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

Abstract

In hereditary diffuse gastric cancer (HDGC), CDH1 germline gene alterations are causative events in 30% of the cases. In 20% of HDGC families, CDH1 germline mutations are of the missense type and the mutation carriers constitute a problem in terms of genetic counseling and surveillance. To access the pathogenic relevance of missense mutations, we have previously developed an in vitro method to functionally characterize them. Pathogenic E-cadherin missense mutants fail to aggregate and become more invasive, in comparison with cells expressing the wild-type (WT) protein. Herein, our aim was to develop a complementary method to unravel the pathogenic significance of E-cadherin missense mutations. We used cells stably expressing WT E-cadherin and seven HDGC-associated mutations (five intracellular and two extracellular) and studied by proximity ligation assays (PLA) how these mutants bind to fundamental regulators of E-cadherin function and trafficking. We focused our attention on the interaction with: p120, b-catenin, PIPKIc and Hakai. We showed that cytoplasmic E-cadherin mutations affect the interaction of one or more binding partners, compromising the E-cadherin stability at the plasma membrane and likely affecting the adhesion complex competence. In the present work, we demonstrated that the study of the interplay between E-cadherin and its binding partners, using PLA, is an easy, rapid, quantitative and highly reproducible technique that can be applied in routine labs to verify the pathogenicity of E-cadherin missense mutants for HDGC diagnosis, especially those located in the intracellular domain of the protein. Keywords: HDGC; E-cadherin; CDH1 mutations; E-cadherin trafficking; E-cadherin binding partners; diagnostic method INTRODUCTIONHereditary diffuse gastric cancer (HDGC) is an autosomal dominant cancer syndrome characterized by a high risk of developing diffuse gastric cancer 1-3 and lobular breast cancer 4-6 during life-time. CDH1 germline gene alterations (mutations or deletions), resulting in E-cadherin inactivation, are the only causative events described till now and were identified in approximately 30% of HDGC cases. 2,3,7 To date, 122 different germline mutations have been described in these families, 8 being the majority of them of the nonsense type, leading to alternative premature termination codons. 3 This type of CDH1 mutant transcripts is commonly downregulated by nonsensemediated decay leading to E-cadherin loss of function 9 and these patients are considered high-risk carriers and are counseled to perform prophylactic total gastrectomy. 10 In about 20% of HDGC families, carriers show CDH1 germline missense mutations 11 and, in contrast to truncating mutations, their pathogenic significance is not straightforward, therefore constituting a problem in terms of genetic counseling and surveillance.In 2004, Fitzgerald and Caldas 10 suggested that the significance of CDH1 missense mutations should be assessed in at least four affected members within a HDGC family in combination with functional and

Genomic landscape and genetic heterogeneity in gastric adenocarcinoma revealed by whole-genome sequencing

“…Two patients (T28 and T62) with diffuse-type GC showed CDH1 germline variant 2494G4A (heterozygous in both PBMC and tumour, corresponds to p.Val832Met), which has previously been described and characterized as pathogenic 20,21 . This is a known variant implicated in hereditary diffuse-type GC.…”

Interaction between Wnt/β-catenin pathway and microRNAs regulates epithelial-mesenchymal transition in gastric cancer (Review)

“…Moreover, both Wnt10A and Wnt10B in GC was tied up with activation of the β-catenin-TCF signaling pathway. CDH1 gene was verified to encode E-cadherin so as to sustain epithelial cell-cell adhesion and suppress tumor invasion while germline gene alterations of CDH1 had a causative role in ~30-50% of hereditary diffuse gastric cancer (HDGC) (61,62). In particular, non-mutated (second) CDH1 allele resulting chiefly from promoter hypermethylation was likely to disintegrate cell adherens junctions and was privy to loss of cell polarity, subsequent to which, β-catenin was activated and HDGC initiated (63,64).…”

Rescue of wild-type E-cadherin expression from nonsense-mutated cancer cells by a suppressor-tRNA

“…This technique has been successfully used by our group to distinguish pathogenic missense E-cadherin mutations from nonpathogenic or WT E-cadherin. 27 Following the same protocol, a striking increase was seen in the number of E-cadherin interactions with b-catenin and p120-catenin when sup-tRNA Arg was coexpressed with 1003int and 1003cDNA ( Figure 3) in comparison with that of cells expressing the same vectors lacking sup-tRNA Arg . These interaction levels were yet lower than those observed for E-cadherin WT-expressing cells.…”