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1.  Evolutionary Analysis of Burkholderia pseudomallei Identifies Putative Novel Virulence Genes, Including a Microbial Regulator of Host Cell Autophagy 
Journal of Bacteriology  2013;195(24):5487-5498.
Burkholderia pseudomallei, the causative agent of melioidosis, contains a large pathogen genome (7.2 Mb) with ∼2,000 genes of putative or unknown function. Interactions with potential hosts and environmental factors may induce rapid adaptations in these B. pseudomallei genes, which can be discerned through evolutionary analysis of multiple B. pseudomallei genomes. Here we show that several previously uncharacterized B. pseudomallei genes bearing genetic signatures of rapid adaptation (positive selection) can induce diverse cellular phenotypes when expressed in mammalian cells. Notably, several of these phenotypes are plausibly related to virulence, including multinuclear giant cell formation, apoptosis, and autophagy induction. Specifically, we show that BPSS0180, a type VI cluster-associated gene, is capable of inducing autophagy in both phagocytic and nonphagocytic mammalian cells. Following infection of macrophages, a B. pseudomallei mutant disrupted in BPSS0180 exhibited significantly decreased colocalization with LC3 and impaired intracellular survival; these phenotypes were rescued by introduction of an intact BPSS0180 gene. The results suggest that BPSS0180 may be a novel inducer of host cell autophagy that contributes to B. pseudomallei intracellular growth. More generally, our study highlights the utility of applying evolutionary principles to microbial genomes to identify novel virulence genes.
doi:10.1128/JB.00718-13
PMCID: PMC3889600  PMID: 24097950
2.  Massively Parallel Sequencing of Patients with Intellectual Disability, Congenital Anomalies and/or Autism Spectrum Disorders with a Targeted Gene Panel 
PLoS ONE  2014;9(4):e93409.
Developmental delay and/or intellectual disability (DD/ID) affects 1–3% of all children. At least half of these are thought to have a genetic etiology. Recent studies have shown that massively parallel sequencing (MPS) using a targeted gene panel is particularly suited for diagnostic testing for genetically heterogeneous conditions. We report on our experiences with using massively parallel sequencing of a targeted gene panel of 355 genes for investigating the genetic etiology of eight patients with a wide range of phenotypes including DD/ID, congenital anomalies and/or autism spectrum disorder. Targeted sequence enrichment was performed using the Agilent SureSelect Target Enrichment Kit and sequenced on the Illumina HiSeq2000 using paired-end reads. For all eight patients, 81–84% of the targeted regions achieved read depths of at least 20×, with average read depths overlapping targets ranging from 322× to 798×. Causative variants were successfully identified in two of the eight patients: a nonsense mutation in the ATRX gene and a canonical splice site mutation in the L1CAM gene. In a third patient, a canonical splice site variant in the USP9X gene could likely explain all or some of her clinical phenotypes. These results confirm the value of targeted MPS for investigating DD/ID in children for diagnostic purposes. However, targeted gene MPS was less likely to provide a genetic diagnosis for children whose phenotype includes autism.
doi:10.1371/journal.pone.0093409
PMCID: PMC3972136  PMID: 24690944
3.  Chaos and Robustness in a Single Family of Genetic Oscillatory Networks 
PLoS ONE  2014;9(3):e90666.
Genetic oscillatory networks can be mathematically modeled with delay differential equations (DDEs). Interpreting genetic networks with DDEs gives a more intuitive understanding from a biological standpoint. However, it presents a problem mathematically, for DDEs are by construction infinitely-dimensional and thus cannot be analyzed using methods common for systems of ordinary differential equations (ODEs). In our study, we address this problem by developing a method for reducing infinitely-dimensional DDEs to two- and three-dimensional systems of ODEs. We find that the three-dimensional reductions provide qualitative improvements over the two-dimensional reductions. We find that the reducibility of a DDE corresponds to its robustness. For non-robust DDEs that exhibit high-dimensional dynamics, we calculate analytic dimension lines to predict the dependence of the DDEs’ correlation dimension on parameters. From these lines, we deduce that the correlation dimension of non-robust DDEs grows linearly with the delay. On the other hand, for robust DDEs, we find that the period of oscillation grows linearly with delay. We find that DDEs with exclusively negative feedback are robust, whereas DDEs with feedback that changes its sign are not robust. We find that non-saturable degradation damps oscillations and narrows the range of parameter values for which oscillations exist. Finally, we deduce that natural genetic oscillators with highly-regular periods likely have solely negative feedback.
doi:10.1371/journal.pone.0090666
PMCID: PMC3965403  PMID: 24667178
4.  Nanostring-Based Multigene Assay to Predict Recurrence for Gastric Cancer Patients after Surgery 
PLoS ONE  2014;9(3):e90133.
