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1.
Nat Genet ; 52(2): 231-240, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932696

RESUMO

Pancreatic adenocarcinoma presents as a spectrum of a highly aggressive disease in patients. The basis of this disease heterogeneity has proved difficult to resolve due to poor tumor cellularity and extensive genomic instability. To address this, a dataset of whole genomes and transcriptomes was generated from purified epithelium of primary and metastatic tumors. Transcriptome analysis demonstrated that molecular subtypes are a product of a gene expression continuum driven by a mixture of intratumoral subpopulations, which was confirmed by single-cell analysis. Integrated whole-genome analysis uncovered that molecular subtypes are linked to specific copy number aberrations in genes such as mutant KRAS and GATA6. By mapping tumor genetic histories, tetraploidization emerged as a key mutational process behind these events. Taken together, these data support the premise that the constellation of genomic aberrations in the tumor gives rise to the molecular subtype, and that disease heterogeneity is due to ongoing genomic instability during progression.


Assuntos
Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/mortalidade , Estudos de Coortes , Feminino , Fator de Transcrição GATA6/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/mortalidade , Fenótipo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Smad4/genética
2.
PLoS One ; 15(1): e0228565, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31999789

RESUMO

Pancreatic cancer (PC) is a highly lethal malignancy, with a 5-year survival rate of 6%. Cancer gene panel testing is expected to allow selection of suitable therapeutic drugs in individual patients with PC and improve their prognosis. Although somatic mutations can be identified in formalin-fixed, paraffin-embedded samples derived from surgical specimen, the rate of surgical indication among patients with PC is only 20%. To acquire genome information with a less invasive method, we used rapid on-site evaluation (ROSE) specimens from endoscopic ultrasound-guided fine-needle aspiration. The present study aimed to retrospectively evaluate the utility of comprehensive cancer gene panel testing with ROSE specimens. DNA was extracted from preserved ROSE specimens of 26 patients diagnosed with PC between 2011 and 2017. DNA sequences of oncogenes and cancer-related genes were determined using the Ion AmpliSeq Comprehensive Caner Panel. We compared KRAS mutations between cancer gene panel testing by next-generation sequencing (NGS) and KRAS mutation analysis by polymerase chain reaction. The mean yield of DNA per extraction from ROSE specimens was 171 ng (range, 34-478 ng). On cancer gene panel testing, we noted KRAS mutations (92%), TP53 mutations (50%), CDKN2A mutations (15%), and SMAD4 mutations (31%). The concordance rate of KRAS mutations between cancer gene panel testing by NGS using ROSE specimens and KRAS mutation analysis by the companion diagnostics using residual materials was 81%. Among five cases of KRAS discordance, three showed KRAS mutations in cancer gene panel testing but not in KRAS mutation analysis. Cancer gene panel testing with ROSE specimens can help stratify unresectable PC patients without additional invasive approaches, and it can be used for therapeutic drug selection.


Assuntos
Aspiração por Agulha Fina Guiada por Ultrassom Endoscópico/métodos , Redes Reguladoras de Genes , Neoplasias Pancreáticas/patologia , Análise de Sequência de DNA/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Inibidor p16 de Quinase Dependente de Ciclina/genética , Feminino , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/genética , Sistemas Automatizados de Assistência Junto ao Leito , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas p21(ras)/genética , Estudos Retrospectivos , Proteína Smad4/genética , Proteína Supressora de Tumor p53/genética
3.
Acta Cytol ; 64(1-2): 103-123, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30970350

RESUMO

Advanced methods of molecular characterization have elucidated the genetic, epigenetic, and proteomic alterations associated with the broad spectrum of pancreatic disease, particularly neoplasia. Next-generation sequencing, in particular, has revealed the genomic diversity among pancreatic ductal adenocarcinoma, neuroendocrine and acinar tumors, solid pseudopapillary neoplasm, and other pancreatico-biliary neoplasms. Differentiating these entities from one another by morphologic analysis alone may be challenging, especially when examining the small quantities of diagnostic material inherent to cytologic specimens. In order to enhance the sensitivity and specificity of pancreatic cytomorphology, multiple diagnostic, prognostic, and predictive ancillary tests have been and continue to be developed. Although a great number of such tests have been developed for evaluation of specimens collected from cystic lesions and strictures, ancillary techniques also play a significant role in the evaluation of cytologic specimens obtained from solid lesions of the pancreas. Furthermore, while some tests have been developed to differentiate diagnostic entities from one another, others have been developed to simply identify dysplasia and malignancy. Ancillary studies are particularly important in the subset of cases for which cytomorphologic analysis provides a result that is equivocal or insufficient to guide clinical management. Selection of appropriate ancillary testing modalities requires familiarity with both their methodology and the molecular basis of the pancreatic diseases for which testing is being performed.


