Biology of CancerSofia MubarikaKuliah S2 FK UGM

Biomolecular and cancerBasic of molecules of lifeHow cancer developedMolecular application in cancer diagnosis Molecular application in cancer treatmentCancer biomarkers

Basic molecules of life

Mathematical Biosciences Institute (Ohio State Univ), 2 October 2003RNAProteinDNADOGMA CENTRAL

Gen : DNA T-A C-GThymine (T) Adenine (A)Cytosine (C) Guanine (G)Over 3 billion base pairs, 30,000 genesHumans are over 99.9% identical

Central Dogma of Molecular BiologyDNARNAProteinTranscriptionTranslationA gene is expressed in 3 steps:1) Transcription: RNA synthesis2) Splicing: removal of intron sequence from RNA3) Translation: Protein synthesis

RNA TranscriptionProtein TranslationDNA RNA ProteinGene Expression

How much DNA do we have?humans have 2 x 23 chromosomesEACH cells contains 6 billion bases DNAthat is 1 meter of DNAa human being has >100.000.000.000.000 cellsthat is 100 billion km of DNA6 billion bases = 6 Gigabyte30.000 - 50.000 genesa lot of junk-DNA contains no code bus has a different function

Cause of cancer? Multiple genetic abnormalities Internal factors : defect genetic/inherited External factors :X RAYSEnvironment : Viral infections : HPV, EBV, HBV, HIVCarcinogenic compounds : food, working area, lifestyle: smoking , alcohol, HIV, HBV, HPV, EBV

RetrovirusesRetroviruses family of RNA viruses that cause cancer in variety of animals and humans.The Retrovirus : 3 main genes gag, pol & env required for virus replication, not play role in cell transformation.a retrovirus can transform cells from normal to cancer if they include a specific gene that is capable of inducing cell transformation this gene is known as Oncogene.

RetrovirusCancerous RetrovirusOncogene

Retrovirus oncogeneTwo main types of oncogenes:Viral oncogene: gene from the retrovirus itselfNon-Viral oncogene (Cellular oncogene): genes derived from the genes of the host cell that are in an inactive form usually. Occasionally if the gene incorporates with the viral genome will form a highly oncogenic virus.Proto-oncogenes: are the form of cellular genes that inactive normally but can incorporate with the viral genome to produce a highly oncogenic virus.

EBV-NPC in IndonesiaMost are Javanese, the 1st among men Hot Spot areas way of life? Salted fish?Special tobacco ingredients?Central Java mapLate stage

EBV-associated tumorsin manEpithelial tissues

Oral hairy leukoplakia 100%

Gastric adenocarcinoma 5-10%

Nasopharyngeal carcinoma,undifferentiated 100%

Salivary gland carcinomas 90%

Epstein-Barr virus Latent Membrane Protein 1 (LMP 1 )JkLRSEBNA 2IkBTRAF-1 LMP1

NCAGDFYWHBQEKMPU1TRAF-3p65/p50Bcl-2- Cell aggregation - Cycline D2- CD 23 - EGFR- CD 18, 21,39,40,44 - tumorigenicity- LFA 1 - CAM (Ca2+ pr- LFA 3 kinase G1- ICAM 1 - vimentin? - MHC Class IIA20blocks apoptosiscellular genes+/-+?-epithelial cell differentiation-+TRADD/TANK

EBV latent membrane protein (LMP1)as an oncoproteinTransform rodent cells, loss of contact inhibitionAnchorage-independent growth

Morphological transformation of human keratinocytes tumour in nude miceInhibit human epithelial cells differentiation

Induction of bcl-2 to protect infected B cells from apoptosisA20 in epithelial cells

-Essential for B-lymphocyte growth transformation

LMP 1NF-kBCox-2Prostaglandin E2VEGF(angiogenesis)EGFRMMP9IL-8(invasivenessIV collagenase)NF-kB(angiogenic factor)

So what .. ( from results??)Clinical application ?? Potential to be developed and further exploredBetter diagnostic approachNon invasive for follow upField epidemiology for early screening/ high riskMolecular epidemiology The profil EBV- NPC to develop vaccine, immunotherapy (LMP1, LMP2, EBNAs)Serology markers.

