Publications by authors named "Martina Veigl"

21 Publications

  • Page 1 of 1

Vascular dimorphism ensured by regulated proteoglycan dynamics favors rapid umbilical artery closure at birth.

Elife 2020 09 10;9. Epub 2020 Sep 10.

Department of Biomedical Engineering, Cleveland Clinic Lerner Research Institute, Cleveland, United States.

The umbilical artery lumen closes rapidly at birth, preventing neonatal blood loss, whereas the umbilical vein remains patent longer. Here, analysis of umbilical cords from humans and other mammals identified differential arterial-venous proteoglycan dynamics as a determinant of these contrasting vascular responses. The umbilical artery, but not the vein, has an inner layer enriched in the hydrated proteoglycan aggrecan, external to which lie contraction-primed smooth muscle cells (SMC). At birth, SMC contraction drives inner layer buckling and centripetal displacement to occlude the arterial lumen, a mechanism revealed by biomechanical observations and confirmed by computational analyses. This vascular dimorphism arises from spatially regulated proteoglycan expression and breakdown. Mice lacking aggrecan or the metalloprotease ADAMTS1, which degrades proteoglycans, demonstrate their opposing roles in umbilical vascular dimorphism, including effects on SMC differentiation. Umbilical vessel dimorphism is conserved in mammals, suggesting that differential proteoglycan dynamics and inner layer buckling were positively selected during evolution.
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http://dx.doi.org/10.7554/eLife.60683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529456PMC
September 2020

5-Fluorouracil Enhances the Antitumor Activity of the Glutaminase Inhibitor CB-839 against -Mutant Colorectal Cancers.

Cancer Res 2020 11 9;80(21):4815-4827. Epub 2020 Sep 9.

Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, Ohio.

encodes the p110α catalytic subunit of PI3K and is frequently mutated in human cancers, including ∼30% of colorectal cancer. Oncogenic mutations in render colorectal cancers more dependent on glutamine. Here we report that the glutaminase inhibitor CB-839 preferentially inhibits xenograft growth of -mutant, but not wild-type (WT), colorectal cancers. Moreover, the combination of CB-839 and 5-fluorouracil (5-FU) induces -mutant tumor regression in xenograft models. CB-839 treatment increased reactive oxygen species and caused nuclear translocation of Nrf2, which in turn upregulated mRNA expression of uridine phosphorylase 1 (UPP1). UPP1 facilitated the conversion of 5-FU to its active compound, thereby enhancing the inhibition of thymidylate synthase. Consistently, knockout of UPP1 abrogated the tumor inhibitory effect of combined CB-839 and 5-FU administration. A phase I clinical trial showed that the combination of CB-839 and capecitabine, a prodrug of 5-FU, was well tolerated at biologically-active doses. Although not designed to test efficacy, an exploratory analysis of the phase I data showed a trend that -mutant patients with colorectal cancer might derive greater benefit from this treatment strategy as compared with WT patients with colorectal cancer. These results effectively demonstrate that targeting glutamine metabolism may be an effective approach for treating patients with -mutant colorectal cancers and warrants further clinical evaluation. SIGNIFICANCE: Preclinical and clinical trial data suggest that the combination of CB-839 with capecitabine could serve as an effective treatment for -mutant colorectal cancers.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642187PMC
November 2020

Relationships between expression of , mitochondrial bioenergetics, and fatigue among patients with prostate cancer.

Cancer Manag Res 2019 18;11:6703-6717. Epub 2019 Jul 18.

Center for Mitochondrial Disease, Department of Pharmacology and Medicine, School of Medicine, Case Western Reserve University, Cleveland, OH, USA.

Cancer-related fatigue (CRF) is the most debilitating symptom with the greatest adverse side effect on quality of life. The etiology of this symptom is still not understood. The purpose of this study was to examine the relationship between mitochondrial gene expression, mitochondrial oxidative phosphorylation, electron transport chain complex activity, and fatigue in prostate cancer patients undergoing radiotherapy (XRT), compared to patients on active surveillance (AS). The study used a matched case-control and repeated-measures research design. Fatigue was measured using the revised Piper Fatigue Scale from 52 patients with prostate cancer. Mitochondrial oxidative phosphorylation, electron-transport chain enzymatic activity, and gene expression were determined using patients' peripheral mononuclear cells. Data were collected at three time points and analyzed using repeated measures ANOVA. The fatigue score was significantly different over time between patients undergoing XRT and AS (<0.05). Patients undergoing XRT experienced significantly increased fatigue at day 21 and day 42 of XRT (<0.01). Downregulated mitochondrial gene (BC1, ubiquinol-cytochrome c reductase, synthesis-like, 0.05) expression, decreased OXPHOS-complex III oxidation (0.05), and reduced activity of complex III were observed over time in patients with XRT. Moreover, increased fatigue was significantly associated with downregulated and decreased complex III oxidation in patients undergoing XRT. Our results suggest that BCS1L and complex III in mitochondrial mononuclear cells are potential biomarkers and feasible therapeutic targets for acute XRT-induced fatigue in this clinical population.
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http://dx.doi.org/10.2147/CMAR.S203317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6645361PMC
July 2019

