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1.
Cancers (Basel) ; 11(9)2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31540244

RESUMO

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.

2.
Sci Rep ; 9(1): 12511, 2019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31467357

RESUMO

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.


Assuntos
Antígenos CD/genética , Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Caderinas/genética , Neoplasias Gástricas/genética , Antineoplásicos/síntese química , Antineoplásicos/química , Neoplasias da Mama/metabolismo , Caderinas/deficiência , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Mutação em Linhagem Germinativa , Humanos , Neoplasias Gástricas/metabolismo
3.
Gastric Cancer ; 22(2): 273-286, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30066183

RESUMO

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.


Assuntos
Caderinas/deficiência , Membrana Celular/metabolismo , Membrana Celular/patologia , Antígenos CD/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caderinas/genética , Linhagem Celular Tumoral , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , Transporte Proteico/fisiologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia
4.
J Biomol Screen ; 20(10): 1286-93, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26384394

RESUMO

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.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Avaliação de Medicamentos/métodos , Determinação de Ponto Final/métodos , Linhagem Celular , Linhagem Celular Tumoral , Sistemas Computacionais , Humanos
5.
Mol Cancer Ther ; 14(5): 1213-23, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25777964

RESUMO

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.


Assuntos
Neoplasias da Mama/genética , Caderinas/deficiência , RNA Interferente Pequeno/farmacologia , Receptores Acoplados a Proteínas G/genética , Neoplasias Gástricas/genética , Antígenos CD , Azepinas/farmacologia , Benzamidas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Descoberta de Drogas , Feminino , Humanos , Imidazóis/farmacologia , Toxina Pertussis/farmacologia , Pirazóis/farmacologia , Piridazinas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/tratamento farmacológico
6.
BMC Cancer ; 14: 552, 2014 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-25079037

RESUMO

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.


Assuntos
Mama/metabolismo , Caderinas/genética , Citoesqueleto/metabolismo , Transição Epitelial-Mesenquimal , Antígenos CD , Sequência de Bases , Mama/citologia , Caderinas/deficiência , Adesão Celular , Linhagem Celular , Feminino , Regulação da Expressão Gênica , Humanos , Análise de Sequência de RNA , Deleção de Sequência
7.
Virus Res ; 181: 27-34, 2014 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-24374268

RESUMO

Noroviruses are an emerging threat to public health, causing large health and economic costs, including at least 200,000 deaths annually. The inability to replicate in cell culture or small animal models has limited the understanding of the interaction between human noroviruses and their hosts. However, an alternative strategy to gain insights into norovirus pathogenesis is to study murine norovirus (MNV-1) that replicates in cultured macrophages. While the innate immune response is central to the resolution of norovirus disease, the adaptive immune response is required for viral clearance. The specific responses of macrophages and dendritic cells to infection drive the adaptive immune response, with chemokines playing an important role. In this study, we have conducted microarray analysis of RAW264.7 macrophages infected with MNV-1 and examined the changes in chemokine transcriptional expression during infection. While the majority of chemokines showed no change, there was specific up-regulation in chemokines reflective of a bias toward a Th1 response, specifically CCL2, CCL3, CCL4, CCL5, CXCL2, CXCL10 and CXCL11. These changes in gene expression were reflected in protein levels as determined by ELISA assay. This virus-induced chemokine response will affect the resolution of infection and may limit the humoral response to norovirus infection.


Assuntos
Quimiocinas/metabolismo , Norovirus/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Quimiocinas/genética , Análise por Conglomerados , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Interferons/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Regulação para Cima , Replicação Viral
8.
Nucleic Acids Res ; 33(4): 1169-81, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15731337

RESUMO

The human hepatitis B virus (HBV) has a compact genome encoding four major overlapping coding regions: the core, polymerase, surface and X. The polymerase initiation codon is preceded by the partially overlapping core and four or more upstream initiation codons. There is evidence that several mechanisms are used to enable the synthesis of the polymerase protein, including leaky scanning and ribosome reinitiation. We have examined the first AUG in the pregenomic RNA, it precedes that of the core. It initiates an uncharacterized short upstream open reading frame (uORF), highly conserved in all HBV subtypes, we designated the C0 ORF. This arrangement suggested that expression of the core and polymerase may be affected by this uORF. Initiation at the C0 ORF was confirmed in reporter constructs in transfected cells. The C0 ORF had an inhibitory role in downstream expression from the core initiation site in HepG2 cells and in vitro, but also stimulated reinitiation at the polymerase start when in an optimal context. Our results indicate that the C0 ORF is a determinant in balancing the synthesis of the core and polymerase proteins.


Assuntos
Códon de Iniciação , Regulação Viral da Expressão Gênica , Produtos do Gene pol/genética , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/genética , Iniciação Traducional da Cadeia Peptídica , RNA Viral/química , Regiões 5' não Traduzidas , Sequência de Bases , Linhagem Celular Tumoral , Sequência Conservada , Genes Reporter , Genoma Viral , Vírus da Hepatite B/classificação , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta
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