Tumor Suppressors Versus Oncogenes The Cancer Phenotype is Usually Recessive R. Weinberg, Cancer Biology Tumor Suppressor Genes The story of Retinoblastoma Retinoblastoma is a cancerous disease 1/20,000 children; 300 per year Average age is 18 months Leukocoria or “white pupil” Treatment: enucleation = eye removal Prognosis is good after enucleation over 90% survival with early detection and treatment Rb is either sporadic or familial - Sporadic cancer in 55-65% of all cases - Sporadic cancers are unilateral Hereditary childhood cancer: - bilateral tumors in ~75% of cases - unilateral tumors in ~25% of cases Children with bilateral (familial) Rb have a high risk of developing non-retinal tumors Familial Sporadic Germ-line mutations in the Rb gene lead to predisposition to cancer In cancer patients with a family history of Retinoblastoma: the inheritance seems to be ? Rb tumors are associated with a deleted region in chromosome 13 Deletion = loss-of-function probably a recessive mutation in the Rb gene Knudson’s “Two Hit” Hypothesis for the Generation of RB Alfred Knudson, PNAS 68:820 (1971) Knudson’s “two hit” hypothesis for the generation of RB Retinoblastoma is inherited as a dominant trait, but it is recessive at the cellular level People with familial Retinoblastoma carry one mutated copy in ALL their cells. Cells that would get a second hit will develop Rb or other cancers later in life. Retinoblastoma is Associated with Loss of Heterozygosity (LOH) at the RB Locus (LOH) (LOH) ~40% of the time the wild type allele is mutated 4% of these are deletions R. Weinberg, Cancer Biology (nondisjunction) Mitotic Nondisjunction Causes (LOH) at the RB Locus ~30% of the time Mitotic Recombination Causes LOH at the RB Locus ~30% of the time Rb = A Tumor Suppressor Gene Predisposition is inherited dominantly, but cancer is not inherited The offspring CANNOT inherit two mutated genes Rb is just one example Inheritance of brca1(lf) mutation results in predisposition for breast cancer How can we clone a tumor-recessive gene? How do we test candidate genes? Oncogenes transform cells into cancerous cells But TSGs are recessive Rb tumors are associated with a deleted region in chromosome 13 Testing a candidate gene Use a fragment of the candidate gene as a probe for hybridization analysis Search for absence of the gene in tumors (hoping both mutated copies are deletions) More on this in Angier book, starting p. 334 Rb gene expression is absent or altered in retinoblastoma tumors Rb tumors WT Friend et al. Nature (86) Other tumors Lee et al. Science (87) Northern blots (mRNA expression) Expression of the RB Gene in RB Mutant Cells Corrects the Cancer No virus RB virus Control virus Parental = WERI-Rb27 cells Lux = control virus infected Rb = RB virus infected Saos-2 cells Saos-2 cells infected with RB retrovirus Tumor formation assayed in “nude” mice Huang et al Science, 242:1563-6 (1988) Bold Predictions, Further Work Dr. David Abramson, RB expert at New York Hospital (ca. 1986, According to Natalie Angier) “I believe that in fifteen years, at the outside, we’ll be able to stop retinoblastoma before it begins. I’m so sure that I’ve already given the drug a name. I call it retino-revert, or retino-prevent. The drug will be an analogue of the natural protein that is missing in retinoblastoma cells … We’ll be able to diagnose a child prenatally and start giving this retino-revert to the mother to prevent retinoblastomas from growing as the fetus is developing. I know I’m going out on a limb with this one, but … Come back to me in 2001 and tell me if I wasn’t right.” pRb: What does it do? pRb is a nuclear protein that undergoes phosphorylation and dephospharylation in concert with the cell cycle RB regulates progression through G1 phase Un- and Hypophosphorylated pRb inhibits the cell from entering a new cell cycle Upon further phosphorylation at the R point, hyper-phosphorylated pRb becomes inert and the cell cycle can proceed Un- and Hypo-phosphorylated Rb inhibits activity of the E2F family of transcription factors Figure 8.23c The Biology of Cancer (© Garland Science 2007) Hyper-phosphorylated Rb cannot bind and inhibit E2F RB/E2F complexes act as transcriptional repressors Releasing Rb from the E2Fs leads to activation of transcription E2Fs have 100s of target genes, mostly involved in DNA replication Rb, the retinoblastoma protein regulates the cell cycle Cell cycle = OFF Rb binds to E2F: no transcription, no entry into S phase Cell cycle = ON Rb does not bind to E2F: transcription and entry into S phase w/o 2 copies of Rb: no cell cycle arrest Sites of RB Missense Mutations in Tumors B A The A and B domain = pocket = where E2F binds Figure 8.23b The Biology of Cancer (© Garland Science 2007) How is Rb activity regulated during the cell cycle? How is Rb activity regulated during the cell cycle? By Cyclin/CDKs Hypo-phosphorylation is catalyzed by cycD-CDK4/6 Hyper-phosphorylation is catalyzed by cycE-CDK2 pRb is hyper-phosphorylated and inhibited (and released from its role as a guardian), only upon cycE expression How is Rb activity regulated during the cell cycle? By Cyclin/CDKs However, E-CDK2 can phosphorylate Rb, only AFTER Rb is phosphorylated by D-CDK4/6 One of the E2F targets: the cycE gene! Transcription of cycE starts a positive feedback loop Rb control of E2F IS the restriction point As E2Fs are necessary for expression of cycE, think how critical negative regulation by Rb is for cell cycle control E2Fs Proliferation Signals The Canonical p16/pRB/E2F “Pathway” The RB Pathway in Human Cancer (p16) Sherr and McCormick, Cancer Cell ·2:103 (2002) How can we study retinoblastoma in the lab? with GEMs: Genetically Engineered Mice Forward versus reverse genetics Forward Genetics: isolate mutants with a specific phenotype and then clone the gene Reverse Genetics: clone the gene and then make a mutant to determine phenotype “Knock Out” your gene in Cultured Pluripotent ES Cells Formation of ES Cells Carrying a Knockout Mutation Positive and Negative Selection of Recombinant ES Cells Making the Mutant Mouse The Phenotype of RB Mutant Mice Homozygous embryos die at day 13.5 massive apoptosis in the CNS anemia caused by lack of blood cells made by the fetal liver therefore RB is not essential for cell division Heterozygous animals are viable they develop pituitary tumors with near 100% penetrance these tumors lose the wild type RB allele also develop thyroid adenomas therefore the Rb-/+ mouse is not a model for retinoblastoma but it is a good model for tumor suppressors And can study it’s mechanism of action genetically Tumorigenesis in RB Mutant Mice Requires E2F Genotype Pituitary Tumors Thyroid Tumors RB (+/-) 19/20 (95%) 10/19 (53%) RB (+/-); E2F1 (+/-) 36/36 (100%) 2/34 (6%) RB (+/-); E2F1 (-/-) 16/26 (62%) 0/22 (0%) Yamasaki et al, Nat. Gen. 18:360 (1998) The Phenotype of RB Mutant Mice Homozygous embryos die at day 13.5 massive apoptosis in the CNS anemia caused by lack of blood cells made by the fetal liver therefore RB is not essential for cell division Heterozygous animals are viable they develop pituitary tumors with near 100% penetrance these tumors lose the wild type RB allele also develop thyroid adenomas therefore the Rb-/+ mouse is not a model for retinoblastoma but it is a good model for tumor suppressors Why don’t Rb-/+ mice get retinoblastoma?????!!!!!!