Gene Expression Changes Accompanying the Duodenal Adenoma-Carcinoma Sequence in Familial Adenomatous Polyposis.

Clin Transl Gastroenterol 2019 06;10(6):e00053

Sanford R. Weiss MD Center for Hereditary Colorectal Neoplasia, Cleveland Clinic, Cleveland, Ohio, USA.

Objectives: Duodenal cancer in familial adenomatous polyposis (FAP) arises from adenomas. Differentially expressed genes (DEGs) in the duodenal adenoma-carcinoma pathway have been identified in murine FAP models, but similar data in patients with FAP are limited. Identifying such changes may have significance in understanding duodenal polyposis therapies and identifying cancer biomarkers. We performed a genome-wide transcriptional analysis to describe the duodenal adenoma-carcinoma sequence and determine changes distinguishing patients with FAP with and without duodenal cancer.

Methods: Transcriptional profiling was performed with the Affymetrix Human Transcriptome Array 2.0 on duodenal biopsies from 12 FAP patients with duodenal cancer (FAP cases) and 12 FAP patients without cancer (FAP controls). DEGs were compared between cancer-normal, adenoma-normal, and cancer-adenoma in FAP cases and between adenomas from FAP cases and FAP controls. Significant results at P < 0.05 were filtered using fold change > 2.

Results: Two hundred twenty-four DEGs were identified at an absolute fold change > 2. In adenoma-normal, downregulation of DEGs involved in metabolism of brush border proteins (LCT), lipids (APOB/A4), reactive oxygen species (GSTA2), and retinol (RBP2) was observed. In the cancer-adenoma comparison, upregulation of DEGs involved in cell invasion/migration (POSTN, SPP1) and downregulation of DEGs involved in Paneth differentiation (DEFA5/6) were observed. In the adenoma-adenoma comparison, downregulation of several DEGs (CLCA1, ADH1C, ANXA10) in FAP case adenomas was observed. DEGs with therapeutic potential include SPP1, which is involved in both cyclooxygenase and epidermal growth factor receptor pathways targeted by the sulindac/erlotinib combination for duodenal polyposis.

Discussion: We describe DEGs in the human duodenal adenoma-carcinoma sequence in FAP, which may have prognostic and therapeutic significance. Validation studies are needed to confirm these findings.
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http://dx.doi.org/10.14309/ctg.0000000000000053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6613862PMC
June 2019

Genomic regions associated with susceptibility to Barrett's esophagus and esophageal adenocarcinoma in African Americans: The cross BETRNet admixture study.

PLoS One 2017 26;12(10):e0184962. Epub 2017 Oct 26.

Division of Gastroenterology and Hepatology, University Hospitals Cleveland Medical Center, Case Western Reserve University School of Medicine, Cleveland, OH, United States of America.

Background: Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) are far more prevalent in European Americans than in African Americans. Hypothesizing that this racial disparity in prevalence might represent a genetic susceptibility, we used an admixture mapping approach to interrogate disease association with genomic differences between European and African ancestry.

Methods: Formalin fixed paraffin embedded samples were identified from 54 African Americans with BE or EAC through review of surgical pathology databases at participating Barrett's Esophagus Translational Research Network (BETRNet) institutions. DNA was extracted from normal tissue, and genotyped on the Illumina OmniQuad SNP chip. Case-only admixture mapping analysis was performed on the data from both all 54 cases and also on a subset of 28 cases with high genotyping quality. Haplotype phases were inferred with Beagle 3.3.2, and local African and European ancestries were inferred with SABER plus. Disease association was tested by estimating and testing excess European ancestry and contrasting it to excess African ancestry.

