Hereditary diffuse gastric cancer is a cancer syndrome caused by germline mutations in the gene for the cell adhesion protein E‐cadherin (CDH1). E‐cadherin plays a central role in the maintenance of ...cell polarity and its loss during tumorigenesis is associated with poorly differentiated cancers and a poor prognosis. Hereditary diffuse gastric cancer is dominated by diffuse‐type gastric adenocarcinoma, often with signet ring cell morphology. Large numbers of stage T1a signet ring cell carcinomas exist in the stomachs of CDH1 mutation carriers from a young age, and these foci sometimes show enrichment to the transition zone between the body and antrum. Generally these signet ring cell carcinomas are hypoproliferative, lack Wnt pathway activation, and are relatively indolent. However, a small proportion of the T1a foci contain cells that are poorly differentiated, display mesenchymal features, and express activated c‐Src and its downstream targets. These same features are observed in more advanced stages of hereditary diffuse gastric cancer progression, suggesting that an epithelial–mesenchymal transition is required for tumor invasion beyond the muscularis mucosae. Hereditary diffuse gastric cancer initiation requires somatic down‐regulation of the second CDH1 allele, which in most cases is caused by DNA promoter hypermethylation. Subsequent to CDH1 down‐regulation, lost polarity in gastric stem or progenitor cells would be predicted to interfere with mitotic spindle orientation and the segregation of cell fate determinants. We predict that this disruption of cell division results in daughter cells being deposited in the lamina propria where their population expands and partially differentiates, resulting in the formation of foci of signet ring cells. (Cancer Sci 2009; 100: 1151–1157)
encodes E-cadherin, a key protein in adherens junctions. Given that E-cadherin is involved in major cellular processes such as embryogenesis and maintenance of tissue architecture, it is no surprise ...that deleterious effects arise from its loss of function. E-cadherin is recognised as a tumour suppressor gene, and it is well established that
genetic alterations cause diffuse gastric cancer and lobular breast cancer-the foremost manifestations of the hereditary diffuse gastric cancer syndrome. However, in the last decade, evidence has emerged demonstrating that
mutations can be associated with lobular breast cancer and/or several congenital abnormalities, without any personal or family history of diffuse gastric cancer. To date, no genotype-phenotype correlations have been observed. Remarkably, there are reports of mutations affecting the same nucleotide but inducing distinct clinical outcomes. In this review, we bring together a comprehensive analysis of
-associated disorders and germline alterations found in each trait, providing important insights into the biological mechanisms underlying E-cadherin's pleiotropic effects. Ultimately, this knowledge will impact genetic counselling and will be relevant to the assessment of risk of cancer development or congenital malformations in
mutation carriers.
E-cadherin is an adherens junction protein that forms homophilic intercellular contacts in epithelial cells while also interacting with the intracellular cytoskeletal networks. It has roles including ...establishment and maintenance of cell polarity, differentiation, migration and signalling in cell proliferation pathways. Its downregulation is commonly observed in epithelial tumours and is a hallmark of the epithelial to mesenchymal transition (EMT).
To improve our understanding of how E-cadherin loss contributes to tumorigenicity, we investigated the impact of its elimination from the non-tumorigenic breast cell line MCF10A. We performed cell-based assays and whole genome RNAseq to characterize an isogenic MCF10A cell line that is devoid of CDH1 expression due to an engineered homozygous 4 bp deletion in CDH1 exon 11.
The E-cadherin-deficient line, MCF10A CDH1-/- showed subtle morphological changes, weaker cell-substrate adhesion, delayed migration, but retained cell-cell contact, contact growth inhibition and anchorage-dependent growth. Within the cytoskeleton, the apical microtubule network in the CDH1-deficient cells lacked the radial pattern of organization present in the MCF10A cells and F-actin formed thicker, more numerous stress fibres in the basal part of the cell. Whole genome RNAseq identified compensatory changes in the genes involved in cell-cell adhesion while genes involved in cell-substrate adhesion, notably ITGA1, COL8A1, COL4A2 and COL12A1, were significantly downregulated. Key EMT markers including CDH2, FN1, VIM and VTN were not upregulated although increased expression of proteolytic matrix metalloprotease and kallikrein genes was observed.