Proliferation of EBV activated blastsLatent reservoir of EBV Infected resting B cellsCTLT cell mediated control of proliferationoropharyngeal epitheliumAg?Infection of circulating B cellsIgA?CorrespondsTo neuronalAxon of neurotropicHerpesvirusVirus replication in EBV cycle in vivo

Develop simple and cheap non-invasive diagnostic and prognostic methods for NPC by monitoring EBV related parametersAim:Serology: anti-EBV IgA NA-based diagnostics: EBV DNA load and/or EBV RNA profile- Serum/plasma- Finger prick- Dried blood spot- Simple and cheap procedure (ELISA, Dipstick, filter) - Screening + Monitoring assay- Blood/plasma- Nasal swab- Simple sampling/extraction (GuSCN) Real-time PCR/NASBA methods Confirmation & monitoring

Molecular Diversity of Epstein-Barr virus IgG and IgA Antibody Responses in Nasopharyngeal Carcinoma:Jajah Fachiroh1, et al. J.Infect.Dis 189 (15-06-04)

EBNA1VCA-p18combinationCut-off value0.12050.22330.3536Sensitivity (%)88.4979.7685.32Specificity (%)80.1370.8690.07

Date 1: 12-09-20012: 03-12-20013: 15-03-20024: 17-10-20025: 16-12-20026: 01-05-20037: 02-07-20038: 05-11-20039: 19-11-2003*

Days 1: 02: 823: 184 CR4: 4005: 4606: 5967: 6588: 784 9: 798* Mx* Lung metastasis (CT/MRI)Case: Female caucasian (age 16): T4N1M0 Treatment Chemoradiation + IFN- > 2 years follow-up R.Mertens et al. Cancer. (1997);80(5):951-9.EBV-serology predictive at >6 months pre-metastasis Note: No increased EBV-DNA in circulation

Grafiek3

2.3651.211

1.8440.834

1.2310.422

0.5610.293

0.4260.203

1.2360.368

2.4320.864

2.8711.564

3.3012.02

VCA-p18 peptide

EAd-extract

Sample number

ELISA OD 450 value

EBV IgA ELISA

Grafiek2

Blad1

123456789

VCA-p182,3651,8441,2310.5610.4261,2362,4322,8713,301

EAd-extr1,2110.8340.4220.2930.2030.3680.8641,5642,020

VCA-p18EAd-extract

12.3651.211

21.8440.834

31.2310.422

40.5610.293

50.4260.203

61.2360.368

72.4320.864

82.8711.564

93.3012.020

Blad1

00

00

00

00

00

00

00

00

00

VCA-p18 peptide

EAd-extract

Sample number

ELISA OD 450 value

EBV IgA ELISA

Blad2

Blad3

EBV Polymorphism polytope vaccine (future)LMP1, 2 and EBNAs) polytope vaccineImmunotherapy Polytope vaccine Decrease the mass tumor of Non Hodgkin lymphoma - clinical trial phase I (Denis Moss et al 2002)

The genome is an organisms complete set of DNA.a bacteria contains about 600,000 DNA base pairshuman and mouse genomes have some 3 billion.human genome has 24 distinct chromosomes.Each chromosome contains many genes. Gene basic physical and functional units of heredity. specific sequences of DNA bases that encode instructions on how to make proteins. Proteins Make up the cellular structurelarge, complex molecules made up of smaller subunits called amino acids.

Proto-Oncogene OncogeneThe proto-oncogene become oncogene by:Mutation:Example: mutation in Ras gene Continuous activation of Ras by (constitutively in the GTP-bound conformation ) Unregulated cell proliferation Cell transformation.

2. Abnormal Activity:Example: Removal of the Regulatory domain in the Raf gene and replaced by gag gene Raf kinase domain consciously active Cell transformationRegulatory DomainProtein Kinase DomainProtein Kinase DomaingagRaf Proto-oncogeneRaf oncogene

3. Gene translocation:Example: c-myc gene is translocated from chromosome 8 to the IgH on the chromosome 14 resulting in abnormal c-myc expression Cell transformation

4. Amplification:Example: Amplification of n-myc neuroblastoma. Amplification of erbB-2 Breast & ovarian carcinomas

How does a Proto-oncogene become an Oncogene?1.Mutation

2. Abnormal Activity3.Gene Translocation4. Amplification

Abnormal Activity

Functions of oncogeneGrowth Factor (example, Epithelium growth factor EGF , and platelet derived growth factor PDGF)Growth Factor Receptor (Example; PDGFR)Signal transudation (example; Ras, Raf, & MEK)Transcription Factor (example; Jun, Fos, Elk-1 & myc)

Oncogenes Oncogene causes cancer by affecting:Cell Proliferation: (example; Ras, Raf, EGF)Cell differentiation (example, PML/RAR that inhibits the differentiation of promyelocyte to granulocyte which will maintain the cell in its active proliferate state)Cell Survival (example; Pl-3/AKT which will activate BCL-2 inhibit Apoptosis & maintain cell survival.

