monitoring of ESFT.

Novel potent anti-STEAP1 bispecific antibody to redirect T cells for cancer immunotherapy

BackgroundThe prognosis for metastatic Ewing sarcoma family of tumors (EFT) is still poor despite high-dose chemotherapy and radiation treatment. Immunotherapies hold promise, but cancer antigen-targeting immunotherapies have largely failed to...

Ewing sarcoma family of tumors-derived small extracellular vesicle proteomics identify potential clinical biomarkers

Purpose: Ewing Sarcoma Family of Tumors (ESFT), the second most common pediatric osseous malignancy, are characterized by the pathognomonic chromosomal EWS-ETS translocation. Outside of tumor biopsy, no clinically relevant ESFT biomarkers exist. Additionally, tumor burden assessment at diagnosis, monitoring of disease responsiveness to therapy, and detection of disease recurrence are limited to radiographic imaging. To identify new, clinically relevant biomarkers we evaluated the proteome of a subset of ESFT-derived small extracellular vesicles (sEVs).Materials and Methods: We performed the first high quality proteomic study of ESFT-derived sEVs from 5 ESFT cell lines representing the most common EWS-ETS fusion types and identified 619 proteins composing the core ESFT sEV proteome. We compared these core proteins to databases of common plasma-based proteins and sEV-associated proteins found within healthy plasma to identify proteins unique or enriched within ESFT.Results: From these analyses, two membrane bound proteins with biomarker potential were selected, CD99/MIC2 and NGFR, to develop a liquid-based assay enriching of ESFT-associated sEVs and detection of sEV mRNA cargo (i.e., EWS-ETS transcripts). We employed this immuno-enrichment approach to diagnosis of ESFT utilizing plasma (250 μl) from both localized and metastatic ESFT pediatric patients and cancer-free controls, and showed significant diagnostic power [AUC = 0.92, p = 0.001 for sEV numeration, with a PPV = 1.00, 95% CI = (0.63, 1.00) and a NPV = 0.67, 95% CI = (0.30, 0.93)].Conclusions: In this study, we demonstrate utilization of circulating ESFT-associated sEVs in pediatric patients as a source of minimally invasive diagnostic and potentially prognostic biomarkers.

Abstract 4141: EWS-FLI regulates mitotic kinesins in Ewing sarcoma family of tumors

Abstract The current situation faced by children and young adults diagnosed with Ewing sarcoma family of tumors (ESFT), the second most common pediatric bone malignancy in the U.S., is bleak. Although the oncogenic phenotype and master regulator of ESFT is driven by an underlying pathognomonic chromosomal translocation (EWS-Ets), therapeutic targeting of this molecular Achilles' heel has been notoriously challenging. For pediatric patients with progressive (refractory) or recurrent ESFT, the overall survival continues to remain poor. To date, no established second-line (salvage) treatment regimen exists for these pediatric sarcoma patients. The failure to improve the overall survival for these patients highlights the need to better understand the molecular points of vulnerability of these diseases, which can be translated into novel treatment strategies. To address this challenge, we have identified KIF11, KIF15 and TPX2, part of the cellular mitotic machinery, as druggable downstream targets of EWS-Ets in ESFT. Our recent studies sought to identify drugs that reverse a gene expression profile similar to the pattern obtained by the silencing of EWS/FLI1 via RNAi approaches (siEWS/FLI1) (Pessetto et al., 2017). This work identified KIF15 (gene rank #1, p=0.00005) and its potential binding partner TPX2 (gene rank #14, p=0.01830) as significantly downregulated genes within this signature. These genes encode KIF15 and TPX2, which are postulated to play a compensatory role in resistance to KIF11 inhibitors in the clinic. We found that KIF11, KIF15 and TPX2 proteins are abundantly expressed in ESFT cell lines as compared with benign controls (7.7-, 5.5-, and 8.5-fold, respectively). We also discovered that RNAi-mediated downregulation of KIF15 increases sensitivity of ESFT cell lines to treatment with KIF11 inhibitors (KIF11i). We have for the first time, shown that silencing EWS-FLI1 (the most common EWS-Ets translocation), in ESFT cells decreases KIF11, KIF15 and TPX2 promoter activity by 99%, 97%, and 87%, respectively. We propose that this activity is a function of EWS-FLI1-mediated E2Fs; EWS-FLI1 disrupts the E2F-regulated proliferation balance such that the suppressive E2F4 transcription factor is displaced by the activating E2F3. Our data show that E2F3 is enriched at KIF15 (8.9-fold) and TPX2 (2.9-fold) promoters. Further, when EWS-FLI1 is siRNA depleted, enrichment at the KIF15 and TPX2 promoters is lost (3.9-fold or 2.6-fold decrease, respectively). These data strongly suggest that aberrant expression of the genes encoding these proteins is controlled by oncogenic EWS-FLI1. Taken together, we have demonstrated that KIF11, KIF15 and TPX2 are aberrantly expressed in ESFT and are putative therapeutic targets. Our ongoing work includes development of KIF11/KIF11/TPX2 axis-targeting compounds toward the goal of implementing pharmacologic approaches to provide new therapies for children diagnosed with these deadly diseases. Citation Format: Rebecca J. Wates, Yan Ma, Jennifer Crow, Glenson Samuel, Andrew K. Godwin. EWS-FLI regulates mitotic kinesins in Ewing sarcoma family of tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4141.

