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A, hierarchical clustering of 19 ESFT cell lines and 10 tumors identifies TC-248 as a BMI-1-low cell line. B, Western blot confirms low expression of BMI-1 protein in TC-248 compared with 2 BMI-1-high cell lines. C, RT-PCR confirms expression of a type 1 EWS-FLI1 fusion in TC-248 cells. D, treatment with an IGF1-R-specific small molecule inhibitor PPP results in growth inhibition of BMI-1-high cells after 72 hours. In contrast, TC-248 cells are significantly less sensitive to PPP (P < 0.0001 compared with each of 4 BMI-1-high cell lines at 500-nmol/L dose). E, growth of TC-248 cells is not significantly inhibited by exposure to the anti-IGF1-R-targeted antibody IMC-A12.
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Ewing sarcoma family tumors (ESFT) are aggressive tumors of putative stem cell origin for which prognostic biomarkers and novel treatments are needed. In several human cancers, high expression of the polycomb protein BMI-1 is associated with poor outcome. We have assessed the potential clinical significance of BMI-1 expression level in ESFT.
BMI-1...
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... showed that BMI-1 is overexpressed in ESFT cell lines in vitro (19). To identify candidate BMI-1-low cell lines, we conducted hierarchical clustering of whole gen- ome expression profiles obtained from the 10 aforemen- tioned primary tumors and 19 ESFT cell lines. Interestingly, only the TC-248 cell line clustered with the BMI-1-low tumors (Fig. 4A). Although no other BMI-1-low cell lines were identified, among BMI-1-high cell lines, transcript expression was found to vary from very high (e.g., TC-32, TC-71, A673) to more moderate (e.g., CHLA-9, TC-466, TTC-487) levels. Reduced expression of BMI-1 in TC-248 cells was confirmed by Western blot (Fig. 4B) and expres- sion of a type 1 ...
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... clustered with the BMI-1-low tumors (Fig. 4A). Although no other BMI-1-low cell lines were identified, among BMI-1-high cell lines, transcript expression was found to vary from very high (e.g., TC-32, TC-71, A673) to more moderate (e.g., CHLA-9, TC-466, TTC-487) levels. Reduced expression of BMI-1 in TC-248 cells was confirmed by Western blot (Fig. 4B) and expres- sion of a type 1 EWS-FLI1 fusion confirmed by RT-PCR (Fig. 4C). Consistent with our hypothesis, TC-248 was significantly less sensitive than BMI-1-high cell lines to the growth inhibitory effect of picropodophyllin (PPP), a selective small molecule inhibitor of IGF1-R (ref. 33; Fig. 4D). Similarly, TC-248 was relatively ...
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... cell lines were identified, among BMI-1-high cell lines, transcript expression was found to vary from very high (e.g., TC-32, TC-71, A673) to more moderate (e.g., CHLA-9, TC-466, TTC-487) levels. Reduced expression of BMI-1 in TC-248 cells was confirmed by Western blot (Fig. 4B) and expres- sion of a type 1 EWS-FLI1 fusion confirmed by RT-PCR (Fig. 4C). Consistent with our hypothesis, TC-248 was significantly less sensitive than BMI-1-high cell lines to the growth inhibitory effect of picropodophyllin (PPP), a selective small molecule inhibitor of IGF1-R (ref. 33; Fig. 4D). Similarly, TC-248 was relatively insensitive to the IGF1-R inhibitory antibody IMC-A12 (Fig. 4E). These in ...
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... in TC-248 cells was confirmed by Western blot (Fig. 4B) and expres- sion of a type 1 EWS-FLI1 fusion confirmed by RT-PCR (Fig. 4C). Consistent with our hypothesis, TC-248 was significantly less sensitive than BMI-1-high cell lines to the growth inhibitory effect of picropodophyllin (PPP), a selective small molecule inhibitor of IGF1-R (ref. 33; Fig. 4D). Similarly, TC-248 was relatively insensitive to the IGF1-R inhibitory antibody IMC-A12 (Fig. 4E). These in vitro studies of ESFT cell lines support our studies of primary human tumors in vivo and suggest that patients whose tumors express low levels of BMI-1 may be less sensitive to IGF1-R-targeted therapy. Prospective testing of ...
