RESUMO
Cytidine deaminase APOBEC3B (A3B) is known to play important roles in creating de novo genomic C-to-T mutations in cancers and contribute to induction of genomic instability. Our study evaluated the roles of A3B in the progression and metastasis of human hepatocellular carcinoma (HCC). Using whole-transcriptome and whole-exome sequencing, and quantitative PCR, we found that A3B was overexpressed in human HCCs and A3B expression was significantly correlated with the proportion of genomic C-to-A and G-to-T mutations. Upon clinicopathological correlation, higher A3B expression was associated with more aggressive tumor behavior. Wild-type A3B (wt-A3B) overexpression in HCC cells promoted cell proliferation, and cell migratory and invasive abilities in vitro, and tumorigenicity and metastasis in vivo. On the other hand, knockdown of A3B suppressed cell proliferation, migratory, and invasive abilities of HCC cells with high endogenous A3B level. However, to our surprise, overexpression of A3B deaminase-dead double mutant (E68A/E255Q) led to similar results as wt-A3B in HCC. Furthermore, overexpression of wt-A3B and mutant A3B both enhanced cell cycle progression in HCC cells. Altogether, our data demonstrated a novel deaminase-independent role of A3B in contributing to HCC tumorigenesis and metastasis.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/patologia , Citidina Desaminase/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/secundário , Antígenos de Histocompatibilidade Menor/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Ciclo Celular , Movimento Celular , Proliferação de Células , Citidina Desaminase/genética , Desaminação , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Antígenos de Histocompatibilidade Menor/genética , Prognóstico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Adjuvant interferon-α (IFN-α) therapy is used to control certain types of cancer in clinics. For hepatocellular carcinoma (HCC), IFN-α therapy is effective in only a subgroup of patients; therefore, identifying biomarkers to predict the response to IFN-α therapy is of high significance and clinical utility. As the induced IFN-stimulated gene expression following IFN-α treatment plays pivotal roles in IFN-α effects, we screened IFN-stimulated gene expression in HCC tissues and found that several IFN-stimulated genes were significantly decreased in HCC. Interestingly, expression of IFN-induced protein with tetratricopeptide repeats (IFIT) family members, including IFIT1, IFIT2, IFIT3, and IFIT5, was decreased in HCC tissues. We further analyzed the expression of IFIT family members in HCC and their roles in patients' responses to IFN-α therapy in two independent randomized controlled IFN-α therapy clinical trials of HCC patients. We found that higher expression of IFIT3, but not other IFITs, in HCC tissues predicts better response to IFN-α therapy, suggesting that IFIT3 may be a useful predictor of the response to IFN-α therapy in HCC patients. Mechanistically, IFIT3 enhanced the antitumor effects of IFN-α by promoting IFN-α effector responses both in vitro and in vivo. IFIT3 could bind signal transducer and activator of transcription 1 (STAT1) and STAT2 to enhance STAT1-STAT2 heterodimerization and nuclear translocation upon IFN-α treatment, thus promoting IFN-α effector signaling. CONCLUSION: Higher IFIT3 expression in HCC tissues predicts better response to IFN-α therapy in HCC patients; IFIT3 promotes IFN-α effector responses and therapeutic effects by strengthening IFN-α effector signaling in HCC. (Hepatology 2017;66:152-166).
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Interferon-alfa/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Adulto , Idoso , Análise de Variância , Biópsia por Agulha , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Estudos de Coortes , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Valor Preditivo dos Testes , Prognóstico , Modelos de Riscos Proporcionais , Medição de Risco , Taxa de Sobrevida , Resultado do TratamentoRESUMO
OBJECTIVE: The nature of the tumour-infiltrating leucocytes (TILs) is known to impact clinical outcome in carcinomas, including hepatocellular carcinoma (HCC). However, the role of tumour-infiltrating B cells (TIBs) remains controversial. Here, we investigate the impact of TIBs and their interaction with T cells on HCC patient prognosis. DESIGN: Tissue samples were obtained from 112 patients with HCC from Singapore, Hong Kong and Zurich and analysed using immunohistochemistry and immunofluorescence. RNA expression of CD19, CD8A, IFNG was analysed using quantitative PCR. The phenotype of freshly isolated TILs was analysed using flow cytometry. A mouse model depleted of mature B cells was used for functional study. RESULTS: Tumour-infiltrating T cells and B cells were observed in close contact with each other and their densities are correlated with superior survival in patients with HCC. Furthermore, the density of TIBs was correlated with an enhanced expression of granzyme B and IFN-γ, as well as with reduced tumour viability defined by low expression of Ki-67, and an enhanced expression of activated caspase-3 on tumour cells. CD27 and CD40 costimulatory molecules and TILs expressing activation marker CD38 in the tumour were also correlated with patient survival. Mice depleted of mature B cells and transplanted with murine hepatoma cells showed reduced tumour control and decreased local T cell activation, further indicating the important role of B cells. CONCLUSIONS: The close proximity of tumour-infiltrating T cells and B cells indicates a functional interaction between them that is linked to an enhanced local immune activation and contributes to better prognosis for patients with HCC.
