Several components of the Wnt signaling cascade have been shown to function either as tumor suppressor proteins or as oncogenes in multiple human cancers, underscoring the relevance of this pathway in oncogenesis and the need for further investigation of Wnt signaling components as potential targets for cancer therapy. Here, using expression profiling analysis as well as in vitro and in vivo functional studies, we show that the Wnt pathway component BCL9 is a novel oncogene that is aberrantly expressed in human multiple myeloma as well as colon carcinoma. We show that BCL9 enhances β-catenin–mediated transcriptional activity regardless of the mutational status of the Wnt signaling components and increases cell proliferation, migration, invasion, and the metastatic potential of tumor cells by promoting loss of epithelial and gain of mesenchymal-like phenotype. Most importantly, BCL9 knockdown significantly increased the survival of xenograft mouse models of cancer by reducing tumor load, metastasis, and host angiogenesis through down-regulation of c-Myc, cyclin D1, CD44, and vascular endothelial growth factor expression by tumor cells. Together, these findings suggest that deregulation of BCL9 is an important contributing factor to tumor progression. The pleiotropic roles of BCL9 reported in this study underscore its value as a drug target for therapeutic intervention in several malignancies associated with aberrant Wnt signaling.
Aspirin use reduces the risk of colorectal neoplasia, at least in part, through inhibition of prostaglandin-endoperoxide synthase 2 (PTGS2, cyclooxygenase 2)-related pathways. Hydroxyprostaglandin dehydrogenase 15-(NAD) (15-PGDH, HPGD) is downregulated in colorectal cancers and functions as a metabolic antagonist of PTGS2. We hypothesized that the effect of aspirin may be antagonized by low 15-PGDH expression in the normal colon. In the Nurses’ Health Study and the Health Professionals Follow-up Study, we collected data on aspirin use and other risk factors every two years and followed up participants for diagnoses of colorectal cancer. Duplication-method Cox proportional, multivariable-adjusted, cause-specific hazards regression for competing risks data was used to compute hazard ratios (HRs) for incident colorectal cancer according to 15-PGDH mRNA expression level measured in normal mucosa from colorectal cancer resections. Among 127,865 participants, we documented 270 colorectal cancer cases that developed during 3,166,880 person-years of follow-up and from which we could assess 15-PGDH expression. Compared with nonuse, regular aspirin use was associated with lower risk of colorectal cancer that developed within a background of colonic mucosa with high 15-PGDH expression (multivariable HR=0.49; 95% CI, 0.34–0.71), but not with low 15-PGDH expression (multivariable HR=0.90; 95% CI, 0.63–1.27) (P for heterogeneity=0.018). Regular aspirin use was associated with lower incidence of colorectal cancers arising in association with high 15-PGDH expression, but not with low 15-PGDH expression in normal colon mucosa. This suggests that 15-PGDH expression level in normal colon mucosa may serve as a biomarker which may predict stronger benefit from aspirin chemoprevention.
colon cancer; rectal cancer; adenocarcinoma; epigenomics; epigenetics
Background & Aims
15-hydroxprostaglandin dehydrogenase (15-PGDH) mediates a colon neoplasia suppressor pathway, acting through metabolic antagonism of cyclooxygenase (COX)-mediated colon carcinogenesis. To determine whether the colon tumor prevention activity of 15-PGDH acts as a constant or variable effect among individuals, we determined whether 15-PGDH levels remains stable over subsite and time in the human colon, determined the extent of differences in 15-PGDH levels between different individuals, and determined whether or not 15-PGDH modulation mediates any part of the anti-colon tumor effect of aspirin.
Using real-time PCR, we measured 15-PGDH mRNA, determining the correlation of 15-PGDH level in replicate colon biopsies, in biopsies from throughout the length of the colon, in repeat biopsies taken 4 months apart, and in paired biopsies of individuals taken before and after aspirin treatment, and by western for 15-PGDH protein in mice.
