O sarcoma de Ewing (SE) é um tumor caracterizado por possuir células indiferenciadas, de origem neuroepitelial. É o segundo tumor ósseo maligno mais comum em crianças e adolescentes. O tratamento consiste na aplicação de quimioterapia neoadjuvante, seguida da cirurgia e a quimioterapia adjuvante ou radioterapia. Contudo, a disseminação deste tumor é muito intensa, e mesmo em pacientes onde a metástase não ocorreu, podem haver células tumorais circulantes. Além disso, a sobrevida de 5 anos que cerca de 70% dos pacientes que não apresentam metástase no momento do diagnóstico atingem, cai para 25% nos pacientes que apresentam metástase. A migração e invasão celular dos tecidos vizinhos e vasos sanguíneos, a adesão celular e a proliferação são processos essenciais para que a metástase tumoral aconteça. Dentre as reguladoras da mobilidade, adesão celular e proliferação estão as GTPases da família Rho, que possuem entre suas quinases efetoras, ROCK1 e ROCK2. Assim, as proteínas ROCK induzem inúmeras respostas celulares que envolvem a regulação de muitas proteínas associadas ao citoesqueleto. Estudos evidenciam que respostas sinalizadas por ROCK agravam fenótipos associados ao câncer e outras doenças. O presente trabalho teve como objetivo verificar a expressão dos genes ROCK1 e ROCK2 em amostras tumorais e linhagens celulares de SE, e os efeitos in vitro da inibição farmacológica de ambas as quinases. O estudo da expressão gênica de ROCK1 e ROCK2 em amostras de pacientes com SE (n=18) revelaram uma hipoexpressão de ambos genes, tanto nas amostras de pacientes com SE quanto nas linhagens celulares SK-ES-1 e RD-ES. Não foi encontrada nenhuma relação das expressões gênicas com os dados clínicos. Contudo, a presença do gene de fusão EWS-FLI1 parece estar associada com menor expressão de ROCK1. Verificou-se que a proliferação celular, a capacidade clonogênica e o ciclo celular não se alteraram significativamente nas linhagens celulares de SE após os diferentes tratamentos com as drogas GSK429286 (inibidor de ROCK1), SR3677 (inibidor de ROCK2) e Hidroxifasudil (pan-inibidor). Porém, apesar de não significativo, observou-se um tênue aumento na migração e invasão celular após o tratamento com as drogas. Nossos dados indicam que ROCK1 e ROCK2 podem possuir um papel na tumorigênese do SE, e ainda estar relacionadas com os mecanismos de migração e invasão celular, processos importantes para a metástase tumoral. / Ewing\'s sarcoma (ES) is characterized by undifferentiated cells of neuroepithelial origin. It is the second most common malignant bone tumor in children and adolescents. Standard treatment consists of preoperative chemotherapy, followed by surgery and postoperative chemotherapy and/or radiotherapy. However, the spread of the tumor is very high and even in patients where the metastasis has not occurred, there may be circulating tumor cells. Furthermore, the 5-year survival which about 70% of patients without metastases at diagnosis achieve, falls to 25% in patients with metastases. Migration and invasion into adjacent tissues and blood vessels, cell adhesion and proliferation are essential for tumor metastasis to occur. Among the regulators of the processes are the Rho family of GTPases, which have as their effector kinases, ROCK1 and ROCK2. Thus, ROCK induce numerous cellular responses involving the regulation of many proteins associated with the cytoskeleton. Studies show that responses regulated by ROCK aggravate phenotypes associated with cancer and other diseases. This study aimed to verify the expression of ROCK1 and ROCK2 genes in ES tumor samples and to evaluate the effects of their pharmacological inhibition in vitro. Gene expression analysis showed lower expression of ROCK in both, patient samples (n=18) and the SK-ES- 1 and RD-ES cell lines. There was no relation of gene expression with clinical data. Nonetheless, the presence of EWS-FLI1 fusion appears to be associated with lower expression of ROCK1. Cell proliferation, clonogenic capacity and the cell cycle were not affected after different treatments with GSK429286 (ROCK1 inhibitor), SR3677 (ROCK2 inhibitor) and Hydroxyfasudil (pan-inhibitor). However, although not significant, there was a tenuous increase in cell migration and invasion following treatment with the drugs. Our data suggest that ROCK1 and ROCK2 might have a role in ES tumorigenesis and may be related im part to migration and invasion cell mechanisms, important processes for tumor metastasis.

