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