Overall, our results demonstrated that E-cadherin loss alone was insufficient to induce an EMT or enhance transforming potential in the non-tumorigenic MCF10A cells but was associated with broad transcriptional changes associated with tissue remodelling.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Hereditary diffuse gastric cancer (HDGC) is the only known cancer syndrome that is dominated by gastric adenocarcinoma. HDGC is caused by germline mutation of the CDH1 gene that encodes the cell ...adhesion protein E-cadherin. Mutation carriers have a more than 70% lifetime risk of developing DGC and an elevated risk of lobular breast cancer. Intestinaltype gastric cancer is not part of the syndrome. Clinical management of HDGC involves predictive genetic testing beginning at or near 16 years of age. It is recommended that mutation carriers undergo prophylactic gastrectomy after about 20 years of age. Anatomical mapping has demonstrated that mutation carriers develop multifocal stage T1a signet ring cell carcinomas, with up to several hundred foci being observed in single stomachs. These foci develop following the somatic inactivation of the second CDH1 allele by mechanisms that include DNA promoter hypermethylation.
Diffuse gastric cancer and lobular breast cancer are aggressive malignancies that are frequently associated with inactivating mutations in the tumor suppressor gene CDH1. Synthetic lethal (SL) ...vulnerabilities arising from CDH1 dysfunction represent attractive targets for drug development. Recently, SLEC-11 (1) emerged as a SL lead in E-cadherin-deficient cells. Here, we describe our efforts to optimize 1. Overall, 63 analogues were synthesized and tested for their SL activity toward isogenic mammary epithelial CDH1-deficient cells (MCF10A-CDH1 –/– ). Among the 26 compounds with greater cytotoxicity, AL-GDa62 (3) was four-times more potent and more selective than 1 with an EC50 ratio of 1.6. Furthermore, 3 preferentially induced apoptosis in CDH1 –/– cells, and Cdh1 –/– mammary and gastric organoids were significantly more sensitive to 3 at low micromolar concentrations. Thermal proteome profiling of treated MCF10A-CDH1 –/– cell protein lysates revealed that 3 specifically inhibits TCOF1, ARPC5, and UBC9. In vitro, 3 inhibited SUMOylation at low micromolar concentrations.
The importance of loss of the cell-cell adhesion molecule E-cadherin (encoded by CDH1) to tumor progression is well established. However, CDH1 germ-line mutations predispose to the cancer ...susceptibility syndrome hereditary diffuse gastric cancer (HDGC), suggesting a role for E-cadherin in tumor initiation. The earliest indications of cancer in the stomachs of CDH1 mutation carriers are microscopic foci of intramucosal signet-ring cell carcinoma (SRCC; designated "eHDGC"). Here, we used N-methyl-N-nitrosourea (MNU) to promote gastric carcinogenesis in wild-type (wt) and cdh1(+/-) mice. MNU induced a variety of gastric tumors; however, intramucosal SRCC developed with an 11 times higher incidence in cdh1(+/-) mice compared with wt mice. The murine SRCC resembled the human eHDGCs in that they were hypoproliferative, lacked nuclear beta-catenin accumulation, and had reduced membrane localization of E-cadherin and its interacting junctional proteins. The down-regulation of E-cadherin in the murine SRCCs confirmed the importance of the second CDH1 hit to the initiation of diffuse gastric cancer. CDH1 promoter hypermethylation has been proposed to be a major second hit in advanced HDGC; however, its contribution to eHDGC was unknown. We thus examined a series of human eHDGC and detected CDH1 promoter methylation in 50% of foci. Promoter methylation was accompanied by reduced wt CDH1 mRNA levels in the foci and had a monoclonal pattern, consistent with an epigenetic initiation of disease. Together, these findings provide compelling evidence for a deficiency in cell-to-cell adhesion being sufficient to initiate diffuse gastric cancer in the absence of hyperproliferation and beta-catenin activation.
