1. WNT-Signalling and possible cures Biologie cellulaire Prof. Dr. Jan De Mey Morgane Perdomini, Raphael Lieberherr, Zrinka Raguz, Anne Thuillier, Anne-Laure du Mesnildot, Sebastian Olnyi

2. 1. Theory partI. Introduction: epidemology, CSCII.Wnt pathway and the development of colon cancerIII. Drug development: problems and possibilities 2. Research partI. Virus-based approachII.ValidationIII. Therapy design and side effectsIV.Personalized therapy 3. 1. Theory partI. Introduction: epidemology, CSCII.Wnt pathway and the development of colon cancerIII. Drug development: problems and possibilities 2. Research partI. Virus-based approachII.ValidationIII. Therapy design and side effectsIV.Personalized therapy 4. Most forms of cancer not related to level of development of countries, but to the lifestyle 8.1million new cases (plus skin cancer) in 1990, 10 million nowadays, 25% of deaths in western countries (2nd after circulatroy disease) Colorectal fourth commonest, but second deadliest in EU survival depends on country Men more affected than women Deprivation decreases mortality, but not incidence 5. Heritated or aquired Mutations familial adenomatous polyposis (FAP): SNP in APC-gene chromosome 18 loss of heterozygosity (LOH) Hereditary nonpolyposis colorectal cancer (HNPCC) common polymorphisms in digestion-enzymes CarcinogensMeIQ, MeIQx, and PhIP, X-ray, Radon, ... Viruses but no virus has been discovered for colorectal cancer yet 6. - Composed of crypts and villis- constantly renewed 7. They have ability of self-renewal and are sufficiently long-living to receive mutations leading to cancer Stem cells involved in tumors are called Cancer Stem Cells (CSC) 2 models of tumor development: stochastic and CSC 8. 1. Theory partI. Introduction: epidemology, CSCII. Wnt pathway and the development ofcolon cancerIII. Drug development: problems and possibilities 2. Research partI. Virus-based approachII.ValidationIII. Therapy design and side effectsIV.Personalized therapy 9. Controls temporal and spatial regulation of cell growth, movement and cell survival Wnt genes: role in epithelial cells proliferation 2 pathways: Planar: Ca2+ involved, contols cellular movement and polarity Canonical: -catenin involved, regulates cell proliferation 10. APC = Adenomatous polyposis coli protein Negative regulator of the Wnt pathway through multiple mechanisms 11. WT APCC- terminally truncated APCCellular processesEffects by WT APCEffects by truncated APCCanonic Wnt signalInhibition Activation of pathwaytranscriptionCell adhesion StimulationWeakening of adhesionCell migrationStimulationStronger stimulationChromosomal segregation Proper segregation Dominant negative: mis-and Mitotic spindle and oritentation segregation: chromosomalorientationinstability (CIN)Cell cycle progressionInhibition of cell Stimulated cell growthgrowth 12. From mutation in stem cells to colorectal cancer Two theories about the origin of adenomas: the bottom-upmodel the top-down modelBottom-up Top-downmodel model 13. From mutation in stem cells tocolorectal cancer Formation of a monocryptal adenoma Crypt fission leads to the spread of mutations 14. 1. Theory partI. Introduction: epidemology, CSCII.Wnt pathway and the development of colon cancerIII. Drug development: problems and possibilities 2.Research partI. Virus-based approachII.ValidationIII. Therapy design and side effectsIV.Personalized therapy 15. Existing and new Non-steroidal anti- inflammatory drugs (NSAIDS) Vitamin A and D Small-molecule inhibitors Antibodies 16. e.g. aspirin, sulindac and indomethacin Regular use reduces incidence and severity of various human cancer FAP / hereditary forms of cancer Effects: Inhibiting proliferation Inducing apoptosis Curbing cancer cell invasion Precise mechanism unique for each drug 17. Suppression of oncogenic AP1 and Wnt pathways Vitamin D derivates interact with vitamin D receptors (VDR) and form a complex Vitamin D VDR transcription factor complex binds -catenin VDR triggers increase of E-cadherin -> relocating -catenin to the cell membrane 18. Drugs designed to disturb -catenin Tcf binding Experiments with single amino acid Tcf or -catenin mutants -> key aa for binding -catenin is a multifunctional protein HTS and in silico screening Other cofactors are also possible targets 19. Culture of stem cells In march 2009 M. CLEVERS developed a method Lack of stem cell marker In 2007 M. CLEVERS found Lgr5 20. 