Growth Of Ewing Sarcoma Cells Research Articles

Abstract 5334: Destabilization of EWS-Fli1 protein by deubiquitinase inhibition in Ewing Sarcoma

Abstract Background: Ewing Sarcoma is a malignant tumor in bone or soft tissue, and typically in teenagers and young adults. Genetically, 85% of Ewing’s Sarcoma contains the chromosomal translocation [t(11;22)(q24;q12)], which fuses EWSR1 gene from chromosome 11 and Fli1 gene from chromosome 22 and generates an aberrant chimeric protein EWS-Fli1. EWS-Fli1 is essential for cell growth and development in EWS-Fli1 positive Ewing Sarcoma cells. Therefore, targeting EWS-Fli1 could be a promising therapeutic strategy for Ewing Sarcoma. Methods and Results: Currently, the compounds of targeting EWS-Fli1 mainly interfere its interaction with partner or DNA. However, it could be a more potent way to manipulate its protein stability. Firstly, the proteasome inhibitor MG132 could retard the degradation of EWS-Fli1 protein in EWS502 cells and A673 cells, and increase its ubiquitination, which hints the degradation of EWS-Fli1 is through the ubiquitin (Ub)-proteasome pathway (UPP). Then, to identify the protein that interact with EWS-Fli1 and modulate its stability, we selected the candidate deubiquitinase according to literatures and the protein homology. And by co-immunoprecipitation assay, we identified a deubiquitinating enzyme (DUB) binding to EWS-Fli1. RNAi-mediated knockdown or inhibition of EWS-Fli1-DUB with a small molecule compound caused a marked increase in EWS-Fli1 ubiquitination, decreased levels of EWS-Fli1 in a proteasome-dependent manner, and further inhibited the growth of Ewing Sarcoma cells. Conclusion: The findings indicate that EWS-Fli1 could be ablated by inhibiting its specific deubiquitinase in Ewing Sarcoma, which could be a novel treatment approach for Ewing Sarcoma. Citation Format: Shan Wang, Ralf Kittler. Destabilization of EWS-Fli1 protein by deubiquitinase inhibition in Ewing Sarcoma [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 5334. doi:10.1158/1538-7445.AM2017-5334

Abstract 696: Inhibition of Ewing sarcoma cell growth by targeting Sp1 and survivin with the small molecule clotam

Abstract Ewing sarcoma (ES) is the most common soft tissue and bone tumor in adolescents and young adults. The survival rate of patients with metastatic disease is poor suggesting an urgent need for the development of more effective therapeutic options. Our aim was to investigate novel targets and identify less toxic agents to improve therapeutic efficacy in ES patients. Specificity protein 1 (Sp1) transcription factor regulates critical genes involved in cell proliferation. Sp1 also mediates the expression of survivin, an inhibitor of apoptosis protein. Overexpression of Sp1 and survivin is linked to aggressiveness and poor prognosis in several cancers; however, their precise association is not adequately evaluated in ES. Previously, we showed that tolfenamic acid (TA) inhibits leukemia, medulloblastoma and neuroblastoma cell proliferation by targeting Sp1. In this study, we investigated the anti-cancer activity of this small molecule using human ES cell lines. CHLA-9 and TC-32 cells were treated with vehicle (DMSO) or TA (2.5-20 µg/ml) and cell viability was measured at 24, 48 and 72 h post-treatment using CellTiter Glo kit. ES cells were treated with vehicle or TA (15 µg/ml) for 48 h. The mRNA and protein expression of Sp1 and survivin was determined by quantitative polymerase chain reaction (qPCR) and Western blot analysis respectively. Flow cytometry was used to measure apoptotic cell population using Annexin-V staining and cell cycle phase distribution with propidium iodide. Markers of apoptosis (caspase 3/7 activity using caspase Glo kit and cleaved PARP by Western blot) and cell cycle arrest (Cyclin D1 and p21 expression by Western blot) were also determined. Sp1 DNA-binding was evaluated by gel shift assay. TA treatment inhibited ES cell viability in a dose and time-dependent manner which is accompanied by the inhibition of Sp1 and survivin protein expression and a 2-3 fold increase in apoptotic markers, caspase 3/7 activity, Annexin-V staining, and c-PARP expression. Interestingly, TA inhibited mRNA expression of survivin but not Sp1, suggesting it has post-translational effects, perhaps proteasome-dependent degradation of Sp1. Gel shift assay revealed a decrease in Sp1 DNA-binding, indicating that TA could directly disrupt the binding of Sp1 with its consensus binding site in the promoter of target genes. Cyclin D1 is crucial for moving the cells from G0/G1 to next phases and induction of p21 is known to inhibit Cyclin D1. In this study, TA up-regulated p21, reduced Cyclin D1 expression and accumulated cells in G0/G1 phase. These results demonstrate that TA induces apoptosis and causes cell cycle arrest in ES cells by targeting Sp1 and survivin. Citation Format: Sagar Shelake, Umesh T. Sankpal, W. Paul Bowman, Matthew Wise, Anish Ray, Riyaz Basha. Inhibition of Ewing sarcoma cell growth by targeting Sp1 and survivin with the small molecule clotam [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 696. doi:10.1158/1538-7445.AM2017-696

Targeting specificity protein 1 transcription factor and survivin using tolfenamic acid for inhibiting Ewing sarcoma cell growth.

