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Josep M Llovet - One of the best experts on this subject based on the ideXlab platform.
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genome wide methylation analysis and epigenetic unmasking identify tumor suppressor genes in hepatocellular carcinoma
Gastroenterology, 2013Co-Authors: Kate Revill, Timothy C Wang, Anja Lachenmayer, Kensuke Kojima, Andrew N Harrington, Yujin Hoshida, Josep M LlovetAbstract:Background & Aims Epigenetic silencing of tumor suppressor genes contributes to the pathogenesis of hepatocellular carcinoma (HCC). To identify clinically relevant tumor suppressor genes silenced by DNA methylation in HCC, we integrated DNA methylation data from human primary HCC samples with data on up-regulation of gene expression after epigenetic unmasking. Methods We performed genome-wide methylation analysis of 71 human HCC samples using the Illumina HumanBeadchip27K array; data were combined with those from microarray analysis of gene re-expression in 4 liver cancer cell lines after their exposure to reagents that reverse DNA methylation (epigenetic unmasking). Results Based on DNA methylation in primary HCC and gene re-expression in cell lines after epigenetic unmasking, we identified 13 candidate tumor suppressor genes. Subsequent validation led us to focus on functionally characterizing 2 candidates, sphingomyelin phosphodiesterase 3 ( SMPD3 ) and neurofilament, heavy polypeptide ( NEFH ), which we found to behave as tumor suppressor genes in HCC. Overexpression of SMPD3 and NEFH by stable transfection of inducible constructs into an HCC cell line reduced cell proliferation by 50% and 20%, respectively (SMPD3, P = .003 and NEFH, P = .003). Conversely, knocking down expression of these genes with small hairpin RNA promoted cell invasion and migration in vitro (SMPD3, P = .0001 and NEFH, P = .022), and increased their ability to form tumors after subcutaneous injection or orthotopic transplantation into mice, confirming their role as tumor suppressor genes in HCC. Low levels of SMPD3 were associated with early recurrence of HCC after curative surgery in an independent patient cohort ( P = .001; hazard ratio = 3.22; 95% confidence interval: 1.6−6.5 in multivariate analysis). Conclusions Integrative genomic analysis identified SMPD3 and NEFH as tumor suppressor genes in HCC. We provide evidence that SMPD3 is a potent tumor suppressor gene that could affect tumor aggressiveness; a reduced level of SMPD3 is an independent prognostic factor for early recurrence of HCC.
Bing Gao - One of the best experts on this subject based on the ideXlab platform.
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Article Identification of Site-Specific Stroke Biomarker Candidates by Laser Capture Microdissection and Labeled Reference Peptide
2016Co-Authors: Tingting Lian, Xu Zhao, Bing GaoAbstract:Abstract: The search to date for accurate protein biomarkers in acute ischemic stroke has taken into consideration the stage and/or the size of infarction, but has not accounted for the site of stroke. In the present study, multiple reaction monitoring using labeled reference peptide (LRP) following laser capture microdissection (LCM) is used to identify site-specific protein biomarker candidates. In middle cerebral artery occlusion (MCAO) rat models, both intact and infarcted brain tissue was collected by LCM, followed by on-film digestion and semi-quantification using triple-quadrupole mass spectrometry. Thirty-four unique peptides were detected for the verification of 12 proteins in both tissue homogenates and LCM-captured samples. Six insoluble proteins, including neurofilament light polypeptide (NEFL), alpha-internexin (INA), microtubule-associated protein 2 (MAP2), myelin basic protein (MBP), myelin proteolipid protein (PLP) and 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNP), were found to be site-specific. Soluble proteins, such as neuron-specific enolase (NSE) and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), and some insoluble proteins, including neurofilament heavy polypeptide (NEFH), glial fibrillary acidic protein (GFAP), microtubule-associated protein ta
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identification of site specific stroke biomarker candidates by laser capture microdissection and labeled reference peptide
