RESUMO
DNA methylation is a major epigenetic mechanism for gene silencing. Whereas methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here, we show that either knockout or catalytic inactivation of the DNA repair enzyme thymine DNA glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific developmentally and hormonally regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.
Assuntos
Metilação de DNA , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Timina DNA Glicosilase/metabolismo , 5-Metilcitosina/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Citidina Desaminase/metabolismo , Citosina/análogos & derivados , Citosina/metabolismo , Feminino , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Timina DNA Glicosilase/genética , Transcrição GênicaRESUMO
BACKGROUND & AIMS: Aberrant DNA methylation is frequent in colorectal cancer (CRC), but underlying mechanisms and pathologic consequences are poorly understood. METHODS: We disrupted active DNA demethylation genes Tet1 and/or Tdg from ApcMin mice and characterized the methylome and transcriptome of colonic adenomas. Data were compared to human colonic adenocarcinomas (COAD) in The Cancer Genome Atlas. RESULTS: There were increased numbers of small intestinal adenomas in ApcMin mice expressing the TdgN151A allele, whereas Tet1-deficient and Tet1/TdgN151A-double heterozygous ApcMin colonic adenomas were larger with features of erosion and invasion. We detected reduction in global DNA hypomethylation in colonic adenomas from Tet1- and Tdg-mutant ApcMin mice and hypermethylation of CpG islands in Tet1-mutant ApcMin adenomas. Up-regulation of inflammatory, immune, and interferon response genes was present in Tet1- and Tdg-mutant colonic adenomas compared to control ApcMin adenomas. This up-regulation was also seen in murine colonic organoids and human CRC lines infected with lentiviruses expressing TET1 or TDG short hairpin RNA. A 127-gene inflammatory signature separated colonic adenocarcinomas into 4 groups, closely aligned with their microsatellite or chromosomal instability and characterized by different levels of DNA methylation and DNMT1 expression that anticorrelated with TET1 expression. Tumors with the CpG island methylator phenotype (CIMP) had concerted high DNMT1/low TET1 expression. TET1 or TDG knockdown in CRC lines enhanced killing by natural killer cells. CONCLUSIONS: Our findings reveal a novel epigenetic regulation, linked to the type of genomic instability, by which TET1/TDG-mediated DNA demethylation decreases methylation levels and inflammatory/interferon/immune responses. CIMP in CRC is triggered by an imbalance of methylating activities over demethylating activities. These mice represent a model of CIMP CRC.
Assuntos
Adenocarcinoma , Adenoma , Neoplasias do Colo , Neoplasias Colorretais , Animais , Humanos , Camundongos , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenoma/genética , Adenoma/patologia , Carcinogênese/genética , Transformação Celular Neoplásica/genética , Neoplasias do Colo/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Ilhas de CpG/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Epigênese Genética , Oxigenases de Função Mista/genética , Fenótipo , Proteínas Proto-Oncogênicas/genéticaRESUMO
Blockchain-based traceability systems are a promising approach because they are decentralized, transparent, and tamper proof; however, if all traceability data are uploaded to a blockchain platform, it may affect the efficiency or even lead to data explosion. Additionally, it is difficult to guarantee the reliability of the original data source of massive Internet of Things (IoT) devices. Furthermore, when different enterprise nodes adopt different data storage structures, the costs that are associated with data sharing will increase. In this paper, we have proposed a trustworthy product traceability system that is based on hyperledger fabric and Electronic Product Code Information Service (EPCIS), which is not only capable of making products traceable, but it can also authenticate and authorize the IoT devices that are used for data collection. First, we adopted the on-chain and off-chain collaborative management mechanism in order to alleviate data explosion on the chain. Second, we proposed a scheme to authenticate and authorize devices based on blockchain. Third, we complied with EPCIS and Core Business Vocabulary (CBV) standards and provided the EPCIS location discovery service in order to improve the interactivity. Finally, we implemented and tested the proposed traceability system and compared it with the existing research. The proposed solution provides product information traceability, data tamper proofing, data confidentiality, and data source reliability.
