RESUMEN
Hemophilia A is a rare bleeding disorder with variable expressivity and allelic heterogeneity. Despite the advancement of prenatal diagnostics and molecular studies, the number of studies reviewing the reproductive choices of hemophilia A carriers and affected individuals remains limited. Through this retrospective review, we hope to gain a deeper understanding of hemophilia A-affected individuals' clinical and molecular characteristics, as well as the reproductive choices of the at-risk couples. A total of 122 individuals harboring likely causative F8 gene alterations from 64 apparently unrelated families attending three centers between 3/2000 and 3/2023 were included in this study. Their clinical and molecular findings as well as reproductive choices were gathered in a clinical setting and verified through the electronic medical record database of the public health system. Forty-seven affected males and 75 female heterozygous carriers were included in the analysis. Among 64 apparently unrelated families, 36 distinct pathogenic/likely pathogenic variants were identified, of which 30.6% (11/36) of variants were novel. While the majority of clinical findings and genotype-phenotype correlations appear to be in accordance with existing literature, female carriers who had no fertility intention were significantly more likely to have affected sons than those who had fertility intention (5/19 vs. 4/5; p = 0.047). Through this retrospective review, we summarized the clinical and molecular characteristics of 122 individuals harboring pathogenic/likely pathogenic F8 variants, as well as their fertility intentions and reproductive outcomes. Further studies are required to look into the considerations involved in reproductive decision-making.
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Hemofilia A , Heterocigoto , Humanos , Hemofilia A/genética , Hemofilia A/patología , Hemofilia A/epidemiología , Femenino , Masculino , Adulto , Mutación/genética , Factor VIII/genética , Estudios Retrospectivos , Estudios de Asociación Genética , FenotipoRESUMEN
OBJECTIVE: Hepatocellular carcinoma (HCC), mostly developed in fibrotic/cirrhotic liver, exhibits relatively low responsiveness to immune checkpoint blockade (ICB) therapy. As myeloid-derived suppressor cell (MDSC) is pivotal for immunosuppression, we investigated its role and regulation in the fibrotic microenvironment with an aim of developing mechanism-based combination immunotherapy. DESIGN: Functional significance of MDSCs was evaluated by flow cytometry using two orthotopic HCC models in fibrotic liver setting via carbon tetrachloride or high-fat high-carbohydrate diet and verified by clinical specimens. Mechanistic studies were conducted in human hepatic stellate cell (HSC)-peripheral blood mononuclear cell culture systems and fibrotic-HCC patient-derived MDSCs. The efficacy of single or combined therapy with anti-programmed death-1-ligand-1 (anti-PD-L1) and a clinically trialled BET bromodomain inhibitor i-BET762 was determined. RESULTS: Accumulation of monocytic MDSCs (M-MDSCs), but not polymorphonuclear MDSCs, in fibrotic livers significantly correlated with reduced tumour-infiltrating lymphocytes (TILs) and increased tumorigenicity in both mouse models. In human HCCs, the tumour-surrounding fibrotic livers were markedly enriched with M-MDSC, with its surrogate marker CD33 significantly associated with aggressive tumour phenotypes and poor survival rates. Mechanistically, activated HSCs induced monocyte-intrinsic p38 MAPK signalling to trigger enhancer reprogramming for M-MDSC development and immunosuppression. Treatment with p38 MAPK inhibitor abrogated HSC-M-MDSC crosstalk to prevent HCC growth. Concomitant with patient-derived M-MDSC suppression by i-BET762, combined treatment with anti-PD-L1 synergistically enhanced TILs, resulting in tumour eradication and prolonged survival in the fibrotic-HCC mouse model. CONCLUSION: Our results signify how non-tumour-intrinsic properties in the desmoplastic microenvironment can be exploited to reinstate immunosurveillance, providing readily translatable combination strategies to empower HCC immunotherapy.
