RESUMO
Non-thyroidal illness syndrome (NTIS), characterized by low serum 3,5,3'-triiodothyronine (T3) with normal l-thyroxine (T4) levels, is associated with malignancy. Decreased activity of type I 5'-deiodinase (DIO1), which converts T4 to T3, contributes to NTIS. T3 binds to thyroid hormone receptor, which heterodimerizes with retinoid X receptor (RXR) and regulates transcription of target genes, such as DIO1. NF-κB activation by inflammatory cytokines inhibits DIO1 expression. The oncogene astrocyte elevated gene-1 (AEG-1) inhibits RXR-dependent transcription and activates NF-κB. Here, we interrogated the role of AEG-1 in NTIS in the context of hepatocellular carcinoma (HCC). T3-mediated gene regulation was analyzed in human HCC cells, with overexpression or knockdown of AEG-1, and primary hepatocytes from AEG-1 transgenic (Alb/AEG-1) and AEG-1 knock-out (AEG-1KO) mice. Serum T3 and T4 levels were checked in Alb/AEG-1 mice and human HCC patients. AEG-1 and DIO1 levels in human HCC samples were analyzed by immunohistochemistry. AEG-1 inhibited T3-mediated gene regulation in human HCC cells and mouse hepatocytes. AEG-1 overexpression repressed and AEG-1 knockdown induced DIO1 expression. An inverse correlation was observed between AEG-1 and DIO1 levels in human HCC patients. Low T3 with normal T4 was observed in the sera of HCC patients and Alb/AEG-1 mice. Inhibition of co-activator recruitment to RXR and activation of NF-κB were identified to play a role in AEG-1-mediated down-regulation of DIO1. AEG-1 thus might play a role in NTIS associated with HCC and other cancers.
Assuntos
Carcinoma Hepatocelular/metabolismo , Moléculas de Adesão Celular/metabolismo , Síndromes do Eutireóideo Doente/metabolismo , Neoplasias Hepáticas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Regulação para Baixo/genética , Síndromes do Eutireóideo Doente/etiologia , Síndromes do Eutireóideo Doente/genética , Regulação Enzimológica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Iodeto Peroxidase/biossíntese , Iodeto Peroxidase/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Glicoproteínas de Membrana/genética , Proteínas de Membrana , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Ligação a RNA , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , Tri-Iodotironina/genética , Tri-Iodotironina/metabolismoRESUMO
Astrocyte elevated gene-1 (AEG-1), also known as MTDH (metadherin) or LYRIC, is an established oncogene. However, the physiological function of AEG-1 is not known. To address this question, we generated an AEG-1 knock-out mouse (AEG-1KO) and characterized it. Although AEG-1KO mice were viable and fertile, they were significantly leaner with prominently less body fat and lived significantly longer compared with wild type (WT). When fed a high fat and cholesterol diet (HFD), WT mice rapidly gained weight, whereas AEG-1KO mice did not gain weight at all. This phenotype of AEG-1KO mice is due to decreased fat absorption from the intestines, not because of decreased fat synthesis or increased fat consumption. AEG-1 interacts with retinoid X receptor (RXR) and inhibits RXR function. In enterocytes of AEG-1KO mice, we observed increased activity of RXR heterodimer partners, liver X receptor and peroxisome proliferator-activated receptor-α, key inhibitors of intestinal fat absorption. Inhibition of fat absorption in AEG-1KO mice was further augmented when fed an HFD providing ligands to liver X receptor and peroxisome proliferator-activated receptor-α. Our studies reveal a novel role of AEG-1 in regulating nuclear receptors controlling lipid metabolism. AEG-1 may significantly modulate the effects of HFD and thereby function as a unique determinant of obesity.
