Colon Cancer And Wnt-signalling
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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. 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
1. First step: retargeting ØMammalian cell binding peptides isolated by phage display
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
Specificity Stop
of cell proliferation
Induction …
of infection and expression
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
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
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. 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
(Eat chicken and fish, fruits and vegetables, brown rice, wholegrain 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, 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
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
1. First step: retargeting ØMammalian cell binding peptides isolated by phage display
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
Specificity Stop
of cell proliferation
Induction …
of infection and expression
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
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
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. 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
(Eat chicken and fish, fruits and vegetables, brown rice, wholegrain 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, 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
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