Genome-wide association studies (GWAS) have identified thousands of genetic variants that influence a variety of diseases and health-related quantitative traits. However, the causal variants underlying the majority of genetic associations remain unknown. The Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Targeted Sequencing Study aims to follow up GWAS signals and identify novel associations of the allelic spectrum of identified variants with cardiovascular related traits.
Methods and Results
The study included 4,231 participants from three CHARGE cohorts: the Atherosclerosis Risk in Communities Study, the Cardiovascular Health Study, and the Framingham Heart Study. We used a case-cohort design in which we selected both a random sample of participants and participants with extreme phenotypes for each of 14 traits. We sequenced and analyzed 77 genomic loci, which had previously been associated with one or more of 14 phenotypes. A total of 52,736 variants were characterized by sequencing and passed our stringent quality control criteria. For common variants (minor allele frequency ≥1%), we performed unweighted regression analyses to obtain p-values for associations and weighted regression analyses to obtain effect estimates that accounted for the sampling design. For rare variants, we applied two approaches: collapsed aggregate statistics and joint analysis of variants using the Sequence Kernel Association Test.
We sequenced 77 genomic loci in participants from three cohorts. We established a set of filters to identify high-quality variants, and implemented statistical and bioinformatics strategies to analyze the sequence data, and identify potentially functional variants within GWAS loci.
genetics; epidemiology; CHARGE; sampling; targeted sequencing
The nuclear receptor peroxisome proliferator-activated receptor-δ/β (PPAR-d) is upregulated in human colorectal cancers, but its role in colonic tumorigenesis remains controversial.
We generated a novel mouse model of intestinally targeted PPAR-d overexpression to simulate PPAR-d upregulation in human colon carcinogenesis. Colon-specific PPAR-d overexpression was confirmed by real-time reverse transcription polymerase chain reaction, immunoblotting, and activity assays. Mice with and without targeted PPAR-d overexpression were tested for azoxymethane (AOM)–induced colonic tumorigenesis. Mouse whole-genome transcriptome microarray analyses were performed to identify PPAR-d target genes to promote tumorigenesis. We used linear models to test for PPAR-d overexpression trend effects on tumor multiplicity. All statistical tests were two-sided.
Targeted PPAR-d overexpression markedly increased colonic tumor incidence (from 0 of 10 wild-type [WT] littermate mice to 9 of 10 mice [P < .001] in 2 FVB/N background mouse lines [villin-PPAR-d-1 and villin-PPAR-d-2] at a 5-mg/kg AOM dose) and multiplicity (number of tumors per mouse per mg/kg dose of AOM increased from 0.47 [95% confidence interval [CI] = 0.22 to 0.72] for the WT littermates to 2.15 [95% CI = 1.90 to 2.40] [P < .001] for the villin-PPAR-d-1 mice and from 0.44 [95% CI = 0.09 to 0.79] for the WT littermates to 1.91 [95% CI = 1.57 to 2.25] [P < .001] for the villin-PPAR-d-2 mice). PPAR-d overexpression reversed resistance to AOM-induced colonic tumorigenesis in C57BL/6 mice. PPAR-d overexpression modulated expression of several novel PPAR-d target genes in normal-appearing colonic epithelial cells of mice with PPAR-d overexpression in a pattern that matched the changes in colonic tumors.
Our finding that PPAR-d upregulation profoundly enhances susceptibility to colonic tumorigenesis should impact the development of strategies of molecularly targeting PPAR-d in cancer and noncancerous diseases.
A first analysis of the genome sequence of the common marmoset (Callithrix jacchus), assembled using traditional Sanger methods and Ensembl annotation, has permitted genomic comparison with apes and that old world monkeys and the identification of specific molecular features a rapid reproductive capacity partly due to may contribute to the unique biology of diminutive The common marmoset has prevalence of this dizygotic primate. twins. Remarkably, these twins share placental circulation and exchange hematopoietic stem cells in utero, resulting in adults that are hematopoietic chimeras.
We observed positive selection or non-synonymous substitutions for genes encoding growth hormone / insulin-like growth factor (growth pathways), respiratory complex I (metabolic pathways), immunobiology, and proteases (reproductive and immunity pathways). In addition, both protein-coding and microRNA genes related to reproduction exhibit rapid sequence evolution. This New World monkey genome sequence enables significantly increased power for comparative analyses among available primate genomes and facilitates biomedical research application.
Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.
Arthropods are the most abundant animals on earth. Among them, insects clearly dominate on land, whereas crustaceans hold the title for the most diverse invertebrates in the oceans. Much is known about the biology of these groups, not least because of genomic studies of the fruit fly Drosophila, the water flea Daphnia, and other species used in research. Here we report the first genome sequence from a species belonging to a lineage that has previously received very little attention—the myriapods. Myriapods were among the first arthropods to invade the land over 400 million years ago, and survive today as the herbivorous millipedes and venomous centipedes, one of which—Strigamia maritima—we have sequenced here. We find that the genome of this centipede retains more characteristics of the presumed arthropod ancestor than other sequenced insect genomes. The genome provides access to many aspects of myriapod biology that have not been studied before, suggesting, for example, that they have diversified receptors for smell that are quite different from those used by insects. In addition, it shows specific consequences of the largely subterranean life of this particular species, which seems to have lost the genes for all known light-sensing molecules, even though it still avoids light.
We undertook a gene identification and molecular characterization project in a large kindred originally clinically diagnosed with SCA-X1. While presenting with ataxia, this kindred also had some unique peripheral nervous system features. The implicated region on the X chromosome was delineated using haplotyping. Large deletions and duplications were excluded by array comparative genomic hybridization. Exome sequencing was undertaken in two affected subjects. The single identified X chromosome candidate variant was then confirmed to co-segregate appropriately in all affected, carrier and unaffected family members by Sanger sequencing. The variant was confirmed to be novel by comparison with dbSNP, and filtering for a minor allele frequency of <1% in 1000 Genomes project, and was not present in the NHLBI Exome Sequencing Project or a local database at the BCM HGSC. Functional experiments on transfected cells were subsequently undertaken to assess the biological effect of the variant in vitro. The variant identified consisted of a previously unidentified non-synonymous variant, GJB1 p.P58S, in the Connexin 32/Gap Junction Beta 1 gene. Segregation studies with Sanger sequencing confirmed the presence of the variant in all affected individuals and one known carrier, and the absence of the variant in unaffected members. Functional studies confirmed that the p.P58S variant reduced the number and size of gap junction plaques, but the conductance of the gap junctions was unaffected. Two X-linked ataxias have been associated with genetic loci, with the first of these recently characterized at the molecular level. This represents the second kindred with molecular characterization of X-linked ataxia, and is the first instance of a previously unreported GJB1 mutation with a dominant and permanent ataxia phenotype, although different CNS deficits have previously been reported. This pedigree has also been relatively unique in its phenotype due to the presence of central and peripheral neural abnormalities. Other X-linked SCAs with unique features might therefore also potentially represent variable phenotypic expression of other known neurological entities.
The expression of 15-lipoxygenase-1 (15-LOX-1) is downregulated in colon cancer and other major cancers, and 15-LOX-1 reexpression in cancer cells suppresses colonic tumorigenesis. Various lines of evidence indicate that 15-LOX-1 expression suppresses premetastatic stages of colonic tumorigenesis; nevertheless, the role of 15-LOX-1 loss of expression in cancer epithelial cells in metastases continues to be debated. Hypoxia, a common feature of the cancer microenvironment, promotes prometastatic mechanisms such as the upregulation of hypoxia-inducible factor (HIF)-1α, a transcriptional master regulator that enhances cancer cell metastatic potential, angiogenesis, and tumor cell invasion and migration. We have, therefore, tested whether restoring 15-LOX-1 in colon cancer cells affects cancer cells' hypoxia response that promotes metastasis. We found that 15-LOX-1 reexpression in HCT116, HT29LMM, and LoVo colon cancer cells inhibited survival, vascular endothelial growth factor (VEGF) expression, angiogenesis, cancer cell migration and invasion, and HIF-1α protein expression and stability under hypoxia. These findings demonstrate that 15-LOX-1 expression loss in cancer cells promotes metastasis and that therapeutically targeting ubiquitous 15-LOX-1 loss in cancer cells has the potential to suppress metastasis.