Assuntos
Carcinoma Ductal Pancreático/diagnóstico , Citodiagnóstico/métodos , Pâncreas/patologia , Neoplasias Pancreáticas/diagnóstico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , MicroRNAs/genética , Pâncreas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Sensibilidade e Especificidade , Proteína Smad4/genética , Proteína Smad4/metabolismo
4.
Cancer Sci ; 111(2): 739-748, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31799787

RESUMO

There are increased opportunities in oncology clinics to identify multiple pancreatic ductal adenocarcinomas (PDAC) that co-occur simultaneously or arise metachronously in the pancreatic parenchyma, yet their pathogenesis remains elusive. We hypothesized that two potential pathways, multicentric carcinogenesis and intrapancreatic metastasis, might contribute to forming multiple PDAC. Among 241 resected cases, we identified 20 cancer nodules from nine patients with multiple PDAC (six with synchronous PDAC, one with metachronous PDAC, and two with both synchronous and metachronous PDAC). Integrated clinical, pathological, and mutational analyses, using TP53 and SMAD4 immunostaining and targeted next-generation sequencing of 50 cancer-related genes, were conducted to examine the intertumor relationships. Four of the nine patients were assessed as having undergone multicentric carcinogenesis because of heterogeneity of immunohistochemical and/or mutation characteristics. In contrast, tumors in the other five patients showed intertumor molecular relatedness. Two of these five patients, available for matched sequencing data, showed two or more shared mutations. Moreover, all the smaller nodules in these five patients showed identical TP53 and SMAD4 expression patterns to the corresponding main tumors. Consequently, these five patients were considered to have undergone intrapancreatic metastasis. None of the five smaller nodules arising from intrapancreatic metastasis was accompanied by pancreatic intraepithelial neoplasia, and three of them were tiny (≤1mm). Patients whose tumors resulted from intrapancreatic metastasis appeared to have higher disease stages and worse outcome than those with tumors from multicentric carcinogenesis. Our results provide insight into pancreatic carcinogenesis, showing that the development of multiple PDAC involves distinct evolutionary paths that potentially affect patient prognosis.


Assuntos
Carcinoma Ductal Pancreático/cirurgia , Neoplasias Primárias Múltiplas/patologia , Segunda Neoplasia Primária/cirurgia , Neoplasias Pancreáticas/cirurgia , Proteína Smad4/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Biomarcadores Tumorais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Análise Mutacional de DNA , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Estadiamento de Neoplasias , Neoplasias Primárias Múltiplas/genética , Neoplasias Primárias Múltiplas/metabolismo , Neoplasias Primárias Múltiplas/cirurgia , Segunda Neoplasia Primária/metabolismo , Segunda Neoplasia Primária/patologia , Pancreatectomia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Análise de Sequência de DNA , Proteína Smad4/genética , Análise de Sobrevida , Proteína Supressora de Tumor p53/genética
5.
Gastroenterology ; 158(1): 238-252, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31585122

RESUMO

BACKGROUND & AIMS: We studied interactions among proteins of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, which interact with microbes, and transforming growth factor beta (TGFB) signaling pathway, which is often altered in colorectal cancer cells. We investigated mechanisms by which CEACAM proteins inhibit TGFB signaling and alter the intestinal microbiome to promote colorectal carcinogenesis. METHODS: We collected data on DNA sequences, messenger RNA expression levels, and patient survival times from 456 colorectal adenocarcinoma cases, and a separate set of 594 samples of colorectal adenocarcinomas, in The Cancer Genome Atlas. We performed shotgun metagenomic sequencing analyses of feces from wild-type mice and mice with defects in TGFB signaling (Sptbn1+/- and Smad4+/-/Sptbn1+/-) to identify changes in microbiota composition before development of colon tumors. CEACAM protein and its mutants were overexpressed in SW480 and HCT116 colorectal cancer cell lines, which were analyzed by immunoblotting and proliferation and colony formation assays. RESULTS: In colorectal adenocarcinomas, high expression levels of genes encoding CEACAM proteins, especially CEACAM5, were associated with reduced survival times of patients. There was an inverse correlation between expression of CEACAM genes and expression of TGFB pathway genes (TGFBR1, TGFBR2, and SMAD3). In colorectal adenocarcinomas, we also found an inverse correlation between expression of genes in the TGFB signaling pathway and genes that regulate stem cell features of cells. We found mutations encoding L640I and A643T in the B3 domain of human CEACAM5 in colorectal adenocarcinomas; structural studies indicated that these mutations would alter the interaction between CEACAM5 and TGFBR1. Overexpression of these mutants in SW480 and HCT116 colorectal cancer cell lines increased their anchorage-independent growth and inhibited TGFB signaling to a greater extent than overexpression of wild-type CEACAM5, indicating that they are gain-of-function mutations. Compared with feces from wild-type mice, feces from mice with defects in TGFB signaling had increased abundance of bacterial species that have been associated with the development of colon tumors, including Clostridium septicum, and decreased amounts of beneficial bacteria, such as Bacteroides vulgatus and Parabacteroides distasonis. CONCLUSION: We found expression of CEACAMs and genes that regulate stem cell features of cells to be increased in colorectal adenocarcinomas and inversely correlated with expression of TGFB pathway genes. We found colorectal adenocarcinomas to express mutant forms of CEACAM5 that inhibit TGFB signaling and increase proliferation and colony formation. We propose that CEACAM proteins disrupt TGFB signaling, which alters the composition of the intestinal microbiome to promote colorectal carcinogenesis.