RESUMO
Malignant mesothelioma (MM) is a therapy-resistant cancer arising primarily from the lining of the pleural and peritoneal cavities. The most frequently altered genes in human MM are cyclin-dependent kinase inhibitor 2A (CDKN2A), which encodes components of the p53 (p14ARF) and RB (p16INK4A) pathways, BRCA1-associated protein 1 (BAP1), and neurofibromatosis 2 (NF2). Furthermore, the p53 gene (TP53) itself is mutated in ~15% of MMs. In many MMs, the PI3K-PTEN-AKT-mTOR signaling node is hyperactivated, which contributes to tumor cell survival and therapeutic resistance. Here, we demonstrate that the inactivation of both Tp53 and Pten in the mouse mesothelium is sufficient to rapidly drive aggressive MMs. PtenL/L ;Tp53L/L mice injected intraperitoneally or intrapleurally with adenovirus-expressing Cre recombinase developed high rates of peritoneal and pleural MMs (92% of mice with a median latency of 9.4 weeks and 56% of mice with a median latency of 19.3 weeks, respectively). MM cells from these mice showed consistent activation of Akt-mTor signaling, chromosome breakage or aneuploidy, and upregulation of Myc; occasional downregulation of Bap1 was also observed. Collectively, these findings suggest that when Pten and Tp53 are lost in combination in mesothelial cells, DNA damage is not adequately repaired and genomic instability is widespread, whereas the activation of Akt due to Pten loss protects genomically damaged cells from apoptosis, thereby increasing the likelihood of tumor formation. Additionally, the mining of an online dataset (The Cancer Genome Atlas) revealed codeletions of PTEN and TP53 and/or CDKN2A/p14ARF in ~25% of human MMs, indicating that cooperative losses of these genes contribute to the development of a significant proportion of these aggressive neoplasms and suggesting key target pathways for therapeutic intervention.
Assuntos
Neoplasias Pulmonares/genética , Mesotelioma/genética , PTEN Fosfo-Hidrolase/genética , Neoplasias Pleurais/genética , Proteína Supressora de Tumor p53/genética , Animais , Carcinogênese/genética , Proliferação de Células/genética , Modelos Animais de Doenças , Humanos , Neoplasias Pulmonares/patologia , Mesotelioma/patologia , Mesotelioma Maligno , Camundongos , PTEN Fosfo-Hidrolase/antagonistas & inibidores , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/patologia , Neoplasias Pleurais/patologia , Transdução de Sinais , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genéticaRESUMO
Oltipraz (OPZ) is a synthetic dithiolethione and is considered a novel activator of nuclear factor E2-related factor 2 (Nrf2). Increasing evidence indicates that Nrf2 protects against cerebral ischemia/reperfusion (I/R) injury by antagonizing ferroptosis and lipid peroxidation. However, the protective effects of OPZ on cerebral I/R injury remain to be elucidated. We investigated the in vitro and in vivo neuroprotective effects of OPZ. Mice were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) to construct an in vivo model and PC12 cells were exposed to oxygen and glucose deprivation/reoxygenation (OGD/R) to establish an in vitro model. OPZ administration reduced the infarct volume and brain water content, and alleviated the neurological deficit of MCAO/R mice. Moreover, OPZ ameliorated MCAO/R-induced oxidative stress by decreasing the levels of 4-HNE and MDA and increasing the activities of SOD and GSH. We also found that OPZ ameliorated MCAO/R-induced ferroptosis by increasing SLC7A11 and GPX4 protein expression and downregulating ACSL4 protein expression. Similarly, the in vitro results revealed that OGD/R-induced oxidative stress and ferroptosis. Finally, mechanistic analysis revealed that OPZ significantly upregulated the Nrf2 expression and Nrf2 knockout (Nrf2 KO) abolished the OPZ-mediated protective effects. Taken together, these findings demonstrate that OPZ ameliorates cerebral I/R injury by suppressing the oxidative stress and ferroptosis.
Assuntos
Ferroptose , Infarto da Artéria Cerebral Média , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2 , Fármacos Neuroprotetores , Estresse Oxidativo , Traumatismo por Reperfusão , Tionas , Tiofenos , Animais , Ferroptose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Tionas/farmacologia , Tionas/uso terapêutico , Células PC12 , Fator 2 Relacionado a NF-E2/metabolismo , Camundongos , Masculino , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Tiofenos/farmacologia , Tiofenos/uso terapêutico , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/patologia , Ratos , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Sistema y+ de Transporte de Aminoácidos/genética , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Modelos Animais de Doenças , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Encéfalo/metabolismo , PirazinasRESUMO
In this randomized controlled trial, 74 patients scheduled for gynecological laparoscopic surgery (American Society of Anesthesiologists grade I/II) were enrolled and randomly divided into two study groups: (i) Group C (control), received sufentanil (0.3 µg/kg) and saline, followed by sufentanil (0.1 µg/kgâh) and saline; and (ii) Group F (OFA), received esketamine (0.15 mg/kg) and lidocaine (2 mg/kg), followed by esketamine (0.1 mg/kgâh) and lidocaine (1.5 mg/kgâh). The primary outcome was the 48-h time-weighted average (TWA) of postoperative pain scores. Secondary outcomes included time to extubation, adverse effects, and postoperative sedation score, pain scores at different time points, analgesic consumption at 48 h, and gastrointestinal functional recovery. The 48-h TWAs of pain scores were 1.32 (0.78) (95% CI 1.06-1.58) and 1.09 (0.70) (95% CI 0.87-1.33) for Groups F and C, respectively. The estimated difference between Groups F and C was - 0.23 (95% CI - 0.58 - 0.12; P = 0.195). No differences were found in any of the secondary outcomes and no severe adverse effects were observed in either group. Balanced OFA with lidocaine and esketamine achieved similar effects to balanced anesthesia with sufentanil in patients undergoing elective gynecological laparoscopic surgery, without severe adverse effects.Clinical Trial Registration: ChiCTR2300067951, www.chictr.org.cn 01 February, 2023.
