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1.  Associations of Mitochondrial Haplogroups B4 and E with Biliary Atresia and Differential Susceptibility to Hydrophobic Bile Acid 
PLoS Genetics  2013;9(8):e1003696.
Mitochondrial dysfunction has been implicated in the pathogenesis of biliary atresia (BA). This study aimed to determine whether a specific mitochondrial DNA haplogroup is implicated in the pathogenesis and prognosis of BA. We determined 40 mitochondrial single nucleotide polymorphisms in 15 major mitochondrial haplogroups by the use of 24-plex PCR and fluorescent beads combined with sequence-specific oligonucleotide probes in 71 patients with BA and in 200 controls in the Taiwanese population of ethnic Chinese background. The haplogroup B4 and E prevalence were significantly lower and higher respectively, in the patients with BA than in the controls (odds ratios, 0.82 [p = 0.007] and 7.36 [p = 0.032] respectively) in multivariate logistic-regression analysis. The 3-year survival rate with native liver was significantly lower in haplogroup E than the other haplogroups (P = 0.037). A cytoplasmic hybrid (cybrid) was obtained from human 143B osteosarcoma cells devoid of mtDNA (ρ0 cell) and was fused with specific mtDNA bearing E and B4 haplogroups donated by healthy Taiwanese subjects. Chenodeoxycholic acid treatment resulted in significantly lower free radical production, higher mitochondrial membrane potential, more viable cells, and fewer apoptotic cybrid B4 cells than parental 143B and cybrid E cells. Bile acid treatment resulted in a significantly greater protective mitochondrial reaction with significantly higher mitochondrial DNA copy number and mitofusin 1 and 2 concentrations in cybrid B4 and parental cells than in cybrid E cells. The results of the study suggested that the specific mitochondrial DNA haplogroups B4 and E were not only associated with lower and higher prevalence of BA respectively, in the study population, but also with differential susceptibility to hydrophobic bile acid in the cybrid harboring different haplogroups.
Author Summary
Mitochondrial dysfunction has been implicated in the pathogenesis of biliary atresia (BA). We determined 40 mitochondrial single nucleotide polymorphisms in different mitochondrial haplogroups in BA patients and controls. The prevalence of haplogroup B4 and E was significantly lower and higher respectively, in the patients with BA than in the controls. The survival rate with native liver was significantly lower in haplogroup E than the other haplogroups. The in vitro study using cybrid cells revealed significantly lower free radical production, higher mitochondrial membrane potential, higher mitochondrial DNA copy number and fewer apoptotic in cybrid B4 cells than cybrid E cells. The study provides a novel insight into the etiopathogenesis and the predictive value of mitochondrial haplogroups in BA.
PMCID: PMC3744426  PMID: 23966875
2.  Integrative Genomics in Combination with RNA Interference Identifies Prognostic and Functionally Relevant Gene Targets for Oral Squamous Cell Carcinoma 
PLoS Genetics  2013;9(1):e1003169.
In oral squamous cell carcinoma (OSCC), metastasis to lymph nodes is associated with a 50% reduction in 5-year survival. To identify a metastatic gene set based on DNA copy number abnormalities (CNAs) of differentially expressed genes, we compared DNA and RNA of OSCC cells laser-microdissected from non-metastatic primary tumors (n = 17) with those from lymph node metastases (n = 20), using Affymetrix 250K Nsp single-nucleotide polymorphism (SNP) arrays and U133 Plus 2.0 arrays, respectively. With a false discovery rate (FDR)<5%, 1988 transcripts were found to be differentially expressed between primary and metastatic OSCC. Of these, 114 were found to have a significant correlation between DNA copy number and gene expression (FDR<0.01). Among these 114 correlated transcripts, the corresponding genomic regions of each of 95 transcripts had CNAs differences between primary and metastatic OSCC (FDR<0.01). Using an independent dataset of 133 patients, multivariable analysis showed that the OSCC–specific and overall mortality hazards ratio (HR) for patients carrying the 95-transcript signature were 4.75 (95% CI: 2.03–11.11) and 3.45 (95% CI: 1.84–6.50), respectively. To determine the degree by which these genes impact cell survival, we compared the growth of five OSCC cell lines before and after knockdown of over-amplified transcripts via a high-throughput siRNA–mediated screen. The expression-knockdown of 18 of the 26 genes tested showed a growth suppression ≥30% in at least one cell line (P<0.01). In particular, cell lines derived from late-stage OSCC were more sensitive to the knockdown of G3BP1 than cell lines derived from early-stage OSCC, and the growth suppression was likely caused by increase in apoptosis. Further investigation is warranted to examine the biological role of these genes in OSCC progression and their therapeutic potentials.