1. Theory partI. Introduction: epidemology, CSCII.Wnt pathway and the development of colon cancerIII. Drug development: problems and possibilities 2. Research partI.Virus-based approachII. ValidationIII. Therapy design and side effectsIV. Personalized therapy 21. Cancer Stem Cells are the best candidates for initiating and maintaining tumors Kill only CSC to avoid apoptosis of normal cells Some specific receptors can be targeted 22. MarkersAdvantagesDisadvantages - present in other Lgr5 - stem cell expression tissues (but rare)- not really specific: also on- present in primary tumors, thendifferentiated CD133down-regulated after epithelial- luminal epithelialmesenchymal transition to cells (prominin) generate CD133- cells, moreaggressive => prevention - CD133- cells => more aggressive tumors - high concentration in colon CSC - Seems to be CD44 - highly tumorigenicpresent in other- CD44- cells: non tumorigenictissues 23. CD44 is a hyaluronate receptor or P-glycoprotein 1 Transmembrane protein Functions: surface adhesion Mediates apoptosis resistance growthfactor/signal transduction pathways 24. non enveloped icosahedral particle capside: hexon (II), penton base (III), fiber (IV), IIIa, VI, VIII and IX 25. 1. First step: retargeting 1 Mammalian cell bindingpeptides isolated by2phage display 35 4 26. 1. First step:retargeting Incorporation into thefiber knob 27. 2. Detargeting Initial fiber knob attachment to cell surface CAR mutation in critical CAR interacting residues Secondary interactions between the RGD motif of the penton and cell surface integrin deletion of the integrin-binding RGD motif 28. Synthetic promoter High specificity High efficiency in tumor cells (high level of -catenin) Totally inactive in cells with normally regulated beta-catenin Functional in adenoviruses 29. siRNA repressing an anti- apoptotic gene, like Bcl2 siRNA repressing a gene implied in the Wnt pathway, like -catenin M protein expression 30. Vesicular stomatitis virus (VSV): negative-stranded RNA virus infects mammals kills tumor cells 830 bp mRNA encodes M protein of 229 aa 31. Induces apoptosis in 2 ways: Activates caspase 9 Inhibits host RNA polymerase I , II, III Inhibits nuclear-cytoplasmic transport of RNA => decrease of transcription initiation factors in cytoplasm 32. 1. Theory partI. Introduction: epidemology, CSCII.Wnt pathway and the development of colon cancerIII. Drug development: problems and possibilities 2. Research partI.Virus-based approachII. ValidationIII. Therapy design and side effectsIV. Personalized therapy 33. Expression of M protein in infected tumor culture Specificity of infection and expression Stop of cell proliferation Induction of apoptosis 34. Procedure culture of normal colon cells and tumor colon cells infection with virus expressing M protein construct purify the protein fraction from the cell samples Immunoblot with specific anti-M protein antibody Immunoblot InfectionProteinextraction M M M Control Tumor 35. Procedure culture of normal colon cells and tumor colon cells infection with virus expressing M protein construct purify the protein fraction from the cell samples Immunoblot with specific anti-M protein antibody Immunoblot InfectionProteinextraction M M M Control Tumor 36. Procedure culture of normal colon cells and tumor colon cells infection with virus expressing M protein construct purify the protein fraction from the cell samples Immunoblot with specific anti-M protein antibody Immunoblot InfectionProteinextraction M M M Control Tumor 37. Procedure culture of normal colon cells and tumor colon cells infection