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Carcinoma Hepatocelular/terapia , Inmunoterapia/métodos , Neoplasias Hepáticas/terapia , Animales , Antígeno B7-H1/antagonistas & inhibidores , Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/inmunología , Reprogramación Celular/inmunología , Ciclopropanos/farmacología , Ciclopropanos/uso terapéutico , Células Estrelladas Hepáticas/inmunología , Humanos , Tolerancia Inmunológica , Cirrosis Hepática/complicaciones , Cirrosis Hepática/patología , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas Experimentales/etiología , Neoplasias Hepáticas Experimentales/inmunología , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas Experimentales/terapia , Masculino , Ratones Endogámicos C57BL , Monocitos/inmunología , Células Supresoras de Origen Mieloide/inmunología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/farmacología , Piridinas/uso terapéutico , Transducción de Señal/fisiología , Células Tumorales Cultivadas , Microambiente Tumoral , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
Emerging evidence indicates that Cullin 4B (CUL4B), a major component of ubiquitin ligase complexes, is over-expressed in diverse cancer types with pro-tumourigenic effects. In this issue of the Journal of Pathology, Yuan and colleagues [6] elucidated the oncogenic activity of CUL4B in hepatocellular carcinoma (HCC) and delineated its role in driving Wnt/ß-catenin signalling. In addition to the stabilization of ß-catenin protein against proteasomal degradation, CUL4B also acts in concert with enhancer of Zeste homologue 2 (EZH2) to concordantly silence multiple Wnt inhibitors. These findings provide significant mechanistic insights into the epigenetic activation of the Wnt/ß-catenin pathway in HCC and shed light on the functional importance of ubiquitination in this intricate regulatory system.
Asunto(s)
Carcinoma Hepatocelular/enzimología , Proteínas Cullin/metabolismo , Neoplasias Hepáticas/enzimología , Vía de Señalización Wnt , beta Catenina/metabolismo , HumanosRESUMEN
Hepatocellular carcinoma (HCC) is a worldwide threat to public health, especially in China, where chronic hepatitis B virus (HBV) infection is found in 80-90% of all HCCs. The HBV-encoded X antigen (HBx) is a trans-regulatory protein involved in virus-induced hepatocarcinogenesis. Although the carboxyl-terminus-truncated HBx, rather than the full-length counterpart, is frequently overexpressed in human HCCs, its functional mechanisms are not fully defined. We investigated the molecular function of a naturally occurring HBx variant which has 35 amino acids deleted at the C-terminus (HBxΔ35). Genome-wide scanning analysis and PCR validation identified growth arrest-specific 2 (GAS2) as a direct target of HBxΔ35 at transcriptional level in human immortalized liver cells. HBxΔ35 was found to bind the promoter region of GAS2 and attenuate its expression to promote hepatocellular proliferation and tumourigenicity. Further functional assays demonstrated that GAS2 induces p53-dependent apoptosis and senescence to counteract HBxΔ35-mediated tumourigenesis. Notably, GAS2 expression was significantly down-regulated in HCCs compared with the corresponding normal tissues. In conclusion, our integrated study uncovered a novel viral mechanism in hepatocarcinogenesis, wherein HBxΔ35 deregulates cell growth via direct silencing of GAS2 and thereby provides a survival advantage for pre-neoplastic hepatocytes to facilitate cancer development.
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Apoptosis , Carcinoma Hepatocelular/metabolismo , Ciclo Celular , Transformación Celular Viral , Senescencia Celular , Silenciador del Gen , Virus de la Hepatitis B/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Microfilamentos/metabolismo , Transactivadores/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Sitios de Unión , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Femenino , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Hepatitis B/complicaciones , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/patogenicidad , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas de Microfilamentos/genética , Regiones Promotoras Genéticas , Transducción de Señal , Factores de Tiempo , Transactivadores/genética , Transcripción Genética , Transfección , Carga Tumoral , Proteína p53 Supresora de Tumor/genética , Proteínas Reguladoras y Accesorias ViralesRESUMEN
The breast cancer associated gene 1 (BRCA1)-A protein complex assembles at DNA damage-induced nuclear foci to facilitate repair of double-stranded breaks. Here, we describe the first systematic comparison of the dynamics, copy number and organization of its core components at foci. We show that the protein pools at individual foci generally comprise a small immobile fraction (â¼20%) and larger mobile fraction (â¼80%), which together occupy the same focal space but exist at different densities. In the mobile fraction, Abraxas (CCDC98) and the heterodimer BARD1-BRCA1 share similar rates of dynamic exchange (complete turnover in â¼500 seconds). In contrast, RAP80, which is required for initial foci assembly, was more dynamic with 25-fold faster turnover at mature foci. In addition, Abraxas, BARD1, BRCA1 and Merit40 (NBA1) were stably retained in the immobile fraction of foci under conditions causing loss of BRCC36 and RAP80, suggesting a shift to RAP80-independent localization after foci formation. These results, combined with our finding that RAP80 (â¼1200 copies per focus) is twofold more abundant than Abraxas/BARD1/BRCA1 at foci, suggest new models defining the dynamic organization of BRCA1-A complex at mature foci, wherein the unusually fast turnover of RAP80 may contribute to its regulation of BRCA1-dependent DNA repair.
