RESUMEN
Hepatocellular carcinomas (HCCs) exhibit a diversity of molecular phenotypes, raising major challenges in clinical management. HCCs detected by surveillance programs at an early stage are candidates for potentially curative therapies (local ablation, resection, or transplantation). In the long term, transplantation provides the lowest recurrence rates. Treatment allocation is based on tumor number, size, vascular invasion, performance status, functional liver reserve, and the prediction of early (<2 years) recurrence, which reflects the intrinsic aggressiveness of the tumor. Well-differentiated, potentially low-aggressiveness tumors form the heterogeneous molecular class of nonproliferative HCCs, characterized by an approximate 50% ß-catenin mutation rate. To define the clinical, pathological, and molecular features and the outcome of nonproliferative HCCs, we constructed a 1,133-HCC transcriptomic metadata set and validated findings in a publically available 210-HCC RNA sequencing set. We show that nonproliferative HCCs preserve the zonation program that distributes metabolic functions along the portocentral axis in normal liver. More precisely, we identified two well-differentiated, nonproliferation subclasses, namely periportal-type (wild-type ß-catenin) and perivenous-type (mutant ß-catenin), which expressed negatively correlated gene networks. The new periportal-type subclass represented 29% of all HCCs; expressed a hepatocyte nuclear factor 4A-driven gene network, which was down-regulated in mouse hepatocyte nuclear factor 4A knockout mice; were early-stage tumors by Barcelona Clinic Liver Cancer, Cancer of the Liver Italian Program, and tumor-node-metastasis staging systems; had no macrovascular invasion; and showed the lowest metastasis-specific gene expression levels and TP53 mutation rates. Also, we identified an eight-gene periportal-type HCC signature, which was independently associated with the highest 2-year recurrence-free survival by multivariate analyses in two independent cohorts of 247 and 210 patients. CONCLUSION: Well-differentiated HCCs display mutually exclusive periportal or perivenous zonation programs. Among all HCCs, periportal-type tumors have the lowest intrinsic potential for early recurrence after curative resection. (Hepatology 2017;66:1502-1518).
Asunto(s)
Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Hígado/patología , Recurrencia Local de Neoplasia/patología , beta Catenina/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/cirugía , Francia/epidemiología , Factor Nuclear 4 del Hepatocito/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/cirugía , Mutación , Recurrencia Local de Neoplasia/genética , Fenotipo , TranscriptomaRESUMEN
BACKGROUND: Aberrant activation of the Wnt/ß-catenin pathway is a major and frequent event in liver cancer, but inhibition of oncogenic ß-catenin signaling has proven challenging. The identification of genes that are synthetically lethal in ß-catenin-activated cancer cells would provide new targets for therapeutic drug design. METHODS: We transfected the parental HuH6 hepatoblastoma cell line with a doxycycline-inducible shRNA against CTNNB1 (gene coding for ß-catenin) to obtain an isogenic cell line pair with or without aberrant ß-catenin signaling. Using this hepatoblastoma isogenic cell line pair, we performed a human kinome-wide siRNA screen to identify synthetic lethal interactions with oncogenic CTNNB1. The phenotypic readouts of the screen were cell proliferation, cell cycle arrest and apoptosis, which were assessed by image-based analysis. In addition, apoptosis was assessed by flow cytometric experiments and immunoblotting. The potential synthetic lethal relationship between candidates genes identified in the screen and oncogenic CTNNB1 was also investigated in a different cellular context, a colorectal HCT116 isogenic cell line pair. RESULTS: We first determined the experimental conditions that led to the efficient expression of shRNA against CTNNB1 and maximal reduction of ß-catenin signaling activity in response to doxycycline treatment. Following high throughput screening in which 687 genes coding for kinases and proteins related to kinases (such as pseudokinases and phosphatases) were targeted, we identified 52 genes required for HuH6 survival. The silencing of five of these genes selectively impaired the viability of HuH6 cells with high ß-catenin signaling: HGS, STRADA, FES, BRAF and PKMYT1. Among these candidates, HGS depletion had