15-Lipoxygenase-1; angiogenesis; HIF-1α; hypoxia
The advent of whole-exome next-generation sequencing (WES) has been pivotal for the molecular characterization of Mendelian disease; however, the clinical application of WES has remained relatively unexplored. We describe our experience with WES as a diagnostic tool in a three-year old female patient with a two-year history of episodic muscle weakness and paroxysmal dystonia who presented following a previous extensive but unrevealing diagnostic work-up. WES was performed on the proband and her two parents. Parental exome data was used to filter de novo genomic events in the proband and suspected mutations were confirmed using di-deoxy sequencing. WES revealed a de novo non-synonymous mutation in exon 21 of the calcium channel gene CACNA1S that has been previously reported in a single patient as a rare cause of atypical hypokalemic periodic paralysis. This was unexpected, as the proband’s original differential diagnosis had included hypokalemic periodic paralysis, but clinical and laboratory features were equivocal, and standard clinical molecular testing for hypokalemic periodic paralysis and related disorders was negative. This report highlights the potential diagnostic utility of WES in clinical practice, with implications for the approach to similar diagnostic dilemmas in the future.
Hypokalemic periodic paralysis; CACNA1S; next generation sequencing; hypotonia
The first generation of genome sequence assemblies and annotations have had a significant impact upon our understanding of the biology of the sequenced species, the phylogenetic relationships among species, the study of populations within and across species, and have informed the biology of humans. As only a few Metazoan genomes are approaching finished quality (human, mouse, fly and worm), there is room for improvement of most genome assemblies. The honey bee (Apis mellifera) genome, published in 2006, was noted for its bimodal GC content distribution that affected the quality of the assembly in some regions and for fewer genes in the initial gene set (OGSv1.0) compared to what would be expected based on other sequenced insect genomes.
Here, we report an improved honey bee genome assembly (Amel_4.5) with a new gene annotation set (OGSv3.2), and show that the honey bee genome contains a number of genes similar to that of other insect genomes, contrary to what was suggested in OGSv1.0. The new genome assembly is more contiguous and complete and the new gene set includes ~5000 more protein-coding genes, 50% more than previously reported. About 1/6 of the additional genes were due to improvements to the assembly, and the remaining were inferred based on new RNAseq and protein data.
Lessons learned from this genome upgrade have important implications for future genome sequencing projects. Furthermore, the improvements significantly enhance genomic resources for the honey bee, a key model for social behavior and essential to global ecology through pollination.
Apis mellifera; GC content; Gene annotation; Gene prediction; Genome assembly; Genome improvement; Genome sequencing; Repetitive DNA; Transcriptome
To characterize the role of rare complete human knockouts in autism spectrum disorders (ASD), we identify genes with homozygous or compound heterozygous loss-of-function (LoF) variants (defined as nonsense and essential splice sites) from exome sequencing of 933 cases and 869 controls. We identify a two-fold increase in complete knockouts of autosomal genes with low rates of LoF variation (≤5% frequency) in cases and estimate a 3% contribution to ASD risk by these events, confirming this observation in an independent set of 563 probands and 4,605 controls. Outside the pseudo-autosomal regions on the X-chromosome, we similarly observe a significant 1.5-fold increase in rare hemizygous knockouts in males, contributing to another 2% of ASDs in males. Taken together these results provide compelling evidence that rare autosomal and X-chromosome complete gene knockouts are important inherited risk factors for ASD.
Expression of 15-lipoxygenase-1 (15-LOX-1) is decreased in many human cancers; however, the mechanistic significance of its decreased expression has been difficult to determine because its mouse homolog 12/15-LOX has opposing functions. We generated a mouse model in which expression of a human 15-LOX-1 transgene was targeted to the intestinal epithelium via the villin promoter. Targeted expression was confirmed by real-time reverse transcription–polymerase chain reaction and immunoblotting. When the 15-LOX-1 transgene was expressed in colonic epithelial cells of two independent mouse lines (B6 and FVB), azoxymethane-inducible colonic tumorigenesis was suppressed (mean number of tumors: wild type [WT] = 8.2, 15-LOX-1+/− = 4.91, 15-LOX-1+/+ = 3.57; WT vs 15-LOX-1+/− two-sided P = .003, WT vs 15-LOX-1+/+ two-sided P < .001; n = 10–14 mice per group). 15-LOX-1 transgene expression was always decreased in the tumors that did develop. In the presence of expression of the 15-LOX-1 transgene, expression of tumor necrosis factor alpha and its target inducible nitric oxide synthase were decreased and activation of nuclear factor-kappa B in colonic epithelial cells was inhibited.
