CANCER Affects 25% of US population Kills 19% of US population (2nd largest killer after heart disease) NOT one disease but 200-300 different defects Etiologic Factors In Cancer: Relative contributions Genetic susceptibility Tobacco Alcohol Diet Infection Occupation Environmental pollution Medications Additional factors ++++ ++++ ++ +++ ++ ++ + + + Source: J. Fraumeni, M.D Inherited Cancer Syndromes Contribute 1-2% of all cancer incidence Rare or unusual cancers, or combinations of cancers, or cancers at an exceptionally early age High lifetime risk of cancer (50-90% or more) May be accompanied by developmental defects 1
Retinoblastoma Hereditary retinoblastoma is an autosomal dominant disorder But cancer requires inactivation of both alleles of single gene 2
Hereditary retinoblastoma is an autosomal dominant disorder Compare with autosomal recessive disorder like cystic fibrosis Carrier - genotype is +/- Genotype is +/- But the tumor is -/- genotype is -/- But cancer requires inactivation of both alleles of single gene This transition requires somatic inactivation of the second - i.e. the wildtype - allele +/+ genotype -/- +/- genotype Somatic mutation This is called the Two-hit Hypothesis The first and second mutations typically occur via different mechanisms The first and second hits typically occur through different mechanisms FIRST HIT - Point mutation Deletion SECOND HIT - Many more possible mechanisms 3
The first and second hits typically occur through different mechanisms FIRST HIT Point mutation Deletion SECOND HIT - Many more possible mechanisms Most of the 2nd hit mechanisms cause Loss of heterozygosity (LOH) of genetic markers in the region of Rb gene Most of the 2nd hit mechanisms cause Loss of heterozygosity (LOH) of genetic markers in the region of Rb gene A Germline genotype- Tumor genotypes- B Rb* WT E e f F A A b Rb* E f a b LOH at all markers a b B Rb* Rb* E E f F LOH at markers around Rb gene 4
There is one additional inactivation mechanism not shown on the previous slide - Changes in methylation status of the t.s. promoter that prevent transcription of the gene This does not give LOH Inspite of this, LOH is a hallmark of many tumor suppressors This has been used to map and clone these genes Retinoblastoma occurs in both hereditary and non-hereditary forms Always Unilateral Two-hit Hypothesis +/+ genotype -/- +/- genotype Somatic mutation 5
Two-hit Hypothesis +/+ genotype -/- +/- genotype Somatic mutation +/+ genotype -/- +/- cell Hereditary form- Somatic mutation Somatic mutation Two-hit Hypothesis +/+ genotype -/- +/- genotype NON-hereditary form- Rb is a common genetic lesion in sporadic tumors Sporadic retinoblastoma Early onset (typical diagnosis - 2 years) Approx 30% of all other tumor types Particularly high incidence in some tumor types (e.g. oesteosarcomas, small cell lung carcinoma) Almost always mutation followed by LOH Late onset (adults) 6
Types of genes which are mutated in cancer- Tumor suppressor genes Oncogenes Oncogenes Tumor formation requires activating mutation (i.e - gain of function) This arises through - Increased or inappropriate expression of normal protein Point mutations that create a mutant protein Most oncogenes have been identified through analysis of viruses and/or tumor cell lines. VIRUSES AND ONCOGENES 1) Retroviruses that encode oncogenes 1900 - ROUS isolated a filtratable agent from chickens that caused cancer. ROUS SARCOMA VIRUS (RSV) 1960 - Tissue culture techniques developed 1970 - Cloned RSV oncogene - v-src 7
Properties of cells in culture Normal Transformed HIGH growth factor dependence REDUCED growth factor dependence Limited lifespan Immortalized Contact inhibited NOT contact inhibited Assays for contact inhibition Normal Transformed 1) Growth on t.c. dishes Monolayer FOCUS formation (Plural - FOCI) Plate single cell suspension in soft agar containing t.c. media 2) Growth in soft agar Single cells don t divide Cells divide to give visible clones 1) Retroviruses that encode oncogenes (i) Rous sarcoma virus (RSV) can transform primary cells RSV Normal rodent cells Infection FOCUS formation 8
1) Retroviruses that encode oncogenes (ii) Mutagenesis separates transformation from other viral functions Wild-type RSV + Chemical mutagen Used a classic temp-sensitive screen Panel of point mutants Identify mutant viruses that have NO defect in viral functions Screen for transformation function using soft agar assays Find one mutant that has wild-type viral functions but CAN T TRANSFORM Since mutagen causes point mutations, suggests SINGLE GENE 1) Retroviruses that encode oncogenes (iii) Cloning RSV oncogene (BISHOP + VARMUS, 1970) Wild-type RSV Deletion mutant-b77 (transformation-defective) ONCOGENE Make cdna Cut WT with restriction enzyme Used a subtractive hybridization screen Denature cdnas Mix 10xMUT:1xWT Re-anneal 1) Retroviruses that encode oncogenes (iii) Cloning RSV oncogene (BISHOP + VARMUS, 1970) Wild-type RSV Deletion mutant-b77 (transformation-defective) ONCOGENE Make cdna Cut WT with restriction enzyme Used a subtractive hybridization screen Clone DNA + enrich for oncogene Denature cdnas Mix 10xMUT:1xWT Re-anneal Gene called v-src 9