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Proteína BRCA1/metabolismo , Núcleo Celular/metabolismo , Daño del ADN , Genes BRCA1 , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Citoplasma/metabolismo , Reparación del ADN , Proteínas de Unión al ADN , Enzimas Desubicuitinizantes , Chaperonas de Histonas , Humanos , Proteínas de la Membrana/metabolismo , Modelos Teóricos , Proteínas Nucleares/metabolismo , ARN Interferente Pequeño/metabolismo , Radiación Ionizante , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Actin, a constituent of the cytoskeleton, is now recognized to function in the nucleus in gene transcription, chromatin remodeling and DNA replication/repair. Actin shuttles in and out of the nucleus through the action of transport receptors importin-9 and exportin-6. Here we have addressed the impact of cell cycle progression and DNA replication stress on actin nuclear localization, through study of actin dynamics in living cells. First, we showed that thymidine-induced G1/S phase cell cycle arrest increased the nuclear levels of actin and of two factors that stimulate actin polymerization: IQGAP1 and Rac1 GTPase. When cells were exposed to hydroxyurea to induce DNA replication stress, the nuclear localization of actin and its regulators was further enhanced. We employed live cell photobleaching assays and discovered that in response to DNA replication stress, GFP-actin nuclear import and export rates increased by up to 250%. The rate of import was twice as fast as export, accounting for actin nuclear accumulation. The faster shuttling dynamics correlated with reduced cellular retention of actin, and our data implicate actin polymerization in the stress-dependent uptake of nuclear actin. Furthermore, DNA replication stress induced a nuclear shift in IQGAP1 and Rac1 with enhanced import dynamics. Proximity ligation assays revealed that IQGAP1 associates in the nucleus with actin and Rac1, and formation of these complexes increased after hydroxyurea treatment. We propose that the replication stress checkpoint triggers co-ordinated nuclear entry and trafficking of actin, and of factors that regulate actin polymerization.
Asunto(s)
Actinas/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Replicación del ADN/efectos de los fármacos , Proteína de Unión al GTP rac1/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Actinas/antagonistas & inhibidores , Actinas/genética , Transporte Activo de Núcleo Celular , Western Blotting , Reparación del ADN/efectos de los fármacos , Recuperación de Fluorescencia tras Fotoblanqueo , Humanos , Hidroxiurea/farmacología , Inmunoprecipitación , Unión Proteica , Transporte de Proteínas , ARN Interferente Pequeño/genética , Transducción de Señal , Timidina/farmacologíaRESUMEN
Smad7 is a principal inhibitor of the TGFß-Smad signalling pathway. We have investigated the functional significance of Smad7 in hepatocellular carcinoma (HCC). Smad7 knockout (KO) and wild-type (WT) mice were injected with diethylnitrosamine (DEN) to induce HCC. The effects of Smad7 on cellular features were examined in HCC cells, using a Smad7 over-expression or deletion approach. Signalling pathway components modulated by Smad7 in HCC were evaluated using luciferase reporter assay and co-immunoprecipitation. Smad7 was down-regulated in human HCCs compared with the adjacent normal tissues (p < 0.001). Smad7 KO mice were more susceptible to DEN-induced HCC than WT mice (78% versus 22%, p < 0.05). HCCs from KO mice displayed a greater proliferation activity (p < 0.05) and a reduced apoptotic index compared with WT littermates (p < 0.05). Deletion of Smad7 promoted cell proliferation in primary cultured HCC cells. In addition, over-expression of Smad7 in HCC cell lines markedly suppressed cell growth (p < 0.0001) and colony formation (p < 0.01). Cell cycle analysis revealed an increase in the G1 phase and a reduction in the S-phase populations, accompanied by up-regulation of p27(Kip1) and down-regulation of cyclin D1. Smad7 increased cell apoptosis (p < 0.01) by mediating an intrinsic [caspase-9, caspase-3 and poly(ADP-ribose) polymerase] apoptotic pathway. Moreover, Smad7 inhibited NF-κB signalling by interacting with TAB2, an upstream activator of NF-κB, and inhibited TGFß signalling by suppressing phosphorylation of Smad3. In conclusion, loss of Smad7 enhances susceptibility to HCC. Smad7 suppresses HCC cell growth by inhibiting proliferation and G1 -S phase transition and inducing apoptosis through attenuation of NF-κB and TGFß signalling. Smad7 acts as a potential tumour suppressor in liver.
