Max Kauer

EWS-FLI1 Suppresses NOTCH-Activated p53 in Ewing's Sarcoma

Although p53 is the most frequently mutated gene in cancer, half of human tumors retain wild-type p53, whereby it is unknown whether normal p53 function is compromised by other cancer-associated alterations. One example is Ewings sarcoma family tumors (ESFT), where 90% express wild-type p53. ESFT are characterized by EWS-FLI1 oncogene fusions. Studying 6 ESFT cell lines, silencing of EWS-FLI1 in a wild-type p53 context resulted in increased p53 and p21(WAF1/CIP1) levels, causing cell cycle arrest. Using a candidate gene approach, HEY1 was linked to p53 induction. HEY1 was rarely expressed in 59 primary tumors, but consistently induced upon EWS-FLI1 knockdown in ESFT cell lines. The NOTCH signaling pathway targets HEY1, and we show NOTCH2 and NOTCH3 to be expressed in ESFT primary tumors and cell lines. Upon EWS-FLI1 silencing, NOTCH3 processing accompanied by nuclear translocation of the activated intracellular domain was observed in all but one p53-mutant cell line. In cell lines with the highest HEY1 induction, NOTCH3 activation was the consequence of JAG1 transcriptional induction. JAG1 modulation by specific siRNA, NOTCH-processing inhibition by either GSI or ectopic NUMB1, and siRNA-mediated HEY1 knockdown all inhibited p53 and p21(WAF1/CIP1) induction. Conversely, forced expression of JAG1, activated NOTCH3, or HEY1 induced p53 and p21(WAF1/CIP1). These results indicate that suppression of EWS-FLI1 reactivates NOTCH signaling in ESFT cells, resulting in p53-dependent cell cycle arrest. Our data link EWS-FLI1 to the NOTCH and p53 pathways and provide a plausible basis both for NOTCH tumor suppressor effects and oncogenesis of cancers that retain wild-type p53.

Hsa-mir-145 is the top EWS-FLI1-repressed microRNA involved in a positive feedback loop in Ewing's sarcoma

EWS-FLI1 is a chromosome translocation-derived chimeric transcription factor that has a central and rate-limiting role in the pathogenesis of Ewings sarcoma. Although the EWS-FLI1 transcriptomic signature has been extensively characterized on the mRNA level, information on its impact on non-coding RNA expression is lacking. We have performed a genome-wide analysis of microRNAs affected by RNAi-mediated silencing of EWS-FLI1 in Ewings sarcoma cell lines, and differentially expressed between primary Ewings sarcoma and mesenchymal progenitor cells. Here, we report on the identification of hsa-mir-145 as the top EWS-FLI1-repressed microRNA. Upon knockdown of EWS-FLI1, hsa-mir-145 expression dramatically increases in all Ewings sarcoma cell lines tested. Vice versa, ectopic expression of the microRNA in Ewings sarcoma cell lines strongly reduced EWS-FLI1 protein, whereas transfection of an anti-mir to hsa-mir-145 increased the EWS-FLI1 levels. Reporter gene assays revealed that this modulation of EWS-FLI1 protein was mediated by the microRNA targeting the FLI1 3′-untranslated region. Mutual regulations of EWS-FLI1 and hsa-mir-145 were mirrored by an inverse correlation between their expression levels in four of the Ewings sarcoma cell lines tested. Consistent with the role of EWS-FLI1 in Ewings sarcoma growth regulation, forced hsa-mir-145 expression halted Ewings sarcoma cell line growth. These results identify feedback regulation between EWS-FLI1 and hsa-mir-145 as an important component of the EWS-FLI1-mediated Ewings sarcomagenesis that may open a new avenue to future microRNA-mediated therapy of this devastating malignant disease.

Notch is active in Langerhans cell histiocytosis and confers pathognomonic features on dendritic cells

Langerhans cell histiocytosis (LCH) is an enigmatic disease defined by the accumulation of Langerhans cell-like dendritic cells (DCs). In the present study, we demonstrate that LCH cells exhibit a unique transcription profile that separates them not only from plasmacytoid and myeloid DCs, but also from epidermal Langerhans cells, indicating a distinct DC entity. Molecular analysis revealed that isolated and tissue-bound LCH cells selectively express the Notch ligand Jagged 2 (JAG2) and are the only DCs that express both Notch ligand and its receptor. We further show that JAG2 signaling induces key LCH-cell markers in monocyte-derived DCs, suggesting a functional role of Notch signaling in LCH ontogenesis. JAG2 also induced matrix-metalloproteinases 1 and 12, which are highly expressed in LCH and may account for tissue destruction in LCH lesions. This induction was selective for DCs and was not recapitulated in monocytes. The results of the present study suggest that JAG2-mediated Notch activation confers phenotypic and functional aspects of LCH to DCs; therefore, interference with Notch signaling may be an attractive strategy to combat this disease.

