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1.
J Pathol ; 254(3): 254-264, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33797756

RESUMO

Hereditary diffuse gastric cancer (HDGC) is a cancer syndrome caused by germline variants in CDH1, the gene encoding the cell-cell adhesion molecule E-cadherin. Loss of E-cadherin in cancer is associated with cellular dedifferentiation and poor prognosis, but the mechanisms through which CDH1 loss initiates HDGC are not known. Using single-cell RNA sequencing, we explored the transcriptional landscape of a murine organoid model of HDGC to characterize the impact of CDH1 loss in early tumourigenesis. Progenitor populations of stratified squamous and simple columnar epithelium, characteristic of the mouse stomach, showed lineage-specific transcriptional programs. Cdh1 inactivation resulted in shifts along the squamous differentiation trajectory associated with aberrant expression of genes central to gastrointestinal epithelial differentiation. Cytokeratin 7 (CK7), encoded by the differentiation-dependent gene Krt7, was a specific marker for early neoplastic lesions in CDH1 carriers. Our findings suggest that deregulation of developmental transcriptional programs may precede malignancy in HDGC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Caderinas/genética , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica/genética , Predisposição Genética para Doença/genética , Neoplasias Gástricas/genética , Animais , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Organoides , Análise de Célula Única , Neoplasias Gástricas/patologia , Transcriptoma
2.
Lancet Oncol ; 21(8): e386-e397, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32758476

RESUMO

Hereditary diffuse gastric cancer (HDGC) is an autosomal dominant cancer syndrome that is characterised by a high prevalence of diffuse gastric cancer and lobular breast cancer. It is largely caused by inactivating germline mutations in the tumour suppressor gene CDH1, although pathogenic variants in CTNNA1 occur in a minority of families with HDGC. In this Policy Review, we present updated clinical practice guidelines for HDGC from the International Gastric Cancer Linkage Consortium (IGCLC), which recognise the emerging evidence of variability in gastric cancer risk between families with HDGC, the growing capability of endoscopic and histological surveillance in HDGC, and increased experience of managing long-term sequelae of total gastrectomy in young patients. To redress the balance between the accessibility, cost, and acceptance of genetic testing and the increased identification of pathogenic variant carriers, the HDGC genetic testing criteria have been relaxed, mainly through less restrictive age limits. Prophylactic total gastrectomy remains the recommended option for gastric cancer risk management in pathogenic CDH1 variant carriers. However, there is increasing confidence from the IGCLC that endoscopic surveillance in expert centres can be safely offered to patients who wish to postpone surgery, or to those whose risk of developing gastric cancer is not well defined.


Assuntos
Síndromes Neoplásicas Hereditárias , Neoplasias Gástricas , Humanos
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.
Mol Cancer ; 17(1): 112, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-30068367

RESUMO

The aim of this study was to uncover the pathogenic relevance and the underlying molecular mechanism of a novel CDH1 variant found in a Hereditary Diffuse Gastric Cancer family (p.L13_L15del), which affects the signal peptide of E-cadherin without changing the remaining predicted sequence. We verified that p.L13_L15del cells yield low levels of E-cadherin, decreased cell adhesion and enhanced cell invasion. Further, we demonstrated that the disruption of the highly conserved hydrophobic core of the signal peptide hampers the binding of cellular components crucial for E-cadherin translation and translocation into the endoplasmic reticulum, constituting a new molecular basis for the loss of a tumour suppressor gene causative of hereditary cancer.


Assuntos
Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Sinais Direcionadores de Proteínas , Neoplasias Gástricas/genética , Adulto , Adesão Celular , Retículo Endoplasmático/metabolismo , Feminino , Variação Genética , Humanos , Masculino , Transporte Proteico , Análise de Sequência de DNA , Neoplasias Gástricas/metabolismo
5.
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
6.
Cancers (Basel) ; 14(7)2022 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-35406381