Assuntos
Antígeno Carcinoembrionário/genética , Carcinogênese/genética , Neoplasias Colorretais/genética , Microbioma Gastrointestinal/fisiologia , Transdução de Sinais/genética , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Antígeno Carcinoembrionário/metabolismo , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais/mortalidade , Modelos Animais de Doenças , Fezes/microbiologia , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Mutação com Ganho de Função , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Metagenômica , Camundongos , Camundongos Transgênicos , Domínios Proteicos/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Esferoides Celulares , Análise de Sobrevida , Fator de Crescimento Transformador beta/metabolismo
6.
J Surg Oncol ; 120(8): 1427-1435, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31729037

RESUMO

BACKGROUND AND OBJECTIVES: Mucinous adenocarcinoma is a distinct subtype of colorectal cancer (CRC) with a worse prognosis when compared with non-mucinous adenocarcinoma. The aim of this study was to compare somatic mutations and copy number alteration (CNA) between mucinous and non-mucinous CRC. METHODS: Data from The Cancer Genome Atlas-colon adenocarcinoma and rectum adenocarcinoma projects were utilized. Mucinous and non-mucinous CRC were compared with regard to microsatellite status, overall mutation rate, the most frequently mutated genes, mutations in genes coding for mismatch repair (MMR) proteins and genes coding for mucin glycoproteins. CNA analysis and pathway analysis was undertaken. RESULTS: Mucinous CRC was more likely to be microsatellite instability-high (MSI-H) and hypermutated. When corrected for microsatellite status the single-nucleotide variation and insertion-deletion rate was similar between the two cohorts. Mucinous adenocarcinoma was more likely to have mutations in genes coding for MMR proteins and mucin glycoproteins. Pathway analysis revealed further differences between the two histological subtypes in the cell cycle, RTK-RAS, transforming growth factor-ß, and TP53 pathways. CONCLUSIONS: Mucinous CRC has some distinct genomic aberrations when compared with non-mucinous adenocarcinoma, many of which are driven by the increased frequency of MSI-H tumors. These genomic aberrations may play an important part in the difference seen in response to treatment and prognosis in mucinous adenocarcinoma.


Assuntos
Adenocarcinoma Mucinoso/genética , Adenocarcinoma/genética , Neoplasias do Colo/genética , Genômica , Neoplasias Retais/genética , Adenocarcinoma/patologia , Adenocarcinoma Mucinoso/patologia , Estudos de Coortes , Neoplasias do Colo/patologia , Variações do Número de Cópias de DNA , Proteínas de Ligação a DNA/genética , Conjuntos de Dados como Assunto , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação INDEL , Instabilidade de Microssatélites , Mucinas/genética , Mutação , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias Retais/patologia , Proteína Smad4/genética , Fator de Crescimento Transformador beta/genética , Proteína Supressora de Tumor p53/genética
7.
Zhonghua Wai Ke Za Zhi ; 57(11): 840-847, 2019 Nov 01.
Artigo em Chinês | MEDLINE | ID: mdl-31694133

