Karl-Ludwig Schaefer

Alternative lengthening of telomeres is associated with chromosomal instability in osteosarcomas

Telomere maintenance is regarded as a key mechanism in overcoming cellular senescence in tumor cells and in most cases is achieved by the activation of telomerase. However there is at least one alternative mechanism of telomere lengthening (ALT) which is characterized by heterogeneous and elongated telomeres in the absence of telomerase activity (TA). We evaluated the prevalence of TA, gene expression of telomerase subunits and ALT in relation to telomere morphology and function in matrix producing bone tumors and in osteosarcoma cell lines and present evidence of a direct association of ALT with telomere dysfunction and chromosomal instability. Telomere fluorescence in situ hybridization (T-FISH) in ALT cells revealed elongated and shortened telomeres, partly in unusual configurations and loci, dicentric marker chromosomes and signal-free chromosome ends. Free ends give rise to end-to-end associations and may induce breakage-fusion-bridge cycles resulting in an increased number of complex chromosomal rearrangements, as detected by multiplex-FISH (M-FISH). We propose that ALT cannot be seen as an equivalent to telomerase activity in telomere maintenance. Its association with telomere dysfunction and chromosomal instability may have major implications for tumor progression.

Impact of EWS-ETS Fusion Type on Disease Progression in Ewing's Sarcoma/Peripheral Primitive Neuroectodermal Tumor: Prospective Results From the Cooperative Euro-E.W.I.N.G. 99 Trial

PURPOSE EWS-ETS fusion genes are the driving force in Ewings sarcoma pathogenesis. Because of the variable breakpoint locations in the involved genes, there is heterogeneity in fusion RNA and protein architecture. Since previous retrospective studies suggested prognostic differences among patients expressing different EWS-FLI1 fusion types, the impact of fusion RNA architecture on disease progression and relapse was studied prospectively within the Euro-E.W.I.N.G. 99 clinical trial. PATIENTS AND METHODS Among 1,957 patients who registered before January 1, 2007, 703 primary tumors were accessible for the molecular biology study. Fusion type was assessed by polymerase chain reaction on frozen (n = 578) or paraffin-embedded materials (n = 125). The primary end point was the time to disease progression or relapse. Results After exclusion of noninformative patients, 565 patients were entered into the prognostic factor analysis comparing type 1 (n = 296), type 2 (n = 133), nontype 1/nontype 2 EWS-FLI1 (n = 91) and EWS-ERG fusions (n = 45). Median follow-up time was 4.5 years. The distribution of sex, age, tumor volume, tumor site, disease extension, or histologic response did not differ between the four fusion type groups. We did not observe any significant prognostic value of the fusion type on the risk of progression or relapse. The only slight difference was that the risk of progression or relapse associated with nontype 1/nontype 2 EWS-FLI1 fusions was 1.38 (95% CI, 0.96 to 2.0) times higher than risk associated with other fusion types, but it was not significant (P = .10). CONCLUSION In contrast to retrospective studies, the prospective evaluation did not confirm a prognostic benefit for type 1 EWS-FLI1 fusions.

Telomerase as a prognostic marker in breast cancer: high‐throughput tissue microarray analysis of hTERT and hTR

Telomerase activity (TA) has been shown to correlate with poor clinical outcome in various tumour entities, indicating that tumours expressing this enzyme may be more aggressive and that TA may be a useful prognostic marker. For breast cancer, however, TA is a controversial prognostic marker; whereas some studies suggest an association between TA and disease outcome, others do not find this association. This study used tissue microarrays (breast carcinoma prognosis arrays) containing 611 samples (each 0.6 mm in diameter) from the tumour centre of paraffin‐embedded breast carcinomas to analyse the catalytic subunit of telomerase, human telomerase reverse‐transcriptase (hTERT), and the internal RNA component (hTR), which are the core components of the telomerase holoenzyme complex. hTERT protein expression was obtained by immunohistochemistry (human anti‐telomerase antibody Ab‐2, Calbiochem), and hTR RNA was measured by radioactive in situ hybridization. hTERT and hTR expression were determined semi‐quantitatively and graded (scores 1–4). Clinical data, such as histological subtype, pT stage, tumour diameter, pN stage, BRE grade, tumour‐specific survival (in months), patients age and others, were available for statistical analysis. A statistically significant correlation was found between tumour‐specific survival (overall survival) and hTERT expression (p < 0.0001) or hTR expression (p = 0.00110). Tumours with higher scores (scores 3, 4) for hTR and/or hTERT were associated with a worse prognosis. In multivariate analysis, hTERT expression was an independent prognostic factor. Previous studies, focusing on analysis of TA in smaller numbers of fresh‐frozen breast carcinomas by the TRAP assay, gave controversial results with respect to TA as a prognostic marker. Using tissue microarrays from 611 breast carcinomas, this study has demonstrated that increased expression levels of the telomerase core components, hTERT and hTR, are associated with lower overall survival. These findings suggest that TA should be included in future validation studies as a prognostic marker in breast cancer. Copyright