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... confirmed by RT-PCR (Fig. 4C). Consistent with our hypothesis, TC-248 was significantly less sensitive than BMI-1-high cell lines to the growth inhibitory effect of picropodophyllin (PPP), a selective small molecule inhibitor of IGF1-R (ref. 33; Fig. 4D). Similarly, TC-248 was relatively insensitive to the IGF1-R inhibitory antibody IMC-A12 (Fig. 4E). These in vitro studies of ESFT cell lines support our studies of primary human tumors in vivo and suggest that patients whose tumors express low levels of BMI-1 may be less sensitive to IGF1-R-targeted therapy. Prospective testing of patient samples is now required to clinically test this novel ...
Citations
... The role of the polycomb protein BMI-1 in Ewing's sarcoma prognosis was studied by Cooper et al. (2011) the author has compared by genetic analysis two samples of bone tumors with an increased or decreased expression of this marker. They concluded that Ewing sarcoma family tumors (ESFT) which do not overexpress BMI-1 represent a novel subclass with a distinct molecular profile and altered activation of, and dependence on, cancer-associated biological pathways (Cooper et al., 2011). ...
... The role of the polycomb protein BMI-1 in Ewing's sarcoma prognosis was studied by Cooper et al. (2011) the author has compared by genetic analysis two samples of bone tumors with an increased or decreased expression of this marker. They concluded that Ewing sarcoma family tumors (ESFT) which do not overexpress BMI-1 represent a novel subclass with a distinct molecular profile and altered activation of, and dependence on, cancer-associated biological pathways (Cooper et al., 2011). ...
Osteosarcoma is the most frequent malignant bone neoplasm, followed by chondrosarcoma and Ewing sarcoma. The diagnosis of bone neoplasms is generally made through histological evaluation of a biopsy. Clinical and radiological features are also important in aiding diagnosis and to complete the staging of bone cancer. In addition to these, there are several non-specific serological or specific molecular markers for bone neoplasms. In bone tumors, molecular markers increase the accuracy of the diagnosis and assist in subtyping bone tumors. Here, we review these markers and discuss their role in the diagnosis and prognosis of the three most frequent malignant bone neoplasms, namely osteosarcoma, chondrosarcoma, and Ewing sarcoma.
... Further studies found that BMI1 is a stem cell gene that determines the proliferative capacity and self-renewal of normal and leukemic stem cells 2 . BMI1 is frequently overexpressed in patients with hematologic [3][4][5] and solid cancers [6][7][8] . Silencing of BMI1 impairs cancer cell proliferation and tumour growth in cancer models [9][10][11][12][13][14][15] , suggesting that BMI1 might represent a valid target for therapeutic intervention 16,17 . ...
BMI1 is a core component of the polycomb repressive complex 1 (PRC1) and emerging data support a role of BMI1 in cancer. The central domain of BMI1 is involved in protein–protein interactions and is essential for its oncogenic activity. Here, we present the structure of BMI1 bound to the polyhomeotic protein PHC2 illustrating that the central domain of BMI1 adopts an ubiquitin-like (UBL) fold and binds PHC2 in a β-hairpin conformation. Unexpectedly, we find that the UBL domain is involved in homo-oligomerization of BMI1. We demonstrate that both the interaction of BMI1 with polyhomeotic proteins and homo-oligomerization via UBL domain are necessary for H2A ubiquitination activity of PRC1 and for clonogenic potential of U2OS cells. Here, we also emphasize need for joint application of NMR spectroscopy and X-ray crystallography to determine the overall structure of the BMI1–PHC2 complex.
... Next we analysed the expression of BMI1 and MEL18, two PRC1 proteins. Some specimens displayed low BMI1 and MEL18 levels (Supplementary Figure S2Aand S2B), accordingly to reported BMI1 expression in many, but not all, ES primary tumors [16]. In embryonic neural tissues BMI1 expression pattern resembled that of RING1B, with low to moderate expression in most of the embryonic neural tissues (Supplementary Figure S2C). ...
Ewing sarcoma (ES) is an aggressive tumor defined by EWSR1 gene fusions that behave as an oncogene. Here we demonstrate that RING1B is highly expressed in primary ES tumors, and its expression is independent of the fusion oncogene. RING1B-depleted ES cells display an expression profile enriched in genes functionally involved in hematological development but RING1B depletion does not induce cellular differentiation. In ES cells, RING1B directly binds the SCN8A sodium channel promoter and its depletion results in enhanced Nav1.6 expression and function. The signaling pathway most significantly modulated by RING1B is NF-κB. RING1B depletion results in enhanced p105/p50 expression, which sensitizes ES cells to apoptosis by FGFR/SHP2/STAT3 blockade. Reduced NaV1.6 function protects ES cells from apoptotic cell death by maintaining low NF-κB levels. Our findings identify RING1B as a trait of the cell-of-origin and provide a potential targetable vulnerability.