Despite the benefits from adjuvant chemotherapy or chemoradiotherapy, approximately one-third of stage II gastric cancer (GC) patients developed recurrences. The aim of this study was to develop and validate a prognostic algorithm for gastric cancer (GCPS) that can robustly identify high-risk group for recurrence among stage II patients. A multi-step gene expression profiling study was conducted. First, a microarray gene expression profiling of archived paraffin-embedded tumor blocks was used to identify candidate prognostic genes (N = 432). Second, a focused gene expression assay including prognostic genes was used to develop a robust clinical assay (GCPS) in stage II patients from the same cohort (N = 186). Third, a predefined cut off for the GCPS was validated using an independent stage II cohort (N = 216). The GCPS was validated in another set with stage II GC who underwent surgery without adjuvant treatment (N = 300). GCPS was developed by summing the product of Cox regression coefficients and normalized expression levels of 8 genes (LAMP5, CDC25B, CDK1, CLIP4, LTB4R2, MATN3, NOX4, TFDP1). A prospectively defined cut-point for GCPS classified 22.7% of validation cohort treated with chemoradiotherapy (N = 216) as high-risk group with 5-year recurrence rate of 58.6% compared to 85.4% in the low risk group (hazard ratio for recurrence = 3.16, p = 0.00004). GCPS also identified high-risk group among stage II patients treated with surgery only (hazard ratio = 1.77, p = 0.0053).
doi:10.1371/journal.pone.0090133
PMCID: PMC3943911  PMID: 24598828
5.  Genome-Wide Saturation Mutagenesis of Burkholderia pseudomallei K96243 Predicts Essential Genes and Novel Targets for Antimicrobial Development 
mBio  2014;5(1):e00926-13.
ABSTRACT
Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the development of new antimicrobial therapies is imperative. To identify drug targets and better understand the complex B. pseudomallei genome, we sought a genome-wide approach to identify lethal gene targets. As B. pseudomallei has an unusually large genome spread over two chromosomes, an extensive screen was required to achieve a comprehensive analysis. Here we describe transposon-directed insertion site sequencing (TraDIS) of a library of over 106 transposon insertion mutants, which provides the level of genome saturation required to identify essential genes. Using this technique, we have identified a set of 505 genes that are predicted to be essential in B. pseudomallei K96243. To validate our screen, three genes predicted to be essential, pyrH, accA, and sodB, and a gene predicted to be nonessential, bpss0370, were independently investigated through the generation of conditional mutants. The conditional mutants confirmed the TraDIS predictions, showing that we have generated a list of genes predicted to be essential and demonstrating that this technique can be used to analyze complex genomes and thus be more widely applied.
IMPORTANCE
Burkholderia pseudomallei is a lethal human pathogen that is considered a potential bioterrorism threat and has limited treatment options due to an unusually high natural resistance to most antibiotics. We have identified a set of genes that are required for bacterial growth and thus are excellent candidates against which to develop potential novel antibiotics. To validate our approach, we constructed four mutants in which gene expression can be turned on and off conditionally to confirm that these genes are required for the bacteria to survive.
doi:10.1128/mBio.00926-13
PMCID: PMC3950516  PMID: 24520057
6.  Protection against Experimental Melioidosis following Immunization with Live Burkholderia thailandensis Expressing a manno-Heptose Capsule 
Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei. It is highly resistant to antibiotic treatment, and there is currently no licensed vaccine. Burkholderia thailandensis is a close relative of Burkholderia pseudomallei but is essentially avirulent in mammals. In this report, we detail the protective efficacy of immunization with live B. thailandensis E555, a strain which has been shown to express an antigenic capsule similar to that of B. pseudomallei. Immunization with E555 induced significant protection against a lethal intraperitoneal B. pseudomallei challenge in a mouse model of infection, with no mice succumbing to infection over the course of the study, even with challenges of up to 6,000 median lethal doses. By comparison, mice immunized with B. thailandensis not expressing a B. pseudomallei-like capsule had significantly decreased levels of protection. E555-immunized mice had significantly higher levels of IgG than mice immunized with noncapsulated B. thailandensis, and these antibody responses were primarily directed against the capsule.
doi:10.1128/CVI.00113-13
PMCID: PMC3697456  PMID: 23677322
7.  Early Time-Dependent Dynamic Changes of TBET and GATA3 mRNA Expressions in Patients with Acute Coronary Syndrome 
Disease markers  2013;35(5):419-429.
Background. T-box expressed in T cells (TBET) and guanine adenine thymine adenine sequence-binding protein 3 (GATA3) play important roles in the differentiation of Th1 and Th2 subsets, which contributes to the progression of acute coronary syndrome (ACS). Objective. This study aimed to investigate the temporal change of TBET/GATA3 mRNA ratio in ACS. Methods. Thirty-three patients suspected of ACS with symptom onset within 24 hours were recruited. Blood samples were taken after arrival at the emergency department and at hourly intervals until the 6th hour. The mRNA expressions of TBET and GATA3 were quantified by a real-time RT-qPCR. Results. The TBET/GATA3 mRNA ratio was elevated dramatically in patients with acute myocardial infarction (AMI) and exhibited biphasic M-shaped release kinetics with two distinct peaks. The ratio was elevated 2 hours after symptom onset, dropped to the lowest level at 10 hours, and rose to the second peak at 14 hours. A similar biphasic M-shaped curve was observed in AMI patients with blood samples taken prior to any intervention. Conclusions. The TBET/GATA3 mRNA ratio was elevated in AMI patients throughout most of the first 20 hours after symptom onset. The biphasic M-shaped release kinetics was more likely to reflect pathophysiological changes rather than treatment effects.
doi:10.1155/2013/139895
PMCID: PMC3810123  PMID: 24223457
8.  Less Is More: Burkholderia pseudomallei and Chronic Melioidosis 
mBio  2013;4(5):e00709-13.