Colonic 15-PGDH levels varied 4.4-fold across the human population. Within individuals, 15-PGDH levels proved highly reproducible (r=0.81 in duplicate biopsies) and stable along the length of the colon, with average 15-PGDH levels deviating by only 17% from rectum to cecum. An individual’s 15-PGDH levels are also highly stable over time, with a median coefficient of variation over a 4-month interval of only 12%. Last, colonic 15-PGDH levels proved resistant to alteration by aspirin, with only a 10% difference in 15-PGDH levels measured before and after aspirin treatment.
15-PGDH levels vary across the population in a stable and reproducible manner, and are resistant to alteration by aspirin. 15-PGDH represents an independent target for modulation by candidate colon tumor chemopreventive agents.
Colon Cancer; 15-PGDH; NSAIDs; Biomarker
Colitis-associated colon cancer (CAC) develops as a result of inflammation-induced epithelial transformation, which occurs in response to inflammatory cytokine-dependent downregulation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and subsequent suppression of prostaglandin metabolism. Agents that both enhance 15-PGDH expression and suppress cyclooxygenase-2 (COX-2) production may more effectively prevent CAC. Synthetic triterpenoids are a class of small molecules that suppress COX-2 as well as inflammatory cytokine signaling. Here, we found that administration of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-C28-methyl ester (CDDO-Me) suppresses CAC in mice. In a spontaneous, inflammation-driven intestinal neoplasia model, deletion of Smad4 specifically in T cells led to progressive production of inflammatory cytokines, including TNF-α, IFN-γ, iNOS, IL-6, IL-1β; as well as activation of STAT1 and STAT3; along with suppression of 15-PGDH expression. Oral administration of CDDO-Me to mice with SMAD4-deficient T cells increased survival and suppressed intestinal epithelial neoplasia by decreasing production of inflammatory mediators and increasing expression of 15-PGDH. Induction of 15-PGDH by CDDO-Me was dose dependent in epithelial cells and was abrogated following treatment with TGF-β signaling inhibitors in vitro. Furthermore, CDDO-Me–dependent 15-PGDH induction was not observed in Smad3–/– mice. Similarly, CDDO-Me suppressed azoxymethane plus dextran sodium sulfate–induced carcinogenesis in wild-type animals, highlighting the potential of small molecules of the triterpenoid family as effective agents for the chemoprevention of CAC in humans.
PIK3CA, which encodes the p110α catalytic subunit of phosphatidylinositol 3-kinase α, is frequently mutated in human cancers. Most of these mutations occur at two hot-spots E545K and H1047R located in the helical domain and the kinase domain, respectively. Here, we report that p110α E545K, but not p110α H1047R, gains the ability to associate with IRS1 independent of the p85 regulatory subunit, thereby rewiring this oncogenic signaling pathway. Disruption of the IRS1-p110α E545K interaction destabilizes the p110α protein, reduces AKT phosphorylation, and slows xenograft tumor growth of a cancer cell line expressing p110α E545K. Moreover, a hydrocarbon-stapled peptide that disrupts this interaction inhibits the growth of tumors expressing p110α E545K.
The purpose of this study is to determine the genetic frequency of GNAS activating mutations in colorectal cancer and the corresponding pathology of GNAS mutant tumors. Oncogenic mutations in GNAS have been described in a number of neoplasms including those of the pituitary, kidney, pancreas, and, more recently, in colon cancer. To ascertain the frequency in colon cancer we employed a sensitive pyrosequencing platform for mutation detection of the R201C and R201H GNAS hotspots in tumor samples representing all clinical stages. We additionally assayed for KRAS and BRAF mutations as previous reports have shown that these often co-occur with activating GNAS mutations. Of the 428 colon tumors assayed, mutations in GNAS were present in 10 of the samples (2.3%), indicating this is a significant, albeit infrequent, mutation in colorectal tumors. Nine GNAS mutant tumors (90%) harbored concomitant activating mutations in either the KRAS or BRAF oncogene, which was significantly greater than the mutation frequency of these genes in the tumor population (56%, p<0.0305). All ten of the GNAS mutant tumors arose in the right (proximal) colon (p<0.007), and 7 of 8 reviewed cases exhibited a marked villous morphology. Taken together, these data indicate that GNAS mutant colon tumors commonly have synchronous mutations in KRAS or BRAF, are right-sided in location, and are associated with a villous morphology.
