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 Olényi

1.
1 Theory part
I. Introduction: epidemology, CSC
II.
II Wnt pathway and the development of colon cancer
III. Drug development: problems and possibilities
2. Research part
I. Virus-based approach
II. Validation
III. Therapy design and side effects
IV. Personalized therapy

1.
1 Theory part
I. Introduction: epidemology, CSC
II. Wnt
II W pathway and the d
h d h development of colon cancer
l f l
III. Drug development: problems and possibilities
2. Research part
I. Virus-based approach
II.
II Validation
III. Therapy design and side effects
IV. Personalized therapy

 Most forms of cancer not related to level of development
of countries, but to the lifestyle
 8.1million new cases ( l skin cancer) in 1990, 10 million
8 ll (plus k ) 990 0 ll
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
 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)
common polymorphisms in digestion-enzymes
 Carcinogens
C i MeIQ, MeIQx, and PhIP, X-ray, Radon, ...
 Viruses
Vi – but no virus has been discovered for colorectal cancer
yet

- Composed of crypts
and villis
- constantly renewed

 They have ability of self-renewal and are
self renewal
sufficiently long-living to receive mutations
leading to ca ce
ead g cancer
 Stem cells involved in tumors are called “Cancer
Cancer
Stem Cells” (CSC)
 2 models of tumor development: stochastic and
CSC

1.
1 Theory part
I. Introduction: epidemology, CSC
II.
II Wnt pathway and the development of colon
cancer
III.
III Drug development: problems and possibilities
2. Research part
I.
I Virus-based
Virus based approach
II. Validation
III
III. Therapy design and side effects
IV. Personalized therapy

 Controls temporal and spatial regulation of cell
C l l d i l l i f ll
growth, movement and cell survival
 Wnt genes: role in epithelial cells proliferation
 2 pathways:
◦ Planar: Ca2 involved, contols cellular movement and
l 2+ l d l ll l d
polarity
◦ Canonical: β catenin involved regulates cell proliferation
β-catenin involved,

 APC = Adenomatous
polyposis coli protein
 Negative regulator of the
Wnt pathway through
multiple mechanisms

WT APC
C-terminally
truncated
APC
Cellular processes Effects by WT APC Effects by truncated APC
Canonic Wnt signal Inhibition Activation of pathway
transcription
Cell adhesion Stimulation Weakening of adhesion
Cell migration Stimulation Stronger stimulation
Chromosomal segregation Proper segregation Dominant negative: mis-
and Mitotic spindle and oritentation segregation: chromosomal
orientation instability (CIN)
Cell cycle progression Inhibition of cell Stimulated cell growth
growth

From mutation in stem cells to
colorectal cancer
Two theories about the
origin of adenomas:
• the “bottom-up”
model
• the “top-down” model
p
Bottom-up Top-down
model model

From mutation in stem cells to
colorectal cancer
• Formation of
a monocryptall
adenoma
• Crypt fission leads
yp
to the spread of
mutations

1.
1 Theory part
I. Introduction: epidemology, CSC
II.
II Wnt pathway and the development of colon cancer
III. Drug development: problems and
possibilities
2. Research part
I.
I Virus-based
Virus based approach
II. Validation
III
III. Therapy design and side effects
IV. Personalized therapy

 Existing and new Non-steroidal anti-
inflammatory drugs (NSAIDS)
 Vitamin A and D
 Small-molecule inhibitors
 Antibodies

 e.g. aspirin, sulindac and indomethacin
 Regular use reduces incidence and severity of various
human cancer
 FAP / hereditary forms of cancer
 Effects:
Inhibiting proliferation
I hibiti lif ti
Inducing apoptosis
Curbing cancer cell invasion
g
 Precise mechanism unique for each drug

 Suppression of oncogenic AP1 and Wnt pathways
 Vitamin D derivates interact with vitamin D
receptors (VDR) and f
d form a complexl
 Vitamin D – VDR transcription factor complex
binds β-catenin
 VDR triggers increase of E-cadherin -> relocating
gg g
β-catenin to the cell membrane

 Drugs designed to disturb β-catenin – Tcf binding
 Experiments with single amino acid Tcf or
β-catenin
β catenin mutants -> key aa for binding
>
 β-catenin i a multifunctional protein
β i is l if i l i
 HTS and in silico screening
 Other cofactors are also possible targets

 Culture of stem cells
In march 2009 M. CLEVERS
developed a method
p
 Lack of stem cell marker
In 2007 M. CLEVERS found
M
Lgr5

1.
1 Theory part
I. Introduction: epidemology, CSC
II.
II Wnt pathway and the development of colon cancer
III. Drug development: problems and possibilities
2.
2 Research part
I. Virus-based approach
II.
II Validation
III. Therapy design and side effects
IV. Personalized therapy

 Cancer Stem Cells are the best candidates for
initiating and maintaining tumors
 Kill only CSC to avoid apoptosis of normal cells
l id i f l ll
 Some specific receptors can be targeted

Markers Advantages Disadvantages
- present in other
Lgr5 - stem cell expression
tissues (but rare)
- not really specific:
also on
- present in p
p primary tumors, then
y , differentiated
CD133 down-regulated after epithelial- luminal epithelial
mesenchymal transition” to cells
(prominin) generate CD133- cells, more
aggressive => prevention - CD133- cells => >
more aggressive
tumors
- high concentration in colon CSC - Seems to be
CD44 - highly tumorigenic p
present in other
- CD44- cells: non tumorigenic tissues