ABSTRACT
The Gram-negative bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. Once considered an esoteric tropical disease confined to Southeast Asia and northern Australia, research on B. pseudomallei has recently gained global prominence due to its classification as a potential bioterrorism agent by countries such as the United States and also by increasing numbers of case reports from regions where it is not endemic. An environmental bacterium typically found in soil and water, assessing the true global prevalence of melioidosis is challenged by the fact that clinical symptoms associated with B. pseudomallei infection are extremely varied and may be confused with diverse conditions such as lung cancer, tuberculosis, or Staphyloccocus aureus infection. These diagnostic challenges, coupled with lack of awareness among clinicians, have likely contributed to underdiagnosis and the high mortality rate of melioidosis, as initial treatment is often either inappropriate or delayed. Even after antibiotic treatment, relapses are frequent, and after resolution of acute symptoms, chronic melioidosis can also occur, and the symptoms can persist for months to years. In a recent article, Price et al. [mBio 4(4):e00388-13, 2013, doi:10.1128/mBio.00388-13] demonstrate how comparative genomic sequencing can reveal the repertoire of genetic changes incurred by B. pseudomallei during chronic human infection. Their results have significant clinical ramifications and highlight B. pseudomallei’s ability to survive in a wide range of potential niches within hosts, through the acquisition of genetic adaptations that optimize fitness and resource utilization.
doi:10.1128/mBio.00709-13
PMCID: PMC3781838  PMID: 24065633
9.  The Condition-Dependent Transcriptional Landscape of Burkholderia pseudomallei 
PLoS Genetics  2013;9(9):e1003795.
Burkholderia pseudomallei (Bp), the causative agent of the often-deadly infectious disease melioidosis, contains one of the largest prokaryotic genomes sequenced to date, at 7.2 Mb with two large circular chromosomes (1 and 2). To comprehensively delineate the Bp transcriptome, we integrated whole-genome tiling array expression data of Bp exposed to >80 diverse physical, chemical, and biological conditions. Our results provide direct experimental support for the strand-specific expression of 5,467 Sanger protein-coding genes, 1,041 operons, and 766 non-coding RNAs. A large proportion of these transcripts displayed condition-dependent expression, consistent with them playing functional roles. The two Bp chromosomes exhibited dramatically different transcriptional landscapes — Chr 1 genes were highly and constitutively expressed, while Chr 2 genes exhibited mosaic expression where distinct subsets were expressed in a strongly condition-dependent manner. We identified dozens of cis-regulatory motifs associated with specific condition-dependent expression programs, and used the condition compendium to elucidate key biological processes associated with two complex pathogen phenotypes — quorum sensing and in vivo infection. Our results demonstrate the utility of a Bp condition-compendium as a community resource for biological discovery. Moreover, the observation that significant portions of the Bp virulence machinery can be activated by specific in vitro cues provides insights into Bp's capacity as an “accidental pathogen”, where genetic pathways used by the bacterium to survive in environmental niches may have also facilitated its ability to colonize human hosts.
Author Summary
Bacterial transcriptomes are dynamic, context-specific and condition-dependent. Infection by the soil bacterium, Burkholderia pseudomallei, causes melioidosis, an often fatal infectious disease of humans and animals. Possessing a large multi-chromosomal genome, B. pseudomallei is able to persist and survive in a multitude of environments. To obtain a comprehensive overview of B. pseudomallei expressed transcripts, we initiated whole-genome transcriptome profiling covering a broad spectrum of conditions and exposures — a so-called “condition compendium”. Using the compendium, we confirmed many previously-annotated genes and operons, and also identified hundreds of novel transcripts including anti-sense transcripts and non-coding RNAs. By systematically examining genes exhibiting highly similar expression patterns, we ascribed putative functions to previously uncharacterized genes, and identified novel regulatory elements controlling these expression patterns. We also used the compendium to elucidate candidate virulence pathways associated with quorum-sensing and infection in mice. Our study showcases the power of a B. pseudomallei condition compendium as a valuable resource for understanding microbial physiology and the pathogenesis of melioidosis.
doi:10.1371/journal.pgen.1003795
PMCID: PMC3772027  PMID: 24068961
10.  Growth Inhibition of Pathogenic Bacteria by Sulfonylurea Herbicides 
Emerging resistance to current antibiotics raises the need for new microbial drug targets. We show that targeting branched-chain amino acid (BCAA) biosynthesis using sulfonylurea herbicides, which inhibit the BCAA biosynthetic enzyme acetohydroxyacid synthase (AHAS), can exert bacteriostatic effects on several pathogenic bacteria, including Burkholderia pseudomallei, Pseudomonas aeruginosa, and Acinetobacter baumannii. Our results suggest that targeting biosynthetic enzymes like AHAS, which are lacking in humans, could represent a promising antimicrobial drug strategy.
doi:10.1128/AAC.02327-12
PMCID: PMC3591922  PMID: 23263008
11.  Genetic and Structural Variation in the Gastric Cancer Kinome Revealed through Targeted Deep Sequencing 
Cancer research  2010;71(1):29-39.