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Carcinoma Hepatocelular/prevención & control , Hepatocitos/metabolismo , Neoplasias Hepáticas Experimentales/prevención & control , Proteína smad7/metabolismo , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Dietilnitrosamina , Fase G1 , Genes Reporteros , Predisposición Genética a la Enfermedad , Células Hep G2 , Hepatocitos/patología , Humanos , Inmunoprecipitación , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Fenotipo , Cultivo Primario de Células , Fase S , Transducción de Señal , Proteína smad7/deficiencia , Proteína smad7/genética , Factores de Tiempo , Transfección , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Obesity is a major risk factor for cancers including hepatocellular carcinoma (HCC) that develops from a background of non-alcoholic fatty liver disease (NAFLD). Hypercholesterolemia is a common comorbidity of obesity. Although cholesterol biosynthesis mainly occurs in the liver, its role in HCC development of obese people remains obscure. Using high-fat high-carbohydrate diet-associated orthotopic and spontaneous NAFLD-HCC mouse models, we found that hepatic cholesterol accumulation in obesity selectively suppressed natural killer T (NKT) cell-mediated antitumor immunosurveillance. Transcriptome analysis of human liver revealed aberrant cholesterol metabolism and NKT cell dysfunction in NAFLD patients. Notably, cholesterol-lowering rosuvastatin restored NKT expansion and cytotoxicity to prevent obesogenic diet-promoted HCC development. Moreover, suppression of hepatic cholesterol biosynthesis by a mammalian target of rapamycin (mTOR) inhibitor vistusertib preceded tumor regression, which was abolished by NKT inactivation but not CD8+ T cell depletion. Mechanistically, sterol regulatory element-binding protein 2 (SREBP2)-driven excessive cholesterol production from hepatocytes induced lipid peroxide accumulation and deficient cytotoxicity in NKT cells, which were supported by findings in people with obesity, NAFLD and NAFLD-HCC. This study highlights mTORC1/SREBP2/cholesterol-mediated NKT dysfunction in the tumor-promoting NAFLD liver microenvironment, providing intervention strategies that invigorating NKT cells to control HCC in the obesity epidemic.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Células T Asesinas Naturales , Enfermedad del Hígado Graso no Alcohólico , Animales , Colesterol/metabolismo , Humanos , Hígado/patología , Mamíferos , Ratones , Monitorización Inmunológica/efectos adversos , Enfermedad del Hígado Graso no Alcohólico/patología , Obesidad/patología , Microambiente TumoralRESUMEN
The liver is an immunologically tolerant organ and a common metastatic site of multiple cancer types. Although a role for cancer cell invasion programs has been well characterized, whether and how liver-intrinsic factors drive metastatic spread is incompletely understood. Here, we show that aberrantly activated hepatocyte-intrinsic cell cycle-related kinase (CCRK) signaling in chronic liver diseases is critical for cancer metastasis by reprogramming an immunosuppressive microenvironment. Using an inducible liver-specific transgenic model, we found that CCRK overexpression dramatically increased both B16F10 melanoma and MC38 colorectal cancer (CRC) metastasis to the liver, which was highly infiltrated by polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs) and lacking natural killer T (NKT) cells. Depletion of PMN-MDSCs in CCRK transgenic mice restored NKT cell levels and their interferon gamma production and reduced liver metastasis to 2.7% and 0.7% (metastatic tumor weights) in the melanoma and CRC models, respectively. Mechanistically, CCRK activated nuclear factor-kappa B (NF-κB) signaling to increase the PMN-MDSC-trafficking chemokine C-X-C motif ligand 1 (CXCL1), which was positively correlated with liver-infiltrating PMN-MDSC levels in CCRK transgenic mice. Accordingly, CRC liver metastasis patients exhibited hyperactivation of hepatic CCRK/NF-κB/CXCL1 signaling, which was associated with accumulation of PMN-MDSCs and paucity of NKT cells compared to healthy liver transplantation donors. In summary, this study demonstrates that immunosuppressive reprogramming by hepatic CCRK signaling undermines antimetastatic immunosurveillance. Our findings offer new mechanistic insights and therapeutic targets for liver metastasis intervention.
