Colon Cancer and WNT-signalling

Colorectal CancerWNT-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 partI. Introduction: epidemology, CSCII. Wnt pathway and the development of colon cancerIII. Drug development: problems and possibilities

2. Research partI. Virus-based approachII. ValidationIII. Therapy design and side effectsIV. Personalized therapy

Structure



Structure

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

Cancer epidemology

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

Most important reasons

- Composed of crypts and villis

- constantly renewed

The intestinal epithelium

Different cells within the intestine

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

Stem cells: Their role in cancer (CSC)

1. Theory partI. Introduction: epidemology, CSC

II. Wnt pathway and the development of colon cancer

III. Drug development: problems and possibilities


Structure

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 = Adenomatous polyposis coli protein

Negative regulator of the Wnt pathway through multiple mechanisms

APC: A critical protein in colorectal cancer

Its role in the Wnt pathway


General functions and structure


General functions and structure

WT APC

C-terminally truncated APC

Cellular processes Effects by WT APC Effects by truncated APC

Canonic Wnt signal transcription

Inhibition Activation of pathway

Cell adhesion Stimulation Weakening of adhesion

Cell migration Stimulation Stronger stimulation

Chromosomal segregation and Mitotic spindle orientation

Proper segregation and oritentation

Dominant negative: mis-segregation: chromosomal instability (CIN)

Cell cycle progression Inhibition of cell growth

Stimulated cell growth

Mutations that are necessary for the development of colorectal cancer

From mutation in stem cells to colorectal cancer

Bottom-up model

Top-down model

Two theories about the origin of adenomas:

• the “bottom-up” model

• the “top-down” model

From mutation in stem cells to colorectal cancer

• Formation of a monocryptal adenoma

• Crypt fission leads to the spread of mutations

1. Theory partI. Introduction: epidemology, CSCII. Wnt pathway and the development of colon cancer

III. Drug development: problems and possibilities


Structure

Existing and new Non-steroidal anti-inflammatory drugs (NSAIDS)

Vitamin A and D

Small-molecule inhibitors

Antibodies

Existing drug approaches

e.g. aspirin, sulindac and indomethacin

Regular use reduces incidence and severity of various human cancer

FAP / hereditary forms of cancer

Effects:Inhibiting proliferationInducing apoptosisCurbing cancer cell invasion

Precise mechanism unique for each drug

Non-steroidal anti-inflammatory drugs (NSAIDS)

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

Other cofactors are also possible targets

Problems for the drug’s development

Culture of stem cellsIn march 2009 M. CLEVERS developed a method

Lack of stem cell markerIn 2007 M. CLEVERS found Lgr5



Structure

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 receptorsMarkers Advantages Disadvantages

Lgr5 - stem cell expression- present in other tissues (but rare)

CD133(prominin)

- present in primary tumors, then down-regulated after epithelial- mesenchymal transition” to

generate CD133- cells, more aggressive => prevention

- not really specific: also on differentiated luminal epithelial cells

- CD133- cells => more aggressive tumors

CD44

- high concentration in colon CSC- highly tumorigenic

- CD44- cells: non tumorigenic

- Seems to be present in other tissues

CD44 description:

CD44 is a hyaluronate receptor or P-glycoprotein 1

Transmembrane protein

Functions:◦ surface adhesion◦ Mediates apoptosis resistance◦ growthfactor/signal transduction

pathways

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

Mammalian cell binding peptides isolated by phage display

1

2

3

4

5


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

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

Functional in adenoviruses

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


2. Research partI. Virus-based approach

II. ValidationIII. Therapy design and side effectsIV. Personalized therapy

Structure

Validation and search for the best dosage

Expression of 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

Control TumorM M

M

InfectionProtein extraction

Immunoblot


Control TumorM M

M


Immunoblot

Procedure



Control TumorM M

M


Immunoblot

Procedure



Control TumorM M

M


Immunoblot

Procedure


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


2. Research partI. Virus-based approachII. Validation

III. Therapy design and side effectsIV. Personalized therapy

Structure

Therapy design: Method of deliveryPossibilities: 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 deliveryPossibilities: 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

Use 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


2. Research partI. Virus-based approachII. ValidationIII. Therapy design and side effects

IV. Personalized therapy

Structure

Personalized medicine

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)

Early diagnosis and indication-tests

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

Prevention adopted to risk assessment

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

Adopted treatment

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

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

Given 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

Additional References

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Colon Cancer and WNT-signalling