Genomic and genetic dissection of pheochromocytona and paraganglioma

Resumen

Pheochromocytoma and paraganglioma (PPGL) are rare neuroendocrine tumors, with a strong genetic component, that comprises fifteen genes so far. The advent of high-throughput technologies have permitted the simultaneous interrogation of thousands biomolecules. As expected, these technologies have already been applied to study PPGL, as well as to many other pathologies. However, some molecular events, such as miRNA expression and DNA methylation, have not yet been explored when this thesis project began in autumn 2009. In the first part of this study, we explored microRNA expression in PPGL, and demonstrated that PPGLs express different miRNA signatures according to genetic background. As a matter of fact, it was possible to identify and validate several miRNAs associated with the primary mutation, as well as miRNAs common among PPGLs, which could potentially be used to guide genetic study. Among the most significant finding was the validation of SDHB-specific miRNA-183/96, which has gained interest lately among the PPGL research community. The second part of this thesis involved the use of bioinformatics integrative study of miRNA-mRNA interactions, which indicated neuronal differentiation as a common theme deregulated in PPGL pathogenesis. This was validated by functional analyses showing miR-183/-96 impeded NGF-induced neuronal differentiation of immature chromaffin cells (PC12 cells). Proteomic studies, initially included to validate our bioinformatics integration methods, also revealed the antagonistic effect of miR-183/96 on NGF-induced chromaffin cell differentiation might results from disruption of growth factor-induced RAS activation as suggested by proteomic analyses. In part three, we analyzed chromosomal alterations in PPGL using SNP-arrays, which enabled us to detect copy neutral events, including chromosome 14 disomy in MAX mutant tumors. We observed and verified previously described chromosomal aberrations in PPGL. Finally, we integrated miRNA, mRNA, SNP-array, and methylation data to obtain a more complete prospective of the molecular events at chromosome 14 in MAX mutant tumors, which the author not only believes it provides the means for the second hit of the MAX gene, but also results the loss of other tumor suppressive genes or gain of imprinted oncogenes. For fourth and final part, we explored DNA methylation patterns in the context of PPGL malignancy, as well as in tumors of diverse genetic backgrounds. Our results demonstrated that DNA methylation patterns differed according to PPGL genotype, and verified previous data showing global hypermethylation in SDHx-related tumors. Most importantly, we identified and validated 52 CpGs associated with malignant behavior in an independent cohort of which forty-eight CpGs showed significant associations with progression free survival. Finally, it was possible to suggest RDBP hypermethylation as a predictor of malignancy, as it was further confirmed in malignant PPGL by pyrosequencing in an independent series of FFPE archival samples. In summary, we have applied numerous high-throughput genomic technologies to study PPGL. These studies have revealed much about the molecular mechanisms behind these tumors, as well demonstrate how genomic technologies, if applied correctly, can compliment and foment research.