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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. 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
- 3. 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
- 4. 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
- 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 MeIQ, MeIQx, and PhIP, X-ray, Radon, ... Viruses – but no virus has been discovered for colorectal cancer yet
- 6. - Composed of crypts and villis - constantly renewed
- 8. 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
- 9. 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
- 10. 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
- 12. APC = Adenomatous polyposis coli protein Negative regulator of the Wnt pathway through multiple mechanisms
- 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
- 16. 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
- 17. From mutation in stem cells to colorectal cancer • Formation of a monocryptal adenoma • Crypt fission leads to the spread of mutations
- 18. 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
- 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 Inducing apoptosis Curbing cancer cell invasion Precise mechanism unique for each drug
- 22. 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
- 24. 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
- 26. Culture of stem cells In march 2009 M. CLEVERS developed a method Lack of stem cell marker In 2007 M. CLEVERS found Lgr5
- 27. 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
- 29. 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
- 32. Markers Advantages Disadvantages - present in other Lgr5 - stem cell expression tissues (but rare) - not really specific: also on - present in primary tumors, then 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 present in other - CD44- cells: non tumorigenic tissues
- 33. CD44 is a hyaluronate receptor or P-glycoprotein 1 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 2 phage display 3 5 4
- 36. 1. First step: retargeting Incorporation into the fiber knob
- 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
- 39. 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
- 40. siRNA repressing an anti- apoptotic gene, like Bcl2 siRNA repressing a gene implied in the Wnt pathway, like β-catenin M protein expression
- 41. Vesicular stomatitis virus (VSV): • negative-stranded RNA virus • infects mammals • kills tumor cells 830 bp mRNA encodes M protein of 229 aa
- 42. 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
- 43. 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
- 44. Expression of M protein in infected tumor culture Specificity of infection and expression Stop of cell 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 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 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 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 Immunoblot Infection Protein extraction M M M Control Tumor
- 49. 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
- 50. 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
- 51. 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
- 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 storage Use of PEG (Polyethylene glycol)
- 55. Virus: • Not replicating in normal cells • CD44 restriction • CTP4: specific promoter • (PEG) Choice of delivery: no systemic application
- 56. 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. 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
- 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 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)
- 60. 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
- 61. 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
- 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 into real treatment is likely more complicated
- 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 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
- 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 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
Notes de l'éditeur
- Other than most other illnesses neither mortality nor cancer cases have direct connection to developmental state of a countryTunesia, Morocco, Egypt, Lybia 69-84, France 142, Germany 141, Switzerland 116, Japan 119, Hungary 256, Mongolia 306 cancer-deaths/100.000 inhabitants
- MeIQ, MeIQx, and PhIP are heterocyclic amine compounds formed when meats and eggs are cooked or grilled at high temperaturesHepaptitis B and Human papillom virusLOH leading to inactivation of SMAD4 (DPC4), and DCC (deleted in colon cancer) tumor suppression gene HNPCC) deficient DNA mismatch repair MSH2, MLH1, and PMS2Digestion enzymes such as cytochrome P450 (CYP), glutathione S-transferase (GST), sulfonotransferase (SULT), N-acetyl transferase 2 (NAT), NADPH-quinone oxidoreductase (NQO1), methyltetrahydrofolate reductase (MTHFR), and microsomal epoxide hydrolase (EPHX1) genesVirus es like like HBV and HPV frequent cause of cancer and there is evidence linking immunodefiency to colon cancer suggesting virus activity, but no proof
- But no VDR in latestagecancers!!!!
- Moleculedatabases
- Currently, much of medical practice is based on \"standards of care\" that are determined by averaging responses across large cohorts. The theory has been that everyone should get the same care based on clinical trials. Personalized Medicine is the concept that managing a patient's health should be based on the individual patient's specific characteristics, including age, gender, height/weight, diet, environment, etc. Recent developments in genetic testing allow the development of \"Genomic Personalized Medicine\" and Predictive Medicine, which is the combination of comprehensive genetic testing with proactive, personalized preventive medicine.In the following slides, potential research- and product-targets for such a holistic approach are marked in red. We would suggest that a pharma company tries to develop (or to license) for each of these targets a cure and markets them together under one brand name, developing and signalling an expertise in the market.
- Familial adenomatouspolyposis (FAP) is an inherited condition in which numerous polyps form mainly in the epithelium of the large intestine. While these polyps start out benign, malignant transformation into colon cancer occurs when not treated.