Transcription factor Specificity protein1 (Sp1) and its downstream target survivin (inhibitor of apoptosisprotein), play major roles in the pathogenesis of various cancers. Ewing Sarcoma (ES) is a common soft tissue/bone tumor in adolescent and young adults. Overexpression of survivin is also linked to the aggressiveness and poor prognosis of ES. Small molecule Tolfenamic acid (TA) inhibits Sp1 and survivin in cancer cells. In this investigation, we demonstrate a strategy to target Sp1 and survivin using TA and positive control Mithramycin A (Mit) to inhibit ES cell growth. Knock down of Sp1 using small interfering RNA (siRNA) resulted in significant (p<0.05) inhibition of CHLA-9 and TC-32 cell growth as assessed by CellTiter-Glo assay kit. TA or Mit treatment caused dose/time-dependent inhibition of cell viability, and this inhibition was correlated with a decrease in Sp1 and survivin protein levels in ES cells. Quantitative PCR results showed that Mit treatment decreased the mRNA expression of both survivin and Sp1, whereas TA diminished only survivin but not Sp1. Proteasome inhibitor restored TA-induced inhibition of Sp1 protein expression suggesting that TA might cause proteasome-dependent degradation. Gel shift assay using ES cell nuclear extract and biotinylated Sp1 consensus oligonucleotides confirmed that both TA and Mit decreased DNA-binding activity of Sp1. These results demonstrate that both Mit and TA reduce expression of Sp1 and survivin, disrupt Sp1 DNA-binding and inhibit ES cell proliferation. This investigation suggests that targeting Sp1 and survivin could be an effective strategy for inhibiting ES cell growth.

Abstract 3201: Insulin-like growth factor 2 (IGF-2) mRNA binding protein 3 predicts poor prognosis and promotes cell proliferation in Ewing sarcoma

Abstract INTRODUCTION: Insulin-like growth factor 2 (IGF-2) mRNA binding protein 3 (IGF2BP3, also known as IMP3) is an oncofetal protein which binds RNA thus influencing the transcript target fate. IGF2BP3 is de novo synthesized in malignancies where it promotes proliferation, drug resistance and metastases. Moreover, IGF2BP3 represents a promising indicator of outcome in several cancers. Ewing sarcoma (EWS) is a rare bone and soft tissue tumor where the IGF system plays a pivotal role. This study aimed to investigate the relevance of IGF2BP3 in EWS and verify its value as a prognostic biomarker. MATERIAL AND METHODS: Total RNA was extracted from two independent cohorts of 30 and 109 snap-frozen tissues from localized primary EWS samples with more than 5 years follow-up. Microarray analysis (Affymetrix) was performed in the cohort of 30 patients while IGF2BP3 expression was analysed by qRT-PCR (Applied Biosystem) in the cohort of 109 cases. Prognostic value of IGF2BP3 was evaluated both in univariate and multivariate analysis. IGF2BP3 expression was assessed by qRT-PCR and western blot in a panel of 14 EWS cell lines. Plasmids for IGF2BP3 silencing or over-expression were transfected in A673 or LAP-35 and IOR/CAR EWS cells, respectively. Soft-agar growth of cell lines with differential IGF2BP3 expression was evaluated. catRAPID express human web server was employed for prediction of novel IGF2BP3/mRNAs interactions. IGF-2 and ABCF1 expression levels were analysed in vitro and in clinical specimens. RESULTS: Microarray results evidenced that IGF2BP3 higher expression correlated with a worse outcome (p-value 0.0002). This data was confirmed in the cohort analysed by qRT-PCR both considering univariate (p-value 0.001) and multivariate analysis (HR:2.83. IC95% [1.22-6.53].p = 0.015). In vitro, higher IGF2BP3 expression correlated with an increased capability of growth in anchorage-independent conditions and IGF2BP3 knockdown significantly decreased soft-agar growth of EWS cells. Oncogenic potential of IGF2BP3 was not found to be mediated through IGF-2-dependent mechanism but ABCF1, an ABC family member with a key role in translation initiation, was predicted as a target of IGF2BP3 by in silico analysis. Accordingly, IGF2BP3 loss- or gain-of-functions approaches induced down- or up-regulation of ABCF1 protein levels, respectively. In addition, a direct correlation between IGF2BP3 and ABCF1 was found in 109 EWS patients (p-value minor than 0.0001). Functional studies on the role of ABCF1 in EWS will follow. CONCLUSIONS: IGF2BP3 represents an indicator of prognosis in EWS therefore its detection may be of help to stratify patients according to risk. We also provided evidences for IGF2BP3 in the regulation of EWS aggressiveness and identified ABCF1 as a new putative mediator of IGF2BP3 malignant effects. (Grants: FIRB RBAP11884M_005 and AIRC IG2013_14049 to KS) Citation Format: Caterina Mancarella, Linda Calzolari, Stefano Ferrari, Davide Donati, Piero Picci, Katia Scotlandi. Insulin-like growth factor 2 (IGF-2) mRNA binding protein 3 predicts poor prognosis and promotes cell proliferation in Ewing sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3201.

Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model.

Ewing sarcoma (ES) involves a tumor-specific chromosomal translocation that produces the EWS-FLI1 protein, which is required for the growth of ES cells both in vitro and in vivo. However, an EWS-FLI1-driven transgenic mouse model is not currently available. Here, we present data from six independent laboratories seeking an alternative approach to express EWS-FLI1 in different murine tissues. We used the Runx2, Col1a2.3, Col1a3.6, Prx1, CAG, Nse, NEFL, Dermo1, P0, Sox9 and Osterix promoters to target EWS-FLI1 or Cre expression. Additional approaches included the induction of an endogenous chromosomal translocation, in utero knock-in, and the injection of Cre-expressing adenovirus to induce EWS-FLI1 expression locally in multiple lineages. Most models resulted in embryonic lethality or developmental defects. EWS-FLI1-induced apoptosis, promoter leakiness, the lack of potential cofactors, and the difficulty of expressing EWS-FLI1 in specific sites were considered the primary reasons for the failed attempts to create a transgenic mouse model of ES.

Abstract B20: In silico and in vitro drug screening identifies new therapeutic approaches targeting DNA double-strand breaks repair in Ewing sarcoma

Abstract Purpose: Ewing sarcoma (EWS) is the second most frequent malignant bone and soft-tissue tumor in children and young adults. More than 85% of EWS expressed the oncogenic transcription factor EWS-FLI1 fusion protein. With current therapies, about 75% of patients with localized disease survive, but patients with metastatic or recurrent disease have a dismal outcome with survival rates less than 20%. Although significant advances have been made in understanding the molecular basis of EWS, little has changed in the treatment of EWS patients over the past four decades. Efforts have been focused on the development of therapeutics targeting the EWS-FLI1 oncoprotein; however, no effective drugs have been developed. Therefore, our goal was to accelerate promising and new combination therapies to EWS patients through drug repurposing and rediscovery, and to expand the treatment options and improve treatment outcomes for patients with this rare and life-threatening disease. Methods: We first employed an integrated gene expression signatures-based bioinformatics approach to identify FDA-approved drugs that are predicted to reverse the EWS disease gene signatures and/or mimic the silencing of the EWS-FLI1 oncogene. We then evaluated the drug hits from the in silico approaches in three benign sarcoma cell lines (Hs 919.T., Hs 822.T., Hs 863.T.) and a panel of six genetically diverse EWS cell lines (A673, TC71, SK-ES-1, RD-ES, CHLA-258 and COG-E-352) by using CellTiter-Blue® Cell Viability Assay. The cytotoxic activities and drug interaction of the most potent drugs in various combinations were further assessed in vitro by using 8x8 matrix dose response assay. The combination index (CI) was calculated by the CompuSyn software according to the Chou and Talalay's method. Cellular apoptosis and DNA damage response after drug treatments in EWS cells were analyzed by Western blot. Results: Twenty-six FDA-approved drugs were identified through in silico prediction approaches (FDR &amp;lt; 0.05). Among them, MS-275, elesclomol, daunorubicin and vindesine are the most potent and selective drugs in killing EWS cells (72 h, IC50 0.004 - 3.65 µM), as compare to the benign sarcoma cells (IC50s in EWS cells were at least 10 times lower than the counterparts in benign controls). The combination of MS-275 (Class I HDAC inhibitor) and elesclomol (a selective mitochondrial reactive oxygen species inducer) was the most robust and synergistic treatment in inhibiting EWS cell growth, with 57%-82% of the combinations showing synergy (CI &amp;lt; 1) in the matrix dose response test across all tested EWS cell lines with either EWS-FLI1 or EWS-ERG fusion. Western blot data indicated that MS-275 and elesclomol induced caspase-dependent apoptosis in EWS cells in both time- and dose-dependent manner. Both MS-275 and elesclomol treatment led to DNA double-strand breaks (DSBs), as evidenced by the phosphorylation of γH2AX. MS-275 treatment inhibited homologous recombination (HR) pathway by downregulating BRCA1 and RAD51, while MS-275 regulated non-homologous end-joining (NHEJ) pathway in a cell type-dependent manner. Conclusions: Our studies revealed that the combination of MS-275 and elesclomol were synergistic and that MS-275 sensitized EWS cells to elesclomol through targeting DNA DSBs repair. This combination therapy may provide an alternative treatment strategy for children and young adults diagnosed with Ewing sarcoma. Citation Format: Yan Ma, Bin Chen, Scott J. Weir, Atul Butte, Andrew K. Godwin. In silico and in vitro drug screening identifies new therapeutic approaches targeting DNA double-strand breaks repair in Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B20.

