E-cadherin Mutations Research Articles

From molecular classification to targeted therapeutics: the changing face of systemic therapy in metastatic gastroesophageal cancer.

Histological classification of adenocarcinoma or squamous cell carcinoma for esophageal cancer or using the Lauren classification for intestinal and diffuse type gastric cancer has limited clinical utility in the management of advanced disease. Germline mutations in E-cadherin (CDH1) or mismatch repair genes (Lynch syndrome) were identified many years ago but given their rarity, the identification of these molecular alterations does not substantially impact treatment in the advanced setting. Recent molecular profiling studies of upper GI tumors have added to our knowledge of the underlying biology but have not led to an alternative classification system which can guide clinician's therapeutic decisions. Recently the Cancer Genome Atlas Research Network has proposed four subtypes of gastric cancer dividing tumors into those positive for Epstein-Barr virus, microsatellite unstable tumors, genomically stable tumors, and tumors with chromosomal instability. Unfortunately to date, many phase III clinical trials involving molecularly targeted agents have failed to meet their survival endpoints due to their use in unselected populations. Future clinical trials should utilize molecular profiling of individual tumors in order to determine the optimal use of targeted therapies in preselected patients.

Abstract 1281: Identification of novel susceptibility genes in familial gastric cancer using next generation sequencing and identity-by-descent mapping

Abstract Gastric cancer is the second most common cause of cancer related deaths worldwide, and approximately 10% of gastric cancer cases show familiar clustering of the disease. It is crucial to identify genes that predispose to familial gastric cancer because such families can undergo screening and cancer risk-reduction approaches, such as prophylactic gastrectomy. Furthermore, identification of new genes is also crucial to advance our understanding of the molecular basis of tumorigenesis. Till date, germline mutations in E-cadherin or CDH1 are the only known cause of Hereditary diffuse gastric cancer (HDGC), a form of familial gastric cancer syndrome. However, CDH1 mutations explain only a subset of familiar gastric cancer cases, indicating that many additional gastric cancer genes are still to be identified. Identification of cancer genes can be greatly facilitated if study samples are collected form isolated populations. These populations have simpler environments and genetic make-ups than that of large outbred groups and show larger founder effects, which allows easier and more tractable gene mapping that can later be portable in the worldwide population. In this study, we combine exome sequencing and identity by descent (IBD) analysis to identify novel, highly penetrant mutations in familial gastric cancer cases recruited in closely related and neighboring isolated populations from the Iberian Peninsula. We have already collected germline and tumor DNA samples from gastric cancer cases that belong to 32 families from the northwest regions of Portugal and Spain. All these cases and families are CDH1 mutation negative, and thus, potentially harbor mutations in unknown genes. We have completed exome sequencing of 16 of the index cases, and our preliminary data analysis has identified several interesting mutations that are in the phase of validation. Moving forward, we plan to complete exome sequencing of the remaining index cases as well as selected relatives from these families and validate mutations in a larger gastric cancer sample set. We will also be performing exome sequencing on tumor DNA that will enable us to identify loss of heterozygocity (LOH), as well as second hits and other molecular signatures in these tumor types. To our knowledge, our gene mapping effort in familial gastric cancer is one of the largest studies of its type, which can help identify novel gastric cancer genes and improve clinical management of gastric cancer cases. Citation Format: Ruta M. Sahasrabudhe, Paul Lott, Clara Ruiz-Ponte, Manuel Teixeira, Luis G. Carvajal-Carmona. Identification of novel susceptibility genes in familial gastric cancer using next generation sequencing and identity-by-descent mapping. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1281. doi:10.1158/1538-7445.AM2014-1281

Review of findings in prophylactic gynaecological specimens in Lynch syndrome with literature review and recommendations for grossing