Viele humane Sarkome sind durch spezifische chromosomale Translokationen oder typische genetische Amplifikationen definiert, welche in der Differentialdiagnostik insbesondere in Fällen, bei denen klinische Daten, Morphologie und Immunhistochemie alleine nicht ausreichend wegweisend sind. Die Formalin-fixiertem Paraffin-eingebetteten (FFPE-) Gewebe von 15 Ewing-Sarkomen, 4 Klarzellsarkomen, 9 Synovialsarkomen, 4 alveolären und 7 embryonalen Rhabdomyosarkomen und 25 Liposarkomen verschiedenen Subtyps wurden mittels Fluoreszenz-in-situ-Hybridisierung (FISH) untersucht um ein Sarkom-spezifisches FISH-Sondenset zur Detektion spezifischer chromosomaler Aberrationen in der Routinediagnostik zu etablieren. Es konnte gezeigt werden, dass die FISH in diesem Aufgabenfeld im Vergleich zur PCR ebenfalls eine hoch effiziente zytogenetische Methode mit hoher Spezifität und hohen positiven Vorhersagewerten mit dem Vorteil der unproblematischen Anwendung an FFPE-Geweben ist. Zur Detektion des Isochromosom 12p , i(12p), als Beispiel für komplexere chromosmale Aberrationen, wurden 7 FFPE-Gewebe aus Keimzelltumoren mit 12p- und 12q-detektierenden FISH-Sonden hybridisiert. Die Detektion des i(12p) konnte im Rahmen dieser Arbeit mittels FISH nicht erreicht werden. Zusammenfassend ist die FISH eine hoch effiziente zytogenetische Methode zur Detektion spezifischer chromosomaler Aberrationen in FFPE-Geweben aus humanen Sarkomen mit hoher Eignung zur Anwendung in der Routinediagnostik. / Many human sarcomas are defined by specific chromosomal translocations or typical genetic amplifications, which can be very useful in differential-diagnosis especially in cases with overlapping clinical data, morphology and results of immunohistochemistry. The formalin-fixed paraffin-embedded tissues of 15 Ewing sarcomas, 4 Clear cell sarcomas, 9 Synovial Sarcomas, 4 Alveolar and 7 Embryonal rhabdomyosarcomas and 25 Liposarcomas of different subtype were tested with fluorescence in situ hybridization (FISH) to establish a sarcoma-specific probe set for the detection of specific chromosomal aberrations in routine diagnostics. The results show that FISH compared to PCR also is an efficient cytogenetic method with high specificity and high positive predictive value especially having the advantage of unproblematic use in formalin-fixed paraffin-embedded tissue. For the detection of the isochromosome 12p, i(12p), as an example for more complex chromosomal aberrations, 7 formalin-fixed paraffin-embedded tissues of Germ Cell Tumors were hybridized with 12p- and 12q-detecting probes. The detection of the i(12p) by using FISH could not be achieved in this study. Concluding FISH is an efficient cytogenetic method for the detection of specific genetic aberrations of human sarcomas in formalin-fixed paraffin-embedded tissue with a high ability in routine diagnostics.

Thesis (D.Min.)--Dallas Theological Seminary, 2006. / Includes abstract. Includes bibliographical references (leaves 235-240).

Thesis (D. Min.)--Dallas Theological Seminary, 2006. / Includes abstract. Includes bibliographical references (leaves 235-240).