the strongest inhibitory effect on cell growth and led to apoptosis specifically in HuH6 with high ß-catenin activity, while HuH6 with low ß-catenin activity were spared. In addition, HGS was identified as a potential synthetic lethal partner of oncogenic CTNNB1 in the HCT116 colorectal isogenic cell line pair. CONCLUSIONS: These results demonstrate the existence of crosstalk between ß-catenin signaling and HGS. Importantly, HGS depletion specifically affected cells with uncontrolled ß-catenin signaling activity in two different types of cancer (Hepatoblastoma HuH6 and colorectal HCT116), and thus may represent a new potential target for novel therapeutic strategies in liver and colorectal cancer.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Hepatoblastoma/genética , Neoplasias Hepáticas/genética , Mutación , Fosfoproteínas/genética , ARN Interferente Pequeño/metabolismo , beta Catenina/antagonistas & inhibidores , Apoptosis , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Células HCT116 , Humanos , Fosfotransferasas/antagonistas & inhibidores , Vía de Señalización Wnt , beta Catenina/genéticaRESUMEN
ERK1/2 is required for certain forms of synaptic plasticity, including the long-term potentiation of synaptic strength. However, the molecular mechanisms regulating synaptically localized ERK1/2 signaling are poorly understood. Here, we show that the MAPK scaffold protein kinase suppressor of Ras 1 (KSR1) is directly phosphorylated by the downstream kinase ERK1/2. Quantitative Western blot analysis further demonstrates that expression of mutated, feedback-deficient KSR1 promotes sustained ERK1/2 activation in HEK293 cells in response to EGF stimulation, compared to a more transient activation in control cells expressing wild-type KSR1. Immunocytochemistry and confocal imaging of primary hippocampal neurons from newborn C57BL6 mice further show that feedback phosphorylation of KSR1 significantly reduces its localization to dendritic spines. This effect can be reversed by tetrodotoxin (1 µM) or PD184352 (2 µM) treatment, further suggesting that neuronal activity and phosphorylation by ERK1/2 lead to KSR1 removal from the postsynaptic compartment. Consequently, electrophysiological recordings in hippocampal neurons expressing wild-type or feedback-deficient KSR1 demonstrate that KSR1 feedback phosphorylation restricts the potentiation of excitatory postsynaptic currents. Our findings, therefore, suggest that feedback phosphorylation of the scaffold protein KSR1 prevents excessive ERK1/2 signaling in the postsynaptic compartment and thus contributes to maintaining physiological levels of synaptic excitability.
Asunto(s)
Plasticidad Neuronal/fisiología , Neuronas/fisiología , Proteínas Quinasas/metabolismo , Sinapsis/fisiología , Secuencia de Aminoácidos , Animales , Benzamidas/farmacología , Sitios de Unión/genética , Western Blotting , Células Cultivadas , Espinas Dendríticas/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Retroalimentación Fisiológica , Células HEK293 , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Datos de Secuencia Molecular , Mutación , Neuronas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Quinasas/genética , Homología de Secuencia de AminoácidoRESUMEN
ß-catenin is a key player in the regulation of gene expression during morphogenesis and tumorigenesis. Although its transactivation often results from stimulation of the Wnt signaling pathway, Wnt-independent regulation of ß-catenin has also been observed in cancer cells. This study discloses a new mechanism for the transactivation of ß-catenin upon EGF receptor activation that relies on the binding of ß-catenin to the PKM2 isoform in the nucleus. This interaction requires phosphorylation of ß-catenin on the Y333 residue by c-Src and the PKM2 domain that binds phosphotyrosine. Importantly, the authors demonstrated that EGF-induced transactivation of ß-catenin is necessary for brain tumor growth and that high levels of c-Src activity, Y333 ß-catenin phosphorylation and nuclear localization of PKM2 altogether correlate with high aggressiveness of tumors in glioblastoma multiforme. Remarkably, this study reveals a novel role for PKM2 in cancer cells where PKM2 appears to be, in addition to its established role in aerobic glycolysis, a major coactivator of ß-catenin transactivation. This nuclear function of PKM2 is shared with other transcription factors such as HIF-1α and OCT4, and highlights the nonmetabolic role of PKM2 during tumorigenesis.
