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Diffuse midline gliomas are among the deadliest human cancers and have had little progress in treatment in the last 50 years. Cell cultures of these tumors have been developed recently, but the degree to which such cultures retain the characteristics of the source tumors is unknown. DNA methylation profiling offers a powerful tool to look at genome-wide epigenetic changes that are biologically meaningful and can help assess the similarity of cultured tumor cells to their in vivo progenitors. Paraffinized diagnostic tissue from three diffuse intrinsic pontine gliomas with H3 K27M mutations was compared with subsequent passages of neurosphere cell cultures from those tumors. Each cell line was passaged 3-4 times and analyzed with DNA methylation arrays and standard algorithms that provided a comparison of diagnostic classification and cluster analysis. All samples tested maintained high classifier scores and clustered within the reference group of H3 K27M-mutant diffuse midline gliomas. There was a gain of 1q in all cell lines, with two cell lines initially manifesting the gain of 1q only during culture. In vitro cell cultures of H3 K27M-mutant gliomas maintain high degrees of similarity in DNA methylation profiles to their source tumor, confirming their fidelity even with some chromosomal changes.
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Cholangiocarcinoma (CCA) is a highly lethal cancer arising from the biliary tract epithelium. The cancer biology of this neoplasm is not well understood. To date, only a few CCA cell lines have been reported, which were mostly developed from Asian patients. In this study, we report and characterize a new intrahepatic CCA cell line, LIV27, derived from a surgically resected tumor in a 67-year-old Caucasian woman with primary sclerosing cholangitis (PSC). LIV27 cells grow well in collagen-coated flasks or plates with a doubling time of 57.8 h at passage 14. LIV27 cells have high tumorigenicity in nude mice and stain positive for CK7 and CK19, markers that differentiate CCA from hepatocellular carcinoma. Karyotype analysis showed that LIV27 is aneuploid. We established a single-locus short tandem repeat profile for the LIV27 cell line. This newly established cell line will be a useful model for studying the molecular pathogenesis of, and developing novel therapies for, cholangiocarcinoma.
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Transforming growth factor-ß (TGF-ß) activates hepatic stellate cells (HSCs), which drive liver fibrosis via the production and deposition of extracellular matrix (ECM). We aimed to elucidate the mechanistic role of sulfatase-2 (SULF2) in liver fibrosis. To this end, we induced liver fibrosis in wild-type (WT) and SULF2 knockout (Sulf2-KO) mice (6-8 weeks-old) via bile duct ligation (BDL), intraperitoneal injection of carbon tetrachloride (CCl4) or thioacetamide (TAA). The levels of fibrosis in the liver sections were assessed via Sirius red and Masson's trichrome staining, immunohistochemistry and immunoblotting for α-smooth muscle actin (α-SMA) and hydroxyproline. To evaluate the interaction between TGF-ß and SULF2, we transfected human HSCs with scrambled control shRNA and shRNA constructs targeting SULF2 and measured α-SMA expression following treatment with TGF-ß1 ligand. We show here that knockout of SULF2 significantly decreases collagen content, as well as bands of bridging fibrosis, as demonstrated by Sirius red, Masson's trichrome and α-SMA staining after BDL, CCl4 and TAA injection in Sulf2-KO versus WT mice. In all three models of liver fibrosis, we observed significantly lower levels of hydroxyproline in the Sulf2-KO mice compared to the WT mice. HSCs with reduced levels of SULF2 failed to significantly express α-SMA and collagen type I following treatment with TGF-ß1. Furthermore, SULF2 co-localizes with TGFBR3 and the in vitro knockdown of SULF2 in HSCs decreases the release of TGF-ß1 from TGFBR3. Together, these data suggest that SULF2 regulates liver fibrosis via the TGF-ß signaling pathway. Pharmacologic inhibition of SULF2 may represent a novel therapeutic approach to improve liver fibrosis.
