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1.
J Pathol ; 263(4-5): 466-481, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38924548

RESUMO

The E3 ubiquitin ligase thyroid hormone receptor interacting protein 12 (TRIP12) has been implicated in pancreatic adenocarcinoma (PDAC) through its role in mediating the degradation of pancreas transcription factor 1a (PTF1a). PTF1a is a transcription factor essential for the acinar differentiation state that is notably diminished during the early steps of pancreatic carcinogenesis. Despite these findings, the direct involvement of TRIP12 in the onset of pancreatic cancer has yet to be established. In this study, we demonstrated that TRIP12 protein was significantly upregulated in human pancreatic preneoplastic lesions. Furthermore, we observed that TRIP12 overexpression varied within PDAC samples and PDAC-derived cell lines. We further demonstrated that TRIP12 was required for PDAC-derived cell growth and for the expression of E2F-targeted genes. Acinar-to-ductal cell metaplasia (ADM) is a reversible process that reflects the high plasticity of acinar cells. ADM becomes irreversible in the presence of oncogenic Kras mutations and leads to the formation of preneoplastic lesions. Using two genetically modified mouse models, we showed that a loss of TRIP12 prevented acini from developing ADM in response to pancreatic injury. With two additional mouse models, we further discovered that a depletion of TRIP12 prevented the formation of KrasG12D-induced preneoplastic lesions and impaired metastasis formation in the presence of mutated KrasG12D and Trp53R172H genes. In summary our study identified an overexpression of TRIP12 from the early stages of pancreatic carcinogenesis and proposed this E3 ubiquitin ligase as a novel regulator of acinar plasticity with an important dual role in initiation and metastatic steps of PDAC. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Assuntos
Células Acinares , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Ubiquitina-Proteína Ligases , Animais , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/enzimologia , Humanos , Células Acinares/patologia , Células Acinares/metabolismo , Células Acinares/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/enzimologia , Metaplasia/patologia , Metaplasia/metabolismo , Plasticidade Celular , Carcinogênese/genética , Carcinogênese/metabolismo , Camundongos , Linhagem Celular Tumoral , Proliferação de Células , Camundongos Knockout , Regulação Neoplásica da Expressão Gênica , Lesões Pré-Cancerosas/patologia , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/enzimologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Transformação Celular Neoplásica/metabolismo , Proteínas de Transporte
2.
Int J Mol Sci ; 21(22)2020 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-33198194

RESUMO

The Thyroid hormone Receptor Interacting Protein 12 (TRIP12) protein belongs to the 28-member Homologous to the E6-AP C-Terminus (HECT) E3 ubiquitin ligase family. First described as an interactor of the thyroid hormone receptor, TRIP12's biological importance was revealed by the embryonic lethality of a murine model bearing an inactivating mutation in the TRIP12 gene. Further studies showed the participation of TRIP12 in the regulation of major biological processes such as cell cycle progression, DNA damage repair, chromatin remodeling, and cell differentiation by an ubiquitination-mediated degradation of key protein substrates. Moreover, alterations of TRIP12 expression have been reported in cancers that can serve as predictive markers of therapeutic response. The TRIP12 gene is also referenced as a causative gene associated to intellectual disorders such as Clark-Baraitser syndrome and is clearly implicated in Autism Spectrum Disorder. The aim of the review is to provide an exhaustive and integrated overview of the different aspects of TRIP12 ranging from its regulation, molecular functions and physio-pathological implications.


Assuntos
Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Animais , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Fácies , Transtornos do Crescimento/genética , Transtornos do Crescimento/metabolismo , Humanos , Hidrocefalia/genética , Hidrocefalia/metabolismo , Deficiência Intelectual Ligada ao Cromossomo X/genética , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Mutação/genética , Neoplasias/genética , Neoplasias/metabolismo , Obesidade/genética , Obesidade/metabolismo
3.
Proc Natl Acad Sci U S A ; 112(7): E738-46, 2015 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-25646470