Suppression of deacetylase SIRT1 mediates tumor suppressive NOTCH response and offers a novel treatment option in metastatic Ewing sarcoma

The developmental receptor NOTCH plays an important role in various human cancers as a consequence of oncogenic mutations. Here we describe a novel mechanism of NOTCH-induced tumor suppression involving modulation of the deacetylase SIRT1, providing a rationale for the use of SIRT1 inhibitors to treat cancers where this mechanism is inactivated because of SIRT1 overexpression. In Ewing sarcoma cells, NOTCH signaling is abrogated by the driver oncogene EWS-FLI1. Restoration of NOTCH signaling caused growth arrest due to activation of the NOTCH effector HEY1, directly suppressing SIRT1 and thereby activating p53. This mechanism of tumor suppression was validated in Ewing sarcoma cells, B-cell tumors, and human keratinocytes where NOTCH dysregulation has been implicated pathogenically. Notably, the SIRT1/2 inhibitor Tenovin-6 killed Ewing sarcoma cells in vitro and prohibited tumor growth and spread in an established xenograft model in zebrafish. Using immunohistochemistry to analyze primary tissue specimens, we found that high SIRT1 expression was associated with Ewing sarcoma metastasis and poor prognosis. Our findings suggest a mechanistic rationale for the use of SIRT1 inhibitors being developed to treat metastatic disease in patients with Ewing sarcoma.

Abstract 1285: A functional liaison between E2F and aberrant ETS oncogenes

Timing, mode and amplitude of gene regulation depend on the tissue specific dynamic composition of transcription factor complexes. Abnormalities of the constituents of the complex are expected to modify the regulatory behavior of the entire module. Here we report a novel E2F-ETS proliferation control module in Ewing9s sarcoma. FLI1 and other ETS factors are frequently found to be fused to EWS in this highly aggressive pediatric cancer of the bone. In a genome-wide localization analysis of EWS/FLI1 as well as E2F3 by ChipSeq, two patterns associated with distinct gene sets and modes of regulation were identified and integrated with gene expression data. EWS/FLI1 significantly colocalized with E2F3 to proximal promoters of activated proliferation genes while EWS/FLI1 binding independent of E2F3 occurred predominantly distant to repressed differentiation genes. We find that this key module of the transcription factor network underlying Ewing9s sarcoma is of general importance and not limited to this specific ETS driven neoplasm. Recently, prostate cancer was shown to frequently harbor fusions of the ERG gene. Here we demonstrate that the ETS/E2F3 complex is also present in VCap prostate cancer cells, identify a common set of genes responsive to this module and computationally show that this set of core-responders is involved in proliferation control and translation. Acknowledgement: This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. 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 1285. doi:1538-7445.AM2012-1285

Abstract 2961: Reactivation of EWS-FLI1 suppressed FOXO1 expression as a novel therapeutic strategy for Ewing's sarcoma

The EWS-FLI1 chimeric protein, characterizing Ewing9s sarcoma (ESFT), is prototypic for an aberrant oncogenic ETS transcription factor. The mechanisms of transcriptional regulation leading to ETS-driven tumorigenesis are poorly understood. In-silico analysis of time-resolved expression data revealed enrichment of recognition motifs for forkhead box (FOX) proteins in EWS-FLI1 repressed promoters. Several FOX genes were found to be bound by EWS-FLI1 in ChIP-seq and transcriptionally affected by EWS-FLI1 knockdown. We therefore hypothesized that EWS-FLI1 exerts an important part of its repressive activity via inhibiting FOX proteins. Upon silencing of EWS-FLI1, both FOXO1 and FOXO3 proteins were strongly induced in ESFT cells consistent with this hypothesis, but only FOXO1 translocated to the nucleus. However, in the presence of EWS-FLI1, FOXO1 is excluded from the nucleus as a consequence of phosphorylation. Nuclear translocation was restored by either inhibition of CDK2, augmented by chemical inhibition of PI3K, or by mutation of CDK2 or AKT phosphorylation sites. Furthermore, ChIP experiments revealed that EWS-FLI1 directly binds the promoter of FOXO1 in vitro suggesting a multi-layered regulation of FOXO1 expression by transcriptional repression and post-translational modification. Functional restoration of nuclear FOXO1 expression in ESFT cells resulted in impaired proliferation and significantly reduced soft agar colony formation ability. A significant overlap between EWS-FLI1 repressed and FOXO1 activated genes was observed. Treatment of ESFT cell lines with Methyl-Seleninic-Acid (MSA) led to re-activation of endogenous FOXO1 in the presence of EWS-FLI1 in a dose- and time-dependent manner, and induced massive cell death, which we found to be at least partially FOXO1-dependent. Taken together, these data confirm our hypothesis that a repressive sub-signature of EWS-FLI1 regulated genes is due to suppression of FOXO1. FOXO1 re-activation by small molecules may constitute a novel therapeutic strategy in the treatment of ESFT. This study was supported by grant 22328-B09 from the Austrian Science Fund FWF. 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 2961. doi:1538-7445.AM2012-2961

