Upload
sol777
View
767
Download
9
Tags:
Embed Size (px)
DESCRIPTION
Presentation about WNT-signalling, Colon Cancer epidemology and its reasons, existing drug approaches and a Virus-based therapy.
Citation preview
WNT-Signalling and possible cures
Biologie cellulaire – Prof. Dr. Jan De MeyMorgane Perdomini, Raphael Lieberherr, Zrinka Raguz, Anne Thuillier, Anne-Laure du Mesnildot, Sebastian Olényi
1 Theory part1. Theory partI. Introduction: epidemology, CSCII Wnt pathway and the development of colon cancerII. Wnt pathway and the development of colon cancerIII. Drug development: problems and possibilities
2. Research part2. Research partI. Virus-based approachII. ValidationIII. Therapy design and side effectsIV. Personalized therapy
1 Theory part1. Theory partI. Introduction: epidemology, CSCII W h d h d l f lII. Wnt pathway and the development of colon cancerIII. Drug development: problems and possibilitiesResearch part2. Research partI. Virus-based approachII ValidationII. ValidationIII. Therapy design and side effectsIV. Personalized therapyIV. Personalized therapy
Most forms of cancer not related to level of development of countries, but to the lifestyle 8 ll ( l k ) 990 0 ll 8.1million new cases (plus skin cancer) in 1990, 10 million nowadays, 25% of deaths in western countries (2nd after circulatroy disease)y
Colorectal fourth commonest, but second deadliest in EU –survival depends on country
Men more affected than women Men more affected than women Deprivation decreases mortality, but not incidence
Heritated or aquired Mutations◦ familial adenomatous polyposis (FAP): SNP in APC-gene◦ chromosome 18 loss of heterozygosity (LOH)◦ Hereditary nonpolyposis colorectal cancer (HNPCC)Hereditary nonpolyposis colorectal cancer (HNPCC)
common polymorphisms in digestion-enzymes
C i Carcinogens MeIQ, MeIQx, and PhIP, X-ray, Radon, ...
Vi Viruses – but no virus has been discovered for colorectal cancer yet
Composed of crypts- Composed of cryptsand villis
- constantly renewed
They have ability of self-renewal and are They have ability of self renewal and are sufficiently long-living to receive mutations leading to canceread g to ca ce
Stem cells involved in tumors are called “Cancer Stem cells involved in tumors are called Cancer Stem Cells” (CSC)
2 models of tumor development: stochastic and CSC
1 Theory part1. Theory partI. Introduction: epidemology, CSCII Wnt pathway and the development of colonII. Wnt pathway and the development of colon
cancerIII Drug development: problems and possibilitiesIII. Drug development: problems and possibilities
2. Research partI Virus based approachI. Virus-based approachII. ValidationIII Therapy design and side effectsIII. Therapy design and side effectsIV. Personalized therapy
C l l d i l l i f ll Controls temporal and spatial regulation of cellgrowth, movement and cell survival
Wnt genes: role in epithelial cells proliferation
2 pathways: l 2 l d l ll l d◦ Planar: Ca2+ involved, contols cellular movement and
polarity◦ Canonical: β catenin involved regulates cell proliferation◦ Canonical: β-catenin involved, regulates cell proliferation
APC = Adenomatous polyposis coli protein
Negative regulator of the Wnt pathway throughWnt pathway through multiple mechanisms
WT APC
C-terminally truncated APCAPC
Cellular processes Effects by WT APC Effects by truncated APC
Canonic Wnt signal Inhibition Activation of pathwayCanonic Wnt signal transcription
Inhibition Activation of pathway
Cell adhesion Stimulation Weakening of adhesion
Cell migration Stimulation Stronger stimulation
Chromosomal segregation and Mitotic spindle
Proper segregation and oritentation
Dominant negative: mis-segregation: chromosomal
orientation instability (CIN)Cell cycle progression Inhibition of cell
growthStimulated cell growth
From mutation in stem cells to From mutation in stem cells to colorectal cancercolorectal cancercolorectal cancercolorectal cancer
Two theories about the origin of adenomas:
• the “bottom-up” model
• the “top-down” modelp
Bottom-up model
Top-down model
From mutation in stem cells to From mutation in stem cells to colorectal cancercolorectal cancercolorectal cancercolorectal cancer
• Formation of la monocryptal
adenoma
• Crypt fission leads ypto the spread of mutationsmutations
1 Theory part1. Theory partI. Introduction: epidemology, CSCII Wnt pathway and the development of colon cancerII. Wnt pathway and the development of colon cancerIII. Drug development: problems and
possibilitiespossibilities2. Research part
I Virus based approachI. Virus-based approachII. ValidationIII Therapy design and side effectsIII. Therapy design and side effectsIV. Personalized therapy
Existing and new Non-steroidal anti- Existing and new Non-steroidal anti-inflammatory drugs (NSAIDS)
