Análise da expressão de marcadores da transição epitélio mesênquima e suas modificações pós-traducionais (PTMs) em lesões endometrióticas

Abstract

The Epithelial-Mesenchymal Transition (EMT) is a potential mechanism in the development of endometriosis due to alterations in cell adhesion molecules and pathways that can lead to enhanced migratory capacity and invasive ability of ectopic endometrium. Post-translational modifications (PTMs) like glycosylation and phosphorylation are crucial in regulating many EMT/cell adhesion markers, and up to date there is no data about the profile of these modifications in endometriosis. Therefore, this study was divided into one retrospective evaluation of variations of EMT markers in endometriotic lesions from different pelvic structures through genic and protein expression analysis; and a prospective mass spectrometry- based analysis of the total proteome and PTMome profile of EMT proteins in peritoneal endometriosis. For the first study, we observed two histologic patterns in the examined endometriotic lesions, a well-differentiated glandular pattern, and an undifferentiated glandular pattern, both coexisting in the same samples. The mRNA expression revealed no difference in E-cadherin expression between control and lesions samples. However, there was downregulation of N-cadherin and Snail mRNA expression, and an upregulation of ZEB1 gene expression in endometriotic lesions. Immunohistochemical analysis revealed a higher frequency of low E-cadherin expression, associated with a higher frequency of high N-cadherin and membrane -catenin expression in endometriosis. All groups had low expression of nuclear -catenin. Vimentin was positive in all samples, with a discreet increased expression in endometriotic lesions. This data corroborates the hypothesis that endometriotic epithelial cells demonstrate a partial EMT phenotype, expressing both epithelial and mesenchymal markers. In the second study, our results showed no major differences in the overall expression profile of the analyzed groups, but the endometriotic lesions presented 276 significant regulations in the total proteome, 2240 in the phosphoproteome and 481 in the sialic acid (SA) N- glycoproteome when compared with the endometrium of endometriosis patients. A deeper analysis of those regulations reveled Gene Ontology (GO) biological processes and KEGG pathways enriched for actin cytoskeleton contraction, positive regulation of cell-substrate adhesion, ECM-receptor interaction and focal adhesion in the total proteome and phosphoproteome. E-cadherin, p120-catenin and Twist 2 presented downregulated phosphosites in endometriotic lesions that can potentially lead to increased cell adhesion. Actin- binding proteins like actinin, filamin A and vinculin presented upregulated phosphosites that could disrupt the cell cytoskeleton and decrease cell adhesion. That balance between increased and decreased cell adhesion in endometriotic lesions provide us important insights about the cellular changes needed for the development and progression of endometriosis. In conclusion our findings highlight the significance of phosphorylation and SA N-glycosylation in preserving the integrity of adherens junctions in endometriosis, and offer new insights for future research into the influence of post-translational modifications on the regulation of epithelial- mesenchymal transition for the establishment of endometriosis.