... The unique specificity of transcriptional targets in Ewing sarcoma, but not other cells may be influenced by it developmental origins as discussed in Chapter 3, but also be affected by the profound dysregulation of epigenetic modifiers in Ewing sarcoma. For instance, Polycomb group (PcG) proteins BMI1 [172][173][174] and EZH2 [21] are overexpressed in Ewing sarcoma. PcG proteins regulated histone modifications that serve to promote activation or repression of numerous genes. ...
Ewing sarcoma is an aggressive bone and soft tissue tumor with a high propensity for metastasis; however, the mechanisms that contribute to this process are poorly understood. The Wnt/beta-catenin signaling pathway is critical for oncogenesis in numerous cancers, and although previous studies implicate a role for this pathway in Ewing sarcoma, its specific function and contribution is unknown. Previous work by our lab revealed that the Wnt-modulatory receptor LGR5 is highly expressed in patients with aggressive disease, and we hypothesized that LGR5 regulates activation of Wnt/beta-catenin signaling. Through investigation of primary tumors, we discovered that focal nuclear beta-catenin is detectable in a subset of Ewing sarcoma patients and strongly associated with LGR5 expression. Patients whose tumors have nuclear beta-catenin or high expression of the downstream Wnt/beta-catenin target LEF1, experienced worse clinical outcomes and overall survival. We next used in vitro and in vivo models to determine the function of Wnt/beta-catenin signaling in Ewing sarcoma. Importantly, we found that LGR5 expression and Wnt activation were highly heterogeneous. We then investigated the downstream effects of Wnt/beta-catenin activation in the most highly Wnt-responsive cells. RNA-sequencing revealed that Wnt/beta-catenin paradoxically inhibits EWS-ETS transcriptional activity, resulting in a phenotypic change from a proliferative to a migratory and metastatic state in vitro and in vivo. In addition, the metastasis-associated molecule Tenascin C was upregulated by Wnt/beta-catenin signaling, and was found to be a mediator of migration in vitro and metastasis in vivo. In the context of the tumor microenvironment, we further found that patient tumors with high Wnt/LEF1 expression had significant correlation with expression of stroma- and angiogenesis-related genes associated with a poor prognosis. Together, these data provide novel avenues of exploration for tumor-microenvironment interactions. In conclusion these findings implicate a critical role for Wnt/beta-catenin-singaling in mediating migration and metastasis. This occurs in part through antagonism of EWS/ETS fusion protein activity and by up-regulation of the metastasis-associated gene Tenascin C. In addition, tumor-microenvironment interactions modulated by Wnt/beta-catenin further contribute to pathogenesis. Together these findings provide exciting new venues for therapeutic investigation.
... Although extremely frequent, overexpression of BMI1 was not a ubiquitous event in UC. It is known that BMI1-negative tumors are a distinct subpopulation in Ewing's sarcoma, with a distinct gene expression profile [39]. Only eight UCs in our original tumor panel and only five in the second TMA panel were BMI1 negative. ...
Urothelial carcinoma (UC) causes significant morbidity and remains the most expensive cancer to treat because of the need for repeated resections and lifelong monitoring for patients with non-muscle-invasive bladder cancer (NMIBC). Novel therapeutics and stratification approaches are needed to improve the outlook for both NMIBC and muscle-invasive bladder cancer. We investigated the expression and effects of B Lymphoma Mo-MLV Insertion Region 1 (BMI1) in UC. BMI1 was found to be overexpressed in most UC cell lines and primary tumors by quantitative real-time polymerase chain reaction and immunohistochemistry. In contrast to some previous reports, no association with tumor stage or grade was observed in two independent tumor panels. Furthermore, upregulation of BMI1 was detected in premalignant bladder lesions, suggesting a role early in tumorigenesis. BMI1 is not located within a common region of genomic amplification in UC. The CDKN2A locus (which encodes the p16 tumor suppressor gene) is a transcriptional target of BMI1 in some cellular contexts. In UC cell lines and primary tissues, no correlation between BMI1 and p16 expression was observed. Retroviral-mediated overexpression of BMI1 immortalized normal human urothelial cells (NHUC) in vitro and was associated with induction of telomerase activity, bypass of senescence, and repression of differentiation. The effects of BMI1 on gene expression were identified by expression microarray analysis of NHUC-BMI1. Metacore analysis of the gene expression profile implicated downstream effects of BMI1 on alpha4/beta1 integrin-mediated adhesion, cytoskeleton remodeling, and CREB1-mediated transcription.
