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Onkogenetika, genetické mechanismy vzniku nádorů. Pavel Vodička 2014 Ústav experimentální medicíny v.v.i AV ČR 1. LF UK Praha.

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Prezentace na téma: "Onkogenetika, genetické mechanismy vzniku nádorů. Pavel Vodička 2014 Ústav experimentální medicíny v.v.i AV ČR 1. LF UK Praha."— Transkript prezentace:

1 Onkogenetika, genetické mechanismy vzniku nádorů. Pavel Vodička 2014 Ústav experimentální medicíny v.v.i AV ČR 1. LF UK Praha

2 VÝCHODISKA Sporadické formy rakoviny jsou charakterizovány polygenní součinností v komplexní interakci s vlivy prostředí (mikro- i makro-) a životního stylu Hodnocení rizika CRC v souvislosti s polymorfismy a haplotypy řady genů s cílem identifikovat relevantní kandidátní geny. Alternativní přístup založený na identifikaci relevantních variant vnímavosti pomocí GWAS. Verifikace a výklad GWAS dat (post-GWAS fine-mapping) cestou meta-analýz kandidátních genů a „next generation“ sekvenováním. Definování jak genetických, tak fenotypických rysů CRC (např. DNA reparační funkční testy)

3 Procento variance Nádorová onemocnění ve Švédsku,Dánsku a Finsku Stomach Colorectum Pancreas Lung Breast (female) Cervix uteri Corpus uteri Ovary Prostate Bladder Leukemia GeneticShared environmentalNonshared environmental Lichtenstein et al, 2000, NEJM %

4 Funkce genů může být ovlivněna:  genovými variantami (polymorfismy)  alteracemi v počtu kopií (amplifikace, delece, duplikace, změny v počtu chromozomů)  změny v struktuře genu, chromosomální struktura (translokace, inverse atd.)  genové mutace - substituce, delece, inserce v kódujícich sekvencích, na hranicích exonů a intronů  epigenetické modifikace (imprinting, DNA metylatce a modifikace histonů - acetylace/deacetylace histonů, metylace nebo fosforylace)

5 Předpoklady 1.Xenobiotika životního prostředí vykazují genotoxický účinek na člověka, hlavně díky přímé vazbě na DNA. 2.Předpokládá se, že genotoxický účinek (poškození DNA a chromosomů) je v souvislosti se vznikem sporadických forem rakoviny. 3.Genetický základ může formovat/ovlivňovat vnímavost jednotlivce k maligním onemocněním a riziku rakoviny. 4.Aparát DNA opravy zachovává integritu genomu tím, že opravuje poškození DNA indukované expozicí potenciálním karcinogenům. Pavel Vodička, Vztah mezi genotypem genů DNA opravy a fenotypem je zásadní pro identifikaci kritických časných markerů karcinogeneze Proč incidence rakoviny mezi silně exponovanými jedinci není dramaticky vyšší než je incidence běžné populace?

6 Kaskáda dějů v genotoxicitě a karcinogeneze XENOBIOTICS REACTIVE METABOLITES PROTEIN ADDUCTS DNA ADDUCTS CYTOTOXICITY, APOPTOSIS DNA SSB Transition lesions, CA, SCE, deletions, chromosomal instability DNA REPAIR MUTAGENESIS HPRT, tumor suppressor genes, oncogenes CARCINOGENESIS INDIVIDUAL SUSCEPTIBILITY Biotransformation and DNA repair genes Additional factors Metabolic activation or deactivation Pavel Vodicka,

7 Mutageny Fyzikální: radiace UV (ultrafialové záření) → T-T, C-C, T-C dimery = chyby v replikaci a transkripci ionizing (rtg, γ) přímý účinek → DNA zlomy nepřímý účinek → ionizace molekul → DNA zlomy Chemické– alkylační činidla - addukty - analoga bazí – chyby v párování bazí - acridinové barviva – inserce - kyselina dusičná –deaminace bazí – chyby v párování bazí přímé mutageny nepřímé mutageny– po metabolické aktivaci (cytochrom dependent oxygenázy) vznikají reaktivní produkty Biologické–viry - virové nukleové kyseliny se integrují do genomu hostitelské buňky

8 METABOLIZMUS fenantrenu

9 styrene styrene oxide (SO) styrene glycol (SG) mandelic acid (MA) phenylglyoxylic acid (PGA) CYP2E1 EPHX1 GSTs PHEMA 1PHEMA 2 CYP2E1*5A, *6, *1B, *1D EPHX1 Tyr113His EPHX1 His139Arg GSTM1 GSTT1 GSTP1 ADH ADH2 *1/*2 ADH3 *1/*2 Metabolizmus styrénu

10 Biomarkers most frequently applied in our Department: 1.Transient biomarkers of carcinogenesis 2.Biomarkers of genomic landscape of cancers 3.Phenotypic biomarker-function of substantial biological systems 4.Biomarkers of epigenetic regulations in carcinogenesis 5.Biomarkers of cancer phenotype 6.Biomarkers of treatment response (genetic, epigenetic, target vs. surrogate) Main requirements for biomarkers Sensitivity Validity Reproducibility Availability Informativness Cost effectiveness Complexity vs Interpretability