Assuntos
Mucosa Intestinal/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Proteínas de Membrana/metabolismo , Aumento de Peso , Tecido Adiposo/metabolismo , Animais , Homeostase , Receptores X do Fígado , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/genética , Obesidade/metabolismo , Receptores Nucleares Órfãos/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Proteínas de Ligação a RNA , Receptores X de Retinoides/metabolismoRESUMO
UNLABELLED: Astrocyte elevated gene-1 (AEG-1) and c-Myc are overexpressed in human hepatocellular carcinoma (HCC) functioning as oncogenes. AEG-1 is transcriptionally regulated by c-Myc, and AEG-1 itself induces c-Myc by activating the Wnt/ß-catenin-signaling pathway. We now document the cooperation of AEG-1 and c-Myc in promoting hepatocarcinogenesis by analyzing hepatocyte-specific transgenic mice expressing either AEG-1 (albumin [Alb]/AEG-1), c-Myc (Alb/c-Myc), or both (Alb/AEG-1/c-Myc). Wild-type and Alb/AEG-1 mice did not develop spontaneous HCC. Alb/c-Myc mice developed spontaneous HCC without distant metastasis, whereas Alb/AEG-1/c-Myc mice developed highly aggressive HCC with frank metastasis to the lungs. Induction of carcinogenesis by N-nitrosodiethylamine significantly accelerated the kinetics of tumor formation in all groups. However, in Alb/AEG-1/c-Myc, the effect was markedly pronounced with lung metastasis. In vitro analysis showed that Alb/AEG-1/c-Myc hepatocytes acquired increased proliferation and transformative potential with sustained activation of prosurvival and epithelial-mesenchymal transition-signaling pathways. RNA-sequencing analysis identified a unique gene signature in livers of Alb/AEG-1/c-Myc mice that was not observed when either AEG-1 or c-Myc was overexpressed. Specifically, Alb/AEG-1/c-Myc mice overexpressed maternally imprinted noncoding RNAs (ncRNAs), such as Rian, Meg-3, and Mirg, which are implicated in hepatocarcinogenesis. Knocking down these ncRNAs significantly inhibited proliferation and invasion by Alb/AEG-1/c-Myc hepatocytes. CONCLUSION: Our studies reveal a novel cooperative oncogenic effect of AEG-1 and c-Myc that might explain the mechanism of aggressive HCC. Alb/AEG-1/c-Myc mice provide a useful model to understand the molecular mechanism of cooperation between these two oncogenes and other molecules involved in hepatocarcinogenesis. This model might also be of use for evaluating novel therapeutic strategies targeting HCC.
Assuntos
Neoplasias Hepáticas Experimentais/etiologia , Proteínas de Membrana/fisiologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Albuminas/análise , Animais , Carcinogênese , Células Cultivadas , Transição Epitelial-Mesenquimal , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Pulmonares/secundário , Proteínas de Membrana/análise , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-myc/análise , Proteínas de Ligação a RNARESUMO
Hepatocellular carcinoma (HCC) is a fatal cancer with no effective therapy. Astrocyte elevated gene-1 (AEG-1) plays a pivotal role in hepatocarcinogenesis and inhibits retinoic acid-induced gene expression and cell death. The combination of a lentivirus expressing AEG-1 shRNA and all-trans retinoic acid (ATRA) profoundly and synergistically inhibited subcutaneous human HCC xenografts in nude mice. We have now developed liver-targeted nanoplexes by conjugating poly(amidoamine) (PAMAM) dendrimers with polyethylene glycol (PEG) and lactobionic acid (Gal) (PAMAM-PEG-Gal) which were complexed with AEG-1 siRNA (PAMAM-AEG-1si). The polymer conjugate was characterized by (1)H-NMR, MALDI, and mass spectrometry; and optimal nanoplex formulations were characterized for surface charge, size, and morphology. Orthotopic xenografts of human HCC cell QGY-7703 expressing luciferase (QGY-luc) were established in the livers of athymic nude mice and tumor development was monitored by bioluminescence imaging (BLI). Tumor-bearing mice were treated with PAMAM-siCon, PAMAM-siCon+ATRA, PAMAM-AEG-1si, and PAMAM-AEG-1si+ATRA. In the control group the tumor developed aggressively. ATRA showed little effect due to high AEG-1 levels in QGY-luc cells. PAMAM-AEG-1si showed significant reduction in tumor growth, and the combination of PAMAM-AEG-1si+ATRA showed profound and synergistic inhibition so that the tumors were almost undetectable by BLI. A marked decrease in AEG-1 level was observed in tumor samples treated with PAMAM-AEG-1si. The group treated with PAMAM-AEG-1si+ATRA nanoplexes showed increased necrosis, inhibition of proliferation, and increased apoptosis when compared to other groups. Liver is an ideal organ for RNAi therapy and ATRA is an approved anticancer agent. Our exciting observations suggest that the combinatorial approach might be an effective way to combat HCC.