The roles of cholecystokinin 2 receptor (CCK2R) in numerous physiologic processes in the gastrointestinal tract and central nervous system are ‘well documented. There has been some evidence that CCK2R alterations play a role in cancers, but the functional significance of these alterations for tumorigenesis is unknown. We have identified six mutations in CCK2R among a panel of 140 colorectal cancers and 44 gastric cancers. We show that these mutations increase receptor activity, activate multiple downstream signaling pathways, increase cell migration, and promote angiogenesis. Our findings suggest that somatic mutations in CCK2R may promote tumorigenesis through deregulated receptor activity and highlight the importance of evaluating CCK2R inhibitors to block both the normal and mutant forms of the receptor.
Protein tyrosine phosphatase non-receptor type 14 (PTPN14) is frequently mutated in a variety of human cancers. However, the cell signaling pathways regulated by PTPN14 largely remain to be elucidated. Here, we identify a list of potential substrates of PTPN14 using a phospho-proteomic approach. We show that p130Cas is a direct substrate of PTPN14 and that PTPN14 specifically regulates p130Cas phosphorylation at tyrosine residue 128 (Y128) in colorectal cancer (CRC) cells. We engineered CRC cells homozygous for a p130Cas Y128F knock-in mutant and found that these cells exhibit significantly reduced migration and colony formation, impaired anchorage-independent growth, slower xenograft tumor growth in nude mice, and have decreased phosphorylation of AKT. Furthermore, we demonstrate that SRC phosphorylates p130Cas Y128 and that CRC cell lines harboring high levels of pY128 Cas are more sensitive to SRC family kinase inhibitor Dasatinib. These findings suggest that p130Cas Y128 phosphorylation may be exploited as a predictive marker for Dasatinib response in cancer patients. In aggregate, our studies reveal a novel signaling pathway that plays an important role in colorectal tumorigenesis.
PTPN14; p130Cas; tumorigenesis; colorectal cancer
Cancer is characterized by gene expression aberrations. Studies have largely focused on coding sequences and promoters, despite the fact that distal regulatory elements play a central role in controlling transcription patterns. Here we utilize the histone mark H3K4me1 to analyze gain and loss of enhancer activity genome wide in primary colon cancer lines relative to normal colon crypts. We identified thousands of variant enhancer loci (VELs) that comprise a signature that is robustly predictive of the in vivo colon cancer transcriptome. Furthermore, VELs are enriched in haplotype blocks containing colon cancer genetic risk variants, implicating these genomic regions in colon cancer pathogenesis. We propose that reproducible changes in the epigenome at enhancer elements drive a unique transcriptional program to promote colon carcinogenesis.
NTRK3 is a member of the neurotrophin receptor family and regulates cell survival. It appears to be a dependence receptor, and thus has the potential to act as an oncogene or as a tumor suppressor gene. NTRK3 is a receptor for NT-3 and when bound to NT-3 it induces cell survival, but when NT-3 free, it induces apoptosis. We identified aberrantly methylated NTRK3 in colorectal cancers through a genome-wide screen for hypermethylated genes. This discovery led us to assess whether NTRK3 could be a tumor suppressor gene in the colon. NTRK3 is methylated in 60% of colon adenomas and 67% of colon adenocarcinomas. NTRK3 methylation suppresses NTRK3 expression. Reconstitution of NTRK3 induces apoptosis in colorectal cancers, if NT-3 is absent. Furthermore, the loss of NTRK3 expression associates with neoplastic transformation in vitro and in vivo. We also found that a naturally occurring mutant NTRK3 found in human colorectal cancer inhibits the tumor suppressor activity of NTRK3. In summary, our findings suggest NTRK3 is a conditional tumor suppressor gene that is commonly inactivated in colorectal cancer by both epigenetic and genetic mechanisms whose function in the pathogenesis of colorectal cancer depends on the expression status of its ligand, NT-3.