RESUMO

Allostatic load (AL) is a measure of overall physiological wear-and-tear over the life course, which could partially be the consequence of early life exposures. AL could allow a better understanding of the potential biological pathways playing a role in the construction of the social gradient in adult health. To explore the biological embedding hypothesis, we examined whether adverse childhood experiences (ACEs) are associated with elevated AL in midlife. We used imputed data on 3,782 women and 3,753 men of the National Child Development Study in Britain followed up seven times. ACEs were measured using prospective data collected at ages 7, 11, and 16. AL was operationalized using data from the biomedical survey collected at age 44 on 14 parameters representing four biological systems. We examined the role of adult health behaviors, body mass index (BMI), and socioeconomic status as potential mediators using a path analysis. ACEs were associated with higher AL for both men and women after adjustment for early life factors and childhood pathologies. The path analysis showed that the association between ACEs and AL was largely explained by early adult factors at age 23 and 33. For men, the total mediated effect was 59% (for two or more ACEs) via health behaviors, education level, and wealth. For women, the mediated effect represented 76% (for two or more ACEs) via smoking, BMI, education level, and wealth. Our results indicate that early psychosocial stress has an indirect lasting impact on physiological wear-and-tear via health behaviors, BMI, and socioeconomic factors in adulthood.


Assuntos
Nível de Saúde , Acontecimentos que Mudam a Vida , Estudos de Coortes , Humanos , Pessoa de Meia-Idade , Reino Unido
4.
Biochim Biophys Acta ; 1853(10 Pt A): 2392-403, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26036346

RESUMO

MUC1 is an oncogenic mucin overexpressed in several epithelial cancers, including pancreatic ductal adenocarcinoma, and is considered as a potent target for cancer therapy. To control cancer progression, miRNAs became very recently, major targets and tools to inhibit oncogene expression. Inhibiting MUC1 using miRNAs appears thus as an attractive strategy to reduce cancer progression. However, potent miRNAs and associated mechanisms regulating MUC1 expression remain to be identified. To this aim, we undertook to study MUC1 regulation by miRNAs in pancreatic cancer cells and identify those with tumor suppressive activity. MiRNAs potentially targeting the 3'-UTR, the coding region, or the 5'-UTR of MUC1 were selected using an in silico approach. Our in vitro and in vivo experiments indicate that miR-29a and miR-330-5p are strong inhibitors of MUC1 expression in pancreatic cancer cells through direct binding to MUC1 3'-UTR. MUC1 regulation by the other selected miRNAs (miR-183, miR-200a, miR-876-3p and miR-939) was found to be indirect. MiR-29a and miR-330-5p are also deregulated in human pancreatic cancer cell lines and tissues and in pancreatic tissues of Kras(G12D) mice. In vitro, miR-29a and miR-330-5p inhibit cell proliferation, cell migration, cell invasion and sensitize pancreatic cancer cells to gemcitabine. In vivo intra-tumoral injection of these two miRNAs in xenografted pancreatic tumors led to reduced tumor growth. Altogether, we have identified miR-29a and miR-330-5p as two new tumor suppressive miRNAs that inhibit the expression of MUC1 oncogenic mucin in pancreatic cancer cells.


Assuntos
Genes Supressores de Tumor , MicroRNAs/biossíntese , Mucina-1/biossíntese , Neoplasias Pancreáticas/metabolismo , RNA Neoplásico/biossíntese , Regiões 5' não Traduzidas/genética , Animais , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Camundongos , MicroRNAs/genética , Mucina-1/genética , Transplante de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , RNA Neoplásico/genética
5.
Mol Ther ; 23(4): 779-89, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25586689

RESUMO

This phase 1 trial was aimed to determine the safety, pharmacokinetics, and preliminary clinical activity of CYL-02, a nonviral gene therapy product that sensitizes pancreatic cancer cells to chemotherapy. CYL-02 was administrated using endoscopic ultrasound in 22 patients with pancreatic cancer that concomitantly received chemotherapy (gemcitabine). The maximum-tolerated dose (MTD) exceeded the maximal feasible dose of CYL-02 and was not identified. Treatment-related toxicities were mild, without serious adverse events. Pharmacokinetic analysis revealed a dose-dependent increase in CYL-02 DNA exposure in blood and tumors, while therapeutic RNAs were detected in tumors. No objective response was observed, but nine patients showed stable disease up to 6 months following treatment and two of these patients experienced long-term survival. Panels of plasmatic microRNAs and proteins were identified as predictive of gene therapy efficacy. We demonstrate that CYL-02 nonviral gene therapy has a favorable safety profile and is well tolerated in patients. We characterize CYL-02 biodistribution and demonstrate therapeutic gene expression in tumors. Treated patients experienced stability of disease and predictive biomarkers of response to treatment were identified. These promising results warrant further evaluation in phase 2 clinical trial.


