RESUMO
BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (iCCA) is a lethal malignancy, with increasing incidence worldwide and limited therapeutic options. Aberrant protein glycosylation is a hallmark of cancer. Here, we thoroughly investigated the possible involvement of fucosylation in cholangiocarcinogenesis. APPROACH AND RESULTS: We discovered that the levels of global fucosylation and members of the fucosylation pathway are ubiquitously upregulated in human iCCA tissues compared to nontumorous surrounding livers and normal biliary cells. In addition, total fucosylation levels correlate with poor patients' prognosis. Furthermore, fucosylation inhibition following 6-alkynylfucose (6AF) administration triggered a dose-dependent decrease in the proliferation and migration of iCCA cell lines. Notably, adding fucose to the cell medium annulled these effects. At the molecular level, 6AF administration or small interfering RNA-mediated silencing of GDP-L-fucose synthetase (FX) and the GDP-fucose transmembrane transporter (SLC35C1), both pivotal players of cellular fucosylation, decreased NOTCH activity, NOTCH1/Jagged1 interaction, NOTCH receptors, and related target genes in iCCA cell lines. In the same cells, EGFR, nuclear factor kappa-light-chain-enhancer of activated B cells p65, and Bcl-xL protein levels diminished, whereas IκBα (a critical cellular NF-κB inhibitor) increased after FX/SLC35C1 knockdown or 6AF administration. In the chick chorioallantoic membrane assay, 6AF treatment profoundly suppresses the growth of iCCA cells. CONCLUSIONS: Elevated global fucosylation characterizes human iCCA, contributing to cell growth and migration through the upregulation of the NOTCH and EGFR/NF-κB pathways. Thus, aberrant fucosylation is a novel pathogenetic player and a potential therapeutic target for human iCCA.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , NF-kappa B/metabolismo , Glicosilação , Prognóstico , Fucose/metabolismo , Colangiocarcinoma/patologia , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Receptores ErbB/metabolismoRESUMO
Background and Objectives: Aberrant upregulation of fatty acid synthase (FASN), catalyzing de novo synthesis of fatty acids, occurs in various tumor types, including human hepatocellular carcinoma (HCC). Although FASN oncogenic activity seems to reside in its pro-lipogenic function, cumulating evidence suggests that FASN's tumor-supporting role might also be metabolic-independent. Materials and Methods: In the present study, we show that FASN inactivation by specific small interfering RNA (siRNA) promoted the downregulation of the S-phase kinase associated-protein kinase 2 (SKP2) and the consequent induction of p27KIP1 in HCC cell lines. Results: Expression levels of FASN and SKP2 directly correlated in human HCC specimens and predicted a dismal outcome. In addition, forced overexpression of SKP2 rendered HCC cells resistant to the treatment with the FASN inhibitor C75. Furthermore, FASN deletion was paralleled by SKP2 downregulation and p27KIP1 induction in the AKT-driven HCC preclinical mouse model. Moreover, forced overexpression of an SKP2 dominant negative form or a p27KIP1 non-phosphorylatable (p27KIP1-T187A) construct completely abolished AKT-dependent hepatocarcinogenesis in vitro and in vivo. Conclusions: In conclusion, the present data indicate that SKP2 is a critical downstream effector of FASN and AKT-dependent hepatocarcinogenesis in liver cancer, envisaging the possibility of effectively targeting FASN-positive liver tumors with SKP2 inhibitors or p27KIP1 activators.
