Genetic and epigenetic mechanisms of tumor predisposition in hereditary non-polyposis colorectal carcinoma and sporadic cancers

Individuals with inherited deficiency in DNA mismatch repair(MMR) (Lynch syndrome) LS are predisposed to different cancers in a non-random fashion. Endometrial cancer (EC) is the most common extracolonic malignancy in LS. LS represents the best characterized form of hereditary nonpolyposis colorectal carcinoma (HNPCC). Other forms of familial non-polyposis colon cancer exist, including familial colorectal cancer type X (FCCX). This syndrome resembles LS, but MMR gene defects are excluded and the predisposition genes are unknown so far. To address why different organs are differently susceptible to cancer development, we examined molecular similarities and differences in selected cancers whose frequency varies in LS individuals. Tumors that are common (colorectal, endometrial, gastric) and less common (brain, urological) in LS were characterized for MMR protein expression, microsatellite instability (MSI), and by altered DNA methylation. We also studied samples of histologically normal endometrium, endometrial hyperplasia,and cancer for molecular alterations to identify potential markers that could predict malignant transformation in LS and sporadic cases. Our results suggest that brain and kidney tumors follow a different pathway for cancer development than the most common LS related cancers.Our results suggest also that MMR defects are detectable in endometrial tissues from a proportion of LS mutation carriers prior to endometrial cancer development. Traditionally (complex) atypical hyperplasia has been considered critical for progression to malignancy. Our results suggest that complex hyperplasia without atypia is equally important as a precursor lesion of malignancy. Tumor profiles from Egypt were compared with colorectal tumors from Finland to evaluate if there are differences specific to the ethnic origin (East vs.West). Results showed for the first time a distinct genetic and epigenetic signature in the Egyptian CRC marked by high methylation of microsatellite stable tumors associated with advanced stage, and low frequency of Wnt signaling activation, suggesting a novel pathway. DNA samples from FCCX families were studied with genome wide linkage analysis using microsatellite markers. Selected genes from the linked areas were tested for possible mutations that could explain predisposition to a large number of colon adenomas and carcinomas seen in these families. Based on the results from the linkage analysis, a number of areas with tentative linkage were identified in family 20. We narrowed down these areas by additional microsatellite markers to found a mutation in the BMPR1A gene. Sequencing of an additional 17 FCCX families resulted in a BMPR1A mutation frequency of 2/18 families (11%). Clarification of the mechanisms of the differential tumor susceptibility in LS increases the understanding of gene and organ specific targets of MMR deficiency. While it is generally accepted that widespread MMR deficiency and consequent microsatellite instability (MSI) drives tumorigenesis in LS, the timing of molecular alterations is controversial. In particular, it is important to know that alterations may occur several years before cancer formation, at stages that are still histologically regarded as normal. Identification of molecular markers that could predict the risk of malignant transformation may be used to improve surveillance and cancer prevention in genetically predisposed individuals. Significant fractions of families with colorectal and/or endometrial cancer presently lack molecular definition altogether. Our findings