What's New in Orthopaedic Oncology.

This article was updated on January, 13, 2016, because of a previous error. The first author's last name was misspelled. Specifically, “Santiago A. Lozano Calderone” should have read “Santiago A. Lozano Calderon.” An erratum has been published: J Bone Joint Surg Am. 2016 Feb 17;98(4):e17. ### Ewing Sarcoma Ewing sarcoma is associated with balanced translocations of the EWS and ETS genes that produce a fusion protein that binds DNA affecting transcription. Two new studies identified targets of the EWS/FLI1 fusion protein. One study showed preferential binding for a DNA region that induced early growth response 2 (EGR2) expression. EGR2 stimulates proliferation, differentiation, and survival of neural crest-derived cells, from which Ewing sarcoma cells are thought to originate. An EGR2 gene knockdown halted Ewing sarcoma cell growth in vitro and in a xenograft model, demonstrating that the EWS/FLI1 fusion protein works through EGR21. A second study showed that ESW/FLI1 targets SLFN11, which is highly expressed in some Ewing sarcomas. Ewing tumors expressing SLFN11 were susceptible to topoisomerase inhibitor treatment2. In a separate genetic study, 116 patient samples of Ewing sarcoma were used to identify somatic mutations. The gene for fibroblast growth factor receptor 1, FGFR1, was noted to have a gain in copy number in 31.7% of primary tumors. Furthermore, RNA interference of FGFR1 expression halted the growth of Ewing sarcoma cells in a xenograft model. The study concluded with a patient example in which treatment with a tyrosine-kinase inhibitor against FGFR1 reduced 18-FDG-PET (18F-deoxyglucose positron emission tomography) activity3. Margulies et al. developed an animal model for Ewing sarcoma treated with radiation therapy. Increased osteoclastic activity without detection of RANKL (receptor activator of nuclear factor κ ligand) in bone affected by the tumor was identified. MCSF (macrophage colony stimulating factor) rather than RANK ligand mediated osteoclastic activity …

Genotoxic stress inhibits Ewing sarcoma cell growth by modulating alternative pre-mRNA processing of the RNA helicase DHX9.

Alternative splicing plays a key role in the DNA damage response and in cancer. Ewing Sarcomas (ES) are aggressive tumors caused by different chromosomal translocations that yield in-frame fusion proteins driving transformation. RNA profiling reveals genes differentially regulated by UV light irradiation in two ES cell lines exhibiting different sensitivity to genotoxic stress. In particular, irradiation induces a new isoform of the RNA helicase DHX9 in the more sensitive SK-N-MC cells, which is targeted to nonsense-mediated decay (NMD), causing its downregulation. DHX9 protein forms a complex with RNA polymerase II (RNAPII) and EWS-FLI1 to enhance transcription. Silencing of DHX9 in ES cells sensitizes them to UV treatment and impairs recruitment of EWS-FLI1 to target genes, whereas DHX9 overexpression protects ES cells from genotoxic stress. Mechanistically, we found that UV light irradiation leads to enhanced phosphorylation and decreased processivity of RNAPII in SK-N-MC cells, which in turn causes inclusion of DHX9 exon 6A. A similar effect on DHX9 splicing was also elicited by treatment with the chemotherapeutic drug etoposide, indicating a more general mechanism of regulation in response to DNA damage. Our data identify a new NMD-linked splicing event in DHX9 with impact on EWS-FLI1 oncogenic activity and ES cell viability.

Abstract 486: BET protein inhibition by JQ1 blocks EWS-FLI1 activity in Ewing sarcoma