Assuntos
Antígenos CD/análise , Linfócitos B/imunologia , Carcinoma Hepatocelular/imunologia , Neoplasias Hepáticas/imunologia , Linfócitos do Interstício Tumoral , Linfócitos T/imunologia , ADP-Ribosil Ciclase 1/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígenos CD19/genética , Antígenos CD20/análise , Linfócitos B/química , Linfócitos B/patologia , Complexo CD3/análise , Antígenos CD40/análise , Antígenos CD8/análise , Antígenos CD8/genética , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/patologia , Caspase 3/análise , Progressão da Doença , Feminino , Expressão Gênica , Granzimas/análise , Humanos , Interferon gama/genética , Antígeno Ki-67/análise , Neoplasias Hepáticas/química , Neoplasias Hepáticas/patologia , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fenótipo , Estudos Retrospectivos , Taxa de Sobrevida , Linfócitos T/química , Linfócitos T/patologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/análise , Adulto JovemRESUMO
OBJECTIVE: We investigated the mutational landscape of mammalian target of rapamycin (mTOR) signalling cascade in hepatocellular carcinomas (HCCs) with chronic HBV background, aiming to evaluate and delineate mutation-dependent mechanism of mTOR hyperactivation in hepatocarcinogenesis. DESIGN: We performed next-generation sequencing on human HCC samples and cell line panel. Systematic mutational screening of mTOR pathway-related genes was undertaken and mutant genes were evaluated based on their recurrence. Protein expressions of tuberous sclerosis complex (TSC)1, TSC2 and pRPS6 were assessed by immunohistochemistry in human HCC samples. Rapamycin sensitivity was estimated by colony-formation assay in HCC cell lines and the treatment was further tested using our patient-derived tumour xenograft (PDTX) models. RESULTS: We identified and confirmed multiple mTOR components as recurrently mutated in HBV-associated HCCs. Of significance, we detected frequent (16.2%, n=18/111) mutations of TSC1 and TSC2 genes in the HCC samples. The spectrum of TSC1/2 mutations likely disrupts the endogenous gene functions in suppressing the downstream mTOR activity through different mechanisms and leads to more aggressive tumour behaviour. Mutational disruption of TSC1 and TSC2 was also observed in HCC cell lines and our PDTX models. TSC-mutant cells exhibited reduced colony-forming ability on rapamycin treatment. With the use of biologically relevant TSC2-mutant PDTXs, we demonstrated the therapeutic benefits of the hypersensitivity towards rapamycin treatment. CONCLUSIONS: Taken together, our findings suggest the significance of previously undocumented mutation-dependent mTOR hyperactivation and frequent TSC1/2 mutations in HBV-associated HCCs. They define a molecular subset of HCC having genetic aberrations in mTOR signalling, with potential significance of effective specific drug therapy.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR/metabolismo , Proteínas Supressoras de Tumor/genética , Adulto , Idoso , Animais , Antibióticos Antineoplásicos/farmacologia , Proteína Axina/genética , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Análise Mutacional de DNA , Proteínas de Ligação a DNA , Feminino , Humanos , Neoplasias Hepáticas/química , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Taxa de Mutação , Transplante de Neoplasias , Proteínas Nucleares/genética , Transdução de Sinais , Sirolimo/farmacologia , Fatores de Transcrição/genética , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Ensaio Tumoral de Célula-Tronco , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/análise , Adulto Jovem , beta Catenina/genéticaRESUMO
BACKGROUND & AIMS: Hepatitis B virus (HBV) integration is common in HBV-associated hepatocellular carcinoma (HCC) and may play an important pathogenic role through the production of chimeric HBV-human transcripts. We aimed to screen the transcriptome for HBV integrations in HCCs. METHODS: Transcriptome sequencing was performed on paired HBV-associated HCCs and corresponding non-tumorous liver tissues to identify viral-human chimeric sites. Validation was further performed in an expanded cohort of human HCCs. RESULTS: Here we report the discovery of a novel pre-mRNA splicing mechanism in generating HBV-human chimeric protein. This mechanism was exemplified by the formation of a recurrent HBV-cyclin A2 (CCNA2) chimeric transcript (A2S), as detected