11 Zlepšení hodnocení expozice Příspěvek ke zpřesnění křivky dávka/účinek při nízkých hladinách expozice. Příspěvek k objasnění mechanismů genotoxicity a karcinogenity Identikace vnímavých jedinců a stanovení stupně vnímavosti Stanovení patogeneze a/nebo prognózy Potřeba validních biomarkerů v časné karcinogenezi Využití biomarkerů :

12 Biomarkery používané v in vivo studiích Comet test – jednořetězcové zlomy DNA Specifické DNA addukty – primární poškození DNA Micronucleus = chromosomální fragment v cytoplazmě erytrocytů nebo binukleárních buněk po zablokování cytokineze cytochalasinem Cytogenetické analýzy buněk kostní dřeně u experimentálních zvířat nebo lidských periferních lymfocytů – chromozomální zlomy nebo přestavba (rearangement) Výměna sesterských chromatid – inkorporace BUdR do DNA během kultivace buněk (pro dva buněčné cykly)– různá substituce obou chromatid = různé zbarvení sesterských chromatid Individuální vnímavost

13 Hlavní místa elektrofilních ataků v nucleosidech

14  N7-gua 45% αN7-gua 29%  N6-ade 6%  N3-ade 6%  N3-ade 3%  N1-ade 2%  N3-ura 2%  N1-hypox 2%  N4-cyt 1%  N6-ade 1%  N2-gua 3% N7-gua 70% N1-ade 17% N3-ade 10% N3-cyt 1% N3-ura 1% N6-ade 1% From: Vodicka et al. Mutat Res 2002; Vodicka et al. Drug Metab Rev 2006; Koskinen and Plna Chem Biol Interact 2002 Spektrum specifických DNA addukt ů styrénu (A) a 1,3 butadienu (B) A B Pavel Vodicka,

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16 Deaminací vzniká hypoxantín a AT-GC tranzice

17 Mutace = permanentní děditelná změna genetického materiálu = změna v sekvenci nucleotidů nebo přestavba DNA v genomu Mutace: spontánní indukované Mutace: somatické germline či gametické

18 Mutace: genomové mutace – změny v počtu chromozomů: a) euploidní změna = znásobení haploidních chromosomálních setů (triploidy, tetraploidy) b) aneuploidy = další chromozom (trisomie) nebo chybějící chromozom (monosomie) chromozomové mutace= strukturální chromozomální aberace– zlomy a výměny chromozomálních segmentů genové mutace = kvalitativní nebo kvantitativní změny v sekvencích DNA

19 GENOVÉ mutace mutace bez jakékoliv změny aminových kyselin (degenerace genetického kódu) „ MISSENSE mutation“ - záměna jedné aminokyseliny za jinou „ NONSENSE mutation“- mutace způsobí jeden ze tří „stop“ kodonů „ ELONGATION mutations“ změna stop kodónu na triplet kódujíci aminovou kyselinu „FRAME SHIFT mutations“- inserce, delece Mutace v rRNA a tRNA genech - chyby v translaci

20 Nejvýznamnější DNA addukty a jejich předpokládaná role v mutagenezi. Vodicka et al. Drug Metab Rev 2006 AdductMutation N7-guanineGC  TA N 2 -guanineGC  TA O 6 -guanineGC  AT 8-OH-guanine GC  TA 8-OH-adenine AT  CG N3-adenineAT  TA N1-adenineAT  GC N 6 -adenine AT  GC N3-cytosineCG  AT, GC  TA -AT  GC -GC  TA, AT  TA Expected Found Expected Found (dominating BS for SO) Found Pavel Vodicka,

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24 SSB MUTATIONS, CHROMOSOMAL ABERRATIONS, CYTOTOXICITY O 6 -ALKYL- TRANSFERASE REPAIR PERSISTANCE ?? REPAIR MUTATIONS, CYTOTOXICITY AT  GC MUTATIONS, CYTOTOXICITY GC  TA MUTATIONS, CYTOTOXICITY REPAIR MISPAIRING, GC  TA, AT  TA MUTATIONS MISPAIRING, MUTATIONS, BLOCK OF REPLICATIONS, CYTOTOXICITY REPAIR EXCISION REPAIR MUTATIONS, CYTOTOXICITY CELL DEATH TUMOUR DEVELOPMENT REPAIR Předpokládané důsledky různých DNA adduktů

25 Hlavní cesty DNA opravy  DNA oprava je komplexní systém obrany (cca. 120 genů) s cílem udržet genomovou integritu a je z hlavních cest předcházejích karcinogenezu.  Interindividuální rozdíly v kapacitě DNA oprav-významný faktor rizika maligních onemocnění (včetně CRC).  CRC-komplexní onemocnění, kde vnímavost může být ovlivněna genetickými variantami v DNA reparačních systémech.