Results: Both datasets, the 54 cases and the 28 cases, identified two admixture regions. An association of excess European ancestry on chromosome 11p reached a 5% genome-wide significance threshold, corresponding to -log10(P) = 4.28. A second peak on chromosome 8q reached -log10(P) = 2.73. The converse analysis examining excess African ancestry found no genetic regions with significant excess African ancestry associated with BE and EAC. On average, the regions on chromosomes 8q and 11p showed excess European ancestry of 15% and 20%, respectively.

Conclusions: Chromosomal regions on 11p15 and 8q22-24 are associated with excess European ancestry in African Americans with BE and EAC. Because GWAS have not reported any variants in these two regions, low frequency and/or rare disease associated variants that confer susceptibility to developing BE and EAC may be driving the observed European ancestry association evidence.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0184962PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5657624PMC
November 2017

A nonrandomized trial of vitamin D supplementation for Barrett's esophagus.

PLoS One 2017 18;12(9):e0184928. Epub 2017 Sep 18.

Department of Medicine, University Hospitals Cleveland Medical Center, Cleveland, Ohio, United States of America.

Background: Vitamin D deficiency may increase esophageal cancer risk. Vitamin D affects genes regulating proliferation, apoptosis, and differentiation and induces the tumor suppressor 15-hydroxyprostaglandin dehydrogenase (PGDH) in other cancers. This nonrandomized interventional study assessed effects of vitamin D supplementation in Barrett's esophagus (BE). We hypothesized that vitamin D supplementation may have beneficial effects on gene expression including 15-PGDH in BE.

Methods: BE subjects with low grade or no dysplasia received vitamin D3 (cholecalciferol) 50,000 international units weekly plus a proton pump inhibitor for 12 weeks. Esophageal biopsies from normal plus metaplastic BE epithelium and blood samples were obtained before and after vitamin D supplementation. Serum 25-hydroxyvitamin D was measured to characterize vitamin D status. Esophageal gene expression was assessed using microarrays.

Results: 18 study subjects were evaluated. The baseline mean serum 25-hydroxyvitamin D level was 27 ng/mL (normal ≥30 ng/mL). After vitamin D supplementation, 25-hydroxyvitamin D levels rose significantly (median increase of 31.6 ng/mL, p<0.001). There were no significant changes in gene expression from esophageal squamous or Barrett's epithelium including 15-PGDH after supplementation.

Conclusion: BE subjects were vitamin D insufficient. Despite improved vitamin D status with supplementation, no significant alterations in gene expression profiles were noted. If vitamin D supplementation benefits BE, a longer duration or higher dose of supplementation may be needed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0184928PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5602627PMC
October 2017

Transcriptomic-metabolomic reprogramming in EGFR-mutant NSCLC early adaptive drug escape linking TGFβ2-bioenergetics-mitochondrial priming.

Oncotarget 2016 Dec;7(50):82013-82027

Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA.

The impact of EGFR-mutant NSCLC precision therapy is limited by acquired resistance despite initial excellent response. Classic studies of EGFR-mutant clinical resistance to precision therapy were based on tumor rebiopsies late during clinical tumor progression on therapy. Here, we characterized a novel non-mutational early adaptive drug-escape in EGFR-mutant lung tumor cells only days after therapy initiation, that is MET-independent. The drug-escape cell states were analyzed by integrated transcriptomic and metabolomics profiling uncovering a central role for autocrine TGFβ2 in mediating cellular plasticity through profound cellular adaptive Omics reprogramming, with common mechanistic link to prosurvival mitochondrial priming. Cells undergoing early adaptive drug escape are in proliferative-metabolic quiescent, with enhanced EMT-ness and stem cell signaling, exhibiting global bioenergetics suppression including reverse Warburg, and are susceptible to glutamine deprivation and TGFβ2 inhibition. Our study further supports a preemptive therapeutic targeting of bioenergetics and mitochondrial priming to impact early drug-escape emergence using EGFR precision inhibitor combined with broad BH3-mimetic to interrupt BCL-2/BCL-xL together, but not BCL-2 alone.
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http://dx.doi.org/10.18632/oncotarget.13307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347670PMC
December 2016

A Cross-sectional Study of KLKB1 and PRCP Polymorphisms in Patient Samples with Cardiovascular Disease.

Front Med (Lausanne) 2016 29;3:17. Epub 2016 Apr 29.

Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA; Department of Medicine, Division of Hematology-Oncology, Case Western Reserve University, Cleveland, OH, USA; University Hospitals Case Medical Center, Cleveland, OH, USA.

