Gianpaolo Suriano

Germline mutations of the E-cadherin(CDH1) and TP53 genes, rather than of RUNX3 and HPP1, contribute to genetic predisposition in German gastric cancer patients

Germline mutations of the cell adhesion molecule E-cadherin have been shown for the first time to underlie a hereditary diffuse type gastric cancer syndrome (HDGC) in Maori families,1 and subsequently have been reported in HDGC patients of various ethnic origins.2–9 Gastric cancer may also be associated with other hereditary tumour syndromes, which are mainly characterised by carcinomas of other organs. One of these syndromes is the HNPCC syndrome (hereditary nonpolyposis colorectal cancer syndrome), which is caused by germline mutations in DNA mismatch repair genes.10 Furthermore, gastric cancer has been observed in the context of the Li Fraumeni syndrome, a rare cancer syndrome due to germline mutations of the TP53 tumour suppressor gene,11–14 as well as in association with the FAP and Peutz–Jeghers syndrome.15 Despite known molecular genetic causes, contributing to a genetic predisposition to gastric cancer, a considerable number of familial cases have been reported that were not attributable to one of this hereditary syndromes, suggesting that unknown susceptibility genes for gastric cancer might exist. Putative tumour suppressor genes that are commonly inactivated in sporadic gastric cancers, such as the RUNX3 or HPP1 genes, are potential candidate susceptibility genes.16,17 In a previous study, we identified one E-cadherin germline mutation among seven diffuse type familial gastric cancer patients, indicating that in addition to E-cadherin , other genes might be involved in genetic predisposition to the disease in this patient group.4 There were three goals of the present study. First, we aimed to extend our analysis of E-cadherin germline mutations to a higher number of patients, to evaluate the contribution of germline mutations in this gene to German familial and early onset gastric cancer patients and to characterise identified missense mutations for their functional relevance. Secondly, we wished to analyse the role …

Helicobacter pylori induces gastric epithelial cell invasion in a c-Met and type IV secretion system-dependent manner

Helicobacter pylori interacts with gastric epithelial cells, activating signaling pathways important for carcinogenesis. In this study we examined the role of H. pylori on cell invasion and the molecular mechanisms underlying this process. The relevance of H. pylori cag pathogenicity island-encoded type IV secretion system (T4SS), CagA, and VacA for cell invasion was also investigated. We found that H. pylori induces AGS cell invasion in collagen type I and in Matrigel invasion assays. H. pylori-induced cell invasion requires the direct contact between bacteria and cancer cells. H. pylori-mediated cell invasion was dependent on the activation of the c-Met receptor and on increased MMP-2 and MMP-9 activity. The abrogation of the c-Met receptor using the specific NK4 inhibitor or the silencing of c-Met expression with small interference RNA suppressed both cell invasion and MMP activity. Studies with different H. pylori strains revealed that cell invasion, c-Met tyrosine phosphorylation, and increased MMP-2 and MMP-9 activity were all dependent on the presence of a functional bacterial T4SS, but not on VacA cytotoxicity. Our findings demonstrate that H. pylori strains with a functional T4SS stimulate gastric epithelial cell invasion through a c-Met-dependent signaling pathway that comprises an increase in MMP-2 and MMP-9 activity.

The intracellular E-cadherin germline mutation V832 M lacks the ability to mediate cell-cell adhesion and to suppress invasion.

E-cadherin germline missense mutations have been shown to be responsible for significant loss of protein activity. A new cytoplasmic E-cadherin germline missense mutation (V832 M) was recently identified in a hereditary diffuse gastric cancer (HDGC) Japanese family. This E-cadherin mutant was cloned in a Chinese hamster ovary cell model system and functionally characterized, in terms of aggregation and invasion. Cells expressing the germline V832M mutant fail to aggregate and invade into collagen, supporting the pathogenic role of this germline missense mutation in gastric cancer. We also tested the ability of this mutation to activate the TCF–LEF trascriptional activity, in comparison with three other E-cadherin missense mutations (T340A, A634V and A617T), associated to loss of E-cadherin function. All the E-cadherin mutants reduced TCF–LEF activation to a similar extent as the wild-type protein, suggesting that the oncogenic effect of the E-cadherin mutants is unlikely to be transmitted through a β-catenin-dependent activation of the WNT pathway.

Molecular pathology of familial gastric cancer, with an emphasis on hereditary diffuse gastric cancer

Gastric cancer is one of the major causes of cancer-related death worldwide. Familial clustering is observed in about 10% of cases; 1–3% of cases are hereditary. In the latter group, a syndrome which has been well characterised is hereditary diffuse gastric cancer; this is specifically associated with CDH1 (E-cadherin) germline mutations in about 30% of families. In this article, the state of the art of familial gastric cancer regarding the clinical, molecular and pathology features is reviewed, as well as the practical aspects for a correct diagnosis and clinical management.