in 12.5% (6 of 48) of HCC patients, but in none of the 22 non-HCC HBV-associated cirrhotic liver samples examined. Upon the integration of HBV into the intron of the CCNA2 gene, the mammalian splicing machinery utilized the foreign splice sites at 282nt. and 458nt. of the HBV genome to generate a pseudo-exon, forming an in-frame chimeric fusion with CCNA2. The A2S chimeric protein gained a non-degradable property and promoted cell cycle progression, demonstrating its potential oncogenic functions. CONCLUSIONS: A pre-mRNA splicing mechanism is involved in the formation of HBV-human chimeric proteins. This represents a novel and possibly common mechanism underlying the formation of HBV-human chimeric transcripts from intronically integrated HBV genome with functional impact. LAY SUMMARY: HBV is involved in the mammalian pre-mRNA splicing machinery in the generation of potential tumorigenic HBV-human chimeras. This study also provided insight on the impact of intronic HBV integration with the gain of splice sites in the development of HBV-associated HCC.
Assuntos
Carcinoma Hepatocelular , Vírus da Hepatite B , Neoplasias Hepáticas , Proteínas de Fusão Oncogênica , Precursores de RNA , Splicing de RNA , Integração Viral , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Ciclina A2/genética , Vírus da Hepatite B/genética , Íntrons , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Proteínas de Fusão Oncogênica/biossíntese , Precursores de RNA/genética , TranscriptomaRESUMO
Viral integration into the human genome upon infection is an important risk factor for various human malignancies. We developed viral integration site detection tool called Virus-Clip, which makes use of information extracted from soft-clipped sequencing reads to identify exact positions of human and virus breakpoints of integration events. With initial read alignment to virus reference genome and streamlined procedures, Virus-Clip delivers a simple, fast and memory-efficient solution to viral integration site detection. Moreover, it can also automatically annotate the integration events with the corresponding affected human genes. Virus-Clip has been verified using whole-transcriptome sequencing data and its detection was validated to have satisfactory sensitivity and specificity. Marked advancement in performance was detected, compared to existing tools. It is applicable to versatile types of data including whole-genome sequencing, whole-transcriptome sequencing, and targeted sequencing. Virus-Clip is available at http://web.hku.hk/~dwhho/Virus-Clip.zip.
Assuntos
Carcinoma Hepatocelular/virologia , Biologia Computacional/métodos , Neoplasias Hepáticas/virologia , Integração Viral , Vírus/metabolismo , Genoma Humano , Hepatite B/virologia , Vírus da Hepatite B/metabolismo , Humanos , Reação em Cadeia da Polimerase , Linguagens de Programação , Análise de Sequência de RNA , Software , TranscriptomaRESUMO
BACKGROUND: Gene set or pathway analysis (GPA) can provide a comprehensive mechanistic overview in delineating molecular pathoetiology of human diseases. Existing tools that can handle for GPA on both mutational and gene expressional aspects are limited. This leads to the development of uGPA (unified Gene Pathway Analyzer). METHODS: uGPA package provides a unified solution to knowledge base-driven competitive GPA that can analyze genome-wide screening data derived from multiple platforms (next generation sequencing or microarray) and sample sources (genomic or transcriptomic) on both mutational and gene expressional perspectives. It allows for a quick assessment of perspective outline of gene sets on the studied disease through enrichment test modeled by cumulative hypergeometric distribution and reports the gene components detected with events (mutation or differential gene expression) in the gene sets. RESULTS: uGPA package has been successfully verified to be applicable on input data generated from NGS-based platforms (transcriptome sequencing and whole-exome sequencing). Genome-wide screening data from other platforms should also apply. CONCLUSIONS: uGPA package delivers a simple, flexible and platform-independent functionality to multiple aspects of GPA. It equips clinicians and scientists with a useful tool in studying molecular mechanism of human diseases using modern high-throughput genome-wide screening strategy, which assists personalized therapy.
