Jozef Ban

Caveolin-1 (CAV1) is a target of EWS/FLI-1 and a key determinant of the oncogenic phenotype and tumorigenicity of Ewing's sarcoma cells.

Tumors of the Ewings sarcoma family (ESFT), such as Ewings sarcoma (EWS) and primitive neuroectodermal tumors (PNET), are highly aggressive malignancies predominantly affecting children and young adults. ESFT express chimeric transcription factors encoded by hybrid genes fusing the EWS gene with several ETS genes, most commonly FLI-1. EWS/FLI-1 proteins are responsible for the malignant phenotype of ESFT, but only few of their transcriptional targets are known. Using antisense and short hairpin RNA-mediated gene expression knockdown, array analyses, chromatin immunoprecipitation methods, and reexpression studies, we show that caveolin-1 (CAV1) is a new direct target of EWS/FLI-1 that is overexpressed in ESFT cell lines and tumor specimens and is necessary for ESFT tumorigenesis. CAV1 knockdown led to up-regulation of Snail and the concomitant loss of E-cadherin expression. Consistently, loss of CAV1 expression inhibited the anchorage-independent growth of EWS cells and markedly reduced the growth of EWS cell-derived tumors in nude mice xenografts, indicating that CAV1 promotes the malignant phenotype in EWS carcinogenesis. Reexpression of CAV1 or E-cadherin in CAV1 knockdown EWS cells rescued the oncogenic phenotype of the original EWS cells, showing that the CAV1/Snail/E-cadherin pathway plays a central role in the expression of the oncogenic transformation functions of EWS/FLI-1. Overall, these data identify CAV1 as a key determinant of the tumorigenicity of ESFT and imply that targeting CAV1 may allow the development of new molecular therapeutic strategies for ESFT patients.

A Molecular Function Map of Ewing's Sarcoma

Background EWS-FLI1 is a chimeric ETS transcription factor that is, due to a chromosomal rearrangement, specifically expressed in Ewings sarcoma family tumors (ESFT) and is thought to initiate the development of the disease. Previous genomic profiling experiments have identified EWS-FLI1–regulated genes and genes that discriminate ESFT from other sarcomas, but so far a comprehensive analysis of EWS-FLI1–dependent molecular functions characterizing this aggressive cancer is lacking. Methodology/Principal Findings In this study, a molecular function map of ESFT was constructed based on an integrative analysis of gene expression profiling experiments following EWS-FLI1 knockdown in a panel of five ESFT cell lines, and on gene expression data from the same platform of 59 primary ESFT. Out of 80 normal tissues tested, mesenchymal progenitor cells (MPC) were found to fit the hypothesis that EWS-FLI1 is the driving transcriptional force in ESFT best and were therefore used as the reference tissue for the construction of the molecular function map. The interrelations of molecular pathways were visualized by measuring the similarity among annotated gene functions by gene sharing. The molecular function map highlighted distinct clusters of activities for EWS-FLI1 regulated genes in ESFT and revealed a striking difference between EWS-FLI1 up- and down-regulated genes: EWS-FLI1 induced genes mainly belong to cell cycle regulation, proliferation, and response to DNA damage, while repressed genes were associated with differentiation and cell communication. Conclusions/Significance This study revealed that EWS-FLI1 combines by distinct molecular mechanisms two important functions of cellular transformation in one protein, growth promotion and differentiation blockage. By taking MPC as a reference tissue, a significant EWS-FLI1 signature was discovered in ESFT that only partially overlapped with previously published EWS-FLI1–dependent gene expression patterns, identifying a series of novel targets for the chimeric protein in ESFT. Our results may guide target selection for future ESFT specific therapies.

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.

EWS-FLI1 target genes recovered from Ewing's sarcoma chromatin.

In all, 85% of Ewings sarcoma family tumors (ESFT), a neoplasm of unknown histogenesis, express EWS-FLI1 transcription factor gene fusions. To characterize direct target genes avoiding artificial model systems, we cloned genomic DNA from ESFT chromatin precipitating with EWS-FLI1. We now present a comprehensive list of 99 putative transcription factor targets identified, for the first time, by a hypothesis-free approach based on physical interaction. Gene-derived chromatin fragments co-precipitating with EWS-FLI1 were nonrandomly distributed over the human genome and localized predominantly to the upstream region and the first two introns of the genes. At least 20% of putative direct EWS-FLI1 targets were neural genes. One-third of genes recovered showed a significant ESFT-specific expression pattern and were found to be altered upon RNAi-mediated knockdown of EWS-FLI1. Among them, MK-STYX, encoding a MAP kinase phosphatase-like protein, was consistently expressed in ESFT. EWS-FLI1 was found to drive MK-STYX expression by binding to a single ETS binding motif within the first gene intron. MK-STYX serves as precedence for successful recovery of direct EWS-FLI1 targets from the authentic ESFT cellular context, the most relevant system to study oncogenic mechanisms for the discovery of new therapeutic targets in this disease.

UVA and UVB radiation differentially regulate vascular endothelial growth factor expression in keratinocyte-derived cell lines and in human keratinocytes.

