1. Molecular taxonomy of pediatric cancers. Neuroblastomas are cancers of neural crest origin with variable prognosis depending on age at presentation, stage, histology, presence of MYCN amplification, chromosomal ploidy and deletion status of 1p36. Very little is known of the molecular mechanisms that confer good or poor prognosis in this and other malignancies. We have recently demonstrated that cancers can be diagnosed on the basis of gene expression profiling using cDNA microarrays and sophisticated pattern recognition algorithms such as Artificial Neural Networks (Nature Medicine. Vol. 7,6: 673-679, June 2001). We will expand this concept further by profiling a series of on neuroblastomas of different stages and prognosis. With this we hope to identify tumor specific expression patterns or """"""""fingerprints,"""""""" that uniquely identify a poor prognostic group, as well as those associated with specific genetic aberrations including NMYC amplification. By these methods, we may be able to classify expression profiles that correlate with prognosis, and hence identify the genes that confer these biological properties. We also plan to develop a database of a wide range of pediatric malignancies from patient tumors, including archival material, xenografts, as well as prospective samples from patients on treatment protocols at the Pediatric Oncology Branch (POB). By these methods I hope to narrow down the list of genes that defines a particular cancer or diagnostic or prognostic group cluster to a minimum number, which can be used to make a smaller microarrays for possible diagnostic purposes. My primary focus for pediatric solid tumors will be neuroblastoma. For these cancers I will do a more detailed molecular /clinical-expression correlation studies. i.e. how does the expression profiles on MYCN amplified tumors compare with non-MYCN amplified samples? How does 17q amplified tumors or those with 1p loss correlate with the same. These are important questions to answer since these molecular markers correlate with prognosis. Can we identify the genes that correlate with stage of the disease which is also a surrogate marker for prognosis. As well as gene expression studies, we will also perform comparative hybridization on the cDNA chips on these cancers. These studies will increase our knowledge of these malignancies by identifying genes that are significant to the biology of these cancers. In addition, by these methods, we may identify secreted proteins that can be used for diagnosis (e.g. AFP for germ cell tumors), as well as following therapy including the monitoring of tumor regression or recurrence. We may also identify new targets for therapy, including immune therapy, or discover novel molecular targets such as death pathway genes, uniquely expressed in these cancers. 2. Identification of the downstream targets of Pax3-FKHR. The second area which I will address, is characterizing the biological/biochemical properties of the t (2; 13)(q35; q14) that is found in ARMS. This translocation results in the fusion of PAX3, a developmental transcription factor required for limb myogenesis and neural crest development, with FKHR, a member of the forkhead family of transcription factors. The resultant PAX3-FKHR gene possesses transforming properties, however, the effects of this chimeric oncogene on gene expression are largely unknown. I have investigated the actions of these transcription factors, by the introduction of both Pax3 and Pax3-FKHR into NIH-3T3 cells, and monitored the resultant gene expression changes with a murine cDNA microarray containing 2225 elements [PNAS (1999), 96, 23, 13264-13269]. We found that PAX3-FKHR, but not PAX3, activated a myogenic transcription program. This included the induction of transcription factors MyoD, Myogenin, Six1, and Slug, as well as a battery of genes involved in several aspects of muscle function. Notable among the induced genes, were the growth factor gene Igf2, its binding protein Igfbp5, and the transforming growth factor b2. Relevance of this model was suggested, by verification that three of these genes (IGFBP5, HSIX1, and Slug) were also expressed in ARMS cell lines. This study demonstrated the profound myogenic properties of PAX3-FKHR and not PAX3 in NIH-3T3 cells. We postulate, that the presence of the PAX3-FKHR gene in a progenitor cell triggers myogenesis, but these cells then fail to terminally differentiate and exit the cell cycle. This then allows the subsequent acquisition of secondary genetic alterations that would lead to the development of fully malignant ARMS. We have identified several candidate genes in microarray experiments that may prevent this terminal differentiation including the DNA-binding protein inhibitors ID-4 and ID-2 as well as SIX1 and FGFR4. We plan to further characterize these genes, both in terms of their temporal expression and their molecular effects. In addition an exciting new development has been the production of a PAX3-FKHR monoclonal antibody. This antibody made to the fusion protein at the breakpoint, does not recognize the native Pax3 or FKHR. It will prove to be an invaluable resource, and one, which I will use to identify the direct targets of PAX3-FKHR, using a combination of chromatin immuno-precipitation and cDNA microarray technology. 