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Cholangiocarcinoma (CCA) has poor prognosis due to late-stage, symptomatic presentation. Altered DNA methylation markers may improve diagnosis of CCA. Reduced-representation bisulfite sequencing was performed on DNA extracted from frozen CCA tissues and matched to adjacent benign biliary epithelia or liver parenchyma. Methylated DNA markers (MDMs) identified from sequenced differentially methylated regions were selected for biological validation on DNA from independent formalin-fixed, paraffin-embedded CCA tumors and adjacent hepatobiliary control tissues using methylation-specific polymerase chain reaction. Selected MDMs were then blindly assayed on DNA extracted from independent archival biliary brushing specimens, including 12 perihilar cholangiocarcinoma, 4 distal cholangiocarcinoma cases, and 18 controls. Next, MDMs were blindly assayed on plasma DNA from patients with extrahepatic CCA (eCCA), including 54 perihilar CCA and 5 distal CCA cases and 95 healthy and 22 primary sclerosing cholangitis controls, balanced for age and sex. From more than 3,600 MDMs discovered in frozen tissues, 39 were tested in independent samples. In the clinical pilot of 16 MDMs on cytology brushings, methylated EMX1 (empty spiracles homeobox 1) had an area under the curve (AUC) of 0.98 (95% confidence interval [CI], 0.95-1.0). In the clinical pilot on plasma, a cross-validated recursive partitioning tree prediction model from nine MDMs was accurate for de novo eCCA (AUC, 0.88 [0.81-0.95]) but not for primary sclerosing cholangitis-associated eCCA (AUC, 0.54 [0.35-0.73]). Conclusion: Next-generation DNA sequencing yielded highly discriminant methylation markers for CCA. Confirmation of these findings in independent tissues, cytology brushings, and plasma supports further development of DNA methylation to augment diagnosis of CCA.
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Sulfatase 2 (SULF2) is a heparan sulfate editing enzyme that regulates the milieu of growth factors and cytokines involved in a variety of cellular processes. We used a murine model of diet-induced steatohepatitis to assess the effect of SULF2 downregulation on the development of nonalcoholic steatohepatitis (NASH) and liver fibrosis. Wild-type B6;129 mice (WT) and Sulf2-knockout B6;129P2-SULF2Gt(PST111)Byg mice (Sulf2-KO) were fed a fast-food diet (FFD) rich in saturated fats, cholesterol, and fructose or a standard chow diet (SC) ad libitum for 9 mo. WT mice on FFD showed a threefold increase in hepatic Sulf2 mRNA expression, and a 2.2-fold increase in hepatic SULF2 protein expression compared with WT mice on SC. Knockout of Sulf2 led to a significant decrease in diet-mediated weight gain and dyslipidemia compared with WT mice on FFD. Knockout of Sulf2 also abrogated diet-induced steatohepatitis and hepatic fibrosis compared with WT mice on FFD. Furthermore, expression levels of the profibrogenic receptors TGFßR2 and PDGFRß were significantly decreased in Sulf2-KO mice compared with WT mice on FFD. Together, our data suggest that knockout of Sulf2 significantly downregulates dyslipidemia, steatohepatitis, and hepatic fibrosis in a diet-induced mouse model of NAFLD, suggesting that targeting of SULF2 signaling may be a potential therapeutic mechanism in NASH.NEW & NOTEWORTHY We report for the first time that in wild-type (WT) mice, fast-food diet (FFD) induced a threefold increase in hepatic Sulf2 mRNA and a 2.2-fold increase in sulfatase 2 (SULF2) protein expression compared with WT mice on standard chow diet (SC). We showed that knockout of SULF2 ameliorates FFD-induced obesity, hyperlipidemia, steatohepatitis, and fibrosis. These data, along with work from other laboratories, suggest that SULF2 may be critical to the ability of the liver to progress to nonalcoholic steatohepatitis and fibrosis in conditions of overnutrition.