Abstract 3180: Preclinical validation of EZH2 as a therapeutic target in pediatric Ewing's sarcoma

Abstract Disease-free survival in relapsed Ewing's Sarcoma Family of Tumors (ESFT) has not improved over the past 25 years. Current standard-of-care (SOC) agents result in 70% survival in patients with localized ESFT; however, for relapsed patients the survival rates remain between 15-20%. Approximately 85% of ESFTs have the chromosomal translocation t(11;22)(q24;q12) which encodes for the oncogenic EWS/FL1 fusion protein. The EWS/FL1 functions as a potent transcription factor leading to the dysregulated expression of genes that promote and maintain tumorigenesis. A major epigenetic regulator that is a downstream target of EWS/FL1 is the enhancer of Zeste Homolog 2 (EZH2). EZH2 is the catalytic component of the polycomb repressor complex 2 (PRC2). Notably, it is overexpressed in ESFT and maintains tumor oncogenicity by tri-methylating histone 3 lysine 27 (H3K27me3) to modulate gene expression. Genome and transcriptome data obtained by the Pediatric Precision Genomics Program at Riley Hospital for Children at Indiana University Health (IUH) indicate that EZH2 is expressed at high levels in ESFT biopsies. Additionally, other groups have reported that high levels of EZH2 protein in ESFT and other cancers correlate with increased chemoresistance to SOC therapy. We are testing tested the hypothesis that EZH2 contributes to chemoresistance in ESFT by regulating critical growth and survival genes. In addition, we are investigating if pharmacological inhibition of EZH2 will enhance sensitivity to the cytotoxic effects of SOC agents. Pediatric primary and relapsed ESFT cell lines and ESFT xenografts were validated for the EWS/FLI, EZH2, and H3K27me3 signatures. In vitro- and in vivo-pharmacodynamic studies of EZH2 inhibition via tazemetostat were conducted to optimize dosing effect. In ESFT cell lines (TC71, A673, CHLA-9, and CHLA-10), tazemetostat dose-response experiments indicated a significant reduction of H3K27me3 by one day post-treatment which was either sustained or completely blocked by 7-days post-treatment compared to vehicle treated (p&amp;lt;0.001). EZH2-mediated gene regulation was confirmed via qPCR. Inhibition via siRNA or tazemetostat resulted in transcriptional up-regulation of p21 (p&amp;lt;0.05) and down-regulation of C-MYC (p&amp;lt;0.01) in all ESFT cell lines. In TC71 sarcoma xenografts, 200-400 mg/kg BID was well tolerated, and decreased H3K27me3 was evident by 5 and 10 days post-treatment (n=4-5 per cohort, vehicle vs treated, p&amp;lt;0.001). While decreased tumor growth was evident at day 5 post-treatment, growth had recovered by day 10. In vitro assays indicate that tazemetostat potentiates SOC agents, etoposide and irinotecan (SN-38), in mediating ESFT growth inhibition (p&amp;lt;0.001). These data provide rationale for systematic investigation of EZH2 inhibition in combination with SOC. New information will be obtained on how to reprogram the epigenome to mitigate therapeutic resistance in ESFT that overexpress EZH2. Citation Format: Pankita H. Pandya, Barbara Bailey, Adily E. Elmi, Heather R. Bates, Courtney N. Hemenway, Anthony L. Sinn, Khadijeh Bijangi-Vishehsaraei, M. Reza Saadatzadeh, Harlan E. Shannon, Jixin Ding, Mark S. Marshall, Michael J. Ferguson, Lijun Cheng, Lang Li, Mary E. Murray, Jamie L. Renbarger, Karen E. Pollok. Preclinical validation of EZH2 as a therapeutic target in pediatric Ewing's sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3180.

Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT

Background:Primary Ewing's sarcoma family of tumours (ESFTs) may respond to chemotherapy, although many patients experience subsequent disease recurrence and relapse. The survival of ESFT cells following chemotherapy has been attributed to the development of resistant disease, possibly through the expression of ABC transporter proteins.Methods:MRP-1 and Pgp mRNA and protein expression in primary ESFTs was determined by quantitative reverse-transcriptase PCR (RT-qPCR) and immunohistochemistry, respectively, and alternative splicing of MRP-1 by RT-PCR.Results:We observed MRP-1 protein expression in 92% (43 out of 47) of primary ESFTs, and cell membrane MRP-1 was highly predictive of both overall survival (P<0.0001) and event-free survival (P<0.0001). Alternative splicing of MRP-1 was detected in primary ESFTs, although the pattern of splicing variants was not predictive of patient outcome, with the exception of loss of exon 9 in six patients, which predicted relapse (P=0.041). Pgp protein was detected in 6% (38 out of 44) of primary ESFTs and was not associated with patient survival.Conclusion:For the first time we have established that cell membrane expression of MRP-1 or loss of exon 9 is predictive of outcome but not the number of splicing events or expression of Pgp, and both may be valuable factors for the stratification of patients for more intensive therapy.

Abstract 1441: Role of a long non-coding RNA in oncogenesis of Ewing sarcoma

Abstract BACKGROUND: Ewing sarcoma family of tumors (EFT) is the second most common primary malignant bone tumor in children and adolescents. It is a highly invasive disease with a 30% 5-year survival rate for patients with metastatic disease. EFT are defined by a chromosomal translocation that encodes for the fusion gene EWS-FLI1 in 90% of cases. This chimeric gene functions as a transcription factor and regulates tumorigenesis-related gene expression. Our hypothesis is that like protein-coding genes, the non-coding genome in EFT is also important for the biology of Ewing sarcoma. This study focuses on one such non-coding locus of the human genome and explores its role in EFT pathogenesis. METHODS: Expression profiles from a panel of 500 tissue samples including over 100 EFTs, other primary tumors, and normal tissues were generated using Affymetrix Human Exon 1.0 ST microarrays. Data were analyzed using institutional software, Genetrix. RNA-Seq data was generated on two EFT cell lines derived from a primary tumor and its metastatic derivative. Data was validated by polymerase chain reaction (PCR) on 33 cell lines including EFTs and other tumors. Functional studies on EFT cell lines were performed using various RNAi techniques and viral transductions. RESULTS: Expression microarray profiles showed unannotated transcripts to be the strongest discriminators of EFT from all other tumor types. One such transcript, AK057037, was the most highly expressed RNA (1.07 x 10−20) in EFT. RNA-Seq analysis and PCR studies revealed that this transcript is a multi-exonic, presumably, long non-coding RNA (lncRNA) with a maximum predicted open reading frame of 137 amino acids that had no homology, protein domains, or characteristic motifs. Validation studies confirmed that this lncRNA was exclusively expressed in EFT. EWS-FLI regulates expression of this transcript by directly binding to its promoter region. Furthermore, functional data based on knocking down and overexpressing the transcript showed that the lncRNA acts as an oncogene in EFT and enhances tumorigenicity by increasing the metastatic potential of the tumor cell lines. RNA-Seq results showed that the metastatic tumor-derived cell line had increased transcriptional activity of the lncRNA than the primary tumor-derived cell line. Chromatin immunoprecipitation assay detected binding of the lncRNA to the polycomb repressor complex 2 (PRC2) proteins. CONCLUSION: This study establishes lncRNA AK057037 as a promoter of EFT pathogenesis. Its association with the PRC2 complex suggests its role in chromatin modification. Targeting this lncRNA may help improve outcomes in children with metastatic Ewing disease. Using AK057037 as an example, we show that the non-coding genome is important in EFT. Further work on the non-coding transcriptome may reveal additional functional RNAs that contribute to the biology of this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1441. doi:1538-7445.AM2012-1441

Redirecting T Cells to Ewing's Sarcoma Family of Tumors by a Chimeric NKG2D Receptor Expressed by Lentiviral Transduction or mRNA Transfection

We explored the possibility to target Ewing's sarcoma family of tumors (ESFT) by redirecting T cells. To this aim, we considered NKG2D-ligands (NKG2D-Ls) as possible target antigens. Detailed analysis of the expression of MICA, MICB, ULBP-1, -2, and -3 in fourteen ESFT cell lines revealed consistent expression of at least one NKG2D-L. Thus, for redirecting T cells, we fused a CD3ζ/CD28-derived signaling domain to the ectodomain of NKG2D, however, opposite transmembrane orientation of this signaling domain and NKG2D required inverse orientation fusion of either of them. We hypothesized that the particularly located C-terminus of the NKG2D ectodomain should allow reengineering of the membrane anchoring from a native N-terminal to an artificial C-terminal linkage. Indeed, the resulting chimeric NKG2D receptor (chNKG2D) was functional and efficiently mediated ESFT cell death triggered by activated T cells. Notably, ESFT cells with even low NKG2D-L expression were killed by CD8pos and also CD4pos cells. Both, mRNA transfection and lentiviral transduction resulted in high level surface expression of chNKG2D. However, upon target-cell recognition receptor surface levels were maintained by tranfected RNA only during the first couple of hours after transfection. Later, target-cell contact resulted in strong and irreversible receptor down-modulation, whereas lentivirally mediated expression of chNKG2D remained constant under these conditions. Together, our study defines NKG2D-Ls as targets for a CAR-mediated T cell based immunotherapy of ESFT. A comparison of two different methods of gene transfer reveals strong differences in the susceptibility to ligand-induced receptor down-modulation with possible implications for the applicability of RNA transfection.