We report on results from whole-exome sequencing (WES) of 1,039 subjects diagnosed with autism spectrum disorders (ASD) and 870 controls selected from the NIMH repository to be of similar ancestry to cases. The WES data came from two centers using different methods to produce sequence and to call variants from it. Therefore, an initial goal was to ensure the distribution of rare variation was similar for data from different centers. This proved straightforward by filtering called variants by fraction of missing data, read depth, and balance of alternative to reference reads. Results were evaluated using seven samples sequenced at both centers and by results from the association study. Next we addressed how the data and/or results from the centers should be combined. Gene-based analyses of association was an obvious choice, but should statistics for association be combined across centers (meta-analysis) or should data be combined and then analyzed (mega-analysis)? Because of the nature of many gene-based tests, we showed by theory and simulations that mega-analysis has better power than meta-analysis. Finally, before analyzing the data for association, we explored the impact of population structure on rare variant analysis in these data. Like other recent studies, we found evidence that population structure can confound case-control studies by the clustering of rare variants in ancestry space; yet, unlike some recent studies, for these data we found that principal component-based analyses were sufficient to control for ancestry and produce test statistics with appropriate distributions. After using a variety of gene-based tests and both meta- and mega-analysis, we found no new risk genes for ASD in this sample. Our results suggest that standard gene-based tests will require much larger samples of cases and controls before being effective for gene discovery, even for a disorder like ASD.
This study evaluates association of rare variants and autism spectrum disorders (ASD) in case and control samples sequenced by two centers. Before doing association analyses, we studied how to combine information across studies. We first harmonized the whole-exome sequence (WES) data, across centers, in terms of the distribution of rare variation. Key features included filtering called variants by fraction of missing data, read depth, and balance of alternative to reference reads. After filtering, the vast majority of variants calls from seven samples sequenced at both centers matched. We also evaluated whether one should combine summary statistics from data from each center (meta-analysis) or combine data and analyze it together (mega-analysis). For many gene-based tests, we showed that mega-analysis yields more power. After quality control of data from 1,039 ASD cases and 870 controls and a range of analyses, no gene showed exome-wide evidence of significant association. Our results comport with recent results demonstrating that hundreds of genes affect risk for ASD; they suggest that rare risk variants are scattered across these many genes, and thus larger samples will be required to identify those genes.
Autism spectrum disorders (ASD) are believed to have genetic and environmental origins, yet in only a modest fraction of individuals can specific causes be identified1,2. To identify further genetic risk factors, we assess the role of de novo mutations in ASD by sequencing the exomes of ASD cases and their parents (n= 175 trios). Fewer than half of the cases (46.3%) carry a missense or nonsense de novo variant and the overall rate of mutation is only modestly higher than the expected rate. In contrast, there is significantly enriched connectivity among the proteins encoded by genes harboring de novo missense or nonsense mutations, and excess connectivity to prior ASD genes of major effect, suggesting a subset of observed events are relevant to ASD risk. The small increase in rate of de novo events, when taken together with the connections among the proteins themselves and to ASD, are consistent with an important but limited role for de novo point mutations, similar to that documented for de novo copy number variants. Genetic models incorporating these data suggest that the majority of observed de novo events are unconnected to ASD, those that do confer risk are distributed across many genes and are incompletely penetrant (i.e., not necessarily causal). Our results support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5 to 20-fold. Despite the challenge posed by such models, results from de novo events and a large parallel case-control study provide strong evidence in favor of CHD8 and KATNAL2 as genuine autism risk factors.