Assuntos
Analgésicos Opioides , Procedimentos Cirúrgicos em Ginecologia , Ketamina , Lidocaína , Dor Pós-Operatória , Sufentanil , Humanos , Sufentanil/administração & dosagem , Sufentanil/efeitos adversos , Feminino , Ketamina/administração & dosagem , Ketamina/efeitos adversos , Lidocaína/administração & dosagem , Lidocaína/efeitos adversos , Adulto , Procedimentos Cirúrgicos em Ginecologia/efeitos adversos , Procedimentos Cirúrgicos em Ginecologia/métodos , Dor Pós-Operatória/tratamento farmacológico , Dor Pós-Operatória/prevenção & controle , Pessoa de Meia-Idade , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/efeitos adversos , Analgésicos Opioides/uso terapêutico , Laparoscopia/efeitos adversos , Laparoscopia/métodos , Anestesia/métodos , Anestesia/efeitos adversos , Anestésicos Locais/administração & dosagem , Medição da DorRESUMO
The human DLX homeobox genes, which are related to Dll (Drosophila distal-less gene), encode transcription factors that are expressed primarily in embryonic development. Recently, DLX5 was reported to act as an oncogene in lymphomas and lung cancers, although the mechanism is not known. The identification of target genes of DLX5 can facilitate our understanding of oncogenic mechanisms driven by overexpression of DLX5. The MYC oncogene is aberrantly expressed in many human cancers and regulates transcription of numerous target genes involved in tumorigenesis. Here we demonstrate by luciferase assay that the MYC promoter is specifically activated by overexpression of DLX5 and that two DLX5 binding sites in the MYC promoter are important for transcriptional activation of MYC. We also show that DLX5 binds to the MYC promoter both in vitro and in vivo and that transfection of a DLX5 expression plasmid promotes the expression of MYC in a dose-dependent manner in mammalian cells. Furthermore, overexpression of DLX5 results in increased cell proliferation by up-regulating MYC. Knockdown of DLX5 in lung cancer cells overexpressing DLX5 resulted in decreased expression of MYC and reduced cell proliferation, which was rescued by overexpression of MYC. Because DLX5 has a restricted pattern of expression in adult tissues, it may serve as a potential therapeutic target for the treatment of cancers that overexpress DLX5.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Neoplasias/genética , Neoplasias/patologia , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Homeodomínio/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Linfoma/enzimologia , Linfoma/genética , Linfoma/patologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/genética , Regulação para Cima/genéticaRESUMO
PI3K/AKT/mTOR pathway hyperactivation is frequent in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL). To model inhibition of mTOR, pre-T-cell lymphoblastic leukemia/lymphoma (pre-T LBL) tumor development was monitored in mice with T lymphocyte-specific, constitutively active AKT (Lck-MyrAkt2) that were either crossed to mTOR knockdown (KD) mice or treated with the mTOR inhibitor everolimus. Lck-MyrAkt2;mTOR KD mice lived significantly longer than Lck-MyrAkt2;mTOR wild-type (WT) mice, although both groups ultimately developed thymic pre-T LBL. An increase in survival was also observed when Lck-MyrAkt2;mTOR WT mice were treated for 8 weeks with everolimus. The transcriptional profiles of WT and KD thymic lymphomas were compared, and Ingenuity Pathway Upstream Regulator Analysis of differentially expressed genes in tumors from mTOR WT versus KD mice identified let-7 and miR-21 as potential regulatory genes. mTOR KD mice had higher levels of let-7a and miR-21 than mTOR WT mice, and rapamycin induced their expression in mTOR WT cells. CDK6 was one of the most downregulated targets of both let-7 and miR21 in mTOR KD tumors. CDK6 overexpression and decreased expression of let-7 in mTOR KD cells rescued a G1 arrest phenotype. Combined mTOR (rapamycin) and CDK4/6 (palbociclib) inhibition decreased tumor size and proliferation in tumor flank transplants, increased survival in an intravenous transplant model of disseminated leukemia compared with single agent treatment, and cooperatively decreased cell viability in human T-ALL/LBL cell lines. Thus, mTOR KD mice provide a model to explore drug combinations synergizing with mTOR inhibitors and can be used to identify downstream targets of inhibition.