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Ciclo Celular , Neoplasias Colorrectales/inmunología , Neoplasias Hepáticas/inmunología , Melanoma Experimental/inmunología , Células Supresoras de Origen Mieloide/inmunología , Células T Asesinas Naturales/inmunología , Microambiente Tumoral , Animales , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundario , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Mouse mammary tumor virus (MMTV) has long been known as a causal agent of breast cancer in mice. To date, varied MMTV-like envelope gene (env) sequences have been identified in up to 74% of human breast cancers. However, the role and origin of these MMTV-like sequences in human breast cancer remain uncertain. Our study was initiated to study the integration of MMTV-like env sequences in human breast cancer. PCR screening has identified 28 (56%) Australian breast cancer specimens and 7 (87.5%) human breast cancer cell lines to be positive for MMTV-like env sequence. In the MCF-7 genome, a fragment containing an MMTV-like env sequence of approximately 1.9 kb plus a downstream rodent-like sequence of approximately 200 bp was found to be integrated into a bacterial-like beta-lactamase sequence by insertional mutagenesis. The identified MMTV-rodent fragment is present in some MCF-7 sublines but absent in the screened specimens and other cell lines. Sporadic mutations found in this fragment indicate it has multiple copies in the MCF-7 genome. Sequence analysis has identified a novel ORF of approximately 1.6 kb which is 94-99% identical to MMTV env genes. RT-PCR was performed on the MCF-7 cDNA but no MMTV-like env transcript was detected. This is the first report to reveal the locus of MMTV-like env sequence in human cells. The MMTV-like env sequence was shown to be distinct from the human endogenous retroviral sequences and is closely related to rodents.
Asunto(s)
Neoplasias de la Mama/genética , Genes env , Virus del Tumor Mamario del Ratón/genética , Animales , Secuencia de Bases , Línea Celular Tumoral , ADN Viral , Humanos , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Mutación , ARN Mensajero/genética , Homología de Secuencia de Ácido Nucleico , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND: Androgen receptor (AR) plays a crucial role as a transcription factor in promoting the development of hepatocellular carcinoma (HCC) which is prone to aberrant chromatin modifications. However, the regulatory effects of AR on epigenetic mediators in HCC remain ill-defined. Enhancer of zeste homolog 2 (EZH2), an oncogene responsible for the tri-methylation of histone H3 at lysine 27 (H3K27me3), was identified to be overexpressed in approximate 70-90% of HCC cases, which prompted us to investigate whether or how AR regulates EZH2 expression. METHODS: Colony formation, soft agar assay, xenograft and orthotopic mouse models were used to determine cell proliferation and tumorigenicity of gene-manipulated HCC cells. Gene regulation was assessed by chromatin immunoprecipitation, luciferase reporter assay, quantitative RT-PCR and immunoblotting. Clinical relevance of candidate proteins in patient specimens was examined in terms of pathological parameters and postsurgical survival rates. FINDINGS: In this study, we found that AR upregulated EZH2 expression by binding to EZH2 promoter and stimulating its transcriptional activity. EZH2 overexpression increased H3K27me3 levels and thereby silenced the expression of Wnt signal inhibitors, resulting in activation of Wnt/ß-catenin signaling and subsequently induction of cell proliferation and tumorigenesis. In a cohort of human HCC patients, concordant overexpression of AR, EZH2, H3K27me3 and active ß-catenin was observed in tumor tissues compared with paired non-tumor tissues, which correlated with tumor progression and poor prognosis. These findings demonstrate a novel working model in which EZH2 mediates AR-induced Wnt/ß-catenin signaling activation through epigenetic modification, and support the application of EZH2-targeted reagents for treating HCC patients.
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Carcinoma Hepatocelular/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Neoplasias Hepáticas/metabolismo , Receptores Androgénicos/metabolismo , Vía de Señalización Wnt , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular , Proteína Potenciadora del Homólogo Zeste 2/genética , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica , Histonas/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Metilación , Ratones Desnudos , Modelos Biológicos , Pronóstico , Transcripción Genética , Regulación hacia Arriba/genética , beta Catenina/metabolismoRESUMEN
Obesity increases the risk of hepatocellular carcinoma (HCC) especially in men, but the molecular mechanism remains obscure. Here, we show that an androgen receptor (AR)-driven oncogene, cell cycle-related kinase (CCRK), collaborates with obesity-induced pro-inflammatory signaling to promote non-alcoholic steatohepatitis (NASH)-related hepatocarcinogenesis. Lentivirus-mediated Ccrk ablation in liver of male mice fed with high-fat high-carbohydrate diet abrogates not only obesity-associated lipid accumulation, glucose intolerance and insulin resistance, but also HCC development. Mechanistically, CCRK fuels a feedforward loop by inducing STAT3-AR promoter co-occupancy and transcriptional up-regulation, which in turn activates mTORC1/4E-BP1/S6K/SREBP1 cascades via GSK3ß phosphorylation. Moreover, hepatic CCRK induction in transgenic mice stimulates mTORC1-dependent G-csf expression to enhance polymorphonuclear myeloid-derived suppressor cell recruitment and tumorigenicity. Finally, the STAT3-AR-CCRK-mTORC1 pathway components are concordantly over-expressed in human NASH-associated HCCs. These findings unveil the dual roles of an inflammatory-CCRK circuitry in driving metabolic and immunosuppressive reprogramming through mTORC1 activation, thereby establishing a pro-tumorigenic microenvironment for HCC development.