with virus expressing M protein construct purify the protein fraction from the cell samples Immunoblot with specific anti-M protein antibody Immunoblot InfectionProteinextraction M M M Control Tumor 38. CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS) Formazan quantity measured at 490nm proportional to number of living cells in culture Procedure tissue culture, plating in 96-well plate infect with virus, use different dosagesexpressing M protein or PBS add MTS read absorbtion at 490nm 39. Mito CaptureTM Apoptosis Detection Kit Cationic dye Healthy cells red fluorescence Apoptotic cells green fluorescence Detection: fluorescence microscopy or flow cytometer Procedure cell culture infect with virus, use differentdosagesexpressing M protein or PBS staining qualitative test: microscope quantitative test: flow cytometer 40. 1. Theory partI. Introduction: epidemology, CSCII.Wnt pathway and the development of colon cancerIII. Drug development: problems and possibilities 2. Research partI.Virus-based approachII. ValidationIII. Therapy design and side effectsIV. Personalized therapy 41. Possibilities: Intravenous injection Systemic distribution: Elevated risk of side effects Non-homogenous distribution in tumor Intratumoral implantation Elevated risk of immune response Intratumoral injection More specific targeting Risks of systemic distribution minimized Non-replicating virus in normal cells CD44 restriction (PEG) 42. Possibilities: Intravenous injection Systemic distribution: Elevated risk of side effects Non-homogenous distribution in tumor Intratumoral implantation Elevated risk of immune response Intratumoral injection More specific targeting Risks of systemic distribution minimized Non-replicating virus in normal cells CD44 restriction (PEG) 43. Aim: Evade neutralizing antibodies Lower clearance ratio Block transduction to liver Easier storageUse of PEG (Polyethylene glycol) 44. Virus: Not replicating in normal cells CD44 restriction CTP4: specific promoter (PEG) Choice of delivery: no systemic application 45. Non-specific infection of other cells CD44 Also present on T cells Might have consequences for immune system Risk of replication in non-cancer cells Non-specific transcription of M protein Liver damage due to systemic distribution 46. 1. Theory partI. Introduction: epidemology, CSCII.Wnt pathway and the development of colon cancerIII. Drug development: problems and possibilities 2. Research partI. Virus-based approachII. ValidationIII. Therapy design and side effectsIV. Personalized therapy 47. Risk factors: Personal or family history of colorectal cancer or adenomatous polyps Personal history of chronic inflammatory bowel disease, such as ulcerative colitis or Crohn's disease Personal or family history of other types of cancer, such as those involving the breast, ovary, uterus, and other organs Regular colonoscopy from the age of 50 (risk- group: 40) on until 75 (85) Gene tests for hereditary non-polyposis colorectal cancer and familial adenomatous polyposis (100% risk) 48. Fighting Inflammatory Bowel Disease (retinoid , Iron III compounds) Avoid risks such as tobbacco (carcinogens, increases polyp sizes), beer or spirits 1-2 glasses of wine/week (resveratrol) Prefer low-fat, low cholesterol, high-fiber-diet (Eat chicken and fish, fruits and vegetables, brown rice, whole-grain bread, and wheat pasta) Sports or at least medium activity Medium sun-bathing to enrich vitamin D 49. Anti-EGFR monoclonal antibodies for tumors without K-ras mutations Gene tests Anti-inflammatory drugs if COX2 present e.g. Aspirin COX2-test Group workout excercises - Exercise books Vitamin D-supply Resveratrol treatment Immune system empowerment and triggering: Vitamin-cure, Folate- supplements, interleukin-12 50. No good treatment available yet Still a lot of research on mechanisms, needed Theory for our virus-based therapy seems simple, but turning it into real treatment is likely more complicated 51. 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