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Hígado Graso/genética , Hígado Graso/patología , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Sulfatasas/genética , Animales , Dieta Occidental , Regulación hacia Abajo , Dislipidemias/genética , Comida Rápida , Femenino , Resistencia a la Insulina , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , ARN Interferente Pequeño/genética , Aumento de Peso/genéticaRESUMEN
The expression of the extracellular sulfatase SULF2 has been associated with increased hepatocellular carcinoma (HCC) growth and poor patient survival. However, the molecular mechanisms underlying SULF2-associated tumor growth remain unclear. To address this gap, here we developed a transgenic mouse overexpressing Sulf2 in hepatocytes under the control of the transthyretin promoter. In this model, Sulf2 overexpression potentiated diethylnitrosamine-induced HCC. Further analysis indicated that the transcription factor GLI family zinc finger 1 (GLI1) mediates Sulf2 expression during HCC development. A cross of the Sulf2-overexpressing with Gli1-knockout mice revealed that Gli1 inactivation impairs SULF2-induced HCC. Transcriptomic analysis revealed that Sulf2 overexpression is associated with signal transducer and activator of transcription 3 (STAT3)-specific gene signatures. Interestingly, the Gli1 knockout abrogated SULF2-mediated induction of several STAT3 target genes, including suppressor of cytokine signaling 2/3 (Socs2/3); Pim-1 proto-oncogene, Ser/Thr kinase (Pim1); and Fms-related tyrosine kinase 4 (Flt4). Human orthologs were similarly regulated by SULF2, dependent on intact GLI1 and STAT3 functions in HCC cells. SULF2 overexpression promoted a GLI1-STAT3 interaction and increased GLI1 and STAT3 enrichment at the promoters of their target genes. Interestingly, the SULF2 overexpression resulted in GLI1 enrichment at select STAT3 consensus sites, and vice versa. siRNA-mediated STAT3 or GLI1 knockdown reduced promoter binding of GLI1 and STAT3, respectively. Finally, chromatin-capture PCR confirmed long-range co-regulation of SOCS2 and FLT3 through changes in promoter conformation. These findings define a mechanism whereby SULF2 drives HCC by stimulating formation of a GLI1-STAT3 transcriptional complex.
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Carcinoma Hepatocelular/etiología , Neoplasias Hepáticas/etiología , Factor de Transcripción STAT3/metabolismo , Sulfatasas/fisiología , Proteína con Dedos de Zinc GLI1/metabolismo , Animales , Carcinogénesis , Humanos , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Unión Proteica , Proto-Oncogenes Mas , Factores de Transcripción STAT , Sulfatasas/metabolismo , TransactivadoresRESUMEN
Autophagy has been shown to be a key cellular event controlling tumor growth in different neoplasms including hepatocellular carcinoma (HCC). Although this biological role of autophagy has been clearly established, the mechanism underlying its regulation remains elusive. Here, we demonstrate a role of sulfatase 2 (SULF2), a 6-O-endosulfatase modulating various growth factors and cytokine-related signaling pathways controlling tumor cell proliferation and survival, in the regulation of autophagy in HCC cells. SULF2 increased autophagosome formation, shown by increased LC3B-II protein and green fluorescent protein-LC3 puncta. Increased fusion between autophagosomes and lysosomes/lysosomal enzymes, higher expression of lysosomal membrane protein, and an increase in autolysosomes were also shown by western blot, immunofluorescence, and electron microscopy of SULF2-expressing cells, indicating enhanced autophagic flux. In contrast, RNA-interference silencing of SULF2 in Huh7 cells induced lysosomal membrane permeabilization with diffuse cytosolic staining of cathepsin D and punctate staining of galectin-3. Analysis of the mechanism showed that inhibition of lysosome-associated protein transmembrane 4 beta (LAPTM4B), a gene induced by SULF2, resulted in decreased autophagosome formation, decreased fusion between autophagosomes and lysosomes, and increased lysosomal membrane permeabilization. Interestingly, down-regulation of LAPTM4B also phenocopies the knockdown of SULF2, significantly reducing cell viability and colony formation. Conclusion: Our results demonstrate a role for SULF2 in the regulation of autophagic flux that is mediated through LAPTM4B induction in HCC cells, and provide a foundation for future translational efforts targeting autophagy in liver malignancies.
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PURPOSE: We investigated the antitumor effect of the casein kinase II (CK2) inhibitor CX-4945 on cholangiocarcinoma (CCA). METHODS: We assessed the effect of CX-4945 alone and/or in combination with gemcitabine and cisplatin on cell viability, colony formation, and apoptosis of CCA cell lines and on in vivo growth of HuCCT1 xenografts. RESULTS: CX-4945 dose-dependently decreased viability of HuCCT1, EGI-1, and Liv27 and decreased phospho-AKT/total AKT and phospho-PTEN/total PTEN ratios. CX-4945 significantly increased caspase 3/7 activity in a dose- and time-dependent manner. CX-4945 significantly enhanced the effect of gemcitabine or cisplatin on HuCCT1, EGI-1, and Liv27 cells and inhibited the phosphorylation of DNA repairing enzymes XRCC1 and MDC1. Further, CX-4945 alone significantly inhibited growth of HuCCT1 mouse xenograft tumors. Combining CX-4945 with gemcitabine and cisplatin was more potent than CX-4945 alone or gemcitabine/cisplatin. The effect of CX-4945 on cell proliferation, apoptosis, the PI3K/AKT pathway, and DNA repair was confirmed in the mouse xenografts. CONCLUSION: CX-4945 has an antiproliferative effect on CCA and enhances the effect of gemcitabine and cisplatin through its inhibitory effect on the PI3K/AKT pathway and DNA repair.