expand the phenotypic spectrum of BMPR1A mutations and, for the first time, link FCCX families to the germline mutation of a specific gene. In particular, our observations encourage screening of additional families with FCCX for BMPR1A mutation, which is necessary in obtaining a reliable estimate of the share of BMPR1A-associated cases among all FCCX families worldwide. Clinically, the identification of predisposing mutations enables targeted cancer prevention in proven mutation carriers and thereby reduces cancer morbidity and mortality in the respective families.Lynchin syndrooma (LS) on perinnöllinen syöpäoireyhtymä, joka altistaa mm. kohtu,- munuais,- virtsarakko ja mahasyöville, sekä aivokasvaimille. Aiemmin LS tunnettiin kliinisen kuvansa perusteella perinnöllisenä ei-polypoottisena paksusuolen syöpänä eli HNPCC-tautina (hereditary non-polypotic colorectal cancer). LS:n tärkeimmät alttiusgeenit ovat DNA:n väärinpariutuneita emäksiä korjaavat MLH1, MSH2 ja MSH6. Po. alttiusgeenien tunnistamisen jälkeen termi LS onkin rajattu siihen HNPCC:n alaryhmään, jossa tiedetään olevan kyseisen korjausgeeniryhmän synnynnäinen virhe. Kliinisin kriteerein määritellyssä HNPCC-taudissa tunnetaan toinenkin alaryhmä, LS:aa muistuttava suvuittainen paksu-ja peräsuolisyöpäoireyhtymä eli FCCX (familial colorectal cancer type X). Sen alttiusgeenit ovat toistaiseksi olleet tuntemattomia, maailmanlaajuisesta etsinnästä huolimatta. Väitöskirjatyön ensimmäisessä osatyössä tutkittiin LS:n harvinaisempia syöpiä (aivokasvaimet, virtsarakko- ja munuaissyövät), joita verrattiin yleisimpiin paksusuoli,- kohtu ja mahasyöpiin. Toisessa osatyössä tutkimme LS-naisten kohtusyövän esiasteita ja vertasimme molekylaarisia tekijöitä satunnaisesti esiintyviin esiasteisiin. LS-naisilla alttius sairastua kohtusyöpään on jopa 60 % eli kaikkein yleisin LS-syövistä. Kolmannessa osatyössä vertasimme suomalaisten ja egyptiläisten lähinnä satunnaisesti esiintyviä paksu-ja peräsuolisyöpiä. Neljännessä osatyössä etsimme kytkentäanalyysin avulla alttiusgeenejä FCCX:ään. Havaitsimme, että LS:aan liittyvät harvinaisemmat syövät eli aivokasvaimet ja munuaissyövät kehittyvät erilaista reittiä kuin yleisimmät paksusuoli,- maha ja kohtusyövät. Löysimme eroja LS-naisten sekä verrokkiaineiston (satunnaisesti esiintyvien) kohtusyövän esiasteiden välillä. LS:ssa voitiin havaita normaalissa kohdun limakalvossa molekyylimuutoksia jopa yli kymmenen vuotta ennen varsinaisen kohtusyövän kehittymistä. Perinteisesti kohtusyövän kehittymisen tärkeimpänä ennusmerkkinä on pidetty atyyppistä kompleksista hyperplasiaa. Oman molekyylikartoituksemme perusteella kompleksinen hyperplasia atypian kanssa tai ilman ennustaa kohtusyövän kehittymistä; nämä tulokset on kuitenkin hyvä varmistaa isommassa aineistossa. Väitöskirjatyön tärkeimpänä löydöksenä voidaan pitää yhden ja samalla ensimmäisen alttiusgeenin löytymistä FCCX-syndroomaan. Aineistomme käsittää n. 20 FCCX-sukua, joista alttiusgeeniä on etsitty jo useita vuosia, tuloksetta. Kytkentäanalyysin antaman positiivisen tuloksen ja sen jälkeisen hienokartoituksen avulla pystyimme kaventamaan kromosomissa 10q23 sijaitsevaa aluetta. Läpisekvensoimme alueelta kolme geeniä ja löysimme kahdesta FCCX-suvusta mutaation BMPR1A-geenistä. BMPR1A geenin on todettu mutatoituneen n. 20 % Juveniilissä polypoosi syndroomassa (JPS), mutta näissä FCCX-suvuissa ei ollut viitteitä JPS:ään. JPS on myös paksusuolisyövän riskitekijä, mutta ilmiasultaan erilainen kuin FCCX. Tämän perusteella voidaan päätellä, että ilmiasultaan erilaiset sairaudet voivat olla kuitenkin saman alttiusgeenin aiheuttamia, mikä antaa aiheen etsiä muista FCCX-suvuista ko. geenin mutaatioita maailmanlaajuisesti

