Análise molecular do gene prkar1a na odontogênese e nos tumores odontogênicos mistos

Abstract

Odontogenic tumours constitute a rare and heterogenous group which share similarities with the tooth germs. The tumors derived from odontogenic epithelium and odontogenic ectomesenchyme with or without hard tissue formation, were classified by the World Health Organization (WHO) as the mixed odontogenic tumours (MOT). Belong to this group of MOT, ameloblastic fibroma (AF), which is reported the malignant transformation of the ectomesenchymal counterpart into ameloblastic fibrosarcoma (AFS), besides ameloblastic fibrodentinoma (AFD) and ameloblastic fibroodontoma (AFO). cAMP-dependent protein kinase (PKA) is the main mediator of cAMP in mammalian cells, and is a tetramer composed of two regulatory subunits and two catalytic. From genes codifing PKA subunities, the only one associated with human neoplasias is PRKAR1A. PRKAR1A is a tumour-suppressor gene encoding PKA regulatory subunit 1 (R1). PRKAR1A is involved in the pathogenesis of odontogenic myxomas, an odontogenic tumour probably derived from the ectomesenchymal tissue, which revealed decreased PRKAR1A protein expression and mutations. As the MOT share this ectomesenchymal origin, together with the participation of the odontogenic epithelium, it is possible that these tumours share similarities with the odontogenic myxomas in their molecular pathogenesis. The aim of this study was to investigate the pattern of expression of PRKAR1A in murine and human odontogenesis as well as investigate the occurrence of molecular alterations of PRKAR1A in AF, AFO, AFD and AFS. We further investigated if Prkar1a+/- mice develop odontogenic tumours. To odontogenesis investigation, mouse and human embryos of different stages of development were processed to analysis of PRKAR1A/Prkar1a and PRKAR2A/Prkar2a mRNA expression by wholemount in situ and radioactive in situ hybridization. The expression of human genes transcripts were also verified by qRT-PCR in human dental germ. To investigate the PRKAR1A molecular alterations in odontogenic tumours, 13 samples of TOM were assessed by qRT-PCR, immunohistochemistry, LOH analysis and direct sequencing. The MOT were laser microdissected and the epithelial component was separated from the mesenchymal part by laser microdissection in 7/13 of cases. Mice Prkar1a+/- heads were paraffin embedded and stained with HE. The genes of R1 and R2 subunits showed expression in stages of odontogenesis in murine and human tooth germs evaluated by in situ or qRT-PCR. The odontogenic tumours analysis demonstrated a significantly decreased PRKAR1A mRNA levels of expression comparing with normal oral mucosa, while the PRKAR2A expression did not show difference between groups. The MOT exhibited a variable imunoexpression pattern of PRKAR1A protein, demonstrating loss of imunoexpression in both components of tumour, mainly in the ectomesenchyme of AF. LOH was seen in most tumours evaluated, in which all benign tumours with loss of imunoexpression we still observed presence of LOH in PRKAR1A locus. Mutations underreported were identified in four samples, being one missense, two synonymous, besides two mutations in 5UTR and four intronic mutations. Prkar1a heterozygous mice did not show evidence of odontogenic tumour formation. We conclude PRKAR1A/Prkar1a and PRKAR2A/Prkar2a are expressed during odontogenesis and the expression of PRKAR1A is decreased in MOT. The decreased in expression of mRNA and protein of PRKAR1A gene occurred together with LOH in gene locus. Although Prkar1a haploinsufficiency in mouse has not been sufficient to cause odontogenic tumours, molecular alterations in PRKAR1A are present in MOT, participating of molecular pathogenesis of these tumours.