Colorectal cancer

1. Colorectal cancer Third most common type of cancer in the western world. Arise from adenomatous polyps in the colon. Risks: age, general life style, food, alcohol, smoking and absence of exercise (>95%) of CC have a sporadic origin Two subtypes of heritable CC : -FAP, involve APC gene -HNPCC, involve MMR genes

2. Symptoms include: bowel habit changes, blood upon defecation, discharge of mucus and urgency amongst others. • Diagnosis is mainly by colonoscopy and genetic testing especially for families with a history of either FAP or HNPCC.

3. Dukes classification commonly used: • A - Tumour confined to the intestinal wall • B - Tumour invading through the intestinal wall • C - With lymph node(s) involvement • D - With distant metastasis • The treatment depends on the stage. • Surgery is the primary treatment • Chemotherapy and/or radiotherapy

4. How to find important genes of CC? Positional cloning • Identify interesting region by generating frequency plots. • FISH • Bac-clones for gained onkogenes. • LOH • microsatelite markers for lost tumor suppressor genes.

5. Gain frequency plot

6. FISH • DNA sequence cloned with BACs in bacteria. • Hybridisation of flourescent marked complementary DNA sequence to a specific region of chromosomes.

7. TPD52 MMP16 CCNE2 ANGPT1

8. TPD52 tumor protein D52 • 8q21 • 81246391..81109658 • in transformation and metastasis • Interactions are unclear • over-expressed in tumor cells

9. MMP16 matrix metallopeptidase 16 • 8q21 • 89408833..89118576 • Activate MMP2 by cleavage • Involved in breakdown of extracellular matrix. • Have been found to be up regulated in CC and important for metastasis.

10. CCNE2 cyclin E2 • 8q22.1 • 95976660..95961628 • cell cycle G1/S transition • Regulatory subunit of CDK2.

11. ANGPT1 angiopoietin 1 • 108579430..108330886 • 8q22.3-q23 • Important for angiogenesis • Protein is a secreted glycoprotein • Binds to endothelial cell-specific tyrosine-protein kinase receptor

12. Loss frequency plot

13. Analysis of TSG inactivation by LOH Mechanisms of inctivation: ”Knudson two hit hypothesis” -Genetic (whole/partial chromosomal loss, mitotic recombination or point mutations) -Epigenetic silencing. Principle of LOH analysis -Regions flanking the tumors undergo LOH alongside the TSG -Use of known polymorphic markers spanning region can lead to the identification of TSG -PCR using specific primers for different allelic variants of markers in the human population can be used to determine the presence or absence of an allele.

14. MNT D17S1798 D17S919 D17S1832 D17S1881 D17S906 GPS2 D17S1353 TP53 D17S1796 D17S945 D17S1803 D17S921 D17S1843 FLCN D17S805 D17S1794 RASD1 D17S793 D17S1871

15. MNT: MAX binding protein -Myc/max/mad complex member -Antagonist of myc -Anti-growth, potential tumor suppressor GPS2: G protein pathway suppressor 2, also known as AMF-1 -protein involved in G protein-mitogen-activated protein kinase (MAPK) signaling cascades. -Suppresses Ras-Mapk activity and interferes with JNK -Augments p53 dependent transcription -Component of the MMR complex

16. FLCN:folliculin -located within the Smith-Magenis syndrome region on chromosome 17p11.2 -Mutations in this gene are associated with Birt-Hogg-Dube syndrome (BHD) -BHD mutations are Inherited and increase risk of developing renal and lung tumors -Mutation present in MSI CC RASD1: RAS, dexamethasone-induced 1 -Ras related protein -prevents aberrant cell growth

17. TP53: tumor protein p53 -Plays key roles in cell cycle regulation and Apoptosis -TF which activates transcription CDKN2A for cell cycle arrest BAX, FAS receptor for apoptosis -most common TSG which is lost in many tumor types -inherited as germ line mutations in some cancer-prone families with Li-Fraumeni syndrome