Plasma kallikrein formed from prekallikrein (PK) produces bradykinin from kininogens and activates factor XII. Plasma PK is activated by factors αXIIa, βXIIa, or prolylcarboxypeptidase (PRCP). A cross-sectional investigation determined if there is an association of PRCP and KLKB1 polymorphisms with cardiovascular disease (CVD). DNA was obtained from 2243 individuals from the Prevention of Events with Angiotensin Converting Enzyme trial. Two PRCP SNPs, rs7104980 and rs2298668, and two KLKB1 SNPs, rs3733402 and rs3087505, were genotyped. Logistic regression models were performed for history of diabetes, myocardial infarction, stroke, angina, angiographic coronary disease, CABG, intermittent claudication, percutaneous transluminal coronary angioplasty (PTCA), and transient ischemic attack. The PRCP SNP rs7104980 increased the odds of having a history of PTCA by 21% [odds ratio (OR) = 1.211; 95% confidence intervals (CI) = (1.008, 1.454)]; P = 0.041, but was non-significant after Bonferroni correction. Alternatively, having the G allele for rs3733402 (KLKB1 gene) decreased the odds of having a history of angiographic coronary disease by 24% [OR = 0.759; 95% CI = (0.622, 0.927)]; P = 0.007 that was statistically significant (P < 0.01) after Bonferroni correction for multiple hypothesis testing. When the best-fit model based on the Akaike information criterion controlled for age, weight, gender, hypertension, and history of angina, the G allele of KLKB1 rs3733402 that is associated with less plasma kallikrein activity correlated with reduced history of CVD.
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http://dx.doi.org/10.3389/fmed.2016.00017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850149PMC
May 2016

A Germline Variant on Chromosome 4q31.1 Associates with Susceptibility to Developing Colon Cancer Metastasis.

PLoS One 2016 11;11(1):e0146435. Epub 2016 Jan 11.

Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.

We tested for germline variants showing association to colon cancer metastasis using a genome-wide association study that compared Ashkenazi Jewish individuals with stage IV metastatic colon cancers versus those with stage I or II non-metastatic colon cancers. In a two-stage study design, we demonstrated significant association to developing metastatic disease for rs60745952, that in Ashkenazi discovery and validation cohorts, respectively, showed an odds ratio (OR) = 2.3 (P = 2.73E-06) and OR = 1.89 (P = 8.05E-04) (exceeding validation threshold of 0.0044). Significant association to metastatic colon cancer was further confirmed by a meta-analysis of rs60745952 in these datasets plus an additional Ashkenazi validation cohort (OR = 1.92; 95% CI: 1.28-2.87), and by a permutation test that demonstrated a significantly longer haplotype surrounding rs60745952 in the stage IV samples. rs60745952, located in an intergenic region on chromosome 4q31.1, and not previously associated with cancer, is, thus, a germline genetic marker for susceptibility to developing colon cancer metastases among Ashkenazi Jews.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0146435PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4709047PMC
July 2016

Induction of KIAA1199/CEMIP is associated with colon cancer phenotype and poor patient survival.

Oncotarget 2015 Oct;6(31):30500-15

Department of Medicine, Case Western Reserve University and Case Medical Center, Cleveland, OH, USA.

Genes induced in colon cancer provide novel candidate biomarkers of tumor phenotype and aggressiveness. We originally identified KIAA1199 (now officially called CEMIP) as a transcript highly induced in colon cancer: initially designating the transcript as Colon Cancer Secreted Protein 1. We molecularly characterized CEMIP expression both at the mRNA and protein level and found it is a secreted protein induced an average of 54-fold in colon cancer. Knockout of CEMIPreduced the ability of human colon cancer cells to form xenograft tumors in athymic mice. Tumors that did grow had increased deposition of hyaluronan, linking CEMIP participation in hyaluronan degradation to the modulation of tumor phenotype. We find CEMIP mRNA overexpression correlates with poorer patient survival. In stage III only (n = 31) or in combined stage II plus stage III colon cancer cases (n = 73), 5-year overall survival was significantly better (p = 0.004 and p = 0.0003, respectively) among patients with low CEMIP expressing tumors than those with high CEMIP expressing tumors. These results demonstrate that CEMIP directly facilitates colon tumor growth, and high CEMIP expression correlates with poor outcome in stage III and in stages II+III combined cohorts. We present CEMIP as a candidate prognostic marker for colon cancer and a potential therapeutic target.
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http://dx.doi.org/10.18632/oncotarget.5921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741547PMC
October 2015

Reply to Ashktorab et al.: Mutational landscape of colon cancers in African Americans.