Stable interference of EWS–FLI1 in an Ewing sarcoma cell line impairs IGF-1/IGF-1R signalling and reveals TOPK as a new target

BACKGROUND: Ewing sarcoma is a paradigm of solid tumour -bearing chromosomal translocations resulting in fusion proteins that act as deregulated transcription factors. Ewing sarcoma translocations fuse the EWS gene with an ETS transcription factor, mainly FLI1. Most of the EWS–FLI1 target genes still remain unknown and many have been identified in heterologous model systems.METHODS: We have developed a stable RNA interference model knocking down EWS–FLI1 in the Ewing sarcoma cell line TC71. Gene expression analyses were performed to study the effect of RNA interference on the genetic signature of EWS–FLI1 and to identify genes that could contribute to tumourigenesis.RESULTS: EWS–FLI1 inhibition induced apoptosis, reduced cell migratory and tumourigenic capacities, and caused reduction in tumour growth. IGF-1 was downregulated and the IGF-1/IGF-1R signalling pathway was impaired. PBK/TOPK (T-LAK cell-originated protein kinase) expression was decreased because of EWS–FLI1 inhibition. We showed that TOPK is a new target gene of EWS–FLI1. TOPK inhibition prompted a decrease in the proliferation rate and a dramatic change in the cells ability to grow in coalescence.CONCLUSION: This is the first report of TOPK activity in Ewing sarcoma and suggests a significant role of this MAPKK-like protein kinase in the Ewing sarcoma biology.

Efficacy of and resistance to anti-IGF-1R therapies in Ewing's sarcoma is dependent on insulin receptor signaling

Identification of patient selection criteria and understanding of the potential mechanisms involved in the development of resistance are crucial for an appropriate and successful design of clinical trials with anti-insulin-like growth factor (IGF)-1R therapies. Few Ewings sarcomas are highly sensitive to IGF-1R targeting and understanding the reason why, may hold the secret to improve successful treatments. In this paper, we show that a major mechanism of resistance to highly specific inhibitors of IGF-1R, either antibodies or tyrosine kinase inhibitors may involve enhanced insulin receptor (IR)-A homodimer formation and IGF-2 production. Resistant cells are able to switch from IGF-1/IGF-1R to IGF-2/IR-A dependency to maintain sustained activation of AKT and ERK1/2, proliferation, migration and metastasis. These cells also showed higher proliferative response to insulin, in keeping with a switch towards insulin pathways sustaining proliferation and malignancy, rather than metabolism. Our findings demonstrate a role for IR-A in eliciting intrinsic and adaptive resistance to anti-IGF-1R therapies. Thus, we indicate that tumors with low IGF-1R:IR ratio are unlikely to greatly benefit from anti-IGF-1R therapies and that the efficacy of anti-IGF-1R therapies should be evaluated in relationship to the IR-A:IGF-1R ratio in cancer cells. Moreover, we provide evidences supporting IR-A as an important target in sarcoma therapy.

Profiling and functional annotation of mRNA gene expression in pediatric rhabdomyosarcoma and Ewing's sarcoma.