Updates in the field of hereditary nonpolyposis colorectal cancer : Expert Review of Gastroenterology & Hepatology

ABSTRACT Introduction Up to one third of colorectal cancers show familial clustering and 5% are hereditary single-gene disorders. Hereditary non-polyposis colorectal cancer comprises DNA mismatch repair-deficient and -proficient subsets, represented by Lynch syndrome (LS) and familial colorectal cancer type X (FCCTX), respectively. Accurate knowledge of molecular etiology and genotype-phenotype correlations are critical for tailored cancer prevention and treatment. Areas covered The authors highlight advances in the molecular dissection of hereditary non-polyposis colorectal cancer, based on recent literature retrieved from PubMed. Future possibilities for novel gene discoveries are discussed. Expert commentary LS is molecularly well established, but new information is accumulating of the associated clinical and tumor phenotypes. FCCTX remains poorly defined, but several promising candidate genes have been discovered and share some preferential biological pathways. Multi-level characterization of specimens from large patient cohorts representing multiple populations, combined with proper bioinformatic and functional analyses, will be necessary to resolve the outstanding questions.Peer reviewe

Analysis of sub-3 nm particle growth in connection with sulfuric acid in a boreal forest

We analyzed nanoparticle growth during new-particle-formation events based on ten years of measurements carried out at a boreal forest site in Hyytiala, Finland, concentrating on the sub-3 nm particles and the role of sulfuric acid in their growth. Growth rates of 1.5-3 nm diameter particles were determined from ion spectrometer measurements and compared with parameterized sulfuric acid concentration and other atmospheric parameters. The calculated growth rates from sulfuric acid condensation were on average 7.4% of the observed growth rates and the two did not correlate. These suggest that neither sulfuric acid monomer condensation nor coagulation of small sulfuric acid clusters was the primary growth mechanism in these atmospheric conditions. Also no clear sign of organic condensation being the single main growth mechanism was seen. These observations are consistent with the hypothesis that several factors have comparative roles in the sub-3 nm growth.Peer reviewe

Loss of p15(INK4b) Expression in colorectal cancer is Linked to Ethnic Origin

Peer reviewe

Sequencing of Lynch syndrome tumors reveals the importance of epigenetic alterations

Genomic instability and epigenetic aberrations are important classifiers of human tumors, yet, their interrelations are poorly understood. We used Lynch syndrome (LS) to address such relationships. Forty-five tumors (11 colorectal adenomas, 18 colorectal carcinomas, and 16 ovarian carcinomas) were profiled for CpG Island Methylator Phenotype (CIMP) and somatic mutations. All tumors showed high-degree microsatellite instability. Panel sequencing of 578 cancer-relevant genes revealed the average number of 1433, 1124, and 657 non-synonymous somatic mutations per colorectal adenoma, colorectal carcinoma, and ovarian carcinoma, respectively. Genes harboring mutations with allele frequency 25 % or higher in at least 31 % of tumors were regarded to be possible drivers. Among 72 and 10 such genes identified in colorectal and ovarian tumors, respectively, the most frequently mutated genes BRD4 and MLL2 (62 % of colorectal tumors) and ARID1A (50 % of ovarian carcinomas) are involved in epigenetic regulation. The total number of somatic mutations or mutant genes per tumor were significantly associated with CIMP. Our results suggest that even in an inherited disease, tumor type-specific epigenetic changes are significant and may result from regulatory changes (CIMP) or structural events (mutations of epigenetic regulatory genes). The findings are clinically relevant since many of the affected pathways can be therapeutically targeted.Peer reviewe

Pseudoexons provide a mechanism for allele-specific expression of APC in familial adenomatous polyposis

Allele-specific expression (ASE) of the Adenomatous Polyposis Coli (APC) gene occurs in up to one-third of families with adenomatous polyposis (FAP) that have screened mutation-negative by conventional techniques. To advance our understanding of the genomic basis of this phenomenon, 54 APC mutation-negative families (21 with classical FAP and 33 with attenuated FAP, AFAP) were investigated. We focused on four families with validated ASE and scrutinized these families by sequencing of the blood transcriptomes (RNA-seq) and genomes (WGS). Three families, two with classical FAP and one with AFAP, revealed deep intronic mutations associated with pseudoexons. In all three families, intronic mutations (c.646-1806T > G in intron 6, c.1408+729A > G in intron 11, and c.1408+731C > T in intron 11) created new splice donor sites resulting in the insertion of intronic sequences (of 127 bp, 83 bp, and 83 bp, respectively) in the APC transcript. The respective intronic mutations were absent in the remaining polyposis families and the general population. Premature stop of translation as the predicted consequence as well as co-segregation with polyposis supported the pathogenicity of the pseudoexons. We conclude that next generation sequencing on RNA and genomic DNA is an effective strategy to reveal and validate pseudoexons that are regularly missed by traditional screening methods and is worth considering in apparent mutation-negative polyposis families.Instituto Multidisciplinario de Biología Celula

Dual Specificity Phosphatase 1 Regulates Human Inducible Nitric Oxide Synthase Expression by p38 MAP Kinase

The role of dual specificity phosphatase 1 (DUSP1) in inducible nitric oxide synthase (iNOS) expression in A549 human pulmonary epithelial cells, J774 mouse macrophages and primary mouse bone marrow-derived macrophages (BMMs) was investigated. iNOS expression was induced by a cytokine mixture (TNF, IFNγ and IL-1β) in A549 cells and by LPS in J774 cells, and it was inhibited by p38 MAPK inhibitors SB202190 and BIRB 796. Stimulation with cytokine mixture or LPS enhanced also DUSP1 expression. Down-regulation of DUSP1 by siRNA increased p38 MAPK phosphorylation and iNOS expression in A549 and J774 cells. In addition, LPS-induced iNOS expression was enhanced in BMMs from DUSP1(−/−) mice as compared to that in BMMs from wild-type mice. The results indicate that DUSP1 suppresses iNOS expression by limiting p38 MAPK activity in human and mouse cells. Compounds that enhance DUSP1 expression or modulate its function may be beneficial in diseases complicated with increased iNOS-mediated NO production