Prophylactic hysterectomy with bilateral salpingo-oophorectomy is being increasingly undertaken in patients with Lynch syndrome (LS). The pathological features in such specimens are not well described and, unlike the SEE-FIM protocol for salpingo-oophorectomy specimens in BRCA1/2 mutation carriers and the gastrectomy grossing protocols for patients with CDH1 (E-cadherin) mutations, guidelines have not been devised for the grossing of prophylactic gynaecological specimens from LS patients. We aimed to review the pathological findings in a series of prophylactic gynaecological specimens from LS patients and develop guidelines for the grossing of these specimens. We reviewed the pathological findings in 25 prophylactic gynaecological specimens from LS patients and audited the grossing protocols in different centres across Ontario, Canada. We found a 32% incidence of endometrial carcinoma or a precursor lesion; the two endometrial cancers identified were low-grade, low-stage endometrioid adenocarcinomas. To address the absence of guidelines for pathological examination, we undertook a literature review of gynaecological malignancies and incidental findings in prophylactic specimens in LS patients. We provide recommendations regarding the grossing of such specimens which includes in-toto examination of the lower uterine segment, endometrium, ovaries and fallopian tubes with representative sampling of the cervix.

Functional Analysis Of The Mutations Of E Cadherin Exon 4-5, Exon 7, Exon 8, Exon 9 And Exon 16 In Sporadic Infiltrating Ductal Carcinoma Of The Breast

E-cadherin mutations are a major step in the metastatic progression. Mutated cadherins contributes to different aspects of cancer progression like drug resistance, angiogenesis, cancer cell invasion and metastasis. Thus cadherins can be regarded as a valuable diagnostic and prognostic indicators as well as potential therapeutic targets of cancer therapy in future. Identification of E-cadherin mutations and characterization of the mutated protein may improve our understanding of how cells escape from their normal location and spread through the body. The present study aimed at analyzing the functional consequences of such E- cadherin mutations in 50 patients with infiltrating ductal carcinoma of the breast.

Persistent Nausea and Vomiting in Pregnancy

Question: A 23-year-old pregnant woman at 27 weeks gestation presented with 10 weeks of persistent nausea, emesis, and a 20-pound weight loss. On examination, she appeared ill and had epigastric tenderness. Laboratory assessment was notable for anemia (hemoglobin level, 7 g/dL). Hyperemesis gravidarum was entertained, but upper endoscopy revealed circumferential, edematous narrowing of the gastric antrum with ulceration (Figure A, arrow). Gastric biopsies were obtained (Figure B). Computed tomography showed intrauterine pregnancy (Figure C, thin arrow) and a thickened gastric wall (Figure C, thick arrow). What is the diagnosis? Look on page 323 for the answer and see the Gastroenterology web site (www.gastrojournal.org) for more information on submitting your favorite image to Clinical Challenges and Images in GI. Gastric biopsy (Figure B) demonstrates adenocarcinoma with signet ring cells (arrows). The thick arrow in Figure C shows circumferential gastric thickening consistent with linitis plastica. The patient subsequently acknowledged her father perished with gastric cancer at age 45 years. Hereditary diffuse gastric cancer (HDGC) was confirmed by CDH-1 gene mutation testing. Vaginal delivery was expedited and chemotherapy initiated. Unfortunately, the patient succumbed to metastatic disease. Gastric cancer during pregnancy is rare, but should be considered in women with hyperemesis late in pregnancy. HDGC is an autosomal-dominant, inherited gastric cancer syndrome characterized by germline mutation of CDH-1 gene, which encodes for the cell-to-cell adhesion protein E-cadherin. It is a relatively rare cancer syndrome but may account for approximately 1% of all cases of gastric carcinoma in populations with a low incidence of gastric cancer such as the United States, Canada, and the United Kingdom.1Blair V. Martin I. Shaw D. et al.Hereditary diffuse gastric cancer: diagnosis and management.Clin Gastroenterol Hepatol. 2006; 4: 262-275Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar Diffuse-type gastric adenocarcinoma with signet-ring cells is the most common cancer in carriers of germline CDH-1 mutations. Genetic testing for mutations in the CDH-1 gene is widely available. However, only 30%–50% of families meeting diagnostic criteria carry a predisposing CDH-1 mutation.1Blair V. Martin I. Shaw D. et al.Hereditary diffuse gastric cancer: diagnosis and management.Clin Gastroenterol Hepatol. 2006; 4: 262-275Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar The revised International Gastric Cancer Linkage Consortium diagnostic criteria for CDH-1 mutation testing in HDGC include (1) 2 gastric cancer cases in family, 1 confirmed diffuse gastric cancer under age 50, (2) 3 confirmed diffuse gastric cancer cases in first- or second-degree relatives independent of age, (3) diffuse gastric cancer in an individual under age of 40, and (4) personal or family history of diffuse gastric cancer and lobular breast cancer, 1 diagnosed under the age of 50.2Fitzgerald R.C. Hardwick R. Huntsman D. et al.Hereditary diffuse gastric cancer: updated consensus guidelines for clinical management and directions for future research.J Med Genet. 2010; 47: 436-444Crossref PubMed Scopus (412) Google Scholar The other malignancy that occurs at increased frequency in HDGC families is lobular breast carcinoma. It is unclear why germline CDH-1 mutation predisposes predominantly to gastric cancer. There is a high lifetime risk for advanced gastric cancer in HDGC of approximately 70% in men and 80% in women by 80 years of age.3Pharoah P. Guilford P. Caldas C. Consortium TIGCLIncidence of gastric cancer and breast cancer in CDH1 (E-cadherin) mutation carriers from hereditary diffuse gastric cancer families.Gastroenterology. 2001; 121: 1348-1353Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar The risk for advanced gastric cancer is age-related and estimated to be <1% at 20 years and increasing to 4% at 30 years.3Pharoah P. Guilford P. Caldas C. Consortium TIGCLIncidence of gastric cancer and breast cancer in CDH1 (E-cadherin) mutation carriers from hereditary diffuse gastric cancer families.Gastroenterology. 2001; 121: 1348-1353Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar Because most patients have advanced stage gastric carcinoma at diagnosis, prophylactic gastrectomy is recommended in asymptomatic CDH-1 mutation carriers over the age of 20 years to prevent the development of this lethal cancer. If prophylactic gastrectomy is declined, intensive gastroscopic surveillance should be performed with the patient informed of the limitations for endoscopic detection of early lesions in HDGC.