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BACKGROUND: The GALAD score is a serum biomarker-based model that predicts the probability of having hepatocellular carcinoma (HCC) in patients with chronic liver disease. We aimed to assess the performance of the GALAD score in comparison with liver ultrasound for detection of HCC. METHODS: A single-center cohort of 111 HCC patients and 180 controls with cirrhosis or chronic hepatitis B and a multicenter cohort of 233 early HCC and 412 cirrhosis patients from the Early Detection Research Network (EDRN) phase II HCC Study were analyzed. RESULTS: The area under the ROC curve (AUC) of the GALAD score for HCC detection was 0.95 [95% confidence interval (CI), 0.93-97], which was higher than the AUC of ultrasound (0.82, P <0.01). At a cutoff of -0.76, the GALAD score had a sensitivity of 91% and a specificity of 85% for HCC detection. The AUC of the GALAD score for early-stage HCC detection remained high at 0.92 (95% CI, 0.88-0.96; cutoff -1.18, sensitivity 92%, specificity 79%). The AUC of the GALAD score for HCC detection was 0.88 (95% CI, 0.85-0.91) in the EDRN cohort. The combination of GALAD and ultrasound (GALADUS score) further improved the performance of the GALAD score in the single-center cohort, achieving an AUC of 0.98 (95% CI, 0.96-0.99; cutoff -0.18, sensitivity 95%, specificity 91%). CONCLUSIONS: The performance of the GALAD score was superior to ultrasound for HCC detection. The GALADUS score further enhanced the performance of the GALAD score. IMPACT: The GALAD score was validated in the United States.
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Biomarcadores de Tumor/sangre , Carcinoma Hepatocelular/diagnóstico , Detección Precoz del Cáncer/métodos , Neoplasias Hepáticas/diagnóstico , Ultrasonografía/métodos , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/diagnóstico por imagen , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Humanos , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Pronóstico , Curva ROCRESUMEN
PURPOSE: Cholangiocarcinoma (CCA) is a malignancy of the bile ducts. The purpose of this discovery study was to identify effective serum markers for surveillance of cholangiocarcinoma. EXPERIMENTAL DESIGN: Using a glycomic method, patients with CCA were determined to have increased levels of alpha-1,3 and alpha-1,6 linked fucosylated glycan. Proteomic analysis of the serum fucosylated proteome identified proteins such as alpha-2-macroglobulin, kininogen, hemopexin, fetuin-A, alpha-1 anti-trypsin, and ceruloplasmin as being hyperfucosylated in HCC. The levels of these glycoproteins in 109 patients with CCA, primary sclerosing cholangitis (PSC), and control patients were compared to the performance of CA-19-9, the current "gold standard" assay for cholangiocarcinoma. RESULTS: Fucosylated Fetuin-A (fc-Fetuin-A) had the best ability to differentiate CCA from PSC, with an AUROC of 0.812 or 0.8665 at differentiating CCA from those with PSC or other liver disease. CA-19-9 had poor ability to differentiate PSC from cholangiocarcinoma (AUROC of 0.625). CONCLUSION AND CLINICAL RELEVANCE: Using glycomic and proteomic methods we identified a set of proteins that contain altered glycan in the sera of those with CCA. One of these proteins, fucosylated Fetuin-A may have value in the surveillance of people at risk for the development of cholangiocarcinoma.