Assuntos
Quinase 6 Dependente de Ciclina/metabolismo , Perfilação da Expressão Gênica/métodos , Serina-Treonina Quinases TOR/metabolismo , Animais , Carcinogênese , Regulação para Baixo , Camundongos , Camundongos TransgênicosRESUMO
Thymine DNA Glycosylase (TDG) is a base excision repair enzyme that acts as a thymine and uracil DNA N-glycosylase on G:T and G:U mismatches, thus protecting CpG sites in the genome from mutagenesis by deamination. In addition, TDG has an epigenomic function by removing the novel cytosine derivatives 5-formylcytosine and 5-carboxylcytosine (5caC) generated by Ten-Eleven Translocation (TET) enzymes during active DNA demethylation. We and others previously reported that TDG is essential for mammalian development. However, its involvement in tumor formation is unknown. To study the role of TDG in tumorigenesis, we analyzed the effects of its inactivation in a well-characterized model of tumor predisposition, the ApcMin mouse strain. Mice bearing a conditional Tdgflox allele were crossed with Fabpl::Cre transgenic mice, in the context of the ApcMin mutation, in order to inactivate Tdg in the small intestinal and colonic epithelium. We observed an approximately 2-fold increase in the number of small intestinal adenomas in the test Tdg-mutant ApcMin mice in comparison to control genotypes (p=0.0001). This increase occurred in female mice, and is similar to the known increase in intestinal adenoma formation due to oophorectomy. In the human colorectal cancer (CRC) TCGA database, the subset of patients with TDG and APC expression in the lowest quartile exhibits an excess of female cases. We conclude that TDG inactivation plays a role in intestinal tumorigenesis initiated by mutation/underexpression of APC. Our results also indicate that TDG may be involved in sex-specific protection from CRC.
RESUMO
Homeobox genes play a critical role in embryonic development, but they have also been implicated in cancer through mechanisms that are largely unknown. While not expressed during normal T-cell development, homeobox transcription factor genes can be reactivated via recurrent chromosomal rearrangements in human T-cell acute leukemia/lymphoma (T-ALL), a malignancy often associated with activated Notch and Akt signaling. To address how epigenetic reprogramming via an activated homeobox gene might contribute to T-lymphomagenesis, we investigated a transgenic mouse model with thymocyte-specific overexpression of the Dlx5 homeobox gene. We demonstrate for the first time that Dlx5 induces T-cell lymphomas with high penetrance. Integrated ChIP-seq and mRNA microarray analyses identified Notch1/3 and Irs2 as direct transcriptional targets of Dlx5, a gene signature unique to lymphomas from Lck-Dlx5 mice as compared to T-cell lymphomas from Lck-MyrAkt2 mice, which were previously reported by our group. Moreover, promoter/enhancer studies confirmed that Dlx5 directly transactivates Notch expression. Notch1/3 expression and Irs2-induced Akt signaling were upregulated throughout early stages of T-cell development, which promoted cell survival during ß-selection of T lymphocytes. Dlx5 was required for tumor maintenance via its activation of Notch and Akt, as tumor cells were highly sensitive to Notch and Akt inhibitors. Together, these findings provide unbiased genetic and mechanistic evidence that Dlx5 acts as an oncogene when aberrantly expressed in T cells, and that it is a novel discovery that Notch is a direct target of Dlx5. These experimental findings provide mechanistic insights about how reactivation of the Dlx5 gene can drive T-ALL by aberrant epigenetic reprogramming of the T-cell genome.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/fisiologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/fisiologia , Linfoma de Células T/patologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Receptor Notch1/genética , Animais , Apoptose , Proliferação de Células , Humanos , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Transdução de Sinais , Ativação Transcricional , Células Tumorais CultivadasRESUMO
OBJECTIVE: Patients with type 2 diabetes (T2DM) often have multiple comorbidities which may impact the selection of antihyperglycemic therapies. The purpose of this study was to quantify the prevalence and co-prevalence of common comorbidities. RESEARCH DESIGN AND METHODS: A retrospective study was conducted using the Quintiles Electronic Medical Record database. Adult patients with T2DM who had ≥1 encounter from July 2014 to June 2015 (index period) with ≥1 year medical history available were included. The index date was defined as the most recent encounter date during the 1 year index period. MAIN OUTCOME MEASURES: Comorbid conditions were assessed using all data available prior to and including the index date. Patient characteristics, laboratory measures, and comorbidities were summarized via descriptive analyses, overall and by subgroups of age (<65, 65-74, 75+ years) and gender. RESULTS: Of the 1,389,016 eligible patients, 53% were female and the median age was 65 years. 97.5% of patients had at least one comorbid condition in addition to T2DM and 88.5% had at least two. The comorbidity burden tended to increase in older age groups and was higher in men than women. The most common conditions in patients with T2DM included hypertension (HTN) in 82.1%; overweight/obesity in 78.2%; hyperlipidemia in 77.2%; chronic kidney disease (CKD) in 24.1%; and cardiovascular disease (CVD) in 21.6%. The highest co-prevalence was demonstrated for the combination of HTN and hyperlipidemia (67.5%), followed by overweight/obesity and HTN (66.0%), overweight/obesity and hyperlipidemia (62.5%), HTN and CKD (22.4%), hyperlipidemia and CKD (21.1%), HTN and CVD (20.2%), hyperlipidemia and CVD (20.1%), overweight/obesity and CKD (19.1%) and overweight/obesity and CVD (17.0%). LIMITATIONS: Limitations include the potential for misclassification/underreporting due to the use of diagnostic codes, drug codes, or laboratory measures for identification of medical conditions. CONCLUSIONS: The vast majority of patients with T2DM have multiple comorbidities. To ensure a comprehensive approach to patient management, the presence of multimorbidity should be considered in the context of clinical decision making.