Primary Gastric Linitis Plastica Presenting as Severe Non-Healing Helicobacter pylori-Associated Gastritis

Purpose: Gastric linitis plastica (GLP) is a rigid and constrictive thickening of the stomach secondary to diffuse infiltration of poorly differentiated adenocarcinoma. The risk factors leading to GLP are poorly understood, and believed to be secondary to underlying genetic predispositions, i.e. E-cadherin mutations. In contrast to intestinal-type gastric adenocarcinomas, diffuse-type GLP is not believed to be related to environmental risk factors, and has not been described as associated with Helicobacter pylori. We describe a case of GLP associated with, and presenting as severe H.pylori-associated gastritis. A 48 year old man with no past medical history was admitted for two weeks of new abdominal fullness with nausea/vomiting. CT abdomen/pelvis demonstrated dilated stomach with H. pyloric thickening. On upper endoscopy, several antral ulcers with surrounding severe pyloric congestion and edema were noted. Antral biopsies showed moderate chronic gastritis with marked reactive changes, and serum H. pylori IgG antibodies were positive. The patient was diagnosed with, and treated for severe H. pylori gastritis with ulcerative inflammation causing partial gastric outlet obstruction. The patient demonstrated slow clinical improvement, and was taken back to endoscopy at two weeks. Persistent ulcerations and congestion was noted, and endoscopic ultrasound (EUS) exam demonstrated diffuse infiltration of the gastric wall with loss of normal five layer anatomy. Pathology from deep bite biopsies and fine needle aspiration showed diffuse poorly differentiated adenocarcinoma. The patient was taken for a subtotal gastrectomy and discharged for outpatient adjuvant chemoradiation. The diffusely infiltrative nature of GLP leads to a rigid stomach that typically manifests endoscopically with a “leather bottle” appearance, and clinically as epigastric pain with nausea/vomiting, anorexia, and weight loss. This type of gastric cancer has a very poor prognosis, occurs in relatively young patients, and has not been associated with H. pylori gastritis and other environmental risk factors. In this unique case, the patient presented with non-healing ulcers with associated partial outlet obstruction that was attributed to an underlying H. pylori infection. This case highlights a number of salient issues: 1.) the inherent difficult diagnosis of GLP due to high frequency of mucosal sparing and false negative biopsies, 2.) the need to re-evaluate non-healing ulcers or patients with H. pylori not responding to traditional therapy, and 3.) the need for re-evaluation of a possible link between H. pylori and GLP.