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Neoplasias de los Conductos Biliares/metabolismo , Biomarcadores de Tumor/metabolismo , Colangiocarcinoma/metabolismo , Fucosa/metabolismo , Proteómica , alfa-2-Glicoproteína-HS/metabolismo , Neoplasias de los Conductos Biliares/sangre , Neoplasias de los Conductos Biliares/diagnóstico , Colangiocarcinoma/sangre , Colangiocarcinoma/diagnóstico , Femenino , Fucosa/sangre , Glicosilación , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Existing antiangiogenic approaches to treat metastatic hepatocellular carcinoma (HCC) are weakly effectual, prompting further study of tumor angiogenesis in this disease setting. Here, we report a novel role for sulfatase 2 (SULF2) in driving HCC angiogenesis. Sulf2-deficient mice (Sulf2 KO) exhibited resistance to diethylnitrosamine-induced HCC and did not develop metastases like wild-type mice (Sulf2 WT). The smaller and less numerous tumors formed in Sulf2 KO mice exhibited a markedly lower microvascular density. In human HCC cells, SULF2 overexpression increased endothelial proliferation, adhesion, chemotaxis, and tube formation in a paracrine fashion. Mechanistic analyses identified the extracellular matrix protein periostin (POSTN), a ligand of αvß3/5 integrins, as an effector protein in SULF2-induced angiogenesis. POSTN silencing in HCC cells attenuated SULF2-induced angiogenesis and tumor growth in vivo The TGFß1/SMAD pathway was identified as a critical signaling axis between SULF2 and upregulation of POSTN transcription. In clinical HCC specimens, elevated levels of SULF2 correlated with increased microvascular density, POSTN levels, and relatively poorer patient survival. Together, our findings define an important axis controlling angiogenesis in HCC and a mechanistic foundation for rational drug development. Cancer Res; 77(3); 632-45. ©2016 AACR.
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Carcinoma Hepatocelular/patología , Moléculas de Adhesión Celular/biosíntesis , Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias Hepáticas/patología , Neovascularización Patológica/patología , Animales , Biomarcadores de Tumor/análisis , Western Blotting , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidad , Inmunoprecipitación de Cromatina , Ensayo de Inmunoadsorción Enzimática , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidad , Ratones , Ratones Noqueados , Neovascularización Patológica/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/fisiología , Proteínas Smad/metabolismo , Sulfatasas , Sulfotransferasas/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
BACKGROUND: The human sulfatase 1 (SULF1) and sulfatase 2 (SULF2) genes modulate cell signaling and homeostasis in many tissues. Gene expression analyses have implicated SULF2 in disease pathogenesis, including Alzheimer's disease (AD), but changes in brain SULF2 expression have not been directly established. OBJECTIVE: To investigate the expression of SULF1 and SULF2 in brain tissues from AD cases and cognitively normal controls. METHODS: Autopsy tissue from AD cases (n = 20) and age-and gender-matched cognitively normal controls (n = 20) were identified from the Mayo Clinic Alzheimer's Disease Patient Registry neuropathology database. Tissue slides were stained for SULF1 and SULF2 protein expression in the hippocampus and frontal lobe and an expression score computed from the proportion of cells stained and the intensity of staining (range 0 [no expression] to 9 [marked expression]). RESULTS: SULF2 expression was reduced in AD cases. Compared to cognitively normal controls, SULF2 expression in AD cases was significantly decreased in the hippocampal Cornu Ammonis (CA) (mean score of 6.5 in cases versus 8.3 in controls; p = 0.003), in the gray matter of the parahippocampal gyrus (5.6 in cases versus 7.6 in controls; p = 0.003), and in the frontal lobe gray matter (5.4 in cases versus 7.4 in controls; p = 0.002). There was no difference in SULF1 expression in the hippocampus or frontal lobe of AD cases and controls. As expected there were no differences in SULF1 or SULF2 expression in white matter in AD cases compared to cognitively normal controls. CONCLUSION: Decresed SULF2 in specific regions of the brain occurs in AD.
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Cholangiocarcinoma is a highly lethal cancer with limited therapeutic options. Recent genomic analysis of cholangiocarcinoma has revealed the presence of fibroblast growth factor receptor 2 (FGFR2) fusion proteins in up to 13% of intrahepatic cholangiocarcinoma (iCCA). FGFR fusions have been identified as a novel oncogenic and druggable target in a number of cancers. In this study, we established a novel cholangiocarcinoma patient derived xenograft (PDX) mouse model bearing an FGFR2-CCDC6 fusion protein from a metastatic lung nodule of an iCCA patient. Using this PDX model, we confirmed the ability of the FGFR inhibitors, ponatinib, dovitinib and BGJ398, to modulate FGFR signaling, inhibit cell proliferation and induce cell apoptosis in cholangiocarcinoma tumors harboring FGFR2 fusions. In addition, BGJ398 appeared to be superior in potency to ponatinib and dovitinib in this model. Our findings provide a strong rationale for the investigation of FGFR inhibitors, particularly BGJ398, as a therapeutic option for cholangiocarcinoma patients harboring FGFR2 fusions.