NTRK3 is a neurotrophin receptor and appears to be a dependence receptor in certain tissues. NTRK3 has been previously shown to be an oncogene in breast cancer and possibly hepatocellular carcinoma. Through a genome-wide methylation screen, we unexpectedly found that NTRK3 is commonly methylated in colorectal cancers but not in normal colon samples, which led us to assess whether NTRK3 could be a tumor suppressor gene in the colon. We now demonstrate that NTRK3 is frequently methylated in colorectal adenomas and cancers. Induced NTRK3 expression in the absence of its ligand, NT-3, causes apoptosis and suppresses in vitro anchorage-independent colony formation and in vivo tumor growth. Reintroduction of NT-3 releases colon cancer cells from NTRK3-mediated apoptosis, which is consistent with NTRK3 being a dependence receptor in the colon. Finally, somatic mutations of NTRK3 have been observed in primary human colorectal cancer. We provide evidence that a subset of these mutations inactivate tumor suppressor activities of NTRK3. These findings suggest that NTRK3 is a conditional tumor suppressor gene in the colon that is inactivated by both genetic and epigenetic mechanisms and whose function in the pathogenesis of colorectal cancer depends on the expression status of its ligand, NT-3.
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 5 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 250x average coverage was obtained with >96% of target bases having at least 30 sequence reads. Results were then compared to previously performed high throughput Sanger sequencing. Using an algorithm of needing a positive call from all 3 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%. 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.
next generation sequencing; colon cancer; driver gene mutations
One of the key questions about genomic alterations in cancer is whether they are functional in the sense of contributing to the selective advantage of tumor cells. The frequency with which an alteration occurs might reflect its ability to increase cancer cell growth, or alternatively, enhanced instability of a locus may increase the frequency with which it is found to be aberrant in tumors, regardless of oncogenic impact. Here we’ve addressed this on a genome-wide scale for cancer-associated focal deletions, which are known to pinpoint both tumor suppressor genes (tumor suppressors) and unstable loci. Based on DNA copy number analysis of over one-thousand human cancers representing ten different tumor types, we observed five loci with focal deletion frequencies above 5%, including the A2BP1 gene at 16p13.3 and the MACROD2 gene at 20p12.1. However, neither RNA expression nor functional studies support a tumor suppressor role for either gene. Further analyses suggest instead that these are sites of increased genomic instability and that they resemble common fragile sites (CFS). Genome-wide analysis revealed properties of CFS-like recurrent deletions that distinguish them from deletions affecting tumor suppressor genes, including their isolation at specific loci away from other genomic deletion sites, a considerably smaller deletion size, and dispersal throughout the affected locus rather than assembly at a common site of overlap. Additionally, CFS-like deletions have less impact on gene expression and are enriched in cell lines compared to primary tumors. We show that loci affected by CFS-like deletions are often distinct from known common fragile sites. Indeed, we find that each tumor tissue type has its own spectrum of CFS-like deletions, and that colon cancers have many more CFS-like deletions than other tumor types. We present simple rules that can pinpoint focal deletions that are not CFS-like and more likely to affect functional tumor suppressors.