Made available in DSpace on 2013-08-07T18:41:31Z (GMT). No. of bitstreams: 1 000438120-Texto+Completo-0.pdf: 2337312 bytes, checksum: 3bcbd6b2d333489c6e0cefb866d0b73c (MD5) Previous issue date: 2012 / Ewing’s sarcoma was first described by James Ewing in 1921 and it is the second most common bone tumor in children and young adults. Both chromosomal breakage and translocation occur in this sarcoma. The EWS gene is localized in chromosome 22 and is involved in this translocation. However, little is known about this gene breaking region and what sequences could be involved in higher chromosomal break susceptibility. In this study we aimed to investigate three SNPs in the EWS gene breaking region in a healthy subjects’ population and in Ewing’s sarcoma patients. Genotyping was performed by TaqMan® assay for allelic discrimination using Real-Time PCR System. We conducted analysis of allelic and genotypic frequencies, as well as association and transmission disequilibrium tests. According to our results, the control group showed similar and different genotypes distribution of all SNPs when compared to other populations studied by different projects, which shows how important it is to know the frequencies of our population. To test the hypothesis that some SNP, SNParrangement or haplotype could influence in the susceptibility to develop Ewing’s sarcoma, we compared affected with non-affected individuals using association studies. The results showed one significant difference: a higher presence of homozygote T-rs4820804 in Ewing’s Sarcoma patients. Transmission Disequilibrium Test (TDT) was performed to compare data from Ewing’s Sarcoma patients and from their families but no statistically significant result was found. In conclusion, we find that the TT-rs4820804 EWS genotype can be associate with Ewing’s sarcoma and that the rs4820804 SNP can be a candidate to understand the EWS breakage susceptibility. / O sarcoma de Ewing foi primeiramente descrito por James Ewing em 1921 e é o segundo tumor ósseo mais frequente em crianças, adolescente e adultos jovens. Neste sarcoma, é comum ocorrer a quebra e a translocação cromossômica. Dentre os genes envolvidos nesta translocação está o gene EWS, localizado no cromossomo 22. Entretanto, pouco se sabe a respeito da região de quebra deste gene e quais sequências poderiam levar a uma maior susceptibilidade a quebra cromossômica. Sendo assim, o objetivo deste trabalho foi investigar três polimorfismos de base única (SNPs) presentes na região de quebra do gene EWS, em uma população de indivíduos saudáveis e em pacientes afetados pelo Sarcoma de Ewing. A genotipagem para os SNPs selecionados foi realizada usando TaqMan SNP Genotyping Assay pelo sistema de PCR em tempo real. Nós realizamos análises de frequências alélicas e genotípicas, assim como um estudo de associação e de desequilíbrio de transmissão.A comparação das frequências alélicas e genotípicas entre as populações deste estudo e entre populações de projetos já publicados mostrou particularidades entre as populações, revelando a importância de se conhecer tais frequências na população de estudo. Para testar a hipótese de que algum SNP, haplótipo ou combinação específica de SNPs poderia influenciar na susceptibilidade ao Sarcoma de Ewing, comparamos afetados com não-afetados realizando estudos de associação cujos resultados mostraram uma única diferença significativa: a maior incidência no genótipo TT-rs4820804 entre os afetados pelo Sarcoma de Ewing. O Teste de Desequilíbrio de Transmissão (TDT) comparou os dados dos pacientes afetados e os dados de seus familiares, mas nenhum resultado significativo foi encontrado. Em conclusão, o genótipo TT-rs4820804 pode estar associado ao Sarcoma de Ewing e o SNP rs4820804 pode ser candidato para auxílio do entendimento da susceptibilidade de quebra do gene EWS.

Made available in DSpace on 2013-08-07T18:41:34Z (GMT). No. of bitstreams: 1 000438127-Texto+Completo-0.pdf: 909961 bytes, checksum: 5f9f40febc8291b363b759367f823d98 (MD5) Previous issue date: 2012 / In this study the SNPs rs640098, rs491714, rs611307 into the FLI1 gene were genotyped in a sample of 201 subjects from southern Brazilian population, in 24 Ewing’s sarcoma patients (geographically matched with control group) and 54 of their family members, including parents and siblings. We performed association studies comparing genotypic frequencies of rs640098, rs491714, rs611307 into the FLI1 gene, and all possible genotype combinations between Ewing’s Sarcoma patients and control group. Of the three SNPs investigated individually, only one of them showed a significant result when compared to the control group; our non-combined analysis revealed a significantly higher presence of homozygote A-rs497714 among Ewing’s Sarcoma patients (p=0. 