Genetic alterations in kinases have been linked to multiple human pathologies. To explore the landscape of kinase genetic variation in gastric cancer (GC), we used targeted, paired-end deep sequencing to analyze 532 protein and phosphoinositide kinases in 14 GC cell lines. We identified 10,604 single-nucleotide variants (SNV) in kinase exons including greater than 300 novel nonsynonymous SNVs. Family-wise analysis of the nonsynonymous SNVs revealed a significant enrichment in mitogen-activated protein kinase (MAPK)-related genes (P < 0.01), suggesting a preferential involvement of this kinase family in GC. A potential antioncogenic role for MAP2K4, a gene exhibiting recurrent alterations in 2 lines, was functionally supported by siRNA knockdown and overexpression studies in wild-type and MAP2K4 variant lines. The deep sequencing data also revealed novel, large-scale structural rearrangement events involving kinases including gene fusions involving CDK12 and the ERBB2 receptor tyrosine kinase in MKN7 cells. Integrating SNVs and copy number alterations, we identified Hs746T as a cell line exhibiting both splice-site mutations and genomic amplification of MET, resulting in MET protein overexpression. When applied to primary GCs, we identified somatic mutations in 8 kinases, 4 of which were recurrently altered in both primary tumors and cell lines (MAP3K6, STK31, FER, and CDKL5). These results demonstrate that how targeted deep sequencing approaches can deliver unprecedented multilevel characterization of a medically and pharmacologically relevant gene family. The catalog of kinome genetic variants assembled here may broaden our knowledge on kinases and provide useful information on genetic alterations in GC.
doi:10.1158/0008-5472.CAN-10-1749
PMCID: PMC3719377  PMID: 21097718
13.  Host Cell Transcriptome Profile during Wild-Type and Attenuated Dengue Virus Infection 
Dengue viruses 1–4 (DENV1-4) rely heavily on the host cell machinery to complete their life cycle, while at the same time evade the host response that could restrict their replication efficiency. These requirements may account for much of the broad gene-level changes to the host transcriptome upon DENV infection. However, host gene function is also regulated through transcriptional start site (TSS) selection and post-transcriptional modification to the RNA that give rise to multiple gene isoforms. The roles these processes play in the host response to dengue infection have not been explored. In the present study, we utilized RNA sequencing (RNAseq) to identify novel transcript variations in response to infection with both a pathogenic strain of DENV1 and its attenuated derivative. RNAseq provides the information necessary to distinguish the various isoforms produced from a single gene and their splice variants. Our data indicate that there is an extensive amount of previously uncharacterized TSS and post-transcriptional modifications to host RNA over a wide range of pathways and host functions in response to DENV infection. Many of the differentially expressed genes identified in this study have previously been shown to be required for flavivirus propagation and/or interact with DENV gene products. We also show here that the human transcriptome response to an infection by wild-type DENV or its attenuated derivative differs significantly. This differential response to wild-type and attenuated DENV infection suggests that alternative processing events may be part of a previously uncharacterized innate immune response to viral infection that is in large part evaded by wild-type DENV.
Author Summary
Dengue is the most common insect-borne viral disease globally. The continued absence of an effective therapy stems from an incomplete understanding of disease pathogenesis, of which the host response to infection is thought to play a central role. While previous studies have described the changes in total gene expression with dengue virus infection, they have not been able to provide any information on the subtle variations of the host RNA. These variations lead to the production of gene isoforms that can have a profound effect on gene function. In the current study, we have used the newly developed technique of RNA sequencing to more accurately interrogate the variations in the host RNA after infection with a wild-type dengue virus or its attenuated derivative. Findings from this study show that there is an extensive amount of previously uncharacterized variation in host RNA response to dengue infection. The response to infection with the wild-type dengue also differs significantly from infection with the vaccine strain. This suggests that variations in the host RNA comprise a part of the host response to viral infection that is in large part evaded by wild-type dengue viruses.