The question of molecular heterogeneity and of tumoral phenotype in cancer remains unresolved. To understand the underlying molecular basis of this phenomenon, we analyzed genome-wide expression data of colon cancer metastasis samples, as these tumors are the most advanced and hence would be anticipated to be the most likely heterogeneous group of tumors, potentially exhibiting the maximum amount of genetic heterogeneity. Casting a statistical net around such a complex problem proves difficult because of the high dimensionality and multi-collinearity of the gene expression space, combined with the fact that genes act in concert with one another and that not all genes surveyed might be involved. We devise a strategy to identify distinct subgroups of samples and determine the genetic/molecular signature that defines them. This involves use of the local sparse bump hunting algorithm, which provides a much more optimal and biologically faithful transformed space within which to search for bumps. In addition, thanks to the variable selection feature of the algorithm, we derived a novel sparse gene expression signature, which appears to divide all colon cancer patients into two populations: a population whose expression pattern can be molecularly encompassed within the bump and an outlier population that cannot be. Although all patients within any given stage of the disease, including the metastatic group, appear clinically homogeneous, our procedure revealed two subgroups in each stage with distinct genetic/molecular profiles. We also discuss implications of such a finding in terms of early detection, diagnosis and prognosis.
local sparse bump hunting; mixture density; class discovery; colon cancer patient subtyping; early diagnosis and prognosis
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.
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).
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).
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.
We have previously established aberrant DNA methylation of Vimentin exon-1 (VIM methylation) as a common epigenetic event in colon cancer and as a biomarker for detecting colon neoplasia. We now examine VIM methylation in neoplasia of the upper gastrointestinal tract.
Using a quantitative real-time Methylation-Specific PCR assay we tested for VIM methylation in archival specimens of esophageal and gastric neoplasia.
We find that acquisition of aberrant VIM methylation is highly common in these neoplasms, but largely absent in controls. The highest frequency of VIM methylation was detected in lesions of the distal esophagus, including 91% of Barrett’s esophagus (BE, n=11), 100% of high grade dysplasia (HGD, n=5), and 81% of esophageal adenocarcinoma (EAC, n=26), but absent in controls (n=9). VIM methylation similarly was detected in 87% of signet ring (n=15) and 53% of intestinal type gastric cancers (n=17). Moreover, in tests of cytology brushings VIM methylation proved detectable in 100% of BE cases (n=7), 100% of HGD cases (n=4), and 83% of EAC cases (n=18), but was absent in all controls (n=5).
These findings establish aberrant VIM methylation as a highly common epigenetic alteration in neoplasia of the upper gastrointestinal tract, and demonstrate that Barrett’s esophagus, even without dysplasia, already contains epigenetic alterations characteristic of adenocarcinoma.
These findings suggest VIM methylation as a biomarker of upper gastrointestinal neoplasia with potential for development as molecular cytology in esophageal screening.
Barrett’s Esophagus; Esophageal Cancer; Gastric Cancer; Vimentin; Methylation
Genetic influences may be discerned in families that have multiple affected members and may manifest as an earlier age of cancer diagnosis. In this study we determine whether cancers develop at an earlier age in multiplex Familial Barrett’s Esophagus (FBE) kindreds, defined by 3 or more members affected by Barrett’s esophagus (BE) or esophageal adenocarcinoma (EAC).
Information on BE/EAC risk factors and family history was collected from probands at eight tertiary care academic hospitals. Age of cancer diagnosis and other risk factors were compared between non-familial (no affected relatives), duplex (two affected relatives), and multiplex (three or more affected relatives) FBE kindreds.
The study included 830 non-familial, 274 duplex and 41 multiplex FBE kindreds with 274, 133 and 43 EAC and 566, 288 and 103 BE cases, respectively. Multivariable mixed models adjusting for familial correlations showed that multiplex kindreds were associated with a younger age of cancer diagnosis (p = 0.0186). Median age of cancer diagnosis was significantly younger in multiplex compared to duplex and non-familial kindreds (57 vs. 62 vs. 63 yrs, respectively, p = 0.0448). Mean body mass index (BMI) was significantly lower in multiplex kindreds (p = 0.0033) as was smoking (p < 0.0001), and reported regurgitation (p = 0.0014).
Members of multiplex FBE kindreds develop EAC at an earlier age compared to non-familial EAC cases. Multiplex kindreds do not have a higher proportion of common risk factors for EAC, suggesting that this aggregation might be related to a genetic factor.
These findings indicate that efforts to identify susceptibility genes for BE and EAC will need to focus on multiplex kindreds.