Proc Natl Acad Sci U S A 2015 Jun 4;112(22):E2853. Epub 2015 May 4.

Case Comprehensive Cancer Center, and Department of Medicine, Case Medical Center, Case Western Reserve University, Cleveland, OH 44106; Department of Pathology.

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http://dx.doi.org/10.1073/pnas.1505059112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4460466PMC
June 2015

Novel recurrently mutated genes in African American colon cancers.

Proc Natl Acad Sci U S A 2015 Jan 12;112(4):1149-54. Epub 2015 Jan 12.

Case Comprehensive Cancer Center, and Department of Medicine, Case Medical Center, Case Western Reserve University, Cleveland, OH 44106; Department of Pathology.

We used whole-exome and targeted sequencing to characterize somatic mutations in 103 colorectal cancers (CRC) from African Americans, identifying 20 new genes as significantly mutated in CRC. Resequencing 129 Caucasian derived CRCs confirmed a 15-gene set as a preferential target for mutations in African American CRCs. Two predominant genes, ephrin type A receptor 6 (EPHA6) and folliculin (FLCN), with mutations exclusive to African American CRCs, are by genetic and biological criteria highly likely African American CRC driver genes. These previously unsuspected differences in the mutational landscapes of CRCs arising among individuals of different ethnicities have potential to impact on broader disparities in cancer behaviors.
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http://dx.doi.org/10.1073/pnas.1417064112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4313860PMC
January 2015

Discovery of common sequences absent in the human reference genome using pooled samples from next generation sequencing.

BMC Genomics 2014 Aug 16;15:685. Epub 2014 Aug 16.

Center for Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, OH, USA.

Background: Sequences up to several megabases in length have been found to be present in individual genomes but absent in the human reference genome. These sequences may be common in populations, and their absence in the reference genome may indicate rare variants in the genomes of individuals who served as donors for the human genome project. As the reference genome is used in probe design for microarray technology and mapping short reads in next generation sequencing (NGS), this missing sequence could be a source of bias in functional genomic studies and variant analysis. One End Anchor (OEA) and/or orphan reads from paired-end sequencing have been used to identify novel sequences that are absent in reference genome. However, there is no study to investigate the distribution, evolution and functionality of those sequences in human populations.

Results: To systematically identify and study the missing common sequences (micSeqs), we extended the previous method by pooling OEA reads from large number of individuals and applying strict filtering methods to remove false sequences. The pipeline was applied to data from phase 1 of the 1000 Genomes Project. We identified 309 micSeqs that are present in at least 1% of the human population, but absent in the reference genome. We confirmed 76% of these 309 micSeqs by comparison to other primate genomes, individual human genomes, and gene expression data. Furthermore, we randomly selected fifteen micSeqs and confirmed their presence using PCR validation in 38 additional individuals. Functional analysis using published RNA-seq and ChIP-seq data showed that eleven micSeqs are highly expressed in human brain and three micSeqs contain transcription factor (TF) binding regions, suggesting they are functional elements. In addition, the identified micSeqs are absent in non-primates and show dynamic acquisition during primate evolution culminating with most micSeqs being present in Africans, suggesting some micSeqs may be important sources of human diversity.

Conclusions: 76% of micSeqs were confirmed by a comparative genomics approach. Fourteen micSeqs are expressed in human brain or contain TF binding regions. Some micSeqs are primate-specific, conserved and may play a role in the evolution of primates.
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http://dx.doi.org/10.1186/1471-2164-15-685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4148959PMC
August 2014

Genetic variation in 15-hydroxyprostaglandin dehydrogenase and colon cancer susceptibility.

PLoS One 2013 22;8(5):e64122. Epub 2013 May 22.

Department of Family Medicine and Community Health, Case Western Reserve University and University Hospitals Case Medical Center, Cleveland, Ohio, USA.

Background: 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is a metabolic antagonist of COX-2, catalyzing the degradation of inflammation mediator prostaglandin E2 (PGE2) and other prostanoids. Recent studies have established the 15-PGDH gene as a colon cancer suppressor.

Methods: We evaluated 15-PDGH as a colon cancer susceptibility locus in a three-stage design. We first genotyped 102 single-nucleotide polymorphisms (SNPs) in the 15-PGDH gene, spanning ∼50 kb up and down-stream of the coding region, in 464 colon cancer cases and 393 population controls. We then genotyped the same SNPs, and also assayed the expression levels of 15-PGDH in colon tissues from 69 independent patients for whom colon tissue and paired germline DNA samples were available. In the final stage 3, we genotyped the 9 most promising SNPs from stages 1 and 2 in an independent sample of 525 cases and 816 controls (stage 3).

