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E-cadherin-deficient cells have synthetic lethal vulnerabilities in plasma membrane organisation, dynamics and function.

ABSTRACT: BACKGROUND:The 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. METHODS:Density 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. 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 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. CONCLUSIONS:E-cadherin loss leads to disturbances in receptor signalling and plasma membrane trafficking and organisation, creating druggable vulnerabilities.

SUBMITTER: Godwin TD

PROVIDER: S-EPMC6394693 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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E-cadherin-deficient cells have synthetic lethal vulnerabilities in plasma membrane organisation, dynamics and function.

Godwin Tanis D TD Kelly S Thomas ST Brew Tom P TP Bougen-Zhukov Nicola M NM Single Andrew B AB Chen Augustine A Stylianou Cassie E CE Harris Lawrence D LD Currie Sophie K SK Telford Bryony J BJ Beetham Henry G HG Evans Gary B GB Black Michael A MA Guilford Parry J PJ

Gastric cancer : official journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association 20180731 2

<h4>Background</h4>The 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.<h4>Methods</h4>Density distributions of cell viability data from a genome-wide RNAi screen of isogenic MCF10A and MCF10A-CDH1<sup>-/-</sup> cells were used to identify protein classes ...[more]

PMID: 30066183

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Project description:BackgroundGenome-wide loss-of-function profiling is widely used for systematic identification of genetic dependencies in cancer cells; however, the poor reproducibility of RNA interference (RNAi) screens has been a major concern due to frequent off-target effects. Currently, a detailed understanding of the key factors contributing to the sub-optimal consistency is still a lacking, especially on how to improve the reliability of future RNAi screens by controlling for factors that determine their off-target propensity.MethodsWe performed a systematic, quantitative analysis of the consistency between two genome-wide shRNA screens conducted on a compendium of cancer cell lines, and also compared several gene summarization methods for inferring gene essentiality from shRNA level data. We then devised novel concepts of seed essentiality and shRNA family, based on seed region sequences of shRNAs, to study in-depth the contribution of seed-mediated off-target effects to the consistency of the two screens. We further investigated two seed-sequence properties, seed pairing stability, and target abundance in terms of their capability to minimize the off-target effects in post-screening data analysis. Finally, we applied this novel methodology to identify genetic interactions and synthetic lethal partners of cancer drivers, and confirmed differential essentiality phenotypes by detailed CRISPR/Cas9 experiments.ResultsUsing the novel concepts of seed essentiality and shRNA family, we demonstrate how genome-wide loss-of-function profiling of a common set of cancer cell lines can be actually made fairly reproducible when considering seed-mediated off-target effects. Importantly, by excluding shRNAs having higher propensity for off-target effects, based on their seed-sequence properties, one can remove noise from the genome-wide shRNA datasets. As a translational application case, we demonstrate enhanced reproducibility of genetic interaction partners of common cancer drivers, as well as identify novel synthetic lethal partners of a major oncogenic driver, PIK3CA, supported by a complementary CRISPR/Cas9 experiment.ConclusionsWe provide practical guidelines for improved design and analysis of genome-wide loss-of-function profiling and demonstrate how this novel strategy can be applied towards improved mapping of genetic dependencies of cancer cells to aid development of targeted anticancer treatments.

Dataset Information

E-cadherin-deficient cells have synthetic lethal vulnerabilities in plasma membrane organisation, dynamics and function.

Publications

E-cadherin-deficient cells have synthetic lethal vulnerabilities in plasma membrane organisation, dynamics and function.

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