Assuntos
Carcinoma Hepatocelular , Inibidor de Quinase Dependente de Ciclina p27 , Neoplasias Hepáticas , Proteínas Quinases Associadas a Fase S , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Humanos , Animais , Camundongos , Linhagem Celular Tumoral , Ácido Graxo Sintases/metabolismo , Ácido Graxo Sintase Tipo I/metabolismo , Ácido Graxo Sintase Tipo I/genética , Regulação para Baixo , MasculinoRESUMO
BACKGROUND & AIMS: YES-associated protein (YAP) aberrant activation is implicated in intrahepatic cholangiocarcinoma (iCCA). Transcriptional enhanced associate domain (TEAD)-mediated transcriptional regulation is the primary signaling event downstream of YAP. The role of Wnt/ß-Catenin signaling in cholangiocarcinogenesis remains undetermined. Here, we investigated the possible molecular interplay between YAP and ß-Catenin cascades in iCCA. METHODS: Activated AKT (Myr-Akt) was coexpressed with YAP (YapS127A) or Tead2VP16 via hydrodynamic tail vein injection into mouse livers. Tumor growth was monitored, and liver tissues were collected and analyzed using histopathologic and molecular analysis. YAP, ß-Catenin, and TEAD interaction in iCCAs was investigated through coimmunoprecipitation. Conditional Ctnnb1 knockout mice were used to determine ß-Catenin function in murine iCCA models. RNA sequencing was performed to analyze the genes regulated by YAP and/or ß-Catenin. Immunostaining of total and nonphosphorylated/activated ß-Catenin staining was performed in mouse and human iCCAs. RESULTS: We discovered that TEAD factors are required for YAP-dependent iCCA development. However, transcriptional activation of TEADs did not fully recapitulate YAP's activities in promoting cholangiocarcinogenesis. Notably, ß-Catenin physically interacted with YAP in human and mouse iCCA. Ctnnb1 ablation strongly suppressed human iCCA cell growth and Yap-dependent cholangiocarcinogenesis. Furthermore, RNA-sequencing analysis revealed that YAP/ transcriptional coactivator with PDZ-binding motif (TAZ) regulate a set of genes significantly overlapping with those controlled by ß-Catenin. Importantly, activated/nonphosphorylated ß-Catenin was detected in more than 80% of human iCCAs. CONCLUSION: YAP induces cholangiocarcinogenesis via TEAD-dependent transcriptional activation and interaction with ß-Catenin. ß-Catenin binds to YAP in iCCA and is required for YAP full transcriptional activity, revealing the functional crosstalk between YAP and ß-Catenin pathways in cholangiocarcinogenesis.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Proteínas de Sinalização YAP , beta Catenina , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/patologia , Carcinogênese , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAP/genética , Proteínas de Sinalização YAP/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is a primary liver tumor with high lethality and increasing incidence worldwide. While tumor resection or liver transplantation is effective in the early stages of the disease, the therapeutic options for advanced HCC remain limited and the benefits are temporary. Thus, novel therapeutic targets and more efficacious treatments against this deadly cancer are urgently needed. Here, we investigated the pathogenetic and therapeutic role of eukaryotic initiation factor 4A1 (eIF4A1) in this tumor type. We observed consistent eIF4A1 upregulation in HCC lesions compared with non-tumorous surrounding liver tissues. In addition, eIF4A1 levels were negatively correlated with the prognosis of HCC patients. In HCC lines, the exposure to various eIF4A inhibitors triggered a remarkable decline in proliferation and augmented apoptosis, paralleled by the inhibition of several oncogenic pathways. Significantly, anti-growth effects were achieved at nanomolar concentrations of the eIF4A1 inhibitors and were further increased by the simultaneous administration of the pan mTOR inhibitor, Rapalink-1. In conclusion, our results highlight the pathogenetic relevance of eIF4A1 in HCC and recommend further evaluation of the potential usefulness of pharmacological combinations based on eIF4A and mTOR inhibitors in treating this aggressive tumor.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Prognóstico , Apoptose , Proliferação de Células , Linhagem Celular TumoralRESUMO
BACKGROUND AND AIMS: Mothers against decapentaplegic homolog (SMAD) 7 is an antagonist of TGF-ß signaling. In the present investigation, we sought to determine the relevance of SMAD7 in liver carcinogenesis using in vitro and in vivo approaches. APPROACH AND RESULTS: We found that SMAD7 is up-regulated in a subset of human HCC samples with poor prognosis. Gene set enrichment analysis revealed that SMAD7 expression correlates with activated yes-associated protein (YAP)/NOTCH pathway and cholangiocellular signature genes in HCCs. These findings were substantiated in human HCC cell lines. In vivo, overexpression of Smad7 alone was unable to initiate HCC development, but it significantly accelerated c-Myc/myeloid cell leukemia 1 (MCL1)-induced mouse HCC formation. Consistent with human HCC data, c-Myc/MCL1/Smad7 liver tumors exhibited an increased cholangiocellular gene expression along with Yap/Notch activation and epithelial-mesenchymal transition (EMT). Intriguingly, blocking of the Notch signaling did not affect c-Myc/MCL1/Smad7-induced hepatocarcinogenesis while preventing cholangiocellular signature expression and EMT, whereas ablation of Yap abolished c-Myc/MCL1/Smad7-driven HCC formation. In mice overexpressing a myristoylated/activated form of AKT, coexpression of SMAD7 accelerated carcinogenesis and switched the phenotype from HCC to intrahepatic cholangiocarcinoma (iCCA) lesions. In human iCCA, SMAD7 expression was robustly up-regulated, especially in the most aggressive tumors, and directly correlated with the levels of YAP/NOTCH targets as well as cholangiocellular and EMT markers. CONCLUSIONS: The present data indicate that SMAD7 contributes to liver carcinogenesis by activating the YAP/NOTCH signaling cascade and inducing a cholangiocellular and EMT signature.