Results: In the first two stages, three SNPs (rs1365611, rs6844282 and rs2332897) were statistically significant (p<0.05) in combined analysis of association with risk of colon cancer and of association with 15-PGDH expression, after adjustment for multiple testing. For one additional SNP, rs2555639, the T allele showed increased cancer risk and decreased 15-PGDH expression, but just missed statistical significance (p-adjusted = 0.063). In stage 3, rs2555639 alone showed evidence of association with an odds ratio (TT compared to CC) of 1.50 (95% CI = 1.05-2.15, p = 0.026).

Conclusions: Our data suggest that the rs2555639 T allele is associated with increased risk of colon cancer, and that carriers of this risk allele exhibit decreased expression of 15-PGDH in the colon.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0064122PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3661460PMC
April 2014

Global mutational profiling of formalin-fixed human colon cancers from a pathology archive.

Mod Pathol 2012 Dec 10;25(12):1599-608. Epub 2012 Aug 10.

J Craig Venter Institute, San Diego, CA, USA.

The advent of Next-Generation sequencing technologies, which significantly increases the throughput and reduces the cost of large-scale sequencing efforts, provides an unprecedented opportunity for discovery of novel gene mutations in human cancers. However, it remains a challenge to apply Next-Generation technologies to DNA extracted from formalin-fixed paraffin-embedded cancer specimens. We describe here the successful development of a custom DNA capture method using Next-Generation for detection of 140 driver genes in five formalin-fixed paraffin-embedded human colon cancer samples using an improved extraction process to produce high-quality DNA. Isolated DNA was enriched for targeted exons and sequenced using the Illumina Next-Generation platform. An analytical pipeline using 3 software platforms to define single-nucleotide variants was used to evaluate the data output. Approximately 250 × average coverage was obtained with >96% of target bases having at least 30 sequence reads. Results were then compared with previously performed high-throughput Sanger sequencing. Using an algorithm of needing a positive call from all three callers to give a positive result, 98% of the verified Sanger sequencing somatic driver gene mutations were identified by our method with a specificity of 90%. In all, 13 insertions and deletions identified by Next-Generation were confirmed by Sanger sequencing. We also applied this technology to two components of a biphasic colon cancer, which had strikingly differing histology. Remarkably, no new driver gene mutation accumulation was identified in the more undifferentiated component. Applying this method to profiling of formalin-fixed paraffin-embedded colon cancer tissue samples yields equivalent sensitivity and specificity for mutation detection as Sanger sequencing of matched cell lines derived from these cancers. This method directly enables high-throughput comprehensive mutational profiling of colon cancer samples, and is easily extendable to enable targeted sequencing from formalin-fixed paraffin-embedded material for other tumor types.
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http://dx.doi.org/10.1038/modpathol.2012.121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3697090PMC
December 2012

How do alignment programs perform on sequencing data with varying qualities and from repetitive regions?

BioData Min 2012 Jun 18;5(1). Epub 2012 Jun 18.

Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, OH, 44106, USA.

Background: Next-generation sequencing technologies generate a significant number of short reads that are utilized to address a variety of biological questions. However, quite often, sequencing reads tend to have low quality at the 3' end and are generated from the repetitive regions of a genome. It is unclear how different alignment programs perform under these different cases. In order to investigate this question, we use both real data and simulated data with the above issues to evaluate the performance of four commonly used algorithms: SOAP2, Bowtie, BWA, and Novoalign.

Methods: The performance of different alignment algorithms are measured in terms of concordance between any pair of aligners (for real sequencing data without known truth) and the accuracy of simulated read alignment.

Results: Our results show that, for sequencing data with reads that have relatively good quality or that have had low quality bases trimmed off, all four alignment programs perform similarly. We have also demonstrated that trimming off low quality ends markedly increases the number of aligned reads and improves the consistency among different aligners as well, especially for low quality data. However, Novoalign is more sensitive to the improvement of data quality. Trimming off low quality ends significantly increases the concordance between Novoalign and other aligners. As for aligning reads from repetitive regions, our simulation data show that reads from repetitive regions tend to be aligned incorrectly, and suppressing reads with multiple hits can improve alignment accuracy.