Highlights of This Issue

Next-generation sequencing (NGS) can be applied to routine formalin-fixed paraffin-embedded clinical samples as a means to further classify the lobular phenotype of invasive breast cancer into tumors with and without E-cadherin (CDH1) mutations. When Ross and colleagues used this approach to evaluate invasive lobular carcinoma (ILC) with CDH1 mutation, they found a significant enrichment of genomic alterations in the targetable HER2 (ERBB2) gene. Of the 22 CDH1-mutated ILCs evaluated in this study, 6 (27%) featured ERBB2 alterations, only 1 of which was a classic gene amplification that was detectable by the routine slide-based ERBB2/HER2 tests in common practice (immunohistochemistry and FISH). The 5 gene amplification-negative ERBB2 alterations detected included 4 base substitutions (mutations) and 1 novel ERBB2-GRB7 gene fusion. Recent evidence suggests that ERBB2 mutations may be sensitive to standard anti-HER2 targeted therapies in breast and other cancers. Thus, given the relatively high incidence of treatable ERBB2 alterations in relapsed and metastatic CDH1-mutated ILC, the use of NGS to identify these alterations which cannot be detected by routine slide-based tests has the potential to change routine clinical practice.Treatment of patients with BRAFV600E colorectal cancer is a critical unmet medical need. To address this deficiency, Coffee and colleagues have developed a novel genetically engineered mouse model for BRAFV600E colorectal cancer. Their results suggest that BRAFV600E tumors are not sensitive to single-agent BRAF inhibition and implicate concurrent signaling through the parallel phosphoinositide 3-kinase (PI3K)/mTOR signaling axis. Using a combination of in vitro and in vivo approaches, the authors show that simultaneous targeting with both a BRAF inhibitor and a PI3K/mTOR inhibitor may be required for successful treatment in this population of colorectal cancer patients.Targeting “undruggable targets,” such as transcription factors, is a challenge. Varsano and colleagues address this task by focusing on altering the transcription factor's subcellular localization. Based on their findings that subcellular localization dictates ATF2 oncogenic (nuclear) or tumor-suppressive (cytosolic) function, they developed a high-content screen and used it to identify small molecules that promote ATF2 nuclear export. The hits identified were shown to sensitize melanoma to cell death along the protein kinase C signaling pathway, which drives ATF2 nuclear localization and oncogenic activity in these tumors. The proof-of-concept screen reported in this study provides the foundation for larger screening campaigns as well as further development and characterization of the small molecules that were identified.Jimeno and colleagues evaluated the Smoothened receptor inhibitor IPI-926 in advanced cancer patients with a focus on basal cell carcinoma of the skin. The uniqueness of this phase I trial is that IPI-926 is a direct derivative of cyclopamine, the plant alkaloid whose discovery enhanced our understanding of the hedgehog pathway (HhP). The HhP is dysregulated in a variety of solid tumors and is proposed to provide key growth and survival signals to tumor cells. IPI-926 was administered with a tolerable toxicity profile in patients with advanced solid tumors and has shown substantial efficacy in patients with basal cell carcinoma.

E-cadherin Polarity Is Determined by a Multifunction Motif Mediating Lateral Membrane Retention through Ankyrin-G and Apical-lateral Transcytosis through Clathrin