RESUMEN
Biogenesis of iron-sulfur clusters (ISC) is essential to almost all forms of life and involves complex protein machineries. This process is initiated within the mitochondrial matrix by the ISC assembly machinery. Cohort and case report studies have linked mutations in ISC assembly machinery to severe mitochondrial diseases. The voltage-dependent anion channel (VDAC) located within the mitochondrial outer membrane regulates both cell metabolism and apoptosis. Recently, the C-terminal truncation of the VDAC1 isoform, termed VDAC1-ΔC, has been observed in chemoresistant late-stage tumor cells grown under hypoxic conditions with activation of the hypoxia-response nuclear factor HIF-1α. These cells harbored atypical enlarged mitochondria. Here, we show for the first time that depletion of several proteins of the mitochondrial ISC machinery in normoxia leads to a similar enlarged mitochondria phenotype associated with accumulation of VDAC1-ΔC. This truncated form of VDAC1 accumulates in the absence of HIF-1α and HIF-2α activations and confers cell resistance to drug-induced apoptosis. Furthermore, we show that when hypoxia and siRNA knock-down of the ISC machinery core components are coupled, the cell phenotype is further accentuated, with greater accumulation of VDAC1-ΔC. Interestingly, we show that hypoxia promotes the downregulation of several proteins (ISCU, NFS1, FXN) involved in the early steps of mitochondrial Fe-S cluster biogenesis. Finally, we have identified the mitochondria-associated membrane (MAM) localized Fe-S protein CISD2 as a link between ISC machinery downregulation and accumulation of anti-apoptotic VDAC1-ΔC. Our results are the first to associate dysfunction in Fe-S cluster biogenesis with cleavage of VDAC1, a form which has previously been shown to promote tumor resistance to chemotherapy, and raise new perspectives for targets in cancer therapy.
Asunto(s)
Resistencia a Antineoplásicos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hierro/metabolismo , Mitocondrias/metabolismo , Eliminación de Secuencia , Azufre/metabolismo , Canal Aniónico 1 Dependiente del Voltaje/metabolismo , Caspasa 3/metabolismo , Activación Enzimática/genética , Técnicas de Silenciamiento del Gen , Células HeLa , Células Hep G2 , Humanos , Mitocondrias/efectos de los fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteolisis/efectos de los fármacos , Hipoxia Tumoral/efectos de los fármacos , Canal Aniónico 1 Dependiente del Voltaje/genéticaRESUMEN
BACKGROUND: Fine tuning of the Wnt/ß-catenin signaling pathway is essential for the proper development and function of the liver. Aberrant activation of this pathway is observed in 20%-40% of hepatocellular carcinomas (HCC). Notum encodes a secreted Wnt deacylase that inhibits Wnt activity and thereby restricts the zone of activation of Wnt/ß-catenin signaling. An important role of NOTUM has been described in development in drosophila, planaria and zebrafish, but its role in the mammalian liver is unknown. Notum is required for spatial control of the Wnt/ß-catenin signaling in several animal models and the Wnt/ß-catenin pathway contributes to liver patterning involved in metabolic zonation. Therefore, Notum may be involved in the liver patterning induced by the Wnt/ß-catenin signaling during the adult stage. METHODOLOGY/PRINCIPAL FINDINGS: We generated a conditional Notum knockout mouse mutant to study the effect of the deletion of Notum in the liver. We show that Notum is a direct target of the Wnt/ß-catenin signaling in the liver. Liver-specific deletion of Notum did not modify liver zonation, but Notum deletion had a long-term effect on mouse physiology. In particular, male mutant mice developed metabolic disorders. CONCLUSION: We show that Notum is not a key actor of Wnt/ß-catenin-dependent liver patterning of adult mice, but has role in liver glucose homeostasis. Male mice deficient in Notum specifically in the liver develop metabolic dysfunctions implicating Notum in the development of Type 2 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2 , Esterasas/genética , Eliminación de Gen , Hepatocitos/enzimología , Hígado/enzimología , Vía de Señalización Wnt/genética , Animales , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/genética , Femenino , Masculino , Ratones , Ratones Mutantes , Especificidad de ÓrganosRESUMEN