A model to infer the pathogenic significance of CDH1 germline missense variants

Germline mutations of the E-cadherin gene (CDH1) are involved in the tumorigenesis of hereditary diffuse gastric cancer (HDGC). Recent studies have highlighted the lifesaving potential of total prophylactic gastrectomy for CDH1 germline mutation carriers. In this regard, CDH1 germline mutations of the missense type represent a clinical burden in genetic counseling, as their pathogenic relevance is not straightforward. In this work, we have outlined a possible multivariate approach to infer the significance of such variants. We reviewed all HDGC-associated E-cadherin germline missense mutations reported to date. The information collected included: co-segregation of the mutation within pedigrees, frequency in healthy population control, recurrence in independent families, and functional in vitro and in silico data. We used the neighbor-joining method to group mutations according to the collected information and assessed the robustness of mutation clusters with a bootstrap test. CDH1 germline missense variants were classified according to the parameters defined in the multivariate analysis. This analysis allowed the distribution of the variants into two distinct groups: neutral variants vs mutations. The model described in this study provides an important tool that can ultimately improve the genetic counseling offered to the carriers of the germline CDH1 missense variants.

α-Catenin mediates initial E-cadherin-dependent cell–cell recognition and subsequent bond strengthening

α-Catenin is essential in cadherin-mediated epithelium development and maintenance of tissues and in cancer progression and metastasis. However, recent studies question the conventional wisdom that α-catenin directly bridges the cadherin adhesion complex to the actin cytoskeleton. Therefore, whether α-catenin plays a direct role in cadherin-dependent cell adhesion is unknown. Here, single-molecule force spectroscopy measurements in cells depleted of α-catenin or expressing the hereditary diffuse gastric cancer associated V832M E-cadherin germ-line missense mutation show that α-catenin plays a critical role in cadherin-mediated intercellular recognition and subsequent multibond formation within the first 300 ms of cell contact. At short contact times, α-catenin mediates a 30% stronger interaction between apposing E-cadherin molecules than when it cannot bind the E-cadherin–β-catenin complex. As contact time between cells increases, α-catenin is essential for the strengthening of the first intercellular cadherin bond and for the ensuing formation of additional bonds between the cells, all without the intervention of actin. These results suggest that a critical decision to form an adhesion complex between 2 cells occurs within an extremely short time span and at a single-molecule level and identify a previously unappreciated role for α-catenin in these processes.

Epidermal growth factor receptor structural alterations in gastric cancer.

BackgroundEGFR overexpression has been described in many human tumours including gastric cancer. In NSCLC patients somatic EGFR mutations, within the kinase domain of the protein, as well as gene amplification were associated with a good clinical response to EGFR inhibitors. In gastric tumours data concerning structural alterations of EGFR remains controversial. Given its possible therapeutic relevance, we aimed to determine the frequency and type of structural alterations of the EGFR gene in a series of primary gastric carcinomas.MethodsDirect sequencing of the kinase domain of the EGFR gene was performed in a series of 77 primary gastric carcinomas. FISH analysis was performed in 30 cases. Association studies between EGFR alterations and the clinical pathological features of the tumours were performed.ResultsWithin the 77 primary gastric carcinomas we found two EGFR somatic mutations and several EGFR polymorphisms in exon 20. Six different intronic sequence variants of EGFR were also found. Four gastric carcinomas showed balanced polysomy or EGFR gene amplification. We verified that gastric carcinoma with alterations of EGFR (somatic mutations or copy number variation) showed a significant increase of tumour size (p = 0.0094) in comparison to wild-type EGFR carcinomas.ConclusionWe demonstrate that EGFR structural alterations are rare in gastric carcinoma, but whenever present, it leads to tumour growth. We considered that searching for EGFR alterations in gastric cancer is likely to be clinically important in order to identify patients susceptible to respond to tyrosine kinase inhibitors.

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

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.

Concurrent hypermethylation of gene promoters is associated with a MSI-H phenotype and diploidy in gastric carcinomas

Changes in the pattern of DNA methylation are among the most common alterations observed in human cancers, such as gastric carcinomas. We analysed in a series of 51 sporadic gastric carcinomas the methylation status of the promoter regions of the hMLH1, CDH1, MGMT and COX2 genes. We aimed to determine the frequency of CpG island hypermethylation and to find out whether the occurrence of concurrent hypermethylation is related to the clinicopathological features of the gastric carcinomas. Using methylation-sensitive restriction analysis/polymerase chain reaction (PCR) and methylation-specific PCR (MSP) strategies, we searched for the presence of hypermethylation on the promoter region of the 4 selected genes. All showed hypermethylation of their promoter regions with frequencies of 37, 51, 61 and 29% for hMLH1, CDH1, MGMT and COX2, respectively. Concurrent hypermethylation was more frequently observed in MSI-H (P=0.0005) and diploid (P=0.029) tumours. Hypermethylation of hMLH1 was associated with MSI-H tumours (P=0.0001), whereas hypermethylation of MGMT was associated with MSI-H (p=0.021) and diploid tumours (p=0.012). Our results indicate that concurrent hypermethylation is a common event in gastric cancer, suggesting that global methylation changes play an important role in the development of sporadic gastric carcinoma. Moreover, inactivation of different gene promoters by hypermethylation is significantly associated with microsatellite instability (MSI-H) and diploidy: hMLH1 determines MSI-H and MGMT the diploid status of gastric carcinomas.

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

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, β-catenin, PIPKIγ 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.