Abstract Ewing sarcoma is the second most common bone cancer in children, with an estimated 5-year survival rate of 20% for metastatic cases. These tumors harbor the characteristic chromosomal translocation t(11;22)(q24;q12), which leads to expression of the oncogenic chimeric transcription factor, EWS-FLI1. EWS-FLI1 drives Ewing sarcoma tumorigenesis, and is an attractive therapeutic target because it is expressed exclusively in tumor cells. However, directly targeting EWS-FLI1 has proved difficult. Thus, despite advances in our understanding of EWS-FLI1 function, treatment options remain limited, and to date, no targeted therapeutics have been developed. In this study, we investigated the effects of the epigenetic modifier drug, JQ1, on Ewing sarcoma pathogenesis. JQ1 is a small molecule inhibitor of the BET family of bromodomain proteins, which bind to acetylated histones and transcription factors to regulate gene expression. JQ1 selectively inhibits these acetylation-dependent BET protein interactions. Recent studies reveal that JQ1 has potent anti-neoplastic activity against multiple cancers, with c-MYC downregulation being a key mechanism by which it induces cell death in several tumor types. Because Ewing sarcoma cells express high levels of c-MYC, we predicted that they would be sensitive to the cytotoxic effects of JQ1. Our results demonstrate that JQ1 indeed inhibits growth of Ewing sarcoma cells, but unexpectedly, it does not function through inhibition of c-MYC. Using multiple patient-derived Ewing sarcoma cell lines, we find that JQ1 induces apoptosis as measured by PARP cleavage. Preliminary data further indicate that JQ1 suppresses growth of Ewing sarcoma xenografts in vivo. Mechanistic studies and microarray analysis reveal that JQ1 inhibits EWS-FLI1 activity, with multiple targets of the oncogene being rapidly downregulated upon JQ1 treatment. Studies are currently underway to identify the specific BET family members required for EWS-FLI1 function, and to determine the mechanism by which they regulate EWS-FLI1 activity. Given the emergence of numerous JQ1-derivative compounds targeting specific BET proteins, it is hopeful that this class of compounds may ultimately be used as targeted therapies for the treatment of Ewing sarcoma. Citation Format: Krista L. Bledsoe, Aaron Stonestrom, Stephan Kadauke, Laura Quick, Robert Young, Gerd A. Blobel, Margaret M. Chou. BET protein inhibition by JQ1 blocks EWS-FLI1 activity in Ewing sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 486. doi:10.1158/1538-7445.AM2015-486

Abstract 490: The Trithorax proteins menin and MLL promote Ewing sarcoma tumorigenicity

Abstract Normal development is governed by covalent modifications to chromatin that are mediated by the reciprocal actions of Polycomb group (PcG) and Trithorax (TrxG) proteins, which promote gene repression and activation, respectively. We recently reported that HOX genes, key developmental transcription factors that are regulated by PcG and TrxG during embryogenesis, are abnormally expressed in Ewing sarcoma (ES). In particular, we found that the posterior HOXD genes, including HOXD13, are markedly overexpressed, and that the promoters of these genes are aberrantly marked with the TrxG-dependent histone mark, H3K4me3. The TrxG proteins MLL and menin cooperate to drive HOX gene expression and promote tumorigenesis in leukemia. We therefore hypothesized that MLL and menin might also contribute to ES pathogenesis. Gene and protein expression were determined by microarray, q-RT-PCR and western blot of ES cell lines and tumors. Lentiviral shRNA constructs were used to knock down HOXD13, menin and MLL. Proliferation, survival, and tumorigenicity of genetically modified cells were assessed in vitro and in vivo. Inhibition of TrxG function was achieved by exposing cells to MI-503, a small molecule inhibitor of menin-MLL interactions. Our results confirm that menin, MLL and HOXD13 are all highly expressed by ES relative to non-malignant tissues and stem cells. Moreover, loss of function studies implicate each as a tumor promoting oncogene. Specifically, knockdown of HOXD13, menin or MLL inhibited cell proliferation and cell death was also induced by loss of either MLL or HOXD13. Colony formation in soft agar was likewise inhibited by MLL and HOXD13 knockdown and tumor formation in vivo was significantly delayed in ES cells with reduced expression of HOXD13. Importantly, knockdown of MLL led to reduced expression of HOXD13 further implicating MLL as a key mediator of HOXD13 over-expression in ES and supporting the hypothesis that, like MLL-fusion-dependent leukemias, HOX deregulation and tumorigenicity in ES are dependent on the TrxG complex. To further test this hypothesis we exposed ES cells to MI-503, an inhibitor of menin-MLL protein-protein interaction that disrupts the TrxG complex. Exposure of ES cells to MI-503 resulted in a dose-dependent inhibition of cell proliferation and marked inhibition of colony formation in soft agar. In contrast, MI-NC, a control compound with similar structure that lacks affinity for the menin-MLL interaction had no effect on ES cell growth or viability. Together these data for the first time demonstrate a key role for menin and MLL in ES pathogenesis and highlight a novel opportunity for therapeutic intervention in these aggressive tumors. Citation Format: Laurie K. Svoboda, Cassondra Cramer, Ashley Harris, Natashay Bailey, Tomek Cierpicki, Jolanta Grembecka, Elizabeth Lawlor. The Trithorax proteins menin and MLL promote Ewing sarcoma tumorigenicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 490. doi:10.1158/1538-7445.AM2015-490

Abstract A75: A new, microRNA-regulated, epigenetic tumor-promoting pathway in Ewing sarcoma

Abstract Ewing Sarcoma is an aggressive pediatric bone and soft tissue malignancy driven by transcription factor oncogenic fusions, of which EWS/Fli1 is the most common. Our studies of the role of microRNAs in EWS/Fli1-driven oncogenesis have uncovered a new tumor-promoting pathway in Ewing Sarcoma. Specifically, we have found that EWS/Fli1 downregulates miR-22, which in turn results in increased expression of the H3K9 histone demethylase, and miR-22 target, KDM3A. Overexpression of miR-22 and stable depletion of KDM3A each results in impaired clonogenic and anchorage-independent growth of Ewing Sarcoma cells. KDM3A depletion further results in impaired tumorigenesis in a xenograft model, upregulation of the repressive H3K9 methyl histone mark, and diminished expression of multiple oncogenes in Ewing Sarcoma. Our studies thus identify a new, potentially targetable, tumor-promoting pathway in Ewing Sarcoma, involving modulation of H3K9 histone methylation downstream of EWS/Fli1. Citation Format: Janet K. Parrish, Marybeth Sechler, Paul Jedlicka. A new, microRNA-regulated, epigenetic tumor-promoting pathway in Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A75.

