Of the sarcomas that occur in children and adolescents, rhabdomyosarcoma is the most common soft tissue sarcoma while Ewings family of tumors are the second most common malignant bone tumors after osteosarcomas. Together, these three tumors constitute 10% of all newly diagnosed cancers in children and adolescents. Almost 95% of the Ewings family of tumors are associated with a chromosomal translocation; 85% have a t(11;22) translocation, 10% have a t(21;22) translocation, generating the EWS-FLI1 and EWS-ERG fusion genes, respectively, and less than 1% harbor a translocation that fuses the EWS gene to a member of the ETS family of transcription factors. In rhabdomyosarcomas, about 70% to 80% of tumors do not harbor a translocation, but 20% are associated with stable chromosomal translocations that result in the formation of transcription factors PAX3-FKHR (t(2;13)) and PAX7-FKHR (t(1;13)). Rhabdomyosarcomas with no fusion gene, in general, have a better prognosis than those associated with the translocation. Fusion-positive rhabdomyosaromas have a molecular signature that is different from their fusion-negative counterparts. Chemotherapeutic regimens have improved the disease-free survival of non-metastatic rhabdomyosarcomas and Ewings sarcomas to 70%; however, cases with metastasis at diagnosis and with recurrences have an unfavorable outcome with overall survival of 20% to 25% in Ewings sarcoma and fusion-positive rhabdomyosarcoma. ❧ As intensified chemotherapy has not been able to improve prognosis, current research strategies are focused at identifying tumor-specific genes, both protein-coding and non-coding, that can be targeted with drugs to improve treatment outcome. These druggable targets or biomarkers should be strongly associated with tumors and should be important in maintaining pathways of tumor progression such as cell survival, growth, angiogenesis, invasion, and metastasis. In addition, there is an urgent need for more predictive biomarkers that can predict prognosis and/or patient response to therapy so that effective treatment strategies can be formulated. This dissertation begins by describing a study that identified gene expression signatures that were associated with fusion-positive rhabdomyosarcomas. Following validation of some of the genes from the expression signature, the most strongly associated gene, neuronal nitric oxide synthase, nNOS, is chosen for further molecular studies. The study identifies high expression of nNOS as a predictor of poor outcome in rhabdomyosarcomas. Further, functional studies identify PAX-FKHR as a regulator of nNOS, while nNOS directed pathogenesis of these fusion-positive tumors by promoting cell survival and tumorigenecity through the NO-cGMP pathway. This study concludes by identifying small molecule inhibitors that can target nNOS to decrease cell growth in fusion-postive rhabdomyosarcomas. The next study helps define the characteristics and functional role of a long non-coding RNA, AK057037, in Ewings family of tumors. The study introduces the mechanism of discovery of cancer-specific biomarkers and their characterization using integrated genomics wherein imformation about the tumor is derived at the genetic and epigenetic levels. The study employs the use of genomic profiles of various primary tumors and normal tissues to understand the exclusive association of AK057037 with Ewings sarcoma. The transcriptional activity, as determined by sequencing the transcribed genome of two tumor cell lines, followed by validation studies helps characterize the transcript. Computational softwares predicted no open reading frame in the transcript, thus confirming it as a non-coding entity. Functional studies with EWS-FLI1 show that the fusion gene directly regulates AK057037, and similar studies with AK057037 indicate its role in promoting metastasis and tumorigenecity in Ewings family of tumors. The final part of the study deals with recognizing its presence in normal development with a hint of its involvement in chromatin regulation. ❧ These studies together highlight the importance of using high-throughput genomic platforms to identify biomarkers that can potentially have diagnostic, predictive, and/or therapeutic importance in cancer biology. Further evaluation of these molecules can help assess their role as predictors of treatment outcomes and underscore the signaling pathways involved in tumor progression such that targeted therapies can be developed.
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