New insights into the genetic basis of colorectal cancer

Colorectal cancer (CRC) is the third most common cancer, and the second most common cause of cancer mortality. The aim of this thesis work was to gain novel insight into the molecular mechanisms behind CRC predisposition, as well as tumor progression and development. Microsatellite instability (MSI) arises due to a defective mismatch repair system and is characteristic for a subset of all CRCs. Here, we aimed to identify novel MSI target genes with potential oncogenic effects. We characterized all genes overexpressed in MSI CRCs and predicted to escape nonsense-mediated decay when mutated. The mitotic checkpoint kinase TTK was identified with protein-elongating mutations in 59% of MSI CRCs. TTK has an essential role in spindle assembly checkpoint (SAC) signaling, however, the mutated protein did not show SAC weakening. While no evidence of oncogenic mechanisms was observed, the high mutation frequency of TTK argues for biological significance. In another screening effort on MSI CRCs, we sought to identify novel oncogenes with activating hotspot mutations. The exomes of 25 tumor and respective healthy colon tissues were sequenced. A total of 15 candidate oncogenes with hotspot mutations were identified. Three genes, ZBTB2, PSRC1 and RANBP2, displayed hotspot mutations also in the validation set of 86 MSI CRCs. Interestingly, the CRC-associated mutant form of ZBTB2 increased cell proliferation. Additional work is needed to further clarify the role of the identified somatic mutations in CRC tumorigenesis. The candidate oncogenes identified in this thesis work might be used to develop personalized tumor profiling and therapy. Inherited susceptibility is estimated to be involved in approximately one-third of all CRCs. However, the great majority of inherited CRC susceptibility remains still molecularly unexplained. A recent systematic sequencing study on CRC reported a set of somatically mutated genes, termed candidate cancer (CAN) genes. We examined the mutational profiles of 15 top-ranked CAN genes for somatic mutations as well as for germline variants in 45 familial CRC cases. In our tumor set, six of the CAN genes were somatically mutated. In germline, three private missense variants were identified in CSMD3, EPHB6 and c10orf137. In another effort, we exome sequenced 96 independent cases with familial CRC. We identified 11 novel candidate CRC susceptibility genes with rare putative LoF variants. Seven loss-of-heterozygosity events, involving four genes, were observed in the data. In each occasion, the losses targeted the wild-type allele (P=0.0078), providing further support that true culprits are among the eleven genes. This study provides an interesting set of candidate predisposing genes, which might explain a subset of common familial CRC. The identified germline variants need to be validated in larger sample sets to provide firm evidence for disease predisposition. Additional work is also needed to characterize the detailed functional and clinical relevance of the identified candidate CRC predisposing genes. This information, then, can ultimately be translated into tools for cancer prevention and early diagnosis in individuals carrying true predisposition alleles.Kolorektal cancer (KRC, cancer i tjock- och ändtarm) är en av de vanligaste cancerformerna i västvärlden. Flera faktorer, både ärftliga och i människans omgivning, påverkar uppkomsten av KRC. En kartläggning av de genetiska förändringarna bakom KRC är viktig för att bättre förstå tumörutvecklingen. Sådan information kan sedan användas för att utveckla effektivare behandlingsmetoder för att minska död och lidande orsakade av KRC. De två huvudsakliga syftena i detta doktorandprojekt var att 1) få ny insikt i de molekylära mekanismerna bakom kolorektal tumörutveckling 2) identifiera nya genetiska faktorer som påverkar ärftlig benägenhet för KRC. En del kolorektala tumörer hör till gruppen mikrosatellit instabila (MSI). Dessa tumörer har ett defekt DNA-reparationssystem vilket leder till ett högt antal mutationer i DNA-sekvensen, speciellt i repetitiva DNA-segment. Syftet med detta projekt var att identifiera nya onkogener i denna tumörtyp. Onkogener är gener som ändrar grundläggande egenskaper hos celler, till exempel tillväxt och differentiering. Genetiska avvikelser i en sådan gen kan leda till malign transformation av cellen. Två olika DNA-sekvenseringsmetoder användes; den så kallade Sanger-dideoxy-metoden samt nästa generations DNA-sekvensering. Ett antal nya onkogener med hög mutationsfrekvens identifierades i kolorektala tumörer av gruppen MSI. Ytterligare studier krävs för att klarlägga hur dessa onkogener påverkar malign transformation av cellen. De identifierade onkogenerna kan möjligtvis användas för att utveckla mer effektiva strategier för individuell tumörprofilering och cancerterapi. Det finns klara bevis för att genetiska faktorer predisponerar för KRC i ungefär vart tredje fall. Ett antal kända ärftliga syndrom medför en ökad risk för KRC. Men dessa syndrom är sällsynta och bidrar bara till en bråkdel av fallen. Än i dag förblir en stor del av den ärftliga benägenheten för KRC molekylärt oförklarad. Det andra syftet med denna avhandling var att identifiera nya gener som ökar benägenheten till familjär KRC (patienter med en förstagradenssläkting med KRC). Även här användes Sanger-dideoxy-metoden samt nästa generations DNA-sekvensering. Flera attraktiva kandidatgener identifierades med protein-kapande DNA-variationer i patienternas könsceller. DNA-variationerna var väldigt sällsynta eller frånvarande från den allmänna populationen. Ytterligare forskningar, med större sampelmaterial, är dock nödvändiga för att bevisa att de identifierande DNA-variationerna påverkar den ärftliga benägenheten till familjär KRC. Identifikationen av nya gener bakom ärftlig KRC möjliggör en förbättrad patientförsörjning med tidig diagnos och förbättrad riskprofilering