Abstract 1413: RUNX3 plays an oncogenic role in Ewing sarcoma cells

Abstract Ewing sarcomas are aggressive, poorly differentiated pediatric tumors of bone and soft tissue. Ewing sarcoma affects one to three people per million every year and is the second most common pediatric bone malignancy after osteosarcoma. Approximately 85% of Ewing sarcomas harbor the EWS-FLI fusion protein, which arises from a chromosomal translocation, t(11:22)(q24:q12). EWS-FLI interacts with numerous lineage-essential transcription factors to maintain mesenchymal progenitors in an undifferentiated state. We previously showed that EWS-FLI binds the osteogenic transcription factor RUNX2, and prevents osteoblast differentiation. RUNX3 is highly homologous to RUNX2 and participates in the mesenchymal-derived bone formation. RUNX3 has been described as both a tumor suppressor and an oncogene in different tumor types. In this study, we investigated the role of RUNX3 in Ewing Sarcoma. RUNX3 was detected in all Ewing sarcoma cells examined (7/7 cell lines, and 4/4 primary tumors), whereas RUNX2 was detected in only 73% of these specimens. Immunoprecipitation experiments revealed that RUNX3 binds to EWS-FLI via its Runt domain, which is &amp;gt;91% identical to the Runt domain in RUNX2. The interaction between RUNX3 and EWS-FLI in cell nuclei was confirmed by immunofluorescence. Moreover, EWS-FLI interactions prevented RUNX3 from activating the transcription of a RUNX-responsive reporter, p6OSE2. To determine the role of RUNX3 in Ewing sarcoma cell growth, we stably suppressed RUNX3 expression in the Ewing sarcoma cell line A673 with shRNAs. In anchorage independent growth assays in soft agar, RUNX3-deficient A673 cells formed smaller colonies than cells expressing scrambled shRNAs. Consistent with the smaller colonies, RUNX3 suppressed cells had increased expression of the cyclin dependent kinase inhibitor p21. Taken together, these results suggest and oncogenic role for RUNX3 in Ewing sarcoma. Citation Format: Krista L. Bledsoe, Meghan E. McGee-Lawrence, Emily T. Camilleri, Andre M. Oliveira, Andre J. van Wijnen, Jennifer J. Westendorf. RUNX3 plays an oncogenic role in Ewing sarcoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1413. doi:10.1158/1538-7445.AM2014-1413

RUNX3 facilitates growth of Ewing sarcoma cells.

Ewing sarcoma is an aggressive pediatric small round cell tumor that predominantly occurs in bone. Approximately 85% of Ewing sarcomas harbor the EWS/FLI fusion protein, which arises from a chromosomal translocation, t(11:22)(q24:q12). EWS/FLI interacts with numerous lineage-essential transcription factors to maintain mesenchymal progenitors in an undifferentiated state. We previously showed that EWS/FLI binds the osteogenic transcription factor RUNX2 and prevents osteoblast differentiation. In this study, we investigated the role of another Runt-domain protein, RUNX3, in Ewing sarcoma. RUNX3 participates in mesenchymal-derived bone formation and is a context dependent tumor suppressor and oncogene. RUNX3 was detected in all Ewing sarcoma cells examined, whereas RUNX2 was detected in only 73% of specimens. Like RUNX2, RUNX3 binds to EWS/FLI via its Runt domain. EWS/FLI prevented RUNX3 from activating the transcription of a RUNX-responsive reporter, p6OSE2. Stable suppression of RUNX3 expression in the Ewing sarcoma cell line A673 delayed colony growth in anchorage independent soft agar assays and reversed expression of EWS/FLI-responsive genes. These results demonstrate an important role for RUNX3 in Ewing sarcoma.

Anchorage-independent growth of Ewing sarcoma cells under serum-free conditions is not associated with stem-cell like phenotype and function.