We report a highly conserved motif in the E-cadherin juxtamembrane domain that determines apical-lateral polarity by conferring both restricted mobility at the lateral membrane and transcytosis of apically mis-sorted protein to the lateral membrane. Mutations causing either increased lateral membrane mobility or loss of apical-lateral transcytosis result in partial mis-sorting of E-cadherin in Madin-Darby canine kidney cells. However, loss of both activities results in complete loss of polarity. We present evidence that residues required for restricted mobility mediate retention at the lateral membrane through interaction with ankyrin-G, whereas dileucine residues conferring apical-lateral transcytosis act through a clathrin-dependent process and function in an editing pathway. Ankyrin-G interaction with E-cadherin is abolished by the same mutations resulting in increased E-cadherin mobility. Clathrin heavy chain knockdown and dileucine mutation of E-cadherin both cause the same partial loss of polarity of E-cadherin. Moreover, clathrin knockdown causes no further change in polarity of E-cadherin with dileucine mutation but does completely randomize E-cadherin mutants lacking ankyrin-binding. Dileucine mutation, but not loss of ankyrin binding, prevented transcytosis of apically mis-sorted E-cadherin to the lateral membrane. Finally, neurofascin, which binds ankyrin but lacks dileucine residues, exhibited partial apical-lateral polarity that was abolished by mutation of its ankyrin-binding site but was not affected by clathrin knockdown. The polarity motif thus integrates complementary activities of lateral membrane retention through ankyrin-G and apical-lateral transcytosis of mis-localized protein through clathrin. Together, the combination of retention and editing function to ensure a high fidelity steady state localization of E-cadherin at the lateral membrane.

MiRNA expression profiling of 51 human breast cancer cell lines reveals subtype and driver mutation-specific miRNAs

IntroductionBreast cancer is a genetically and phenotypically complex disease. To understand the role of miRNAs in this molecular complexity, we performed miRNA expression analysis in a cohort of molecularly well-characterized human breast cancer cell lines to identify miRNAs associated with the most common molecular subtypes and the most frequent genetic aberrations.MethodsUsing a microarray carrying LNA™ modified oligonucleotide capture probes), expression levels of 725 human miRNAs were measured in 51 breast cancer cell lines. Differential miRNA expression was explored by unsupervised cluster analysis and was then associated with the molecular subtypes and genetic aberrations commonly present in breast cancer.ResultsUnsupervised cluster analysis using the most variably expressed miRNAs divided the 51 breast cancer cell lines into a major and a minor cluster predominantly mirroring the luminal and basal intrinsic subdivision of breast cancer cell lines. One hundred and thirteen miRNAs were differentially expressed between these two main clusters. Forty miRNAs were differentially expressed between basal-like and normal-like/claudin-low cell lines. Within the luminal-group, 39 miRNAs were associated with ERBB2 overexpression and 24 with E-cadherin gene mutations, which are frequent in this subtype of breast cancer cell lines. In contrast, 31 miRNAs were associated with E-cadherin promoter hypermethylation, which, contrary to E-cadherin mutation, is exclusively observed in breast cancer cell lines that are not of luminal origin. Thirty miRNAs were associated with p16INK4 status while only a few miRNAs were associated with BRCA1, PIK3CA/PTEN and TP53 mutation status. Twelve miRNAs were associated with DNA copy number variation of the respective locus.ConclusionLuminal-basal and epithelial-mesenchymal associated miRNAs determine the subdivision of miRNA transcriptome of breast cancer cell lines. Specific sets of miRNAs were associated with ERBB2 overexpression, p16INK4a or E-cadherin mutation or E-cadherin methylation status, which implies that these miRNAs may contribute to the driver role of these genetic aberrations. Additionally, miRNAs, which are located in a genomic region showing recurrent genetic aberrations, may themselves play a driver role in breast carcinogenesis or contribute to a driver gene in their vicinity. In short, our study provides detailed molecular miRNA portraits of breast cancer cell lines, which can be exploited for functional studies of clinically important miRNAs.

Expression of E-cadherin and KRAS mutation may serve as biomarkers of cetuximab-based therapy in metastatic colorectal cancer