Conclusions: This study provides a systematic comparison of commonly used alignment algorithms in the context of sequencing data with varying qualities and from repetitive regions. Our approach can be applied to different sequencing data sets generated from different platforms. It can also be utilized to study the performance of other alignment programs.
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http://dx.doi.org/10.1186/1756-0381-5-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3414812PMC
June 2012

H3K4me3 inversely correlates with DNA methylation at a large class of non-CpG-island-containing start sites.

Genome Med 2012 May 28;4(5):47. Epub 2012 May 28.

Department of Genetics and Genome Sciences, Case Western Reserve University, 10900 Euclid Ave, Cleveland, OH 44106, USA.

Background: In addition to mutations, epigenetic silencing of genes has been recognized as a fundamental mechanism that promotes human carcinogenesis. To date, characterization of epigenetic gene silencing has largely focused on genes in which silencing is mediated by hypermethylation of promoter-associated CpG islands, associated with loss of the H3K4me3 chromatin mark. Far less is known about promoters lacking CpG-islands or genes that are repressed by alternative mechanisms.

Methods: We performed integrative ChIP-chip, DNase-seq, and global gene expression analyses in colon cancer cells and normal colon mucosa to characterize chromatin features of both CpG-rich and CpG-poor promoters of genes that undergo silencing in colon cancer.

Results: Epigenetically repressed genes in colon cancer separate into two classes based on retention or loss of H3K4me3 at transcription start sites. Quantitatively, of transcriptionally repressed genes that lose H3K4me3 in colon cancer (K4-dependent genes), a large fraction actually lacks CpG islands. Nonetheless, similar to CpG-island containing genes, cytosines located near the start sites of K4-dependent genes become DNA hypermethylated, and repressed K4-dependent genes can be reactivated with 5-azacytidine. Moreover, we also show that when the H3K4me3 mark is retained, silencing of CpG island-associated genes can proceed through an alternative mechanism in which repressive chromatin marks are recruited.

Conclusions: H3K4me3 equally protects from DNA methylation at both CpG-island and non-CpG island start sites in colon cancer. Moreover, the results suggest that CpG-rich genes repressed by loss of H3K4me3 and DNA methylation represent special instances of a more general epigenetic mechanism of gene silencing, one in which gene silencing is mediated by loss of H3K4me3 and methylation of non-CpG island promoter-associated cytosines.
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http://dx.doi.org/10.1186/gm346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3506913PMC
May 2012

DNA mismatch repair (MMR)-dependent 5-fluorouracil cytotoxicity and the potential for new therapeutic targets.

Br J Pharmacol 2009 Oct 23;158(3):679-92. Epub 2009 Sep 23.

Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, USA.

The metabolism and efficacy of 5-fluorouracil (FUra) and other fluorinated pyrimidine (FP) derivatives have been intensively investigated for over fifty years. FUra and its antimetabolites can be incorporated at RNA- and DNA-levels, with RNA level incorporation provoking toxic responses in human normal tissue, and DNA-level antimetabolite formation and incorporation believed primarily responsible for tumour-selective responses. Attempts to direct FUra into DNA-level antimetabolites, based on mechanism-of-action studies, have led to gradual improvements in tumour therapy. These include the use of leukovorin to stabilize the inhibitory thymidylate synthase-5-fluoro-2'-deoxyuridine 5' monophoshate (FdUMP)-5,10-methylene tetrahydrofolate (5,10-CH(2)FH(4)) trimeric complex. FUra incorporated into DNA also contributes to antitumour activity in preclinical and clinical studies. This review examines our current state of knowledge regarding the mechanistic aspects of FUra:Gua lesion detection by DNA mismatch repair (MMR) machinery that ultimately results in lethality. MMR-dependent direct cell death signalling or futile cycle responses will be discussed. As 10-30% of sporadic colon and endometrial tumours display MMR defects as a result of human MutL homologue-1 (hMLH1) promoter hypermethylation, we discuss the use and manipulation of the hypomethylating agent, 5-fluorodeoxycytidine (FdCyd), and our ability to manipulate its metabolism using the cytidine or deoxycytidylate (dCMP) deaminase inhibitors, tetrahydrouridine or deoxytetrahydrouridine, respectively, as a method for re-expression of hMLH1 and re-sensitization of tumours to FP therapy.
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http://dx.doi.org/10.1111/j.1476-5381.2009.00423.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2765589PMC
October 2009

A role for DNA mismatch repair in sensing and responding to fluoropyrimidine damage.