International Journal of Molecular Sciences, 2015Co-Authors: Tingting Lian, Xu Zhao, Bing GaoAbstract:The search to date for accurate protein biomarkers in acute ischemic stroke has taken into consideration the stage and/or the size of infarction, but has not accounted for the site of stroke. In the present study, multiple reaction monitoring using labeled reference peptide (LRP) following laser capture microdissection (LCM) is used to identify site-specific protein biomarker candidates. In middle cerebral artery occlusion (MCAO) rat models, both intact and infarcted brain tissue was collected by LCM, followed by on-film digestion and semi-quantification using triple-quadrupole mass spectrometry. Thirty-four unique peptides were detected for the verification of 12 proteins in both tissue homogenates and LCM-captured samples. Six insoluble proteins, including neurofilament light polypeptide (NEFL), alpha-internexin (INA), microtubule-associated protein 2 (MAP2), myelin basic protein (MBP), myelin proteolipid protein (PLP) and 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNP), were found to be site-specific. Soluble proteins, such as neuron-specific enolase (NSE) and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), and some insoluble proteins, including neurofilament heavy polypeptide (NEFH), glial fibrillary acidic protein (GFAP), microtubule-associated protein tau (MAPT) and tubulin β-3 chain (TUBB3), were found to be evenly distributed in the brain. Therefore, we conclude that some insoluble protein biomarkers for stroke are site-specific, and would make excellent candidates for the design and analysis of relevant clinical studies in the future.
Kate Revill - One of the best experts on this subject based on the ideXlab platform.
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genome wide methylation analysis and epigenetic unmasking identify tumor suppressor genes in hepatocellular carcinoma
Gastroenterology, 2013Co-Authors: Kate Revill, Timothy C Wang, Anja Lachenmayer, Kensuke Kojima, Andrew N Harrington, Yujin Hoshida, Josep M LlovetAbstract:Background & Aims Epigenetic silencing of tumor suppressor genes contributes to the pathogenesis of hepatocellular carcinoma (HCC). To identify clinically relevant tumor suppressor genes silenced by DNA methylation in HCC, we integrated DNA methylation data from human primary HCC samples with data on up-regulation of gene expression after epigenetic unmasking. Methods We performed genome-wide methylation analysis of 71 human HCC samples using the Illumina HumanBeadchip27K array; data were combined with those from microarray analysis of gene re-expression in 4 liver cancer cell lines after their exposure to reagents that reverse DNA methylation (epigenetic unmasking). Results Based on DNA methylation in primary HCC and gene re-expression in cell lines after epigenetic unmasking, we identified 13 candidate tumor suppressor genes. Subsequent validation led us to focus on functionally characterizing 2 candidates, sphingomyelin phosphodiesterase 3 ( SMPD3 ) and neurofilament, heavy polypeptide ( NEFH ), which we found to behave as tumor suppressor genes in HCC. Overexpression of SMPD3 and NEFH by stable transfection of inducible constructs into an HCC cell line reduced cell proliferation by 50% and 20%, respectively (SMPD3, P = .003 and NEFH, P = .003). Conversely, knocking down expression of these genes with small hairpin RNA promoted cell invasion and migration in vitro (SMPD3, P = .0001 and NEFH, P = .022), and increased their ability to form tumors after subcutaneous injection or orthotopic transplantation into mice, confirming their role as tumor suppressor genes in HCC. Low levels of SMPD3 were associated with early recurrence of HCC after curative surgery in an independent patient cohort ( P = .001; hazard ratio = 3.22; 95% confidence interval: 1.6−6.5 in multivariate analysis). Conclusions Integrative genomic analysis identified SMPD3 and NEFH as tumor suppressor genes in HCC. We provide evidence that SMPD3 is a potent tumor suppressor gene that could affect tumor aggressiveness; a reduced level of SMPD3 is an independent prognostic factor for early recurrence of HCC.