Novel treatment strategies for Ewing sarcoma aim to eliminate residual tumor cells that have maintained the capacity to reinitiate tumor growth after intensive conventional therapy. Preclinical models that more closely mimic in vivo tumor growth than standard monolayer cultures are needed. Sphere formation under anchorage-independent, serum-free conditions has been proposed to enrich for cells with tumor-initiating, stem cell-like properties in various solid cancers. In the present study, we assessed the phenotype and functional stem cell characteristics of Ewing sarcoma spheres. Spheres were generated under serum-free culture conditions from four Ewing sarcoma cell lines and four relapse tumor biopsies. Standard monolayer cultures were established as controls. Median levels of surface expression of the Ewing sarcoma marker CD99 as well as the supposed stem cell marker CD133 and the neural crest marker CD57 were comparable between spheres and monolayers. Ewing sarcoma spheres from individual tumors failed to continuously self-renew by secondary sphere formation. They contained variable proportions of side populations (SPs). Sphere culture did not enhance the in vivo tumorigenicity of Ewing sarcoma cells in a murine xenograft model. We conclude that sphere formation under serum-free conditions is not a reliable tool to enrich for cells with stem cell characteristics in Ewing sarcoma. By mimicking the anchorage-independent, multicellular growth of Ewing sarcoma micrometastases, in vitro sphere growth may still add value as a preclinical tool to evaluate the efficacy of novel therapeutics.

Dual targeting of EWS-FLI1 activity and the associated DNA damage response with trabectedin and SN38 synergistically inhibits Ewing sarcoma cell growth.

The goal of this study is to optimize the activity of trabectedin for Ewing sarcoma by developing a molecularly targeted combination therapy. We have recently shown that trabectedin interferes with the activity of EWS-FLI1 in Ewing sarcoma cells. In this report, we build on this work to develop a trabectedin-based combination therapy with improved EWS-FLI1 suppression that also targets the drug-associated DNA damage to Ewing sarcoma cells. We demonstrate by siRNA experiments that EWS-FLI1 drives the expression of the Werner syndrome protein (WRN) in Ewing sarcoma cells. Because WRN-deficient cells are known to be hypersensitive to camptothecins, we utilize trabectedin to block EWS-FLI1 activity, suppress WRN expression, and selectively sensitize Ewing sarcoma cells to the DNA-damaging effects of SN38. We show that trabectedin and SN38 are synergistic, demonstrate an increase in DNA double-strand breaks, an accumulation of cells in S-phase and a low picomolar IC50. In addition, SN38 cooperates with trabectedin to augment the suppression of EWS-FLI1 downstream targets, leading to an improved therapeutic index in vivo. These effects translate into the marked regression of two Ewing sarcoma xenografts at a fraction of the dose of camptothecin used in other xenograft studies. These results provide the basis and rationale for translating this drug combination to the clinic. In addition, the study highlights an approach that utilizes a targeted agent to interfere with an oncogenic transcription factor and then exploits the resulting changes in gene expression to develop a molecularly targeted combination therapy.

Clinical and biological significance of hepatoma-derived growth factor in Ewing's sarcoma

We sought to investigate the clinicopathological significance and biological function of hepatoma-derived growth factor (HDGF) in Ewing's sarcoma. Our results showed that HDGF expression is up-regulated in Ewing's sarcoma. Nuclear HDGF expression is significantly associated with tumour volume (p < 0.001), metastases at diagnosis (p < 0.001), low overall survival rate (p < 0.001) and low disease-free survival rate (p < 0.001). HDGF knock-down results in significant reduction of Ewing's sarcoma cell growth, proliferation and enhances tumourigenesis, both in vitro and in vivo. Meanwhile, HDGF knock-down causes cell cycle arrest and enhanced sensitization to serum starvation-induced apoptosis. Furthermore, recombinant HDGF promotes proliferation and colony formation of Ewing's sarcoma cells. Ninety-eight candidate HDGF downstream genes were identified in Ewing's sarcoma cells using cDNA microarray analysis. In addition, we found that HDGF knock-down inhibited FLI1 expression in Ewing's sarcoma cells at the mRNA and protein levels. Our findings suggest that HDGF exhibits oncogenic properties and may be a novel prognostic factor in Ewing's sarcoma. Targeting HDGF might be a potential therapeutic strategy for Ewing's sarcoma.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 2035: Anti-epileptic drug targets Ewing sarcoma.