Assuntos
Terapia Genética , Neoplasias Pancreáticas/terapia , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/metabolismo , Distribuição Tecidual
6.
J Biol Chem ; 289(51): 35593-604, 2014 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-25355311

RESUMO

Pancreas transcription factor 1a (PTF1a) plays a crucial role in the early development of the pancreas and in the maintenance of the acinar cell phenotype. Several transcriptional mechanisms regulating expression of PTF1a have been identified. However, regulation of PTF1a protein stability and degradation is still unexplored. Here, we report that inhibition of proteasome leads to elevated levels of PTF1a and to the existence of polyubiquitinated forms of PTF1a. We used the Sos recruitment system, an alternative two-hybrid system method to detect protein-protein interactions in the cytoplasm and to map the interactome of PTF1a. We identified TRIP12 (thyroid hormone receptor-interacting protein 12), an E3 ubiquitin-protein ligase as a new partner of PTF1a. We confirmed PTF1a/TRIP12 interaction in acinar cell lines and in co-transfected HEK-293T cells. The protein stability of PTF1a is significantly increased upon decreased expression of TRIP12. It is reduced upon overexpression of TRIP12 but not a catalytically inactive TRIP12-C1959A mutant. We identified a region of TRIP12 required for interaction and identified lysine 312 of PTF1a as essential for proteasomal degradation. We also demonstrate that TRIP12 down-regulates PTF1a transcriptional and antiproliferative activities. Our data suggest that an increase in TRIP12 expression can play a part in PTF1a down-regulation and indicate that PTF1a/TRIP12 functional interaction may regulate pancreatic epithelial cell homeostasis.


Assuntos
Proteínas de Transporte/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Western Blotting , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Proliferação de Células , Citoplasma/metabolismo , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Neoplasias Pancreáticas/patologia , Ligação Proteica , Estabilidade Proteica , Proteólise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
7.
Commun Biol ; 7(1): 1065, 2024 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-39215188

RESUMO

Cytidine deaminase (CDA) converts cytidine and deoxycytidine into uridine and deoxyuridine as part of the pyrimidine salvage pathway. Elevated levels of CDA are found in pancreatic tumors and associated with chemoresistance. Recent evidence suggests that CDA has additional functions in cancer cell biology. In this work, we uncover a novel role of CDA in pancreatic cancer cell metabolism. CDA silencing impairs mitochondrial metabolite production, respiration, and ATP production in pancreatic cancer cells, leading to a so-called Pasteur effect metabolic shift towards glycolysis. Conversely, we find that CDA expression promotes mitochondrial biogenesis and oxidative phosphorylation, independently of CDA deaminase activity. Furthermore, we observe that patient primary cells overexpressing CDA are more sensitive to mitochondria-targeting drugs. Collectively, this work shows that CDA plays a non-canonical role in pancreatic cancer biology by promoting mitochondrial function, which could be translated into novel therapeutic vulnerabilities.


Assuntos
Citidina Desaminase , Mitocôndrias , Biogênese de Organelas , Neoplasias Pancreáticas , Humanos , Citidina Desaminase/metabolismo , Citidina Desaminase/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Mitocôndrias/metabolismo , Linhagem Celular Tumoral , Fosforilação Oxidativa , Glicólise
8.
Cancer Res ; 84(7): 1013-1028, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38294491

RESUMO

Cytidine deaminase (CDA) functions in the pyrimidine salvage pathway for DNA and RNA syntheses and has been shown to protect cancer cells from deoxycytidine-based chemotherapies. In this study, we observed that CDA was overexpressed in pancreatic adenocarcinoma from patients at baseline and was essential for experimental tumor growth. Mechanistic investigations revealed that CDA localized to replication forks where it increased replication speed, improved replication fork restart efficiency, reduced endogenous replication stress, minimized DNA breaks, and regulated genetic stability during DNA replication. In cellular pancreatic cancer models, high CDA expression correlated with resistance to DNA-damaging agents. Silencing CDA in patient-derived primary cultures in vitro and in orthotopic xenografts in vivo increased replication stress and sensitized pancreatic adenocarcinoma cells to oxaliplatin. This study sheds light on the role of CDA in pancreatic adenocarcinoma, offering insights into how this tumor type modulates replication stress. These findings suggest that CDA expression could potentially predict therapeutic efficacy and that targeting CDA induces intolerable levels of replication stress in cancer cells, particularly when combined with DNA-targeted therapies. SIGNIFICANCE: Cytidine deaminase reduces replication stress and regulates DNA replication to confer resistance to DNA-damaging drugs in pancreatic cancer, unveiling a molecular vulnerability that could enhance treatment response.