Using Affymetrix oligonucleotide microarrays, we analyzed mRNA gene expression patterns of 12 primary pediatric rhabdomyosarcomas (RMS) and 11 Ewings sarcomas (EWS), which belong to the small round blue cell tumors (SRBCTs). Diagnostic classification of these cancers is frequently complicated by the highly similar appearance in routine histology, and additional molecular markers could significantly improve tumor classification. A combination of three independent statistical approaches (t‐test, SAM, k‐nearest neighborhood analysis) resulted in 101 highly significant probe sets that clearly discriminate between EWS and RMS. We identified novel marker transcripts that have not been previously associated with either RMS or EWS yet, including CITED2, glypican 3 (GPC3), and cyclin D1 (CCND1). Expression levels for selected candidate genes were validated by quantitative real‐time reverse‐transcription PCR. Furthermore, to identify biologically meaningful trends, functional annotations were assigned to 946 genes differentially expressed between EWS and RMS (t‐test). Genes involved in protein biosynthesis (n = 28) and complex assembly (n = 9), lipid metabolism (n = 23), energy generation (n = 22), and mRNA processing (n = 11) were expressed significantly higher in EWS. Thus, functional annotation of tumor‐specific genes reveals detailed insights into tumor biology and differentiation‐specific expression patterns and gives important clues related to the possible cellular origin of these pediatric tumors. Supplementary material for this article is available at the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020‐7136/suppmat/index.html.

Microarray analysis of Ewing's sarcoma family of tumours reveals characteristic gene expression signatures associated with metastasis and resistance to chemotherapy

In Ewings sarcoma family of tumours (ESFT), the clinically most adverse prognostic parameters are the presence of tumour metastasis at time of diagnosis and poor response to neoadjuvant chemotherapy. To identify genes differentially regulated between metastatic and localised tumours, we analysed 27 ESFT specimens using Affymetrix microarrays. Functional annotation of differentially regulated genes revealed 29 over-represented pathways including PDGF, TP53, NOTCH, and WNT1-signalling. Regression of primary tumours (n=20) induced by polychemotherapy was found to be correlated with the expression of genes involved in angiogenesis, apoptosis, ubiquitin proteasome pathway, and PI3 kinase and p53 pathways. These findings could be confirmed by in vitro cytotoxicity assays. A set of 46 marker genes correctly classifies these 20 tumours as responding versus non-responding. We conclude that expression signatures of initial tumour biopsies can help to identify ESFT patients at high risk to develop tumour metastasis or to suffer from a therapy refractory cancer.

Genetic imbalances revealed by comparative genomic hybridization in osteosarcomas.

Osteosarcomas are the most frequent bone sarcomas. The molecular chromosomal aberrations in osteosarcomas were analyzed by comparative genomic hybridization (CGH). We studied 47 frozen tumors (41 primary samples, 6 relapses) in osteosarcoma patients registered in the Cooperative Osteosarcoma Study (COSS) protocol. Genomic imbalances were detected in 40 of 41 primary tumors and 6 of 6 relapsed tumors. Gains were more frequent than losses (ratio of 1.3:1). The median number of changes was 16 and 12 in primary and relapsed osteosarcomas, respectively. The median number of aberrations in primary high‐grade osteosarcomas (17.0) was significantly higher than in low‐ or intermediate‐grade osteosarcoma subtypes (3.0) (p = 0.038). The most frequent gains included 8q, 1p21‐p31 and 1q21‐q24, and the most frequent losses were 10q, 5q and 13q. High‐level gains were observed on 8q23‐q24, 17p13 and 1q21‐q24. A gain of 19p (p < 0.001) or loss of 9p (p = 0.027) was more frequent in poor responders than in good responders. Univariate analysis revealed that patients with primary metastases (p = 0.002), poor histologic responses (p = 0.005), high‐level gains of 19p (p = 0.012) or losses of 13q14 (p = 0.042) had significantly lower event‐free survival (EFS), whereas patients with a loss of 5q (p = 0.007) or a loss of 10q21‐22 (p = 0.017) had significantly higher EFS than patients without these aberrations. Multivariate analysis demonstrated that primary metastasis, loss of 13q14 and loss of 5q were independent prognostic factors. The findings of our study seem to be useful for evaluating the prognosis of patients and may finally lead to treatment strategies based on genetic background of osteosarcoma.

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.