Assuntos
Neoplasias dos Ductos Biliares/genética , Carcinoma Hepatocelular/genética , Colangiocarcinoma/genética , Neoplasias Hepáticas/genética , Proteína Smad7/genética , Idoso , Animais , Neoplasias dos Ductos Biliares/mortalidade , Neoplasias dos Ductos Biliares/patologia , Carcinogênese/genética , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/cirurgia , Linhagem Celular Tumoral , Colangiocarcinoma/mortalidade , Colangiocarcinoma/patologia , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Hepatectomia , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Masculino , Camundongos , Pessoa de Meia-Idade , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Receptores Notch/metabolismo , Proteína Smad7/metabolismo , Regulação para Cima , Proteínas de Sinalização YAP/metabolismoRESUMO
BACKGROUND & AIMS: Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is upregulated in many tumor types and is a promising target for cancer therapy. Herein, we elucidated the functional role of FAK in intrahepatic cholangiocarcinoma (iCCA) development and progression. METHODS: Expression levels and activation status of FAK were determined in human iCCA samples. The functional contribution of FAK to Akt/YAP murine iCCA initiation and progression was investigated using conditional Fak knockout mice and constitutive Cre or inducible Cre mice, respectively. The oncogenic potential of FAK was further examined via overexpression of FAK in mice. In vitro cell line studies and in vivo drug treatment were applied to address the therapeutic potential of targeting FAK for iCCA treatment. RESULTS: FAK was ubiquitously upregulated and activated in iCCA lesions. Ablation of FAK strongly delayed Akt/YAP-driven mouse iCCA initiation. FAK overexpression synergized with activated AKT to promote iCCA development and accelerated Akt/Jag1-driven cholangiocarcinogenesis. Mechanistically, FAK was required for YAP(Y357) phosphorylation, supporting the role of FAK as a central YAP regulator in iCCA. Significantly, ablation of FAK after Akt/YAP-dependent iCCA formation strongly suppressed tumor progression in mice. Furthermore, a remarkable iCCA growth reduction was achieved when a FAK inhibitor and palbociclib, a CDK4/6 inhibitor, were administered simultaneously in human iCCA cell lines and Akt/YAP mice. CONCLUSIONS: FAK activation contributes to the initiation and progression of iCCA by inducing the YAP proto-oncogene. Targeting FAK, either alone or in combination with anti-CDK4/6 inhibitors, may be an effective strategy for iCCA treatment. LAY SUMMARY: We found that the protein FAK (focal adhesion kinase) is upregulated and activated in human and mouse intrahepatic cholangiocarcinoma samples. FAK promotes intrahepatic cholangiocarcinoma development, whereas deletion of FAK strongly suppresses its initiation and progression. Combined FAK and CDK4/6 inhibitor treatment had a strong anti-cancer effect in in vitro and in vivo models. This combination therapy might represent a valuable and novel treatment against human intrahepatic cholangiocarcinoma.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/efeitos adversos , Proteínas de Sinalização YAP/efeitos dos fármacos , Animais , California , Colangiocarcinoma/etiologia , Estudos de Coortes , Modelos Animais de Doenças , Proteína-Tirosina Quinases de Adesão Focal/administração & dosagem , Camundongos , Transdução de Sinais/efeitos dos fármacos , Proteínas de Sinalização YAP/administração & dosagemRESUMO
The Italian law of December 2010 establishes new criteria and parameters for the evaluation of faculty members. The parameters are represented by the number of articles published in journals listed in the main international data banks, the total number of citations and the h index. Candidates with qualifications at least in two out of three parameters may access the national competitions for associate or full professor and apply for an academic appointment. This system developed with the aim to fight nepotism and promote meritocracy, progressively led to the deterioration of the Italian university system. Since promotion in academia is strictly dependent on publications the faculty members found the solution to get over this system by organizing themselves into large consortia or small groups with the purpose of sharing authorship in scientific publications. In this way parameter thresholds may be easily reached and even surpassed. An Italian adagio says: "Fatta la Legge, Trovato l'Inganno"; "Every law has its Loophole". However, there is no science without ethics and researchers must stay away from any kind of compromise.