26 Repair Pathways

27 Cell cycle DNA damage sensing DNA repair onkogeny Klíčová role opravy DNA v nádorové patogenezi hormony insulin, estrogen Růstové factory receptors, signal transducers EGFR, IGF-1, MAPK cytokiny a molekuly zánětu TGF1 / Smad3, interleukins stárnutí epigenetické faktory Chronický zánět a stres mikroprostředí obezitaexcesívní exo/endo poškození DNA

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29 Telomerová dysfunkce/udržování „DNA damage response mechanisms“ Control of apoptosis Cell cycle checkpoint control DNA damage sensing DNA repair DNA damage signaling Telomere maintenance Damage processing Double-strand break repair Martínez and Blasco, Aging Cell Oct;9(5):

30 Bázově-excizní oprava damaged base removed by glycosylase glycosylase AP endonuclease polymerase, ligase apurinic site removed by specific endonuclease gap filled by DNA polymerase and sealed with ligase Obr. : R.Štětina

31 endonuclease exonuclease polymerase ligase Nucleotidová excizní oprava Incision near to the damaged site by endonuclease The piece with the damaged base is removed by exonuclease DNA polymerase fils the gap The process is completed by sealing of strands by the ligase Obr. : R.Štětina

32 Opravy chybného párování bází řízené metylací  Opravy chybného párování bází řízené metylací (mismatch repair, MMR) hrají klíčovou roli v zachování genomické stability. MMR opravuje chyby v replikaci DNA, zahrnující nesprávně zařazané nukleotidy, inzerce a delece. A dále chyby v průběhu mitotické nebo meiotické rekombinace.  Složky systému MMR jsou také zahrnuty v odpovědi na poškození DNA, buněčném cyklu, apoptóze a remodelaci chromatinu.  Geny MMR jsou důležité jak v etiologii familiárního CRC, tak v souboru sporadicky se vyskytujících nádorů. Dysfunkce MMR vede k nárůstu nestability v mikrosatelitních sekvencích (MSI) napříč genomem.

33 Původ dvouřetězcových zlomů (DSB):: endogenní: oxidativní metabolizmus topoizomerázy (jednořetězcové zlomy-SSB,DSB) chyby v DNA replikaci nebo opravě DNA rekombinace –“ crossing over“ v meiose V(D)J rekombinace, „class switching“immunoglobulinových genů exogenní: radiace (ionizujíci, ultrafialové), chemikálie, restrikční endonukleázy DSB jsou indukovány přímo – ionizujícím zářením nebo nepřímo–UV zářením, chemikáliemi + enzymatic ká oprava→ SSB(single-strand breaks) → DSB (double-strand breaks)

34 Opravy dvouřetězcových zlomů: HR = homologní rekombinace - potřeba sesterské chromatidy (v G2, S fázích buněčného cyklu) - či přítomnost homologního chromosomu (meiotic recombination) NHEJ = nonhomologous end joining – hlavně vn G0, G1 - bez přítomnosti homologního templátu – „error prone“ Oba typy: - eliminace DSB - or mutation and chromosomal aberration – consequence of erroneous repair (HR, NHEJ) Řada genů zodpovědných za HR: e.g. BRCA1, BRCA2 XRCC1,XRCC2,NBS1,Rad geny etc.

35 PŘÍKLADY Z GENETICKÉ TOXICOLOGIE A MOLEKULÁRNÍ EPIDEMIOLOGIE Molekulárně epidemiologické studie definují biomarkery, jež charakterizují interní expozici, biologicky účinnou dávku xenobotik jakož i biologický účinek (často prediktivní pro vznik rakoviny) a variabilitu v individuální vnímavosti.

36 O 6 addukty v granulocytech a lymfocytech exponovaných a kontrolních osob Intraindividual variability: F=0.28, P=0.872

37 1-THB-AdeninOVe DNA addukty ve vztahu k GSTM1 a GSTT1 genotyp ů m u osob exponovaných 1,3-BD GSTM1 GSTT1 Kombinace genotypů GSTM1 + GSTT1 Pavel Vodicka,

38 Styrene1,3-ButadienePropylene oxide MouseHumanRatHumanRatHuman Exposure (mg/m 3 ) a /115 b 660 c d ≈10 a 1-alkyl-adenine b Amino-terminal valine (pmol/g) Ratio inStyrene1,3-ButadienePropylene oxide Hemoglobin adducts in humans alkyl-adenine adducts in animals alkyl-adenine adducts in humans a,b – exposures corresponding to Hemoglobin and N1-Ade adducts determinations, c – inhalation exposure for 5 days, d – inhalation exposure for 20 days; Values recalculated to make 1-Ade adducts formation comparable Pavel Vodicka, Comparison of Animal and Human DNA and Hemoglobin Adducts after Styrene, 1,3-Butadiene and Propylene oxide exposures

39 DNA adduct proportion: a lesson from animal experiments 40-fold excess of 7-alkylG as compared to 1-alkylA in lungs 1-alkylA 5-fold lower in liver than in lungs, whereas 7-alkylG undetected (!)-efficient repair in liver? Several-fold higher mEH expression in liver? Only persisting 7-alkylG in lungs in comparison to 7-alkylG excreted in urine (spontaneous depurination, BER) accounts for 0.5% of the total 7-G alkylation. Relevance for malignant transformation? The total 7-G alkylation of styrene, calculated as a percentage of the total styrene uptake, accounts for %. Relevance for malignant transformation? Assumption to organ-specific DNA repair Naturally occuring mutational rate ranges from 1-10/10 9 dNp. Pavel Vodicka Styrene inhalation in NMRI mice: Vodicka et al. Toxicol Appl Pharmacol, 2006

40 Různé kombinace genotypů v souvislosti s poškozením DNA A B A B P=0.002 P=0.005 A= EPHX1 (high activity), GSTT1 (positive) vs. B= EPHX1 (low activity), GSTT1 (null) A= EPHX1 (high activity), GSTM1 (positive) vs. B= EPHX1 (low activity), GSTM1 (null)