Abstract B18: The transcribed non-coding genome modulates the biology of Ewing sarcoma family of tumors

Abstract Introduction: Ewing sarcoma family of tumors (EFT) is the second most common primary malignant bone tumor in children and adolescents. It features a characteristic chromosomal translocation; the most common being (t11;22)(q24;q12), which encodes for the chimeric EWS-FLI1 oncoprotein. The disease is highly invasive with a 30% 5-year survival rate for patients with metastatic disease. This study identifies and characterizes a non-coding locus that is unique to EFTs and mediates at least in part the role of the chimeric oncogene in EFT pathogenesis. This particular locus suggests that better understanding of the functional non-coding transcriptome of this developmental cancer can potentially assist in discovering biomarkers that improve the diagnosis and therapeutic management of the disease. Methods: Expression profiles from a panel of 500 tissue samples including over 100 EFTs, other primary tumors, and normal tissues were generated using Affymetrix Human Exon 1.0 ST microarrays. Data were analyzed using institutional software, Genetrix. RNAseq data was generated on two EFT cell lines derived from a primary tumor and its metastatic derivative. Data was validated by PCR on 33 cell lines including EFTs and other tumors. Functional studies on EFT cell lines were performed using various RNAi techniques and viral transductions. Additional characterization of the RNA was done by cellular fractionation studies and northern blots. Results: Expression microarray profiles showed unannotated transcripts to be the strongest discriminators of EFTs from all other tumor types. One such transcript, AK057037, was the most highly expressed RNA (1.07 × 10−20 in EFTs. In silico modeling and RNASeq analyses revealed that this transcript is a multi-exonic, presumably, long non-coding RNA (lncRNA) with a maximum predicted open reading frame of 137 amino acids that had no homology, protein domains, or characteristic motifs. Additionally, multiple variants of the transcript exist in both the nucleus and the cytoplasm. Validation studies confirmed that this lncRNA was exclusively expressed in EFTs. The EFT-specific oncoprotein, EWS-FLI1, regulates expression of this transcript by directly binding to its promoter region. Furthermore, functional data based on knocking down and overexpressing the transcript shows that AK057037 acts as an oncogene in EFT and enhances tumorigenicity by increasing the metastatic potential of the tumor cell lines. Conclusion: The study established that lncRNA AK057037 promotes EFT pathogenesis. Its exclusive presence in EFT suggests that expression of this lncRNA is tissue-specific and highly regulated. Targeting AK057037 in these tumors may help improve outcomes in children with metastatic disease. Further work on the non-coding transcriptome may reveal additional functional RNAs that contribute to the biology of this disease. Citation Format: Sheetal A. Mitra, Daniel H. Wai, William A. May, Jonathan D. Buckley, Philipp Kapranov, Robert J. Arceci, Timothy J. Triche. The transcribed non-coding genome modulates the biology of Ewing sarcoma family of tumors [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B18.

STAT3 Regulates Proliferation and Immunogenicity of the Ewing Family of TumorsIn Vitro

The Ewing sarcoma family of tumors (ESFT) represents an aggressive spectrum of malignant tumour types with common defining histological and cytogenetic features. To evaluate the functional activation of signal transducer and activator of transcription 3 (STAT3) in ESFT, we evaluated its activation in primary tissue sections and observed the functional consequences of its inhibition in ESFT cell lines. STAT3 was activated (tyrosine 705-phosphorylated) in 18 out of 31 primary tumours (58%), either diffusely (35%) or focally (23%). STAT3 was constitutively activated in 3 out of 3 ESFT cell lines tested, and its specific chemical inhibition resulted in complete loss of cell viability. STAT3 inhibition in ESFT cell lines was associated with several consistent changes in chemokine profile suggesting a role of STAT3 in ESFT in both cell survival and modification of the cellular immune environment. Together these data support the investigation of STAT3 inhibitors for the Ewing family of tumors.