Assuntos
Carcinoma Hepatocelular/terapia , Moléculas de Adesão Celular/antagonistas & inibidores , Neoplasias Hepáticas/terapia , Nanopartículas/administração & dosagem , RNA Interferente Pequeno/genética , Tretinoína/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Moléculas de Adesão Celular/genética , Proliferação de Células/efeitos dos fármacos , Terapia Combinada , Terapia Genética , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Nanopartículas/química , Proteínas de Ligação a RNA , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Despite the prevalence of HCC, there is no effective, systemic treatment. The transcription factor LSF is a promising protein target for chemotherapy; it is highly expressed in HCC patient samples and cell lines, and promotes oncogenesis in rodent xenograft models of HCC. Here, we identify small molecules that effectively inhibit LSF cellular activity. The lead compound, factor quinolinone inhibitor 1 (FQI1), inhibits LSF DNA-binding activity both in vitro, as determined by electrophoretic mobility shift assays, and in cells, as determined by ChIP. Consistent with such inhibition, FQI1 eliminates transcriptional stimulation of LSF-dependent reporter constructs. FQI1 also exhibits antiproliferative activity in multiple cell lines. In LSF-overexpressing cells, including HCC cells, cell death is rapidly induced; however, primary or immortalized hepatocytes are unaffected by treatment with FQI1. The highly concordant structure-activity relationship of a panel of 23 quinolinones strongly suggests that the growth inhibitory activity is due to a single biological target or family. Coupled with the striking agreement between the concentrations required for antiproliferative activity (GI(50)s) and for inhibition of LSF transactivation (IC(50)s), we conclude that LSF is the specific biological target of FQIs. Based on these in vitro results, we tested the efficacy of FQI1 in inhibiting HCC tumor growth in a mouse xenograft model. As a single agent, tumor growth was dramatically inhibited with no observable general tissue cytotoxicity. These findings support the further development of LSF inhibitors for cancer chemotherapy.
Assuntos
Benzodioxóis/farmacologia , Carcinoma Hepatocelular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/metabolismo , Quinolonas/farmacologia , Fatores de Transcrição/metabolismo , Animais , Proliferação de Células , Sobrevivência Celular , Ensaios de Seleção de Medicamentos Antitumorais , Genes Reporter , Hepatócitos/citologia , Humanos , Concentração Inibidora 50 , Camundongos , Modelos Químicos , Células NIH 3T3 , Transplante de Neoplasias , Oncogenes , Relação Estrutura-Atividade , Ativação TranscricionalRESUMO
Hepatocellular carcinoma (HCC) is a highly fatal disease mandating development of novel, targeted therapies to elicit prolonged survival benefit to the patients. Insulin-like growth factor-binding protein-7 (IGFBP7), a secreted protein belonging to the IGFBP family, functions as a potential tumor suppressor for HCC. In the present study, we evaluated the therapeutic efficacy of a replication-incompetent adenovirus expressing IGFBP7 (Ad.IGFBP7) in human HCC. Ad.IGFBP7 profoundly inhibited viability and induced apoptosis in multiple human HCC cell lines by inducing reactive oxygen species (ROS) and activating a DNA damage response (DDR) and p38 MAPK. In orthotopic xenograft models of human HCC in athymic nude mice, intravenous administration of Ad.IGFBP7 profoundly inhibited primary tumor growth and intrahepatic metastasis. In a nude mice subcutaneous model, xenografts from human HCC cells were established in both flanks and only left-sided tumors received intratumoral injection of Ad.IGFBP7. Growth of both left-sided injected tumors and right-sided uninjected tumors were markedly inhibited by Ad.IGFBP7 with profound suppression of angiogenesis. These findings indicate that Ad.IGFBP7 might be a potent therapeutic eradicating both primary HCC and distant metastasis and might be an effective treatment option for terminal HCC patients.
Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/terapia , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/terapia , Adenoviridae/genética , Animais , Carcinoma Hepatocelular/genética , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos Nus , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The transcription factor late SV40 factor (LSF) is overexpressed in human hepatocellular carcinoma (HCC) fostering a highly aggressive and metastatic phenotype. Angiogenesis is an essential component of cancer aggression and metastasis and HCC is a highly aggressive and angiogenic cancer. In the present studies, we analyzed the molecular mechanism of LSF-induced angiogenesis in HCC. Employing human umbilical vein endothelial cells (HUVEC) differentiation assay and chicken chorioallantoic membrane (CAM) assay we document that stable LSF overexpression augments and stable dominant negative inhibition of LSF (LSFdn) abrogates angiogenesis by human HCC cells. A quest for LSF-regulated factors contributing to angiogenesis, by chromatin immunoprecipitation-on-chip (ChIP-on-chip) assay, identified matrix metalloproteinase-9 (MMP-9) as a direct target of LSF. MMP-9 expression and enzymatic activity were higher in LSF-overexpressing cells and lower in LSFdn-expressing cells. Deletion mutation analysis identified the LSF-responsive regions in the MMP-9 promoter and ChIP assay confirmed LSF binding to the MMP-9 promoter. Inhibition of MMP-9 significantly abrogated LSF-induced angiogenesis as well as in vivo tumorigenesis, thus reinforcing the role of MMP-9 in facilitating LSF function. The present findings identify a novel target of LSF contributing to its oncogenic properties.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica , Metaloproteinase 9 da Matriz/biossíntese , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Elementos de Resposta , Fatores de Transcrição/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/enfermagem , Linhagem Celular Tumoral , Embrião de Galinha , Membrana Corioalantoide/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Neovascularização Fisiológica/genética , Deleção de Sequência , Fatores de Transcrição/genética , Transplante Heterólogo , Regulação para Cima/genéticaRESUMO
Staphylococcal nuclease domain-containing 1 (SND1) is a multifunctional protein that is overexpressed in multiple cancers, including hepatocellular carcinoma (HCC). Stable overexpression of SND1 in Hep3B cells expressing a low level of SND1 augments, whereas stable knockdown of SND1 in QGY-7703 cells expressing a high level of SND1 inhibits establishment of xenografts in nude mice, indicating that SND1 promotes an aggressive tumorigenic phenotype. In this study we analyzed the role of SND1 in regulating tumor angiogenesis, a hallmark of cancer. Conditioned medium from Hep3B-SND1 cells stably overexpressing SND1 augmented, whereas that from QGY-SND1si cells stably overexpressing SND1 siRNA significantly inhibited angiogenesis, as analyzed by a chicken chorioallantoic membrane assay and a human umbilical vein endothelial cell differentiation assay. We unraveled a linear pathway in which SND1-induced activation of NF-κB resulted in induction of miR-221 and subsequent induction of angiogenic factors Angiogenin and CXCL16. Inhibition of either of these components resulted in significant inhibition of SND1-induced angiogenesis, thus highlighting the importance of this molecular cascade in regulating SND1 function. Because SND1 regulates NF-κB and miR-221, two important determinants of HCC controlling the aggressive phenotype, SND1 inhibition might be an effective strategy to counteract this fatal malady.