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Carcinoma Hepatocelular/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Neoplasias Hepáticas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Obesidad/metabolismo , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/inmunología , Línea Celular Tumoral , Quinasas Ciclina-Dependientes/genética , Femenino , Células Hep G2 , Humanos , Tolerancia Inmunológica/genética , Tolerancia Inmunológica/inmunología , Inflamación/genética , Inflamación/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/inmunología , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Obesidad/genética , Obesidad/inmunología , Interferencia de ARN , Tratamiento con ARN de Interferencia , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
INTRODUCTION: Many of the DNA sequence variants identified in the breast cancer susceptibility gene BRCA1 remain unclassified in terms of their potential pathogenicity. Both multifactorial likelihood analysis and functional approaches have been proposed as a means to elucidate likely clinical significance of such variants, but analysis of the comparative value of these methods for classifying all sequence variants has been limited. METHODS: We have compared the results from multifactorial likelihood analysis with those from several functional analyses for the four BRCA1 sequence variants A1708E, G1738R, R1699Q, and A1708V. RESULTS: Our results show that multifactorial likelihood analysis, which incorporates sequence conservation, co-inheritance, segregation, and tumour immunohistochemical analysis, may improve classification of variants. For A1708E, previously shown to be functionally compromised, analysis of oestrogen receptor, cytokeratin 5/6, and cytokeratin 14 tumour expression data significantly strengthened the prediction of pathogenicity, giving a posterior probability of pathogenicity of 99%. For G1738R, shown to be functionally defective in this study, immunohistochemistry analysis confirmed previous findings of inconsistent 'BRCA1-like' phenotypes for the two tumours studied, and the posterior probability for this variant was 96%. The posterior probabilities of R1699Q and A1708V were 54% and 69%, respectively, only moderately suggestive of increased risk. Interestingly, results from functional analyses suggest that both of these variants have only partial functional activity. R1699Q was defective in foci formation in response to DNA damage and displayed intermediate transcriptional transactivation activity but showed no evidence for centrosome amplification. In contrast, A1708V displayed an intermediate transcriptional transactivation activity and a normal foci formation response in response to DNA damage but induced centrosome amplification. CONCLUSION: These data highlight the need for a range of functional studies to be performed in order to identify variants with partially compromised function. The results also raise the possibility that A1708V and R1699Q may be associated with a low or moderate risk of cancer. While data pooling strategies may provide more information for multifactorial analysis to improve the interpretation of the clinical significance of these variants, it is likely that the development of current multifactorial likelihood approaches and the consideration of alternative statistical approaches will be needed to determine whether these individually rare variants do confer a low or moderate risk of breast cancer.
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Biomarcadores de Tumor/análisis , Neoplasias de la Mama/química , Neoplasias de la Mama/genética , Genes BRCA1 , Mutación Missense , Alanina , Arginina , Neoplasias de la Mama/patología , Centrosoma , ADN Complementario , Análisis Factorial , Femenino , Predisposición Genética a la Enfermedad , Ácido Glutámico , Glutamina , Glicina , Humanos , Inmunohistoquímica , Queratinas/análisis , Técnicas de Amplificación de Ácido Nucleico , Plásmidos , Polimorfismo de Nucleótido Simple , Valor Predictivo de las Pruebas , Receptores de Estrógenos/análisis , Medición de Riesgo , Factores de Riesgo , Análisis de Secuencia de ADN , ValinaRESUMEN
We propose a new method for determining the target genes of transcriptional enhancers in specific cells and tissues. It combines global trends across many samples and sample-specific information, and considers the joint effect of multiple enhancers. Our method outperforms existing methods when predicting the target genes of enhancers in unseen samples, as evaluated by independent experimental data. Requiring few types of input data, we are able to apply our method to reconstruct the enhancer-target networks in 935 samples of human primary cells, tissues and cell lines, which constitute by far the largest set of enhancer-target networks. The similarity of these networks from different samples closely follows their cell and tissue lineages. We discover three major co-regulation modes of enhancers and find defense-related genes often simultaneously regulated by multiple enhancers bound by different transcription factors. We also identify differentially methylated enhancers in hepatocellular carcinoma (HCC) and experimentally confirm their altered regulation of HCC-related genes.