Heterogeneous Role of the Glutathione Antioxidant System in Modulating the Response of ESFT to Fenretinide in Normoxia and Hypoxia

Glutathione (GSH) is implicated in drug resistance mechanisms of several cancers and is a key regulator of cell death pathways within cells. We studied Ewing's sarcoma family of tumours (ESFT) cell lines and three mechanistically distinct anticancer agents (fenretinide, doxorubicin, and vincristine) to investigate whether the GSH antioxidant system is involved in the reduced sensitivity to these chemotherapeutic agents in hypoxia. Cell viability and death were assessed by the trypan blue exclusion assay and annexin V-PI staining, respectively. Hypoxia significantly decreased the sensitivity of all ESFT cell lines to fenretinide-induced death, whereas the effect of doxorubicin or vincristine was marginal and cell-line-specific. The response of the GSH antioxidant system in ESFT cell lines to hypoxia was variable and also cell-line-specific, although the level of GSH appeared to be most dependent on de novo biosynthesis rather than recycling. RNAi-mediated knockdown of key GSH regulatory enzymes γ-glutamylcysteine synthetase or glutathione disulfide reductase partially reversed the hypoxia-induced resistance to fenretinide, and increasing GSH levels using N-acetylcysteine augmented the hypoxia-induced resistance in a cell line-specific manner. These observations are consistent with the conclusion that the role of the GSH antioxidant system in modulating the sensitivity of ESFT cells to fenretinide is heterogeneous depending on environment and cell type. This is likely to limit the value of targeting GSH as a therapeutic strategy to overcome hypoxia-induced drug resistance in ESFT. Whether targeting the GSH antioxidant system in conjunction with other therapeutics may benefit some patients with ESFT remains to be seen.

High Expression of Complement Component 5 (C5) at Tumor Site Associates with Superior Survival in Ewing's Sarcoma Family of Tumour Patients

Background. Unlike in most adult-onset cancers, an association between typical paediatric neoplasms and inflammatory triggers is rare. We studied whether immune system-related genes are activated and have prognostic significance in Ewing's sarcoma family of tumors (ESFTs). Method. Data analysis was performed on gene expression profiles of 44 ESFT patients, 11 ESFT cell lines, and 18 normal skeletal muscle samples. Differential expression of 238 inflammation and 299 macrophage-related genes was analysed by t-test, and survival analysis was performed according to gene expression. Results. Inflammatory genes are activated in ESFT patient samples, as 38 of 238 (16%) inflammatory genes were upregulated (P < 0.001) when compared to cell lines. This inflammatory gene activation was characterized by significant enrichment of macrophage-related gene expression with 58 of 299 (19%) of genes upregulated (P < 0.001). High expression of complement component 5 (C5) correlated with better event-free (P = 0.01) and overall survival (P = 0.004) in a dose-dependent manner. C5 and its receptor C5aR1 expression was verified at protein level by immunohistochemistry on an independent ESFT tumour tissue microarray. Conclusion. Immune system-related gene activation is observed in ESFT patient samples, and prognostically significant inflammatory genes (C5, JAK1, and IL8) for ESFT were identified.

Notch signalling is off and is uncoupled from HES1 expression in Ewing's sarcoma

Notch can act as an oncogene or as a tumour suppressor and thus can either promote or inhibit tumour cell growth. To establish Notch status in Ewing's sarcoma family of tumours (ESFT), we investigated the Notch pathway by gene expression profiling meta-analysis or immunohistochemistry in samples obtained from 96 and 24 ESFT patients, respectively. We found that although Notch receptors were highly expressed, Notch did not appear to be active, as evidenced by the absence of Notch receptors in cell nuclei. In contrast, we show that Notch receptors known to be active in colon adenocarcinoma, hepatocarcinoma, and pancreatic carcinoma stain cell nuclei in these tumours. High expression of the Notch effector HES1 transcription factor, usually used as a surrogate marker for active Notch, was also restricted to outside of the nucleus in the majority of ESFT, and analysis of HES1 gene targets indicated HES1 to be transcriptionally inactive. Neither forced activation nor pharmacological or genetic blocking of Notch affected HES1 expression in ESFT cells, indicating HES1 expression to be uncoupled from the Notch pathway. Additional functional studies in ESFT cell lines confirmed Notch to be switched off. Finally, unlike experiments in which HES1 expression was modulated, experimental activation of Notch in ESFT cell lines via several means blocked cell proliferation and reduced their clonogenic potential in soft agar. These indicate that HES1 is uncoupled from Notch in ESFT, that EWS-FLI1-mediated inhibition of Notch contributes to ESFT aggressive cell growth, and support a role for Notch in ESFT tumour suppression, at least partly through the Notch effector HEY1.

