Endoplasmic reticulum quality control: a new mechanism of E-cadherin regulation and its implication in cancer

Abstract

E-cadherin is critical for the maintenance of tissue architecture and is a major component of adherens junctions. Its role in tumour development is well established, with many human carcinomas exhibiting E-cadherin loss at the invasive front. In many invasive carcinomas, the mechanisms leading to the loss of E-cadherin remains elusive. Here, we hypothesize that mechanisms of protein quality control play a key role in E-cadherin regulation. As a cell model system, we used CHO cells stably expressing E-cadherin germline missense mutations R749W and E757K, which are associated with hereditary diffuse gastric cancer. An abnormal pattern of E-cadherin expression was observed, with protein accumulating mainly in the endoplasmic reticulum (ER). We demonstrated that E-cadherin missense mutants are subjected to Endoplasmic Reticulum Quality Control (ERQC) and that their loss is due to ER-associated degradation. Treatment of these mutant cells with specific chemical chaperones restored E-cadherin to the cell membrane and rescued its function. We show that ERQC plays a major role in E-cadherin regulation and propose that overcoming this regulation may represent an approach to rescue E-cadherin expression and functionality in cancer.

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

“…14 Using functional in vitro assays, we demonstrated that cells expressing pathogenic CDH1 missense mutations fail to aggregate and become more invasive, in comparison with cells expressing wild-type (WT) E-cadherin, 5,12,[15][16][17][18][19][20][21][22] supporting their pathogenic relevance.…”

“…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).…”

“…5,12,[15][16][17][18][19]21 The slow aggregation assay showed that all the mutants have an impact on the cell-cell adhesion ability but the type of aggregation showed distinct phenotypes (Figure 7a). Comparing with the compact aggregates formed by the WT E-cadherin-expressing cells, some mutants display smaller cellular aggregates, such as mutants T340A, A634V, R749W, P799R and V832M, and others exhibited a close-to-isolated phenotype, as observed for mutants E757K and E781D, very similar to the phenotype of Mock cells.…”

“…21 These mutants raised a special interest because they affect the juxtamembrane domain of E-cadherin, reported as crucial for E-cadherin traffic dynamics. 23,24 E-cadherin expression and stability at the cell surface of epithelial cells are tightly regulated by post-translational mechanisms, including exocytosis and endocytosis.…”

“…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.…”