RESUMO
The CTNNB1 gene, encoding ß-catenin, is frequently mutated in hepatocellular carcinoma (HCC, â¼30%) and in hepatoblastoma (HB, >80%), in which DLK1/DIO3 locus induction is correlated with CTNNB1 mutations. Here, we aim to decipher how sustained ß-catenin activation regulates DLK1/DIO3 locus expression and the role this locus plays in HB and HCC development in mouse models deleted for Apc (ApcΔhep) or Ctnnb1-exon 3 (ß-cateninΔExon3) and in human CTNNB1-mutated hepatic cancer cells. We identified an enhancer site bound by TCF-4/ß-catenin complexes in an open conformation upon sustained ß-catenin activation (DLK1-Wnt responsive element [WRE]) and increasing DLK1/DIO3 locus transcription in ß-catenin-mutated human HB and mouse models. DLK1-WRE editing by CRISPR-Cas9 approach impaired DLK1/DIO3 locus expression and slowed tumor growth in subcutaneous CTNNB1-mutated tumor cell grafts, ApcΔhep HB and ß-cateninΔExon3 HCC. Tumor growth inhibition resulted either from increased FADD expression and subsequent caspase-3 cleavage in the first case or from decreased expression of cell cycle actors regulated by FoxM1 in the others. Therefore, the DLK1/DIO3 locus is an essential determinant of FoxM1-dependent cell proliferation during ß-catenin-driven liver tumorigenesis. Targeting the DLK1-WRE enhancer to silence the DLK1/DIO3 locus might thus represent an interesting therapeutic strategy to restrict tumor growth in primary liver cancers with CTNNB1 mutations.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Humanos , Camundongos , beta Catenina/genética , beta Catenina/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Carcinogênese/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Cateninas/genética , Cateninas/metabolismo , Proliferação de Células/genética , Neoplasias Hepáticas/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Regulação para CimaRESUMO
Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is one of the leading causes of cancer-related deaths worldwide. The multitarget inhibitor sorafenib is a first-line treatment of patients with advanced unresectable HCC. Recent clinical studies have evidenced that patients treated with sorafenib together with the antidiabetic drug metformin have a survival disadvantage compared with patients receiving sorafenib only. Here, we examined whether a clinically relevant dose of metformin (50 mg/kg per day) could influence the antitumoral effects of sorafenib (15 mg/kg per day) in a subcutaneous xenograft model of human HCC growth using two different sequences of administration, i.e., concomitant versus sequential dosing regimens. We observed that the administration of metformin 6 hours prior to sorafenib was significantly less effective in inhibiting tumor growth (15.4% tumor growth inhibition) than concomitant administration of the two drugs (59.5% tumor growth inhibition). In vitro experiments confirmed that pretreatment of different human HCC cell lines with metformin reduced the effects of sorafenib on cell viability, proliferation, and signaling. Transcriptomic analysis confirmed significant differences between xenografted tumors obtained under the concomitant and the sequential dosing regimens. Taken together, these observations call into question the benefit of parallel use of metformin and sorafenib in patients with advanced HCC and diabetes, as the interaction between the two drugs could ultimately compromise patient survival. SIGNIFICANCE STATEMENT: When drugs are administered sequentially, metformin alters the antitumor effect of sorafenib, the reference treatment for advanced hepatocellular carcinoma, in a preclinical murine xenograft model of liver cancer progression as well as in hepatic cancer cell lines. Defective activation of the AMP-activated protein kinase pathway as well as major transcriptomic changes are associated with the loss of the antitumor effect. These results echo recent clinical work reporting a poorer prognosis for patients with liver cancer who were cotreated with metformin and sorafenib.