Abstract
BackgroundThe E-cadherin gene (CDH1) is frequently mutated in diffuse gastric cancer and lobular breast cancer, and germline mutations predispose to the cancer syndrome Hereditary Diffuse Gastric Cancer. We are taking a synthetic lethal approach to identify druggable vulnerabilities in CDH1-mutant cancers.MethodsDensity distributions of cell viability data from a genome-wide RNAi screen of isogenic MCF10A and MCF10A-CDH1−/− cells were used to identify protein classes affected by CDH1 mutation. The synthetic lethal relationship between selected protein classes and E-cadherin was characterised by drug sensitivity assays in both the isogenic breast MCF10A cells and CDH1-isogenic gastric NCI-N87. Endocytosis efficiency was quantified using cholera toxin B uptake. Pathway metagene expression of 415 TCGA gastric tumours was statistically correlated with CDH1 expression.ResultsMCF10A-CDH1−/− cells showed significantly altered sensitivity to RNAi inhibition of groups of genes including the PI3K/AKT pathway, GPCRs, ion channels, proteosomal subunit proteins and ubiquitinylation enzymes. Both MCF10A-CDH1−/− and NCI-N87-CDH1−/− cells were more sensitive than wild-type cells to compounds that disrupted plasma membrane composition and trafficking, but showed contrasting sensitivities to inhibitors of actin polymerisation and the chloride channel inhibitor NS3728. The MCF10A-CDH1−/− cell lines showed reduced capacity to endocytose cholera toxin B. Pathway metagene analysis identified 20 Reactome pathways that were potentially synthetic lethal in tumours. Genes involved in GPCR signalling, vesicle transport and the metabolism of PI3K and membrane lipids were strongly represented amongst the candidate synthetic lethal genes.ConclusionsE-cadherin loss leads to disturbances in receptor signalling and plasma membrane trafficking and organisation, creating druggable vulnerabilities.
Highlights
CDH1 is a tumour suppressor gene that encodes E-cadherin, a homophilic cell-to-cell adhesion protein that is localised to the adherens junction on the basolateral surface of epithelial cells
To elucidate the mechanisms underpinning synthetic lethality in E-cadherin-deficient cells, we have extended our analysis of the siRNA screen data to include genes with the reverse effect to synthetic lethality, that is, genes whose siRNAs reduced the viability of MCF10A cells more than MCF10A-CDH1−/− cells
We hypothesise that disorganisation of the cortical cytoskeleton in E-cadherindeficient cells undermines the efficiency of these different processes, establishing numerous vulnerabilities that can be exposed with specific RNAi and chemical antagonists
Summary
CDH1 is a tumour suppressor gene that encodes E-cadherin, a homophilic cell-to-cell adhesion protein that is localised to the adherens junction on the basolateral surface of epithelial cells. To elucidate the mechanisms underpinning E-cadherin’s diverse SL interactions and the subsequent vulnerabilities in CDH1-null cancers, we have extended our analysis of isogenic cell line pairs and applied a novel bioinformatic approach that statistically queries tumour genome-wide expression data for potential SL pathways. Results MCF10A-CDH1−/− cells showed significantly altered sensitivity to RNAi inhibition of groups of genes including the PI3K/AKT pathway, GPCRs, ion channels, proteosomal subunit proteins and ubiquitinylation enzymes. Both MCF10ACDH1−/− and NCI-N87-CDH1−/− cells were more sensitive than wild-type cells to compounds that disrupted plasma membrane composition and trafficking, but showed contrasting sensitivities to inhibitors of actin polymerisation and the chloride channel inhibitor NS3728. Conclusions E-cadherin loss leads to disturbances in receptor signalling and plasma membrane trafficking and organisation, creating druggable vulnerabilities
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