Asunto(s)
Elementos de Facilitación Genéticos , Epigénesis Genética , Regulación de la Expresión Génica , Redes Reguladoras de Genes/genética , Transcripción Genética , Carcinoma Hepatocelular/genética , Línea Celular , Metilación de ADN , Genes Relacionados con las Neoplasias , Humanos , Células K562 , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Fosfoproteínas/biosíntesis , Fosfoproteínas/genética , Cultivo Primario de Células , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/genética , Telomerasa/biosíntesis , Telomerasa/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The adenomatous polyposis coli (APC) tumor suppressor is a multifunctional regulator of Wnt signaling and acts as a mobile scaffold at different cellular sites. APC was recently found to stimulate microtubule (MT) growth at the interphase centrosome; however, little is known about its dynamics and localization at this site. To address this, we analysed APC dynamics in fixed and live cells by fluorescence microscopy. In detergent-extracted cells, we discovered that APC was only weakly retained at the centrosome during interphase suggesting a rapid rate of exchange. This was confirmed in living cells by fluorescence recovery after photobleaching (FRAP), which identified two pools of green fluorescent protein (GFP)-APC: a major rapidly exchanging pool (~86%) and minor retained pool (~14%). The dynamic exchange rate of APC was unaffected by C-terminal truncations implicating a targeting role for the N-terminus. Indeed, we mapped centrosome localization to N-terminal armadillo repeat (ARM) domain amino acids 334-625. Interestingly, the rate of APC movement to the centrosome was stimulated by intact MTs, and APC dynamics slowed when MTs were disrupted by nocodazole treatment or knockdown of γ-tubulin. Thus, the rate of APC recycling at the centrosome is enhanced by MT growth, suggesting a positive feedback to stimulate its role in MT growth.
RESUMEN
Non-alcoholic fatty liver disease (NAFLD), characterized by fat accumulation in liver, is closely associated with central obesity, over-nutrition and other features of metabolic syndrome, which elevate the risk of developing hepatocellular carcinoma (HCC). The Wnt/ß-catenin signaling pathway plays a significant role in the physiology and pathology of liver. Up to half of HCC patients have activation of Wnt/ß-catenin signaling. However, the mutation frequencies of CTNNB1 (encoding ß-catenin protein) or other antagonists targeting Wnt/ß-catenin signaling are low in HCC patients, suggesting that genetic mutations are not the major factor driving abnormal ß-catenin activities in HCC. Emerging evidence has demonstrated that obesity-induced metabolic pathways can deregulate chromatin modifiers such as histone deacetylase 8 to trigger undesired global epigenetic changes, thereby modifying gene expression program which contributes to oncogenic signaling. This review focuses on the aberrant epigenetic activation of Wnt/ß-catenin in the development of NAFLD-associated HCC. A deeper understanding of the molecular mechanisms underlying such deregulation may shed light on the identification of novel druggable epigenetic targets for the prevention and/or treatment of HCC in obese and diabetic patients.