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Diabetes Mellitus/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Metformina/administração & dosagem , Sorafenibe/administração & dosagem , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Esquema de Medicação , Interações Medicamentosas , Quimioterapia Combinada , Feminino , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Metformina/farmacologia , Camundongos , Transdução de Sinais/efeitos dos fármacos , Sorafenibe/farmacologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Membrane receptor intracellular trafficking and signalling are frequently altered in cancers. Our aim was to investigate whether clathrin-dependent trafficking modulates signalling of the ErbB receptor family in response to amphiregulin (AR), EGF, heparin-binding EGF-like growth factor (HB-EGF) and heregulin-1ß (HRG). Experiments were performed using three hepatocellular carcinoma (HCC) cell lines, Hep3B, HepG2 and PLC/PRF/5, expressing various levels of EGFR, ErbB2 and ErbB3. Inhibition of clathrin-mediated endocytosis (CME), by down-regulating clathrin heavy chain expression, resulted in a cell- and ligand-specific pattern of phosphorylation of the ErbB receptors and their downstream effectors. Clathrin down-regulation significantly decreased the ratio between phosphorylated EGFR (pEGFR) and total EGFR in all cell lines when stimulated with AR, EGF, HB-EGF or HRG, except in HRG-stimulated Hep3B cells in which pEGFR was not detectable. The ratio between phosphorylated ErbB2 and total ErbB2 was significantly decreased in clathrin down-regulated Hep3B cells stimulated with any of the ligands, and in HRG-stimulated PLC/PRF/5 cells. The ratio between phosphorylated ErbB3 and total ErbB3 significantly decreased in clathrin down-regulated cell lines upon stimulation with EGF or HB-EGF. STAT3 phosphorylation levels significantly increased in all cell lines irrespective of stimulation, while that of AKT remained unchanged, except in AR-stimulated Hep3B and HepG2 cells in which pAKT was significantly decreased. Finally, ERK phosphorylation was insensitive to clathrin inhibition. Altogether, our observations indicate that clathrin regulation of ErbB signalling in HCC is a complex process that likely depends on the expression of ErbB family members and on the autocrine/paracrine secretion of their ligands in the tumour environment.
Assuntos
Carcinoma Hepatocelular/metabolismo , Clatrina/metabolismo , Neoplasias Hepáticas/metabolismo , Receptor ErbB-2/metabolismo , Transdução de Sinais , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Imunofluorescência , Humanos , Ligantes , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/patologia , Receptor ErbB-3/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Metabolic diseases such as obesity and type 2 diabetes are recognized as independent risk factors for hepatocellular carcinoma (HCC). Hyperinsulinemia, a hallmark of these pathologies, is suspected to be involved in HCC development. The molecular adapter growth factor receptor binding protein 14 (Grb14) is an inhibitor of insulin receptor catalytic activity, highly expressed in the liver. To study its involvement in hepatocyte proliferation, we specifically inhibited its liver expression using a short hairpin RNA strategy in mice. Enhanced insulin signaling upon Grb14 inhibition was accompanied by a transient induction of S-phase entrance by quiescent hepatocytes, indicating that Grb14 is a potent repressor of cell division. The proliferation of Grb14-deficient hepatocytes was cell-autonomous as it was also observed in primary cell cultures. Combined Grb14 down-regulation and insulin signaling blockade using pharmacological approaches as well as genetic mouse models demonstrated that Grb14 inhibition-mediated hepatocyte division involved insulin receptor activation and was mediated by the mechanistic target of rapamycin complex 1-S6K pathway and the transcription factor E2F1. In order to determine a potential dysregulation in GRB14 gene expression in human pathophysiology, a collection of 85 human HCCs was investigated. This revealed a highly significant and frequent decrease in GRB14 expression in hepatic tumors when compared to adjacent nontumoral parenchyma, with 60% of the tumors exhibiting a reduced Grb14 mRNA level. CONCLUSION: Our study establishes Grb14 as a physiological repressor of insulin mitogenic action in the liver and further supports that dysregulation of insulin signaling is associated with HCC. (Hepatology 2017;65:1352-1368).