Vitamin A and D
Small-molecule inhibitors
Antibodies
e.g. aspirin, sulindac and indomethacin e.g. aspirin, sulindac and indomethacin
Regular use reduces incidence and severity of various hhuman cancer
FAP / hereditary forms of cancer FAP / hereditary forms of cancer
Effects:I hibiti lif tiInhibiting proliferationInducing apoptosisCurbing cancer cell invasiong
Precise mechanism unique for each drug
Suppression of oncogenic AP1 and Wnt pathways
Vitamin D derivates interact with vitamin D (VDR) d f lreceptors (VDR) and form a complex
Vitamin D – VDR transcription factor complex binds β-catenin
VDR triggers increase of E-cadherin -> relocating gg gβ-catenin to the cell membrane
Drugs designed to disturb β-catenin – Tcf binding
Experiments with single amino acid Tcf or β catenin mutants > key aa for bindingβ-catenin mutants -> key aa for binding
β i i l if i l i β-catenin is a multifunctional protein
HTS and in silico screening
Other cofactors are also possible targets
Culture of stem cellsIn march 2009 M. CLEVERS developed a methodp
Lack of stem cell markerIn 2007 M CLEVERS foundIn 2007 M. CLEVERS found Lgr5
1 Theory part1. Theory partI. Introduction: epidemology, CSCII Wnt pathway and the development of colon cancerII. Wnt pathway and the development of colon cancerIII. Drug development: problems and possibilities
2 Research part2. Research partI. Virus-based approachII ValidationII. ValidationIII. Therapy design and side effectsIV. Personalized therapyIV. Personalized therapy
Cancer Stem Cells are the best candidates for initiating and maintaining tumorsinitiating and maintaining tumors
Kill l CSC id i f l ll Kill only CSC to avoid apoptosis of normal cells
Some specific receptors can be targeted
Markers Advantages Disadvantages
Lgr5 - stem cell expression - present in other tissues (but rare)
- present in primary tumors, then
- not really specific: also on differentiated
CD133(prominin)
p p y ,down-regulated after epithelial-mesenchymal transition” to generate CD133- cells, more
differentiated luminal epithelial cellsCD133- cells >aggressive => prevention - CD133 cells => more aggressive tumors
CD44- high concentration in colon CSC- highly tumorigenic
- Seems to be present in other
- CD44- cells: non tumorigenicptissues
CD44 is a hyaluronate receptoror P-glycoprotein 1g y p
Transmembrane protein Transmembrane protein
Functions: Functions:◦ surface adhesion
Mediates apoptosis resistance◦ Mediates apoptosis resistance◦ growthfactor/signal transduction
pathwayspathways
• non enveloped icosahedral “particle”
• capside: hexon (II), penton base (III), fiber (IV), IIIa, VI, VIII and IX
1. First step: retargeting 1
Mammalian cell binding peptides isolated by phage
2display 2
3
5
4
1 First step: retargeting1. First step: retargeting
Incorporation into the fiber knob
2. Detargeting
• Initial fiber knob attachment to cell surface CAR mutation in critical CAR interacting residues
• Secondary interactions between the RGD motif of the pentonand cell surface integrinand cell surface integrin deletion of the integrin-binding RGD motif
Synthetic promoter High specificity High efficiency in tumor cells (high level of β-catenin)g y ( g β ) Totally inactive in cells with normally regulated beta catenin regulated beta-catenin Functional in adenoviruses
siRNA repressing an anti- siRNA repressing an anti-apoptotic gene, like Bcl2
siRNA repressing a gene impliedin the Wnt pathway like β-in the Wnt pathway, like βcatenin
M protein expression
Vesicular stomatitis Vesicular stomatitisvirus (VSV):• negative-stranded g
RNA virus • infects mammals• kills tumor cells• kills tumor cells
830 bp mRNA pencodes M protein of 229 aa
Ind ces apoptosis in 2 a s Induces apoptosis in 2 ways:• Activates caspase 9• Inhibits host RNA polymerase I II III• Inhibits host RNA polymerase I , II, III
Inhibits nuclear-cytoplasmic transport of RNA => decrease of transcription initiation factors in cytoplasmdecrease of transcription initiation factors in cytoplasm
1 Theory part1. Theory partI. Introduction: epidemology, CSCII Wnt pathway and the development of colon cancerII. Wnt pathway and the development of colon cancerIII. Drug development: problems and possibilities
2 Research part2. Research partI. Virus-based approachII ValidationII. ValidationIII. Therapy design and side effectsIV. Personalized therapyIV. Personalized therapy
Expression of M protein in infected tumor cultureculture
Specificity of infection and expression Specificity of infection and expression
Stop of cell proliferationp p
Induction of apoptosis
…
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
The image part with relationship ID rId4 was not found in the file.