... Supervised analyses of the data using strategies designed to test specific pre-determined hypotheses may uncover differences that would not be evident with unsupervised clustering methods. As examples, supervised comparison of gene expression profiles between BMI-1 over-expressing and BMI-1 negative ES revealed differences in pathway activation not evident from unsupervised analyses agnostic to BMI-1 status [46]. Likewise, subtle differences in gene expression are detectable between tumours with different EWS-ETS fusions, but these differences are only apparent when supervised analysis of the data is performed [23]. ...
Relapse of Ewing sarcoma (ES) can occur months or years after initial remission, and salvage therapy for relapsed disease is usually ineffective. Thus, there is great need to develop biomarkers that can predict which patients are at risk for relapse so that therapy and post-therapy evaluation can be adjusted accordingly. For this study, we performed whole genome expression profiling on two independent cohorts of clinically annotated ES tumours in an effort to identify and validate prognostic gene signatures. ES specimens were obtained from the Children's Oncology Group and whole genome expression profiling performed using Affymetrix Human Exon 1.0 ST arrays. Lists of differentially expressed genes between survivors and non-survivors were used to identify prognostic gene signatures. An independent cohort of tumours from the Euro-Ewing cooperative group was similarly analysed as a validation cohort. Unsupervised clustering of gene expression data failed to segregate tumours based on outcome. Supervised analysis of survivors versus non-survivors revealed a small number of differentially expressed genes and several statistically significant gene signatures. Gene-specific enrichment analysis demonstrated that integrin and chemokine genes were associated with survival in tumours where stromal contamination was present. Tumours that did not harbour stromal contamination showed no association of any genes or pathways with clinical outcome. Our results reflect the challenges of performing RNA-based assays on archived bone tumour specimens. In addition, they reveal a key role for tumour stroma in determining ES prognosis. Future biological and clinical investigations should focus on elucidating the contribution of tumour:micro-environment interactions on ES progression and response to therapy.
... The prognosis of patients suffering from ESFTs is still poor. Prognostic biomarkers and novel treatment strategies are needed to cure disseminated Ewing's sarcoma [18,78]. New therapy strategies are the different forms of immuno- therapy, where ESFT-specific antigens have to be identified as targets for cytotoxic T-lymphocytes [74]. ...
Ewing`s sarcoma (ES) family of tumors (ESFTs) are round cell tumors of bone and soft tissues, afflicting children and young adults. This review summarizes the present findings about ES cancer stem cell (CSC) targeted therapy: prognostic factors, chromosomal translocations, initiation, epigenetic mechanisms, candidate cell of ES origin (Mesenchymal stem cells (MSCs) and Neural crest stem cells (NCSCs)). The ES CSC model, histopathogenesis, histogenesis, pathogenesis, ES mediated Hematopoietic stem progenitor cells (HSPCs) senescence are also discussed. Reviewed is ESFTs therapy concerning CSCs, radiotherapy, risk of subsequent neoplasms, stem cell (SC) support, promising therapeutic targets for ES CSCs (CSC markers, immune targeting, RNAi phenotyping screens, proposed new drugs), candidate EWS-FLI1 target genes and further directions (including human embryonic stem cells (hESCs)). Bone marrow-derived human MSCs are permissive for EWS-FLI1 expression with transition to ESFT-like cellular phenotype. ESFTs are genetically related to NCSC, permissive for EWS-FLI1 expression and susceptible to oncogene-induced immortalization. Primitive neuroectodermal features and MSC origin of ESFTs provide a basis of immune targeting. The microRNAs profile of ES CSCs is shared by ESCs and CSCs from divergent tumor types. Successful reprogramming of differentiated human somatic cells into a pluripotent state allow creation of patient- and disease-specific SCs. The functional role of endogenous EWS at stem cell level on both senescence and tumorigenesis is link between cancer and aging. The regulatory mechanisms of oncogenic activity of EWS fusions could provide new prognostic biomarkers, therapeutic opportunities and tumor-specific anticancer agents against ESFTs.