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinoma Hepatocelular/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Neoplasias Hepáticas/fisiopatologia , Receptor de Insulina/metabolismo , Animais , Carcinoma Hepatocelular/epidemiologia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Células Cultivadas , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/epidemiologia , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: Sorafenib is effective in hepatocellular carcinoma (HCC), but patients ultimately present disease progression. Molecular mechanisms underlying acquired resistance are still unknown. Herein, we characterise the role of tumour-initiating cells (T-ICs) and signalling pathways involved in sorafenib resistance. DESIGN: HCC xenograft mice treated with sorafenib (n=22) were explored for responsiveness (n=5) and acquired resistance (n=17). Mechanism of acquired resistance were assessed by: (1) role of T-ICs by in vitro sphere formation and in vivo tumourigenesis assays using NOD/SCID mice, (2) activation of alternative signalling pathways and (3) efficacy of anti-FGF and anti-IGF drugs in experimental models. Gene expression (microarray, quantitative real-time PCR (qRT-PCR)) and protein analyses (immunohistochemistry, western blot) were conducted. A novel gene signature of sorafenib resistance was generated and tested in two independent cohorts. RESULTS: Sorafenib-acquired resistant tumours showed significant enrichment of T-ICs (164 cells needed to create a tumour) versus sorafenib-sensitive tumours (13â 400 cells) and non-treated tumours (1292 cells), p<0.001. Tumours with sorafenib-acquired resistance were enriched with insulin-like growth factor (IGF) and fibroblast growth factor (FGF) signalling cascades (false discovery rate (FDR)<0.05). In vitro, cells derived from sorafenib-acquired resistant tumours and two sorafenib-resistant HCC cell lines were responsive to IGF or FGF inhibition. In vivo, FGF blockade delayed tumour growth and improved survival in sorafenib-resistant tumours. A sorafenib-resistance 175 gene signature was characterised by enrichment of progenitor cell features, aggressive tumorous traits and predicted poor survival in two cohorts (n=442 patients with HCC). CONCLUSIONS: Acquired resistance to sorafenib is driven by T-ICs with enrichment of progenitor markers and activation of IGF and FGF signalling. Inhibition of these pathways would benefit a subset of patients after sorafenib progression.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Fatores de Crescimento de Fibroblastos/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Niacinamida/análogos & derivados , Compostos de Fenilureia/uso terapêutico , Somatomedinas/metabolismo , Idoso , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/genética , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Niacinamida/uso terapêutico , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor IGF Tipo 1 , Receptores de Somatomedina/antagonistas & inibidores , Receptores de Somatomedina/metabolismo , Transdução de Sinais , Somatomedinas/antagonistas & inibidores , Somatomedinas/genética , Sorafenibe , Esferoides Celulares , Taxa de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AIMS: To investigate the status of somatostatin receptors (SSTRs) in resected hepatocellular carcinoma (HCC). METHODS AND RESULTS: Transcript and protein levels of SSTR2, SSTR3 and SSTR5 were investigated, with real-time polymerase chain reaction (PCR) and manual and automated immunohistochemistry (IHC), in 53 resected HCCs and paired non-tumour tissues. SSTR1, SSTR4, SSTR5TMD4 and SSTR5TMD5 were analysed with real-time PCR. SSTR3 and SSTR5 transcripts were expressed in ~25% of HCCs, but not in adjacent non-tumour tissues. SSTR1 and SSTR2 transcripts were overexpressed in 42% and 32% of HCCs, respectively. SSTR4, SSTR5TMD4 and SSTR5TMD5 were not detected. Membrane staining for SSTR2 was detected in 38% of HCCs, whereas SSTR5 protein was detectable in only 11% of HCCs. SSTR3 protein was detected in the majority of HCCs and adjacent non-tumour liver tissues, but membrane staining was <20% of that in HCCs. The results obtained with the two IHC methods were highly correlated (P < 0.0001). Statistical analyses also showed a positive correlation between SSTR2 membrane staining and cytokeratin 19 expression (P = 0.04), serum α-fetoprotein level (P = 0.002), and poor differentiation (P = 0.05). CONCLUSIONS: Membrane SSTR2 is detected reliably in HCCs by IHC, and is a potential therapeutic target, as it is coexpressed with markers of poor prognosis.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Receptores de Somatostatina/biossíntese , Biomarcadores Tumorais/análise , Carcinoma Hepatocelular/mortalidade , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Somatostatina/análiseRESUMO
Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer and is one of the leading causes of cancer-related death. The risk factors for HCC include cirrhosis, chronic viral hepatitis, heavy alcohol intake and metabolic diseases such as obesity, type 2 diabetes and metabolic syndrome. Insulin resistance is a common denominator of all of these conditions and is tethered to hyperinsulinaemia. Here, we give an overview of the recent advances linking hyperinsulinaemia to HCC development and progression. In particular, we summarise the underlying causes of hyperinsulinaemia in the setting of chronic liver diseases. We present epidemiological evidence linking metabolic diseases to HCC risk and HCC-related mortality, as well as the pathogenic cellular and molecular mechanisms explaining this relation. A better understanding of the mechanisms by which insulin participates in HCC biology might ultimately provide novel opportunities for prevention and treatment.