Infection Protein
Immunoblot
M
extraction
Control TumorM M
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
The image part with relationship ID rId4 was not found in the file.
Infection Protein
Immunoblot
M
extraction
Control TumorM M
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
The image part with relationship ID rId4 was not found in the file.
Infection Protein
Immunoblot
M
extraction
Control TumorM M
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
The image part with relationship ID rId4 was not found in the file.
Infection Protein
Immunoblot
M
extraction
Control TumorM M
CellTiter 96® AQ Non-Radioactive Cell Proliferation Assay (MTS)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
i f t ith i diff t d• infect with virus, use different dosages expressing M protein or PBS
• add MTS• read absorbtion at 490nmread absorbtion at 490nm
Mit C t TM A t i D t ti KitMito CaptureTM Apoptosis Detection Kit Cationic dye Healthy cells red fluorescence Healthy cells red fluorescence Apoptotic cells green fluorescence Detection: fluorescence microscopy or flow cytometer
Procedure• cell culture• infect with virus use differentinfect with virus, use different
dosages expressing M protein or PBS
• stainingl• qualitative test: microscope
• quantitative test: flow cytometer
1 Theory part1. Theory partI. Introduction: epidemology, CSCII Wnt pathway and the development of colon cancerII. Wnt pathway and the development of colon cancerIII. Drug development: problems and possibilities
2 Research part2. Research partI. Virus-based approachII. ValidationII. ValidationIII. Therapy design and side effectsIV. Personalized therapyIV. Personalized therapy
Possibilities: Intravenous injection◦ Systemic distribution: Elevated risk of side
effects◦ Non-homogenous distribution in tumor
Intratumoral implantation Intratumoral implantation◦ Elevated risk of immune response
Intratumoral injection Intratumoral injection◦ More specific targeting◦ Risks of systemic distribution minimized Non-replicating virus in normal cells CD44 restriction (PEG)
Possibilities: Intravenous injection◦ Systemic distribution: Elevated risk of side
effects◦ Non-homogenous distribution in tumor
Intratumoral implantation Intratumoral implantation◦ Elevated risk of immune response
Intratumoral injection Intratumoral injection◦ More specific targeting◦ Risks of systemic distribution minimized Non-replicating virus in normal cells CD44 restriction (PEG)
Aim: ◦ Evade neutralizing antibodies◦ Lower clearance ratio◦ Block transduction to liver
E i◦ Easier storage
Use of PEG (Polyethylene glycol)Use of PEG (Polyethylene glycol)
Virus:• Not replicating in normal cells• Not replicating in normal cells• CD44 restriction• CTP4: specific promoterC spec c p o ote• (PEG)
Choice of delivery: no systemic application
Non specific infection of other cells Non-specific infection of other cells• CD44• Also present on T cells• 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
1 Theory part1. Theory partI. Introduction: epidemology, CSCII Wnt pathway and the development of colon cancerII. Wnt pathway and the development of colon cancerIII. Drug development: problems and possibilities
2 Research part2. Research partI. Virus-based approachII. ValidationII. ValidationIII. Therapy design and side effectsIV. Personalized therapyIV. Personalized therapy
Risk factors: Risk factors: • Personal or family history of colorectal cancer or
adenomatous polyps• Personal history of chronic inflammatory bowel disease, suchPersonal 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 organsg , y, , g
Regular colonoscopy from the age of 50 (risk-group: 40) on until 75 (85)40) on until 75 (85)
Gene tests for hereditary non-polyposis colorectal y p ypcancer and familial adenomatous polyposis (100% risk)
Fighting Inflammatory Bowel Disease Fighting Inflammatory Bowel Disease(retinoid , Iron III compounds)
Avoid risks such as tobbacco (carcinogens Avoid risks such as tobbacco (carcinogens, increases polyp sizes), beer or spirits
1-2 glasses of wine/week (resveratrol) 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-(Eat chicken and fish, fruits and vegetables, brown rice, wholegrain bread, and wheat pasta)
Sports or at least medium activity Medium sun-bathing to enrich vitamin D
Anti-EGFR monoclonal antibodies for tumors Anti-EGFR monoclonal antibodies for tumors without K-ras mutations – Gene tests