Abstract 2478: Jagged 2 signalling is active in Langerhans cell histiocytosis and confers pathognomonic features on dendritic cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Langerhans cell histiocytosis (LCH) is an enigmatic disease defined by the accumulation of eponymous Langerhans-cell-like cells of unknown origin. It is a rare disease that may affect any age group, although its most severe clinical course affects predominantly young children. The clinical manifestations range from single lesions that can resolve spontaneously to systemic disease that requires intensive chemotherapy and bone marrow transplantation. Here we demonstrate that LCH cells exhibit a unique transcription profile that clearly separates them from indigenous dendritic cells (DCs), i.e. Langerhans cells, myeloid dendritic cells, and plasmacytoid dendritic cells, indicating a distinct DC entity that occurs under pathologic conditions. Molecular analysis revealed that LCH cells selectively expressed NOTCH ligand Jagged 2 (JAG2) and were the only DCs that coexpressed both NOTCH ligand and receptor. In line, activated NOTCH was detected in LCH but not control biopsies. Furthermore, we show that JAG2 signalling induces key LCH markers CD1a and Langerin on MoDCs, suggesting a role of NOTCH signalling in LCH ontogenesis. Interestingly, JAG2 also strongly induced matrix-metalloproteinase (MMP) 1 in MoDCs, which is highly expressed in LCH lesions and mediates massive tissue destruction and remodelling. The MMP induction by JAG2 was selective for DCs and not recapitulated in monocytes. Together these findings strongly suggest that Jagged2 mediated NOTCH activation confers phenotypic and functional aspects of LCH to DCs. Thus, interference with NOTCH signalling may prove an attractive target to combat 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 2478. doi:1538-7445.AM2012-2478

Abstract 1190: SIRT1 links tumor suppressive NOTCH signaling to p53

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Besides its important role in organismal development, NOTCH receptor signaling exerts tissue specific proliferative or antiproliferative functions. While the oncogenic role of NOTCH has been extensively investigated due to its constitutive activation in T-cell leukemias and several epithelial cancers, little is known about NOTCH mediated tumor suppression. We have previously reported that in Ewings sarcoma, a pediatric bone tumor driven by the chimeric ETS oncogene EWS-FLI1, auto-stimulatory NOTCH signaling is suppressed and that reactivation results in p53 and consequently growth inhibitory p21 induction via activation of the NOTCH effector HEY1. We now demonstrate that HEY1-mediated p53 stimulation is accompanied by C-terminal p53 acetylation as a consequence of downregulation of nuclear deacetylase sirtuin 1 (SIRT1). We found that both EWS-FLI1 and HEY1 bind to the SIRT1 promoter with opposite transcriptional consequences. Thus, knockdown of EWS-FLI1 and ectopic HEY1 expression resulted in similar SIRT1 modulation and p53 acetylation which could be reversed by ectopically expressed SIRT1. Consistent with these results, treatment of Ewings sarcoma cell lines with the sirtuin inhibitor Tenovin 6 resulted in massive cell death. Immunohistochemical analysis of more than 310 Ewings sarcoma samples identified moderate (25%-50% positive nuclei) to strong (>50% positive nuclei) SIRT1 expression in 35% of cases. Additional 20% showed sporadic positivity (between 10-25% positive nuclei). The involvement of SIRT1 in tumor suppressive NOTCH signaling is not restricted to Ewings sarcoma, but also relevant at least to B-cell malignancies and some normal tissues, since we found that in several B-cell leukemia and lymphoma cell lines and in keratinocytes HEY1 was able to lead to activating p53 acetylation in a SIRT1 suppression-dependent manner. Supported by grants from the Austrian Science Fund (P22328-B09) and the European Comission (EU-FP7 STREP 259348). 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 1190. doi:1538-7445.AM2012-1190