RESUMO

Objective: To examine the prognostic value of four important driver gene mutations in patients with radical resection of pancreatic cancer. Methods: The clinical data and follow-up data of pancreatic cancer patients undergoing radical pancreatectomy and targeted sequencing from January 2016 to March 2018 at Department of Hepato-Biliary-Pancreatic Surgery, Changhai Hospital were retrospectively analyzed.There were 159 males and 88 females,aged of (60.8±8.7)years(range:33-83 years) and preoperative CA19-9 of (492.4±496.6)kU/L(range: 2-1 200 kU/L). One hundred and fifty nine cases of tumors were located in the head and 88 cases in the body and tail of the pancreas. After univariate analysis of clinical pathological factors (including gender, age, preoperative CA19-9, tumor location, tumor differentiation, pathological T and N stage, Micr. perineural invasion, Micr. lympho-vascular invasion, resection margin), the variable whose P<0.1 was included in COX regression model with four important driver gene mutations to find which mutation was related to prognosis independently. The number of gene mutations and KRAS subgroups were analyzed by Kaplan-Meier curve. Results: Among 247 patients,the number of KRAS,TP53, SMAD4 and CDKN2A mutations was 212 cases(85.8%), 160 cases(64.8%), 66 cases(26.7%) and 44 cases(17.8%),respectively.KRAS mutation was correlated with the tumor differentiation and pathological T stage (χ(2)=24.570/6.690, P=0.000/0.035), TP53 mutation was correlated with the tumor differentiation and the resected margin(χ(2)=5.500/4.620, P=0.019/0.032), and CDKN2A mutation was correlated with gender(χ(2)=16.574, P=0.000).COX regression model analysis showed that only KRAS mutation was an independent risk factor for disease free survival and overall survival(HR=1.776, 95%CI: 1.079-2.923, P=0.024; HR=1.923, 95%CI: 1.016-3.639, P=0.045); KRAS(G12D) mutation was associated with shorter OS(P=0.007). Conclusion: KRAS and its subgroup KRAS(G12D) mutation can be used as a prognostic index for patients with radical resection of pancreatic cancer.


Assuntos
Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos Glicosídicos Associados a Tumores/sangue , Biomarcadores Tumorais/análise , Inibidor p16 de Quinase Dependente de Ciclina/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Pancreatectomia , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/patologia , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)/genética , Estudos Retrospectivos , Proteína Smad4/genética , Proteína Supressora de Tumor p53/genética
8.
Genes (Basel) ; 10(10)2019 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-31569425

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is the most common and aggressive type of pancreatic cancer. The five-year survival rate of PDAC is very low (less than 8%), which is associated with the late diagnosis, high metastatic potential, and resistance to therapeutic agents. The identification of better prognostic or therapeutic biomarker may have clinical benefits for PDAC treatment. SMAD4, a central mediator of transforming growth factor beta (TGFß) signaling pathway, is considered a tumor suppressor gene. SMAD4 inactivation is frequently found in PDAC. However, its role in prognosis and therapeutics of PDAC is still unclear. In this study, we applied bioinformatics approaches, and integrated publicly available resources, to investigate the role of SMAD4 gene deletion in PDAC. We found that SMAD4 deletion was associated with poorer disease-free, but not overall, survival in PDAC patients. Cancer hallmark enrichment and pathway analysis suggested that the upregulation of cell cycle-related genes in SMAD4-deleted PDAC. Chemotherapy response profiling of PDAC cell lines and patient-derived organoids revealed that SMAD4-deleted PDAC was sensitive to gemcitabine, the first-line treatment for PDAC, and specific cell cycle-targeting drugs. Taken together, our study provides an insight into the prognostic and therapeutic roles of SMAD4 gene deletion in PDAC, and SMAD4 gene copy numbers may be used as a therapeutic biomarker for PDAC treatment.


Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/genética , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pancreáticas/genética , Proteína Smad4/genética , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Ciclo Celular , Linhagem Celular Tumoral , Biologia Computacional/métodos , Desoxicitidina/uso terapêutico , Deleção de Genes , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Células Tumorais Cultivadas
9.
Gastroenterology ; 157(6): 1599-1614.e2, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31585123

RESUMO

BACKGROUND & AIMS: Patterns of genetic alterations characterize different molecular subtypes of human gastric cancer. We aimed to establish mouse models of these subtypes. METHODS: We searched databases to identify genes with unique expression in the stomach epithelium, resulting in the identification of Anxa10. We generated mice with tamoxifen-inducible Cre recombinase (CreERT2) in the Anxa10 gene locus. We created 3 mouse models with alterations in pathways that characterize the chromosomal instability (CIN) and the genomically stable (GS) subtypes of human gastric cancer: Anxa10-CreERT2;KrasG12D/+;Tp53R172H/+;Smad4fl/f (CIN mice), Anxa10-CreERT2;Cdh1fl/fl;KrasG12D/+;Smad4fl/fl (GS-TGBF mice), and Anxa10-CreERT2;Cdh1fl/fl;KrasG12D/+;Apcfl/fl (GS-Wnt mice). We analyzed tumors that developed in these mice by histology for cell types and metastatic potential. We derived organoids from the tumors and tested their response to chemotherapeutic agents and the epithelial growth factor receptor signaling pathway inhibitor trametinib. RESULTS: The gastric tumors from the CIN mice had an invasive phenotype and formed liver and lung metastases. The tumor cells had a glandular morphology, similar to human intestinal-type gastric cancer. The gastric tumors from the GS-TGFB mice were poorly differentiated with diffuse morphology and signet ring cells, resembling human diffuse-type gastric cancer. Cells from these tumors were invasive, and mice developed peritoneal carcinomatosis and lung metastases. GS-Wnt mice developed adenomatous tooth-like gastric cancer. Organoids derived from tumors of GS-TGBF and GS-Wnt mice were more resistant to docetaxel, whereas organoids from the CIN tumors were more resistant to trametinib. CONCLUSIONS: Using a stomach-specific CreERT2 system, we created mice that develop tumors with morphologic similarities to subtypes of human gastric cancer. These tumors have different patterns of local growth, metastasis, and response to therapeutic agents. They can be used to study different subtypes of human gastric cancer.