Assuntos
Carcinoma Hepatocelular/metabolismo , Hiperinsulinismo/metabolismo , Resistência à Insulina , Neoplasias Hepáticas/metabolismo , Transdução de Sinais , Animais , Carcinoma Hepatocelular/patologia , Diabetes Mellitus Tipo 2/complicações , Modelos Animais de Doenças , Progressão da Doença , Hepatite C/complicações , Humanos , Hiperinsulinismo/patologia , Insulina/fisiologia , Cirrose Hepática/complicações , Neoplasias Hepáticas/patologia , Síndrome Metabólica/complicações , Camundongos , Hepatopatia Gordurosa não Alcoólica/complicações , Obesidade/complicações , Ratos , Fatores de RiscoRESUMO
UNLABELLED: Intrahepatic cholangiocarcinoma (CCA) is characterized by an abundant desmoplastic environment. Poor prognosis of CCA has been associated with the presence of alpha-smooth muscle actin (α-SMA)-positive myofibroblasts (MFs) in the stroma and with the sustained activation of the epidermal growth factor receptor (EGFR) in tumor cells. Among EGFR ligands, heparin-binding epidermal growth factor (HB-EGF) has emerged as a paracrine factor that contributes to intercellular communications between MFs and tumor cells in several cancers. This study was designed to test whether hepatic MFs contributed to CCA progression through EGFR signaling. The interplay between CCA cells and hepatic MFs was examined first in vivo, using subcutaneous xenografts into immunocompromised mice. In these experiments, cotransplantation of CCA cells with human liver myofibroblasts (HLMFs) increased tumor incidence, size, and metastatic dissemination of tumors. These effects were abolished by gefitinib, an EGFR tyrosine kinase inhibitor. Immunohistochemical analyses of human CCA tissues showed that stromal MFs expressed HB-EGF, whereas EGFR was detected in cancer cells. In vitro, HLMFs produced HB-EGF and their conditioned media induced EGFR activation and promoted disruption of adherens junctions, migratory and invasive properties in CCA cells. These effects were abolished in the presence of gefitinib or HB-EGF-neutralizing antibody. We also showed that CCA cells produced transforming growth factor beta 1, which, in turn, induced HB-EGF expression in HLMFs. CONCLUSION: A reciprocal cross-talk between CCA cells and myofibroblasts through the HB-EGF/EGFR axis contributes to CCA progression.