Assuntos
Autoria , Ética em Pesquisa , Docentes de Medicina/ética , Editoração/ética , Pesquisadores/ética , Pesquisa , Comportamento Social , Bibliometria , Emprego , Docentes de Medicina/legislação & jurisprudência , Regulamentação Governamental , Humanos , Itália , Seleção de Pessoal/ética , Seleção de Pessoal/normas , Publicações , UniversidadesRESUMO
Amplification and/or activation of the c-Myc proto-oncogene is one of the leading genetic events along hepatocarcinogenesis. The oncogenic potential of c-Myc has been proven experimentally by the finding that its overexpression in the mouse liver triggers tumor formation. However, the molecular mechanism whereby c-Myc exerts its oncogenic activity in the liver remains poorly understood. Here, we demonstrate that the mammalian target of rapamycin complex 1 (mTORC1) cascade is activated and necessary for c-Myc-dependent hepatocarcinogenesis. Specifically, we found that ablation of Raptor, the unique member of mTORC1, strongly inhibits c-Myc liver tumor formation. Also, the p70 ribosomal S6 kinase/ribosomal protein S6 and eukaryotic translation initiation factor 4E-binding protein 1/eukaryotic translation initiation factor 4E signaling cascades downstream of mTORC1 are required for c-Myc-driven tumorigenesis. Intriguingly, microarray expression analysis revealed up-regulation of multiple amino acid transporters, including solute carrier family 1 member A5 (SLC1A5) and SLC7A6, leading to robust uptake of amino acids, including glutamine, into c-Myc tumor cells. Subsequent functional studies showed that amino acids are critical for activation of mTORC1 as their inhibition suppressed mTORC1 in c-Myc tumor cells. In human hepatocellular carcinoma specimens, levels of c-Myc directly correlate with those of mTORC1 activation as well as of SLC1A5 and SLC7A6. CONCLUSION: Our current study indicates that an intact mTORC1 axis is required for c-Myc-driven hepatocarcinogenesis; thus, targeting the mTOR pathway or amino acid transporters may be an effective and novel therapeutic option for the treatment of hepatocellular carcinoma with activated c-Myc signaling. (Hepatology 2017;66:167-181).
Assuntos
Carcinogênese/efeitos dos fármacos , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Complexos Multiproteicos/genética , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose/genética , Biópsia por Agulha , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular , Modelos Animais de Doenças , Genes myc , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Fosfoproteínas/metabolismo , Fosforilação , Modelos de Riscos Proporcionais , Proto-Oncogene Mas , Distribuição Aleatória , Transdução de Sinais/genética , Estatísticas não Paramétricas , Serina-Treonina Quinases TOR/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND: Celiac disease (CD) occurs in subjects positive for HLA-DQ2 and/or DQ8 gene loci at any age following ingestion of gluten-containing food. An increased permeability of the mucosa allows interactions between gliadin macromolecules and genetic factors. It has been observed that Helicobacter pylori has the ability to modulate the integrity of the duodenal epithelium. We aimed to determine whether H. pylori infection may enhance the occurrence of CD in genetically susceptible subjects. MATERIALS AND METHODS: This was a prospective observational study. Patients undergoing upper endoscopy for any reason and positive for HLA-DQ2 and/or DQ8 haplotypes with or without CD were included. H. pylori infection was defined as a positive gastric histopathology and/or 13C-urea breath test. Prevalence of infection was compared between enrolled subjects with and without CD. Multiple logistic regression analysis, adjusting odds ratios for patient age, gender, smoking habit, residency, body mass index, and assumption of nonsteroidal anti-inflammatory drugs (NSAIDs) and proton-pump inhibitors (PPIs) were performed. RESULTS: A total of 397 genetically susceptible individuals (mean age: 37.7 ± 15.3 years; 86% women) were enrolled between October 2014 and October 2017. There were 265 (68%) patients with a diagnosis of CD. Overall, the prevalence of H. pylori infection was 33% and was similar in patients with and without CD (32% vs 36%). Adjustment for all covariates did not reveal any significant association, although adjusted odds ratio (OR) for CD was higher in female (OR = 1.302), in patients H. pylori positive (OR = 1.277), followed by use of NSAIDs (OR = 1.126), respectively. The use of PPIs appeared to be mildly protective against CD (OR = 0.644). CONCLUSION: Our study did not reveal any significant relationship between H. pylori and CD risk, even taking into account other confounders. More importantly, our findings do not support a "protective" role of H. pylori infection against CD, as previously reported. Therefore, there are no reasons to avoid eradication of H. pylori also in subject genetically susceptible for CD.