About 20% hepatocellular carcinomas (HCCs) display wild-type ß-catenin, enhanced Wnt signaling, hepatocyte dedifferentiation and bad outcome, suggesting a specific impact of Wnt signals on HCC stem/progenitor cells. To study Wnt-specific molecular pathways, cell fates and clinical outcome, we fine-tuned Wnt/ß-catenin signaling in liver progenitor cells, using the prototypical Wnt ligand Wnt3a. Cell biology assays and transcriptomic profiling were performed in HepaRG hepatic progenitors exposed to Wnt3a after ß-catenin knockdown or Wnt inhibition with FZD8_CRD. Gene expression network, molecular pathology and survival analyses were performed on HCCs and matching non-tumor livers from 70 patients by real-time PCR and tissue micro-array-based immunohistochemistry. Wnt3a reprogrammed liver progenitors to replicating fibrogenic myofibroblast-like cells displaying stem and invasive features. Invasion was inhibited by 30 nM FZD7 and FZD8 CRDs. Translation of these data to human HCCs revealed two tight gene networks associating cell surface Wnt signaling, stem/progenitor markers and mesenchymal commitment. Both networks were linked by Hyaluronan And Proteoglycan Link Protein 1 (HAPLN1), that appeared de novo in aggressive HCCs expressing cytoplasmic ß-catenin and stem cell markers. HAPLN1 was independently associated with bad overall and disease-free outcome. In vitro, HAPLN1 was expressed de novo in EPCAM¯/NCAM+ mesoderm-committed progenitors, upon spontaneous epithelial-mesenchymal transition and de-differentiation of hepatocyte-like cells to liver progenitors. In these cells, HAPLN1 knockdown downregulated key markers of mesenchymal cells, such as Snail, LGR5, collagen IV and α-SMA. In conclusion, HAPLN1 reflects a signaling network leading to stemness, mesenchymal commitment and HCC progression.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/metabolismo , Mesodermo/metabolismo , Proteoglicanos/metabolismo , Células Madre/metabolismo , Proteínas Wnt/metabolismo , Anciano , Diferenciación Celular , Línea Celular , Línea Celular Tumoral , Análisis por Conglomerados , Proteínas de la Matriz Extracelular/genética , Femenino , Fibroblastos/metabolismo , Estudios de Seguimiento , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Ligandos , Hígado/metabolismo , Hígado/patología , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Proteoglicanos/genética , Transducción de Señal , Células Madre/citología , Análisis de Matrices TisularesRESUMEN
Hepatocellular adenomas (HCA) are benign liver tumors predominantly developed in women using oral contraceptives. Here, exome sequencing identified recurrent somatic FRK mutations that induce constitutive kinase activity, STAT3 activation, and cell proliferation sensitive to Src inhibitors. We also found uncommon recurrent mutations activating JAK1, gp130, or ß-catenin. Chromosome copy number and methylation profiling revealed patterns that correlated with specific gene mutations and tumor phenotypes. Finally, integrative analysis of HCAs transformed to hepatocellular carcinoma revealed ß-catenin mutation as an early alteration and TERT promoter mutations as associated with the last step of the adenoma-carcinoma transition. In conclusion, we identified the genomic diversity in benign hepatocyte proliferation, several therapeutic targets, and the key genomic determinants of the adenoma-carcinoma transformation sequence.