Esophageal adenocarcinoma; Barrett’s esophagus; genetics; family history
There is a critical need to identify molecular markers that can reliably aid in stratifying esophageal adenocarcinoma (EAC) risk in patients with Barrett's esophagus. MicroRNAs (miRNA/miR) are one such class of biomolecules. In the present cross-sectional study, we characterized miRNA alterations in progressive stages of neoplastic development, i.e., metaplasia–dysplasia–adenocarcinoma, with an aim to identify candidate miRNAs potentially associated with progression. Using next generation sequencing (NGS) as an agnostic discovery platform, followed by quantitative real-time PCR (qPCR) validation in a total of 20 EACs, we identified 26 miRNAs that are highly and frequently deregulated in EACs (≥4-fold in >50% of cases) when compared to paired normal esophageal squamous (nSQ) tissue. We then assessed the 26 EAC-derived miRNAs in laser microdissected biopsy pairs of Barrett's metaplasia (BM)/nSQ (n = 15), and high-grade dysplasia (HGD)/nSQ (n = 14) by qPCR, to map the timing of deregulation during progression from BM to HGD and to EAC. We found that 23 of the 26 candidate miRNAs were deregulated at the earliest step, BM, and therefore noninformative as molecular markers of progression. Two miRNAs, miR-31 and –31*, however, showed frequent downregulation only in HGD and EAC cases suggesting association with transition from BM to HGD. A third miRNA, miR-375, showed marked downregulation exclusively in EACs and in none of the BM or HGD lesions, suggesting its association with progression to invasive carcinoma. Taken together, we propose miR-31 and –375 as novel candidate microRNAs specifically associated with early- and late-stage malignant progression, respectively, in Barrett's esophagus.
Mutations in the chromatin remodeling gene ARID1A have recently been identified in the majority of ovarian clear cell carcinomas. To determine the prevalence of mutations in other tumor types, we evaluated 759 malignant neoplasms including those of the pancreas, breast, colon, stomach, lung, prostate, brain and blood (leukemias). We identified truncating mutations in 6% of the neoplasms studied; non-truncating somatic mutations were identified in an additional 0.4% of neoplasms. Mutations were most commonly found in gastrointestinal samples with 12 of 119 (10%) colorectal and 10 of 100 (10%) gastric neoplasms, respectively, harboring changes. More than half of the mutated colorectal and gastric cancers displayed microsatellite instability and the mutations in these tumors were out-of-frame insertions or deletions at mononucleotide repeats. Mutations were also identified in 2% to 8% of tumors of the pancreas, breast, brain (medulloblastomas), prostate, and lung, and none of these tumors displayed microsatellite instability. These findings suggest that the aberrant chromatin remodeling consequent to ARID1A inactivation contributes to a variety of different types of neoplasms.
ARID1A; cancer; chromatin remodeling
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.
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.
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.
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.
Next generation sequencing; Alignment; Sequencing quality; SOAP2; Bowtie; BWA; Novoalign
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.
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.
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.
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.
The presence of hundreds of copies of mitochondrial (mt) DNA in each human cell poses a challenge for complete characterization of mtDNA genomes by conventional sequencing technologies1. Here, we describe digital sequencing of mtDNA genomes using massively parallel sequencing-by-synthesis. Though the mtDNA of human cells is considered to be homogeneous, we found widespread heterogeneity (heteroplasmy) in the mtDNA of normal human cells. Moreover, the frequency of heteroplasmic variants among different tissues of the same individual varied considerably. In addition to the variants identified in normal tissues, cancer cells harbored additional homoplasmic and heteroplasmic mutations that could also be detected in patient plasma. These studies provide new insights into the nature and variability of mtDNA sequences and have intriguing implications for mitochondrial processes during embryogenesis, cancer biomarker development, and forensic analysis. In particular, they demonstrate that individual humans are characterized by a complex mixture of related mitochondrial genotypes rather than a single genotype.