Assuntos
Adenocarcinoma , Citidina Desaminase , Inibidores da Síntese de Ácido Nucleico , Neoplasias Pancreáticas , Humanos , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Citidina Desaminase/metabolismo , DNA , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Replicação do DNA , Inibidores da Síntese de Ácido Nucleico/uso terapêutico
9.
Med Sci (Paris) ; 29(11): 991-7, 2013 Nov.
Artigo em Francês | MEDLINE | ID: mdl-24280502

RESUMO

Point mutations of the Kras oncogene induce in cancerous cells an uncontrolled increase of cell proliferation and invasiveness. Mutation of Kras appears early during the process of the pancreatic carcinogenesis and is the most frequent genetic alteration in pancreatic adenocarcinoma (75 to 95 % of cases) as well as in precancerous lesions such as PanIN and IMPN. These latter lesions and tumour microenvironment are reproduced in transgenic models developed in mice. These models are induced on the basis of Kras mutation (Pdx1-Cre ; Kras(G12D) mice) associated or not to the inactivation of tumour suppressor genes (TP53, DPC4, INK4A). Kras mutation assay is easily performed in human biological samples, especially in the cellular material sampled in pancreatic masses under endoscopic ultrasound by fine needle aspiration biopsy. In the near future, searching for Kras mutation could be useful in clinical practice either for positive diagnosis of pancreatic adenocarcinoma in case of unconclusive/doubtful cytopathological analysis or for the differential diagnosis with chronic pancreatitis especially in its pseudotumoural form.


Assuntos
Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Biópsia por Agulha Fina , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)
10.
Int J Mol Sci ; 14(7): 15029-58, 2013 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-23873296

RESUMO

DNA methylation is a major epigenetic modification that is strongly involved in the physiological control of genome expression. DNA methylation patterns are largely modified in cancer cells and can therefore be used to distinguish cancer cells from normal tissues. This review describes the main technologies available for the detection and the discovery of aberrantly methylated DNA patterns. It also presents the different sources of biological samples suitable for DNA methylation studies. We discuss the interest and perspectives on the use of DNA methylation measurements for cancer diagnosis through examples of methylated genes commonly documented in the literature. The discussion leads to our consideration for why DNA methylation is not commonly used in clinical practice through an examination of the main requirements that constitute a reliable biomarker. Finally, we describe the main DNA methylation inhibitors currently used in clinical trials and those that exhibit promising results.


Assuntos
Metilação de DNA , Neoplasias/diagnóstico , Biomarcadores/sangue , Biomarcadores/metabolismo , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Humanos , Neoplasias/metabolismo
11.
Cell Death Dis ; 14(10): 692, 2023 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-37863914

RESUMO

Transforming growth factor ß (TGFß) pathway is a master regulator of cell proliferation, differentiation, and death. Deregulation of TGFß signalling is well established in several human diseases including autoimmune disorders and cancer. Thus, understanding molecular pathways governing TGFß signalling may help better understand the underlying causes of some of those conditions. Here, we show that a HECT domain E3 ubiquitin ligase TRIP12 controls TGFß signalling in multiple models. Interestingly, TRIP12 control of TGFß signalling is completely independent of its E3 ubiquitin ligase activity. Instead, TRIP12 recruits SMURF2 to SMAD4, which is most likely responsible for inhibitory monoubiquitination of SMAD4, since SMAD4 monoubiquitination and its interaction with SMURF2 were dramatically downregulated in TRIP12-/- cells. Additionally, genetic inhibition of TRIP12 in human and murine cells leads to robust activation of TGFß signalling which was rescued by re-introducing wildtype TRIP12 or a catalytically inactive C1959A mutant. Importantly, TRIP12 control of TGFß signalling is evolutionary conserved. Indeed, genetic inhibition of Drosophila TRIP12 orthologue, ctrip, in gut leads to a reduced number of intestinal stem cells which was compensated by the increase in differentiated enteroendocrine cells. These effects were completely normalised in Drosophila strain where ctrip was co-inhibited together with Drosophila SMAD4 orthologue, Medea. Similarly, in murine 3D intestinal organoids, CRISPR/Cas9 mediated genetic targeting of Trip12 enhances TGFß mediated proliferation arrest and cell death. Finally, CRISPR/Cas9 mediated genetic targeting of TRIP12 in MDA-MB-231 breast cancer cells enhances the TGFß induced migratory capacity of these cells which was rescued to the wildtype level by re-introducing wildtype TRIP12. Our work establishes TRIP12 as an evolutionary conserved modulator of TGFß signalling in health and disease.