Loss of terminal cell differentiation promotes tumorigenesis. 15-LOX-1 contributes to terminal cell differentiation in normal cells. The mechanistic significance of 15-LOX-1 expression loss in human cancers to terminal cell differentiation suppression is unknown. In a screen of 128 cancer cell lines representing more than 20 types of human cancer, we found that 15-LOX-1 mRNA expression levels were markedly lower than levels in terminally differentiated cells. Relative expression levels of 15-LOX-1 (relative to the level in terminally differentiated primary normal human-derived bronchial epithelial cells) were lower in 79% of the screened cancer cell lines than relative expression levels of p16 (INK4A), which promotes terminal cell differentiation and is considered one of the most commonly lost tumor suppressor genes in cancer cells. 15-LOX-1 was expressed during terminal differentiation in three-dimensional air-liquid interface cultures, and 15-LOX-1 expression and terminal differentiation occurred in immortalized non-transformed bronchial epithelial but not lung cancer cell lines. 15-LOX-1 expression levels were lower in human tumors than paired normal lung epithelia. Short hairpin RNA-mediated downregulation of 15-LOX-1 in Caco-2 cells blocked enterocyte-like differentiation, disrupted tight junction formation, and blocked E-cadherin and ZO-1 localization to the cell wall membrane. 15-LOX-1 episomal expression in Caco-2 and HT-29 colon cancer cells induced differentiation. Our findings indicate that 15-LOX-1 downregulation in cancer cells is an important mechanism for terminal cell differentiation dysregulation and support the potential therapeutic utility of 15-LOX-1 re-expression to inhibit tumorigenesis.
15-lipoxygenase-1; terminal cell differentiation; tumorigenesis
We sequenced 8 melanoma exomes to identify novel somatic mutations in metastatic melanoma. Focusing on the MAP3K family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9. Structural modelling predicts that mutations in the kinase domain may affect the activity and regulation of MAP3K5/9 protein kinases. The position of the mutations and loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely inactivating. In vitro kinase assay shows reduction in kinase activity in MAP3K5 I780F and MAP3K9 W333X mutants. Overexpression of MAP3K5 or MAP3K9 mutant in HEK293T cells reduces phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA leads to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.
Enrichment of loci by DNA hybridization-capture, followed by high-throughput sequencing, is an important tool in modern genetics. Currently, the most common targets for enrichment are the protein coding exons represented by the consensus coding DNA sequence (CCDS). The CCDS, however, excludes many actual or computationally predicted coding exons present in other databases, such as RefSeq and Vega, and non-coding functional elements such as untranslated and regulatory regions. The number of variants per base pair (variant density) and our ability to interrogate regions outside of the CCDS regions is consequently less well understood.
We examine capture sequence data from outside of the CCDS regions and find that extremes of GC content that are present in different subregions of the genome can reduce the local capture sequence coverage to less than 50% relative to the CCDS. This effect is due to biases inherent in both the Illumina and SOLiD sequencing platforms that are exacerbated by the capture process. Interestingly, for two subregion types, microRNA and predicted exons, the capture process yields higher than expected coverage when compared to whole genome sequencing. Lastly, we examine the variation present in non-CCDS regions and find that predicted exons, as well as exonic regions specific to RefSeq and Vega, show much higher variant densities than the CCDS.
We show that regions outside of the CCDS perform less efficiently in capture sequence experiments. Further, we show that the variant density in computationally predicted exons is more than 2.5-times higher than that observed in the CCDS.
Next-generation DNA sequencing is opening new avenues for genetic association studies in common diseases that, like deep vein thrombosis (DVT), have a strong genetic predisposition still largely unexplained by currently identified risk variants. In order to develop sequencing and analytical pipelines for the application of next-generation sequencing to complex diseases, we conducted a pilot study sequencing the coding area of 186 hemostatic/proinflammatory genes in 10 Italian cases of idiopathic DVT and 12 healthy controls.