doi:10.1371/journal.pntd.0002107
PMCID: PMC3597485  PMID: 23516652
14.  mTORC1 inhibition restricts inflammation-associated gastrointestinal tumorigenesis in mice 
Gastrointestinal cancers are frequently associated with chronic inflammation and excessive secretion of IL-6 family cytokines, which promote tumorigenesis through persistent activation of the GP130/JAK/STAT3 pathway. Although tumor progression can be prevented by genetic ablation of Stat3 in mice, this transcription factor remains a challenging therapeutic target with a paucity of clinically approved inhibitors. Here, we uncovered parallel and excessive activation of mTOR complex 1 (mTORC1) alongside STAT3 in human intestinal-type gastric cancers (IGCs). Furthermore, in a preclinical mouse model of IGC, GP130 ligand administration simultaneously activated mTORC1/S6 kinase and STAT3 signaling. We therefore investigated whether mTORC1 activation was required for inflammation-associated gastrointestinal tumorigenesis. Strikingly, the mTORC1-specific inhibitor RAD001 potently suppressed initiation and progression of both murine IGC and colitis-associated colon cancer. The therapeutic effect of RAD001 was associated with reduced tumor vascularization and cell proliferation but occurred independently of STAT3 activity. We analyzed the mechanism of GP130-mediated mTORC1 activation in cells and mice and revealed a requirement for JAK and PI3K activity but not for GP130 tyrosine phosphorylation or STAT3. Our results suggest that GP130-dependent activation of the druggable PI3K/mTORC1 pathway is required for inflammation-associated gastrointestinal tumorigenesis. These findings advocate clinical application of PI3K/mTORC1 inhibitors for the treatment of corresponding human malignancies.
doi:10.1172/JCI65086
PMCID: PMC3561832  PMID: 23321674
15.  Whole-genome reconstruction and mutational signatures in gastric cancer 
Genome Biology  2012;13(12):R115.
Background
Gastric cancer is the second highest cause of global cancer mortality. To explore the complete repertoire of somatic alterations in gastric cancer, we combined massively parallel short read and DNA paired-end tag sequencing to present the first whole-genome analysis of two gastric adenocarcinomas, one with chromosomal instability and the other with microsatellite instability.
Results
Integrative analysis and de novo assemblies revealed the architecture of a wild-type KRAS amplification, a common driver event in gastric cancer. We discovered three distinct mutational signatures in gastric cancer - against a genome-wide backdrop of oxidative and microsatellite instability-related mutational signatures, we identified the first exome-specific mutational signature. Further characterization of the impact of these signatures by combining sequencing data from 40 complete gastric cancer exomes and targeted screening of an additional 94 independent gastric tumors uncovered ACVR2A, RPL22 and LMAN1 as recurrently mutated genes in microsatellite instability-positive gastric cancer and PAPPA as a recurrently mutated gene in TP53 wild-type gastric cancer.
Conclusions
These results highlight how whole-genome cancer sequencing can uncover information relevant to tissue-specific carcinogenesis that would otherwise be missed from exome-sequencing data.
doi:10.1186/gb-2012-13-12-r115
PMCID: PMC4056366  PMID: 23237666
16.  Workshop on Treatment of and Postexposure Prophylaxis for Burkholderia pseudomallei and B. mallei Infection, 2010 
The US Public Health Emergency Medical Countermeasures Enterprise convened subject matter experts at the 2010 HHS Burkholderia Workshop to develop consensus recommendations for postexposure prophylaxis against and treatment for Burkholderia pseudomallei and B. mallei infections, which cause melioidosis and glanders, respectively. Drugs recommended by consensus of the participants are ceftazidime or meropenem for initial intensive therapy, and trimethoprim/sulfamethoxazole or amoxicillin/clavulanic acid for eradication therapy. For postexposure prophylaxis, recommended drugs are trimethoprim/sulfamethoxazole or co-amoxiclav. To improve the timely diagnosis of melioidosis and glanders, further development and wide distribution of rapid diagnostic assays were also recommended. Standardized animal models and B. pseudomallei strains are needed for further development of therapeutic options. Training for laboratory technicians and physicians would facilitate better diagnosis and treatment options.
doi:10.3201/eid1812.120638
PMCID: PMC3557896  PMID: 23171644
Burkholderia pseudomallei; melioidosis; Burkholderia mallei; glanders; drug therapy; postexposure prophylaxis; ceftazidime; carbapenems; trimethoprim/sulfamethoxazole; combination; amoxicillin/potassium clavulanate; clavulanic acid bacteria; antibiotic; antibacterial drugs; antimicrobial drugs; bacteria; Suggested citation for this article: Lipsitz R; Garges S; Aurigemma R; Baccam P; Blaney DD; Cheng AC; et al. Workshop on treatment of and postexposure prophylaxis for Burkholderia pseudomallei and B. mallei infection; 2010. Emerg Infect Dis [Internet]. 2012 Dec [date cited]. http://dx.doi.org/10.3201/eid1812.120638
18.  Genetic and bioinformatic analyses of the expression and function of PI3K regulatory subunit PIK3R3 in an Asian patient gastric cancer library 
BMC Medical Genomics  2012;5:34.