Assuntos
Doença Celíaca/epidemiologia , Doença Celíaca/metabolismo , Antígenos HLA-DQ/metabolismo , Infecções por Helicobacter/epidemiologia , Infecções por Helicobacter/metabolismo , Adulto , Feminino , Haplótipos/genética , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto JovemRESUMO
BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (ICC) is a lethal malignancy without effective treatment options. MLN0128, a second generation pan-mTOR inhibitor, shows efficacy for multiple tumor types. We evaluated the therapeutic potential of MLN0128 vs. gemcitabine/oxaliplatin in a novel ICC mouse model. METHODS: We established a novel ICC mouse model via hydrodynamic transfection of activated forms of AKT (myr-AKT) and Yap (YapS127A) protooncogenes (that will be referred to as AKT/YapS127A). Genetic approaches were applied to study the requirement of mTORC1 and mTORC2 in mediating AKT/YapS127A driven tumorigenesis. Gemcitabine/oxaliplatin and MLN0128 were administered in AKT/YapS127A tumor-bearing mice to study their anti-tumor efficacy in vivo. Multiple human ICC cell lines were used for in vitro experiments. Hematoxylin and eosin staining, immunohistochemistry and immunoblotting were applied for the characterization and mechanistic study. RESULTS: Co-expression of myr-AKT and YapS127A promoted ICC development in mice. Both mTORC1 and mTORC2 complexes were required for AKT/YapS127A ICC development. Gemcitabine/oxaliplatin had limited efficacy in treating late stage AKT/YapS127A ICC. In contrast, partial tumor regression was achieved when MLN0128 was applied in the late stage of AKT/YapS127A cholangiocarcinogenesis. Furthermore, when MLN0128 was administered in the early stage of AKT/YapS127A carcinogenesis, it led to disease stabilization. Mechanistically, MLN0128 efficiently inhibited AKT/mTOR signaling both in vivo and in vitro, inducing strong ICC cell apoptosis and only marginally affecting proliferation. CONCLUSIONS: This study suggests that mTOR kinase inhibitors may be beneficial for the treatment of ICC, even in tumors that are resistant to standard of care chemotherapeutics, such as gemcitabine/oxaliplatin-based regimens, especially in the subset of tumors exhibiting activated AKT/mTOR cascade. Lay summary: We established a novel mouse model of intrahepatic cholangiocarcinoma (ICC). Using this new preclinical model, we evaluated the therapeutic potential of mTOR inhibitor MLN0128 vs. gemcitabine/oxaliplatin (the standard chemotherapy for ICC treatment). Our study shows the anti-neoplastic potential of MLN0128, suggesting that it may be superior to gemcitabine/oxaliplatin-based chemotherapy for the treatment of ICC, especially in the tumors exhibiting activated AKT/mTOR cascade.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias dos Ductos Biliares/tratamento farmacológico , Colangiocarcinoma/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Neoplasias dos Ductos Biliares/etiologia , Neoplasias dos Ductos Biliares/patologia , Proteínas de Ciclo Celular , Colangiocarcinoma/etiologia , Colangiocarcinoma/patologia , Feminino , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Alvo Mecanístico do Complexo 2 de Rapamicina/fisiologia , Camundongos , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Proteínas de Sinalização YAPRESUMO
BACKGROUND & AIMS: Although it is well established that fatty acids (FA) are indispensable for the proliferation and survival of cancer cells in hepatocellular carcinoma (HCC), inhibition of Fatty Acid Synthase (FASN) cannot completely repress HCC cell growth in culture. Thus, we hypothesized that uptake of exogenous FA by cancer cells might play an important role in the development and progression of HCC. Lipoprotein lipase (LPL) is the enzyme that catalyses the hydrolysis of triglycerides into free fatty acids (FFA) and increases the cellular uptake of FA. METHODS: We used immunohistochemistry and quantitative reverse transcription real-time polymerase chain reaction to evaluate LPL expression in human and mouse HCC samples. Using lipoprotein-deficient medium as well as siRNAs against LPL and/or FASN, we investigated whether human HCC cells depend on both endogenous and exogenous fatty acids for survival in vitro. RESULTS: We found that LPL is upregulated in mouse and human HCC samples. High expression of LPL in human HCC samples is associated with poor prognosis. In HCC cell lines, silencing of FASN or LPL or culturing the cells in lipoprotein-deficient medium significantly decreased cell proliferation. Importantly, when FASN suppression was coupled to concomitant LPL depletion, the growth restraint of cell lines was further augmented. CONCLUSIONS: The present study strongly suggests that both de novo synthetized and exogenous FA play a major role along hepatocarcinogenesis. Thus, combined suppression of LPL and FASN might be highly beneficial for the treatment of human HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Ácido Graxo Sintase Tipo I/metabolismo , Ácidos Graxos/metabolismo , Lipase Lipoproteica/metabolismo , Neoplasias Hepáticas/metabolismo , Animais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ácido Graxo Sintase Tipo I/genética , Humanos , Lipase Lipoproteica/genética , Neoplasias Hepáticas/patologia , Camundongos , Análise Multivariada , Modelos de Riscos Proporcionais , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Triglicerídeos/metabolismo , Regulação para CimaRESUMO