Screening of Finnish RAD51C founder mutations in prostate and colorectal cancer patients

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3 '-UTR poly(T/U) repeat of EWSR1 is altered in microsatellite unstable colorectal cancer with nearly perfect sensitivity

Approximately 15 % of colorectal cancers exhibit instability of short nucleotide repeat regions, microsatellites. These tumors display a unique clinicopathologic profile and the microsatellite instability status is increasingly used to guide clinical management as it is known to predict better prognosis as well as resistance to certain chemotherapeutics. A panel of five repeats determined by the National Cancer Institute, the Bethesda panel, is currently the standard for determining the microsatellite instability status in colorectal cancer. Recently, a quasimonomorphic mononucleotide repeat 16T/U at the 3' untranslated region of the Ewing sarcoma breakpoint region 1 gene was reported to show perfect sensitivity and specificity in detecting mismatch repair deficient colorectal, endometrial, and gastric cancers in two independent populations. To confirm this finding, we replicated the analysis in 213 microsatellite unstable colorectal cancers from two independent populations, 148 microsatellite stable colorectal cancers, and the respective normal samples by PCR and fragment analysis. The repeat showed nearly perfect sensitivity for microsatellite unstable colorectal cancer as it was altered in 212 of the 213 microsatellite unstable (99.5 %) and none of the microsatellite stable colorectal tumors. This repeat thus represents the first potential single marker for detecting microsatellite instability.Peer reviewe

Eleven Candidate Susceptibility Genes for Common Familial Colorectal Cancer

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Mendelian randomisation implicates hyperlipidaemia as a risk factor for colorectal cancer.

While elevated blood cholesterol has been associated with an increased risk of colorectal cancer (CRC) in observational studies, causality is uncertain. Here we apply a Mendelian randomisation (MR) analysis to examine the potential causal relationship between lipid traits and CRC risk. We used single nucleotide polymorphisms (SNPs) associated with blood levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) as instrumental variables (IV). We calculated MR estimates for each risk factor with CRC using SNP-CRC associations from 9,254 cases and 18,386 controls. Genetically predicted higher TC was associated with an elevated risk of CRC (odds ratios (OR) per unit SD increase = 1.46, 95% confidence interval [CI]: 1.20-1.79, P=1.68x10−4). The pooled ORs for LDL, HDL, and TG were 1.05 (95% CI: 0.92-1.18, P=0.49), 0.94 (95% CI: 0.84-1.05, P= 0.27), and 0.98 (95% CI: 0.85-1.12, P=0.75) respectively. A genetic risk score for 3-hydoxy-3-methylglutaryl-coenzyme A reductase (HMGCR) to mimic the effects of statin therapy was associated with a reduced CRC risk (OR=0.69, 95% CI: 0.49-0.99, P=0.046). This study supports a causal relationship between higher levels of TC with CRC risk, and a further rationale for implementing public health strategies to reduce the prevalence of hyperlipidaemia. This article is protected by copyright. All rights reserved

Characterization of the colorectal cancer–associated enhancer MYC-335 at 8q24: the role of rs67491583

Recent genome-wide association studies have identified multiple regions at 8q24 that confer susceptibility to many cancers. In our previous work, we showed that the colorectal cancer (CRC) risk variant rs6983267 at 8q24 resides within a TCF4 binding site at the MYC-335 enhancer, with the risk allele G having a stronger binding capacity and Wnt responsiveness. Here, we searched for other potential functional variants within MYC-335. Genetic variation within MYC-335 was determined in samples from individuals of European, African, and Asian descent, with emphasis on variants in putative transcription factor binding sites. A 2-bp GA deletion rs67491583 was found to affect a growth factor independent (GFI) binding site and was present only in individuals with African ancestry. Chromatin immunoprecipitation performed in heterozygous cells showed that the GA deletion had an ability to reduce binding of the transcriptional repressors GFI1 and GFI1b. Screening of 1,027 African American colorectal cancer cases and 1,773 healthy controls did not reveal evidence for association (odds ratio: 1.17, 95% confidence interval: 0.97–1.41, P = 0.095). In this study, rs67491583 was identified as another functional variant in the CRC-associated enhancer MYC-335, but further studies are needed to establish the role of rs67491583 in the colorectal cancer predisposition of African Americans