Background
While there is strong evidence for phosphatidylinositol 3-kinase (PI3K) involvement in cancer development, there is limited information about the role of PI3K regulatory subunits. PIK3R3, the gene that encodes the PI3K regulatory subunit p55γ, is over-expressed in glioblastoma and ovarian cancers, but its expression in gastric cancer (GC) is not known. We thus used genetic and bioinformatic approaches to examine PIK3R3 expression and function in GC, the second leading cause of cancer mortality world-wide and highly prevalent among Asians.
Methods
Primary GC and matched non-neoplastic mucosa tissue specimens from a unique Asian patient gastric cancer library were comprehensively profiled with platforms that measured genome-wide mRNA expression, DNA copy number variation, and DNA methylation status. Function of PIK3R3 was predicted by IPA pathway analysis of co-regulated genes with PIK3R3, and further investigated by siRNA knockdown studies. Cell proliferation was estimated by crystal violet dye elution and BrdU incorporation assay. Cell cycle distribution was analysed by FACS.
Results
PIK3R3 was significantly up-regulated in GC specimens (n = 126, p < 0.05), and 9.5 to 15% tumors showed more than 2 fold increase compare to the paired mucosa tissues. IPA pathway analysis showed that PIK3R3 promoted cellular growth and proliferation. Knockdown of PIK3R3 decreased the growth of GC cells, induced G0/G1 cell cycle arrest, decreased retinoblastoma protein (Rb) phosphorylation, cyclin D1, and PCNA expression.
Conclusion
Using a combination of genetic, bioinformatic, and molecular biological approaches, we showed that PIK3R3 was up-regulated in GC and promoted cell cycle progression and proliferation; and thus may be a potential new therapeutic target for GC.
doi:10.1186/1755-8794-5-34
PMCID: PMC3479415  PMID: 22876838
19.  Intrinsic Subtypes of Gastric Cancer, Based on Gene Expression Pattern, Predict Survival and Respond Differently to Chemotherapy 
Gastroenterology  2011;141(2):476-485.e11.
BACKGROUND & AIMS
Gastric cancer (GC) is a heterogeneous disease comprising multiple subtypes that each have distinct biological properties and effects in patients. We sought to identify new, intrinsic subtypes of GC by gene expression analysis of a large panel of GC cell lines. We tested if these subtypes might be associated with differences patient survival times and responses to various standard-of-care cytotoxic drugs.
METHODS
We analyzed gene expression profiles for 37 GC cell lines to identify intrinsic GC subtypes. These subtypes were validated in primary tumors from 521 patients in 4 independent cohorts, where the subtypes were determined by either expression profiling or subtype-specific immunohistochemical markers (LGALS4, CDH17). In vitro sensitivity to 3 chemotherapy drugs (5-FU, cisplatin, oxaliplatin) was also assessed.
RESULTS
Unsupervised cell line analysis identified 2 major intrinsic genomic subtypes (G-INT and G-DIF), that had distinct patterns of gene expression. The intrinsic subtypes, but not subtypes based on Lauren’s histopathologic classification, were prognostic of survival, based on univariate and multivariate analysis in multiple patient cohorts. The G-INT cell lines were significantly more sensitive to 5-FU and oxaliplatin, but more resistant to cisplatin, than the G-DIF cell lines. In patients, intrinsic subtypes were associated with survival time following adjuvant, 5-FU based therapy.
CONCLUSIONS
Intrinsic subtypes of GC, based on distinct patterns of expression, are associated with patient survival and response to chemotherapy. Classification of GC based on intrinsic subtypes might be used to determine prognosis and customize therapy.
doi:10.1053/j.gastro.2011.04.042
PMCID: PMC3152688  PMID: 21684283
Microarray analysis; pharmacogenomics; mRNA; stomach; carcinogenesis
20.  A Burkholderia pseudomallei Toxin Inhibits Helicase Activity of Translation Factor eIF4A 
Science (New York, N.Y.)  2011;334(6057):821-824.
The structure of BPSL1549, a protein of unknown function from Burkholderia pseudomallei reveals a similarity to E. coli cytotoxic necrotizing factor 1. We found that BPSL1549 acted as a potent cytotoxin against eukaryotic cells and was lethal when administered to mice. Expression levels of bpsl1549 correlate with conditions expected to promote or suppress pathogenicity. BPSL1549 promotes deamidation of Gln339 of the translation initiation factor eIF4A, abolishing its helicase activity and inhibiting translation. We propose to name BPSL1549 Burkholderia Lethal Factor 1 (BLF1).
doi:10.1126/science.1211915
PMCID: PMC3364511  PMID: 22076380
21.  Development and Validation of Burkholderia pseudomallei-Specific Real-Time PCR Assays for Clinical, Environmental or Forensic Detection Applications 
PLoS ONE  2012;7(5):e37723.