Vascular endothelial growth factor (VEGF) is a central regulator of neoangiogenesis in inflammatory and neoplastic conditions. Ultraviolet irradiation is one of the mainstays of dermatological therapy for various inflammatory skin diseases. In the present study we have compared the effects of UV irradiation on the production of VEGF by keratinocytes (KC) and by the KC‐derived cell lines A431 and HaCaT. Irradiation of A431 and HaCaT cells with both UVA (10 J/cm2and 20 J/cm2) and UVB (8 mj/cm2and 16 mj/cm2) led to strong upregulation of VEGF mRNA and protein. Induction of VEGF by UVA and UVB in these cells was mediated by different pathways, i.e. the generation of free radicals and the secretion of (a) soluble factor(s), respectively. Unlike KC‐derived cell lines, no increase in VEGF production was observed in KC in primary culture after irradiation with the same UV doses. Increasing the irradiation dose in these cells of UVA to 40 J/cm2led to a marked decrease in soluble VEGF, whereas doses as high as 32 mj/cm2UVB only minimally affected VEGF levels. Reduction of VEGF production by KC might contribute to the effect of UVA irradiation in inflammatory skin diseases. The differential response of primary KC and autonomously growing KC‐derived cell lines to the induction of VEGF by UV light could favor neoangiogenesis in the vicinity of epidermal tumor cells in vivo, thereby endowing them with a growth advantage over normal cells.

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.

Intercohort Gene Expression Co-analysis Reveals Chemokine Receptors as Prognostic Indicators in Ewing's Sarcoma

Purpose: We report a novel analytic method, named intercohort co-analysis or Ican, which aids in the discovery of genes with predictive value for the progression or outcome of diseases from small-size cohorts. We tested this premise in Ewings sarcoma (ES), a highly metastatic cancer of bone and soft tissues that lacks validated molecular metastasis and prognostic indicators. Experimental Design: To uncover genes significantly expressed in ES patient subsets, we first determined a nonarbitrary gene expression significance cutoff based on expression levels in validated expressing and nonexpressing tissues. We next searched for genes that were consistently significantly expressed in several ES cohort and cell line datasets. Significantly expressed genes were independently validated by quantitative reverse transcription-PCR in an additional ES cohort. Results: Analysis of ES cohorts revealed marked intercohort gene expression variability. After filtering out the intercohort variability, CXCR4 and CXCR7 were found to be consistently associated with specific ES subsets. Pairwise analyses showed CXCR4 to correlate with ES metastases, and CXCR4 and CXCR7 to patient survival, but not with several other clinicopathological variables. Conclusion: Ican is a powerful novel method to identifying genes consistently associated with particular disease states in cancers for which large cohorts are not available, currently the case of most cancers. We report for the first time that high CXCR4 expression preferentially associates with metastatic ES, and that of CXCR7 with poor patient survival. Clin Cancer Res; 16(14); 3769–78. ©2010 AACR.

Hypoxia Modulates EWS-FLI1 Transcriptional Signature and Enhances the Malignant Properties of Ewing's Sarcoma Cells In vitro

Hypoxia is an important condition in the tumor cell microenvironment and approximately 1% to 1.5% of the genome is transcriptionally responsive to hypoxia with hypoxia-inducible factor-1 (HIF-1) as a major mediator of transcriptional activation. Tumor hypoxia is associated with a more aggressive phenotype of many cancers in adults, but data on pediatric tumors are scarce. Because, by immunohistochemistry, HIF-1alpha expression was readily detectable in 18 of 28 primary Ewings sarcoma family tumors (ESFT), a group of highly malignant bone-associated tumors in children and young adults, we studied the effect of hypoxia on ESFT cell lines in vitro. Intriguingly, we found that EWS-FLI1 protein expression, which characterizes ESFT, is upregulated by hypoxia in a HIF-1alpha-dependent manner. Hypoxia modulated the EWS-FLI1 transcriptional signature relative to normoxic conditions. Both synergistic as well as antagonistic transcriptional effects of EWS-FLI1 and of hypoxia were observed. Consistent with alterations in the expression of metastasis-related genes, hypoxia stimulated the invasiveness and soft agar colony formation of ESFT cells in vitro. Our data represent the first transcriptome analysis of hypoxic ESFT cells and identify hypoxia as an important microenvironmental factor modulating EWS-FLI1 expression and target gene activity with far-reaching consequences for the malignant properties of ESFT.

Characterization of a cDNA clone, encoding a 70 kDa heat shock protein from the dermatophyte pathogen Trichophyton rubrum.

Trichophyton rubrum is an anthropophilic fungus causing up to 90% of chronic cases of dermatophytosis. To characterize T. rubrum proteins at the molecular level, we established a cDNA library of this pathogen. Here we describe a recombinant cDNA clone identical to eukaryotic 70kDa heat-shock proteins (HSPs). Western blot analysis using an anti HSP70 monoclonal antibody detected a recombinant fusion protein in Escherichia coli transformed with the expression vector containing the cloned cDNA insert. Southern blot analysis of T. rubrum genomic DNA detected no other members of the HSP70 gene family. Further analysis revealed the presence of two introns within the ORF of the HSP70 gene. In Northern blot analysis, the cDNA clone was hybridized to a RNA species of about 3.5kb which was constitutively expressed by cells cultured at 27 degrees C and was strongly up-regulated after culture at 37 degrees C. In summary, we have cloned the first member of the HSP family of dermatophytes and characterized it as a member of the Dnak subfamily of 70kDa HSPs.

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 Ewings 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. Copyright