Variation at 2q35 (PNKD and TMBIM1) influences colorectal cancer risk and identifies a pleiotropic effect with inflammatory bowel disease

To identify new risk loci for colorectal cancer (CRC), we conducted a meta-analysis of seven genome-wide association studies (GWAS) with independent replication, totalling 13 656 CRC cases and 21 667 controls of European ancestry. The combined analysis identified a new risk association for CRC at 2q35 marked by rs992157 (P = 3.15 x 10(-8), odds ratio = 1.10, 95% confidence interval = 1.06-1.13), which is intronic to PNKD (paroxysmal non-kinesigenic dyskinesia) and TMBIM1 (transmembrane BAX inhibitor motif containing 1). Intriguingly this susceptibility single-nucleotide polymorphism (SNP) is in strong linkage disequilibrium (r(2) = 0.90, D' = 0.96) with the previously discovered GWAS SNP rs2382817 for inflammatory bowel disease (IBD). Following on from this observation we examined for pleiotropy, or shared genetic susceptibility, between CRC and the 200 established IBD risk loci, identifying an additional 11 significant associations (false discovery rate [FDR]) <0.05). Our findings provide further insight into the biological basis of inherited genetic susceptibility to CRC, and identify risk factors that may influence the development of both CRC and IBD.Peer reviewe

Mendelian randomisation analysis strongly implicates adiposity with risk of developing colorectal cancer

Background: Observational studies have associated adiposity with an increased risk of colorectal cancer (CRC). However, such studies do not establish a causal relationship. To minimise bias from confounding we performed a Mendelian randomisation (MR) analysis to examine the relationship between adiposity and CRC. Methods: We used SNPs associated with adult body mass index (BMI), waist-hip ratio (WHR), childhood obesity and birth weight as instrumental variables in a MR analysis of 9254 CRC cases and 18 386 controls. Results: In the MR analysis, the odds ratios (ORs) of CRC risk per unit increase in BMI, WHR and childhood obesity were 1.23 (95% CI: 1.02-1.49, P = 0.033), 1.59 (95% CI: 1.08-2.34, P = 0.019) and 1.07 (95% CI: 1.03-1.13, P = 0.018), respectively. There was no evidence for association between birth weight and CRC (OR = 1.22, 95% CI: 0.89-1.67, P = 0.22). Combining these data with a concurrent MR-based analysis for BMI and WHR with CRC risk (totalling to 18 190 cases, 27 617 controls) provided increased support, ORs for BMI and WHR were 1.26 (95% CI: 1.10-1.44, P = 7.7 x 10(-4)) and 1.40 (95% CI: 1.14-1.72, P = 1.2 x 10(-3)), respectively. Conclusions: These data provide further evidence for a strong causal relationship between adiposity and the risk of developing CRC highlighting the urgent need for prevention and treatment of adiposity.Peer reviewe

Comprehensive Evaluation of Protein Coding Mononucleotide Microsatellites in Microsatellite-Unstable Colorectal Cancer

Approximately 15% of colorectal cancers exhibit microsatellite instability (MSI), which leads to accumulation of large numbers of small insertions and deletions (indels). Genes that provide growth advantage to cells via loss-of-function mutations in micro-satellites are called MSI target genes. Several criteria to define these genes have been suggested, one of them being simple mutation frequency. Microsatellite mutation rate, however, depends on the length and nucleotide context of the microsatellite. Therefore, assessing the general impact of mismatch repair deficiency on the likelihood of mutation events is paramount when following this approach. To identify MSI target genes, we developed a statistical model for the somatic background indel mutation rate of microsatellites to assess mutation significance. Exome sequencing data of 24 MSI colorectal cancers revealed indels at 54 million mononucleotide microsatellites of three or more nucleotides in length. The top 105 microsatellites from 71 genes were further analyzed in 93 additional MSI colorectal cancers. Mutation significance and estimated clonality of mutations determined the most likely MSI target genes to be the aminoadipate-semialdehyde dehydrogenase AASDH and the solute transporter SLC9A8. Our findings offer a systematic profiling of the somatic background mutation rate in protein-coding mononucleotide microsatellites, allowing a full cataloging of the true targets of MSI in colorectal cancer. (C) 2017 AACR.Peer reviewe

CTCF/cohesin-binding sites are frequently mutated in cancer

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