The bacterium Burkholderia pseudomallei causes melioidosis, a rare but serious illness that can be fatal if untreated or misdiagnosed. Species-specific PCR assays provide a technically simple method for differentiating B. pseudomallei from near-neighbor species. However, substantial genetic diversity and high levels of recombination within this species reduce the likelihood that molecular signatures will differentiate all B. pseudomallei from other Burkholderiaceae. Currently available molecular assays for B. pseudomallei detection lack rigorous validation across large in silico datasets and isolate collections to test for specificity, and none have been subjected to stringent quality control criteria (accuracy, precision, selectivity, limit of quantitation (LoQ), limit of detection (LoD), linearity, ruggedness and robustness) to determine their suitability for environmental, clinical or forensic investigations. In this study, we developed two novel B. pseudomallei specific assays, 122018 and 266152, using a dual-probe approach to differentiate B. pseudomallei from B. thailandensis, B. oklahomensis and B. thailandensis-like species; other species failed to amplify. Species specificity was validated across a large DNA panel (>2,300 samples) comprising Burkholderia spp. and non-Burkholderia bacterial and fungal species of clinical and environmental relevance. Comparison of assay specificity to two previously published B. pseudomallei-specific assays, BurkDiff and TTS1, demonstrated comparable performance of all assays, providing between 99.7 and 100% specificity against our isolate panel. Last, we subjected 122018 and 266152 to rigorous quality control analyses, thus providing quantitative limits of assay performance. Using B. pseudomallei as a model, our study provides a framework for comprehensive quantitative validation of molecular assays and provides additional, highly validated B. pseudomallei assays for the scientific research community.
doi:10.1371/journal.pone.0037723
PMCID: PMC3356290  PMID: 22624061
22.  PORCN Moonlights in a Wnt-Independent Pathway That Regulates Cancer Cell Proliferation 
PLoS ONE  2012;7(4):e34532.
Porcupine (PORCN) is a membrane-bound O-acyl transferase that is required for the palmitoylation of Wnt proteins, and that is essential in diverse Wnt pathways for Wnt-Wntless (WLS) binding, Wnt secretion, and Wnt signaling activity. We tested if PORCN was required for the proliferation of transformed cells. Knockdown of PORCN by multiple independent siRNAs results in a cell growth defect in a subset of epithelial cancer cell lines. The growth defect is transformation-dependent in human mammary epithelial (HMEC) cells. Additionally, inducible PORCN knockdown by two independent shRNAs markedly reduces the growth of established MDA-MB-231 cancers in orthotopic xenografts in immunodeficient mice. Unexpectedly, the proliferation defect resulting from loss of PORCN occurs in a Wnt-independent manner, as it is rescued by re-expression of catalytically inactive PORCN, and is not seen after RNAi-mediated knockdown of the Wnt carrier protein WLS, nor after treatment with the PORCN inhibitor IWP. Consistent with a role in a Wnt-independent pathway, knockdown of PORCN regulates a distinct set of genes that are not altered by other inhibitors of Wnt signaling. PORCN protein thus appears to moonlight in a novel signaling pathway that is rate-limiting for cancer cell growth and tumorigenesis independent of its enzymatic function in Wnt biosynthesis and secretion.
doi:10.1371/journal.pone.0034532
PMCID: PMC3324524  PMID: 22509316
23.  A comprehensive survey of genomic alterations in gastric cancer reveals systematic patterns of molecular exclusivity and co-occurrence among distinct therapeutic targets 
Gut  2012;61(5):673-684.
Objective
Gastric cancer is a major gastrointestinal malignancy for which targeted therapies are emerging as treatment options. This study sought to identify the most prevalent molecular targets in gastric cancer and to elucidate systematic patterns of exclusivity and co-occurrence among these targets, through comprehensive genomic analysis of a large panel of gastric cancers.
Design
Using high-resolution single nucleotide polymorphism arrays, copy number alterations were profiled in a panel of 233 gastric cancers (193 primary tumours, 40 cell lines) and 98 primary matched gastric non-malignant samples. For selected alterations, their impact on gene expression and clinical outcome were evaluated.
Results
22 recurrent focal alterations (13 amplifications and nine deletions) were identified. These included both known targets (FGFR2, ERBB2) and also novel genes in gastric cancer (KLF5, GATA6). Receptor tyrosine kinase (RTK)/RAS alterations were found to be frequent in gastric cancer. This study also demonstrates, for the first time, that these alterations occur in a mutually exclusive fashion, with KRAS gene amplifications highlighting a clinically relevant but previously underappreciated gastric cancer subgroup. FGFR2-amplified gastric cancers were also shown to be sensitive to dovitinib, an orally bioavailable FGFR/VEGFR targeting agent, potentially representing a subtype-specific therapy for FGFR2-amplified gastric cancers.