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 the cancer syndrome hereditary diffuse gastric cancer (HDGC). CDH1-deficient gastric cancers exhibit high AKT serine/threonine kinase 3 (AKT3) expression, but specific drugs against this AKT isoform are not available. We therefore used two publicly available datasets to identify AKT3-associated genes which could be used to indirectly target AKT3. Reactome analysis identified an enrichment of extracellular matrix remodelling genes in AKT3-high gastric cancers. Of the 51 genes that were significantly correlated with AKT3 (but not AKT1), discoidin domain receptor tyrosine kinase 2 (DDR2) showed the strongest positive association. Treatment of isogenic human cells and mouse gastric and mammary organoids with dasatinib, a small molecule inhibitor of multiple kinases including SRC, BCR-ABL and DDR2, preferentially slowed the growth and induced apoptosis of E-cadherin-deficient cells. Dasatinib treatment also preferentially slowed the growth of gastric and mammary organoids harbouring both Cdh1 and Tp53 mutations. In organoid models, dasatinib treatment was associated with decreased phosphorylation of total AKT, with a stronger effect seen in Cdh1-deficient organoids. Treatment with combinations of dasatinib and an inhibitor of AKT, MK2206, enhanced the effect of dasatinib in breast MCF10A cells. In conclusion, targeting the DDR2-SRC-AKT3 axis with dasatinib represents a promising approach for the chemoprevention and chemotherapy of gastric and breast cancers lacking E-cadherin.

7.
Cancers (Basel) ; 14(1)2021 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-35008266

RESUMO

Germline inactivating variants of CDH1 are causative of hereditary diffuse gastric cancer (HDGC), a cancer syndrome characterized by an increased risk of both diffuse gastric cancer and lobular breast cancer. Because loss of function mutations are difficult to target therapeutically, we have taken a synthetic lethal approach to identify targetable vulnerabilities in CDH1-null cells. We have previously observed that CDH1-null MCF10A cells exhibit a reduced rate of endocytosis relative to wildtype MCF10A cells. To determine whether this deficiency is associated with wider vulnerabilities in vesicle trafficking, we screened isogenic MCF10A cell lines with known inhibitors of autophagy, endocytosis, and sphingolipid metabolism. Relative to wildtype MCF10A cells, CDH1-/- MCF10A cells showed significantly greater sensitivity to several drugs targeting these processes, including the autophagy inhibitor chloroquine, the endocytosis inhibitors chlorpromazine and PP1, and the sphingosine kinase 1 inhibitor PF-543. Synthetic lethality was confirmed in both gastric and mammary organoid models of CDH1 loss, derived from CD44-Cre/Cdh1fl/fl/tdTomato mice. Collectively, these results suggest that both sphingolipid metabolism and vesicle trafficking represent previously unrecognised druggable vulnerabilities in CDH1-null cells and may lead to the development of new therapies for HDGC.

8.
Cancers (Basel) ; 14(1)2021 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-35008338

RESUMO

Inactivating germline mutations in the CDH1 gene (encoding the E-cadherin protein) are the genetic hallmark of hereditary diffuse gastric cancer (HDGC), and somatic CDH1 mutations are an early event in the development of sporadic diffuse gastric cancer (DGC) and lobular breast cancer (LBC). In this study, histone deacetylase (HDAC) inhibitors were tested for their ability to preferentially inhibit the growth of human cell lines (MCF10A and NCI-N87) and murine organoids lacking CDH1 expression. CDH1-/- breast and gastric cells were more sensitive to the pan-HDAC inhibitors entinostat, pracinostat, mocetinostat and vorinostat than wild-type cells, with an elevated growth inhibition that was, in part, attributable to increased apoptosis. CDH1-null cells were also sensitive to more class-specific HDAC inhibitors, but compared to the pan-inhibitors, these effects were less robust to genetic background. Increased sensitivity to entinostat was also observed in gastric organoids with both Cdh1 and Tp53 deletions. However, the deletion of Tp53 largely abrogated the sensitivity of the Cdh1-null organoids to pracinostat and mocetinostat. Finally, entinostat enhanced Cdh1 expression in heterozygous Cdh1+/- murine organoids. In conclusion, entinostat is a promising drug for the chemoprevention and/or treatment of HDGC and may also be beneficial for the treatment of sporadic CDH1-deficient cancers.

9.
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.

10.
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
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