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Antineoplásicos/farmacología , Neoplasias de los Conductos Biliares/tratamiento farmacológico , Colangiocarcinoma/tratamiento farmacológico , Proteínas del Citoesqueleto/metabolismo , Fusión Génica , Neoplasias Pulmonares/tratamiento farmacológico , Compuestos de Fenilurea/farmacología , Pirimidinas/farmacología , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Bencimidazoles/farmacología , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/metabolismo , Neoplasias de los Conductos Biliares/patología , Proliferación Celular/efectos de los fármacos , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Colangiocarcinoma/secundario , Proteínas del Citoesqueleto/genética , Humanos , Imidazoles/farmacología , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Piridazinas/farmacología , Quinolonas/farmacología , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Sphingosine kinase 2 (Sphk2) has an oncogenic role in cancer. A recently developed first-in-class Sphk2 specific inhibitor ABC294640 displays antitumor activity in many cancer models. However, the role of Sphk2 and the antitumor activity of its inhibitor ABC294640 are not known in cholangiocarcinoma. We investigated the potential of targeting Sphk2 for the treatment of cholangiocarcinoma. We found that Sphk2 is overexpressed in five established human cholangiocarcinoma cell lines (WITT, HuCCT1, EGI-1, OZ and HuH28) and a new patient-derived cholangiocarcinoma cell line (LIV27) compared to H69 normal cholangiocytes. Inhibition of Sphk2 by ABC294640 inhibited proliferation and induced caspase-dependent apoptosis. Furthermore, we found that ABC294640 inhibited STAT3 phosphorylation, one of the key signaling pathways regulating cholangiocarcinoma cell proliferation and survival. ABC294640 also induced autophagy. Inhibition of autophagy by bafilomycin A1 or chloroquine potentiated ABC294640-induced cytotoxicity and apoptosis. In addition, ABC294640 in combination with sorafenib synergistically inhibited cell proliferation of cholangiocarcinoma cells. Strong decreases in STAT3 phosphorylation were observed in WITT and HuCCT1 cells exposed to the ABC294640 and sorafenib combination. These findings provide novel evidence that Sphk2 may be a rational therapeutic target in cholangiocarcinoma. Combinations of ABC294640 with sorafenib and/or autophagy inhibitors may provide novel strategies for the treatment of cholangiocarcinoma.
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Adamantano/análogos & derivados , Autofagia/efectos de los fármacos , Neoplasias de los Conductos Biliares/patología , Colangiocarcinoma/patología , Niacinamida/análogos & derivados , Compuestos de Fenilurea/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Piridinas/farmacología , Adamantano/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de los Conductos Biliares/tratamiento farmacológico , Neoplasias de los Conductos Biliares/metabolismo , Proliferación Celular/efectos de los fármacos , Colangiocarcinoma/tratamiento farmacológico , Colangiocarcinoma/metabolismo , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Niacinamida/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Sorafenib , Células Tumorales CultivadasRESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC) is the second most frequent cause of cancer death worldwide. Sulfatase 1 (SULF1) functions as a tumor suppressor in HCC cell lines in vitro, but also has an oncogenic effect in some HCCs in vivo. AIM: To examine the mechanisms regulating SULF1 and its function in HCC. METHODS: First, SULF1 mRNA and protein expression were examined. Second, we examined SULF1 gene copy number in HCC cells. Third, we assessed whether DNA methylation or methylation and/or acetylation of histone marks on the promoter regulate SULF1 expression. Finally, we examined the effect of 5-Aza-dC on sulfatase activity and drug-induced apoptosis. RESULTS: SULF1 mRNA was down-regulated in 9/11 HCC cell lines but only 6/10 primary tumors. SULF1 mRNA correlated with protein expression. Gene copy number assessment by fluorescence in situ hybridization showed intact SULF1 alleles in low SULF1 expressing cell lines. CpG island methylation in the SULF1 promoter and two downstream CpG islands did not show an inverse correlation between DNA methylation and SULF1 expression. However, chromatin immunoprecipitation showed that the SULF1 promoter acquires a silenced chromatin state in low SULF1-expressing cells through an increase in di/trimethyl-K9H3 and trimethyl-K27H3 and a concomitant loss of activating acetyl K9, K14H3 marks. 5-Aza-dC restored SULF1 mRNA expression in SULF1-negative cell lines, with an associated increase in sulfatase activity and sensitization of HCC cells to cisplatin-induced apoptosis. CONCLUSION: SULF1 gene silencing in HCC occurs through histone modifications on the SULF1 promoter. Restoration of SULF1 mRNA expression by 5-Aza-dC sensitized HCC cells to drug-induced apoptosis.