3. Molecular Mechanisms of Drugs using cDNA Microarrays. A third area which I will focus on, is the monitoring of gene expression changes impacted on by drugs. The choice and design of many chemotherapeutic agents, currently used in cancer treatment, has been traditionally empirical in nature. The molecular mechanisms of their actions are not well understood, and their mode of action is often indiscriminate targeting, both of tumor and normal cells. As a model system and with collaboration with Dr Carol Thiele, I have investigated the gene expression alterations during neural differentiation of the neuroblastoma (NB) cell line SMS-KCNR by retinoic acid (RA), using cDNA Microarrays. Neuroblastoma is the most frequently occurring extra-cranial solid tumor of childhood, and has the highest rate of spontaneous regression of any human cancer. RA is known to stimulate morphological neural differentiation of NB. It has been shown to enhance neurite extension, increase membrane excitability, induce neurotransmitter enzymes and reduce tumorogenicity, as well as improve prognosis for high stage disease. SMS-KCNR neuroblastoma cells (containing 1p del and N-Myc amplification), were treated with all-trans retinoic acid (ATRA), or the solvent ethanol (control). RNA was harvested at 0, 2, 6, 16, 30, 48, 80, and 96 hours and 8, 12, 18, and 22 days following ATRA treatment. Gene expression profiles at these time points were compared with time point 0. By this method we are currently identifying pathways critical genes in neuronal differentiation using the analysis tools mentioned above. In addition, we have developed a novel gene-clustering algorithm, based on pre-defined profile templates, to identify expression profiles that drive differentiation. This in vitro model is being translated to an in vivo model of neuroblastoma with collaborations with Dr John Wiggonton. I plan als to devote a fellow to the development of tools for the analysis and mining of gene expression data including the development of pattern recognition algortithms. The combined approaches outlined in this proposed program will allow a comprehensive analysis of pediatric tumor genomes. 4.1 4. Investigation of the role of the tyrosine kinase receptors FGF4R, IGF1R and KIT in pediatric malignancies. Many of the embryonal tumors of childhood have upregulation or increased expression of tyrosine kinase receptors. These genes are upstream of the AKT survival and growth pathways. FGFR4, is expressed during myogenesis and prevents terminal differentiation in myocytes and was recently found by us to be highly expressed only in RMS, and not in normal muscle by cDNA microarays. The relatively strong differential expression of FGFR4 in RMS was confirmed by immunostaining of tissue microarrays. This suggests that, while FGFR4 expression in RMS may be of biological and therapeutic interest. Using a dominant negative FGFR4 we plan to determine by microarrays and in vivo experimentation the role of this gene in oncogenesis in collaboration with Dr Merlino nad Dr Khanna. IGF2 is highly expressed in rhabdomyosarcoma, and neuroblastoma both of which express the IGF1R, we plan with collaboration with Dr Barrett to utilize a dominant negative to the IFG1R to investigate the role of this gene in oncognesis and the metastatic process. Ewings sarcoma is known to express the wild type KIT. In parallel with clinical trials on the use of Gleevac in Ewings sarcoma we plan to investigate the molecular effects of inhibitors in the KIT-AKT pathway (STI571 and 1L-6-Hydroxymethyl- chiro -inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate ) both in the recently established gastro intestinal stromal tumor cell line (GIST 882) which expresses a constitutively activated mutant KIT gene, and in Ewing's sarcoma. By these experiments we hope to perform a comprehensive analysis of these pathways in these malignancies.
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