Assuntos
Modelos Animais de Doenças , Mucosa Gástrica/patologia , Loci Gênicos/genética , Neoplasias Gástricas/genética , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Anexinas/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Transformação Celular Neoplásica/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Integrases/genética , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Smad4/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Proteína Supressora de Tumor p53/genética
10.
Mol Med Rep ; 20(4): 3735-3745, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31485652

RESUMO

The aberrant expression of tumor suppressor Smad4 often occurs in colorectal cancer (CRC), and this phenomenon is believed to be associated with drug resistance. The present study aimed to investigate the effects of Smad4 on the sensitivity of CRC cells to cetuximab, and the possible mechanism underlying such an effect. A total of 629 colorectal adenocarcinoma cases were downloaded from The Cancer Genome Atlas (TCGA) database, and a Smad4 mutation rate of ~21% was demonstrated among the cases. Low expression of Smad4 was present in CRC tissues analyzed by TCGA and in four CRC cell lines, as determined by reverse transcription­quantitative PCR (RT­qPCR) and western blot analysis. Cell Counting kit­8 (CCK­8) was used to measure the effects of different concentrations of cetuximab on SW480 cell viability at 24 and 48 h. The results demonstrated that treatment of SW480 cells with 20 µg/ml cetuximab for 48 h markedly reduced cell viability. In addition, plasmids were transfected into SW480 cells to induce Smad4 silencing or overexpression. Silencing Smad4 attenuated the sensitivity of SW480 CRC cells to cetuximab; this effect was reflected in increased cell viability and slightly increased migration and invasion, as determined by CCK­8, wound scratch and Transwell analyses. RT­qPCR and western blotting was performed to assess the expression levels of apoptosis­ and epithelial­mesenchymal transition (EMT)­related genes. Silencing Smad4 partly reversed the effects of cetuximab on the mRNA and protein expression levels of vimentin, Bax/Bcl­2 and E­cadherin. However, Smad4 overexpression enhanced SW480 cell sensitivity to cetuximab. In conclusion, Smad4 may serve a vital role in the sensitivity of CRC cells to chemotherapeutic drugs by promoting EMT.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Cetuximab/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteína Smad4/genética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Invasividade Neoplásica/genética , Invasividade Neoplásica/prevenção & controle
11.
Bioengineered ; 10(1): 282-291, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31311401

RESUMO

Transforming growth factor (TGF)-ß1 plays a crucial role in the epithelial-to-mesenchymal transition (EMT) in many cancer types and in thyroid cancers. Epigallocatechin-3-gallate (EGCG), the most important ingredient in the green tea, has been reported to possess antioxidant and anticancer activities. However, the cellular and molecular mechanisms explaining its action have not been completely understood. In this study, we found that EGCG significantly suppresses EMT, invasion and migration in anaplastic thyroid carcinoma (ATC) 8505C cells in vitro by regulating the TGF-ß/Smad signaling pathways. EGCG significantly inhibited TGF-ß1-induced expression of EMT markers (E-cadherin reduction and vimentin induction) in 8505C cells in vitro. Treatment with EGCG completely blocked the phosphorylation of Smad2/3, translocation of Smad4. Taken together, these results suggest that EGCG suppresses EMT and invasion and migration by blocking TGFß/Smad signaling pathways.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Catequina/análogos & derivados , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Transdução de Sinais/efeitos dos fármacos , Células Epiteliais da Tireoide/efeitos dos fármacos , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/antagonistas & inibidores , Caderinas/genética , Caderinas/metabolismo , Catequina/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Humanos , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Células Epiteliais da Tireoide/metabolismo , Células Epiteliais da Tireoide/patologia , Fator de Crescimento Transformador beta1/farmacologia , Vimentina/agonistas , Vimentina/genética , Vimentina/metabolismo
12.
Curr Med Sci ; 39(4): 534-540, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31346987

RESUMO

Bcl6, a critical pro-oncogene of human B-cell lymphomas, can promote tumor progress. Previous studies have found that Bcl6 participates in hypoxia injury in cardiomyocytes. However, the effect of Bcl6 on cardiac fibroblasts is still unclear. The aim of this study was to elucidate the functional role of Bcl6 in cardiac fibroblast activation and function. The neonatal rat cardiac fibroblasts were isolated and cultured. First, transforming growth factor ß1 (TGFß1) was used to stimulate fibroblast activation. A decreased expression level of Bcl6 was observed in fibroblasts after stimulation with TGFß1. Then, cells were transfected with adenovirus Bcl6 to overexpress Bcl6. The results showed that Bcl6 overexpression induced decreased proliferation and reduced activation of fibroblasts which were stimulated with TGFß1. It was found that activated smad2 and smad3 were not changed by overexpressing Bcl6, but smad4 was decreased. Furthermore, co-immunoprecipitation results showed that Bcl6 directly bound to smad4, and induced down-regulation of smad4. At last, smad4 activator could counteract the anti-fibroblast effects of Bcl6. In conclusion, Bcl6 may negatively regulate cardiac fibroblast activation and function by directly binding to smad4.