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Genetic risk factors for cholangiocarcinoma (CCA) remain poorly understood. We assessed the effect of single-nucleotide polymorphisms (SNPs) of genes modulating inflammation or carcinogenesis on CCA risk and survival. We conducted a case-control, candidate gene association study of 370 CCA patients and 740 age-, sex-, and residential area-matched healthy controls. Eighteen functional or putatively functional SNPs in nine genes were genotyped. The log-additive genotype effects of SNPs on CCA risk and survival were determined using logistic regression and the log-rank test, respectively. Initial analysis identified significant associations between SNP rs2143417 and rs689466 in cyclooxygenase 2 (COX-2) and CCA risk, after adjusting for multiple comparisons (cutoff of P = 0.0028). However, these findings were not replicated in another independent cohort of 212 CCA cases and 424 matched controls. No significant association was found between any SNP and survival of CCA patients. Although COX-2 has been shown to contribute to cholangiocarcinogenesis, the COX-2 SNPs tested were not associated with risk of CCA. This study shows a lack of association between variants of genes related to inflammation and carcinogenesis and CCA risk and survival. Other factors than these genetic variants may play more important roles in CCA risk and survival.
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Neoplasias de los Conductos Biliares/epidemiología , Carcinogénesis/genética , Colangiocarcinoma/epidemiología , Inflamación/genética , Anciano , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/mortalidad , Estudios de Casos y Controles , Colangiocarcinoma/genética , Colangiocarcinoma/mortalidad , Colangitis Esclerosante/genética , Ciclooxigenasa 2/genética , Citocinas/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Polimorfismo de Nucleótido Simple , Factores de RiesgoRESUMEN
Growing evidence suggests that pretransplant alpha-fetoprotein (AFP) predicts outcomes of hepatocellular carcinoma (HCC) patients treated with liver transplantation. We aimed to determine whether pretransplant AFP, Lens culinaris agglutinin-reactive alpha-fetoprotein (AFP-L3), and des-gamma-carboxyprothrombin (DCP) predicted HCC recurrence after transplantation. A retrospective cohort study of 313 HCC patients undergoing transplantation between 2000 and 2008 was conducted, and 48 (15.3%) developed recurrence during a median follow-up of 90.8 months. The 127 patients with available serum drawn before transplantation were included; they included 86 without recurrence and 41 with recurrence. Serum was tested for AFP, AFP-L3%, and DCP in a blinded fashion with the µTASWako i30 immunoanalyzer. All biomarkers were significantly associated with HCC recurrence. The hazard ratios (HRs) were 3.5 [95% confidence interval (CI), 1.9-6.7; P < 0.0001] for DCP ≥ 7.5 ng/mL and 2.8 (95% CI, 1.4-5.4; P = 0.002) for AFP ≥ 250 ng/mL. The HR increased to 5.2 (95% CI, 2.3-12.0; P < 0.0001) when AFP ≥ 250 ng/mL and DCP ≥7.5 ng/mL were considered together. When they were combined with the Milan criteria, the HR increased from 2.6 (95% CI, 1.4-4.7; P = 0.003) for outside the Milan criteria to 8.6 (95% CI, 3.0-24.6; P < 0.0001) for outside the Milan criteria and AFP ≥ 250 ng/mL and to 7.2 (95% CI, 2.8-18.1; P < 0.0001) for outside the Milan criteria and DCP ≥7.5 ng/mL. Our findings suggest that biomarkers are useful for predicting the risk of HCC recurrence after transplantation. Using both biomarkers and the Milan criteria may be better than using the Milan criteria alone in optimizing the decision of liver transplantation eligibility.
