Builder.ai Founder Sachin Dev Duggal's Strategic Approach to Create an Innova...
Coloncancer3
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 Olényi
2. 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
3. 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
4. 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
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
Carcinogens
C i MeIQ, MeIQx, and PhIP, X-ray, Radon, ...
Viruses
Vi – but no virus has been discovered for colorectal cancer
yet
6. - Composed of crypts
and villis
- constantly renewed
7.
8. 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
9. 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
10. 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,
11.
12. APC = Adenomatous
polyposis coli protein
Negative regulator of the
Wnt pathway through
multiple mechanisms
13.
14. 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
15.
16. 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
17. From mutation in stem cells to
colorectal cancer
• Formation of
a monocryptall
adenoma
• Crypt fission leads
yp
to the spread of
mutations
18. 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
19. Existing and new Non-steroidal anti-
inflammatory drugs (NSAIDS)
Vitamin A and D
Small-molecule inhibitors
Antibodies
20. 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
21.
22. 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
23.
24. 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
25.
26. 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
27. 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
28.
29. 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
30.
31.
32. 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
33. CD44 is a hyaluronate receptor
or P-glycoprotein 1
gy p
Transmembrane protein
Functions:
◦ surface adhesion
◦ Mediates apoptosis resistance
◦ growthfactor/signal transduction
pathways
34. • non enveloped icosahedral “particle”
• capside: hexon (II), penton base (III), fiber (IV),
IIIa, VI, VIII and IX
35. 1. First step: retargeting 1
Mammalian cell binding
peptides isolated by phage
2
display
3
5
4
37. 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
38.
39. 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
40. 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
41. Vesicular stomatitis
virus (VSV):
• negative-stranded
g
RNA virus
• infects mammals
• kills tumor cells
830 bp mRNA
p
encodes M protein of
229 aa
42. 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
43. 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
44. Expression of M protein in infected tumor
culture
Specificity of infection and expression
Stop of cell p
p proliferation
Induction of apoptosis
…
45. 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
46. 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
47. 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
48. 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
49. 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
50. 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
51. 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
52. 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)
53. 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)
54. Aim:
◦ Evade neutralizing antibodies
◦ Lower clearance ratio
◦ Block transduction to liver
◦ Easier
E i storage
Use of PEG (Polyethylene glycol)
55. Virus:
• Not replicating in normal cells
• CD44 restriction
• C
CTP4: specific promoter
spec c p o ote
• (PEG)
Choice of delivery: no systemic application
56. 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
57. 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
58.
59. 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)
60. 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
61. 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
62. 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
63.
64. 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
65. 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