Assuntos
Carcinoma Hepatocelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/metabolismo , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Núcleo Celular/metabolismo , Quimiocina CXCL16 , Quimiocinas CXC/biossíntese , Endonucleases , Ensaio de Imunoadsorção Enzimática/métodos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Metástase Neoplásica , Neovascularização Patológica , RNA Interferente Pequeno/metabolismo , Receptores Depuradores/biossíntese , Ribonuclease Pancreático/biossínteseRESUMO
UNLABELLED: Astrocyte elevated gene-1 (AEG-1) is a key contributor to hepatocellular carcinoma (HCC) development and progression. To enhance our understanding of the role of AEG-1 in hepatocarcinogenesis, a transgenic mouse with hepatocyte-specific expression of AEG-1 (Alb/AEG1) was developed. Treating Alb/AEG-1, but not wild-type (WT) mice, with N-nitrosodiethylamine resulted in multinodular HCC with steatotic features and associated modulation of expression of genes regulating invasion, metastasis, angiogenesis, and fatty acid synthesis. Hepatocytes isolated from Alb/AEG-1 mice displayed profound resistance to chemotherapeutics and growth factor deprivation with activation of prosurvival signaling pathways. Alb/AEG-1 hepatocytes also exhibited marked resistance toward senescence, which correlated with abrogation of activation of a DNA damage response. Conditioned media from Alb/AEG-1 hepatocytes induced marked angiogenesis with elevation in several coagulation factors. Among these factors, AEG-1 facilitated the association of factor XII (FXII) messenger RNA with polysomes, resulting in increased translation. Short interfering RNA-mediated knockdown of FXII resulted in profound inhibition of AEG-1-induced angiogenesis. CONCLUSION: We uncovered novel aspects of AEG-1 functions, including induction of steatosis, inhibition of senescence, and activation of the coagulation pathway to augment aggressive hepatocarcinogenesis. The Alb/AEG-1 mouse provides an appropriate model to scrutinize the molecular mechanism of hepatocarcinogenesis and to evaluate the efficacy of novel therapeutic strategies targeting HCC.
Assuntos
Carcinoma Hepatocelular/genética , Moléculas de Adesão Celular/genética , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Neoplasias Hepáticas/genética , Neovascularização Patológica/genética , Animais , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Senescência Celular/genética , Dietilnitrosamina , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Fator XII/genética , Fator XII/metabolismo , Ácidos Graxos/biossíntese , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Fluoruracila/farmacologia , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/irrigação sanguínea , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Proteínas de Membrana , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica/genética , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Polirribossomos , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNARESUMO
Hepatocellular carcinoma (HCC) is a highly aggressive cancer with no currently available effective treatment. Understanding of the molecular mechanism of HCC development and progression is imperative for developing novel, effective, and targeted therapies for this lethal disease. In this article, we document that the cellular transcription factor Late SV40 Factor (LSF) plays an important role in HCC pathogenesis. LSF protein was significantly overexpressed in human HCC cells compared to normal hepatocytes. In 109 HCC patients, LSF protein was overexpressed in >90% cases, compared to normal liver, and LSF expression level showed significant correlation with the stages and grades of the disease. Forced overexpression of LSF in less aggressive HCC cells resulted in highly aggressive, angiogenic, and multiorgan metastatic tumors in nude mice. Conversely, inhibition of LSF significantly abrogated growth and metastasis of highly aggressive HCC cells in nude mice. Microarray studies revealed that as a transcription factor, LSF modulated specific genes regulating invasion, angiogenesis, chemoresistance, and senescence. The expression of osteopontin (OPN), a gene regulating every step in tumor progression and metastasis, was robustly up-regulated by LSF. It was documented that LSF transcriptionally up-regulates OPN, and loss-of-function studies demonstrated that OPN plays an important role in mediating the oncogenic functions of LSF. Together, these data establish a regulatory role of LSF in cancer, particularly HCC pathogenesis, and validate LSF as a viable target for therapeutic intervention.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias Hepáticas/metabolismo , Oncogenes , Fatores de Transcrição/metabolismo , Animais , Carcinoma Hepatocelular/irrigação sanguínea , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células , Células Cultivadas , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/irrigação sanguínea , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Metástase Neoplásica , Transplante de Neoplasias , Osteopontina/genética , Osteopontina/metabolismo , Interferência de RNA , Ratos , Análise Serial de Tecidos , Fatores de Transcrição/genética , Transcrição Gênica , Regulação para CimaRESUMO
UNLABELLED: There is virtually no effective treatment for advanced hepatocellular carcinoma (HCC) and novel targets need to be identified to develop effective treatment. We recently documented that the oncogene Astrocyte elevated gene-1 (AEG-1) plays a seminal role in hepatocarcinogenesis. Employing yeast two-hybrid assay and coimmunoprecipitation followed by mass spectrometry, we identified staphylococcal nuclease domain containing 1 (SND1), a nuclease in the RNA-induced silencing complex (RISC) facilitating RNAi-mediated gene silencing, as an AEG-1 interacting protein. Coimmunoprecipitation and colocalization studies confirmed that AEG-1 is also a component of RISC and both AEG-1 and SND1 are required for optimum RISC activity facilitating small interfering RNA (siRNA) and micro RNA (miRNA)-mediated silencing of luciferase reporter gene. In 109 human HCC samples SND1 was overexpressed in ≈74% cases compared to normal liver. Correspondingly, significantly higher RISC activity was observed in human HCC cells compared to immortal normal hepatocytes. Increased RISC activity, conferred by AEG-1 or SND1, resulted in increased degradation of tumor suppressor messenger RNAs (mRNAs) that are target of oncomiRs. Inhibition of enzymatic activity of SND1 significantly inhibited proliferation of human HCC cells. As a corollary, stable overexpression of SND1 augmented and siRNA-mediated inhibition of SND1 abrogated growth of human HCC cells in vitro and in vivo, thus revealing a potential role of SND1 in hepatocarcinogenesis. CONCLUSION: We unravel a novel mechanism that overexpression of AEG-1 and SND1 leading to increased RISC activity might contribute to hepatocarcinogenesis. Targeted inhibition of SND1 enzymatic activity might be developed as an effective therapy for HCC.
