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 subconfluent 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.
Chronic obstructive pulmonary disease (COPD) affects the lives of an ever-growing number of people worldwide. The lack of understanding surrounding the pathophysiology of the disease and its progression has led to COPD becoming the third leading cause of death worldwide. COPD is incurable, with current treatments only addressing associated symptoms and sometimes slowing its progression, thus highlighting the need to develop novel treatments. However, this has been limited by the lack of experimental standardization within the respiratory disease research area. A lack of coherent animal models that accurately represent all aspects of COPD clinical presentation makes the translation of promising in vitro data to human clinical trials exceptionally challenging. Here, we review current knowledge within the COPD research field, with a focus on current COPD animal models. Moreover, we include a set of advantages and disadvantages for the selection of pre-clinical models for the identification of novel COPD treatments.
The CDH1 gene, which encodes the cell-to-cell adhesion protein E-cadherin, is frequently mutated in lobular breast cancer (LBC) and diffuse gastric cancer (DGC). However, because E-cadherin is a tumor suppressor protein and lost from the cancer cell, it is not a conventional drug target. To overcome this, we have taken a synthetic lethal approach to determine whether the loss of E-cadherin creates druggable vulnerabilities. We first conducted a genome-wide siRNA screen of isogenic MCF10A cells with and without CDH1 expression. Gene ontology analysis demonstrated that G-protein-coupled receptor (GPCR) signaling proteins were highly enriched among the synthetic lethal candidates. Diverse families of cytoskeletal proteins were also frequently represented. These broad classes of E-cadherin synthetic lethal hits were validated using both lentiviral-mediated shRNA knockdown and specific antagonists, including the JAK inhibitor LY2784544, Pertussis toxin, and the aurora kinase inhibitors alisertib and danusertib. Next, we conducted a 4,057 known drug screen and time course studies on the CDH1 isogenic MCF10A cell lines and identified additional drug classes with linkages to GPCR signaling and cytoskeletal function that showed evidence of E-cadherin synthetic lethality. These included multiple histone deacetylase inhibitors, including vorinostat and entinostat, PI3K inhibitors, and the tyrosine kinase inhibitors crizotinib and saracatinib.Together, these results demonstrate that E-cadherin loss creates druggable vulnerabilities that have the potential to improve the management of both sporadic and familial LBC and DGC. Mol Cancer Ther; 14(5); 1213-23. Ó2015 AACR.
The CDH1 gene, encoding the cell adhesion protein E-cadherin, is one of the most frequently mutated genes in gastric cancer and inactivating germline CDH1 mutations are responsible for hereditary diffuse gastric cancer syndrome (HDGC). Using cell viability assays, we identified that breast (MCF10A) and gastric (NCI-N87) cells lacking CDH1 expression are more sensitive to allosteric AKT inhibitors than their CDH1-expressing isogenic counterparts. Apoptosis priming and total apoptosis assays in the isogenic MCF10A cells confirmed the enhanced sensitivity of E-cadherin-null cells to the AKT inhibitors. In addition, two of these inhibitors, ARQ-092 and MK2206, preferentially targeted mouse-derived gastric Cdh1−/− organoids for growth arrest. AKT protein expression and activation (as measured by phosphorylation of serine 473) were differentially regulated in E-cadherin-null MCF10A and NCI-N87 cells, with downregulation in the normal breast cells, but upregulation in the gastric cancer cells. Bioinformatic analysis of the TCGA STAD dataset revealed that AKT3, but not AKT1 or AKT2, is upregulated in the majority of E-cadherin-deficient gastric cancers. In conclusion, allosteric AKT inhibitors represent a promising class of drugs for chemoprevention and chemotherapy of cancers with E-cadherin loss.
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.
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.
Interstitial lung diseases such as idiopathic pulmonary fibrosis (IPF) are caused by persistent micro-injuries to alveolar epithelial tissues accompanied by aberrant repair processes. IPF is currently treated with pirfenidone and nintedanib, compounds which slow the rate of disease progression but fail to target underlying pathophysiological mechanisms. The DNA repair protein 8-oxoguanine DNA glycosylase-1 (OGG1) has significant roles in the modulation of inflammation and metabolic syndromes. Currently, no pharmaceutical solutions targeting OGG1 have been utilized in the treatment of IPF. In this study we show Ogg1-targeting siRNA mitigates bleomycin-induced pulmonary fibrosis in male mice, highlighting OGG1 as a tractable target in lung fibrosis. The small molecule OGG1 inhibitor, TH5487, decreases myofibroblast transition and associated pro-fibrotic gene expressions in fibroblast cells. In addition, TH5487 decreases levels of pro-inflammatory mediators, inflammatory cell infiltration, and lung remodeling in a murine model of bleomycin-induced pulmonary fibrosis conducted in male C57BL6/J mice. OGG1 and SMAD7 interact to induce fibroblast proliferation and differentiation and display roles in fibrotic murine and IPF patient lung tissue. Taken together, these data suggest that TH5487 is a potentially clinically relevant treatment for IPF but further study in human trials is required.
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