Assuntos
Colangiocarcinoma/patologia , Receptores ErbB/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Hepáticas/patologia , Miofibroblastos/metabolismo , Animais , Neoplasias dos Ductos Biliares , Ductos Biliares Intra-Hepáticos , Linhagem Celular Tumoral , Colangiocarcinoma/fisiopatologia , Progressão da Doença , Gefitinibe , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Neoplasias Hepáticas/fisiopatologia , Camundongos , Quinazolinas/uso terapêutico , Transdução de Sinais , Células Estromais/metabolismoRESUMO
BACKGROUND & AIMS: Sorafenib is the standard of care for the treatment of advanced hepatocellular carcinoma (HCC). However, primary and acquired resistance is observed in patients. We examined whether gefitinib, which inhibits both epidermal growth factor receptor (EGFR) and HER-3 phosphorylation, could improve HCC cell response to sorafenib. METHODS: Sorafenib and gefitinib were tested in HCC tumor xenografts and in sorafenib-sensitive and sorafenib-resistant HCC cell lines. Biomarkers relevant to the HER system were analyzed by Western blotting and ELISA. RNA interference was used to downregulate the HER system. Amphiregulin concentrations were measured by ELISA in sera from patients under sorafenib treatment. RESULTS: Sorafenib combined with gefitinib significantly inhibited tumor growth in mice and reduced cell viability in vitro compared to single agents. In cell lines cultured in 10% serum or treated with EGF, sorafenib alone inhibited phospho-STAT3 while it maintained or even increased phospho-ERK and/or phospho-AKT. The paradoxical effects of sorafenib were prevented by gefitinib or by downregulation of EGFR and HER-3 expression. In cells with acquired resistance to sorafenib, aberrant activation of EGFR/HER-3 receptors as well as overexpression of several EGFR ligands were observed. These enhanced autocrine/paracrine loops led to the constitutive activation of ERK and AKT and conferred increased sensitivity to gefitinib. Increased serum concentrations of amphiregulin were observed in 10 out of 14 patients under sorafenib treatment compared to baselines. CONCLUSIONS: Signaling pathways controlled by EGFR and HER-3 restrict sorafenib effects both in naive and sorafenib-resistant HCC cells. Consequently, gefitinib cooperates with sorafenib to increase antiproliferative response and to prevent resistance.
Assuntos
Antineoplásicos/farmacologia , Benzenossulfonatos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Receptores ErbB/metabolismo , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Piridinas/farmacologia , Receptor ErbB-3/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Anfirregulina , Animais , Antineoplásicos/uso terapêutico , Benzenossulfonatos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Família de Proteínas EGF , Receptores ErbB/antagonistas & inibidores , Feminino , Gefitinibe , Glicoproteínas/metabolismo , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Niacinamida/análogos & derivados , Compostos de Fenilureia , Piridinas/uso terapêutico , Quinazolinas/farmacologia , Sorafenibe , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hepatocellular carcinoma (HCC) is the most common type of liver cancer. HCC is particularly aggressive and is one of the leading causes of cancer mortality. In recent decades, the epidemiological landscape of HCC has undergone significant changes. While chronic viral hepatitis and excessive alcohol consumption have long been identified as the main risk factors for HCC, non-alcoholic steatohepatitis (NASH), paralleling the worldwide epidemic of obesity and type 2 diabetes, has become a growing cause of HCC in the US and Europe. Here, we review the recent advances in epidemiological, genetic, epigenetic and pathogenic mechanisms as well as experimental mouse models that have improved the understanding of NASH progression toward HCC. We also discuss the clinical management of patients with NASH-related HCC and possible therapeutic approaches.
Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Dieta Hiperlipídica/efeitos adversos , Estresse do Retículo Endoplasmático , Predisposição Genética para Doença , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/genéticaRESUMO
PURPOSE: The aim of this study was to investigate the association between expression of insulin-like growth factor-1 receptor (IGF1R) and its ligand, IGF-II, and disease-free survival (DFS) in patients with stage III colon cancer (CC). METHODS: In this retrospective study we included consecutive patients who underwent curative surgery for stage III CC. IGF1R and IGF-II/IGF2 status were evaluated in tumour samples by immunohistochemistry and quantitative real-time PCR (qRT-PCR). Associations of markers with DFS were analysed using Cox proportional hazards models. RESULTS: Hundred and fifty-one CC patients were included (median age, 66.6 years; female, 54.3%). Low levels of IGF1R and IGF-II protein expression were observed in 16.1% and 10.7% of the cases, respectively. No significant differences in clinicopathological characteristics between patients with tumours expressing low IGF1R or IGF-II protein levels and those with high levels were observed. A low IGF1R protein expression was found to be significantly associated with a shorter DFS (HR 3.32; 95% CI, 1.7-6.31; p = 0.0003), while no association was observed between IGF-II protein expression and DFS (HR 0.91; 95% CI, 0.28-2.96; p = 0.87). In a multivariate analysis, IGF1R protein status remained an independent prognostic factor for DFS (HR 2.73; 95% CI, 1.40-5.31; p = 0.003). Furthermore, we found that neither IGF1R nor IGF2 mRNA expression levels as measured by qRT-PCR correlated with the respective protein expression levels as assessed by immunohistochemistry. Neither of the mRNA expression levels was significantly associated with DFS. CONCLUSIONS: From our data we conclude that low IGF1R protein expression represents a poor prognostic biomarker in stage III colon cancer.
Assuntos
Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , RNA Mensageiro/genética , Receptor IGF Tipo 1/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , RNA Mensageiro/metabolismo , Receptor IGF Tipo 1/biossíntese , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Despite the intensive efforts to identify the molecular events responsible for the emergence of liver cancer, hepatocellular carcinoma (HCC) remains a major health problem in the world. Thus, the identification of new therapeutic opportunities is a short-term necessity. These last few decades, non-coding RNAs appeared as interesting therapeutic strategies with their pleiotropic inhibitory action in the cell itself but also in recipient cells via their secretion into extracellular vesicles. This short review recapitulates recent advancements concerning non-coding RNAs and their deregulations in liver cancer.
Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , RNA Longo não Codificante , HumanosRESUMO
The CRISPR technology is rapidly transforming the field of liver research by its versatility and easy use. In vivo gene editing of hepatocytes in adult mice can be achieved using a broad toolbox for both fundamental research and development of therapeutic strategies for future clinical applications. Recent studies showed that CRISPR has a real potential to treat hereditary liver diseases as well as virally induced pathologies. This short review recapitulates very recent advancements regarding the use of CRISPR in liver research and therapy.
Assuntos
Pesquisa Biomédica/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Neoplasias Hepáticas/terapia , Humanos , Neoplasias Hepáticas/genéticaRESUMO
Hepatocellular carcinoma (HCC) is one of the most common and deadly neoplasms. Insulin receptor (IR) exists in two isoforms, IR-A and IR-B, the latter being predominantly expressed in normal adult hepatocytes while IR-A is overexpressed in HCC to the detriment of IR-B. This study evaluated the biological functions associated with IR-A overexpression in HCC in relation to expression of its ligand IGF-II. The value of INSRA:INSRB ratio which was increased in Ë70% of 85 HCC was associated with stem/progenitor cell features such as cytokeratin-19 and α-fetoprotein and correlated with shorter patient survival. IGF2 mRNA upregulation was observed in 9.4% of HCC and was not associated with higher INSRA:INSRB ratios. Ectopic overexpression of IR-A in two HCC cell lines presenting a strong autocrine IGF-II secretion loop or not stimulated cell migration and invasion. In cells cultured as spheroids, IR-A overexpression promoted gene programs related to stemness, inflammation and cell movement. IR-A also increased cell line tumorigenicity in vivo after injection to immunosuppressed mice and the sphere-forming cells made a significant contribution to this effect. Altogether, these results demonstrate that IR-A is a novel player in HCC progression.