A molecular-barcoding strategy was used to multiplex DNA target capture and sequencing, while retaining individual sequence information. Genomic libraries with barcode sequence-tags were pooled (in pools of 8 or 16 samples) and enriched for target DNA sequences. Sequencing was performed on ABI SOLiD-4 platforms. We produced > 12 gigabases of raw sequence data to sequence at high coverage (average: 42X) the 700-kilobase target area in 22 individuals. A total of 1876 high-quality genetic variants were identified (1778 single nucleotide substitutions and 98 insertions/deletions). Annotation on databases of genetic variation and human disease mutations revealed several novel, potentially deleterious mutations. We tested 576 common variants in a case-control association analysis, carrying the top-5 associations over to replication in up to 719 DVT cases and 719 controls. We also conducted an analysis of the burden of nonsynonymous variants in coagulation factor and anticoagulant genes. We found an excess of rare missense mutations in anticoagulant genes in DVT cases compared to controls and an association for a missense polymorphism of FGA (rs6050; p = 1.9 × 10-5, OR 1.45; 95% CI, 1.22-1.72; after replication in > 1400 individuals).
We implemented a barcode-based strategy to efficiently multiplex sequencing of hundreds of candidate genes in several individuals. In the relatively small dataset of our pilot study we were able to identify bona fide associations with DVT. Our study illustrates the potential of next-generation sequencing for the discovery of genetic variation predisposing to complex diseases.
Deep vein thrombosis; venous thromboembolism; next-generation sequencing; target capture; multiplexing; FGA; rs6025; heamostateome; DVT; VTE
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. To explore the genetic origins of this cancer, we used whole exome sequencing and gene copy number analyses to study 32 primary tumors. Tumors from patients with a history of tobacco use had more mutations than did tumors from patients who did not use tobacco, and tumors that were negative for human papilloma virus (HPV) had more mutations than did HPV-positive tumors. Six of the genes that were mutated in multiple tumors were assessed in up to 88 additional HNSCCs. In addition to previously described mutations in TP53, CDKN2A, PIK3CA and HRAS, we identified mutations in FBXW7 and NOTCH1. Interestingly, nearly 40% of the 28 mutations identified in NOTCH1 were predicted to truncate the gene product, suggesting that NOTCH1 may function as a tumor suppressor gene rather than an oncogene in this tumor type.
Molecular targeting for apoptosis induction is being developed for better treatment of cancer. Down-regulation of 15-lipoxygenase-1 (15-LOX-1) is linked to colorectal tumorigenesis. Re-expression of 15-LOX-1 in cancer cells by pharmaceutical agents induces apoptosis. Antitumorigenic agents can also induce apoptosis via other molecular targets. Whether restoring 15-LOX-1 expression in cancer cells is therapeutically sufficient to inhibit colonic tumorigenesis remains unknown. We tested this question by using an adenoviral delivery system to express 15-LOX-1 in in vitro and in vivo models of colon cancer. We found that a) the adenoviral vector 5/3 fiber modification enhanced 15-LOX-1 gene transduction in various colorectal cancer cell lines, b) the adenoviral vector delivery restored 15-LOX-1 expression and enzymatic activity to therapeutic levels in colon cancer cell lines, and c) 15-LOX-1 expression down-regulated the expression of the antiapoptotic proteins XIAP and BcL-XL, activated caspase-3, triggered apoptosis, and inhibited cancer cell survival in vitro and the growth of colon cancer xenografts in vivo. Thus, selective molecular targeting of 15-LOX-1 expression is sufficient to re-establish apoptosis in colon cancer cells and inhibit tumorigenesis. These data provide the rationale for further development of therapeutic strategies to molecularly target 15-LOX-1 for treating colonic tumorigenesis.
Transcriptional suppression of 15-lipoxygenase-1 (15-LOX-1) helps enable human colorectal cancer cells escape apoptosis, a critical mechanism for colonic tumorigenesis. GATA-6 is strongly expressed in vitro in cancer cells; its downregulation by pharmaceuticals is associated with reversal of 15-LOX-1 transcriptional suppression. The mechanistic contribution of GATA-6 overexpression to colonic tumorigenesis, especially concerning 15-LOX-1 transcriptional suppression, remains unknown. We tested whether GATA-6 is differentially overexpressed in human colorectal cancers and whether reversing GATA-6 overexpression in colon cancer cells is sufficient to restore 15-LOX-1 expression and influence cell proliferation or apoptosis. The expression of GATA-6 RNA and protein was measured in paired human colorectal cancer and normal tissues from two separate patient groups. We used GATA-6 small interfering RNA transfection to downregulate GATA-6 expression and examine the effects of this downregulation on 15-LOX-1 expression, cell proliferation, and apoptosis in Caco-2 and HCT-116 colon cancer cells with and without the nonsteroidal anti-inflammatory drug NS-398 or the histone deacetylase inhibitor sodium butyrate. GATA-6 mRNA and protein expressions were higher in cancer than normal epithelia of the colon. GATA-6 knockdown was insufficient by itself but contributed significantly to restoring 15-LOX-1 expression and inducing apoptosis by NS-398 or sodium butyrate. Maintaining 15-LOX-1 transcriptional silencing in cancer cells is a multifactorial process involving GATA-6 overexpression and other regulatory events.