1) Retroviruses that encode oncogenes (iv) v-src is present in genome of other species that have never seen the virus (BISHOP + VARMUS, 1970) Conducted hybridization experiments using v-src or DNA from the deletion mutant B77 as probes. Probe Sp. Chicken Quail Duck Mouse B77 ++++ - - - v-src ++++ ++++ ++++ - Now known that c-src is present in mouse and humans Identification of human oncogenes 1) Screen for cellular homologues of viral oncogenes. Convention - viral gene called v-onc (e.g. v-src) cellular homologue called c-onc (e.g. c-src) More than 50 viral ocogenes have been identified and shown to have cellular homologues In some, but not all, cases the c-onc is found to be involved in human tumors. Usually the v-onc contains a number of changes (point-mutations and/or sometimes deletions) compared with c-onc that increases its transforming potential. This aids identification of key functional domains and activities. Identification of human oncogenes 2) Screen directly for cellular oncogenes. Weinberg, 1982 Human bladder carcinoma After 3 rounds- DNA Make a library of DNA from foci. 3T3 cells (Untransformed, murine cell line) DNA from foci Lots of human DNA Repeat 2X Isolate human sequences by screening for Alu repetitive sequences. Get a single human gene - ras. This was also independently identified as a viral oncogene. More about ras - lecture 2 10
VIRUSES AND ONCOGENES 1) Retroviruses that encode oncogenes 2) Retroviral activation of cellular oncogenes Retrovirus structure- gag pol env Long terminal repeats (LTRs) Potent transcriptional enhancers Can activate the expression of genes that lie either up- or downstream Called INSERTIONAL MUTAGENESIS E.g. - Avian leukosis virus known to activate c-myc expression Amplification of c-myc often occurs in human tumors (esp. leukemia) VIRUSES AND TUMOR SUPPRESSORS Another class of viruses (called the SMALL DNA TUMOR VIRUSES) encode oncoproteins that INACTIVATE tumor suppressors SMALL DNA TUMOR VIRUSES Virus Oncoproteins Human Papilloma Viruses (HPV) E7 E6 Adenoviruses E1A E1B Polyoma viruses (e.g. SV40) Large T antigen (Tag) 11
SMALL DNA TUMOR VIRUSES Virus Oncoproteins Human Papilloma Viruses (HPV) E7 E6 Adenoviruses E1A E1B Polyoma viruses (e.g. SV40) Large T antigen (Tag) Tumor suppressor prb SMALL DNA TUMOR VIRUSES Virus Oncoproteins Human Papilloma Viruses (HPV) E7 E6 Adenoviruses E1A E1B Polyoma viruses (e.g. SV40) Large T antigen (Tag) Tumor suppressor prb p53 More about both prb and p53 In the later lectures Human Papilloma Viruses (HPV) HPV infection can cause cervical cancer Relative risk of cervical cancer increases with number of sexual partners (i.e. it can be a sexually transmitted disease). There are both HIGH RISK (e.g. HPV16 and HPV18) and LOW RISK varients of HPV. Risk correlates with differences in the sequence of the E6 and E7 proteins and their ability to inactivate p53 and prb 12
Cancer is a multi-step process Each mutation must give the cell a selective advantage! COLORECTAL CANCER (CRC) Pathology of the gut It takes 3-6 days for the cells reach the tip They are extruded into the gut lumen and die Differentiation Cell proliferation Stem cells Colonic crypts Pathology of the gut If you disrupt the balance of cell proliferation versus differentiation and/or death you get polyps These are benign lesions Everyone develops them The numbers increase with age 13
At low frequency, the benign polyps can progress through various phases (defined by histological analysis of patient samples) and can ultimately become malignant tumors (carcinomas) The various transitions correlate with the acquisition of genetic lesions. These tend to occur in a defined order. The affected genes are now known. Several were identified through analysis of heredity cancer syndromes 14
Familial Colon Cancer Syndromes FAMILIAL ADENOMATOUS POLYPOSIS (FAP) Cancer of the bowel and duodenum Near 100% penetrance They develop 100 s of polyps at an early age HEREDITARY NON-POLYPOSIS COLON CANCER (HNPCC) Early onset colon and endometrial cancers 75-90% penetrance No increase in the number of polyps 15
Also see APC loss in >60% of sporadic CRC APC loss typically detected in the polyps - I.e. it is an early event - called the INITIATING MUTATION This explains why patients with FAP have 100s of polyps Familial Colon Cancer Syndromes FAMILIAL ADENOMATOUS POLYPOSIS (FAP) Cancer of the bowel and duodenum Near 100% penetrance They develop 100 s of polyps at an early age HEREDITARY NON-POLYPOSIS COLON CANCER (HNPCC) Early onset colon and endometrial cancers 75-90% penetrance No increase in the number of polyps 16
DNA from HNPCC tumors shows Microsatellite Instability (MI) Get dramatic instability in simple repeats (e.g. CACACA) This was a known phenotype of yeast that have defects in missmatch repair enzymes HNPCC patients map to at least 3 different linkage groups 2 major groups - MSH2 (orthologue of muts) - MLH1 (orthologue of mutl). These genes are part of the machinery that recognize and repair DNA mutations. Thus loss of MSH2 or MLH1 cause an increase in the frequency with which the cells acquire mutations - I.e. help other tumorigenic mutations to arise. Types of genes which are mutated in cancer- Tumor suppressor genes Oncogenes MSH2 and MLH1 represent a special class of t.s. genes called the mutator genes 17
For CRC you still need the initiating mutation (usually APC). Thus, patients with HNPCC have normal number of polyps but many more lesions at later stages. Cancer is a multi-step process The order of the mutations is important Each mutation must give the cell a selective advantage! Typically, need to disrupt a variety of cellular processes 1) Growth factor signaling pathways 2) Cell cycle control 3) Apoptosis 4) Cellular lifespan 5) Interaction with surrounding cells 6) Angiogenesis 18