0065; Chi square Test). In all other tested clusters, we always noticed a higher rate of homozygote A-rs497714 among Ewing’s Sarcoma patients independent of the other SNP-arrangements and/or haplotype combinations. In addition, we performed transmission disequilibrium tests comparing data from Ewing’s Sarcoma patients and from their families (parents and siblings), but no statistically significant result was found. In conclusion, the present study provides evidence statistically founded that the AA-rs497714 FLI1 genotype can associated with Ewing's sarcoma. And that this polymorphism can be clinically useful as a potential genetic marker to the prognostic of risk to develop this cancer or to provide insights into FLI1 chromosome breakage context of tumorigenesis. / Neste estudo, os SNPs rs640098, rs491714, rs611307 no gene FLI1 foram genotipados em uma amostra de 201 indivíduos da população do sul do Brasil, e em 24 pacientes portadores de Sarcoma Ewing (geograficamente comparado com grupo controle) e 54 de seus familiares, incluindo pais e irmãos. Realizamos estudos de associação, comparando as freqüências genotípicas do rs640098 e rs491714 e rs611307 no gene FLI1, e todas as combinações possíveis entre o genótipo de pacientes Sarcoma de Ewing e grupo controle. Dos três SNPs investigados individualmente, apenas um deles apresentou um resultado significativo quando comparado com o grupo controle; nossa análise não-combinada revelou uma presença significativamente maior de homozigoto A-rs497714 entre os pacientes de Sarcoma Ewing (p = 0,0065; Chi quadrado). Em todos os outros grupos testados, foi notada uma maior taxa de homozigoto A-rs497714 entre os pacientes com Sarcoma de Ewing, independentemente dos outros arranjos e / ou combinações de SNP e haplótipos. Além disso, foram realizados testes de desequilíbrio de transmissão, comparando dados de pacientes portadores de Sarcoma de Ewing e de suas famílias (pais e irmãos), mas nenhum resultado estatisticamente significativo foi encontrado. Em conclusão, o presente estudo fornece evidências estatisticamente fundada de que o genótipo AA-rs497714 FLI1 pode associado ao sarcoma de Ewing. E que este polimorfismo pode ser clinicamente útil como um potencial marcador genético para o prognóstico de risco para desenvolver este câncer ou para fornecer insights no contexto de quebras cromossômicas de tumorigênese no gene FLI1.

Ewing Sarcoma (ES) is an aggressive pediatric solid tumor. Even though overall-survival for localized patients is approximately 70%, the overall-survival for high risk ES patients has not improved in the last 20 years. Therefore, there is a need for exploration of new therapeutic agents in ES. Recent evidence has demonstrated that ES cells behave like BRCA-deficient tumor types which renders them sensitive to PARP inhibitors in vitro and in vivo. However, a phase II study of the efficacy of single-agent PARP inhibition in patients with relapsed ES did not significantly improve outcome. As single-agent therapy is rarely expected to result in significant clinical responses, in this study, we plan to validate potential targeted combination therapies with PARP inhibitors in ES. Since ES appears to demonstrated BRCA-deficient biology with impaired homologous recombination, cells are expected to be sensitive to both PARP inhibitors and ATR inhibitors, drugs which have a role in regulating DNA damage and impairing homologous recombination. In breast cancer and ovarian cell lines with genetic BRCA-deficiency, PARP and ATR inhibitors have synergistic activity. We hypothesize that these inhibitors will also have synergistic anti-Ewing activity. Furthermore, we recognize that ES cells demonstrate remarkably quiet genomes suggesting that there is minimal ongoing DNA-damage when cells are growing unperturbed. Therefore, we also plan to test the effect of adding low-dose genotoxic chemotherapy to induce additional sensitivity to the combination of PARP and ATR inhibitors in ES. The specific aims of this study were to explore the possible anti-tumor effect of PARP inhibitors combined with ATR inhibitors in ES cell lines, and to explore whether low dose genotoxic chemotherapy with SN38 can potentiate the anti-tumor effect of combined PARP and ATR inhibition in ES cell lines. We studied the anti-Ewing Sarcoma effect of the combination of a PARP inhibitor, AZD2281, and an ATR inhibitor, AZD6738, across a range of doses with and without low doses of a DNA damaging agent, SN38 (irinotecan metabolite), in two ES cell lines. We analyzed synergy by determining the Combination Index (CI) and Fractional Inhibition (FA) of each combination. We found that the ATR inhibitor, AZD6738, was synergistic across large range of concentrations when combined with the PARP inhibitor, AZD2281, in ES cell lines. We also found that treatment of cells with low doses of SN38 increases ES cell sensitivity to treatment with the PARP inhibitor and ATR inhibitor combination. This study provides preclinical support for additional studies exploring these combinations in ES. Given the low number of pediatric patients with ES compared to adult cancer patients, there will be limited attempts in combining these agents in clinical trials. Therefore, the development of an in vivo trial testing the safety and efficacy of this combination in ES mouse models is proposed. / 2020-07-03T00:00:00Z