Assuntos
Carcinoma Hepatocelular/etiologia , Moléculas de Adesão Celular/fisiologia , Neoplasias Hepáticas/etiologia , Proteínas Nucleares/fisiologia , Complexo de Inativação Induzido por RNA/fisiologia , Animais , Endonucleases , Humanos , Proteínas de Membrana , Camundongos , Proteínas de Ligação a RNA , Células Tumorais CultivadasRESUMO
Astrocyte elevated gene-1 (AEG-1) is overexpressed in >90% of human hepatocellular carcinoma (HCC) patients and plays a significant role in mediating aggressive progression of HCC. AEG-1 is known to augment invasion, metastasis, and angiogenesis, and we now demonstrate that AEG-1 directly contributes to another important hallmark of aggressive cancers, that is, resistance to chemotherapeutic drugs, such as 5-fluorouracil (5-FU). AEG-1 augments expression of the transcription factor LSF that regulates the expression of thymidylate synthase (TS), a target of 5-FU. In addition, AEG-1 enhances the expression of dihydropyrimidine dehydrogenase (DPYD) that catalyzes the initial and rate-limiting step in the catabolism of 5-FU. siRNA-mediated inhibition of AEG-1, LSF, or DPYD significantly increased the sensitivity of HCC cells to 5-FU in vitro and a lentivirus delivering AEG-1 siRNA in combination with 5-FU markedly inhibited growth of HCC cells xenotransplanted in athymic nude mice when compared to either agent alone. The present studies highlight 2 previously unidentified genes, AEG-1 and LSF, contributing to chemoresistance. Inhibition of AEG-1 might be exploited as a therapeutic strategy along with 5-FU-based combinatorial chemotherapy for HCC, a highly fatal cancer with currently very limited therapeutic options.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Moléculas de Adesão Celular/genética , Proteínas de Ligação a DNA/genética , Fluoruracila/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Fatores de Transcrição/genética , Animais , Carcinoma Hepatocelular/genética , Moléculas de Adesão Celular/antagonistas & inibidores , Linhagem Celular Tumoral , DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Di-Hidrouracila Desidrogenase (NADP)/fisiologia , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Antígeno Ki-67/análise , Neoplasias Hepáticas/genética , Proteínas de Membrana , Camundongos , Proteínas de Ligação a RNA , Timidilato Sintase/genética , Fatores de Transcrição/fisiologiaRESUMO
BACKGROUND & AIMS: Understanding the molecular pathogenesis of hepatocellular carcinoma (HCC) would facilitate development of targeted and effective therapies for this fatal disease. We recently demonstrated that the cellular transcription factor Late SV40 Factor (LSF) is overexpressed in more than 90% of human HCC cases, compared to the normal liver, and plays a seminal role in hepatocarcinogenesis. LSF transcriptionally upregulates osteopontin (OPN) that plays a significant role in mediating the oncogenic function of LSF. The present study aims at a better understanding of LSF function by analyzing the signaling pathway modulated by LSF. METHODS: Phospho-receptor tyrosine kinase (RTK) array was performed to identify which receptor tyrosine kinases are activated by LSF. Immunohistochemical analysis using tissue microarray was performed to establish correlation among LSF, OPN, and phospho-c-Met levels in HCC patients. Co-immunoprecipitation analysis was performed to check OPN-induced CD44 and c-Met interaction. Inhibition studies using chemicals and siRNAs were performed in vitro and in vivo using nude mice xenograft models to establish the importance of c-Met activation in mediating LSF function. RESULTS: Secreted OPN, induced by LSF, activates c-Met via a potential interaction between OPN and its cell surface receptor CD44. A significant correlation was observed among LSF, OPN, and activated c-Met levels in HCC patients. Chemical or genetic inhibition of c-Met resulted in profound abrogation of LSF-mediated tumorigenesis and metastasis in nude mice xenograft studies. CONCLUSIONS: The present findings elucidate a novel pathway of c-Met activation during hepatocarcinogenesis and support the rationale of using c-Met inhibitors as potential HCC therapeutics.