 CD44 is a hyaluronate receptor
or P-glycoprotein 1
gy p
 Transmembrane protein
 Functions:
◦ surface adhesion
◦ Mediates apoptosis resistance
◦ growthfactor/signal transduction
pathways

• 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
2
display
3
5
4

1.
1 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 penton
and 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
beta catenin
 Functional in adenoviruses

 siRNA repressing an anti-
apoptotic gene, like Bcl2
 siRNA repressing a gene implied
in the Wnt pathway like β-
pathway, β
catenin
 M protein expression

 Vesicular stomatitis
virus (VSV):
• negative-stranded
g
RNA virus
• infects mammals
• kills tumor cells
 830 bp mRNA
p
encodes M protein of
229 aa

 Induces
Ind ces apoptosis in 2 ways:
a s
• Activates caspase 9
• Inhibits host RNA polymerase I , II III
II,
Inhibits nuclear-cytoplasmic transport of RNA =>
decrease of transcription initiation factors in cytoplasm

1.
1 Theory part
I. Introduction: epidemology, CSC
II.
II Wnt pathway and the development of colon cancer
III. Drug development: problems and possibilities
2.
2 Research part
I. Virus-based approach
II.
II Validation
III. Therapy design and side effects
IV. Personalized therapy

 Expression of M protein in infected tumor
culture
 Specificity of infection and expression
 Stop of cell p
p proliferation
 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.
Immunoblot
Infection
Protein
extraction
M
M M
Control Tumor

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.
Immunoblot
Infection
Protein
extraction
M
M M
Control Tumor

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.
Immunoblot
Infection
Protein
extraction
M
M M
Control Tumor

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.
Immunoblot
Infection
Protein
extraction
M
M M
Control Tumor

CellTiter 96® AQueous Non Radioactive Cell Proliferation Assay (MTS)
Non-Radioactive
Formazan quantity measured at 490nm  proportional to number of living cells in
culture
Procedure
• tissue culture, plating in 96-well plate
• i f t with virus, use diff
infect ith i different d
t dosages
 expressing M protein or PBS
• add MTS
• read absorbtion at 490nm

Mito CaptureTM A
Mit C t TM Apoptosis D t ti
t i 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 different
virus
dosages
 expressing M protein or PBS
• staining
l
• qualitative test: microscope
• quantitative test: flow cytometer

1.
1 Theory part
I. Introduction: epidemology, CSC
II.
II Wnt pathway and the development of colon cancer
III. Drug development: problems and possibilities
2.
2 Research part
I. Virus-based approach
II. Validation
III. Therapy design and side effects
IV. Personalized therapy

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)

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)

 Aim:
◦ Evade neutralizing antibodies
◦ Lower clearance ratio
◦ Block transduction to liver
◦ Easier
E i storage
Use of PEG (Polyethylene glycol)

 Virus:
• Not replicating in normal cells
• CD44 restriction
• C
CTP4: specific promoter
spec c p o ote
• (PEG)
 Choice of delivery: no systemic application

 Non-specific
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

1.
1 Theory part
I. Introduction: epidemology, CSC
II.
II Wnt pathway and the development of colon cancer
III. Drug development: problems and possibilities
2.
2 Research part
I. Virus-based approach
II. Validation
III. Therapy design and side effects
IV. Personalized therapy

 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
g , y, , g
 Regular colonoscopy from the age of 50 (risk-group:
40) on until 75 (85)
 Gene tests for hereditary non-polyposis colorectal
y p yp
cancer and familial adenomatous polyposis (100%
risk)

 Fighting Inflammatory Bowel Disease
(retinoid , Iron III compounds)
 Avoid risks such as tobbacco (carcinogens
(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-
fish vegetables rice, whole
grain bread, and wheat pasta)
 Sports or at least medium activity
 Medium sun-bathing to enrich vitamin D

 Anti-EGFR monoclonal antibodies for tumors
without K-ras mutations – Gene tests
 Anti-inflammatory
Anti inflammatory drugs if COX2 present – e g
e.g.
Aspirin – COX2-test
 Group workout excercises - Exercise books
 Vitamin D-supply
 Resveratrol t
R t l treatment
t t
 Immune system empowerment and triggering:
Vi
Vitamin-cure, F l
i Folate-supplements, i
l l ki
interleukin-
12

 No good treatment available yet
 Still a lot of research on mechanisms, … needed
 Theory for our virus-based therapy seems simple,
but turning it i t real t
b tt i into l treatment i lik l more
t t is likely
complicated

 Mining the Wnt pathway for cancer therapeutics; Barker et al.;
Nature 2006
 Tracking Down the Stem Cells of the Intestine: Strategies to Identify
Adult St
Ad lt Stem Cells; Barker et al. Gastroenterology 2007
C ll B k t l G t t l
 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 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
 Effect of Vesicular S
Eff f V i l Stomatitis Vi
i i Virus M i P
Matrix Protein on T
i Transcription Di
i i Directed b H
d by Host RNA P l
Polymerases I II and III M Ah d et al., J
I, II, d III, M. Ahmed l Journal of Vi l
l f Virology, O
October 1998
b
 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
niche. T. al.
 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
Top down morphogenesis of colorectal tumors Shih et al. PNAS, 2000
tumors, al PNAS
 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
Epidemiology, P. al
 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
screening, J. Sidney,
 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