Assuntos
Doença/genética , Genes/genética , Genoma Humano/genética , Sequenciamento de Nucleotídeos em Larga Escala , Expressão Gênica , HumanosRESUMO
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the most common human cancers. Recently, emerging evidence has suggested the role of long non-coding RNAs (lncRNAs) in human carcinogenesis. In this study, we aimed to investigate the expression and functional implications of lncRNAs in human HCC. METHODS: Eighty-eight well-annotated lncRNAs were profiled in primary HCC by quantitative RT-PCR. Functional relevance of lncRNAs was elucidated in HCC cell lines and nude mice models. The regulatory relationship between miRNA and lncRNA was predicted in silico and further validated by luciferase reporter assay and expression analysis. RESULTS: In our profiling study, HOTTIP was identified as the most significantly up-regulated lncRNA in human HCCs, even in early stage of HCC formation. Functionally, knock-down of HOTTIP attenuated HCC cell proliferation in vitro and markedly abrogated tumourigenicity in vivo. In addition, knock-down of HOTTIP also inhibited migratory ability of HCC cells and significantly abrogated lung metastasis in orthotopic implantation model in nude mice. HOTTIP is an antisense lncRNA mapped to the distal end of the HOXA gene cluster. Knock-down of HOTTIP significantly suppressed the expression of a number of HOXA genes. Furthermore, we identified miR-125b as a post-transcriptional regulator of HOTTIP. Ectopic expression of miR-125b reduced HOTTIP-coupled luciferase activity and suppressed the endogenous level of HOTTIP. Moreover, in human HCCs, HOTTIP expression negatively correlated with that of miR-125b. CONCLUSIONS: HOTTIP is a novel oncogenic lncRNA, which negatively regulated by miR-125b. Overexpression of HOTTIP contributes to hepatocarcinogenesis by regulating the expression of its neighbouring protein-coding genes.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Animais , Linhagem Celular Tumoral , Movimento Celular , Modelos Animais de Doenças , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ativação Transcricional , Regulação para CimaRESUMO
Immunoevasion is an emerging hallmark of cancer. Impairment of natural killer (NK) cytotoxicity is a mechanism to evade host immunosurveillance. Granulin-epithelin precursor (GEP) is a hepatic oncofetal protein regulating growth, invasion, and chemoresistance in hepatocellular carcinoma (HCC). We examined the role of GEP in conferring HCC cells the ability to evade NK cytotoxicity. In HCC cell lines, GEP overexpression reduced, whereas GEP suppression enhanced sensitivity to NK cytotoxicity. GEP downregulated surface expression of MHC class I chain-related molecule A (MICA), ligand for NK stimulatory receptor NK group 2 member D (NKG2D), and upregulated human leukocyte antigen-E (HLA-E), ligand for NK inhibitory receptor CD94/NKG2A. Functionally, GEP augmented production of soluble MICA, which suppressed NK activation. Matrix metalloproteinase (MMP)2 and MMP9 activity was involved partly in the GEP-regulated MICA shedding from HCC cells. In primary HCCs (n = 80), elevated GEP (P < 0.001), MICA (P < 0.001), and HLA-E (P = 0.089) expression was observed when compared with those in nontumor (n = 80) and normal livers (n = 10). Serum GEP (P = 0.010) and MICA (P < 0.001) levels were higher in patients with HCC (n = 80) than in healthy individuals (n = 30). High serum GEP and/or MICA levels were associated with poor recurrence-free survival (log-rank test, P = 0.042). Importantly, GEP blockade by mAbs sensitized HCC cells to NK cytotoxicity through MICA. In summary, GEP rendered HCC cells resistant to NK cytotoxicity by modulating MICA expression, which could be reversed by GEP blockade using antibody. Serum GEP and MICA levels are prognostic factors and can be used to stratify patients for targeted therapy.
Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Citotoxicidade Imunológica/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica/efeitos dos fármacos , Modelos Animais de Doenças , Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Metaloproteinases da Matriz/metabolismo , Camundongos , Progranulinas , Antígenos HLA-ERESUMO
In hepatocellular carcinoma (HCC), biomarkers for prediction of prognosis and response to immunotherapy such as interferon-α (IFN-α) would be very useful in the clinic. We found that expression of retinoic acid-inducible gene-I (RIG-I), an IFN-stimulated gene, was significantly downregulated in human HCC tissues. Patients with low RIG-I expression had shorter survival and poorer response to IFN-α therapy, suggesting that RIG-I is a useful prognosis and IFN-α response predictor for HCC patients. Mechanistically, RIG-I enhances IFN-α response by amplifying IFN-α effector signaling via strengthening STAT1 activation. Furthermore, we found that RIG-I deficiency promotes HCC carcinogenesis and that hepatic RIG-I expression is lower in men than in women. RIG-I may therefore be a tumor suppressor in HCC and contribute to HCC gender disparity.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/metabolismo , RNA Helicases DEAD-box/metabolismo , Interferon-alfa/uso terapêutico , Neoplasias Hepáticas/metabolismo , Adulto , Idoso , Animais , Biomarcadores Tumorais/análise , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/mortalidade , Proteína DEAD-box 58 , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Xenoenxertos , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/mortalidade , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Imunológicos , Caracteres Sexuais , Transdução de Sinais/fisiologia , Análise Serial de Tecidos , Proteínas Supressoras de Tumor/metabolismo , Adulto JovemRESUMO
UNLABELLED: Random integration of hepatitis B virus (HBV) DNA into the host genome is frequent in human hepatocellular carcinoma (HCC) and this leads to truncation of the HBV DNA, particularly at the C-terminal end of the HBV X protein (HBx). In this study, we investigated the frequency of this natural C-terminal truncation of HBx in human HCCs and its functional significance. In 50 HBV-positive patients with HCC, full-length HBx was detected in all nontumorous livers. However, full-length HBx was found in only 27 (54%) of the HCC tumors, whereas natural carboxylic acid (COOH)-truncated HBx was found in the remaining 23 (46%) tumors. Upon clinicopathological analysis, the presence of natural COOH-truncated HBx significantly correlated with the presence of venous invasion, a hallmark of metastasis (P = 0.005). Inducible stable expression of the COOH-truncated HBx protein (with 24 amino acids truncated at the C-terminal end) enhanced the cell-invasive ability of HepG2 cells, as compared to full-length HBx, using the Matrigel cell-invasion assay. It also resulted in increased C-Jun transcriptional activity and enhanced transcription of matrix metalloproteinase 10 (MMP10), whereas activation of the MMP10 promoter by COOH-truncated HBx was abolished when the activator protein 1-binding sites on the MMP10 promoter were mutated. Furthermore, silencing of MMP10 by short interfering RNA in HBxΔC1-expressing HepG2 cells resulted in significant reduction of cell invasiveness. CONCLUSIONS: Our data suggest that COOH truncation of HBx, particularly with 24 amino acids truncated at the C-terminal end, plays a role in enhancing cell invasiveness and metastasis in HCC by activating MMP10 through C-Jun.
Assuntos
Carcinoma Hepatocelular/virologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neoplasias Hepáticas/virologia , Metaloproteinase 10 da Matriz/metabolismo , Transativadores/metabolismo , Adulto , Idoso , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Expressão Gênica , Células Hep G2 , Humanos , Fígado/patologia , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteínas Virais Reguladoras e AcessóriasRESUMO
BACKGROUND AND AIM: Granulin-epithelin precursor (GEP) has previously been reported to control cancer growth, invasion, chemo-resistance, and served as novel therapeutic target for cancer treatment. However, the nature and characteristics of GEP interacting partner remain unclear. The present study aims to identify and characterize the novel predominant interacting partner of GEP using co-immunoprecipitation and mass spectrometry. METHODS AND RESULTS: Specific anti-GEP monoclonal antibody was used to capture GEP and its interacting partner from the protein extract of the liver cancer cells Hep3B. The precipitated proteins were analyzed by SDS-PAGE, followed by mass spectrometry and the protein identity was demonstrated to be tropomyosin 3 (TPM3). The interaction has been validated in additional cell models using anti-TPM3 antibody and immunoblot to confirm GEP as the interacting partner. GEP and TPM3 expressions were then examined by real-time quantitative RT-PCR in clinical samples, and their transcript levels were significantly correlated. Elevated TPM3 levels were observed in liver cancer compared with the adjacent non-tumorous liver, and patients with elevated TPM3 levels were shown to have poor recurrence-free survival. Protein expression of GEP and TPM3 was observed only in the cytoplasm of liver cancer cells by immunohistochemical staining. CONCLUSIONS: TPM3 is an interacting partner of GEP and may play an important role in hepatocarcinogenesis.