Assuntos
Proteínas de Transporte , Fator de Crescimento Transformador beta , Animais , Humanos , Camundongos , Proteínas de Transporte/metabolismo , Drosophila/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
12.
Cancers (Basel) ; 13(24)2021 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-34944818

RESUMO

BACKGROUND: Pancreatic adenocarcinoma (PDAC) is a deadly cancer with an extremely poor prognosis. MUC4 membrane-bound mucin is neoexpressed in early pancreatic neoplastic lesions and is associated with PDAC progression and chemoresistance. In cancers, microRNAs (miRNAs, small noncoding RNAs) are crucial regulators of carcinogenesis, chemotherapy response and even metastatic processes. In this study, we aimed at identifying and characterizing miRNAs activated downstream of MUC4-associated signaling in pancreatic adenocarcinoma. MiRnome analysis comparing MUC4-KD versus Mock cancer cells showed that MUC4 inhibition impaired miR-210-3p expression. Therefore, we aimed to better understand the miR-210-3p biological roles. METHODS: miR-210-3p expression level was analyzed by RT-qPCR in PDAC-derived cell lines (PANC89 Mock and MUC4-KD, PANC-1 and MiaPACA-2), as well as in mice and patients tissues. The MUC4-miR-210-3p regulation was investigated using luciferase reporter construct and chromatin immunoprecipitation experiments. Stable cell lines expressing miR-210-3p or anti-miR-210-3p were established using CRISPR/Cas9 technology or lentiviral transduction. We evaluated the biological activity of miR-210-3p in vitro by measuring cell proliferation and migration and in vivo using a model of subcutaneous xenograft. RESULTS: miR-210-3p expression is correlated with MUC4 expression in PDAC-derived cells and human samples, and in pancreatic PanIN lesions of Pdx1-Cre; LstopL-KrasG12D mice. MUC4 enhances miR-210-3p expression levels via alteration of the NF-κB signaling pathway. Chromatin immunoprecipitation experiments showed p50 NF-κB subunit binding on miR-210-3p promoter regions. We established a reciprocal regulation since miR-210-3p repressed MUC4 expression via its 3'-UTR. MiR-210-3p transient transfection of PANC89, PANC-1 and MiaPACA-2 cells led to a decrease in cell proliferation and migration. These biological effects were validated in cells overexpressing or knocked-down for miR-210-3p. Finally, we showed that miR-210-3p inhibits pancreatic tumor growth and proliferation in vivo. CONCLUSION: We identified a MUC4-miR-210-3p negative feedback loop in early-onset PDAC, but also revealed new functions of miR-210-3p in both in vitro and in vivo proliferation and migration of pancreatic cancer cells, suggesting a complex balance between MUC4 pro-oncogenic roles and miR-210-3p anti-tumoral effects.

13.
Cancers (Basel) ; 13(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799792

RESUMO

Pancreatic ducal adenocarcinoma is classically diagnosed in the 7th decade, but approximately 10% of patients are diagnosed under 55 years (y.o.). While the genomic and transcriptomic landscapes of late-onset tumors (LOT) have been described, little is known about early-onset tumors (EOT). Ageing is known to impact DNA methylation and proteome integrity through carbonylation-related oxidative damages. We therefore aimed to assess the global molecular features of EOT. We compared 176 EOT (≤55 y.o.) and 316 LOT (≥70 y.o.) from three distinct surgical cohorts at the clinical/genomic/epigenomic/transcriptomic level. Furthermore, we assessed oxidative stress responses and oxidative proteome damages using 2D gel electrophoresis followed by mass spectrometry protein identification. There was no consistent clinical difference between EOT and LOT across the three cohorts. The mutational landscape of key driver genes and the global methylation profile were similar in the two groups. LOT did display age-related features such as enriched DNA repair gene signatures and upregulation of oxidative stress defenses together with increased proteome carbonylation. However, these age-related differences were more preeminent in non-tumor tissues while tumor proteome and proteome damages were fairly comparable. In conclusion, this multi-omics comparison showed that EOT harbor a comparable molecular profile to that of LOT.