DNA methyltransferase 1 (DNMT1) is the primary enzyme that maintains DNA methylation. We describe a previously unknown mode of regulation of DNMT1 protein stability through the coordinated action of an array of DNMT1-associated proteins. DNMT1 was destabilized by acetylation by the acetyltransferase Tip60, which triggered ubiquitination by the E3 ligase UHRF1, thereby targeting DNMT1 for proteasomal degradation. In contrast, DNMT1 was stabilized by histone deacetylase 1 (HDAC1) and the deubiquitinase HAUSP (herpes virus–associated ubiquitin-specific protease). Analysis of the abundance of DNMT1 and Tip60, as well as the association between HAUSP and DNMT1, suggested that during the cell cycle the initiation of DNMT1 degradation was coordinated with the end of DNA replication and the need for DNMT activity. In human colon cancers, the abundance of DNMT1 correlated with that of HAUSP. HAUSP knockdown rendered colon cancer cells more sensitive to killing by HDAC inhibitors both in tissue culture and in tumor xenograft models. Thus, these studies provide a mechanism-based rationale for the development of HDAC and HAUSP inhibitors for combined use in cancer therapy.
Familial aggregation of esophageal adenocarcinomas, esophagogastric junction adenocarcinomas, and their precursor Barrett’s esophagus has been termed Familial Barrett’s Esophagus (FBE). Numerous studies documenting increased familial risk for these diseases raise the hypothesis that there may be an inherited susceptibility to the development of BE and its associated cancers. In this study, using segregation analysis for a binary trait as implemented in S.A.G.E. 6.0.1, we analyzed data on 881singly ascertained pedigrees in order to determine whether FBE is caused by a common environmental or genetic agent and, if genetic, to identify the mode of inheritance of FBE. The inheritance models were compared by likelihood ratio tests and Akaike’s A Information Criterion. Results indicated that random environmental and/or multifactorial components were insufficient to fully explain the familial nature of FBE, but rather there is segregation of a major type transmitted from one generation to the next (p-value < 10−10). An incompletely dominant inheritance model together with a polygenic component fits the data best. For this dominant model, the estimated penetrance of the dominant allele is 0.1005 (95% confidence interval, CI: 0.0587 to 0.1667) and the sporadic rate is 0.0012 (95% CI: 0.0004 to 0.0042), corresponding to a relative risk of 82.53 (95% CI: 28.70 to 237.35), or odds ratio of 91.63 (95% CI: 32.01 to 262.29). This segregation analysis provides epidemiological evidence in support of one or more rare autosomally inherited dominant susceptibility allele(s) in FBE families, and hence motivates linkage analyses.
familial esophageal adenocarcinomas; complex segregation analysis; dominant major gene inheritance; polygenic component; likelihood; AIC; unified model
Colorectal cancer is the second leading cause of cancer mortality in adult Americans and is caused by both genetic and environmental risk factors. We have replicated our originally reported linkage signal at 9q22-31 by fine mapping an independent collection of colon cancer families. Then, using a custom array of single nucleotide polymorphisms (SNPs) densely spaced across the candidate region, we performed both single-SNP and moving-window association analyses to identify a colon neoplasia risk haplotype. We isolated the association effect to a five SNP haplotype centered around 98.15 megabases (Mb) on chromosome 9q. This haplotype is in strong linkage disequilibrium with the haplotype block containing HABP4 and may be a surrogate for the effect of this CD30 Ki-1 antigen. It is also in close proximity to the GALNT12, which has been recently shown to be altered in colon tumors. Finally, we used a predictive modeling algorithm to demonstrate the contribution of this risk haplotype and surrounding candidate genes in distinguishing between colon cancer cases and healthy controls. The ability to replicate this finding, the strength of the haplotype association (OR=3.68) and the accuracy of our prediction model (~60%) all strongly support the presence of a locus for familial colon cancer on chromosome 9q.
colon cancer; linkage analysis; association analysis; risk; family cancer syndrome