O sarcoma de Ewing (SE) é um tumor caracterizado por possuir células indiferenciadas, de origem neuroepitelial. É o segundo tumor ósseo maligno mais comum em crianças e adolescentes. O tratamento consiste na aplicação de quimioterapia neoadjuvante, seguida da cirurgia e a quimioterapia adjuvante ou radioterapia. Contudo, a disseminação deste tumor é muito intensa, e mesmo em pacientes onde a metástase não ocorreu, podem haver células tumorais circulantes. Além disso, a sobrevida de 5 anos que cerca de 70% dos pacientes que não apresentam metástase no momento do diagnóstico atingem, cai para 25% nos pacientes que apresentam metástase. A migração e invasão celular dos tecidos vizinhos e vasos sanguíneos, a adesão celular e a proliferação são processos essenciais para que a metástase tumoral aconteça. Dentre as reguladoras da mobilidade, adesão celular e proliferação estão as GTPases da família Rho, que possuem entre suas quinases efetoras, ROCK1 e ROCK2. Assim, as proteínas ROCK induzem inúmeras respostas celulares que envolvem a regulação de muitas proteínas associadas ao citoesqueleto. Estudos evidenciam que respostas sinalizadas por ROCK agravam fenótipos associados ao câncer e outras doenças. O presente trabalho teve como objetivo verificar a expressão dos genes ROCK1 e ROCK2 em amostras tumorais e linhagens celulares de SE, e os efeitos in vitro da inibição farmacológica de ambas as quinases. O estudo da expressão gênica de ROCK1 e ROCK2 em amostras de pacientes com SE (n=18) revelaram uma hipoexpressão de ambos genes, tanto nas amostras de pacientes com SE quanto nas linhagens celulares SK-ES-1 e RD-ES. Não foi encontrada nenhuma relação das expressões gênicas com os dados clínicos. Contudo, a presença do gene de fusão EWS-FLI1 parece estar associada com menor expressão de ROCK1. Verificou-se que a proliferação celular, a capacidade clonogênica e o ciclo celular não se alteraram significativamente nas linhagens celulares de SE após os diferentes tratamentos com as drogas GSK429286 (inibidor de ROCK1), SR3677 (inibidor de ROCK2) e Hidroxifasudil (pan-inibidor). Porém, apesar de não significativo, observou-se um tênue aumento na migração e invasão celular após o tratamento com as drogas. Nossos dados indicam que ROCK1 e ROCK2 podem possuir um papel na tumorigênese do SE, e ainda estar relacionadas com os mecanismos de migração e invasão celular, processos importantes para a metástase tumoral. / Ewing\'s sarcoma (ES) is characterized by undifferentiated cells of neuroepithelial origin. It is the second most common malignant bone tumor in children and adolescents. Standard treatment consists of preoperative chemotherapy, followed by surgery and postoperative chemotherapy and/or radiotherapy. However, the spread of the tumor is very high and even in patients where the metastasis has not occurred, there may be circulating tumor cells. Furthermore, the 5-year survival which about 70% of patients without metastases at diagnosis achieve, falls to 25% in patients with metastases. Migration and invasion into adjacent tissues and blood vessels, cell adhesion and proliferation are essential for tumor metastasis to occur. Among the regulators of the processes are the Rho family of GTPases, which have as their effector kinases, ROCK1 and ROCK2. Thus, ROCK induce numerous cellular responses involving the regulation of many proteins associated with the cytoskeleton. Studies show that responses regulated by ROCK aggravate phenotypes associated with cancer and other diseases. This study aimed to verify the expression of ROCK1 and ROCK2 genes in ES tumor samples and to evaluate the effects of their pharmacological inhibition in vitro. Gene expression analysis showed lower expression of ROCK in both, patient samples (n=18) and the SK-ES- 1 and RD-ES cell lines. There was no relation of gene expression with clinical data. Nonetheless, the presence of EWS-FLI1 fusion appears to be associated with lower expression of ROCK1. Cell proliferation, clonogenic capacity and the cell cycle were not affected after different treatments with GSK429286 (ROCK1 inhibitor), SR3677 (ROCK2 inhibitor) and Hydroxyfasudil (pan-inhibitor). However, although not significant, there was a tenuous increase in cell migration and invasion following treatment with the drugs. Our data suggest that ROCK1 and ROCK2 might have a role in ES tumorigenesis and may be related im part to migration and invasion cell mechanisms, important processes for tumor metastasis.