Abstract Ewing sarcoma is a highly aggressive and metastatic bone or soft tissue tumor that affects children and adolescents. It is characterized by chromosomal translocations involving EWS gene and one of the ETS (Erythroblastosis virus E26 transformation-specific) families of transcription factors (ERG, FLI1, ETV1, ETV4, E1AF and FEV). All of the fusion genes juxtapose the EWS gene to an ETS related gene. Previously our laboratory has discovered several ETS oncogenes that have shown to play major roles in human cancers. ERG (ETS Related Gene), an important member of the ETS family genes, discovered by Drs. Reddy and Rao is shown to be involved in Ewing sarcoma. Our laboratory has previously demonstrated that ERG protein functions as a sequence specific transcriptional activator. Our preliminary results have shown that ERG and aberrant EWS-ERG proteins inhibit RXR transcriptional activation and apoptosis. These results suggest that transcriptional inhibition may play a major role in apoptosis function. Using a cell based assay system developed in our laboratory that measures the inhibitory activities of ERG proteins, we screened for known drugs that can reverse the inhibitory effects of RXR transcriptional activity. Since HDACs regulate nuclear receptors, we hypothesized that anti-epileptic drug, valproic acid and trichostatin-A (HDAC inhibitors) may reverse the inhibitory properties of EWS-ERG oncoprotein on RXR transcriptional activity and they can be used as therapeutic agents in Ewing sarcoma. Methods: Cell based assays were used to measure the RXR transcriptional activation function and in vivo assays were used to study the cell viability and apoptosis activity. Results: (1) Anti-epileptic drugs, valproic acid (VPA) and trichostatin-A (TSA) have shown to reverse the inhibitory effect of EWS-ERG on RXR transcriptional activity. (2) VPA and TSA inhibit the cell growth of Ewing sarcoma cells expressing EWS-ERG onco-protein. (3) VPA induce apoptosis in Ewing sarcoma cells. (4) VPA induce the expression of RXR target genes. Conclusion: Ewing sarcoma requires highly intensive chemotherapy along with surgery and/or radiation. Current therapies are associated with significant short and long term side effects. Hence new therapeutic approaches are needed. The above results suggest that the anti-epileptic drug VPA could be used as a potential treatment option for regulation of the function of EWS-ERG onco-protein. Thus, a promising drug with a new potential will have profound impact on prevention, management and treatment of Ewing family of cancers. Citation Format: Shubhalaxmi Kayarthodi, Yasuo Fujimura, Kunchala Rungsrisuriyachai, Jinbo Fang, Veena Rao, Shyam P. Reddy. Anti-epileptic drug targets Ewing sarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2035. doi:10.1158/1538-7445.AM2013-2035

Abstract 4176: MicroRNA-193b is growth-inhibitory in Ewing sarcoma, and represses ErbB4.

Abstract Ewing sarcoma, the second most common cancer of bone and soft tissue in adolescents and young adults, is an aggressive malignancy with poor long-term outcome. Ewing sarcoma is driven primarily by the EWS/Fli-1 fusion oncoprotein, which arises from the recurrent t(11,22) translocation. Our laboratory has been interested in identifying microRNAs (miRs) with growth-modulating properties in Ewing sarcoma, manipulation of which could potentially be used for novel therapies, as shown in preclinical studies for other cancers. We have previously shown that replacement of select miRs, normally repressed by EWS/Fli1, can inhibit the growth of Ewing sarcoma cells. To identify additional, and more potent, miRs with growth-suppressive properties in EWS, we have performed a phenotype-based, secondary miniscreen in Ewing sarcoma cell lines. This screen, using transfection of miR mimics into the EWS cell lines A673 and SK-ES-1, followed by growth assays as read-out, identified miR-193b as a strong candidate growth suppressive miR in Ewing sarcoma. Subsequent cloning and stable overexpression of endogenous miR-193b verified the growth suppressive phenotype. Examination of candidate targets further identified ErbB4 as potently repressed upon miR-193b overexpression. ErbB4 has recently been shown to play a key role in Ewing sarcoma, specifically as a driver of metastasis and as a mediator of resistance to cytotoxic chemotherapy, key biological parameters that define high-risk disease. With the need for therapy expansion in pediatric EWS, miR-193b replacement, through ErbB4 inhibition, may be a way to help control high-risk disease. Citation Format: Colin Moore, Paul Jedlicka. MicroRNA-193b is growth-inhibitory in Ewing sarcoma, and represses ErbB4. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4176. doi:10.1158/1538-7445.AM2013-4176

BIRC5 expression is a poor prognostic marker in Ewing sarcoma

BIRC5 (Survivin), an inhibitor of apoptosis protein (IAP), is over-expressed in several human cancers and increased expression is associated with poor prognosis. The goal of the current study was to evaluate the role of BIRC5 in Ewing sarcoma (ES), the second most common pediatric bone sarcoma. BIRC5 protein expression was determined in ES cell lines using Western Blot analysis. Functional role of survivin on growth and viability of ES cells was assessed by siRNA knockdown of BIRC5 and by using a small molecule inhibitor YM155. Immunohistochemical analysis for BIRC5 protein was performed on patient tumor samples using an anti-survivin antibody. The degree of BIRC5 protein expression was correlated with clinical parameters and patient outcome. BIRC5 is over-expressed in a panel of ES cell lines. Gene silencing of BIRC5 in the ES cell line TC-71 decreases cell growth by more than 50% for each BIRC5 siRNA construct compared to non-silencing siRNA control constructs. YM155 also reduces ES cell growth and viability with an EC(50) ranging from 2.8 to 6.2 nM. BIRC5 protein is expressed in majority of the ES tumor samples with minimal expression in normal tissue (P < 0.005). Tumors with more than 50% expression are associated with worse overall survival than tumors with less than 50% expression (Hazard Ratio: 6.05; CI: 1.7-21.4; P = 0.04). BIRC5 is over-expressed in ES cell lines and tumor samples. Further, it plays an important role in cell growth and viability in vitro. Higher degree of expression in patients is an independent poor prognostic factor.