Assuntos
Diabetes Mellitus Tipo 2 , Idoso , Doenças Cardiovasculares/complicações , Doenças Cardiovasculares/epidemiologia , Comorbidade , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/epidemiologia , Feminino , Humanos , Hiperlipidemias/complicações , Hiperlipidemias/epidemiologia , Hipertensão/complicações , Hipertensão/epidemiologia , Masculino , Pessoa de Meia-Idade , Obesidade/complicações , Obesidade/epidemiologia , Sobrepeso/complicações , Sobrepeso/epidemiologia , Prevalência , Estudos RetrospectivosRESUMO
Individuals harboring inherited heterozygous germline mutations in BAP1 are predisposed to a range of benign and malignant tumor types, including malignant mesothelioma, melanoma, and kidney carcinoma. However, evidence to support a tumor-suppressive role for BAP1 in cancer remains contradictory. To test experimentally whether BAP1 behaves as a tumor suppressor, we monitored spontaneous tumor development in three different mouse models with germline heterozygous mutations in Bap1, including two models in which the knock-in mutations are identical to those reported in human BAP1 cancer syndrome families. We observed spontaneous malignant tumors in 54 of 93 Bap1-mutant mice (58%) versus 4 of 43 (9%) wild-type littermates. All three Bap1-mutant models exhibited a high incidence and similar spectrum of neoplasms, including ovarian sex cord stromal tumors, lung and mammary carcinomas, and spindle cell tumors. Notably, we also observed malignant mesotheliomas in two Bap1-mutant mice, but not in any wild-type animals. We further confirmed that the remaining wild-type Bap1 allele was lost in both spontaneous ovarian tumors and mesotheliomas, resulting in the loss of Bap1 expression. Additional studies revealed that asbestos exposure induced a highly significant increase in the incidence of aggressive mesotheliomas in the two mouse models carrying clinically relevant Bap1 mutations compared with asbestos-exposed wild-type littermates. Collectively, these findings provide genetic evidence that Bap1 is a bona fide tumor suppressor gene and offer key insights into the contribution of carcinogen exposure to enhanced cancer susceptibility. Cancer Res; 76(9); 2836-44. ©2016 AACR.
Assuntos
Genes Supressores de Tumor , Mutação em Linhagem Germinativa , Síndromes Neoplásicas Hereditárias , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Animais , Hibridização Genômica Comparativa , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Predisposição Genética para Doença/genética , Genótipo , Heterozigoto , Imuno-Histoquímica , Microdissecção e Captura a Laser , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Malignant mesotheliomas are highly aggressive tumors usually caused by exposure to asbestos. Germline-inactivating mutations of BAP1 predispose to mesothelioma and certain other cancers. However, why mesothelioma is the predominate malignancy in some BAP1 families and not others, and whether exposure to asbestos is required for development of mesothelioma in BAP1 mutation carriers are not known. To address these questions experimentally, we generated a Bap1(+/-) knockout mouse model to assess its susceptibility to mesothelioma upon chronic exposure to asbestos. Bap1(+/-) mice exhibited a significantly higher incidence of asbestos-induced mesothelioma than wild-type (WT) littermates (73% vs. 32%, respectively). Furthermore, mesotheliomas arose at an accelerated rate in Bap1(+/-) mice than in WT animals (median survival, 43 weeks vs. 55 weeks after initial exposure, respectively) and showed increased invasiveness and proliferation. No spontaneous mesotheliomas were seen in unexposed Bap1(+/-) mice followed for up to 87 weeks of age. Mesothelioma cells from Bap1(+/-) mice showed biallelic inactivation of Bap1, consistent with its proposed role as a recessive cancer susceptibility gene. Unlike in WT mice, mesotheliomas from Bap1(+/-) mice did not require homozygous loss of Cdkn2a. However, normal mesothelial cells and mesothelioma cells from Bap1(+/-) mice showed downregulation of Rb through a p16(Ink4a)-independent mechanism, suggesting that predisposition of Bap1(+/-) mice to mesothelioma may be facilitated, in part, by cooperation between Bap1 and Rb. Drawing parallels to human disease, these unbiased genetic findings indicate that BAP1 mutation carriers are predisposed to the tumorigenic effects of asbestos and suggest that high penetrance of mesothelioma requires such environmental exposure.