Dipeptidyl Peptidases as Survival Factors in Ewing Sarcoma Family of Tumors: IMPLICATIONS FOR TUMOR BIOLOGY AND THERAPY

Ewing sarcoma family of tumors (ESFT) is a group of aggressive pediatric malignancies driven by the EWS-FLI1 fusion protein, an aberrant transcription factor up-regulating specific target genes, such as neuropeptide Y (NPY) and its Y1 and Y5 receptors (Y5Rs). Previously, we have shown that both exogenous NPY and endogenous NPY stimulate ESFT cell death via its Y1 and Y5Rs. Here, we demonstrate that this effect is prevented by dipeptidyl peptidases (DPPs), which cleave NPY to its shorter form, NPY(3-36), not active at Y1Rs. We have shown that NPY-induced cell death can be abolished by overexpression of DPPs and enhanced by their down-regulation. Both NPY treatment and DPP blockade activated the same cell death pathway mediated by poly(ADP-ribose) polymerase (PARP-1) and apoptosis-inducing factor (AIF). Moreover, the decrease in cell survival induced by DPP inhibition was blocked by Y1 and Y5R antagonists, confirming its dependence on endogenous NPY. Interestingly, similar levels of NPY-driven cell death were achieved by blocking membrane DPPIV and cytosolic DPP8 and DPP9. Thus, this is the first evidence of these intracellular DPPs cleaving releasable peptides, such as NPY, in live cells. In contrast, another membrane DPP, fibroblast activation protein (FAP), did not affect NPY actions. In conclusion, DPPs act as survival factors for ESFT cells and protect them from cell death induced by endogenous NPY. This is the first demonstration that intracellular DPPs are involved in regulation of ESFT growth and may become potential therapeutic targets for these tumors.

Identification of an Inhibitor of the EWS-FLI1 Oncogenic Transcription Factor by High-Throughput Screening

Chromosomal translocations generating oncogenic transcription factors are the hallmark of a variety of tumors, including many sarcomas. Ewing sarcoma family of tumors (ESFTs) are characterized by the t(11;22)(q24;q12) translocation that generates the Ewing sarcoma breakpoint region 1 and Friend leukemia virus integration 1 (EWS-FLI1) fusion transcription factor responsible for the highly malignant phenotype of this tumor. Although continued expression of EWS-FLI1 is believed to be critical for ESFT cell survival, a clinically effective small-molecule inhibitor remains elusive likely because EWS-FLI1 is a transcription factor and therefore widely felt to be "undruggable." We developed a high-throughput screen to evaluate more than 50 000 compounds for inhibition of EWS-FLI1 activity in TC32 ESFT cells. We used a TC32 cell-based luciferase reporter screen using the EWS-FLI1 downstream target NR0B1 promoter and a gene signature secondary screen to sort and prioritize the compounds. We characterized the lead compound, mithramycin, based on its ability to inhibit EWS-FLI1 activity in vitro using microarray expression profiling, quantitative reverse transcription-polymerase chain reaction, and immunoblot analysis, and in vivo using immunohistochemistry. We studied the impact of this inhibition on cell viability in vitro and on tumor growth in ESFT xenograft models in vivo (n = 15-20 mice per group). All statistical tests were two-sided. Mithramycin inhibited expression of EWS-FLI1 downstream targets at the mRNA and protein levels and decreased the growth of ESFT cells at half maximal inhibitory concentrations between 10 (95% confidence interval [CI] = 8 to 13 nM) and 15 nM (95% CI = 13 to 19 nM). Mithramycin suppressed the growth of two different ESFT xenograft tumors and prolonged the survival of ESFT xenograft-bearing mice by causing a decrease in mean tumor volume. For example, in the TC32 xenograft model, on day 15 of treatment, the mean tumor volume for the mithramycin-treated mice was approximately 3% of the tumor volume observed in the control mice (mithramycin vs control: 69 vs 2388 mm(3), difference = 2319 mm(3), 95% CI = 1766 to 2872 mm(3), P < .001). Mithramycin inhibits EWS-FLI1 activity and demonstrates ESFT antitumor activity both in vitro and in vivo.

Abstract 1936: Notch signaling is switched off and displays properties of a tumor suppressor in Ewing's sarcoma

Abstract Notch can act as an oncogene or as a tumor suppressor and thus can either promote or inhibit tumor cell growth. To establish Notch status in Ewing's sarcoma family of tumors (ESFT), we investigated the Notch pathway by gene expression profiling meta-analysis or immunohistochemistry in samples obtained from 96 and 24 ESFT patients, respectively. We find that although Notch receptors were highly expressed, Notch did not appear to be active as evidenced by the absence of Notch receptors in cell nuclei. High expression of the Notch effector HES1 transcription factor, usually used as a surrogate marker for active Notch, was also restricted to outside of the nucleus in the majority of tumors, and analysis of HES1 gene targets indicated HES1 to be transcriptionally inactive. Neither forced activation nor pharmacological or genetic blocking of Notch affected HES1 expression in ESFT cells, indicating HES1 expression to be uncoupled from the Notch pathway. Additional functional studies in ESFT cell lines confirmed Notch to be switched off. Finally, unlike experiments in which HES1 expression was modulated, experimental activation of Notch in ESFT cell lines via several means blocked cell proliferation. These indicate that HES1 is uncoupled from Notch in ESFT, that EWS-FLI1-mediated inhibition of Notch contributes to ESFT aggressive cell growth, and supports a role for Notch in ESFT tumor suppression, at least partly through the Notch effector HEY1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1936. doi:10.1158/1538-7445.AM2011-1936