Oncogene 2003 Oct;22(47):7376-88

Laboratory of Molecular Stress Responses, Department of Radiation Oncology, Case Western Reserve University, Biomedical Research Building 326-East, 2109 Adelbert Road, Cleveland, OH 44106-4942, USA.

The phenomenon of damage tolerance, whereby cells incur DNA lesions that are nonlethal, largely ignored, but highly mutagenic, appears to play a key role in carcinogenesis. Typically, these lesions are generated by alkylation of DNA or incorporation of base analogues. This tolerance is usually a result of the loss of specific DNA repair processes, most often DNA mismatch repair (MMR). The availability of genetically matched MMR-deficient and -corrected cell systems allows dissection of the consequences of this unrepaired damage in carcinogenesis as well as the elucidation of cell cycle checkpoint responses and cell death consequences. Recent data indicate that MMR plays an important role in detecting damage caused by fluorinated pyrimidines (FPs) and represents a repair system that is probably not the primary system for detecting damage caused by these agents, but may be an important system for correcting key mutagenic lesions that could initiate carcinogenesis. In fact, clinical studies have shown that there is no benefit of FP-based adjuvant chemotherapy in colon cancer patients exhibiting microsatellite instability, a hallmark of MMR deficiency. MMR-mediated damage tolerance and futile cycle repair processes are discussed, as well as possible strategies using FPs to exploit these systems for improved anticancer therapy.
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http://dx.doi.org/10.1038/sj.onc.1206941DOI Listing
October 2003

Reduced expression of NFAT-associated genes in UCB versus adult CD4+ T lymphocytes during primary stimulation.

Blood 2003 Dec 28;102(13):4608-17. Epub 2003 Aug 28.

Case Western Reserve University, 11100 Euclid Ave, Wearn 433, Cleveland, OH 44106-5065, USA.

The cellular and molecular mechanisms underlying the blunted allo-responsiveness of umbilical cord blood (UCB) T cells have not been fully elucidated. Protein expression of NFATc2 (nuclear factor of activated T cells c2), a critical transcription factor necessary for up-regulation of multiple cytokines known to amplify T-cell allogeneic responses, is reduced in UCB T cells. Affymetrix oligonucleotide microarrays were used to compare gene expression of primary purified CD4+ UCB T cells to adult peripheral blood CD4+ T cells (AB) at baseline, 6, and 16 hours of primary stimulation. NFAT-regulated genes exhibited lower expression in UCB CD4+ T cells including the following: granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin 3 (IL-3), IL-4, IL-5, IL-13, IL-2 receptor alpha (IL-2Ralpha; CD25), CD40L, and macrophage inflammatory protein 1 alpha (MIP-1alpha). Transcription factors involved in the NFAT pathway including C/EBPbeta, JunB, and Fosl1 (Fra-1), as well as Th1- and Th2-related transcription factors STAT4 (signal transducers and activators of transcription 4), T-bet, and c-maf showed reduced expression in UCB compared with AB during primary stimulation. Reduced cytokine, chemokine, and receptor expression was also found in UCB. Gene array data were confirmed using RNase protection assays, flow cytometry, and quantitative multiplexed cytokine measurements. Reduced global expression of NFAT-associated genes, as well as cytokines and chemokines, in UCB CD4+ T cells may contribute to the decreased graft-versus-host disease (GVHD) observed after UCB transplantation.
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http://dx.doi.org/10.1182/blood-2003-05-1732DOI Listing
December 2003

Mismatch repair and drug responses in cancer.

Drug Resist Updat 1999 Oct;2(5):295-306

Department of Medicine, Case Western Reserve University, University Hospitals of Cleveland, Cleveland, OH

Defects in mismatch repair contribute to development of approximately 15% of colon cancers and to origination of endometrial, gastric and other cancers. Tumors with defects in mismatch repair exhibit marked resistance to alkylators and a variety of anticancer agents that modify DNA to create substrates for the mismatch repair system. These altered drug responses appear to derive from requirements for mismatch repair proteins in signalling apoptosis, altered cell cycle checkpoint behaviour and/or loss of mismatch repair dependent toxicity arising from futile repair cycling. Altered repair mechanisms for mismatched substrates in mismatch repair defective tumors provide both challenges for development of tumor-phenotype-screening methodologies to assure appropriate therapy is administered for these cancers and foci for development of new therapy approaches that capitalize on modified drug responses in mismatch repair- defective cells. Copyright 1999 Harcourt Publishers Ltd.
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http://dx.doi.org/10.1054/drup.1999.0099DOI Listing
October 1999