Assuntos
Miofibroblastos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteína Smad4/genética , Fator de Crescimento Transformador beta1/genética , Animais , Animais Recém-Nascidos , Proliferação de Células/genética , Regulação da Expressão Gênica/genética , Humanos , Miofibroblastos/efeitos dos fármacos , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Ratos , Proteína Smad2/genética , Proteína Smad3/genética , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
13.
Biofactors ; 45(5): 750-762, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31188510

RESUMO

Chronic kidney disease (CKD) is one of the major global health concerns and is responsible for end-stage renal disease (ESRD) complications. Inflammation plays a pivotal role in the progression of CKD. In the present study, we evaluated the renoprotective effects of a potent immunomodulator steroidal lactone, Withaferin A (WfA), in an animal model of renal injury (unilateral ureteral obstruction, UUO) and further investigated if the inhibition of inflammatory signaling can be a useful approach to reduce renal injury. Animals were randomly divided into five groups: Sham control, UUO control, WfA control, WfA low dose (1 mg/kg), and WfA high dose (3 mg/kg). Oxidative stress was measured by the estimation of reduced glutathione and lipid peroxidation levels. H&E and Picrosirius Red staining were performed to assess the extent of histological damage and collagen deposition. Furthermore, the molecular mechanism of the WfA effects was explored by immunohistochemistry, enzyme-linked immunosorbent assay, multiplex analysis of transforming growth factor ß (TGF-ß) pathway, and an array of inflammatory cytokines/chemokines. Interestingly, our pharmacological intervention significantly attenuated tissue collagen, inflammatory signaling, and macrophage signaling. WfA intervention abrogated the inflammatory signaling as evident from the modulated levels of chemokines and cytokines. The levels of TGF-ß along with downstream signaling molecules were also attenuated by WfA treatment as revealed by inhibition in the expression of TGF-ß1, TGF-ß2, p-Smad2, p-Smad3, total Smad4, p-Akt, and p-ERK. We, to the best of our knowledge, prove for the first time that WfA has potential renoprotective activity against UUO-induced nephropathy due to its outstanding anti-inflammatory properties.


Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Obstrução Ureteral/tratamento farmacológico , Vitanolídeos/farmacologia , Animais , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glutationa/agonistas , Glutationa/metabolismo , Inflamação , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/metabolismo , Resultado do Tratamento , Obstrução Ureteral/genética , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia
14.
Mol Med Rep ; 19(6): 4980-4988, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059017

RESUMO

Deficiency of surfactant proteins (SPs) is the main cause of respiratory distress syndrome (RDS) and chronic lung diseases. Our previous study demonstrated that miR­431 was differentially expressed between infants with RDS and infants without RDS using microarray analysis. However, the potential role of miR­431 in the development of lung function is still unknown. In the present study, the morphological characteristics of lung tissues and the expression levels of miR­431 were examined at three time points of rat lung development [gestational days 19 and 21 (E19, and E21) and postnatal day (P3)]. The protein and mRNA levels of SMAD4 and SPs (SP­A, SP­B, SP­C and SP­D) were also validated by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blot analysis, respectively. The expression levels of miR­431 were gradually decreased over time periods of E19, E21 and P3, as determine using RT­qPCR and fluorescence in situ hybridization. Dual luciferase­reporter assays revealed that SMAD4 is a direct target of miR­431. The mRNA and protein expression levels of SMAD4 and SPs increased gradually in rat lung tissues from E19 to P3. The order of magnitude was as follows: E19, E21 and P3. The present study demonstrated that the expression level of miR­431 decreased in the order of E19, E21 and P3 during rat lung development. The target gene of miR­431, SMAD4, was negatively regulated by miR­431, and its expression levels in the rat lung tissue increased from E19 to the P3. Surfactant synthesis was further increased over the E19 to P3 time period. Further studies are required to determine how miR­431 regulates pulmonary surfactant synthesis by targeting SMAD4.