Conclusion
The study demonstrates the existence of five distinct gastric cancer patient subgroups, defined by the signature genomic alterations FGFR2 (9% of tumours), KRAS (9%), EGFR (8%), ERBB2 (7%) and MET (4%). Collectively, these subgroups suggest that at least 37% of gastric cancer patients may be potentially treatable by RTK/RAS directed therapies.
doi:10.1136/gutjnl-2011-301839
PMCID: PMC3322587  PMID: 22315472
Barrett's carcinoma; Barrett's metaplasia; cancer; cell signalling; chemotherapy; colorectal cancer screening; copy number alterations; gastric cancer; gastric carcinoma; gastric pre-cancer; gastrointestinal cancer; gastrointesinal endoscopy; gastroscopy; gene expression; gene mutation; molecular pathology; oesophageal cancer; receptor tyrosine kinases; targeted therapies
24.  A Densely Interconnected Genome-Wide Network of MicroRNAs and Oncogenic Pathways Revealed Using Gene Expression Signatures 
PLoS Genetics  2011;7(12):e1002415.
MicroRNAs (miRNAs) are important components of cellular signaling pathways, acting either as pathway regulators or pathway targets. Currently, only a limited number of miRNAs have been functionally linked to specific signaling pathways. Here, we explored if gene expression signatures could be used to represent miRNA activities and integrated with genomic signatures of oncogenic pathway activity to identify connections between miRNAs and oncogenic pathways on a high-throughput, genome-wide scale. Mapping >300 gene expression signatures to >700 primary tumor profiles, we constructed a genome-wide miRNA–pathway network predicting the associations of 276 human miRNAs to 26 oncogenic pathways. The miRNA–pathway network confirmed a host of previously reported miRNA/pathway associations and uncovered several novel associations that were subsequently experimentally validated. Globally, the miRNA–pathway network demonstrates a small-world, but not scale-free, organization characterized by multiple distinct, tightly knit modules each exhibiting a high density of connections. However, unlike genetic or metabolic networks typified by only a few highly connected nodes (“hubs”), most nodes in the miRNA–pathway network are highly connected. Sequence-based computational analysis confirmed that highly-interconnected miRNAs are likely to be regulated by common pathways to target similar sets of downstream genes, suggesting a pervasive and high level of functional redundancy among coexpressed miRNAs. We conclude that gene expression signatures can be used as surrogates of miRNA activity. Our strategy facilitates the task of discovering novel miRNA–pathway connections, since gene expression data for multiple normal and disease conditions are abundantly available.
Author Summary
MicroRNAs (miRNAs) are naturally occurring small RNA molecules of ∼22 nucleotides that regulate gene expression. Recent studies have shown that miRNAs can behave as important components of cellular signaling pathways, as pathway regulators or pathway targets. Currently however, only a few miRNAs have been functionally linked to specific signaling pathways, raising the need for novel approaches to accelerate the identification of miRNA–pathway connections. Here, we show that gene expression signatures, previously used to reflect patterns of pathway activation, can also be used to represent miRNA activities. Using this approach, we constructed a genome-wide miRNA–pathway network predicting the associations of 276 human miRNAs to 26 oncogenic pathways. The miRNA–pathway network confirmed a host of previously reported miRNA/pathway associations and uncovered several novel associations that were subsequently experimentally validated. Besides being the first study to conceptually demonstrate that expression signatures can act as surrogates of miRNA activity, our study provides a large database of candidate pathway-modulating miRNAs, which researchers interested in a particular pathway (e.g. Ras, Myc) are likely to find useful. Moreover, because this approach solely employs gene expression, it is immediately applicable to the thousands of microarray data sets currently available in the public domain.
doi:10.1371/journal.pgen.1002415
PMCID: PMC3240594  PMID: 22194702
25.  Paradoxical relationship between chromosomal instability and survival outcome in cancer 
Cancer research  2011;71(10):3447-3452.
Chromosomal instability (CIN) is associated with poor prognosis in human cancer. However, in certain animal tumour models elevated CIN negatively impacts upon organism fitness, and is poorly tolerated by cancer cells. To better understand this seemingly contradictory relationship between CIN and cancer cell biological fitness and its relationship with clinical outcome, we applied the CIN70 expression signature, which correlates with DNA-based measures of structural chromosomal complexity and numerical chromosomal instability in vivo, to gene expression profiles of 2125 breast tumours from 13 published cohorts. Tumours with extreme CIN, defined as the highest quartile CIN70 score, were predominantly of the estrogen receptor (ER) negative, basal-like phenotype and displayed the highest chromosomal structural complexity and chromosomal numerical instability. We found that the extreme CIN/ER-negative tumours were associated with improved prognosis relative to tumours with intermediate CIN70 scores in the third quartile. We also observed this paradoxical relationship between CIN and prognosis in ovarian, gastric and non-small cell lung cancer, with poorest outcome in tumours with intermediate, rather than extreme, CIN70 scores. These results suggest a non-monotonic relationship between gene signature expression and hazard ratio for survival outcome, which may explain the difficulties encountered in the identification of prognostic expression signatures in ER negative breast cancer. Furthermore, the data are consistent with the intolerance of excessive CIN in carcinomas and provide a plausible strategy to define distinct prognostic patient cohorts with ER-negative breast cancer. Inclusion of a surrogate measurement of CIN may improve cancer risk stratification and future therapeutic approaches.
doi:10.1158/0008-5472.CAN-10-3667
PMCID: PMC3096721  PMID: 21270108

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