14.
J Hepatol ; 53(5): 880-8, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20801538

RESUMO

BACKGROUND & AIMS: KLF6 protein is a transcription factor that plays important functions in hepatocellular carcinoma (HCC), which is one of the leading causes of death by cancer worldwide. Previous studies showed the existence of three splice variants of KLF6, termed SV1, SV2, and SV3. An increased SV1/KLF6 mRNA ratio in HCC was already described. In this study, we aimed to investigate the expression of the SV2 variant in HCC samples and its role in hepatic cells. METHODS: We measured the expression of the SV2 variant in HCC and adjacent tissue samples by q-RT-PCR. We established IHH and HepG2 stable cell lines over-expressing the SV2 variant and measured cell growth and apoptotic rate. RESULTS: We observed a reduced expression of the SV2 variant in HCC samples versus surrounding tissues and normal liver. Interestingly, our findings demonstrate that the over-expression of the SV2 variant in IHH and HepG2 cells leads to a significant reduction of proliferation associated with cell death by apoptosis. We further demonstrate that the SV2 expression leads to an induction of the cell-cycle-controlling p21(CIP/WAF1) and the pro-apoptotic Bax genes, mediated by the p53 protein. We show further that the SV2 expression in IHH and HepG2 cells induces their sensitivity to the anti-cancer drug, gemcitabine. CONCLUSION: We reveal a reduced expression of the SV2 variant of KLF6 in HCC samples and describe anti-proliferative and pro-apoptotic functions for this variant in hepatic cells.


Assuntos
Apoptose , Carcinoma Hepatocelular/patologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/fisiologia , Neoplasias Hepáticas/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Processamento Alternativo , Carcinoma Hepatocelular/tratamento farmacológico , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/análise , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Regulação para Baixo , Células Hep G2 , Humanos , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Neoplasias Hepáticas/tratamento farmacológico , Isoformas de Proteínas , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteína Supressora de Tumor p53/fisiologia , Proteína X Associada a bcl-2/análise , Gencitabina
15.
Clin Chem ; 56(4): 603-12, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20093556

RESUMO

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has the poorest overall prognosis among gastrointestinal cancers; however, curative resection in early-stage PDAC greatly improves survival rates, indicating the importance of early detection. Because abnormal microRNA production is commonly detected in cancer, we investigated noninvasive precursor pancreatic intraepithelial neoplasia (PanIN) lesions for microRNA production as a potential early biomarker of PDAC. METHODS: Pathologists identified and classified ductal lesions. We extracted total RNA from laser-capture microdissected PanIN tissue samples from a conditional KRAS(G12D) mouse model (n = 29) or of human origin (n = 38) (KRAS is v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog). MicroRNA production was quantified by quantitative real-time PCR. Internal controls included 5S and U6 RNAs. RESULTS: Production of microRNAs miR-21, miR-205, and miR-200 paralleled PanIN progression in the KRAS(G12D) mouse model, compared with microRNA production in samples of nonpathologic ducts. miR-21 demonstrated the highest relative concentrations in the precursor lesions. Interestingly, miR-205 and miR-21 up-regulation preceded phenotypic changes in the ducts. The production of microRNAs miR-21, miR-221, miR-222, and let-7a increased with human PanIN grade, with peak production occurring in hyperplastic PanIN-2/3 lesions. In situ hybridization analysis indicated miR-21 production to be concentrated in pathologic ductal cells. miR-21 production was regulated by KRAS(G12D) and epidermal growth factor receptor in PDAC-derived cell lines. CONCLUSIONS: Aberrant microRNA production is an early event in the development of PanIN. Our findings indicate that miR-21 warrants further investigation as a marker for early detection of PDAC.


Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/genética , MicroRNAs/genética , Neoplasias Pancreáticas/genética , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Animais , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/cirurgia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos Knockout , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/cirurgia , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade
16.
Clin Chem ; 56(7): 1107-18, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20431052

RESUMO

BACKGROUND: The poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is accounted for by the absence of early diagnostic markers and effective treatments. MicroRNAs inhibit the translation of their target mRNAs. The production of microRNAs is strongly altered in cancers, but the causes of these alterations are only partially known. DNA hypermethylation is a major cause of gene inactivation in cancer. Our aims were to identify microRNAs whose gene expression is inactivated by hypermethylation in PDAC and to determine whether this hypermethylation-mediated repression is an early event during pancreatic carcinogenesis. We also sought to investigate whether these differentially methylated regions can serve as a diagnostic marker for PDAC. METHODS: MicroRNA production was measured by microarray hybridization and reverse-transcription quantitative PCR. The level of DNA methylation was measured by bisulfite mapping and semiquantitative methylation-specific PCR. RESULTS: We identified 29 microRNAs encoded by genes whose expression is potentially inactivated by DNA hypermethylation. We focused our study on microRNA 148a (miR-148a) and found its production to be repressed, not only in PDAC samples but also in preneoplastic pancreatic intraepithelial neoplasia (PanIN) lesions. More importantly, we found that hypermethylation of the DNA region encoding miR-148a is responsible for its repression, which occurs in PanIN preneoplastic lesions. Finally, we show that the hypermethylated DNA region encoding miR-148a can serve as an ancillary marker for the differential diagnosis of PDAC and chronic pancreatitis (CP). CONCLUSIONS: We show that the hypermethylation of the DNA region encoding miR-148a is responsible for its repression in PDAC precursor lesions and can be a useful tool for the differential diagnosis of PDAC and CP.


Assuntos
Carcinoma Ductal Pancreático/diagnóstico , Metilação de DNA , MicroRNAs/biossíntese , Neoplasias Pancreáticas/diagnóstico , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Diagnóstico Diferencial , Regulação para Baixo , Inativação Gênica , Humanos , Camundongos , Camundongos Mutantes , MicroRNAs/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Pancreatite Crônica/diagnóstico , Reação em Cadeia da Polimerase , Lesões Pré-Cancerosas/diagnóstico , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia
17.
Mol Cell Biol ; 27(1): 395-410, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17030625

RESUMO

DNA methylation is a major determinant of epigenetic inheritance. DNA methyltransferase 1 (DNMT1) is the enzyme responsible for the maintenance of DNA methylation patterns during cell division, and deregulated expression of DNMT1 leads to cellular transformation. We show herein that AU-rich element/poly(U)-binding/degradation factor 1 (AUF1)/heterogeneous nuclear ribonucleoprotein D interacts with an AU-rich conserved element in the 3' untranslated region of the DNMT1 mRNA and targets it for destabilization by the exosome. AUF1 protein levels are regulated by the cell cycle by the proteasome, resulting in cell cycle-specific destabilization of DNMT1 mRNA. AUF1 knock down leads to increased DNMT1 expression and modifications of cell cycle kinetics, increased DNA methyltransferase activity, and genome hypermethylation. Concurrent AUF1 and DNMT1 knock down abolishes this effect, suggesting that the effects of AUF1 knock down on the cell cycle are mediated at least in part by DNMT1. In this study, we demonstrate a link between AUF1, the RNA degradation machinery, and maintenance of the epigenetic integrity of the cell.


Assuntos
DNA (Citosina-5-)-Metiltransferases/fisiologia , Metilação de DNA , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/fisiologia , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Sequência de Bases , Ciclo Celular , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , Epigênese Genética , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular
18.
Endocrinology ; 149(6): 3137-47, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18325993