Assuntos
Carcinoma Hepatocelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Hepáticas/metabolismo , Recidiva Local de Neoplasia , Tropomiosina/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Citoplasma/metabolismo , Intervalo Livre de Doença , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/patologia , Progranulinas , Ligação Proteica , Mapeamento de Interação de Proteínas , Precursores de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estatísticas não Paramétricas , Tropomiosina/genéticaRESUMO
Hepatitis B virus encoded X antigen (HBx) is a trans-regulatory protein that alters the activity of selected transcription factors and cytoplasmic signal transduction pathways. HBx transcriptionally up-regulates the expression of a unique gene, URG11, which in turn transcriptionally up-regulates ß-catenin, thereby contributing importantly to hepatocarcinogenesis. HBx and URG11 also alter the expression of multiple microRNAs, and by miRNA array analysis, both were shown to promote the expression of miR-148a. Elevated miR-148a was also seen in HBx positive liver samples from infected patients. To study the function of miR-148a, anti-148a was introduced into HepG2 and Hep3B cells stably expressing HBx or stably over-expressing URG11. Anti-miR-148a suppressed cell proliferation, cell cycle progression, cell migration, anchorage independent growth in soft agar and subcutaneous tumor formation in SCID mice. Introduction of anti-miR-148a increased PTEN protein and mRNA expression, suggesting that PTEN was targeted by miR-148a. Anti-miR-148a failed to suppress PTEN expression when co-transfected with reporter gene mutants in the 3'UTR of PTEN mRNA. Introduction of anti-miR-148a also resulted in depressed Akt signaling by HBx and URG11, resulting in decreased expression of ß-catenin. Thus, miR-148a may play a central role in HBx/URG11 mediated HCC, and may be an early diagnostic marker and/or therapeutic target associated with this tumor type.
Assuntos
Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/virologia , Hepatite B/metabolismo , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/virologia , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , Adulto , Idoso , Animais , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Humanos , Masculino , Camundongos , Camundongos SCID , MicroRNAs/antagonistas & inibidores , Pessoa de Meia-Idade , PTEN Fosfo-Hidrolase/biossíntese , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transativadores/análise , Transativadores/biossíntese , Proteínas Virais Reguladoras e Acessórias , beta Catenina/biossínteseRESUMO
OBJECTIVE: Hepatocellular carcinoma (HCC) is a heterogeneous disease with poor prognosis and limited methods for predicting patient survival. The nature of the immune cells that infiltrate tumours is known to impact clinical outcome. However, the molecular events that regulate this infiltration require further understanding. Here the ability of immune genes expressed in the tumour microenvironment to predict disease progression was investigated. METHODS: Using quantitative PCR, the expression of 14 immune genes in resected tumour tissues from 57 Singaporean patients was analysed. The nearest-template prediction method was used to derive and test a prognostic signature from this training cohort. The signature was then validated in an independent cohort of 98 patients from Hong Kong and Zurich. Intratumoural components expressing these critical immune genes were identified by in situ labelling. Regulation of these genes was analysed in vitro using the HCC cell line SNU-182. RESULTS: The identified 14 immune-gene signature predicts patient survival in both the training cohort (p=0.0004 and HR=5.2) and the validation cohort (p=0.0051 and HR=2.5) irrespective of patient ethnicity and disease aetiology. Importantly, it predicts the survival of patients with early disease (stages I and II), for whom classical clinical parameters provide limited information. The lack of predictive power in late disease stages III and IV emphasises that a protective immune microenvironment has to be established early in order to impact disease progression significantly. This signature includes the chemokine genes CXCL10, CCL5 and CCL2, whose expression correlates with markers of T helper 1 (Th1), CD8(+) T and natural killer (NK) cells. Inflammatory cytokines (tumour necrosis factor α, interferon γ) and Toll-like receptor 3 ligands stimulate intratumoural production of these chemokines which drive tumour infiltration by T and NK cells, leading to enhanced cancer cell death. CONCLUSION: A 14 immune-gene signature, which identifies molecular cues driving tumour infiltration by lymphocytes, accurately predicts survival of patients with HCC especially in early disease.