Assuntos
Amianto/toxicidade , Mutação em Linhagem Germinativa , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/genética , Mesotelioma/etiologia , Mesotelioma/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Animais , Modelos Animais de Doenças , Epigenômica , Feminino , Predisposição Genética para Doença , Genótipo , Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , Mesotelioma Maligno , Camundongos , Camundongos Knockout , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismoRESUMO
Hypoxia-inducible factor-1 α (HIF-1α) is an important prognostic factor in ovarian carcinoma. Hypoxia contributes to tumor progression and is involved in the epithelial-mesenchymal transition (EMT). Twist2 is an EMT regulator, however, it remains poorly understood in ovarian carcinoma. The present study evaluated the expression of HIF-1α and Twist2 and further investigated whether Twist2 is involved in hypoxia-induced apoptosis in ovarian cancer. A series of matched paraffin-embedded tissue sections from human primary ovarian cancer and normal ovarian tissues were examined through immunohistochemical analysis, a Twist2-overexpressing stable ovarian cancer cell line was established and deferoxamine (DFO) was introduced to simulate hypoxic conditions. DFO-induced apoptosis was examined by fluorescence microscopy, MTT assays and flow cytometry. In addition, a western blot analysis was performed to examine the molecular mechanism(s) of action. Twist2 increased in epithelial ovarian cancers associated with HIF-1α expression. The acquired expression of Twist2 was able to promote the survival of ovarian cancer cells through Akt phosphorylation under DFO-induced hypoxic stress. The results suggest that Twist2 activates the PI-3K-Akt pathway to protect cells from apoptosis in a hypoxic environment. Moreover, Twist2 may be involved in the HIF-1α signaling pathway in ovarian cancer.
RESUMO
BACKGROUND: Twist2 has been shown to promote human tumor invasion as in breast cancer and cervical cancer. However, whether Twist2 promotes human ovarian cancer progression remains to be elucidated. Here, we investigate the role of Twist2 in ovarian cancer invasion and metastasis as well as the underlying molecular mechanisms. METHODS: Twist2 expression was detected by Immunohistochemistry (IHC) on tissue microarray of human ovarian cancers with scoring procedure according to the staining intensity and pattern. Twist2 gene was stably introduced into SKOV-3 ovarian cancer cells to examine the changes of cellular morphology, motility, invasiveness, and EMT molecular markers. RESULTS: Twist2 expression is significantly increased in ovarian cancers along with the FIGO disease stage, indicating that Twist2 may be associated with ovarian cancer metastasis. Overexpression of Twist2 induced the EMT phenotype including downregulation of E-cadherin, and upregulation of N-cadherin and ß-catenin in human ovarian cancer cells, suggesting that Twist2 might promote ß-catenin release from the E-cadherin/ß-catenin complex through inhibition of E-cadherin. Thus, ß-catenin degradation was inhibited due to inhibition of APC, and the Wnt/ß-catenin pathway was then activated by nuclear ß-catenin accumulation, which may activate transcription of downstream target genes to promote tumor invasion and metastasis. Collectively, these data indicated that ß-catenin is involved in Twist2-induced EMT in ovarian cancer. CONCLUSION: Our data indicates that upregulation of Twist2 is correlated with the FIGO stage in human ovarian cancers. In this report, we demonstrated that nuclear ß-catenin is accumulated in Twist2-induced EMT cells to facilitates ovarian cancer invasion and metastasis.