Assuntos
Pulmão/crescimento & desenvolvimento , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , Animais , Animais Recém-Nascidos , Sequência de Bases , Feminino , Pulmão/metabolismo , Pulmão/patologia , MicroRNAs/genética , Microscopia Eletrônica , Gravidez , Proteínas Associadas a Surfactantes Pulmonares/genética , Proteínas Associadas a Surfactantes Pulmonares/metabolismo , Ratos , Ratos Sprague-Dawley , Alinhamento de Sequência , Proteína Smad4/química , Proteína Smad4/genética , Proteína Smad4/metabolismo
15.
Virchows Arch ; 475(1): 39-47, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31056731

RESUMO

Our study was done in order to identify novel molecular markers to predict which locally advanced rectal cancers (LARCs) might be resistant to neoadjuvant chemoradiotherapy (nCRT). Seventy-four patients with LARCs treated with nCRT were collected. Pathological evaluation after nCRT was performed according to the tumor regression grading (TRG) system. Next-generation sequencing kit including 279 exons of 59 genes was performed on Illumina Miseq Platform. Sanger sequencing was performed to confirm some mutations. Four of the tumors (4/74, 5.4%) had BRAF mutation, which presented in one TRG 2 tumor and three TRG 3 tumors but was not observed in TRG 0-1 tumors. Higher mutational frequency of BRAF gene in TRG 3 tumors (3/12, 25%) was found in comparison with the TRG 0-2 tumors (1/62, 1.6%; p = 0.012). Eight tumors (8/74, 10.8%) harbored SMAD4 mutations, which was mutated across all TRG groups. However, SMAD4 mutated more in TRG 3 tumors (4/12, 33.3%) compared with that in TRG 0-2 tumors (4/62, 6.5%; p = 0.020). The patients with BRAF-mutated LARCs had shorter progression-free survival (PFS) (p = 0.045) and shorter overall survival (OS) (p = 0.000) than the BRAF wild-type (WT) ones. The patients with SMAD4-mutated tumors had shorter PFS than the WT cases (p = 0.008). BRAF and SMAD4 genetic mutations might be important molecular markers to predict resistance to nCRT and poor prognosis in LARCs. More cases are needed to confirm these findings in the near future.


Assuntos
Biomarcadores Tumorais/genética , Quimiorradioterapia Adjuvante , Resistencia a Medicamentos Antineoplásicos/genética , Mutação , Terapia Neoadjuvante , Proteínas Proto-Oncogênicas B-raf/genética , Tolerância a Radiação/genética , Neoplasias Retais/terapia , Proteína Smad4/genética , Adulto , Idoso , Quimiorradioterapia Adjuvante/efeitos adversos , Análise Mutacional de DNA , Progressão da Doença , Éxons , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Terapia Neoadjuvante/efeitos adversos , Gradação de Tumores , Estadiamento de Neoplasias , Fenótipo , Intervalo Livre de Progressão , Neoplasias Retais/genética , Neoplasias Retais/mortalidade , Neoplasias Retais/patologia , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo
16.
Mol Carcinog ; 58(9): 1648-1655, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31140647

RESUMO

SMAD4 is a potent tumor suppressor and a central mediator of the TGFß signaling pathway. SMAD4 genetic loss is frequent in squamous cell carcinomas (SCCs). Reports of SMAD4 expression in SCCs vary significantly possibly due to inter-tumor heterogeneity or technical reasons. SMAD4 loss is an initiation event for SCCs. In tumor epithelial cells, SMAD4 loss causes increased proliferation, decreased apoptosis, and "Brca-like" genomic instability associated with DNA repair defects. SMAD4 loss also plays a role in the expansion of cancer stem cells. Epithelial SMAD4 loss causes overexpression of TGFß that is released into the tumor microenvironment and contributes to SCC progression through proinflammatory and immune evasive mechanisms. SMAD4 loss, while not a direct therapeutic target, is associated with multiple targetable pathways that require further therapeutic studies. Altogether, SMAD4 loss is a potential biomarker in SCCs that should be further studied for its values in prognostic and therapeutic predictions. Such information will potentially guide future biomarker-driven clinical trial designs and improve SCC patient outcomes.


Assuntos
Carcinoma de Células Escamosas/genética , Proteína Smad4/genética , Animais , Biomarcadores Tumorais/genética , Reparo do DNA/genética , Células Epiteliais/patologia , Genes Supressores de Tumor/fisiologia , Humanos , Transdução de Sinais/genética , Microambiente Tumoral/genética
17.
Nat Commun ; 10(1): 1665, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971692

RESUMO

Lung cancer is the leading cause of cancer-related deaths worldwide. Tumor suppressor genes remain to be systemically identified for lung cancer. Through the genome-wide screening of tumor-suppressive transcription factors, we demonstrate here that GATA4 functions as an essential tumor suppressor in lung cancer in vitro and in vivo. Ectopic GATA4 expression results in lung cancer cell senescence. Mechanistically, GATA4 upregulates multiple miRNAs targeting TGFB2 mRNA and causes ensuing WNT7B downregulation and eventually triggers cell senescence. Decreased GATA4 level in clinical specimens negatively correlates with WNT7B or TGF-ß2 level and is significantly associated with poor prognosis. TGFBR1 inhibitors show synergy with existing therapeutics in treating GATA4-deficient lung cancers in genetically engineered mouse model as well as patient-derived xenograft (PDX) mouse models. Collectively, our work demonstrates that GATA4 functions as a tumor suppressor in lung cancer and targeting the TGF-ß signaling provides a potential way for the treatment of GATA4-deficient lung cancer.