Tumour Suppressor GenesTumour Suppressor genes: are genes that act to inhibit cell proliferation and tumour development.

If Tumor Suppresor Gene was Mutated Inactivated It will lead to cell transformation OR

Hereditary MutationNon-hereditary Mutation

Function of Tumour Suppressor geneAntagonize the action of oncogene. (ex.PTEN which converts PIPIII to PIPII because PIPIII will activate Pl-3/AKT which will activate BCL-2 that will inhibit apoptosis and induce cell transformation)

AKTBCL-2Inhibit apoptosis & induce cell transformation PI-3

2. Transcription factorsRepressor transcription factors: example; WT1 is a repressor that appears to suppress transcription factor ( Insulin like growth factor) which will contribute in the development of tumour.Activator transcription factors: example; SMAD family that are activated by TGF-, leading to inhibition of cell proliferation.Function of Tumour Suppressor gene

3. Regulate cell cycle :Rb gene: that inhibits the cell cycle in the G1 phase decrease cell proliferation.INK-4 gene: that produces P16 that inhibits cdk4/cyclin D action ( to phosphorylate Rb gene to inactivate its action)P53: that produces P21 that has the same action of P16 in inhibiting the action of cdk4/cyclin D

Function of Tumour Suppressor gene

4. Induce apoptosis:P53 release will increase Bax form holes in the mitochondria release cytochrom c activate apoptosis

Function of Tumour Suppressor gene

Cancer DetectionCancer detection :Clinical detection by mammogram, coloscopy etcMolecular detection by SerotypeRestriction fragment length polymorphism (RFLP)PCRWestern Blot

Cancer Treatment Chemotherapy:Deals with DNA damage, & has affinity to all proliferating cells not specifying if it was a cancer cell or not.Inhibiting AngiogenesisInhibit blood flow/supply to the tumour cellsDecrease franesylation of RasDecrease activation of Ras, because Ras mutation causes most cancers.Monoclonal Antibody

Biomarkers and Prevention?Folate and vit B12 decreased breast Ca risk (Canc Epid Biomarkers Prev 2006;15(3):443-448)Genetic variation of Nucleotide excission repair (NER) and cancer risk ( Canc Epid Biomarkers Prev 2006;15:536-542)Lipid profile :Monosaturates lipid elevated and low ratio w6/w3 fatty acid decreased breast ca risk ( Canc Epid Biomarkers Prev 2006;15:416-421)

Molecular target in cancer therapyAnti tyrosine kinase: Gleevec, IressaAnti VEGFEGFR inhibitor etcNeed to enhance translational research into early IRT-MTA (Interdiscilinary Research Teams) for Molecular Target assesment

100500% EnergyLow-fat meat ChickenEggsFishFruitVegetables (carrots)NutsHoney100500% EnergyFruitVegetablesBeansMeatChickenFishGrainMilk/-productsIsolated CarbohydratesIsolated Fat/Oil Alcohol1.200.000 Generations between feast en faminePaleolithic era2-3 Generations in energy abundanceModern TimesThe same genes The changed diet

Mutch, FASEB 2005Nutrigenomics and nutrigenetics: two sides of a coinFor personalized nutrition:

effects of diet on body-metabolism

influence of genotype on nutritionally related diseases

must be considered

**NutrigenomicsTarget Genes Mechanisms PathwaysSignatures Profiles BiomarkersFoods NutritionMolecular Nutrition & GenomicsNutritional Systems BiologyIdentification of dietary signalsIdentification of dietary sensorsIdentification of target genesReconstruction of signaling pathways Measurement of stress signaturesIdentification of early biomarkersSmall research groups Small budgetsLarge research consortia Big moneyComplexity

**Problem 1: Nutrition tasty + complexIndonesia ?? - no change life style 2030 obesity predicted about 50% 50% of those DM

Bioactive ComponentsCellularDivisionCompoundActivationApoptosisDNARepairDifferentiationImmunityInflammationHormonalHomeostasisCellularEnergeticsDigestionBioactiveComponents

**Optimal NutritionLifestyleIndividual genotype Functional phenotypeProblem 2:Our gene passports and nutritionAA AB BBEat right for your genotype??

**Predisposition GenotypePrognostic markersDiagnostic markersNutritional Systems Biology

*Im betting that an intimate understanding of cell cycle regulation and apoptosis is key to understanding the process of carcinogenesis.If nothing else, the main idea emphasized here is that the coupling between the cell cycle and apoptosis how these processes are coordinated is key.This talk will focus on the links and controls of the onset of the cell cycle and apoptosis.***********