Assuntos
Núcleo Celular/metabolismo , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/metabolismo , Proteínas Repressoras/biossíntese , Proteína 1 Relacionada a Twist/biossíntese , beta Catenina/metabolismo , Transporte Ativo do Núcleo Celular/genética , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/patologia , Feminino , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Proteínas Repressoras/genética , Proteína 1 Relacionada a Twist/genética , Regulação para Cima/genética , beta Catenina/genéticaRESUMO
BACKGROUND: Twist2 (Dermo1) has been shown to mediate the epithelial-mesenchymal transition (EMT) to promote tumor invasion and even metastasis. However, the involvement of EMT in breast cancer progression is highly debated, partially due to clinical observations showing that the majority of human breast carcinoma metastases express E-cadherin and maintain their epithelial morphology. The molecular mechanism by which Twist2 participates in EMT of breast cancer in vivo remains poorly understood. METHODS: We examined Twist2 expression pattern in human breast carcinomas by western blot and tissue microarray, and analyzed Twist2 cellular localization by confocal microscopy, cell fractionation and other approaches. RESULTS: Twist2 expression was significantly increased in breast cancer. Cytoplasmic Twist2 positive cancer cells expressing E-cadherin on the cellular membrane were mainly located at tumor center of primary carcinomas and lymph metastases, while cancer cells with nuclear Twist2 clearly showed loss of E-cadherin and were detected at the invasive front in ductal breast carcinomas. In addition, ectopically stable-expressed Twist2 was found to localize in the cytoplasm of cancer cells. Collectively, these data indicate that upregulation of cytoplasmic Twist2 is correlated with tumor histological type and tumor metastasis in human breast cancers. CONCLUSION: The differential cellular distribution of Twist2 may be associated with tumor progression. The cytoplasmic Twist2 in cancer cells at tumor center of primary carcinomas and lymph metastases contributes to the maintenance of epithelial cancer characteristics expressing E-cadherin in a noninvasive state, while the nuclear Twist2 at the cancer invasion front activates EMT to deprive epithelial property of neoplastic cells, thus facilitating invasion and metastasis. These findings suggest that heterogeneous expression of Twist2 in tumors may have a functional link to tumor progression.
Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Repressoras/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Caderinas/biossíntese , Citoplasma/metabolismo , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Perfilação da Expressão Gênica , Humanos , Microscopia Confocal/métodos , Metástase Neoplásica , Fatores de Transcrição da Família Snail , Frações Subcelulares/metabolismo , Fatores de Transcrição/biossíntese , Regulação para CimaRESUMO
Group I p21-activated kinases (PAK) are important effectors of the small GTPases Rac and Cdc42, which regulate cell motility/migration, survival, proliferation, and gene transcription. Hyperactivation of these kinases have been reported in many tumor types, making PAKs attractive targets for therapeutic intervention. PAKs are activated by growth factor-mediated signaling and are negatively regulated by the tumor suppressor neurofibromatosis type 2 (NF2)/Merlin. Thus, tumors characterized by NF2 inactivation would be expected to show hyperactivated PAK signaling. On the basis of this rationale, we evaluated the status of PAK signaling in malignant mesothelioma, an aggressive neoplasm that is resistant to current therapies and shows frequent inactivation of NF2. We show that group I PAKs are activated in most mesotheliomas and mesothelioma cell lines and that genetic or pharmacologic inhibition of PAKs is sufficient to inhibit mesothelioma cell proliferation and survival. We also identify downstream effectors and signaling pathways that may contribute mechanistically to PAK-related tumorigenesis. Specifically, we show that inhibition of PAK results in attenuation of AKT and Raf-MAPK signaling and decreased tumor cell viability. Collectively, these data suggest that pharmacologic inhibition of group I PAKs may have therapeutic efficacy in tumors characterized by PAK activation.
Assuntos
Dissulfetos/farmacologia , Regulação Neoplásica da Expressão Gênica/genética , Mesotelioma/genética , Naftóis/farmacologia , Neurofibromina 2/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Técnicas de Silenciamento de Genes , Humanos , Mesotelioma/tratamento farmacológico , Mesotelioma/metabolismo , Camundongos , Neurofibromina 2/antagonistas & inibidores , Neurofibromina 2/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismoRESUMO
The CDKN2A/ARF locus encompasses overlapping tumor suppressor genes p16(INK4A) and p14(ARF), which are frequently co-deleted in human malignant mesothelioma (MM). The importance of p16(INK4A) loss in human cancer is well established, but the relative significance of p14(ARF) loss has been debated. The tumor predisposition of mice singly deficient for either Ink4a or Arf, due to targeting of exons 1α or 1ß, respectively, supports the idea that both play significant and nonredundant roles in suppressing spontaneous tumors. To further test this notion, we exposed Ink4a(+/-) and Arf(+/-) mice to asbestos, the major cause of MM. Asbestos-treated Ink4a(+/-) and Arf(+/-) mice showed increased incidence and shorter latency of MM relative to wild-type littermates. MMs from Ink4a(+/-) mice exhibited biallelic inactivation of Ink4a, loss of Arf or p53 expression and frequent loss of p15(Ink4b). In contrast, MMs from Arf(+/-) mice exhibited loss of Arf expression, but did not require loss of Ink4a or Ink4b. Mice doubly deficient for Ink4a and Arf, due to deletion of Cdkn2a/Arf exon 2, showed accelerated asbestos-induced MM formation relative to mice deficient for Ink4a or Arf alone, and MMs exhibited biallelic loss of both tumor suppressor genes. The tumor suppressor function of Arf in MM was p53-independent, since MMs with loss of Arf retained functional p53. Collectively, these in vivo data indicate that both CDKN2A/ARF gene products suppress asbestos carcinogenicity. Furthermore, while inactivation of Arf appears to be crucial for MM pathogenesis, the inactivation of both p16(Ink4a) and p19(Arf) cooperate to accelerate asbestos-induced tumorigenesis.
