You are on page 1of 65

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. II. III. IV. Virus-based approach Validation Therapy design and side effects 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. II. III. IV. Virus-based approach Validation Therapy design and side effects 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 Viruses
cancer yet

MeIQ, MeIQx, and PhIP, X-ray, Radon, ...

– but no virus has been discovered for colorectal

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. II. III. IV. Virus-based approach Validation Therapy design and side effects 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

Cterminally 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 model

Top-down 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. II. Introduction: epidemology, CSC Wnt pathway and the development of colon cancer

III. Drug development: problems and possibilities 2. Research part
I. II. III. IV. Virus-based approach Validation Therapy design and side effects Personalized therapy

Existing drug approaches

Existing and new Non-steroidal antiinflammatory 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-cellspecific receptors
1. First step: retargeting
ØMammalian cell binding peptides isolated by phage display
1

2

3

5 4

Entry through cancer-cellspecific receptors
1. First step: retargeting
ØIncorporation into the fiber knob

Entry through cancer-cellspecific 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 antiapoptotic 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
 Expression

of M protein in infected tumor culture of infection and expression

 Specificity  Stop

of cell proliferation of apoptosis

 Induction …

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 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 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 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 M Control Tumor

Detection of cell proliferation in target cells
CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS)

Formazan quantity measured at 490nm culture

proportional to number of living cells in

•tissue culture, plating in 96-well plate •infect with virus, use different dosages −expressing M protein or PBS •add MTS •read absorbtion at 490nm

Procedure

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

•cell culture •infect with virus, use different dosages −expressing M protein or PBS •staining •qualitative test: microscope •quantitative test: flow cytometer

Procedure

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. II. Virus-based approach 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 indicationtests

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 Sports or at least medium activity  Medium sun-bathing to enrich vitamin D

(Eat chicken and fish, fruits and vegetables, brown rice, wholegrain bread, and wheat pasta)

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, interleukin12

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

  

                   