41 XPD (AA), EPHX1 (low activity) vs. XPD (CC), EPHX1 (high activity) Různé kombinace genotypů v souvislosti s poškozením DNA AB B= XPD (CC), XPG (GC+CC), XPC (AA) A=XPD (AA), XPG (GG), XPC (CC) P=0.016 P=0.006 vs. A B

42 Modified from A.D. Kligerman, Y. Hu / Chemico-Biological Interactions 166 (2007) Block of strand elongation Exogenous factors Působení různých exogenních faktorů v indukci DNA poškození Pavel Vodicka

43 CHA: control and exposed populations Frequency of chromosomal damage Healthy controls (N = 751) compared with healthy exposed subjects (N = 1028)

44 CHA: control and exposed population - distribution Distribution of frequencies of CAs (N = 1779)

45 CHA: control and exposed population - genotypes

46 CHA: control and exposed population - confounders Binary logistic regression models – chromosomal damage and genotype variants (adjusted odds ratio) CAs aOR95% C.I. For ORP-value Smoking – Occupational exposure – EPHX1 high activity genotype – CTA aOR95% C.I. For ORP-value Occupational exposure – XPD23 k751Q (T/G) –

47 Cyclin D1 (CCND1) G870A genotyp, hlavní konfoundery a frekvence ChA. VariablePersonsSignificanceOR 95% C.I. for OR LowerUpper Age (continuous) Sex (M/F)370/ Smoking (S/NS)250/ Exposed/Unexposed553/ CCND1_GG CCND1_GA CCND1_AA

48 Frekvence chromozomálních aberací (%) a CCND1 genotyp

49 BER kapacita a XRCC1 polymorphismus u zdravých osob Pavel Vodicka,

50 Results from functional studies: Vodicka et al,Carcinogenesis 2007 In a healthy population (n=244) associations between DNA repair polymorphisms and individual DNA repair capacity have been investigated. Significant associations have been found for XRCC1 Arg399Gln and hOGG1 Ser326Cys and for different BER polymorphisms in combination. A significant decrease in the capacity to repair DNA oxidative damage has been associated with variant alleles in hOGG1 Ser326Cys and APE1 Asn148Glu in combination (P=0.018). OGG1 Ser326Cys genotype OGG1 OGG1 X APE1 8-OH-dG Base Excision Repair : (Modified from Friedberg, 2004)

51 P=0.080 P=0.006 Vztah mezi 8-hydroxy-2´-deoxyguanosine adduckty v lymfocytech a hOGG1 Ser326Cys polymorfismem Pavel Vodicka, Variantní alela Ser souvisí se sníženou funkcí oxoguanine glycosylasy 1 (Luna et al., Nucleic Acids Res. 2005)

52 Slyskova J et al, Environ Mol Mutagen, 2011; Vodicka P et al, Carcinogenesis, 2007 Incision (tail DNA%) Influence of genetic variability on DNA repair

53 Influence of biological and lifestyle factors on DNA repair Betatp Sex Fruit intake XPG Asn1104His Betatp Sex Fruit intake Oxidative lesions + strand breaks NER 340 healthy individuals : 180 women / 160 men with similar age distribution : 150 food items + 12 antioxidants measured in plasma Slyskova J et al, DNA Repair, accepted

54 Examples from humans: DNA damage and DNA repair according to exposure, Ist sampling

55 The DNA repair activity in all animals is calculated as a difference between the initial levels of SSBs, measured immediately after γ-irradiation, and the levels of SSBs detected after 40 min. of incubation. Pavel Vodicka DNA repair kinetic: a lesson from animal study 2 γ-irradiation specific DNA repair activity in liver cells of mice, during and after the period of inhalation of 1,3-butadiene, and of unexposed control mice at particular time intervals

56 Examples from humans: DNA repair rates in relation to exposure and smoking habit In general healthy population: DNA repair capacity vs smoking R=0.168, P=0.006 Butadiene exposure

57 Clonal nature of tumors – from a single cell CARCINOGENESIS =multistep process – genetic + environmental factors Multiple mutations (growth controlling genes) Multiple causes and mechanisms Environmental factors: chemical carcinogens UV, ionizing radiation tumor viruses – RNA, DNA viruses Life-style Mutations – role in initiation of carcinogenesis

58 Tentative multistep carcinogenesis process, as designed for CRC, and potential points for biomarker recruitment General concept adopted and modified from Gareca et al., (2003) Eur. J. Cancer 39, Accumulation of DNA damage Attempt for DNA replication Genetic DNA mutations Signalling pathways Increased proliferation CRC Clinical presentation XME CYP, GST DNA repair enzymes TS, oncogenes APC, K-ras, p53 catenin COX-2 Polyps number& histology of polyps Adenoma formation Dysplasia Invasive cancer DNA adducts Grade of displasia Pavel Vodicka

59 Genetické změny v progresi CRC

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63 Journal.cambridge.com Knudson´s two-hit hypothesis of loss of function of tumor suppressor gene

64 Genomic instability The failure to transmit an accurate copy of the entire genome from one cell to its two daughter cells. Genomic instability can be divided into 2 categories Chromosomal instability (CIN) Microsatellite instability CIN has been linked to aneuploidy and CAs, LOH Cancer cells frequently exhibit CAs as translocations, deletions, or gene amplification – an importatnt source of chromosomal instability CINs are related to abnormalities of DNA metabolism, DNA repair, cell cycle control, apoptosis

