Identification of molecular variations involved in pituitary tumorigenesis based on exomic sequencing and transcriptome

• Grant number: 22/04883-3
• Support Opportunities: Scholarships in Brazil - Post-Doctorate
• Effective date (Start): May 01, 2022
• Effective date (End): February 28, 2023
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Principal Investigator: Margaret de Castro
• Grantee: Junier Marrero Gutiérrez
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 14/03989-6 - Uncovering pathophysiological and molecular mechanisms involved in tumorigenesis by platforms for next-generation sequencing (NGS), AP.TEM

Abstract

Molecular studies using approaches based on candidate genes and studies of linkage in familial diseases have contributed, but not completely clarified, the molecular pathogenesis of sporadic pituitary tumors. Pituitary adenomas are among the most frequent intracranial neoplasms, exhibiting hormonal hyperproduction or compressions in approximately 1:1000 individuals. It is interesting to note that, data from our group and literature published so far have been successful in ruling out the involvement of genes orof candidate signaling pathways in this process (Wnt pathway, the protocogenes MYC, BCL1, FGF4/HSTF1, SEA, and HER2 and the TP53 tumor suppressor gene). On the other hand, few findings positive results have been reported: genetic or epigenetic alterations have already been described in some tumor subtypes, such as hypermethylation of the human RAS gene in GH-secreting adenomas, prolactin and ACTH. Dysregulation of genes involved in the cell cycle, including over expression of the PTTG gene has been demonstrated in corticotrophic tumors. Mutations in the GNAS gene occur in about 30% of GH-secreting tumors. Mutations in the MEN1 gene are found in tumors pituitary glands of patients with MEN1 while mutations in the CDKN1B gene (p27) occur inpatients with MEN4. Inactivating germline mutations of the AIP gene are frequent in patients with familial but rare somatotrophinomas in sporadic tumors. The differential expression of miRNAs have been associated with pituitary tumorigenesis. Additionally, the role of long RNAs non-coding proteins (lncRNAs), such as MEG3, in pituitary tumorigenesis has been pointed out. Finally, a study by our group points to Ribosomal Proteins (RP) as possible candidates in corticotrophic tumors. Objectives: Build and analyze exome and transcriptome libraries of pituitary tumor samples and evaluate potential epigenetic factors involved. Material and Methods: Samples from 130 ACTH-producing pituitary tumors will be used; producers of GH and non-secretors, in addition to Craniopharyngiomas. Tumor DNA and RNA will be extracted and genomic. Exome-seq and RNA-seq libraries will be constructed, which will be submitted to sequencing on an Illumina platform. The generated data will be analyzed through computational tools and analyzed in a supervised way aiming at the identification of changes in involved pathways/genes. The findings of interest will be validated through prediction of functional effect (in silico), automated direct sequencing (DNA-seq), quantitative PCRin real time (qPCR; RNA-seq), integrated analysis of variants with clinical and tumor phenotype, confirmation of functional effect (ex vivo, in vitro and in vivo). Perspectives: the discovery of new gene variants or genes differentially expressed in the pituitary tumors and Craniopharyngiomas by genome-wide sequencing may elucidate new mechanisms involved in pituitary tumorigenesis. The validation of alterations found will enable the identification of new diagnostic molecular markers and and pave the way for the development of new therapeutic targets. Goals: 1. Build exome-seq, methyl-seq, Chip-seq and RNA-seq libraries from DNA and RNA from samples of pituitary tumors and Craniopharyngiomas; 2. Analyze, in a supervised way, the data obtained through computational tools; 3. Validate the changes of interest observed through in silico, in vitro, and in vivo studies; 4. Search for therapeutic strategies aimed at alterations validated through in-house studies silica, in vitro and in vivo. (AU)