Cetuximab (Cmab), a chimeric monoclonal antibody for targeting the epidermal growth factor receptor, has become one of the standard treatments for metastatic colorectal cancer (mCRC). However, only a small proportion of patients respond to Cmab, and it has been reported that KRAS mutation is a negative biomarker of response to Cmab therapy. The aim of this study was to detect additional biomarkers of response to Cmab therapy in patients with mCRC. We evaluated the effects of Cmab therapy in 36 patients with mCRC according to the Response Evaluation Criteria in Solid Tumors, and classified patients who achieved complete response, partial response or stable disease as responders, and patients who achieved progressive disease as non-responders. We retrospectively examined the difference between the two groups using KRAS analysis and immunohistochemistry to determine the expression of E-cadherin, p53 and Ki67. Nineteen patients were responders, while 17 patients were non-responders. KRAS status and expression of E-cadherin were significantly correlated with the effect of Cmab therapy. Moreover, the expression of E-cadherin was significantly correlated with the effect of Cmab therapy in KRAS wild-type patients. In KRAS mutant-type patients, the expression of E-cadherin did not significantly correlate with the effect of Cmab therapy, but all responders with KRAS mutant-type tumors expressed E-cadherin. Our results indicate that the expression of E-cadherin detected by immunohistochemistry may be a positive predictor of Cmab-based therapy in mCRC, and that a combination of E-cadherin immunohistochemistry and KRAS analysis may be a more sensitive biomarker than KRAS analysis alone.

Molecular Profiling of Gastric Cancer: Toward Personalized Cancer Medicine

Molecular Profiling of Gastric Cancer: Toward Personalized Cancer Medicine

Research progress of familial gastric cancer

The etiology of familial gastric cancer (FGC) are E-cadherin (CDH1) mutations,helicobacter pylori infection,etc.The median age of patients with FGC is 50-65 years old.The male-female sex ratio is 2 ∶ 1.The most of tumors of FGC locate in distal stomach and present diffuse type in histological classification.It is reported that the people who has a family history of gastric cancer especially for those parents has an increased risk of gastric cancer.It is difficult to find FGC in early stage by current technology.The preventive methods include eradication of helicobacter pylori and prophylactic total gastrectomy,etc. Key words: Stomach neoplasms; Family; Heredity

Molecular and Survival Differences between Familial and Sporadic Gastric Cancers

Mismatch repair (MMR) and germline E-cadherin (CDH1) mutations are two of the major pathways of carcinogenesis in familial gastric cancer (GC). A total of 260 sporadic and 66 familial GC patients were enrolled and molecular and survival differences were compared. Familial GC patients had earlier onset and were diagnosed at an earlier stage and had both a better 5-year overall survival rate and 3-year disease-free survival rate compared with sporadic GC patients. Only in diffuse type GC, the MSI-H phenotype and abnormal MMR protein expression were significantly higher in familial GC than in sporadic GC. In MSI-H GC, MLH1 promoter methylation was slightly higher in sporadic GC than familial GC (50% versus 23.1%), while the frequency of MMR gene mutation was slightly higher in familial GC than in sporadic GC (15.4% versus 3.1%). All of the patients with MMR gene mutation had diffuse type GC. Among familial GC patients with CDH1 mutation, most patients (72.3%) had diffuse type GC. In summary, for familial GC patients, we recommend screening of MSI status and CDH1 mutation especially for diffuse type GC. Because of the low incidence, mutation analysis of MMR gene might be considered in MSI-H familial GC with diffuse type only.

Unimpaired Skin Carcinogenesis in Desmoglein 3 Knockout Mice

The contribution of adherens junction inactivation, typically by downregulation or mutation of the transmembrane core component E-cadherin, to cancer progression is well recognized. In contrast, the role of the desmosomal cadherin components of the related cell-cell adhesion junction, the desmosome, in cancer development has not been well explored. Here, we use mouse models to probe the functional role of desmosomal cadherins in carcinogenesis. Because mice lacking the desmosomal cadherin Desmoglein 3 (Dsg3) have revealed a crucial role for Dsg3 in cell-cell adhesion in stratified epithelia, we investigate the consequence of Dsg3 loss in two models of skin carcinogenesis. First, using Dsg3−/− keratinocytes, we show that these cells display adhesion defects in vitro and compromised tumor growth in allograft assays, suggesting that Dsg3 enables tumor formation in certain settings. In contrast, using an autochthonous model for SCC development in response to chronic UVB treatment, we discover a surprising lack of enhanced tumorigenesis in Dsg3−/− mice relative to controls, unlike mice lacking the desmosomal component Perp. Accordingly, there is no defect in the apoptotic response to UVB or enhanced immune cell infiltration upon Dsg3 loss that could promote tumorigenesis. Thus, Dsg3 does not display a clear function as a tumor suppressor in these mouse skin cancer models. Continued unraveling of the roles of Dsg3 and other desmosomal constituents in carcinogenesis in different contexts will be important for ultimately improving cancer diagnosis, prognostication, and treatment.