Assuntos
Antígenos CD/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Células-Tronco Neoplásicas/patologia , Receptor de Insulina/metabolismo , Animais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Xenoenxertos , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Isoformas de ProteínasRESUMO
Purpose: Cholangiocarcinoma (CCA) is a desmoplastic tumor of the biliary tree in which epidermal growth factor receptor (EGFR) is overexpressed and contributes to cancer progression. Although EGFR has been envisaged as a target for therapy, treatment with tyrosine kinase inhibitors (TKI) such as erlotinib did not provide therapeutic benefit in patients with CCA, emphasizing the need to investigate resistance mechanisms against EGFR inhibition.Experimental Design: Resistant CCA cells to EGFR inhibition were obtained upon long-time exposure of cells with erlotinib. Cell signaling, viability, migration, and spheroid growth were determined in vitro, and tumor growth was evaluated in CCA xenograft models.Results: Erlotinib-resistant CCA cells displayed metastasis-associated signatures that correlated with a marked change in cell plasticity associated with an epithelial-mesenchymal transition (EMT) and a cancer stem cell (CSC)-like phenotype. Resistant cells exhibited an upregulation of insulin receptor (IR) and insulin-like growth factor (IGF) 1 receptor (IGF1R), along with an increase in IGF2 expression. IR/IGF1R inhibition reduced EMT and CSC-like traits in resistant cells. In vivo, tumors developed from resistant CCA cells were larger and exhibited a more prominent stromal compartment, enriched in cancer-associated fibroblasts (CAF). Pharmacological coinhibition of EGFR and IR/IGF1R reduced tumor growth and stromal compartment in resistant tumors. Modeling of CCA-CAF crosstalk showed that IGF2 expressed by fibroblasts boosted IR/IGF1R signaling in resistant cells. Furthermore, IR/IGF1R signaling positively regulated fibroblast proliferation and activation.Conclusions: To escape EGFR-TKI treatment, CCA tumor cells develop an adaptive mechanism by undergoing an IR/IGF1R-dependent phenotypic switch, involving a contribution of stromal cells. Clin Cancer Res; 24(17); 4282-96. ©2018 AACR.
Assuntos
Colangiocarcinoma/tratamento farmacológico , Fator de Crescimento Insulin-Like II/genética , Receptor de Insulina/genética , Receptores de Somatomedina/genética , Animais , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Xenoenxertos , Humanos , Camundongos , Miofibroblastos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptor IGF Tipo 1 , Transdução de Sinais/efeitos dos fármacosRESUMO
Insulin receptor (IR) plays a key role in the control of glucose homeostasis; however, the regulation of its cellular expression remains poorly understood. Here we show that the amount of biologically active IR is regulated by the cleavage of its ectodomain, by the ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), in a glucose concentration-dependent manner. In vivo studies demonstrate that BACE1 regulates the amount of IR and insulin signaling in the liver. During diabetes, BACE1-dependent cleavage of IR is increased and the amount of IR in the liver is reduced, whereas infusion of a BACE1 inhibitor partially restores liver IR. We suggest the potential use of BACE1 inhibitors to enhance insulin signaling during diabetes. Additionally, we show that plasma levels of cleaved IR reflect IR isoform A expression levels in liver tumors, which prompts us to propose that the measurement of circulating cleaved IR may assist hepatic cancer detection and management.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Antígenos CD/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Fígado/metabolismo , Receptor de Insulina/metabolismo , Animais , Diabetes Mellitus/metabolismo , Feminino , Glucose/química , Glicosilação , Células HEK293 , Humanos , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/metabolismo , Domínios Proteicos , Transdução de SinaisRESUMO
Recent evidence indicates that growth hormone (GH) is involved in liver regeneration. To test whether insulin-like growth factor I (IGF-I) mediates this effect, we studied liver regeneration induced by partial hepatectomy in liver-specific IGF type 1 receptor knockout (LIGFREKO) mice. The absence of IGF-1R caused a significant decrease in hepatocyte proliferation in males (-52%), but not in females, as assessed by Ki67 immunohistochemistry. Cyclin D1 and cyclin A protein levels in the livers of LIGFREKO males were only half those in controls, indicating that cyclin induction during liver regeneration is dependent on IGF-1R signaling. Analyzing the signaling cascade initiated by IGF-1R, we observed a lack of IRS-1 induction in LIGFREKO livers. In contrast, the induction of IRS-2 synthesis was similar in LIGFREKO and control groups, suggesting the existence of differential regulation of IRS synthesis during liver regeneration. Regenerating livers from LIGFREKO animals also showed significantly less activated ERKs than controls. Our findings demonstrate that IGF-1R makes a significant contribution to liver regeneration. Using the LIGFREKO model, we provide new evidence that IGF-1R/IRS-1/ERK signaling may be the intracellular pathway controlling the cell cycle via cyclin D1 and cyclin A in the regenerating liver.
