E-cadherin germline missense mutations and cell phenotype: evidence for the independence of cell invasion on the motile capabilities of the cells

Abstract

In Hereditary Diffuse Gastric Cancer syndrome, E-cadherin germline mutations of the missense type harbour significant functional consequences. In this study, we have characterised the effect of T340A, A617T, A634V and V832M E-cadherin germline missense mutations on cell morphology, motility and proliferation. Wild-type E-cadherin and A617T expressing cells have an epithelial-like morphology, with polarised cells migrating unidirectionally. T340A and A634V expressing cells, fibroblast-like, have a high motile phenotype. We show that this phenotype is dependent on an increased level of active RhoA. V832M expressing cells grow in piled-up structure of round cells, as an effect of the disturbance of the binding between alpha-catenin and beta-catenin. The destabilisation of the adhesion complex is shown to hamper the motile capabilities of these cells. We did not observe any effect of the E-cadherin mutations on cell proliferation. We show the existence of a genotype-phenotype correlation between different E-cadherin mutations and cell behaviour. However, we demonstrate that the ability of cells expressing the different E-cadherin mutations to invade is independent on their motile capabilities, providing evidence that motility is neither necessary nor sufficient for cells to invade. Our data give new insights into the understanding of the mechanisms linking invasion and E-cadherin mutations in diffuse gastric cancer.

The importance of E-cadherin binding partners to evaluate the pathogenicity of E-cadherin missense mutations associated to HDGC

“…21,47 Surprisingly, the mutant V832M, which was described to interact normally with b-catenin but not with a-catenin, 36,48 shows decreased association with b-catenin. This can be due to the increased sensibility and accuracy of PLA to detect protein-protein interactions when compared with co-immunoprecipitation technique.…”

“…E-cadherin mutants T340A, A634V, R749W, E757K, E781D, P799R and V832M found in the HDGC context, 16,18,19,21,[34][35][36][37][38] were constructed by site-directed mutagenesis in the entry vector CDH1pENTR 221 (Clone ID: IOH46767, Invitrogen, Grand Island, NY, USA), following the protocol described by Wang and Wilkinson. 41 By LR recombination reaction, the open reading frame was subcloned in the pEF6/Myc-His vector (Invitrogen).…”

“…50 Cells expressing E-cadherin mutations located at the extracellular domain are more motile, conferring a more aggressive in vitro phenotype, than those affecting the E-cadherin intracellular portion. 36,50 For that reason, E-cadherin mutations localized at the intracellular domain pose additional problems in clinical terms and make it urgent to improve the accuracy of the in vitro methods. Being aware of this, the structural E-cadherin model was recently updated and now covers the prodomain, the extracellular domain and the catenin binding domain.…”

“…We selected a panel of seven HDGC missense mutations (five intracellular and two extracellular) that have been proven to be pathogenic, 16,18,19,21,[34][35][36][37][38] and studied the ability of the mutant proteins to interact with direct E-cadherin binding partners using proximity ligation assays (PLA), a new tool to detect in situ proteinprotein interactions. 39,40 Fundamentally, PLA is a method where protein-recognition events are converted into detectable DNA molecules.…”

Frequent accumulation of nuclear E-cadherin and alterations in the Wnt signaling pathway in esophageal squamous cell carcinomas

“…[8][9][10] However, previous studies have reported that none of the studied germline E-cadherin mutations is responsible for an increase in the b-catenin/Lef transcriptional activity. 11,12 E-Cadherin Cytoplasmic Domain Induces Cyclin D1, AP-1 and STAT1/STAT2 Promoter Activities…”