Caracterização da expressão dos genes da via de sinalização WNT e do processo de transição epitélio-mesênquima durante a embriogênese de rim e suas implicações em tumores de Wilms / Gene expression characterization of WNT signaling pathway and epithelial-mesenchymal transition during kidney embryogenesis and their implications in Wilms tumors

RESUMO

O desenvolvimento do rim envolve interações complexas entre as células epiteliais e mesenquimais. Uma interrupção em qualquer passo crucial da progressão morfogenética pode levar a má formação renal e doenças, incluindo câncer. O tumor de Wilms (TW) ou nefroblastoma é originado pela interrupção da diferenciação das células do blastema metanéfrico resultando em um tumor composto por três componentes histológicos, blastema, epitélio e estroma... Nesse estudo, para identificar as alterações moleculares do início da diferenciação do rim e que são recapituladas no TW, a expressão de genes pertencentes às vias de transdução de sinal foi avaliada em células do blastema metanéfrico de quatro estágios temporais da diferenciação em camundongos (de 15,5 dias pós-coito até o rim completamente diferenciado), e em células do blastema de TWs e de rins diferenciados em humanos... O RNA total das células capturadas a laser foi amplificado e hibridado. Foram selecionados 18 genes, cuja expressão no início da diferenciação do rim foi recapitulada no TW, dos quais 11 foram menos expressos no tumor e apresentaram expressão crescente durante a nefrogênese e 7 foram mais expressos no tumor e apresentaram expressão decrescente durante a nefrogênese ... As alterações da expressão desses genes devem ser eventos precoces da interrupção da diferenciação que leva a transformação maligna. Adicionalmente, combinações de trios de genes foram avaliadas como marcadores preditivos de recaída em TW. O melhor trio composto por IGF2, TESK1 e PAX2, foi capaz de discriminar corretamente 85,71% das amostras quanto a recaída e não recaída. Esse estudo contribuiu para a identificação de novos genes associados com o aparecimento do TW, e como são alterações moleculares precoces, provavelmente são desencadeadoras do aparecimento do TW. Dessa forma, são candidatos apropriados para serem testados como alvos terapêuticos e marcadores moleculares para auxiliar a prática diária do TW.

ABSTRACT

Kidney development involves complex interactions between epithelial and mesenchymal cells. A disruption at any crucial step of the morphogenetic progression may lead to kidney malformations and diseases, including cancer. Wilms tumor (WT) or nephroblastoma originates from the metanephric blastema cells, which were unable to complete the differentiation process, resulting in a tumor composed by three histological components, blastema, epithelia and stroma. Blastemal component presents molecular characteristics which are highly similar to the earliest stages of kidney development, suggesting that it retains the key molecular events responsible for WT onset. Deregulation of signal transduction pathways has showed to be an important factor for interruption of renal differentiation, and probably for the WT onset. In this study, aiming to identify the early molecular alterations potentially involved in WT arising, the expression pattern of genes belonging to the transduction signaling pathways was evaluated in cells of the metanephric blastema in four temporal differentiation stages from 15,5 days post-coitum to the fully differentiated kidney in mice, and in blastemal cells of WT and human differentiated kidneys. To evaluate nephrogenesis in mice and WT in humans, we developed a model that allows the hybridization of target molecules of both species in the same cDNA microarray platform. Three hundred and twenty-six stretches of orthologous genes from humans and mice that share high similarity at nucleotide level were immobilized on a glass platform. Wnt signaling pathway, phosphatidylinositol (PI3K) and genes related to epithelial-mesenchymal transition and epithelial-mesenchymal (EMT/MET), associated to the embryonic development, were among the evaluated pathways. Blastemal cells were laser captured from 24 WT samples at four stages of nephrogenesis. Total RNA from these samples was amplified and hybridized in the platform. Eighteen genes were selected, whose expression in the initial of kidney differentiation were also observed in WT, where 11 were down-regulated in WT and reported an increasing expression during nephrogenesis and 7 were up-regulated in WT and presented a decreasing expression during nephrogenesis. Non-supervised hierarchical clustering based on the expression of 18 genes grouped differentiated kidneys from both species, human and murine, and discriminated both from WT samples, which were grouped with the earliest kidney stages, validating the model proposed by this study. The 18 genes were assessed in independent groups of samples at mRNA and protein levels. Twelve out of 18 genes were selected for validation by quantitative RT-PCR in the initial sample set and the expression of 9 (75%) genes was confirmed. From the 9 genes, 5 (55.6%) were validated in an independent group of samples. At protein level, 8 genes were evaluated by immunohistochemistry in WT, during nephrogenesis and in differentiated kidneys in humans. Quantitative evaluation of protein expression was consistent with the results of mRNA expression for 5 (62.5%) genes. This study identified WNT5B, PI3KCA, FZD2, GRK7, TESK1 and TIMP3, which had not been previously associated to WT. Gene expression alterations may be the early events of the interruption of cell differentiation that leads to malignant transformation. Additionally, combinations of trios of genes were tested as predictors of WT relapse revealing IGF2, TESK1 and PAX2 as the best trio because it was able to correctly discriminate 85.71% of the samples. This study contributes to the identification of new genes associated to the WT onset and to the early molecular alterations, which are likely to trigger the WT arising. In this way, these genes are candidates to be tested for therapeutic targets and also for molecular markers to assist the daily WT practice.