Author Summary
Neck lymph node metastasis is the most important prognostic factor in oral squamous cell carcinoma (OSCC). To identify genes associated with this critical step of OSCC progression, we compared DNA copy number aberrations and gene expression differences between tumor cells found in metastatic lymph nodes versus those in non-metastatic primary tumors. We identified 95 transcripts (87 genes) with metastasis-specific genome abnormalities and gene expression. Tested in an independent cohort of 133 OSCC patients, the 95 gene signature was an independent risk factor of disease-specific and overall death, suggesting a disease progression phenotype. We knocked down the expression of over-amplified genes in five OSCC cell lines. Knockdown of 18 of the 26 tested genes suppressed the cell growth in at least one cell line. Interestingly, cell lines derived from late-stage OSCC were more sensitive to the knockdown of G3BP1 than cell lines derived from early-stage OSCC. The knockdown of G3BP1 increased programmed cell death in the p53-mutant but not wild-type OSCC cell lines. Taken together, we demonstrate that CNA–associated transcripts differentially expressed in carcinoma cells with an aggressive phenotype (i.e., metastatic to lymph nodes) can be biomarkers with both prognostic information and functional relevance. Moreover, results suggest that G3BP1 is a potential therapeutic target against late-stage p53-negative OSCC.
PMCID: PMC3547824  PMID: 23341773
3.  Genomic Profiling Identifies GATA6 as a Candidate Oncogene Amplified in Pancreatobiliary Cancer 
PLoS Genetics  2008;4(5):e1000081.
Pancreatobiliary cancers have among the highest mortality rates of any cancer type. Discovering the full spectrum of molecular genetic alterations may suggest new avenues for therapy. To catalogue genomic alterations, we carried out array-based genomic profiling of 31 exocrine pancreatic cancers and 6 distal bile duct cancers, expanded as xenografts to enrich the tumor cell fraction. We identified numerous focal DNA amplifications and deletions, including in 19% of pancreatobiliary cases gain at cytoband 18q11.2, a locus uncommonly amplified in other tumor types. The smallest shared amplification at 18q11.2 included GATA6, a transcriptional regulator previously linked to normal pancreas development. When amplified, GATA6 was overexpressed at both the mRNA and protein levels, and strong immunostaining was observed in 25 of 54 (46%) primary pancreatic cancers compared to 0 of 33 normal pancreas specimens surveyed. GATA6 expression in xenografts was associated with specific microarray gene-expression patterns, enriched for GATA binding sites and mitochondrial oxidative phosphorylation activity. siRNA mediated knockdown of GATA6 in pancreatic cancer cell lines with amplification led to reduced cell proliferation, cell cycle progression, and colony formation. Our findings indicate that GATA6 amplification and overexpression contribute to the oncogenic phenotypes of pancreatic cancer cells, and identify GATA6 as a candidate lineage-specific oncogene in pancreatobiliary cancer, with implications for novel treatment strategies.
Author Summary
Pancreatic cancer is a devastating disease, having among the lowest survival rates of any cancer. A better understanding of the molecular basis of pancreatic cancer may lead to improved rationale therapies. We report here the discovery of amplification (i.e. extra copies) of the GATA6 gene in many human pancreatic cancers. GATA6 is a regulator of gene expression and functions in the development of the normal pancreas. Our findings indicate that its amplification and aberrant overexpression contribute to pancreatic cancer development. GATA6 joins a growing list of cancer genes with key roles in normal human development but pathogenic roles in cancer when aberrantly expressed. Our discovery of GATA6 amplification provides a new foothold into understanding the pathogenic mechanisms underlying pancreatic cancer, and suggests new strategies for therapy by targeting GATA6 or the genes it regulates.
PMCID: PMC2413204  PMID: 18535672

Results 1-3 (3)