Adriana Rebelo - One of the best experts on this subject based on the ideXlab platform.
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Cryptic amyloidogenic elements in mutant NEFH causing Charcot-Marie-Tooth 2 trigger aggresome formation and neuronal death
Acta Neuropathologica Communications, 2017Co-Authors: Arnaud Jacquier, Raoul Juntas-morales, Marianne Giroux, Valérie Castellani, Cecile Delorme, Odile Dubourg, Edwige Belotti, Guilhem Sole, Claude-alain Maurage, Adriana RebeloAbstract:Neurofilament heavy chain (NEFH) gene was recently identified to cause autosomal dominant axonal Charcot-Marie-Tooth disease (CMT2cc). However, the clinical spectrum of this condition and the physio-pathological pathway remain to be delineated. We report 12 patients from two French families with axonal dominantly inherited form of CMT caused by two new mutations in the NEFH gene. A remarkable feature was the early involvement of proximal muscles of the lower limbs associated with pyramidal signs in some patients. Nerve conduction velocity studies indicated a predominantly motor axonal neuropathy. Unique deletions of two nucleotides causing frameshifts near the end of the NEFH coding sequence were identified: in family 1, c.3008_3009del (p.Lys1003Argfs*59), and in family 2 c.3043_3044del (p.Lys1015Glyfs*47). Both frameshifts lead to 40 additional amino acids translation encoding a cryptic amyloidogenic element. Consistently, we show that these mutations cause protein aggregation which are recognised by the autophagic pathway in motoneurons and triggered caspase 3 activation leading to apoptosis in neuroblastoma cells. Using electroporation of chick embryo spinal cord, we confirm that NEFH mutants form aggregates in vivo and trigger apoptosis of spinal cord neurons. Thus, our results provide a physiological explanation for the overlap between CMT and amyotrophic lateral sclerosis (ALS) clinical features in affected patients.
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Additional file 2: Figure S2. of Cryptic amyloidogenic elements in mutant NEFH causing Charcot-Marie-Tooth 2 trigger aggresome formation and neuronal death
2017Co-Authors: Arnaud Jacquier, Raoul Juntas-morales, Marianne Giroux, Valérie Castellani, Cecile Delorme, Odile Dubourg, Edwige Belotti, Guilhem Sole, Claude-alain Maurage, Adriana RebeloAbstract:eGFP-NEFH WT form filamentous network in vitro and in ovo. A. monomeric eGFP tag NEFH WT expression can form visible filamentous network in SH-EP under lower expression condition when transfected at low concentration (optimal recommended concentration diluted four time). eGFP-NEFH WT form filamentous network in spinal motoneuron in vitro (B) and in vivo (C). Scale bar 10 μm. (TIFF 156 kb
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Additional file 3: Figure S3. of Cryptic amyloidogenic elements in mutant NEFH causing Charcot-Marie-Tooth 2 trigger aggresome formation and neuronal death
2017Co-Authors: Arnaud Jacquier, Raoul Juntas-morales, Marianne Giroux, Valérie Castellani, Cecile Delorme, Odile Dubourg, Edwige Belotti, Guilhem Sole, Claude-alain Maurage, Adriana RebeloAbstract:NEFH mutations modify cell morphology in vitro. A Mutant NEFH expression induces morphological changes as seen on 10× microscopic images. Scale bar represent 100 μm. B-C. Quantification of the average shape factor and radius of transfected SH-EP cells. Values represent means in percent +/− standard deviation of at least 15 fields (Cells analyzed >1000 per condition) and analyzed by Kruskal-Wallis one way ANOVA on ranks test followed by Dunn’s methods (*P
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Additional file 4: Figure S4. of Cryptic amyloidogenic elements in mutant NEFH causing Charcot-Marie-Tooth 2 trigger aggresome formation and neuronal death
2017Co-Authors: Arnaud Jacquier, Raoul Juntas-morales, Marianne Giroux, Valérie Castellani, Cecile Delorme, Odile Dubourg, Edwige Belotti, Guilhem Sole, Claude-alain Maurage, Adriana RebeloAbstract:Representative primary motoneuron in its entirety in vitro. A Non transfected motoneuron revealed by SMI-32 staining. B. Magnetofected motoneuron with eGFP tag NEFH WT or mutated form, without counterstaining. (PNG 156 kb
David Sidransky - One of the best experts on this subject based on the ideXlab platform.