The cell-cell adhesion protein E-cadherin (CDH1) is a tumor suppressor that is required to maintain cell adhesion, cell polarity and cell survival signalling. Somatic mutations in CDH1 are common in ...diffuse gastric cancer (DGC) and lobular breast cancer (LBC). In addition, germline mutations in CDH1 predispose to the autosomal dominant cancer syndrome Hereditary Diffuse Gastric Cancer (HDGC). One approach to target cells with mutations in specific tumor suppressor genes is synthetic lethality. To identify novel synthetic lethal compounds for the treatment of cancers associated with E-cadherin loss, we have undertaken a high-throughput screening campaign of ~114,000 lead-like compounds on an isogenic pair of human mammary epithelial cell lines - with and without CDH1 expression. This unbiased approach identified 12 novel compounds that preferentially harmed E-cadherin-deficient cells. Validation of these compounds using both real-time and end-point viability assays identified two novel compounds with significant synthetic lethal activity, thereby demonstrating that E-cadherin loss creates druggable vulnerabilities within tumor cells. In summary, we have identified novel synthetic lethal compounds that may provide a new strategy for the prevention and treatment of both sporadic and hereditary LBC and DGC.
Purpose We investigated whether the RNA assay uRNA® and its derivative Cx bladder ® have greater sensitivity for the detection of bladder cancer than cytology, NMP22™ BladderChek™ and NMP22™ ELISA, ...and whether they are useful in risk stratification. Materials and Methods A total of 485 patients presenting with gross hematuria but without a history of urothelial cancer were recruited prospectively from 11 urology clinics in Australasia. Voided urine samples were obtained before cystoscopy. The sensitivity and specificity of the RNA tests were compared to cytology and the NMP22 assays using cystoscopy as the reference. The ability of Cx bladder to distinguish between low grade, stage Ta urothelial carcinoma and more advanced urothelial carcinoma was also determined. Results uRNA detected 41 of 66 urothelial carcinoma cases (62.1% sensitivity, 95% CI 49.3–73.8) compared with NMP22 ELISA (50.0%, 95% CI 37.4–62.6), BladderChek (37.9%, 95% CI 26.2–50.7) and cytology (56.1%, 95% CI 43.8–68.3). Cx bladder, which was developed on the study data, detected 82%, including 97% of the high grade tumors and 100% of tumors stage 1 or greater. The cutoffs for uRNA and Cx bladder were prespecified to give a specificity of 85%. The specificity of cytology was 94.5% (95% CI 91.9–96.5), NMP22 ELISA 88.0%, (95% CI 84.6–91.0) and BladderChek 96.4% (95% CI 94.2–98.0). Cx bladder distinguished between low grade Ta tumors and other detected urothelial carcinoma with a sensitivity of 91% and a specificity of 90%. Conclusions uRNA and Cx bladder showed improved sensitivity for the detection of urothelial carcinoma compared to the NMP22 assays. Stratification with Cx bladder provides a potential method to prioritize patients for the management of waiting lists.
Cell viability assays fulfill a central role in drug discovery studies. It is therefore important to understand the advantages and disadvantages of the wide variety of available assay methodologies. ...In this study, we compared the performance of three endpoint assays (resazurin reduction, CellTiter-Glo, and nuclei enumeration) and two real-time systems (IncuCyte and xCELLigence). Of the endpoint approaches, both the resazurin reduction and CellTiter-Glo assays showed higher cell viabilities when compared directly to stained nuclei counts. The IncuCyte and xCELLigence real-time systems were comparable, and both were particularly effective at tracking the effects of drug treatment on cell proliferation at sub-confluent growth. However, the real-time systems failed to evaluate contrasting cell densities between drug-treated and control-treated cells at full growth confluency. Here, we showed that using real-time systems in combination with endpoint assays alleviates the disadvantages posed by each approach alone, providing a more effective means to evaluate drug toxicity in monolayer cell cultures. Such approaches were shown to be effective in elucidating the toxicity of synthetic lethal drugs in an isogenic pair of MCF10A breast cell lines.
PDF