Assuntos
Hepatócitos/citologia , Hepatócitos/metabolismo , Regeneração Hepática , Fígado/metabolismo , Receptor IGF Tipo 1/deficiência , Receptor IGF Tipo 1/metabolismo , Animais , Proliferação de Células , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica , Genótipo , Proteínas Substratos do Receptor de Insulina , Fígado/citologia , Masculino , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptor IGF Tipo 1/genética , Caracteres SexuaisRESUMO
BACKGROUND/AIM: The aim of this study was to assess the incidence of MSI in a large series of human hepatocellular carcinomas (HCC) with various etiologies. MATERIALS AND METHODS: The MSI status was determined by polymerase chain reaction (PCR) using 5 mononucleotide and 13 CAn dinucleotide repeats. RESULTS: None of the 122 HCC samples displayed an MSI-High phenotype, as defined by the presence of alterations at more than 30% of the microsatellite markers analyzed. Yet, limited microsatellite instability consisting in the insertion or deletion of a few repeat motifs was detected in 32 tumor samples (26.2%), regardless of the etiology of the underlying liver disease. MSI tended to be higher in patients with cirrhosis (p=0.051), possibly reflecting an impact of the inflammatory context in this process. CONCLUSION: Based on a large series of HCC with various etiologies, our study allowed us to definitely conclude that MSI is not a hallmark of HCC.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Instabilidade de Microssatélites , Repetições de Microssatélites/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/patologia , Feminino , Humanos , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Adulto JovemAssuntos
Benzenossulfonatos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Fator de Transcrição STAT3/efeitos dos fármacos , Animais , Humanos , Niacinamida/análogos & derivados , Compostos de Fenilureia , SorafenibeRESUMO
BACKGROUND: The heregulin-1ß/HER3-driven pathway is implicated in several epithelial malignancies and its blockade is currently undergoing clinical investigation. Paradoxically, the status and the regulation of this pathway is poorly known in hepatocellular carcinoma (HCC). METHODS: Using 85 HCC obtained after tumour resection, heregulin-1ß and HER3 expression was evaluated by real-time RT-PCR, ELISA and/or immunohistochemistry. Statistics were performed to analyze associations between gene expression and clinicopathological parameters. The effects of insulin on the heregulin-1ß/HER3 pathway was investigated in four HCC cell lines. RESULTS: HER3 mRNA was upregulated in 52 % of tumours, while heregulin-1ß mRNA was downregulated in 82 %. Hepatitis B and C viral infections were respectively associated with high and low HER3 mRNA expression. No association was seen between neither HER3 or heregulin-1ß mRNA and prognostic factors, survival or recurrence. Immunohistochemistry showed predominant cytoplasmic staining of HER3 in tumours but the staining was nonreproducible. HER3 mRNA and protein levels were not correlated in liver tissues. In HCC cells, insulin promoted HER3 proteasomal degradation and inhibited heregulin-1ß stimulation of cell migration. HER3 and insulin receptor co-immunoprecipitated in these cells. The loss of insulin receptor expression by RNA interference sensitized cells to heregulin-1ß-induced AKT phosphorylation. CONCLUSIONS: Autocrine heregulin-1ß loop is uncommon in HCC and HER3 mRNA expression is differentially influenced by hepatitis viruses. Insulin is a negative regulator of HER3 protein expression and function in HCC cells. Altogether these data may explain why HER3 and heregulin-1ß expression have no prognostic value and suggest that HCC patients are unlikely to derive benefit from HER3-targeted monotherapies.