65 Sources of genomic instability Nucleotide-excision instability (NIN): Loss of nucleotide excision repair activity Microsatellite instability (MSI): Deficiency of mismatch repair system Chromosomal instability (CIN):  Aneuploidy  Translocations  Insertions  Deletions  Amplifications Mutator phenotype Loss of cell cycle control (promoting CIN) Defects in mitotic apparatus (propagating CIN) Defects in DNA repair (inducing CIN) Pavel Vodicka

66 From H. Rajagopalan, C. Lengauer / Nature 432 (2004) Some processes implicated in the process of aneuploidy Pavel Vodicka

67 CIN Presence of multiple structural or numerical chromosome changes in tumour cells, in practice, often inferred from finding aneuploidy and/or polyploidy It is believed that CIN has a genetic basis The molecular basis of CIN has remained mysterious (many mechanisms have been postulated to be responsible for CIN) 1.Mutations in the mitotic spindle checkpoint; the genes that ensure the proper segregation of duplicated chromosomes 2.Tetraploidization of the genome caused by endoreduplication followed by loss of chromosomes due to instability of tetraploid genome 3.Mutations in the process of duplication of the centrosome 4.Formation of double strand breaks also generates CA 5.Impaired replication fork progression and increased DNA replication stress – CIN in colorectal cancer cell lines; additonally, new CIN-suppressor genes identified (MCD 4/PIGN, MEX3C and KIAA0222) Burrell at al.,Nature, 494, 2013,

68 -% of aberrant cells (ACs), chromosomal aberrations (CAs), chromatid type aberrations (CTA), chromosom type aberrations (CSA) Vodicka et al., Carcinogenesis 2010 CHROMOSOMAL DAMAGE ACCORDING TO THE TYPE OF MALIGNANCY Pavel Vodicka

69 Chromosomal aberrations and cancer risk - distributions Distribution of frequencies of ACs

70 CHA: control and exposed population - distributions Distribution of frequencies of CAs (N = 1780)

71 TNM and Grading characteristics in solid tumors TNM I +II vs. TMN III + IV CRCLung cancer Breast cancer TestP-value Percentage of ACs in relation to the TNM categories Mann-Whitney U test Percentage of CAs in relation to the TNM categories Mann-Whitney U test Percentage of CTAin relation to the TNM categories Mann-Whitney U test Percentage of CSA in relation to the TNM categoriesis Mann-Whitney U test The significance level at 0.05 Grading G+G2 vs. G3+G4 CRCLung cancer Breast cancer Null HypothesisTestP-value Percentage of ACs in relation to the categories of grading Mann-Whitney U test Percentage of CAs in relation to the categories of grading Mann-Whitney U test Percentage of CTA in relation to the categories of grading Mann-Whitney U test Percentage of CSA in relation to the categories of grading Mann-Whitney U test The significance level at 0.05

72 Tom van Wezel, Mutagenesis, 2012

73 Repair Pathways

74 Schematic representation of the DNA repair and Growth arrest signaling pathways. Examples of the molecule known to act on the regulatory pathways are shown

75 Implication of DNA repair modulations in cancer. DNA repair downregulation can contribute to genomic instability, which promotes malignant transformation of cells, and leads to cellular sensitivity to DNA damaging therapy. DNA repair upregulation can contribute to genomic stability, which lead to acquired resistance to the DNA damaging therapy.

76 Bleomycine Challenge Assay – DSB Repair

77 Bleomycin challenge

78 Mismatch repair pathway The DNA mismatch repair (MMR) system plays a key role in maintenance of genomic stability. MMR edits mismatches and insertions/deletions generated during DNA replication and mitotic/meiotic recombination. The components of MMR machinery are also involved in DNA damage response, cell cycle, apoptosis, chromatin remodeling and antibody diversity. MMR genes are of importance as „high penetrance“ genes in the aetiology of familial CRC (HNPCC syndrome) and a set of sporadic cancers. MMR disfunction leads to gross instability in microsatellite sequences throughout genome (MSI). A limited knowledge has been available on the role of MMR genes as „low-penetrance“ genes in sporadic CRC risk

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80 CpG island methylation of the hMLH1 promoter region vs MLH1 expression by IHC We analyzed colon biopsies of 105 CRC patients We found hypermethylation in 8 tumors with MSI-H status. Hypermethylated tumors localized in right colon only (4x caecum C18.0. and 4x ascendens C18.2) There is a strong association between promoter metylation of hMLH1 and reduced protein expression (R=-0.75, P<0.0001).

81 Expression of MMR genes mRNA expression levels of MMR genes in tumor and healthy tissues in the study subjects  fold lower mRNA levels of EXO1 gene were observed in tumor tissues when compared with adjacent healthy mucosa  significantly higher expression levels of MSH3 gene were observed in colon tumors when compared to adjacent healthy mucosa; 1.18 fold change

82 Fold change differences in expression level of MMR genes in colon tumors relative to rectal tumors

83 Ovarial cancer

84 Dlouhé delece – předbežné výsledky

85 Sumární změny v MLH1 genu: MLH1+ gain of the exonMLH1- loss of the exon

86 Dráhy DNA oprav  DNA repair is a complex system of defenses evolved to protect the genomic integrity and involved in the process preventing carcinogenesis.  Interindividual differences in DNA repair capacities are important determinants of cancer risk including CRC.  CRC represents a complex disease where susceptibility may be influenced by genetic polymorphisms in the DNA repair system.

