Abstract
E-cadherin is critical for the maintenance of tissue architecture due to its role in cell-cell adhesion. E-cadherin mutations are the genetic cause of Hereditary Diffuse Gastric Cancer (HDGC) and missense mutations represent a clinical burden, due to the uncertainty of their pathogenic role. In vitro and in vivo, most mutations lead to loss-of-function, although the causal factor is unknown for the majority. We hypothesized that destabilization could account for the pathogenicity of E-cadherin missense mutations in HDGC, and tested our hypothesis using in silico and in vitro tools. FoldX algorithm was used to calculate the impact of each mutation in E-cadherin native-state stability, and the analysis was complemented with evolutionary conservation, by SIFT. Interestingly, HDGC patients harbouring germline E-cadherin destabilizing mutants present a younger age at diagnosis or death, suggesting that the loss of native-state stability of E-cadherin accounts for the disease phenotype. To elucidate the biological relevance of E-cadherin destabilization in HDGC, we investigated a group of newly identified HDGC-associated mutations (E185V, S232C and L583R), of which L583R is predicted to be destabilizing. We show that this mutation is not functional in vitro, exhibits shorter half-life and is unable to mature, due to premature proteasome-dependent degradation, a phenotype reverted by stabilization with the artificial mutation L583I (structurally tolerated). Herein we report E-cadherin structural models suitable to predict the impact of the majority of cancer-associated missense mutations and we show that E-cadherin destabilization leads to loss-of-function in vitro and increased pathogenicity in vivo.
Highlights
E-cadherin is a cell-cell adhesion glycoprotein comprised of five extracellular cadherin-type repeats, one transmembrane region and a highly conserved cytoplasmic tail [1,2]
Collection of E-cadherin sequence variants and PDBs E-cadherin variants associated to Hereditary Diffuse Gastric Cancer (HDGC) or EODGC were collected from the literature, and somatic variants were collected from the Catalogue of Somatic Mutations in Cancer (COSMIC) database
In vitro functional assays were performed for the abovementioned HDGC/EODGC mutations, and we found a perfect correlation between the functionality in vitro and the presence/ absence of structural impact: E185V and S232C retain the adhesive function of E-cadherin, and are able to form tight cellular aggregates, while L583R exhibits a clear scattered pattern, resembling Mock cells (Figure 3A), indicating that E185V and S232C are non-pathogenic and L583R is pathogenic
Summary
E-cadherin is a cell-cell adhesion glycoprotein comprised of five extracellular cadherin-type repeats, one transmembrane region and a highly conserved cytoplasmic tail [1,2]. E-cadherin is expressed primarily in epithelial cells and is the major component of Adherens Junctions (AJ). These junctions cluster, via homophilic interactions, through the extracellular domains of calciumdependent E-cadherin molecules, on the surface of homotypic neighbour cells. The role of E-cadherin in tumour development is well described, and its loss of expression is a hallmark in carcinomas [3]. Loss of E-cadherin expression is frequently associated to genetic events such as splice site and truncation mutations caused by insertions, deletions, and nonsense mutations, in addition to missense mutations [5]. Missense mutations are found in these two types of sporadic cancer and in synovial sarcomas [7]
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