Abstract 1182: A large non-coding RNA that is characteristic of Ewing sarcoma family of tumors

Abstract Introduction: Ewing sarcoma is the second most common primary malignant bone tumor in children and adolescents. Ewing sarcoma family of tumors (EFT) feature a characteristic chromosomal translocation t(11;22)(q24;q12), which encodes for the chimeric EWS-FLI1 oncoprotein. A minority of EFT have additional translocation variants partnering EWS with other members of the ETS family of transcription factors. This disease is highly invasive with a 30% 5-year survival rate for patients with metastatic disease. This study aimed to identify and validate candidate biomarker(s), both protein-coding and non-coding, that are unique to EFT and can potentially assist in objective diagnosis and therapeutic management. Methods: Using Affymetrix Human Exon 1.0 ST microarrays, genome-wide expression profiles were generated on a panel of 500 tissue samples including over 100 Ewing sarcomas, other primary tumors, normal tissues and stem cells. Whole transcriptome sequencing (RNASeq) was also performed on 2 cell lines. Microarray data was validated by RT-PCR. EFT cell lines were transfected with siRNA against EWS-FLI1 and subjected to retinoic acid (RA)-induced differentiation to monitor expression changes in EFT-specific transcripts. Cellular fractionation studies and northern blots were performed to characterize location and size of the candidate transcript, respectively. Results: Expression microarray profiles identified both genes and candidate non-coding RNAs (ncRNAs) that were strongly associated with EFT, including the candidate ncRNA AK057037. In silico modeling and RNASeq of this transcript revealed a large, intergenic, non-coding 2,641 base, 8-exon RNA (lincRNA) with a predicted open reading frame of 137 amino acids with no homology, protein domains, or characteristic motifs. RT-PCR data from 33 tumor cell lines validated over-expression of this unique and exclusive transcript in EFT. Alternative splicing of both exons and introns as well as extended forms of the transcript were also identified. Northern blot confirmed multiple variants of the transcript in addition to the known 2.6 kb region. Cellular fractionation indicated that the transcript was abundantly present in the nucleus. EWS-FLI1 knockdown in EFT cells reduced its expression. AK057037 was detected during early differentiation of human embryonic stem cells to neural crest stem cells (NCSC), putative cells of EFT origin. Furthermore, RA-induced differentiation of EFT cell lines decreased the transcript's expression, corroborating the hypothesis that AK057037 contributes to the primitive NCSC-like phenotype of EFT. Conclusions: AK057037 is highly and exclusively expressed in EFT, and its expression is regulated by EWS-FLI1. This multi-exon transcript with multiple splice variants has characteristic features of a lincRNA. Its strong association with EFT and expression in NCSC indicates that it may be a key component of Ewing sarcoma pathogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1182. doi:10.1158/1538-7445.AM2011-1182

Abstract 167: Identification of miR-34a as a prognostic biomarker of Ewing sarcoma family of tumors