... A robust biology effort has been established in ES with 626 patients enrolled on the AEWS02B1 study and 275 patients enrolled on the AEWS07B1 study. Selected successes of this effort include an evaluation of the significance of translocation type on patient outcome [23], a molecular profiling study identifying ES that do not over-express BMI-1 as a distinct molecular subclass [102] as well as the development of tissue resources such as microarrays containing tumor samples that are linked to therapeutic studies and can be used for evaluation of prognostic factors. ...
In the US, approximately 650 children are diagnosed with osteosarcoma and Ewing sarcoma (ES) each year. Five-year survival ranges from 65% to 75% for localized disease and <30% for patients with metastases. Recent findings include interval-compressed five drug chemotherapy improves survival with localized ES. In osteosarcoma a large international trial investigating the addition of ifosfamide/etoposide or interferon to standard therapy has completed accrual. For ES an ongoing trial explores the addition of cyclophosphamide/topotecan to interval-compressed chemotherapy. Trials planned by the Children's Oncology Group will investigate new target(s) including IGF-1R and mTOR in ES, and RANKL and GD2 in osteosarcoma. Pediatr Blood Cancer © 2012 Wiley Periodicals, Inc.
... ERG has also been reported to be overexpressed in Ewing's sarcomas [42][43][44][45]. We thus analyzed the available gene expression profiling studies of Ewing's sarcoma tumors for TDRD1 and ERG expression [46,47]. In contrast to prostate cancer, there was no co-expression of ERG and TDRD1 in any of these studies (r 2 = 0.03 and 0.02, respectively). ...
Background
Overexpression of ERG transcription factor due to genomic ERG-rearrangements defines a separate molecular subtype of prostate tumors. One of the consequences of ERG accumulation is modulation of the cell’s gene expression profile. Tudor domain-containing protein 1 gene (TDRD1) was reported to be differentially expressed between TMPRSS2:ERG-negative and TMPRSS2:ERG-positive prostate cancer. The aim of our study was to provide a mechanistic explanation for the transcriptional activation of TDRD1 in ERG rearrangement-positive prostate tumors.
Methodology/Principal Findings
Gene expression measurements by real-time quantitative PCR revealed a remarkable co-expression of TDRD1 and ERG (r2 = 0.77) but not ETV1 (r2<0.01) in human prostate cancer in vivo. DNA methylation analysis by MeDIP-Seq and bisulfite sequencing showed that TDRD1 expression is inversely correlated with DNA methylation at the TDRD1 promoter in vitro and in vivo (ρ = −0.57). Accordingly, demethylation of the TDRD1 promoter in TMPRSS2:ERG-negative prostate cancer cells by DNA methyltransferase inhibitors resulted in TDRD1 induction. By manipulation of ERG dosage through gene silencing and forced expression we show that ERG governs loss of DNA methylation at the TDRD1 promoter-associated CpG island, leading to TDRD1 overexpression.
Conclusions/Significance
We demonstrate that ERG is capable of disrupting a tissue-specific DNA methylation pattern at the TDRD1 promoter. As a result, TDRD1 becomes transcriptionally activated in TMPRSS2:ERG-positive prostate cancer. Given the prevalence of ERG fusions, TDRD1 overexpression is a common alteration in human prostate cancer which may be exploited for diagnostic or therapeutic procedures.
... ESFTs have high propensity to metastasize in bone, bone marrow, and lung. ESFTs are aggressive round cell tumors of putative stem cell origin, for which prognostic biomarkers and novel treatments are needed [2,3]. ESFTs are chemotherapy-sensitive cancers, and even patients with metastatic disease commonly achieve remission. ...
Ewing’s sarcoma (EWS) protein is a member of the TET (TLS/EWS/TAF15) family of RNA and DNA-binding proteins with unknown cellular role. EWS protein is encoded by the EWS oncogene on chromosome 22q12, a target of chromosomal translocations in Ewing’s sarcoma tumors. The exact mechanism of EWS participation in gene expression and pathogenesis of the resulting cancers is not defined. The binding partners of native EWS and EWS fusion proteins (EFPs) are described schematically in a model, an attempt to link the transcription with the splicing. The experimental data about the partnerships of EWS and EFPs are summarized, which may lead to better understanding of their function.
