Dana Ohana (Presenter)
Leiden University Medical Centre
Bio: In 2007 I started studying at the VU-University in Amsterdam where I did a bachelor's degree in Pharmaceutical sciences followed by a master's degree Drug Discovery and Safety. In January 2013 I began my PhD research project in the Center of Proteomics and Metabolomics at the Leiden University Medical Center. The focus of my PhD-project is the development, validation and application of novel high-throughput proteomics methods for both clinical and research applications. At the moment I am working with a transgenic zebrafish model system for Ewing sarcoma to discover more about the origin of the disease and to be able to treat patients with Ewing sarcoma more effectively.
Authorship: D. Ohana(1), W. van der Ent(2), H.P. Spaink(2), B.E. Snaar-Jagalska(2), Magnus Palmblad(1)
(1)Center for proteomics and metabolomics, Leiden University Medical Center, Leiden, The Netherlands (2) Institute of biology, Leiden University, Leiden, The Netherlands
| Short Abstract Ewing sarcoma, a pediatric bone sarcoma, is characterized by a reciprocal translocation event between EWSR1 and a gene of the ETS family. A binary transgenic zebrafish model for Ewing’s sarcoma has been recently developed, which expresses EWSR1-ERG neuronally and GFP for monitoring. A bottom-up proteomics approach was performed on embryos expressing EWSR1-ERG and on wild type embryos. Spectral counting was used to calculate the changes between the mutated and wild type fish. A variety of the up and downregulated proteins were involved in pathways, such as oncogenesis, transcription and translation and cellular respiration, which were repeatedly associated with Ewing’s sarcoma. |
Long Abstract
Ewing sarcoma is a pediatric sarcoma affecting bone. A quarter of the patients have metastases at the time of diagnosis, and these patients have a 2-year-event-free survival rate of 20% (1). This poor prognosis shows the necessity of more research for new effective treatments. The disease is characterized by a reciprocal translocation event between EWSR1 and a member of the ETS family of genes. The EWSR1-FLI1 fusion gene is found in 85% of the patients and in another 10% the EWSR1-ERG fusion gene was detected (2). The resulting chimeric protein causes transcriptional deregulation, and the effects of these mutations on transcriptomic level have been investigated extensively. A lot less is known about the effects on the proteome after introduction or knockdown of EWSR1-ETS. Despite the fact that the key feature of the disease is known, no viable murine model has been developed yet (3). A binary transgenic zebrafish model for Ewing sarcoma has been recently established, next to the in vitro and xenotransplantation models (4). The model has expression of human EWSR1-ERG, driven by the conditional silent UAS promotor, which requires a Gal4 element for activation (5). Crossing of zebrafish expressing Gal4 under a neuronal promoter with UAS-GFP-EWS-ERG zebrafish develops offspring with localized neuronal EWSR1-ERG expression and can be monitored by GFP expression. Developmental malformations can be observed from 1 day post fertilization (dpf) and onwards.
A standardized bottom-up proteomics approach was performed on duplicates of embryos of 3dpf expressing EWSR1-ERG and on duplicates of wild type embryos, to investigate the proteome after introduction of the human EWSR1-ERG oncogene. Spectral counting, a label free method quantification method, was used for quantification of the most abundant proteins. Label-free spectral counting is an increasingly popular method for quantitative proteomics, being far less expensive than using zebrafish labeled with stable isotopes.
Evaluation of the obtained protein expression data was done by correlation of the duplicates. For the wild type control fish the R2=0.994 and for the EWSR1-ERG fish R2=0.962. Subsequently the fold change between the mutated and wild type fish were calculated, and various proteins were consistently upregulated and downregulated. An annotation tool was used for analysis of the various proteins and the pathways these proteins are involved (6). The up and downregulated proteins, for example upregulated WNT5a and downregulated NOTCH2, were involved in a variety of pathways and processes, such oncogenesis, transcription and translation and cellular respiration, which were repeatedly associated with Ewing sarcoma. On protein level some down and upregulation of proteins, like the downregulation of the NOTCH2, could be confirmed by literature (7) .
References & Acknowledgements:
References
(1).Meyers, P. A.; Krailo, M. D.; Ladanyi, M.; Chan, K. W.; Sailer, S. L.; Dickman, P. S.; Baker, D. L.; Davis, J. H.; Gerbing, R. B.; Grovas, A.; Herzog, C. E.; Lindsley, K. L.; Liu-Mares, W.; Nachman, J. B.; Sieger, L.; Wadman, J.; Gorlick, R. G., High-dose melphalan, etoposide, total-body irradiation, and autologous stem-cell reconstitution as consolidation therapy for high-risk Ewing's sarcoma does not improve prognosis. J Clin Oncol 2001, 19 (11), 2812-20.
(2).Turc-Carel, C.; Aurias, A.; Mugneret, F.; Lizard, S.; Sidaner, I.; Volk, C.; Thiery, J. P.; Olschwang, S.; Philip, I.; Berger, M. P.; et al., Chromosomes in Ewing's sarcoma. I. An evaluation of 85 cases of remarkable consistency of t(11;22)(q24;q12). Cancer Genet Cytogenet 1988, 32 (2), 229-38.
(3).Lin, P. P.; Pandey, M. K.; Jin, F.; Xiong, S.; Deavers, M.; Parant, J. M.; Lozano, G., EWS-FLI1 induces developmental abnormalities and accelerates sarcoma formation in a transgenic mouse model. Cancer Res 2008, 68 (21), 8968-75.
(4).Ent, W. v. d. In vivo modelling of Ewing sarcoma in zebrafish (thesis). Leiden University, 2015.
(5).Brand, A. H.; Perrimon, N., Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 1993, 118 (2), 401-15.
(6).(a) Huang, D. W.; Sherman, B. T.; Tan, Q.; Collins, J. R.; Alvord, W. G.; Roayaei, J.; Stephens, R.; Baseler, M. W.; Lane, H. C.; Lempicki, R. A., The DAVID Gene Functional Classification Tool: a novel biological module-centric algorithm to functionally analyze large gene lists. Genome Biol 2007, 8 (9), R183; (b) Huang, D. W.; Sherman, B. T.; Tan, Q.; Kir, J.; Liu, D.; Bryant, D.; Guo, Y.; Stephens, R.; Baseler, M. W.; Lane, H. C.; Lempicki, R. A., DAVID Bioinformatics Resources: expanded annotation database and novel algorithms to better extract biology from large gene lists. Nucleic Acids Res 2007, 35 (Web Server issue), W169-75.
(7).Ban, J.; Aryee, D. N.; Fourtouna, A.; van der Ent, W.; Kauer, M.; Niedan, S.; Machado, I.; Rodriguez-Galindo, C.; Tirado, O. M.; Schwentner, R.; Picci, P.; Flanagan, A. M.; Berg, V.; Strauss, S. J.; Scotlandi, K.; Lawlor, E. R.; Snaar-Jagalska, E.; Llombart-Bosch, A.; Kovar, H., Suppression of deacetylase SIRT1 mediates tumor-suppressive NOTCH response and offers a novel treatment option in metastatic Ewing sarcoma. Cancer Res 2014, 74 (22), 6578-88.
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