Assuntos
Carcinoma Hepatocelular/imunologia , Quimiocinas/imunologia , Neoplasias Hepáticas/imunologia , Linfócitos do Interstício Tumoral/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD8-Positivos/imunologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/cirurgia , Quimiocina CCL2/metabolismo , Quimiocina CCL5/metabolismo , Quimiocina CXCL10/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Células Matadoras Naturais/imunologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Células Th1/imunologia , Receptor 3 Toll-Like/imunologia , Adulto JovemRESUMO
Hepatitis B virus (HBV) is a major etiologic agent of chronic liver disease and hepatocellular carcinoma (HCC). HBV-encoded X antigen, HBx, and pathways implicated in the self-renewal of stem cells contribute to HCC, but it is not clear whether HBx expression promotes "stemness." Thus, experiments were designed to test the hypothesis that HBx triggers malignant transformation by promoting properties that are characteristic of cancer stem cells (CSC). To test this hypothesis, HepG2 cells were stably transduced with HBx and then assayed for phenotypic and molecular characteristics of "stemness." The relationship between HBx and "stemness"-associated markers was also evaluated by immunohistochemical staining of liver and tumor tissue sections from HBV-infected patients. The results showed that Oct-4, Nanog, Klf-4, ß-catenin, and epithelial cell adhesion molecule (EpCAM) were activated by HBx in vitro and in vivo. EpCAM was detected in the nuclei of human HCC cells from infected patients. HBx promotes "stemness" by activating ß-catenin and epigenetic upregulation of miR-181, both of which target EpCAM. HBx expression was also associated with depressed levels of E-cadherin. Moreover, HBx stimulated cell migration, growth in soft agar, and spheroid formation. This work is the first to propose that HBV promotes "stemness" in the pathogenesis of HCC. HBx-associated upregulated expression of multiple "stemness" markers supports the hypothesis that HBx contributes to hepatocarcinogenesis, at least in part, by promoting changes in gene expression that are characteristics of CSCs.
Assuntos
Neoplasias Hepáticas/metabolismo , Células-Tronco Neoplásicas/citologia , Transativadores/metabolismo , Adulto , Idoso , Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Epigênese Genética , Molécula de Adesão da Célula Epitelial , Feminino , Vírus da Hepatite B/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Virais Reguladoras e Acessórias , beta Catenina/metabolismoRESUMO
The middle hepatic vein may be included in right liver living donor liver transplantation (LDLT) to optimize hepatic venous outflow. We studied the graft's ability to relieve portal hypertension and accommodate portal hyperperfusion with portal manometry and ultrasonic flowmetry. Surgical outcomes with respect to portal hemodynamometry were also investigated. The ages of the recipients and donors for 46 consecutive LDLT procedures were 50 (range, 16-66 years) and 31 years (range, 18-54 years), respectively. The graft to standard liver volume ratio was 47.4% (range, 32.4%-69.0%). The hospital mortality rate was 4.4% as 2 recipients died from a subarachnoid hemorrhage and sepsis. The portal pressure dropped by 8 mm Hg (range, -7 to 19 mm Hg) from 23 (range, 8-37 mm Hg) to 14 mm Hg (range, 10-26 mm Hg) after graft implantation. The portal inflow positively correlated with the portal pressure before native liver hepatectomy (R(2) = 0.305, P = 0.001) and not with the graft size. The portal inflow increased from 81 mL/minute/100 g (range, 35-210 mL/minute/100 g) before donor right hepatectomy to 318 mL/minute/100 g (range, 102-754 mL/minute/100 g) after graft implantation. The graft portal inflow had a positive linear correlation with the recipient portal pressure before native liver total hepatectomy (R(2) = 0.261, P = 0.001) but not after graft implantation, and it had a negative correlation with the graft to standard liver volume ratio (R(2) = 0.247, P = 0.001). Only 1 of the graft biopsies showed moderate sinusoidal congestion. Twelve recipients had Clavien grade 2+ complications that were not related to the portal inflow and pressure or graft size. Right liver LDLT including the middle hepatic vein effectively lowered the recipient portal pressure by allowing unimpeded venous outflow.
