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Colorectal Cancer

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
Structure
1. Theory part
I. Introduction: epidemology, CSC
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
Structure
1. Theory part
I. Introduction: epidemology, CSC
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
Cancer epidemology
 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
Most important reasons
 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 MeIQ, MeIQx, and PhIP, X-ray, Radon, ...

 Viruses – but no virus has been discovered for colorectal


cancer yet
The intestinal epithelium
- Composed of crypts
and villis

- constantly renewed
Different cells within the
intestine
Stem cells: Their role in cancer
(CSC)
 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
Structure
1. Theory part
I. Introduction: epidemology, CSC
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
Wnt pathway
 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: β-catenin involved, regulates cell
proliferation
Canonical Wnt pathway
APC: A critical protein in colorectal cancer
Its role in the Wnt pathway

 APC = Adenomatous
polyposis coli protein

 Negative regulator of the


Wnt pathway through
multiple mechanisms
APC: A critical protein in colorectal cancer
General functions and structure
APC: A critical protein in colorectal cancer
General functions and structure

WT APC

C-
terminally
truncated
APC
Mutations that are necessary for
the development of colorectal
cancer
From mutation in stem cells to
colorectal cancer

Two theories about the


origin of adenomas:

•the “bottom-up”
model

•the “top-down” model

Bottom-up Top-down
model model
From mutation in stem cells to
colorectal cancer
•Formation of
a monocryptal
adenoma

• Crypt fission leads


to the spread of
mutations
Structure
1. Theory part
I. Introduction: epidemology, CSC
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
Existing drug approaches
 Existing and new Non-steroidal anti-
inflammatory drugs (NSAIDS)

 Vitamin A and D

 Small-molecule inhibitors

 Antibodies
Non-steroidal anti-inflammatory
drugs (NSAIDS)
 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


Vitamin D
 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 β-catenin

 VDR triggers increase of E-cadherin ->


relocating β-catenin to the cell membrane
Small-molecule inhibitors
 Drugs designed to disturb β-catenin – Tcf
binding

 Experiments with single amino acid Tcf or


β-catenin mutants -> key aa for binding

 β-catenin is a multifunctional protein

 HTS and in silico screening


Problems for the drug’s
development

 Culture of stem cells


In march 2009 M. CLEVERS
developed a method
 Lack of stem cell marker

In 2007 M. CLEVERS found


Lgr5
Structure
1. Theory part
I. Introduction: epidemology, CSC
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
Goal in therapy
Goal in therapy:
to kill only the CSC

 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


Virus-based approach
Different target receptors
CD44 description:

 CD44 is a hyaluronate receptor


or P-glycoprotein 1

 Transmembrane protein

 Functions:
◦ surface adhesion
◦ Mediates apoptosis resistance
◦ growthfactor/signal transduction
Adenoviral virion (Ad5)
•non enveloped icosahedral “particle”

•capside: hexon (II), penton base (III), fiber (IV),


IIIa, VI, VIII and IX
Entry through cancer-cell-
specific receptors

1. First step: retargeting 1

ØMammalian cell binding


peptides isolated by phage
2
display

4
Entry through cancer-cell-
specific receptors
1. First step: retargeting

ØIncorporation into the


fiber knob
Entry through cancer-cell-
specific receptors
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
Virus-based approach
CTP4 promoter

 Synthetic promoter

 High specificity

 High efficiency in tumor cells (high level of β-catenin)

 Totally inactive in cells with normally

 regulated beta-catenin
Different strategies
 siRNA repressing an anti-
apoptotic gene, like Bcl2

 siRNA repressing a gene


implied in the Wnt pathway,
like β-catenin

 M protein expression
Choice of insert

 Vesicular stomatitis
virus (VSV):
• negative-stranded
RNA virus
• infects mammals
• kills tumor cells

 830 bp mRNA
encodes M protein of
229 aa
M protein
 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
Structure
1. Theory part
I. Introduction: epidemology, CSC
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
Validation and search for the
best dosage
 Expressionof M protein in infected
tumor culture
 Specificity of infection and expression
 Stop of cell proliferation
 Induction of apoptosis
…
Expression of the M protein and
its specifity to colon cancer
tissue 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

Infection
Protein
extraction

M M
Control Tumor
Expression of the M protein and
its specifity to colon cancer
tissue 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

Infection
Protein
extraction

M M
Control Tumor
Expression of the M protein and
its specifity to colon cancer
tissue 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

Infection
Protein
extraction

M M
Control Tumor
Expression of the M protein and
its specifity to colon cancer
tissue 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

Infection
Protein
extraction

M M
Control Tumor
Detection of cell proliferation in
target cells
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 dosages
−expressing M protein or PBS
•add MTS
•read absorbtion at 490nm
Detection of apoptosis in target
cells
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 different
dosages
−expressing M protein or PBS
•staining
•qualitative test: microscope
•quantitative test: flow cytometer
Structure
1. Theory part
I. Introduction: epidemology, CSC
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
Therapy design: Method of
delivery
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)
Therapy design: Method of
delivery
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)
Shielding?
 Aim:
◦ Evade neutralizing antibodies
◦ Lower clearance ratio
◦ Block transduction to liver
◦ Easier storage

ØUse of PEG (Polyethylene glycol)


Securities: avoid side effects

 Virus:
• Not replicating in normal cells
• CD44 restriction
• CTP4: specific promoter
• (PEG)

 Choice of delivery: no systemic application


Possible side effects
 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


Structure
1. Theory part
I. Introduction: epidemology, CSC
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
Perso
naliz
ed
medi
cine
Early diagnosis and indication-
tests
 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)
Prevention adopted to risk
assessment
 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
Adopted treatment
 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
Conclusion
 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
Given References
 Mining the Wnt pathway for cancer therapeutics; Barker et al.;
Nature 2006
 Tracking Down the Stem Cells of the Intestine: Strategies to
Identify Adult 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 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
Additional References
 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 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
 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
 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
 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
 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