INTRODUCTION: The prevalence of H. pylori infection is high in underdeveloped countries and is associated with growth retardation. In the first half of the 20th century, Sardinia was an underdeveloped region; however, more recent development resulted in a decline in H. pylori infection. Because body height is correlated with health and nutritional status in childhood, the association among H. pylori infection and height was explored. MATERIALS AND METHODS: A retrospective observational study was conducted involving patients undergoing endoscopy for dyspepsia from 2002 to 2012. H. pylori status was assessed by histology plus the rapid urease test or 13Carbon-urea breath test. RESULTS: Body height and H. pylori status were assessed in 5045 adult patients: 3257 (64.6%) were women. Patients born after 1950 showed a significant increase in height (average 3.22 cm) compared to patients born before 1950 (163.93 vs 160 cm; 95% confidence interval, CI = 2.74-3.70 cm) (p < .0001). H. pylori-infected patients were nearly 1 cm shorter than uninfected patients (95% CI = -1.35 to·-0.09 cm) (p = .012). The multivariate linear regression analysis showed male gender, birth cohort, and occupational categories to be strongly associated with height, while the weak effect of H. pylori infection disappeared. CONCLUSIONS: Our results demonstrate a strong secular trend related to body height in Sardinia with a minimal influence of H. pylori infection.
Assuntos
Estatura , Deficiências do Desenvolvimento/epidemiologia , Dispepsia/complicações , Infecções por Helicobacter/complicações , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos Transversais , Dispepsia/patologia , Feminino , Infecções por Helicobacter/patologia , Humanos , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Prevalência , Estudos RetrospectivosRESUMO
AIM OF THE STUDY: The aim of the present study was to define the frequency of organ-specific and non-organ-specific autoantibodies in a cohort of Latent Autoimmune Diabetes in Adults (LADA) patients and to test whether multiple antibodies positivity could be a predictor of early insulin dependence. MATERIALS AND METHODS: We enrolled 210 LADA and 210 type 2 diabetes mellitus (T2D) patients. In all subjects anti-islet antigen-2 (IA-2Ab), anti-thyroperoxidase (TPOAb), anti-zinc transporter 8 (ZnT8Ab), anti-nuclear (ANA), anti-parietal cell (APCA), anti-smooth muscle (ASMA), anti-mitochondrial (AMA), anti-liver kidney microsomes (LKM), and anti-reticulin (ARA) circulating antibodies were assessed. RESULTS: The frequency of TPOAb, ZnT8Ab, APCA, and IA-2Ab positivity was, respectively, detected in 40.0%, 32.4%, 24.7%, and 9.5% of LADA patients, whereas their frequency was significantly lower in T2D patients (11.4%, 1.9%, 9.5%, and 0.0%, respectively, p < 0.001). The frequency of ANA was the same in both groups whereas the frequency of ASMA, ARA, AMA, and LKM was very low (range 0.0-3.3%). The presence of TPOAb associated with ZnT8Ab, IA-2Ab, or APCA allows one to predict the progression of disease with a high specificity but low sensibility. CONCLUSIONS: LADA patients show an increased frequency of organ- and non-organ-specific antibodies. Consequently, a screening is worthwhile in these patients. The simultaneous presence of TPOAb with ZnT8, IA-2Ab, or APCA may help differentiate clinical phenotypes and predict faster insulin dependence in LADA patients.
Assuntos
Autoanticorpos/sangue , Diabetes Mellitus Tipo 2/sangue , Diabetes Autoimune Latente em Adultos/sangue , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Brainstem degeneration in Parkinson's disease (PD) may explain the occurrence of many non-motor symptoms in this condition. Purposes of the present work were to investigate brainstem function in PD through a battery of vestibular-evoked myogenic potentials (VEMP) allowing a comprehensive brainstem exploration and to correlate VEMP findings with symptoms related to brainstem involvement. Cervical (cVEMP), masseter (mVEMP) and ocular (oVEMP) VEMPs were investigated in 24 PD patients and compared with those recorded in 24 age-matched controls. Presence of symptoms ascribable to brainstem dysfunction, such as daytime sleepiness, REM sleep behavior disorder and depression, was investigated through Epworth Sleepiness Scale, Parkinson's Disease Sleep Scale, REM Sleep Disorder Screening Questionnaire (RBD-SQ) and Geriatric Depression Scale. Postural instability was additionally assessed through mini-BESTest. The frequency of alteration of VEMPs in patients was 83.3 % when considering the whole set and 41.7 % for cVEMP, 66.7 % for mVEMP and 45.8 % for oVEMP. This was significantly different from controls, with absence being the prevalent alteration in PD. A significant inverse correlation between the number of altered VEMPs and mini-BESTest and a direct correlation with RBD-SQ were found. The VEMP battery under study allowed the identification of brainstem dysfunctions in PD patients, which correlated with clinical tests suggestive of postural and REM sleep disorders. VEMPs might represent a valuable tool of brainstem assessment in PD.