87  While exposure in healthy subjects may result in compensatory increase in DNA excision repair,  Individuals with defective DNA repair are at severely increased risk of developing cancer and other diseases  What do we measure by applying functional repair assays in surrogate (blood) cells and in target (tumor) tissues? FOCUSING ON DNA REPAIR CAPACITY IN CANCER PATIENTS

88 DNA repair capacity is multifactorial biologival process NER-DRC phenotype Transcript Proteín > >> ERCC1 mRNA ERCC1 > >> XPA mRNA XPA > >> XPB mRNA XPB > >> XPC mRNA XPC > >> XPD mRNA XPD > >> XPE mRNA XPE > >> XPF mRNA XPF > >> XPG mRNA XPG Gene SNP 6 SNPs 9 SNPs 8 SNPs 15 SNPs 13 SNPs 14 SNPs 7 SNPs 13 SNPs Precursors, availability Epigenetic regulation Transcription regulation Enzyme activation Enzymme inhibition External exposure Nutrition < < < Biological factors Other interfering proteins Environmental variability Mechanism Source Genetic variability < Life style

89 DNA DAMAGE IN INCIDENT CRC PATIENTS VS CONTROLS P < N=69

90 P < N=69 NER CAPACITY IN INCIDENT SPORADIC CRC PATIENTS

91 NER CAPACITY IN CRC PATIENTS STRATIFIED FOR STAGING OF THE TUMOR

92 NER GENE EXPRESSION LEVELS IN CRC PATIENTS AND CONTROLS

93 A D C B BER RATES IN VARIOUS TISSUES of CRC PATIENTS

94 A D C B NER RATES IN VARIOUS TISSUES of CRC PATIENTS

95 GenePathwayFold changep-value NEIL1BER APEX1BER OGG1BER PARP1BER LIG3BER MUTYHBER MPGBER NTHL1BER CSBNER CCNHNER XPANER XPDNER LIG1NER MNAT1NER RPA3NER CDK7NER XPFNER RPA2NER XPBNER DDB1NER ERCC1NER DDB2NER RAD23BNER Expression profile of the genes involved in excision repair

96 48 controls 39 CRC patients Peripheral blood DNA repair in relation to therapy Slyskova J et al, Molecular Carcinogenesis, in press Surgery Chemotherapy T0T1T2 6 months ~ 64 years

97 p=0.009 p=0.001 N=38 N=33 p=0.038 p=0.032 N=38 N=33

98 40 DNA repair genes → Slyskova J et al, Molecular Carcinogenesis, in press CRC T0 Controls DNA repair in relation to therapy + T1 + T2

99 Oxaliplati n NER repairBER repair 5-fluorouracil Source: Clinical Colorectal Cancer CIG Media LP Source: Longley D. Natu Introduction

100 Questions to be addressed Mechanism? Cancer temporary suppression of DNA repair WHERE? Target, surrogate? tail DNA % NER Lower repair = Good responders Better prognosis Advanced side effects Higher repair = Poor responders Worse prognosis Milder side effects ?

101 Department of Molecular Biology of Cancer Institute of Experimental Medicine, Academy of Sciences of Czech Republic Website: Prague (CZ) P. Vodicka V. Vymetalkova L. Vodickova J. Slyskova P. Procházka M. Svoboda L.Bielik, M. Kroupa A. Rejhová S. Vodenkova K. Jiraskova Collaborating clinicians M. Schneiderová L. Lipská, M. Levý T. Büchler Pilsen (CZ) Václav Liška and collegues Hradec Kralove (CZ) Rudolf Stetina This work is supported by GACR P304/10/1286, P304/12/1585 and IGA NR ACKNOWLEDGEMENTS I would like to express my deep gratitude to all co-authors, particularly for their friendship and valuable contribution London, Oxford (UK) Ian Tomlinson Richard Houlston Pisa and Torino (I) Stefano Landi Federica Gemignani Barbara Pardini Alessio Naccarati Heidelberg (D) Kari Hemminki Asta Foersti Rajiv Kumar Justo L. Bermejo Federico Canzian Daniele Campa Öröbro, Sweden Torbjorn Nilsson

102 D ě kuji Vám za pozornost

103 Cytogenetic analysis in patients with newly diagnosed colorectal and breast cancer and healthy controls – results CRC Controls (n=298) Patients (n=100) P-value Age (years) 56,9±13,563,0±10,2 <0,0001 Aberrant cells (%) 1,82±1,322,14±1,44(*) 0,056 Total aberrations (%) 1,95±1,472,27±1,65(*) 0,087 CTA (%) 1,11±0,991,45±1,28* 0,030 CSA (%) 0,84±1,130,82±1,01 0,838 BREAST Controls (n=123) Patients (n=123) P-value Age (years) 65,2±16,259,7±10,2 0,015 Aberrant cells (%) 1,98±1,41 2,52±1,53** 0,002 Total aberrations (%) 2,08±1,522,64±1,60** 0,002 CTA (%) 1,23±1,091,54±1,33(*) 0,082 CSA (%) 0,85±0,991,11±0,99* 0,023 statistically significant difference between patients and controls *p≤0,05 (*) the borderline of statistical significance statistically significant difference between patients and controls *p≤0,05, **p≤0,01 (*) the borderline of statistical significance

104 Chromosomal damage (the percentage of aberrant cells, total aberrations, CTA and CSA) in patients with newly diagnosed CRC and breast cancer and healthy controls