Assuntos
Amianto/efeitos adversos , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Loci Gênicos/genética , Mesotelioma/genética , Mesotelioma/patologia , Lesões Pré-Cancerosas/patologia , Proteína Supressora de Tumor p14ARF/deficiência , Alelos , Animais , Cromossomos de Mamíferos/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inativação Gênica , Humanos , Camundongos , Lesões Pré-Cancerosas/genética , Proteína Supressora de Tumor p14ARF/metabolismoRESUMO
The oncogene v-akt was isolated from a retrovirus that induced murine thymic lymphomas. Transgenic mice expressing a constitutively activated form of the cellular homologue Akt2 specifically in immature T cells develop spontaneous thymic lymphomas. We hypothesized that tumors from these mice might exhibit oncogenic chromosomal rearrangements that cooperate with activated Akt2 in lymphomagenesis. Cytogenetic analysis revealed a recurrent clonal inversion of chromosome 6, inv(6), in thymic lymphomas from multiple transgenic founder lines, including one line in which 15 of 15 primary tumors exhibited this same rearrangement. Combined fluorescence in situ hybridization, PCR, and DNA sequence analyses showed that the distal inv(6) breakpoint resides at the T-cell receptor beta chain locus, Tcrb. The proximal breakpoint maps to a region near a locus comprising the linked homeobox/transcription factor genes Dlx5 and Dlx6. Expression analysis of genes translocated to the vicinity of the Tcrb enhancer revealed that Dlx5 and Dlx6 are overexpressed in tumors exhibiting the inv(6). Experimental overexpression of Dlx5 in mammalian cells resulted in enhanced cell proliferation and increased colony formation, and clonogenic assays revealed cooperativity when both Dlx5 and activated Akt2 were coexpressed. In addition, DLX5, but not DLX6, was found to be abundantly expressed in three of seven human T-cell lymphomas tested. These findings suggest that the Dlx5 can act as an oncogene by cooperating with Akt2 to promote lymphomagenesis.
Assuntos
Inversão Cromossômica , Regulação Neoplásica da Expressão Gênica , Genes Homeobox , Proteínas de Homeodomínio/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Linfoma de Células T/genética , Proteínas Proto-Oncogênicas c-akt/genética , Fatores de Transcrição/genética , Animais , Proliferação de Células , Humanos , Linfoma de Células T/patologia , Camundongos , Camundongos Transgênicos , Modelos BiológicosRESUMO
During 3T3-L1 preadipocyte differentiation induction, the insulin-stimulated insulin-like growth factor-1 (IGF-1) receptor signal is responsible for the induction of adipocyte differentiation. Treatment with inhibitors of phosphatidylinositol 3-kinase, LY294002 or wortmannin, leads to the complete blockade of adipocyte differentiation in 3T3-L1 preadipocytes. Of the three factors (1-methyl-3-isobutylxanthine, dexamethasone, and insulin) inducing 3T3-L1 preadipocyte differentiation, only insulin was able to activate the phosphatidylinositol 3-kinase-protein kinase B/Akt signal cascade. In 3T3-L1 preadipocytes, protein kinase B/Akt 1 RNA interference not only suppressed the expression of protein kinase B/Akt 1 but also blocked hormone-induced adipocyte differentiation. In these protein kinase B/Akt 1 RNA interference cells, the signal molecules upstream of protein kinase B/Akt 1, such as IGF-1 receptor and insulin receptor substrate-1, were normally activated by insulin stimulation, whereas insulin-stimulated phosphorylation of forkhead transcription factor (FKHR), which is a downstream molecule of PKB/Akt 1, was blocked. Thus, protein kinase B/Akt 1 is an important signal mediator in IGF-1 receptor signal cascade for inducing adipocyte differentiation.