- Inflammatory bowel disease (IBD) is a group of inflammatory conditions of the large intestine and small intestine. The major types of IBD are Crohn's disease and ulcerative colitis. It significantly increases, as all inflammations, the risk of cancer. There are different medicaments in the field for this, and better to be developed.http://www.faqs.org/patents/app/20080255069First, inhaled or swallowed tobacco smoke transports carcinogens to the colon. Second, tobacco use seems to increase polyp size.In general, the bigger the polyp, the greater the chance it will become cancerous. Studies indicate that 12% of fatal colorectal cancers may be attributable to smoking.BeerA study published in the American Journal of Gastroenterology found that people who drank more than one beer a day were more likely to develop colorectal cancer than non-beer drinkers. Another study (published in the journal Gut) provided more details, adding that drinking beer didn't impact colon cancer risk, but could increase rectal cancer risk. The researchers determined that drinking one beer a day more than doubled the risk of developing rectal cancer.SpiritsIf you like spirits, you're not going to like this. And if you don't know what spirits are, they're drinks like gin, bourbon, and vodka. A study published in the journal Gut found that drinking two glasses of spirits a day more than doubled the risk of developing rectal cancer. Think doubling your risk is bad? Researchers at Stony Brook University determined that drinking nine or more servings of spirits a week on a regular basis tripled colon cancer risk.WineThe same researchers who reported that spirits could triple colon cancer risk, also determined that drinking a glass or two of wine each week could decrease colon cancer risk by as much as 60%. Researchers at Stony Brook University in New York have released findings of a study that indicates people who drink three or more glasses of red wine a week reduce the chances that they will develop colon cancerDiets high in fat and cholesterol (especially from animal sources) have been linked to an increased risk of colon cancer. Low-fiber diets have also been associated with increased risk, but the research isn't as clear. This recommendation is independent of weight. Even if you can eat steak and liver at every meal and stay trim, you're still increasing your risk of developing colon cancer by virtue of what you're eating. A balanced diet seems to be the way to go.Since diabetics have an increased risk of developing colon cancer, it's also a good idea to avoid food choices that are known to lead to diabetes. In general, these include high-glycemic foods like sugary snacks and refined carbohydrates, often referred to as \"white\" foods. Examples include white sugar, white rice, white bread, and white pasta. Less-processed versions like brown rice, whole-grain bread, and wheat pasta are healthy substitutes.Research has shown that a sedentary lifestyle contributes to colon cancer development.Resveratrol induces cell death in colorectal cancer cells by a novel pathway involving lysosomal cathepsin DNicol F. Trincheri, Giuseppina Nicotra, Carlo Follo, Roberta Castino and Ciro Isidoro*
- The finding that patients with colorectal cancer who do not respond to treatment with panitumumab ( Vectibix, Amgen) and cetuximab ( Erbitux, Bristol-Myers Squibb) have tumors with KRAS mutations is a practice-changing discovery, says Jose Baselga, MD, from Valld'Hebron University Hospital, in Barcelona, Spain. Both drugs are antibodies that inhibit epidermal growth factor receptor (EGFR), and they are expensive. \"Patients with KRAS mutations have zero response to these drugs,\" Dr. Baselga told Medscape Oncology. Not using these expensive products in such patients would save money, while restricting the use of these drugs to patients who have tumors without KRAS mutations \"enriches the likelihood that these patients will respond to therapy,\" he said.When presenting his doctoral thesis, Murdani said that the nuclear factor NF-kB was found in 73.5 percent of cases *72 patients* while enzyme COX-2 was present in almost 50 percent of cases *48 patients*. Both markers, he added, were significantly higher in cancer tissues than when compared with normal mucosa.As inflammation is the common cause of nonhereditary colorectal cancer in Indonesia, the use of anti-inflammatory medications such as aspirin during treatment could assist patients, a study has found.Participating in regular group exercise classes can help women with early breast cancer feel better, both physically and mentally, while they're undergoing treatment, according to a study in the British Medical Journal.Patients diagnosed with colon cancer who had abundant vitamin D in their blood were less likely to die during a follow-up period than those who were deficient in the vitamin, according to a new study by scientists at Dana-Farber Cancer Institute.Regression of colon cancer and induction of antitumor immunity by intratumoral injection of adenovirus expressing interleukin-12 Guillermo Mazzolini, Cheng Qian, Xiaoming Xie, Yonglian Sun, Juan J Lasarte, Marek Drozdzik and Jesús Prietocase-control study using data from the Scripps Green Hospital in La Jolla, Calif., The AAPS JournalPharmacogenomics: The promise of personalized medicine [Cancer Research 66, 3992-3995, April 15, 2006]© 2006 American Association for Cancer ResearchPriority ReportsKRAS Mutation Status Is Predictive of Response to Cetuximab Therapy in Colorectal Cancer Astrid Lièvre1,3, Jean-Baptiste Bachet3, Delphine Le Corre1, Valérie Boige4, Bruno Landi2, Jean-François Emile3, Jean-François Côté1,2, Gorana Tomasic4, Christophe Penna3, Michel Ducreux4, Philippe Rougier3, Frédérique Penault-Llorca5 and Pierre Laurent-Puig1,2Karapetis CS, Khambata-Ford S, Jonker DJ, et al. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med. 2008;359:1757-1765. Cancer Epidemiology Biomarkers & Prevention 17, 543, March 1, 2008. doi: 10.1158/1055-9965.EPI-07-2615© 2008 American Association for Cancer ResearchMeta-analysis of Colorectal Cancer Gene Expression Profiling Studies Identifies Consistently Reported Candidate BiomarkersSimon K. Chan1,2, Obi L. Griffith1,3, Isabella T. Tai1,4 and Steven J.M. Jones1,2,3