Assuntos
Fator de Transcrição GATA4/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Neoplasias Pulmonares/genética , Proteínas Wnt/metabolismo , Células A549 , Animais , Senescência Celular/genética , Regulação para Baixo , Feminino , Fator de Transcrição GATA4/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Pulmão/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Prognóstico , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Transdução de Sinais/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Regulação para Cima , Proteínas Wnt/genética , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Nat Genet ; 51(5): 777-785, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30988513

RESUMO

BMP/SMAD signaling is a crucial regulator of intestinal differentiation1-4. However, the molecular underpinnings of the BMP pathway in this context are unknown. Here, we characterize the mechanism by which BMP/SMAD signaling drives enterocyte differentiation. We establish that the transcription factor HNF4A acts redundantly with an intestine-restricted HNF4 paralog, HNF4G, to activate enhancer chromatin and upregulate the majority of transcripts enriched in the differentiated epithelium; cells fail to differentiate on double knockout of both HNF4 paralogs. Furthermore, we show that SMAD4 and HNF4 function via a reinforcing feed-forward loop, activating each other's expression and co-binding to regulatory elements of differentiation genes. This feed-forward regulatory module promotes and stabilizes enterocyte cell identity; disruption of the HNF4-SMAD4 module results in loss of enterocyte fate in favor of progenitor and secretory cell lineages. This intersection of signaling and transcriptional control provides a framework to understand regenerative tissue homeostasis, particularly in tissues with inherent cellular plasticity5.


Assuntos
Enterócitos/citologia , Enterócitos/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Proteína Smad4/metabolismo , Animais , Sítios de Ligação/genética , Células CACO-2 , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Elementos Facilitadores Genéticos , Fator 4 Nuclear de Hepatócito/deficiência , Fator 4 Nuclear de Hepatócito/genética , Humanos , Camundongos , Camundongos Knockout , Transdução de Sinais , Proteína Smad4/deficiência , Proteína Smad4/genética
20.
Genome Med ; 11(1): 19, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30914061

RESUMO

BACKGROUND: Genome-wide association studies (GWAS) have identified several hundred susceptibility loci for type 2 diabetes (T2D). One critical, but unresolved, issue concerns the extent to which the mechanisms through which these diverse signals influencing T2D predisposition converge on a limited set of biological processes. However, the causal variants identified by GWAS mostly fall into a non-coding sequence, complicating the task of defining the effector transcripts through which they operate. METHODS: Here, we describe implementation of an analytical pipeline to address this question. First, we integrate multiple sources of genetic, genomic and biological data to assign positional candidacy scores to the genes that map to T2D GWAS signals. Second, we introduce genes with high scores as seeds within a network optimization algorithm (the asymmetric prize-collecting Steiner tree approach) which uses external, experimentally confirmed protein-protein interaction (PPI) data to generate high-confidence sub-networks. Third, we use GWAS data to test the T2D association enrichment of the "non-seed" proteins introduced into the network, as a measure of the overall functional connectivity of the network. RESULTS: We find (a) non-seed proteins in the T2D protein-interaction network so generated (comprising 705 nodes) are enriched for association to T2D (p = 0.0014) but not control traits, (b) stronger T2D-enrichment for islets than other tissues when we use RNA expression data to generate tissue-specific PPI networks and (c) enhanced enrichment (p = 3.9 × 10- 5) when we combine the analysis of the islet-specific PPI network with a focus on the subset of T2D GWAS loci which act through defective insulin secretion. CONCLUSIONS: These analyses reveal a pattern of non-random functional connectivity between candidate causal genes at T2D GWAS loci and highlight the products of genes including YWHAG, SMAD4 or CDK2 as potential contributors to T2D-relevant islet dysfunction. The approach we describe can be applied to other complex genetic and genomic datasets, facilitating integration of diverse data types into disease-associated networks.


Assuntos
Algoritmos , Diabetes Mellitus Tipo 2/genética , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla/métodos , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Predisposição Genética para Doença , Humanos , Polimorfismo de Nucleotídeo Único , Proteína Smad4/genética , Proteína Smad4/metabolismo , Transcriptoma
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