RESUMO

Somatostatin is a neuropeptide that inhibits exocrine and endocrine secretions of several hormones and negatively regulates cell proliferation. These events are mediated through somatostatin engagement on one of five G protein-coupled receptors named SSTR1 to STTR5. Somatostatin binding to SSTR2 mediates predominantly antisecretory and antiproliferative effects; two important biological activities in the gastroenteropancreatic endocrine and exocrine system. Herein we demonstrate novel regulatory sequences for human (h) SSTR2 transcription. By genomic DNA sequence analysis, we reveal two CpG islands located 3.8 kb upstream from the transcription start site. We identify a novel transcription start site and a promoter region within one of these CpG islands. We demonstrate that two epigenetic modifications, DNA methylation and histone acetylation, regulate the activation of hSSTR2 upstream promoter. Furthermore, we show that the transcription from this upstream promoter region directly correlates to hSSTR2 mRNA expression in various human cell lines. A combined treatment of a demethylating agent, 5-aza-2-deoxycytidine and a histone deacetylase inhibitor, trichostatin A, leads to increased expression of hSSTR2 mRNA in cell lines in which the CpG island is methylated. The epigenetic regulation of this promoter region results in differential expression of hSSTR2 mRNA in human cell lines. This study reveals the existence of a novel upstream promoter for the hSSTR2 gene that is regulated by epigenetic modifications, suggesting for complex control of the hSSTR2 transcription.


Assuntos
Cromossomos Humanos Par 17 , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Receptores de Somatostatina/genética , Sequência de Bases , Cromatina/genética , Cromatina/ultraestrutura , Metilação de DNA , Fosfatos de Dinucleosídeos , Éxons , Genes Reporter , Humanos , Luciferases/genética , Dados de Sequência Molecular , Plasmídeos , Deleção de Sequência , Homologia de Sequência do Ácido Nucleico
19.
Biol Proced Online ; 10: 47-57, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19048127

RESUMO

DNA methyltransferase 1 (DNMT1) is the enzyme responsible for the maintenance of DNA methylation patterns during cell division. DNMT1 expression is tightly regulated within the cell cycle. Our previous study showed that the binding of a protein with an apparent size of approximately 40 kDa on DNMT1 3'-UTR triggered the destabilization of DNMT1 mRNA transcript during G(o)/G(1) phase. Using RNA affinity capture with the 3'-UTR of DNMT1 mRNA and matrix-assisted laser desorption-time of flight tandem mass spectrometry (MALDI-TOF-MS-MS) analysis, we isolated and identified AUF 1 (AU-rich element ARE:poly-(U)-binding/degradation factor) as the binding protein. We then validated the role of this protein in the destabilization of DNMT1 mRNA. In this report, we detail the different approaches used for the isolation, the identification of a RNA binding protein and the validation of its role.

20.
J Gastroenterol Hepatol ; 23(1): 78-86, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18171345

RESUMO

BACKGROUND AND AIMS: Macronodules (MN) occurring in cirrhosis are considered to be precursor lesions for hepatocellular carcinoma (HCC). However, early molecular events in hepatocellular carcinogenesis are poorly understood. The aim of this study was to compare gene expression profiling between cirrhotic tissues, MN, and HCC, to identify genes early involved in liver carcinogenesis. METHODS: Tissues were obtained from explanted livers: nine cirrhosis, 10 MN, and seven HCC. Total RNAs were extracted by RNeasy and reverse transcribed with labelled [(33)P]-alpha ATP. Hybridations were performed on Atlas Human Cancer 1.2 membranes (1176 genes). RESULTS: A two-way hierarchical clustering algorithm successfully isolated specific gene expression profiles when comparing MN, cirrhosis, and HCC. A total of 16 and 14 genes were up- and down-expressed, respectively, in HCC as compared to cirrhotic tissues. The molecular signature of MN was characterized by the down-expression of 23 and 42 genes as compared to cirrhosis and HCC, respectively. Among them, Klf6 was down-expressed in all MN samples whereas it was over-expressed in cirrhosis and HCC. This result was confirmed at RNA level by quantitative real time-polymerase chain reaction and at protein level by Western blotting. However, no mutation in the exon 2 of Klf6 was detected. CONCLUSION: We identified a molecular signature of MN characterized by a down-expression of several genes. One of them, Klf6 was found to be down-expressed in all MN without evidence of somatic mutations in the exon 2. This gene could be involved at an early stage of hepatocarcinogenesis.


Assuntos
Carcinoma Hepatocelular/genética , Fatores de Transcrição Kruppel-Like/genética , Cirrose Hepática/genética , Neoplasias Hepáticas/genética , Proteínas Proto-Oncogênicas/genética , Adulto , Idoso , Carcinoma Hepatocelular/metabolismo , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Humanos , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/biossíntese , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/biossíntese
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