Made available in DSpace on 2014-08-20T13:32:57Z (GMT). No. of bitstreams: 1 tese_helena_strelow_thurow.pdf: 807973 bytes, checksum: 9ce5f5a742c1cda1d087be4eaaf27eae (MD5) Previous issue date: 2011-07-15 / A proteína p53,codificada pelo gene TP53, vem sendo estudada há mais de 30 anos e já foi denominada de "guardiã do genoma.

Background: Despite multi-modality therapy, the majority of patients with metastatic or recurrent Ewing sarcoma (ES), the second most common pediatric bone malignancy, will die of their disease. ES tumors express aberrantly activated ETS transcription factors through translocations that fuse the EWS gene to ETS family genes FLI1 or ERG. The aberrant activation of ETS transcription factors promotes malignant transformation and proliferation. While, FLI1 or ERG cannot be readily targeted, there is an opportunity to deploy functional genomics screens, to develop novel therapeutic approaches by identifying targetable liabilities in EWS/FLI1 dependent tumors. Materials and Methods: We performed a near whole-genome pooled shRNA screen in a panel of five EWS/FLI1 dependent Ewing sarcoma cell lines and one EWS/ERG cell line to identify essential genes. Essential genes were defined as those genes whose loss resulted in reduced viability selectively in ES cells compared to non-Ewing cancer cell lines. Essential hits were subsequently validated with genomic knockdown and chemical inhibition in vitro, followed by validation of the on-target effect of chemical inhibition. Next, we determined the in vivo effects of small-molecule inhibition on survival and tumor growth in NOD scid gamma (NSG) mice with established subcutaneous ES xenografts. Results: Top hits in our screen that could be readily targeted by small-molecule inhibitors, and thus have potential for rapid clinical validation, were selected for further investigation. These hits included IKBKE, CCND1 and CDK4. IKBKΕ, a non-canonical IKK with an oncogenic role in breast cancer, was one of the top kinase hits in the screen. IKBKΕ shares significant homology to TBK1, another non-canonical IKK that is essential in k-RAS dependent lung cancer. We validated IKBKE through small-molecule inhibition of IKBKE/TBK1 and shRNA based knockdown. Ewing sarcoma cell lines are sensitive to low micromolar concentrations of two IKBKE/TBK1 inhibitors (CYT387 and MRT67307). Additionally, in a panel of ES cell lines, knockdown of IKBKE resulted in decreased growth and impaired colony formation. These observations, paired with impairment of NF-κB nuclear localization following CYT387 treatment suggests that non-canonical IKK mediated signaling may be essential in Ewing sarcoma. We further validated these results through inhibition of IKBKE/TBK1 in in vivo xenograft models treated with 100 mg/kg/day of CYT387. Treatment over the course of twenty-nine days resulted in a significant increase in survival (p-value = 0.0231) and a significant decrease (p-value = 0.036) in tumor size after fifteen days of treatment. CDK4 and CCND1 are highly expressed in Ewing sarcoma as compared to other tumor types. shRNA mediated knockdown of CDK4 and CCND1 resulted in impaired viability and anchorage independent growth. Furthermore, treatment of Ewing sarcoma cell lines with a highly selective CDK4/6 inhibitor, LEE011, resulted in decreased viability (IC50 range of 0.26-18.06 μM), potent G1 arrest in six of eight EWS/FLI1 containing Ewing sarcoma lines tested and apoptosis in a panel of four highly sensitive lines. Administration of 75 mg/kg/day and 250 mg/kg/day of LEE011 in NSG mice with Ewing xenografts resulted in significant impairment of tumor growth, (p-value <0.001 for both treatment arms), as compared to vehicle control. Conclusions: These studies suggest a role for the targeting of IKBKE and CDK 4/6 in Ewing sarcoma, findings with immediate clinical relevance for patients with this malignancy, because small-molecule inhibitors of these proteins have already entered clinical trial for other disease indications.