105 Distribution of the frequency of aberrant cells in patients with newly diagnosed colorectal and breast cancer and healthy controls (expressed in %) 0 – 1% aberrant cells low frequency of aberrant cells 2% aberrant cells middle frequency of aberrant cells 3 a ≥4% aberrant cells high frequency of aberrant cells

106 Binary logistic regression analysis – determination of the cancer risk influenced by chromosomal damage and other factors (age and smoking) – aOR 100 patients vs. 298 controls aOR95%CIP-value Aberrant cells (%)1,1560, ,3710,097 Age (years)1,0381, ,058 ≤0,0001 Smoking1,0651, ,1310,038 Total aberrations (%)1,1170, ,2980,149 Age (years)1,0381, ,058≤0,0001 Smoking1,0661, ,1320,035 CTA (%)1,2951, ,5960,016 Age (years)1,0391, ,058≤0,0001 Smoking1,0631, ,1290,047 CSA (%)0,9520, ,1840,656 Age (years)1,0401, ,059≤0,0001 Smoking1,0671, ,1320, patients vs. 123 controls aOR95%CIP-value Aberrant cells (%)1,3351, ,6000, 002 Age (years)0,9690, ,988 0,002 Smoking1,1041, ,1980,018 Total aberrations (%)1,3151, ,5620,002 Age (years)0,9680, ,9870,001 Smoking1,1021, ,1960,020 CTA (%)1,1940, ,4800,106 Age (years)0,9730, ,9920,005 Smoking1,0961, ,189 0,028 CSA (%)1,5671, ,0830,002 Age (years)0,9640, ,984≤0,0001 Smoking1,1181, ,2150,008 CRCBreast cancer

107 Opravy chybného párování bází řízené metylací  Opravy chybného párování bází řízené metylací (mismatch repair, MMR) hrají klíčovou roli v zachování genomické stability. MMR opravuje chyby v replikaci DNA, zahrnující nesprávně zařazané nukleotidy, inzerce a delece. A dále chyby v průběhu mitotické nebo meiotické rekombinace.  Složky systému MMR jsou také zahrnuty v odpovědi na poškození DNA, buněčném cyklu, apoptóze a remodelaci chromatinu.  Geny MMR jsou důležité jak v etiologii familiárního CRC, tak v souboru sporadicky se vyskytujících nádorů. Dysfunkce MMR vede k nárůstu nestability v mikrosatelitních sekvencích (MSI) napříč genomem.

108 Metylation of MLH1 in CRC patients 12 CRC samples showed higher level of methylation than 2.5% When setting „cutoff“ for methylation to 15% – strong association with MSI-H 7 healthy tissues showed methylation higher than 2.5% - often in pairs with methylated CRC sample

109

110 MLH1 protein expression - colon ascendens and flexura hepatica [%][%]

111 [%][%] N MLH1 protein expression - colon ascendant and flexura hepatica

112 There is a association between promoter metylation of MLH1 and protein expression of MLH1 R = R 2 = 0.52 P = Expression of MLH1 in CRC patients

113 Long deletions – preliminary results

114 All changes in MLH1 gene: MLH1+ gain of the exonMLH1- loss of the exon

115 DNA repair pathway  DNA repair is a complex system of defenses evolved to protect the genomic integrity and involved in the process preventing carcinogenesis.  Interindividual differences in DNA repair capacities are important determinants of cancer risk including CRC.  CRC represents a complex disease where susceptibility may be influenced by genetic polymorphisms in the DNA repair system.

116 DNA damage in controls and CRC incident patients at the time of diagnosis P < N=69

117 P < N=69 NER capacity in controls and CRC incident patients at the time of diagnosis

118 NER capacity in CRC patients stratified for staging of the tumor

119 NER gene expression levels in CRC patients and controls

120 BER hladiny v různých tkáních CRC pacientů

121 NER hladiny v různých tkáních CRC pacientů

122 Vztah mezi NER a BER hodnotami v nádorové tkáni R=0.32 p=0.007 R 2 = 0.10

123 Survival or Apoptosis, that’s the problem in cancer therapy and for individual health. The determination either survival or apoptosis is due to the balance between DNA damage and the DNA repair levels in cells.

124 Děkuji Vám za pozornost

125 Arguments for genomic instability as the engine of tumorgenesis Tumors harbour too many mutations to be explained by anything other than underlying genomic instability The probability of a tumour acquiring enough mutations for the full, malignant phenotype is too low unless the cells have an unstable genome Humans and model organisms with inherent genomic instability are prone to tumors In some tumours, there is direct evidence that some pathways that are involved in maintaining genomic integrity are defective

126 the studies ON MULTIPLE LOW-RISK VARIANTS Sporadic forms of cancer are hallmarked by multigenic features in a complex interaction with environmental/life-style factors To assess the CRC risk in association with polymorphisms, and haplotypes in several pathways to identify relevant candidate genes. To participate on the alternative approach to identify relevant susceptibility loci using GWAS. To verify and refine GWAS (post-GWAS fine-mapping) data by candidate gene meta-analyses and by next generation sequencing Definition of both genetic and phenotypic landscape of CRC (e.g. DNA repair functional tests)