Asunto(s)
Adenoma de Células Hepáticas/genética , Carcinoma Hepatocelular/genética , Transformación Celular Neoplásica/genética , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/genética , Proteínas Tirosina Quinasas/genética , Adenoma de Células Hepáticas/enzimología , Adenoma de Células Hepáticas/patología , Animales , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Transformación Celular Neoplásica/patología , Metilación de ADN , Activación Enzimática , Perfilación de la Expresión Génica , Humanos , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Ratones , Mutación , Células 3T3 NIH , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factores de Riesgo , TransfecciónRESUMEN
Biogenesis of iron-sulfur (Fe-S) clusters in mammals involves a complex mitochondrial machinery that provides inorganic sulfide and iron for their assembly and insertion into apo-proteins. Mechanisms of Fe-S cluster assembly are just being unraveled, and regulation of the genes of this machinery remains unknown. In this study, we report that expression of two essential components of the Fe-S machinery, the cysteine desulfurase Nfs1 and its scaffold protein partner IscU, is down-regulated at both mRNA and protein levels when murine macrophages are physiologically stimulated with IFN-gamma and LPS. Regulation did not rely on cluster disassembly or NO production because exposure of cells to exogenous sources of NO did not alter Nfs1 expression, while it converted cytosolic Fe-S aconitase into its apo-form and because macrophages from NOS2 deficient mice displayed Nfs1 down-regulation. While IFN-gamma alone induced Nfs1 protein instability, LPS triggered a delayed decline of Nfs1, rather involving transcriptional events or mRNA instability. Also, the expression of IscU was down-regulated in IFN-gamma- and/or LPS-stimulated macrophages independently of NO, pointing to a general mechanism for marshalling the regulation of the Fe-S cluster assembly machinery in macrophages exposed to inflammatory stimuli.
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Liasas de Carbono-Azufre/metabolismo , Interferón gamma/administración & dosificación , Proteínas Hierro-Azufre/metabolismo , Lipopolisacáridos/administración & dosificación , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Animales , Liasas de Carbono-Azufre/administración & dosificación , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Ratones , Ratones Endogámicos C57BLRESUMEN
In prokaryotes and yeast, the general mechanism of biogenesis of iron-sulfur (Fe-S) clusters involves activities of several proteins among which IscS and Nfs1p provide, through cysteine desulfuration, elemental sulfide for Fe-S core formation. Although these proteins have been well characterized, the role of their mammalian homolog in Fe-S cluster biogenesis has never been evaluated. We report here the first functional study that implicates the putative cysteine desulfurase m-Nfs1 in the biogenesis of both mitochondrial and cytosolic mammalian Fe-S proteins. Depletion of m-Nfs1 in cultured fibroblasts through small interfering RNA-based gene silencing significantly inhibited the activities of mitochondrial NADH-ubiquinone oxidoreductase (complex I) and succinate-ubiquinone oxidoreductase (complex II) of the respiratory chain, as well as aconitase of the Krebs cycle, with no alteration in their protein levels. Activity of cytosolic xanthine oxidase, which holds a [2Fe-2S] cluster, was also specifically reduced, and iron-regulatory protein-1 was converted from its [4Fe-4S] aconitase form to its apo- or RNA-binding form. Reduction of Fe-S enzyme activities occurred earlier and more markedly in the cytosol than in mitochondria, suggesting that there is a mechanism that primarily dedicates m-Nfs1 to the biogenesis of mitochondrial Fe-S clusters in order to maintain cell survival. Finally, depletion of m-Nfs1, which conferred on apo-IRP-1 a high affinity for ferritin mRNA, was associated with the down-regulation of the iron storage protein ferritin.