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Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/cirugía , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/cirugía , Trasplante de Hígado/métodos , Recurrencia Local de Neoplasia/diagnóstico , Anciano , Biomarcadores/metabolismo , Carcinoma Hepatocelular/patología , Estudios de Cohortes , Femenino , Humanos , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/patología , Lectinas de Plantas/química , Modelos de Riesgos Proporcionales , Precursores de Proteínas/metabolismo , Protrombina/metabolismo , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Transducción de Señal , Tomografía Computarizada por Rayos X/métodos , alfa-Fetoproteínas/metabolismoRESUMEN
UNLABELLED: In vitro studies have proposed a tumor suppressor role for sulfatase 1 (SULF1) in hepatocellular carcinoma (HCC); however, high expression in human HCC has been associated with poor prognosis. The reason underlying this paradoxical observation remains to be explored. Using a transgenic (Tg) mouse model overexpressing Sulf1 (Sulf1-Tg), we assessed the effects of SULF1 on the diethylnitrosamine model of liver carcinogenesis. Sulf1-Tg mice show a higher incidence of large and multifocal tumors with diethylnitrosamine injection compared to wild-type mice. Lung metastases were found in 75% of Sulf1-Tg mice but not in wild-type mice. Immunohistochemistry, immunoblotting, and reporter assays all show a significant activation of the transforming growth factor-ß (TGF-ß)/SMAD transcriptional pathway by SULF1 both in vitro and in vivo. This effect of SULF1 on the TGF-ß/SMAD pathway is functional; overexpression of SULF1 promotes TGF-ß-induced gene expression and epithelial-mesenchymal transition and enhances cell migration/invasiveness. Mechanistic analyses demonstrate that inactivating mutation of the catalytic site of SULF1 impairs the above actions of SULF1 and diminishes the release of TGF-ß from the cell surface. We also show that SULF1 expression decreases the interaction between TGF-ß1 and its heparan sulfate proteoglycan sequestration receptor, TGFßR3. Finally, using gene expression from human HCCs, we show that patients with high SULF1 expression have poorer recurrence-free survival (hazard ratio 4.1, 95% confidence interval 1.9-8.3; P = 0.002) compared to patients with low SULF1. We also found strong correlations of SULF1 expression with TGF-ß expression and with several TGF-ß-related epithelial-mesenchymal transition genes in human HCC. CONCLUSION: Our study proposes a novel role of SULF1 in HCC tumor progression through augmentation of the TGF-ß pathway, thus defining SULF1 as a potential biomarker for tumor progression and a novel target for drug development for HCC.
Asunto(s)
Carcinoma Hepatocelular/etiología , Neoplasias Hepáticas/etiología , Proteínas Smad/fisiología , Sulfotransferasas/fisiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Masculino , Ratones , Activación TranscripcionalRESUMEN
BACKGROUND AND AIMS: Brivanib is a selective inhibitor of vascular endothelial growth factor receptor (VEGFR) and fibroblast growth factor receptor (FGFR) tyrosine kinases, which are both involved in mechanisms of liver fibrosis. We hypothesized that inhibition of VEGFR and FGFR by brivanib would inhibit liver fibrosis. We therefore examined the effect of brivanib on liver fibrosis in three mouse models of fibrosis. METHODS: In vivo, we induced liver fibrosis by bile duct ligation (BDL), chronic carbon tetrachloride (CCl4), and chronic thioacetamide (TAA) administration. Liver fibrosis was examined by immunohistochemistry and Western immunoblotting. In vitro, we used LX-2 human hepatic stellate cells (HSCs) to assess the effect of brivanib on stellate cell proliferation and activation. RESULTS: After in vivo induction with BDL, CCl4, and TAA, mice treated with brivanib showed reduced liver fibrosis and decreased expression of collagen Iα1 and α-smooth muscle actin in the liver. In vitro, brivanib decreased proliferation of HSCs induced by platelet-derived growth factor (PDGF), VEGF, and FGF. Brivanib also decreased stellate cell viability and inhibited PDGFBB-induced phosphorylation of its cognate receptor. CONCLUSION: Brivanib reduces liver fibrosis in three different animal models and decreases human hepatic stellate cell activation. Brivanib may represent a novel therapeutic approach to treatment of liver fibrosis and prevention of liver cancer.