Assuntos
Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/metabolismo , Osteopontina/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Receptores de Hialuronatos/metabolismo , Camundongos , Camundongos Nus , Modelos Biológicos , Transplante de Neoplasias , Transdução de Sinais , Transplante HeterólogoRESUMO
AEG-1 is an oncogene that is overexpressed in all cancers, including hepatocellular carcinoma. AEG-1 plays a seminal role in promoting cancer development and progression by augmenting proliferation, invasion, metastasis, angiogenesis and chemoresistance, all hallmarks of aggressive cancer. AEG-1 mediates its oncogenic function predominantly by interacting with various protein complexes. AEG-1 acts as a scaffold protein, activating multiple protumorigenic signal transduction pathways, such as MEK/ERK, PI3K/Akt, NF-κB and Wnt/ß-catenin while regulating gene expression at transcriptional, post-transcriptional and translational levels. Our recent studies document that AEG-1 is fundamentally required for activation of inflammation. A comprehensive and convincing body of data currently points to AEG-1 as an essential component critical to the onset and progression of cancer. The present review describes the current knowledge gleaned from patient and experimental studies as well as transgenic and knockout mouse models, on the impact of AEG-1 on hepatocarcinogenesis.
RESUMO
The staphylococcal nuclease and tudor domain containing 1 (SND1) is a multifunctional protein overexpressed in breast, prostate, colorectal and hepatocellular carcinomas and malignant glioma. Molecular studies have revealed the multifaceted activities of SND1 involved in regulating gene expression at transcriptional as well as post-transcriptional levels. Early studies identified SND1 as a transcriptional co-activator. SND1 is also a component of RNA-induced silencing complex (RISC) thus mediating RNAi function, a regulator of mRNA splicing, editing and stability, and plays a role in maintenance of cell viability. Such diverse actions allow the SND1 to modulate a complex array of molecular networks, thereby promoting carcinogenesis. Here, we describe the crucial role of SND1 in cancer development and progression, and highlight SND1 as a potential target for therapeutic intervention.
Assuntos
Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias/genética , Proteínas Nucleares/genética , Carboxipeptidases/genética , Endonucleases , Humanos , Terapia de Alvo Molecular , Neoplasias/patologia , Proteínas Nucleares/biossíntese , Interferência de RNA , Splicing de RNA/genéticaRESUMO
Hepatocellular carcinoma (HCC) is a lethal malignancy with high mortality and poor prognosis. Oncogenic transcription factor Late SV40 Factor (LSF) plays an important role in promoting HCC. A small molecule inhibitor of LSF, Factor Quinolinone Inhibitor 1 (FQI1), significantly inhibited human HCC xenografts in nude mice without harming normal cells. Here we evaluated the efficacy of FQI1 and another inhibitor, FQI2, in inhibiting endogenous hepatocarcinogenesis. HCC was induced in a transgenic mouse with hepatocyte-specific overexpression of c-myc (Alb/c-myc) by injecting N-nitrosodiethylamine (DEN) followed by FQI1 or FQI2 treatment after tumor development. LSF inhibitors markedly decreased tumor burden in Alb/c-myc mice with a corresponding decrease in proliferation and angiogenesis. Interestingly, in vitro treatment of human HCC cells with LSF inhibitors resulted in mitotic arrest with an accompanying increase in CyclinB1. Inhibition of CyclinB1 induction by Cycloheximide or CDK1 activity by Roscovitine significantly prevented FQI-induced mitotic arrest. A significant induction of apoptosis was also observed upon treatment with FQI. These effects of LSF inhibition, mitotic arrest and induction of apoptosis by FQI1s provide multiple avenues by which these inhibitors eliminate HCC cells. LSF inhibitors might be highly potent and effective therapeutics for HCC either alone or in combination with currently existing therapies.