E-cadherin promotor methylation and mutation are inversely related to motility capacity of breast cancer cells

Inactivation of the tumor suppressor E-cadherin is an important event during breast tumorigenesis, as its decreased expression is linked to aggressiveness and metastasis. However, the relationship between the different modes of E-cadherin inactivation (mutation versus promotor hypermethylation) and breast cancer cell behavior is incompletely understood. The high correlation between E-cadherin inactivation status and cell morphology in vitro suggests different biological roles for the two inactivation modes during breast tumorigenesis. Because E-cadherin has been linked to cell invasion and metastasis, and cell motility is a crucial prerequisite to form metastases, we here compared the cell motility capacities of breast cancer cell lines with known E-cadherin status. Using barrier migration assays and time-lapse microscopy, we analyzed the migratory capacity of nine well-characterized human breast cancer cell lines (MDA-MB-231, MCF-7, T47D, BT549, MPE600, CAMA-1, SUM159PT, SUM52PE, and SK-BR-3). This subset was chosen based on E-cadherin gene status (wild-type, mutated, and promotor hypermethylated): three cell lines of each group. In addition, cell proliferation assays were performed for all conditions, to dissect migratory from proliferative effects. In this study, we demonstrate an overt association between the mode of E-cadherin inactivation and cell migration. Promotor hypermethylated E-cadherin cell lines showed a higher migration capacity, while cell lines with mutated E-cadherin were less motile compared to wild-type E-cadherin cell lines. Migration induction by fibronectin and basic fibroblast growth factor did not alter the cell motility association differences. Cell proliferation assays showed that the associations found were not caused by proliferation differences. Inhibition and overexpression of E-cadherin as well as DNA demethylation confirmed the relationship between E-cadherin and breast cancer cell motility. Our results demonstrate an association between the mode of E-cadherin inactivation and migration of breast cancer cells, which justifies more detailed research on the role of E-cadherin inactivation in cell migration and metastasis.

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.

821 Analysis of Germline E-cadherin Mutations in Brazilian Patients With Hereditary Diffuse Gastric Cancer

821 Analysis of Germline E-cadherin Mutations in Brazilian Patients With Hereditary Diffuse Gastric Cancer

E-Cadherin Destabilization Accounts for the Pathogenicity of Missense Mutations in Hereditary Diffuse Gastric Cancer

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.

Advances in gastric cancer prevention.

Gastric cancer is a multifactorial neoplastic pathology numbering among its causes both environmental and genetic predisposing factors. It is mainly diffused in South America and South-East Asia, where it shows the highest morbility percentages and it is relatively scarcely diffused in Western countries and North America. Although molecular mechanisms leading to gastric cancer development are only partially known, three main causes are well characterized: Helicobacter pylori (H. pylori) infection, diet rich in salted and/or smoked food and red meat, and epithelial cadherin (E-cadherin) mutations. Unhealthy diet and H. pylori infection are able to induce in stomach cancer cells genotypic and phenotypic transformation, but their effects may be crossed by a diet rich in vegetables and fresh fruits. Various authors have recently focused their attention on the importance of a well balanced diet, suggesting a necessary dietary education starting from childhood. A constant surveillance will be necessary in people carrying E-cadherin mutations, since they are highly prone in developing gastric cancer, also within the inner stomach layers. Above all in the United States, several carriers decided to undergo a gastrectomy, preferring changing their lifestyle than living with the awareness of the development of a possible gastric cancer. This kind of choice is strictly personal, hence a decision cannot be suggested within the clinical management. Here we summarize the key points of gastric cancer prevention analyzing possible strategies referred to the different predisposing factors. We will discuss about the effects of diet, H. pylori infection and E-cadherin mutations and how each of them can be handled.

Expression of E-cadherin and Kras mutation may be biomarker of Cetuximab based therapy in metastatic colorectal cancer

Expression of E-cadherin and Kras mutation may be biomarker of Cetuximab based therapy in metastatic colorectal cancer