Anti inflammatory drugs if COX2 present e g Anti-inflammatory drugs if COX2 present – e.g. Aspirin – COX2-test
Group workout excercises Exercise books Group workout excercises - Exercise books Vitamin D-supply
R t l t t t Resveratrol treatment Immune system empowerment and triggering:
Vi i F l l i l kiVitamin-cure, Folate-supplements, interleukin-12
No good treatment available yet No good treatment available yet Still a lot of research on mechanisms, … needed
Theory for our virus-based therapy seems simple, b t t i it i t l t t t i lik lbut turning it into real treatment is likely more complicated
Mining the Wnt pathway for cancer therapeutics; Barker et al.; Mining the Wnt pathway for cancer therapeutics; Barker et al.; Nature 2006
Tracking Down the Stem Cells of the Intestine: Strategies to Identify Ad lt St C ll B k t l G t t l 2007Adult Stem Cells; Barker et al. Gastroenterology 2007
Mechanisms of Disease: from stem cells to colorectal cancer, Donald et al., Nature Clinical Practice 2006
An Antagonist of Dishevelled Protein-Protein Interaction Suppresses B-Catenin–Dependent Tumor Cell Growth Fujii et al., Cancer Res 2007Cancer Res 2007
Small-molecule antagonists of the oncogenic Tcf/-catenin protein complex; Lepourcelet et al., Cancer Cell 2004
Colon cancer stem cells; Ricci-Vitiani et al. Gut 2008
Induction of apoptosis and tumor regression by vesicular stomatitis virus in the presence of gemcitabine in lung cancer, L. Q et al., Int J Cancer. 2004 Eff f V i l S i i Vi M i P i T i i Di d b H RNA P l I II d III M Ah d l J l f Vi l O b 1998 Effect of Vesicular Stomatitis Virus Matrix Protein on Transcription Directed by Host RNA Polymerases I, II, and III, M. Ahmed et al., Journal of Virology, October 1998
A promising cancer gene therapy agent based on the matrix protein of vesicular stomatitis virus, J. Zhao et al., The FASEB Journal Prognostic Markers for Colorectal Cancer: Expression of P53 and BCL2, H.Pereira et al., world journal of surgery Delivery of Viral Vectors to Tumor Cells: Extracellular Transport, Systemic Distribution, and Strategy for Improvement, Y. Wang et al., Annales of biomedical engineering,
2006 Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche T Sato et al Nature 2009 Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche. T. Sato et al. Nature, 2009 Adenomous polyposis coli (APC): a multi-functional tumor suppressor gene. K. Aoki et al. Journal of cell science, 2007. Non-traditional roles for the Adenomous polyposis coli (APC) tumor suppressor protein. C. Hanson gene, 2005. Current Advances and Future Challenges in Adenoviral Vector Biology and Targeting, K. Campos, Curr Gene Ther. 2007 June Reprogrammed viruses as cancer therapeutics: targeted, armed and shielded, Cattaneo et al., Nature, 2008 Top down morphogenesis of colorectal tumors Shih et al PNAS 2000 Top-down morphogenesis of colorectal tumors, Shih et al. PNAS, 2000 identification of stem cells in small intestine and colon by marker gene Lgr5, Clevers 2007 Optimization of a synthetic beta-catenin-dependant promoter for tumor-specific cancer gene therapy, Wrighton 2004 Nutrigenetics and nutraceuticals: the next wave riding on personalized medicine, M. Subbiah, Translational Research 2007 Cancer epidemiology in the last century and the next decade, J. Peto, Nature 2001 ABC of colorectal cancer Epidemiology P Boyle et al BMJ 2000 ABC of colorectal cancer Epidemiology, P. Boyle et al., BMJ 2000 Wnt signaling and cancer, P. Polakus, Genes Dev. 2000 Therapeutic potential of resveratrol: the in vivo evidence, JA Baur, Nat Rev Drug Discov 5 A Comparative Case-Control Study of Colorectal Cancer and Adenoma, I. Kato, Cancer science 2005 Dietary vitamin D and calcium and risk of colorectal cancer: 19-year prospective study in men, C. Garland et al., The Lancet 1985 Colorectal cancer screening J Sidney Best Practice & Research Clinical Gastroenterology 2007 Colorectal cancer screening, J. Sidney, Best Practice & Research Clinical Gastroenterology 2007 Regression of colon cancer and induction of antitumor immunity by intratumoral injection of adenovirus expressing interleukin-12 G. Mazzolini, Nature 1999 KRAS Mutation Status Is Predictive of Response to Cetuximab Therapy in Colorectal Cancer, A. Lièvre. Cancer Research 2006 Survival in colorectal cancer: impact of body mass and exercise, N. Hall, Gut 2006