Assuntos
Tronco Encefálico/fisiopatologia , Doença de Parkinson/fisiopatologia , Transtorno do Comportamento do Sono REM/fisiopatologia , Potenciais Evocados Miogênicos Vestibulares/fisiologia , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Villages in the island of Sardinia in the Mediterranean that display exceptional longevity are clustered within a defined mountainous region. Because of their unique location we hypothesize that these villages had a unique infectious disease exposure relevant to the observed successful longevity. These highland villages had a significant exposure to malaria in the first half of the 20th century after which malaria was eliminated due to vector control mechanisms. In addition, there is likely a high incidence of Helicobacter pylori infections among shepherds in Sardinia, the primary occupation of many living in the LBZ, as well as helminth infections among children. This suggests that individuals living in the LBZ had a unique infectious disease exposure. Specifically, we hypothesize that the continued high exposure of residents in the LBZ to these infectious agents prior to the 1950s lead to the generation of a uniquely trained (or imprinted) immune system. Once some of these diseases were eliminated in the latter half of the century, individuals within the LBZ were equipped with a trained immune system that was uniquely capable of not only responding effectively to common infections but also responding in a manner that maximized maintaining tissue health. In addition, there are lifestyle factors that also favor such a trained immune system. This hypothesis may help explain the slow progression of chronic immune mediated diseases as well as other chronic non-transmissible age-related diseases seen in the Sardinian LBZ and serve as a template for future studies that support or refute this hypothesis.
RESUMO
Sex differences in aging manifest in disparities in disease prevalence, physical health, and lifespan, where women tend to have greater longevity relative to men. However, in the Mediterranean Blue Zones of Sardinia (Italy) and Ikaria (Greece) are regions of centenarian abundance, male-female centenarian ratios are approximately one, diverging from the typical trend and making these useful regions in which to study sex differences of the oldest old. Additionally, these regions can be investigated as examples of healthy aging relative to other populations. DNA methylation (DNAm)-based predictors have been developed to assess various health biomarkers, including biological age, Pace of Aging, serum interleukin-6 (IL-6), and telomere length. Epigenetic clocks are biological age predictors whose deviation from chronological age has been indicative of relative health differences between individuals, making these useful tools for interrogating these differences in aging. We assessed sex differences between the Horvath, Hannum, GrimAge, PhenoAge, Skin and Blood, and Pace of Aging predictors from individuals in two Mediterranean Blue Zones and found that men displayed positive epigenetic age acceleration (EAA) compared to women according to all clocks, with significantly greater rates according to GrimAge (ß = 3.55; p = 1.22 × 10-12), Horvath (ß = 1.07; p = 0.00378) and the Pace of Aging (ß = 0.0344; p = 1.77 × 10-08). Other DNAm-based biomarkers findings indicated that men had lower DNAm-predicted serum IL-6 scores (ß = -0.00301, p = 2.84 × 10-12), while women displayed higher DNAm-predicted proportions of regulatory T cells than men from the Blue Zone (p = 0.0150, 95% Confidence Interval [0.00131, 0.0117], Cohen's d = 0.517). All clocks showed better correlations with chronological age in women from the Blue Zones than men, but all clocks showed large mean absolute errors (MAE >30 years) in both sexes, except for PhenoAge (MAE <5 years). Thus, despite their equal survival to older ages in these Mediterranean Blue Zones, men in these regions remain biologically older by most measured DNAm-derived metrics than women, with the exception of the IL-6 score and proportion of regulatory T cells.