127 Simplified scheme showing a putative effect of low penetrance variants on CRC susceptibility 1 SNP / 2SNPs/…. Haplotypes Gene 1 Pathway 2 Cell cycle Pathway 3 Metabolism Pathway 4 Immune response Other Pathways Cancer Risk 1 SNP / 2SNPs/…. Haplotypes Gene 2 1 SNP / 2SNPs/…. Haplotypes Gene 3 1 SNP / 2SNPs/…. Haplotypes ……… 1 SNP / 2SNPs/…. Haplotypes Gene n +/- External environmental influence Individual Susceptibility Internal environmental influence Pathway 1 DNA Repair NER, BER, DSB, MMR, Rev. Repair

128 TP53 haplotype analysis Global P –value for haplotype effect < Yellow Significant ORs for individual haplotypes are in bold (Yellow= increased risk, Blue= decreased risk) TP53 Ins 11951_ Ex4 +119G> C IVS7 +72C>T Ex G>A ControlsCRCOR (95% CI) A1A1 GCG Referent A2A2 CCG ( ) A1A1 CCG ( ) A1A1 CCA ( ) A2A2 GCG ( ) A1A1 CTG ( ) A1A1 GTG ( ) A1A1 GCA ( ) Polakova et al., Human Mut 2009

129 16 CRC susceptibility loci identified by GWAS 8q24.21 (rs ), 18q21 (rs , rs and rs ), 15q13.3 (rs and rs10318), 10p14 (rs ), 8q23.3 (rs ) 1 1q41 (rs , rs ), 3q26.2 (rs ), 12q13.13 (rs , rs ) and 20q13.33 (rs ), 14q22.2 (rs ), 16q22.1 (rs ), 19q13.11 (rs ) and 20p12.3 (rs and rs ) 2 11q23.1 (rs ) 3 1 Tomlinson et al., Nature Genetics Houlston et al., Nature Genetics Pittman et al., Human Molecular Genetics 2008

130 SNPs at 16 genetic loci associated with CRC risk rs * (1q41), rs (3q26.2), rs (8q23.3), rs (8q24.21), rs (10p14), rs * (11q23.1), rs (12q13.13), rs #, rs (14q22.2), rs † (15q13.3), rs (16q22.1), rs (18q21.1), rs (19q13.11), rs † and rs † (20p12.3) and rs # (20q13.33). Statisticaly significant associations with specific phenotypes are highlighted *microsatelite stable rectal disease † microsatelite stable colonic disease # microsatelite instability colonic disease Lubbe et al., Carcinogenesis 2011

131 Gene promoter variant CASP N ins/del (rs ) verified on 13T European CRC cases and 14T controls: no association with sporadic cases (Peterlongo et al. In preparation) None of gene variants in APC D188V (rs459552), MLH1 I219V (rs ), MSH6 P92P (rs ) and D180D (rs ) and MUTYH V22M (rs ) and Q338H (rs ) was associated with sporadic CRC risk on the same population as above (Picelli et al. In preparation) Two pairs of tagging SNPs at 1q41 (rs and rs ) and 12q13.13 (rs and rs ) were associated with CRC. However, neither pair fully captured the association. The study shows intrinsic difficulties of post-GWAS fine-mapping studies ( Spain et al. Human Molec Genetics, Epub Nov )

132 Recent research shows a pendulum swinging from GWAS to next generation candidate gene studies-capturing variants with low frequency and small effect Post-GWAS fine mapping-identification of candidate functional variants Meta-analyses of candidate functional variants Tomlinson et al. Mutagenesis 2012 in press Other approaches to identify functional consequences of relevant pathways (examples shown above) Slyskova et al. Mutagenesis 2012 in press

133 Různé kombinace genotypů v souvislosti s chromosomálním poškozením P=0.008 P=0.002 A= EPHX1 (high activity), GSTM1 (positive) vs. B= EPHX1 (low activity), GSTM1 (null) A B A B A= EPHX1 (high activity), GSTT1 (positive) vs. B= EPHX1 (low activity), GSTT1 (null)

134 A= XPD (AA), EPHX1 (low activity) vs. B= XPD (CC), EPHX1 (high activity) A=XPD (AA+AC), XPG (GG), XPC (CC), XRCC1 (GA+AA) Různé kombinace genotypů v souvislosti s chromosomálním poškozením * B= XPD (CC), XPG (GC+CC), XPC (AA+AC), XRCC1 (GG+GA) P=0.004 P=0.007 vs. A B A B

135 Associace mezi kapacitou opravy oxidativního DNA poškození, hOGG1 a APE1 genotypy * * OGG1 Ser326Cys genotype A decrease in the capacity to repair DNA oxidative damage has been associated with variant alleles in hOGG1 Ser326Cys and APE1 Asn148Glu in combination (P=0.018). Pavel Vodicka

136 Examples from humans: DNA damage and DNA repair according to exposure, Ist sampling

137 The DNA repair activity in all animals is calculated as a difference between the initial levels of SSBs, measured immediately after γ-irradiation, and the levels of SSBs detected after 40 min. of incubation. Pavel Vodicka DNA repair kinetic: a lesson from animal study 2 γ-irradiation specific DNA repair activity in liver cells of mice, during and after the period of inhalation of 1,3-butadiene, and of unexposed control mice at particular time intervals

138 Clonal nature of tumors – from a single cell CARCINOGENESIS =multistep process – genetic + environmental factors Multiple mutations (growth controlling genes) Multiple causes and mechanisms Environmental factors: chemical carcinogens UV, ionizing radiation tumor viruses – RNA, DNA viruses Life-style Mutations – role in initiation of carcinogenesis


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