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Abstract 5067: Neurofilament heavy polypeptide regulates the Akt-β-catenin pathway in human esophageal squamous cell carcinoma
Cellular and Molecular Biology, 2010Co-Authors: Myoung Sook Kim, Xiaofei Chang, Cynthia Lebron, Jatin K. Nagpal, Juna Lee, Yiping Huang, Keishi Yamashita, Barry Trink, Edward A. Ratovitski, David SidranskyAbstract:Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH) in a significant proportion of primary esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused up-regulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/β-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of β-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/β-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5067.
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Neurofilament Heavy Polypeptide Regulates the Akt-β-Catenin Pathway in Human Esophageal Squamous Cell Carcinoma
PloS one, 2010Co-Authors: Myoung Sook Kim, Xiaofei Chang, Cynthia Lebron, Jatin K. Nagpal, Juna Lee, Yiping Huang, Keishi Yamashita, Barry Trink, Edward A. Ratovitski, David SidranskyAbstract:Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH) in a significant proportion of primary esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused up-regulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/β-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of β-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/β-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways.
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Neurofilament heavy polypeptide regulates the Akt-beta-catenin pathway in human esophageal squamous cell carcinoma.
Public Library of Science (PLoS), 2010Co-Authors: Myoung Sook Kim, Xiaofei Chang, Cynthia Lebron, Jatin K. Nagpal, Juna Lee, Yiping Huang, Keishi Yamashita, Barry Trink, Edward A. Ratovitski, David SidranskyAbstract:Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH) in a significant proportion of primary esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused up-regulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/beta-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of beta-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/beta-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways
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Neurofilament Heavy Polypeptide Regulates the Akt-b- Catenin Pathway in Human Esophageal Squamous Cell Carcinoma
2009Co-Authors: Myoung Sook Kim, Xiaofei Chang, Cynthia Lebron, Jatin K. Nagpal, Juna Lee, Yiping Huang, Barry Trink, Edward A. Ratovitski, David SidranskyAbstract:Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH) in a significant proportion of primary esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused upregulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/b-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of b-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/b-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways
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Promoter hypermethylation of the neurofilament heavy chain gene in human esophageal cancer
Cancer Research, 2007Co-Authors: Xiaofei Chang, Myoung Sook Kim, Keishi Yamashita, Jin Baek, Yutaka Tokumaru, Chulso Moon, David SidranskyAbstract:2860 Epigenetic silencing of tumor suppressor genes in neoplasia has been recognized as a common mechanism of cancer development. Genes which exhibit frequent cancer-specific methylation may have tumor suppressive activities and hold value as biomarkers for cancer diagnosis. Pharmacological unmasking in esophageal squamous cell carcinoma (ESCC) cell lines uncovered the neurofilament heavy chain (NEFH) gene as a candidate methylated gene. NEFH expression was silenced in ESCC cell lines and reactivated by the demethylating agent, 5-aza-2’-deoxycytidine. Quantitative methylation-specific PCR detected NEFH promoter hypermethylation in 34/70 (48.5%) primary human ESCC and absent or minimal levels in normal corresponding tissues. Moreover, overexpression of NEFH in ESCC cell lines effectively inhibited the formation of cell colonies (44% compared to control vector). NEFH silencing may, thus, provide a selective growth advantage during ESCC tumorigenesis. Furthermore, NEFH methylation shows promise as a biomarker for ESCC due to its common occurrence and high cancer cell specificity.