Assuntos
Hepatectomia , Veias Hepáticas/cirurgia , Hipertensão Portal/cirurgia , Transplante de Fígado , Doadores Vivos , Pressão na Veia Porta , Veia Porta/cirurgia , Adolescente , Adulto , Biópsia , Velocidade do Fluxo Sanguíneo , Feminino , Veias Hepáticas/fisiopatologia , Hong Kong , Mortalidade Hospitalar , Humanos , Hipertensão Portal/mortalidade , Hipertensão Portal/fisiopatologia , Modelos Lineares , Transplante de Fígado/efeitos adversos , Transplante de Fígado/mortalidade , Masculino , Manometria , Pessoa de Meia-Idade , Veia Porta/fisiopatologia , Estudos Prospectivos , Fluxo Sanguíneo Regional , Reologia , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
Cadherin is an important cell adhesion molecule that plays paramount roles in organ development and the maintenance of tissue integrity. Dysregulation of cadherin expression is often associated with disease pathology including tissue dysplasia, tumor formation, and metastasis. Cadherin-17 (CDH17), belonging to a subclass of 7D-cadherin superfamily, is present in fetal liver and gastrointestinal tract during embryogenesis, but the gene becomes silenced in healthy adult liver and stomach tissues. It functions as a peptide transporter and a cell adhesion molecule to maintain tissue integrity in epithelia. However, recent findings from our group and others have reported aberrant expression of CDH17 in major gastrointestinal malignancies including hepatocellular carcinoma (HCC), stomach and colorectal cancers, and its clinical association with tumor metastasis and advanced tumor stages. Furthermore, alternative splice isoforms and genetic polymorphisms of CDH17 gene have been identified in HCC and linked to an increased risk of HCC. CDH17 is an attractive target for HCC therapy. Targeting CDH17 in HCC can inhibit tumor growth and inactivate Wnt signaling pathway in concomitance with activation of tumor suppressor genes. Further investigation on CDH17-mediated oncogenic signaling and cognate molecular mechanisms would shed light on new targeting therapy on HCC and potentially other gastrointestinal malignancies.
Assuntos
Caderinas/fisiologia , Carcinoma Hepatocelular/etiologia , Neoplasias Hepáticas/etiologia , Animais , Biomarcadores Tumorais/análise , Caderinas/análise , Caderinas/química , Caderinas/genética , Carcinoma Hepatocelular/tratamento farmacológico , Epigênese Genética , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Isoformas de Proteínas , Transdução de SinaisRESUMO
Small hepatocellular carcinomas (HCCs) can be effectively cured by surgery with good clinical outcomes. However, the conventional AFP marker is ineffective in detecting small tumors. Here we employed a proteomic profiling approach to identify a candidate marker for HCC (Assuntos
Biomarcadores Tumorais/sangue
, Carcinoma Hepatocelular/sangue
, Neoplasias Hepáticas/sangue
, Proteômica/métodos
, Vimentina/sangue
, Adulto
, Idoso
, Área Sob a Curva
, Carcinoma Hepatocelular/diagnóstico
, Carcinoma Hepatocelular/patologia
, Linhagem Celular Tumoral
, Estudos de Coortes
, Diagnóstico Precoce
, Ensaio de Imunoadsorção Enzimática
, Feminino
, Humanos
, Neoplasias Hepáticas/diagnóstico
, Neoplasias Hepáticas/patologia
, Masculino
, Pessoa de Meia-Idade
, Sensibilidade e Especificidade
RESUMO
Functional neuroendocrine tumors are often low-grade malignant neoplasms that can be cured by surgery if detected early, and such detection may in turn be accelerated by the recognition of neuropeptide hypersecretion syndromes. Uniquely, however, relief of peptic symptoms induced by hypergastrinemia is now available from acid-suppressive drugs such as proton-pump inhibitors (PPIs). Here we describe a clinical case in which time to diagnosis from the onset of peptic symptoms was delayed more than 10 years, in part reflecting symptom masking by continuous prescription of the PPI omeprazole. We propose diagnostic criteria for this under-recognized new clinical syndrome, and recommend that physicians routinely measure serum gastrin levels in persistent cases of PPI-dependent dyspepsia unassociated with H. pylori.