Abstract Clinical outcomes of Ewing sarcoma family of tumors (ESFT) were improved after the introduction of multimodal therapies including intensive chemotherapy. However current therapeutic approaches still leave us the problems including short-term toxicities of chemotherapy and serious late adverse effects; second malignancy and fatal cardiac failure even for long survivors. Therefore, we are in the urgent need to develop the novel biomarkers for an appropriate stratification of patients at diagnosis to establish more effective and less invasive therapy for all ESFT patients. MicroRNAs (miRNAs) are newly-recognized small non gene-coding RNA family that regulate gene expression through translational suppression. More recently, they have been increasingly accepted as playing crucial roles in pathogenesis of most human malignancy as well as normal cell differentiation. However, little is known regarding to the possible role of miRNAs on sarcoma including ESFT. Here we focus on the whole profiling of the miRNA expressions in ESFT for the aim to elucidate the novel miRNA biomarkers in the treatment of ESFT. Randomly selected 34 primary biopsy samples of ESFT have been analyzed for complete miRNA expression profiles. All of the patients were treated at Rizzoli Institute from 1991 to 2008 with multimodal therapies. Microarray and statistical analyses were carried out using Agilent's miRNA microarray kit (v2) and GeneSpring GX analysis software, respectively. To identify miRNAs associated with the prognosis of ESFT patients we first compared the expression profiles of 21 patients who relapsed within 5 years with 13 patients who remained free of disease throughout the follow-up. As a result, we identified 16 miRNAs differentially expressed between the two groups (fold change &amp;gt; 1.5, p&amp;lt;0.1). We have also performed univariate Cox regression analysis to elucidate the miRNAs, whose expression level are related to the length of event-free survival (EFS) and/or overall survival (OS) and found out 15 miRNAs and 32 miRNAs that significantly correlated with EFS and OS, respectively. We then validated these data with quantitative real time PCR using extended series of 49 primary biopsy samples. After these validation, we have confirmed that low expressions of miR-34a were strongly associated with worse prognosis (P&amp;lt;0.001, Logrank test) and remained most significant biomarker after multivariate analyses. Moreover expression levels of miR-34a using real time PCR were highly correlated with the level of corresponding microarray data (Pearson correlation test: p&amp;lt;0.0001). Over-expression of miR-34a inhibited the colony formation in the soft agar and enhanced chemosensitivities of ESFT cell lines. In conclusion, we have identified miR-34a as a powerful biomarker to distinguish the prognosis of ESFT patients through the whole miRNA profiling analyses (grants from EU project Eurobonet and Italian Association for Cancer Research). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 167. doi:10.1158/1538-7445.AM2011-167

Ecteinascidin 743 Interferes with the Activity of EWS-FLI1 in Ewing Sarcoma Cells

ET-743 (trabectedin; Yondelis) is approved in Europe for the treatment of soft tissue sarcomas. Emerging phase 1 and 2 clinical data have shown high response rates in myxoid liposarcoma in part owing to the inhibition of the FUS-CHOP transcription factor. In this report, we show that modulation of specific oncogenic transcription factors by ET-743 may extend to other tumor types. We demonstrate that, among a panel of pediatric sarcomas, Ewing sarcoma family of tumors (ESFTs) cell lines bearing the EWS-FLI1 transcription factor are the most sensitive to treatment with ET-743 compared with osteosarcoma, rhabdomyosarcoma, and synovial sarcoma. We show that ET-743 reverses a gene signature of induced downstream targets of EWS-FLI1 in two different ESFT cell lines (P = .001). In addition, ET-743 directly suppresses the promoter activity of a known EWS-FLI1 downstream target NR0B1 luciferase reporter construct without changing the activity of a constitutively active control in ESFT cells. Furthermore, the effect is specific to EWS-FLI1, as forced expression of EWS-FLI1 in a cell type that normally lacks this fusion protein, HT1080 cells, induces the same NR0B1 promoter, but this activation is completely blocked by ET-743 treatment. Finally, we used gene set enrichment analysis to confirm that other mechanisms of ET-743 are active in ESFT cells. These results suggest a particular role for ET-743 in the treatment of translocation-positive tumors. In addition, the modulation of EWS-FLI1 makes it a novel targeting agent for ESFT and suggests that further development of this compound for the treatment of ESFT is warranted.

Caveolin-1 Modulates the Ability of Ewing's Sarcoma to Metastasize

Metastasis is the final stage of tumor progression and is thought to be responsible for up to 90% of deaths associated with solid tumors. Caveolin-1 (CAV1) regulates multiple cancer-associated processes related to malignant tumor progression. In the present study, we tested the hypothesis that CAV1 modulates the metastatic ability of cells from the Ewing's sarcoma family of tumors (ESFT). First, we analyzed the expression of CAV1 by immunostaining a tissue microarray containing 43 paraffin-embedded ESFT tumors with known EWS translocations. Even though no evidence was found for a significant association between CAV1 expression and stage, size or tumor site, all metastatic samples (10 of 10) had significantly high CAV1 expression, suggesting that high CAV1 content could positively contribute to enhance ESFT metastasis. To determine the effect of CAV1 on the migratory and invasive capabilities of ESFT cells, we knocked down CAV1 expression in TC252 and A673 cells by stably transfecting a previously validated shRNA construct. In vitro, migration and invasion assays showed that for both cell lines, CAV1 knocked-down cells migrated and invaded significantly less (P ≤ 0.01) than control cells. Moreover, control A673 cells introduced into BALB/c nude mice by tail vein injection strongly colonized the lungs. In contrast, animals injected with CAV1 knocked-down cells showed either no incidence of metastasis or developed lung metastases after a significant delay (P < 0.0001). Finally, we show that the molecular mechanisms by which CAV1 carries out its key role in regulating ESFT metastasis involve matrix metalloproteinase production and activation as well as the control of the expression of SPARC, a known determinant of lung colonization.