Assuntos
Antineoplásicos/farmacologia , Benzodioxóis/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Proteínas de Ligação a DNA/antagonistas & inibidores , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Quinolonas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Dietilnitrosamina , Relação Dose-Resposta a Droga , Genes myc , Predisposição Genética para Doença , Humanos , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Camundongos Transgênicos , Mitose/efeitos dos fármacos , Terapia de Alvo Molecular , Neovascularização Patológica , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is a vicious and highly vascular cancer with a dismal prognosis. It is a life-threatening illness worldwide that ranks fifth in terms of cancer prevalence and third in cancer deaths. Most patients are diagnosed at an advanced stage by which time conventional therapies are no longer effective. Targeted molecular therapies, such as the multikinase inhibitor sorafenib, provide a modest increase in survival for advanced HCC patients and display significant toxicity. Thus, there is an immense need to identify novel regulators of HCC that might be targeted effectively. The insulin-like growth factor (IGF) axis is commonly abnormal in HCC. Upon activation, the IGF axis controls metabolism, tissue homeostasis, and survival. Insulin-like growth factor-binding protein 7 (IGFBP7) is a secreted protein of a family of low-affinity IGF-binding proteins termed "IGFBP-related proteins" that have been identified as a potential tumor suppressor in HCC. IGFBP7 has been implicated in regulating cellular proliferation, senescence, and angiogenesis. In this review, we provide a comprehensive discussion of the role of IGFBP7 in HCC and the potential use of IGFBP7 as a novel biomarker for drug resistance and as an effective therapeutic strategy.
RESUMO
Staphylococcal nuclease domain containing-1 (SND1) is overexpressed in human hepatocellular carcinoma (HCC) patients and promotes tumorigenesis by human HCC cells. We now document that SND1 increases angiotensin II type 1 receptor (AT1R) levels by increasing AT1R mRNA stability. This results in activation of ERK, Smad2 and subsequently the TGFß signaling pathway, promoting epithelial-mesenchymal transition (EMT) and migration and invasion by human HCC cells. A positive correlation was observed between SND1 and AT1R expression levels in human HCC patients. Small molecule inhibitors of SND1, alone or in combination with AT1R blockers, might be an effective therapeutic strategy for late-stage aggressive HCC.
RESUMO
Activation of the oncogene AEG-1 (MTDH, LYRIC) has been implicated recently in the development of hepatocellular carcinoma (HCC). In mice, HCC can be initiated by exposure to the carcinogen DEN, which has been shown to rely upon activation of NF-κB in liver macrophages. Because AEG-1 is an essential component of NF-κB activation, we interrogated the susceptibility of mice lacking the AEG-1 gene to DEN-induced hepatocarcinogenesis. AEG-1-deficient mice displayed resistance to DEN-induced HCC and lung metastasis. No difference was observed in the response to growth factor signaling or activation of AKT, ERK, and ß-catenin, compared with wild-type control animals. However, AEG-1-deficient hepatocytes and macrophages exhibited a relative defect in NF-κB activation. Mechanistic investigations showed that IL6 production and STAT3 activation, two key mediators of HCC development, were also deficient along with other biologic and epigenetics findings in the tumor microenvironment, confirming that AEG-1 supports an NF-κB-mediated inflammatory state that drives HCC development. Overall, our findings offer in vivo proofs that AEG-1 is essential for NF-κB activation and hepatocarcinogenesis, and they reveal new roles for AEG-1 in shaping the tumor microenvironment for HCC development.