Abstract 2198: A functional ETS/E2F module in cancers expressing ETS fusion genes

Ewing9s sarcoma (ESFT) is a highly aggressive pediatric cancer characterized by the chimeric ETS transcription factor EWS-FLI1 as a result of a chromosomal translocation. By whole genome gene expression and ChIP-seq analyses we obtained evidence that EWS-FLI1 directly binds to 50% of E2F target genes in ESFT. Furthermore, several E2F factors were themselves found to be directly regulated by EWS-FLI1. Therefore, we studied the functional interaction of EWS-FLI1 with E2F. Ten arbitrarily chosen EWS-FLI1/E2F candidate targets including E2F3, RAD51, and ATAD2 were chosen for an in-depth promoter activity study by luciferase reporter gene assays in combination with mutation analysis of transcription factor binding motifs. By ChIP-PCR we confirmed direct EWS-FLI1 promoter binding, and observed decreased reporter activity for all ten studied promoters upon knockdown of EWS-FLI1 by RNAi. Furthermore, the study of promoter occupancy by different E2F family members revealed that silencing of EWS-FLI1 results in the exchange of EWS-FLI1 induced, activating E2F3 by constitutively expressed repressive E2F4 on the promoters of there jointly regulated target genes. Importantly, testing E2F3 promoter occupancy on wildtype and ETS motif mutated promoter constructs in ESFT cell lines revealed that binding of E2F3 to its target promoters is dependent on an intact ETS binding site. Mutation of the ETS motif resulted in reduced binding of E2F3. These data are consistent with a model in which EWS-FLI1 actively recruits an activating E2F factor thereby replacing a repressing E2F factor and in which EWS-FLI1 binding is essential for E2F binding. Strikingly, the functional E2F/ETS transcriptional module detected in ESFT for the chimeric ETS factor EWS-FLI1 was found to be also relevant to TMPRSS2-ERG expressing prostate cancer cells, but not active in HeLa cells. Supported by grants from the Austrian Science Fund (P22328-B09), and the European Comission (EU-FP7 STREP 259348). R. Schwentner is a recipient of a DOC-fFORTE-fellowship of the Austrian Academy of Sciences. 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 2198. doi:1538-7445.AM2012-2198

Abstract 1949: Positive feedback regulation between EWS-FLI1 and miR-145 in Ewing's sarcoma

The EWS-FLI1 chimeric protein is a potent tumor specific transcriptional regulator and the molecular cause of Ewing9s sarcoma family of tumors (ESFT). We had previously hypothesized that, since EWS-FLI1 is the major driver of aberrant gene expression in ESFT, there should be an inverse correlation between the changes in gene expression observed in ESFT cell lines in response to RNAi-mediated EWS-FLI1 knockdown and the corresponding gene expression in primary tumors relative to the tissue of origin. We found that mesenchymal progenitor cells (MPC) fit this assumption best and established an ESFT specific EWS-FLI1 transcriptional signature on the mRNA level. Following the same approach, we now studied the EWS-FLI1 dependent regulation of microRNAs (miRNAs) in ESFT. Genome-wide miRNA analysis was performed after shRNA-mediated stable (5 cell lines) and inducible (1 cell line) EWS-FLI1 knockdown, as well as in 6 primary ESFT and 6 MPC samples using the stem-loop reverse transcription quantitative PCR platform. Among miRNAs consistently affected by EWS-FLI1 silencing and inversely expressed in primary tumors compared to MPCs was hsa-miR-145 which we found upregulated upon EWS-FLI1 silencing. Since several target sites exist for this miRNA within the FLI1 3′UTR, we asked if hsa-miR-145 is capable of modulating EWS-FLI1 expression in ESFT cells. Upon ectopic expression of pri-miR-145 in wildtype p53 ESFT cells, strong modulation of EWS-FLI1 protein levels, but not RNA levels, was observed. In p53 mutant cell lines this effect was less pronounced consistent with a recently reported role of p53 in miR-145 maturation. Preliminary studies suggest that hsa-miR-145 is indirectly regulated by EWS-FLI1 by a mechanism that may involve the NOTCH signaling pathway. Our results identify a positive feed-back loop between EWS-FLI1 and hsa-miR-145 expression in ESFT. Supported by the 6th framework program of the European Commission, (STREP “E.E.T. Pipeline“ contract LSHC-CT-2006-037260) and grant P20665-B12 of the Austrian Science Fund FWF. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1949.