transcriptional regulation; colon cancer; apoptosis
Terminal differentiation is an important event for maintaining normal homeostasis in the colorectal epithelium, and the loss of apoptosis is an important mechanism underlying colorectal tumorigenesis. The very limited current data on the role of lipoxygenase (LOX) metabolism in tumorigenesis suggest that the oxidative metabolism of linoleic and arachidonic acid possibly shifts from producing antitumorigenic 15-LOX-1 and 15-LOX-2 products to producing pro-tumorigenic 5-LOX and 12-LOX products. We examined whether this shift occurs in vitro in the human colon cancer cell line Caco-2 in association with the loss of terminal differentiation and apoptosis or in vivo during the formation of colorectal adenomas in patients with familial adenomatous polyposis (FAP). Restoring terminal differentiation and apoptosis of Caco-2 cells increased mRNA levels of 5-LOX, 15-LOX-2 and 15-LOX-1, but the only significant increases in protein expression and enzymatic activity were of 15-LOX-1. In FAP patients, 15-LOX-1 expression and activity were significantly down-regulated in adenomas (versus in paired non-neoplastic epithelial mucosa), whereas 5-LOX and 15-LOX-2 protein expressions and enzymatic activities were not. We conducted a validation study with immunohistochemical testing in a second group of FAP patients; 15-LOX-1 expression was down-regulated in colorectal adenomas (versus in non-neoplastic epithelial mucosa) in 87% (13/15) of this group. We confirmed the mechanistic relevance of these findings by demonstrating that ectopically restoring 15-LOX-1 expression re-established apoptosis in Caco-2 cells. Therefore, 15-LOX-1 down-regulation rather than a shift in the balance of LOXs is likely the dominant alteration in LOX metabolism that contributes to colorectal tumorigenesis through repressing apoptosis.
Lipoxygenases; colon cancer; apoptosis
Peroxisome proliferator–activated receptors (PPARs) are transcription factors that strongly influence molecular events in normal and cancer cells. PPAR-beta/delta overexpression suppresses the activity of PPAR-gamma and -alpha. This interaction has been questioned, however, by studies with synthetic ligands of PPARs in PPAR-beta/delta–null cells, and it is not known whether an interaction between PPAR-beta/delta and -gamma exists, especially in relation to the signaling by natural PPAR ligands. Oxidative metabolites of linoleic and arachidonic acids are natural ligands of PPARs. 13-S-hydroxyoctadecadienoic acid (13-S-HODE), the main product of 15-lipoxygenase-1 (15-LOX-1) metabolism of linoleic acid, downregulates PPAR-beta/delta. We tested (a) whether PPAR-beta/delta expression modulates PPAR-gamma activity in experimental models of the loss and gain of PPAR-beta/delta function in colon cancer cells and (b) whether 15-LOX-1 formation of 13-S-HODE influences the interaction between PPAR-beta/delta and PPAR-gamma. We found that (a) 15-LOX-1 formation of 13-S-HODE promoted PPAR-gamma activity, (b) PPAR-beta/delta expression suppressed PPAR-gamma activity in models of both loss and gain of PPAR-beta/delta function, (c) 15-LOX-1 activated PPAR-gamma by downregulating PPAR-beta/delta , and (d) 15-LOX-1 expression induced apoptosis in colon cancer cells via modulating PPAR-beta/delta suppression of PPAR-gamma. These findings elucidate a novel mechanism of the signaling by natural ligands of PPARs, which involves modulating the interaction between PPAR-beta/delta and PPAR-gamma.