RESUMO
BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive primary liver tumor with increasing incidence worldwide, dismal prognosis, and few therapeutic options. Mounting evidence underlines the role of the Hippo pathway in this disease; however, the molecular mechanisms whereby the Hippo cascade contributes to cholangiocarcinogenesis remain poorly defined. METHODS: We established novel iCCA mouse models via hydrodynamic transfection of an activated form of transcriptional coactivator with PDZ-binding motif (TAZ), a Hippo pathway downstream effector, either alone or combined with the myristoylated AKT (myr-AKT) protooncogene, in the mouse liver. Hematoxylin and eosin staining, immunohistochemistry, electron microscopy, and quantitative real-time RT-PCR were applied to characterize the models. In addition, in vitro cell line studies were conducted to address the growth-promoting roles of TAZ and its paralog YAP. RESULTS: Overexpression of TAZ in the mouse liver triggered iCCA development with very low incidence and long latency. In contrast, co-expression of TAZ and myr-AKT dramatically increased tumor frequency and accelerated cancer formation in mice, with 100% iCCA incidence and high tumor burden by 10 weeks post hydrodynamic injection. AKT/TAZ tumors faithfully recapitulated many of the histomolecular features of human iCCA. At the molecular level, the development of the cholangiocellular lesions depended on the binding of TAZ to TEAD transcription factors. In addition, inhibition of the Notch pathway did not hamper carcinogenesis but suppressed the cholangiocellular phenotype of AKT/TAZ tumors. Also, knockdown of YAP, the TAZ paralog, delayed cholangiocarcinogenesis in AKT/TAZ mice without affecting the tumor phenotype. Furthermore, human preinvasive and invasive iCCAs and mixed hepatocellular carcinoma/iCCA displayed widespread TAZ activation and downregulation of the mechanisms protecting TAZ from proteolysis. CONCLUSIONS: Overall, the present data underscore the crucial role of TAZ in cholangiocarcinogenesis.
Assuntos
Neoplasias dos Ductos Biliares , Carcinoma Hepatocelular , Colangiocarcinoma , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Neoplasias dos Ductos Biliares/genética , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Carcinoma Hepatocelular/patologia , Colangiocarcinoma/patologia , Via de Sinalização Hippo , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteínas de Sinalização YAPRESUMO
Human longevity may be found in single individuals as well as in the population as a whole ("population longevity"). Longevity Blue Zones (LBZs), which are areas with an unusually high number of oldest old, have been identified in Sardinia and the Greek island of Ikaria. We compared the lifestyle, health status and some genetic markers of the LBZ populations with those of reference populations from Italy and Greece; the data were extracted from the GEHA database. In the LBZs, the proportion of individuals who never married or were married and still living with their spouse was significantly greater. Nonagenarians males and females with a high selfâperception of optimism and/or a high score for self-rated health were also found in larger proportions in LBZs. Among the variables with lower frequency were the proportion of the widowed, the percentage of subjects who had suffered a stroke and the frequency of Apoε4 and Apoε2 and the TT genotype of FOXO3A gene. Compared to behavioral and health indicators, the impact of genetic factors might be relatively less important in the LBZs. Nevertheless, further research is needed to identify potential epigenetic traits that might play a predominant role due to the interaction between genetics and the human and physical environments.
Assuntos
Nível de Saúde , Envelhecimento Saudável , Estilo de Vida , Longevidade/fisiologia , Nonagenários , Otimismo , Idoso de 80 Anos ou mais , Bases de Dados Genéticas/estatística & dados numéricos , Epigenômica/métodos , Feminino , Perfil Genético , Grécia/epidemiologia , Envelhecimento Saudável/genética , Envelhecimento Saudável/psicologia , Humanos , Itália/epidemiologia , Masculino , Nonagenários/fisiologia , Nonagenários/psicologia , Nonagenários/estatística & dados numéricos , AutoimagemRESUMO
Metabolic reprogramming is a hallmark of many cancer types, including hepatocellular carcinoma (HCC). Identifying the critical players in this process might be crucial for the generation of novel and effective anti-neoplastic therapies. In the present investigation, we determined the importance of carbohydrate responsive element binding protein (ChREBP), a central player in the regulation of lipid and glucose metabolism in the liver, on the development of HCC in in vitro and in vivo models. We found that genetic deletion of ChREBP (that will be referred to as ChREBPKO mice) strongly delays or impairs hepatocarcinogenesis driven by AKT or AKT/c-Met overexpression in mice, respectively. In contrast, HCC development was found to be completely unaffected by ChREBP depletion in mice co-expressing AKT and N-Ras protooncogenes. In mouse and human HCC cell lines, suppression of ChREBP via specific small interfering RNAs (siRNAs) resulted in decreased proliferation and induction of apoptosis. Of note, these cellular events were strongly augmented by concomitant inhibition of the mitogen-activated protein kinase (MAPK) pathway. The present data indicate that ChREBP activity might be required or dispensable for HCC growth, depending on the oncogenes involved. In particular, the activation of Ras/MAPK signaling might represent a possible mechanism of resistance to ChREBP depletion in this tumor type. Additional studies are needed to unravel the molecular mechanisms rendering HCC cells insensitive to ChREBP suppression.