RESUMEN
The flagellated protozoan Giardia intestinalis is one of the most prevalent human-infective parasites with a worldwide distribution. This parasite must encyst to complete the life cycle and N-acetylgalactosamine is produced from endogenous glucose for cyst wall synthesis during the transformation. UDP-N-acetylglucosamine pyrophosphorylase in G. intestinalis (GiUAP, EC 2.7.7.23) is the fourth enzyme in the inducible pathway of N-acetylgalactosamine biosynthesis, catalysing the conversion of N-acetylglucosamine-1-P to UDP-N-acetylglucosamine. In this study the gene GiUAP was cloned and sequenced from the Portland 1 strain using PCR techniques. It has an ORF of approximately 1.3 kb and contains no introns. BLAST and ClustalW analysis of the deduced amino acid sequence revealed significant similarities to other eukaryotic UAPs with putative active sites identified. Southern hybridization showed that GiUAP exists as a single-copy gene and it was shown to have two transcripts by RT-PCR and Northern hybridization. RLM-RACE identified both 5' and 3' untranslated regions and suggested the transcripts exist as a 5'-capped mRNA, with the use of two tandem polyadenylation sites to generate two unusually long giardial 3' untranslated regions of approximately 522 bp and approximately 3 kb. Moreover, a recombinant protein (rGiUAP) was expressed in E. coli and subjected to physical characterizations. Surprisingly the results obtained in this study were significantly different from those reported for the GiUAP in MR4 strain, suggesting this gene is under different transcription control in different strains of G. intestinalis. This report describes the molecular characterization of GiUAP and provides an opportunity to explore the control of gene expression during encystation of the parasite.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Giardia lamblia/enzimología , Nucleotidiltransferasas/genética , Sistemas de Lectura Abierta/fisiología , Proteínas Protozoarias/genética , Regiones no Traducidas 3'/genética , Regiones no Traducidas 5'/genética , Acetilgalactosamina/biosíntesis , Animales , Secuencia de Bases , Pared Celular/enzimología , Pared Celular/genética , Dosificación de Gen , Giardia lamblia/genética , Giardiasis/enzimología , Giardiasis/genética , Glucosa/metabolismo , Humanos , Intrones/genética , Datos de Secuencia Molecular , Nucleotidiltransferasas/metabolismo , Poliadenilación/fisiología , Reacción en Cadena de la Polimerasa/métodos , Proteínas Protozoarias/metabolismoRESUMEN
BARD1 is a breast cancer tumor suppressor with multiple domains and functions. BARD1 comprises a tandem BRCT domain at the C-terminus, and this sequence has been reported to target BARD1 to distinct subcellular locations such as nuclear DNA breakage sites and the centrosome through binding to regulatory proteins such as HP1 and OLA1, respectively. We now identify the BRCT domain as a binding site for p53. We first confirmed previous reports that endogenous BARD1 binds to p53 by immunoprecipitation assay, and further show that BARD1/p53 complexes locate at mitochondria suggesting a cellular location for p53 regulation of BARD1 apoptotic activity. We used a proximity ligation assay to map three distinct p53 binding sequences in human BARD1, ranging from weak (425-525) and modest (525-567) to strong (551-777 comprising BRCT domains). Deletion of the BRCT sequence caused major defects in the ability of BARD1 to (1) bind p53, (2) localize to the cytoplasm and mitochondria, and (3) induce Bax oligomerization and apoptosis. Our data suggest that BARD1 can move to mitochondria independent of p53, but subsequently associates with p53 to induce Bax clustering in part by decreasing mitochondrial Bcl-2 levels. We therefore identify a role for the BRCT domain in stimulating BARD1 nuclear export and mitochondrial localization, and in assembling mitochondrial BARD1/p53 complexes to regulate specific activities such as apoptotic function.
Asunto(s)
Apoptosis , Neoplasias de la Mama/metabolismo , Citoplasma/metabolismo , Mitocondrias/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Citoplasma/genética , Roturas del ADN , Femenino , Humanos , Células MCF-7 , Mitocondrias/genética , Mitocondrias/patología , Estructura Terciaria de Proteína , Transporte de Proteínas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Eliminación de Secuencia , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Rapid assembly of DNA damage response (DDR) proteins at nuclear "repair" foci is a hallmark response of ionizing radiation (IR)-treated cells. The ubiquitin E3 ligases RNF8 and RNF168 are critical for foci formation, and here we aim to determine their dynamic mobility and abundance at individual foci in living cells. To this end, YFP-tagged RNF8 and RNF168 were expressed at physiological levels in MCF-7 cells, then analyzed by fluorescence recovery after photobleaching (FRAP) assays, nuclear retention measurement, and virus-like particles (VLPs)-based quantification. The results showed that RNF8 and RNF168 were both highly dynamic at IR-induced foci. Intriguingly, RNF8 displayed remarkably faster in vivo association/dissociation rates than RNF168, and RNF8-positive IR-foci were less resistant to detergent extraction. In addition, copy number assay revealed that RNF168 was two-fold more abundant than RNF8 at foci. Collectively, we show for the first time that RNF8 moves on-and-off nuclear DNA repair foci more than six-fold as quickly as RNF168. The faster kinetics of RNF8 recruitment explains why RNF8 is generally observed at DNA-breaks prior to RNF168. Moreover, our finding that RNF8 is less abundant than RNF168 identifies RNF8 as a rate-limiting determinant of focal repair complex assembly.