Tumor Suppressor Genes Dr. W. Edward Mercer Departments of Microbiology/Immunology, Biochemistry/Molecular Biology Jefferson Medical College, Kimmel Cancer Center Thomas Jefferson University http://www4.kimmelcancercenter.org/courses/mercer/documentlistREVIEWonly.php Cancer a Malady of Genes Tumor Suppressor Genes Gatekeepers and Caretakers “Gatekeepers” are the genes that directly control cell birth and cell death. RB, p53, PTEN, APC, BRCA1 and BRCA2 “Caretakers” do not directly control cell birth or cell death but rather control the rate of mutations of other genes, including gatekeeper genes. XP-A, ATM, hMSH1, hMLH2, hPMS1,hPMS2, WRN-H CELL CYCLE CHECKPOINTS CYCLIN A/ cdk2 S-phase checkpoint S G1/S checkpoint CYCLIN E / cdk2 p53 G1 G2 G2/M checkpoint CYCLIN D / cdk4,5,6 RB1 p53 M CYCLIN B/ cdc2 Cyclin-dependent kinases (CDKs) play an important role in Cell Cycle transitions Importance of the cell cycle and apoptosis in tissue homeostasis. TISSUE HOMEOSTASIS The balance between cell birth and cell death! Homeostasis Lost Hyperplasia TUMOR Tumor growth kinetics is different for gatekeepers and caretakers When a gatekeeper gene is altered through mutation, the rate of cell birth exceeds that of cell death, and a tumor is initiated. When a caretaker gene is altered, the cell accumulates mutations at a high rate and the process of tumorigenesis is accelerated. A raise in mutation rate may make tumorigenesis faster. The emergence and survival of a tumor is most likely a form “DARWINIAN MICROEVOLUTION” Survival of the fittest!!! Most Caretakers genes are inhibitors of the cell cycle First inhibitor of cell cycle cloned as a classical TSG retinoblastoma gene (RB1) Eye tumors Second site primary tumors – osteosarcomas Pineal glad tumors Knudson’s 2-hit mutation model for retinoblastoma. Figure 7.7 The Biology of Cancer (© Garland Science 2007) Retinoblastoma Appearance Retinoblastoma may be unifocal or multifocal. About 60% of patients have unilateral RB with a mean age of diagnosis of 24 months; About 40% have bilateral RB with a mean age of diagnosis of 15 months. Secondary tumors – osteosarcomas and rhabdomyosarcomas 4.4% have secondary tumor in 10 years, 18.3% in 20 years; 26.1 % in 30 years 70% of patients have point mutations in RB1 gene; 10% -- partial deletion of RB1 gene; 20% -- causes unknown; appearance is the same Structure of the RB1 gene RB1 gene covers 180-kb in 13q14 region The 27 exons of RB1 range in size from 31 to 1,889 base pairs. The translated product of RB1 (p105-RB1) consists of 928 amino acids. About 80-85% of mutations result in a premature termination codon. Mutations are scattered throughout exon 1 to exon 25 of the RB1 gene and its promoter region. About 80% of de novo germline mutations are paternal in origin. The reason for this is unknown Structure of RB1 protein DNA tumor virus oncoproteins: SV40 large T, HPV E7 and Ad E1A also bind the A/B pocket and block RB function in cell cycle regulation The RB gene Family TSG,s Human pRB/p105 Human p107 Human pRb2/p130 P.P. Claudio et al., Genome Biology, 2002 All three members of pRB family have similar domain structure and genomic organization Cell-cycle dependent phosphorylation of Rb Hyper-phosphorylated Rb p p Rb p p p p Restriction point p Rb p phase p G1 phase G2 phase M G0 Hypo-phosphorylated Rb Rb S Phosphorylation of Rb allows cells to transit the restriction point and enter S phase Quiescent cells p p p Rb http://www3.kumc.edu/jcalvet/PowerPoint/23 phase p p p Rb p p p p Rb p Molecular Mechanisms of RB1 How does RB1 control cell cycle progression? RB1 protein sequesters a transcription factor called E2F. The E2F family of transcription factors are required for transcriptional activation of genes needed for DNA metabolism/synthesis. Genes such as thymidine synthatase, dihydrofolate reductase, DNA polymerase alpha, and others DNA synthesis not allowed DNA synthesis allowed E2F1 P P Р E2F1 Р P P Р P P pRB P P Р pRB P P Р Р PР P PР P Cyclin D/CDK4 Cyclin complexes are not active pRB less phosphorylated E2F1 bound to pocket Cyclin Е/СDK2 Cyclin complexes are active pRB more phosphorylated E2F1 is free to promote cell cycle THE p53 TUMOR SUPPRESSOR PROTEIN Most Frequently Inactivated Gene in Cancer To Date: Over 30,000 Journal Citations p53 is involved in multiple cellular processes/pathways Apoptosis Cell cycle arrest p53 p53 DNA repair Negative regulation Senescence TP53 functions as a tumor suppressor. Colorectal cancers – progress in discrete steps: Loss of Heterozygosity Definition of a tumor suppressor loss one allele mutate the other ie: absence of normal protein Knudsen’s two hit hypothesis Mutations in p53 identified Science 244:217 www.p53.iarc.fr/index.html chromosome 17 p53 Li-Fraumeni Syndrome - an inherited predisposition to cancer - various kinds of cancers occur: osteosarcomas, soft tissue sarcomas, breast cancer - multiple tumors in the same individual - 70% of families inherit a mutation in p53 Science 250:1233; Nature 348:747 (1990) Anatomy of Human p53 Gene Functional domains of p53 protein p53 protein binds p53 as a tetramer p53 is a DNA damage response protein ”Guardian of the Genome” Ultraviolet light Ionizing radiation Drugs (bleomycin, 5-FU, adriamycin) Hypoxia Oncogenes (eg. Ras, Myc, SV40-T) Transfection and other forms of cellular stress; including “growth factor deprivation” induces a p53 response often resultin in apoptosis. Post translational modifications and increased p53 protein stability play a role. Early events in p53 research This is a benchmark paper because it was the first p53 inducible gene identified and the first CDK inhibitor to be identified. Biological Activities of p53 Protein 1. Wild type p53 is a transcription factor. - positive and negative effects. - most closely correlated with tumor suppression. 2. Wild type p53 is important role in cell cycle control. -ectopic expression of wtp53 during the Go to Sphase transition blocks cells in G1. -ectopic expression of wtp53 during the S-phase blocks cells in G2-phase. 3. Wild type p53 can induce apoptosis. -transcriptional activation of pro-apoptotic genes Figure 9.4 The Biology of Cancer (© Garland Science 2007) p53 hotspot mutations Mutant p53 Protein in Colorectal Tumors p53 mutations in colon cancer Good Result!! Progression to Cancer Good Result!! p21 WAF1/CIP - a universal inhibitor of CDKs originally - isolated in one of three ways: a p53 inducible transcript (WAF1); interaction with CDK2 (CIP1); mRNA over expressed in senescent cells (SD1) - binds to CDK1, CDK2,3,4 and 6 - normal fibroblasts found in a quaternary complex, throughout the cell cycle, with CDK and PCNA (proliferating cell nuclear antigen) - complex has activity in proliferating cells but is inhibited by the addition of more p21 to the complex WAF1 chromosome localization Chromosome 6p El-Deiry et al. Cell (1993) 75:817. p21WAF1: a p53-inducible gene p53 binding sequences in promoter Cip-1/Kip family of CDK inhibitors Inhibits Cdk2–cyclin E and cyclin A -- cdk2 complexes Block G1/S transition P21 Cip1=Waf1=Sdi1 Up-regulated in senescence and differentiation P27 Kip1 Regulated by growth inhibitory cytokines and by contact inhibition P57 Kip2 p21 in stress response and apoptosis p21 counteracts the apoptotic process p21 protects colorectal carcinoma and melanoma cells from p53-induced apoptosis !!! Heat shock DNA damage TNF-α p21WAF1 Some cascades ASK1 (MEKK5) Kinase SAPK (JNK) Kinase p38 Classical apoptotic response Inhibit activation of pro-enzyme Caspase 3 Can degrade p21 itself INK4-family of CDK inhibitors p16Ink4a p15Ink4b p18Ink4c p19Ink4d compete with D-type cyclins for binding to the CDK subunit The inhibitory action of the Ink4 proteins is largely dependent on the presence of pRb in the cell. When RB1 is damaged cyclin E expression is already increased and inhibition of cyclin D-CDK4 complexes does not inhibit S-phase entry p16 Ink4a Most studied gene in the family because of three reasons: -- Its mutations are common in hereditary and sporadic melanoma samples -- Only this gene out of whole family fulfills all criteria for being tumor suppressor -- Very special genomic structure of this gene p16 INK4a gene structure Actually two genes here Indirect regulation of Rb and p53 Sherr, C. (2001) Nature Reviews Molecular Cell Biology 2:731-737 INK4a in sporadic cancer Mutations of INK4a-encoding gene in sporadic cancers are rare. Major mechanism of inactivation of this gene in primary tumors is a small (<200 kb) deletions of both copies of INK4a PRIME EXAMPLES: Pancreatic cancer: 30% deletions, 32% deletion + mutation, 13% deletion+methylation Head and neck squamous cell carcinoma (HNSCC): 27% deletions, 11% deletion + mutation, 30% deletion + methylation In HNSCC p16 mutations arise very early -in progression of benign hyperplasia to carcinoma in situ The ARF Tumor Suppressor ARF inactivation reduces p53-dependent apoptosis ARF, Mdm2, p53 low levels Nucleolus ARF p53 Mdm2 Nucleus Cytoplasm Mdm2 targets p53 for degradation ARF induction: p53 induced, activated, nucleoplasmic, and stable Novel mechanism of preventing p53 turnover ARF sequesters Mdm2 in the Nucleolus ARF connects RB and p53 pathways http://www.mskcc.org/mskcc/ shared/images/brain_cancer_illus.jpg Table 7.1 part 1 of 2 The Biology of Cancer (© Garland Science 2007) Translational Research Basic Biomedical Science Molecular Genetic and Biochemical Pathways Insight into the molecular processes involving cell cycle control and apoptotic pathways is critically-important for the design of novel therapeutic agents for cancer intervention.