MCB 252 Topic 4 RAS Genetics and Cancer Prof David Rivier MCB 252 Spring 2015 Topic 4 Reading Lodish pages 734 744 Topic 4 Basics of Genetics Some Cancer Genetics Initial Cell Biology of Ras Identification of Pathway Components in Fruit Flies and Yeast Ordering genes and protein on a pathway How Does Ras Contribute to Cancer Growth Factor EGF TKR EGFR Grb2 SOS Ras Raf What is the mutant form of Ras that contributes to cancer Mutant Ras stuck in GTP form Is the oncogenic form dominant or recessive to wild type MAPK What would be the phenotype of a cell that could not make any Ras protein Transcription Entry into cell cycle proliferation MEK Thinking about Genetics Thinking about Genetics Lodish 5 2 Thinking about Genetics Genotype RAS WT RAS WT Phenotype Divides when signaled RAS CA RAS WT Divides without signal RAS CA RAS CA Divides without signal ras RAS WT Divides when signaled ras ras Cannot Divide RAS WT Wild type RAS CA Constitutively active also referred to in textbook as RasD ras completely inactive gene null phenotype Thinking about Genetics Genotype RAS WT RAS WT Divides Phenotype when signaled RAS CA RAS WT Divides without signal RAS CA RAS CA Divides without signal ras RAS WT Divides when signaled ras ras Cannot Divide Dominant mutations are often Gain of Function Mutations Here Ras has gained the ability to signal in the absence of the signaling molecule Recessive mutations are typically Loss of Function Mutations Here Ras has lost the ability to signal in the presence of the signaling molecule Thinking about Genetics Genotype Phenotype RAS WT RAS WT Divides when signaled RAS CA RAS WT Divides without signal RAS CA RAS CA Divides without signal ras RAS WT Divides when signaled ras ras Cannot Divide Gain of Function Mutations and Loss of Function Mutations typically have opposite phenotypes Here the Gain of Function Ras allele leads to constitutive cell division whereas Loss of Function of both alleles of Ras leads to the inability to divide Thinking about Genetics Genotype Phenotype RAS WT RAS WT Divides when signaled RAS CA RAS WT Divides without signal RAS CA RAS CA Divides without signal ras RAS CA Divides when signaled ras ras Cannot Divide The Null Phenotype is the phenotype that corresponds to the complete loss of function of both copies of the gene The null phenotype is typically the most extreme form of the loss of function phenotype The null phenotype is typically opposite to the gain of function phenotype Topic 4 Basics of Genetics Some Cancer Genetics Initial Cell Biology of Ras Identification of Pathway Components in Fruit Flies and Yeast Ordering genes and protein on a pathway Genetics of Cancer Cancer often results from mutations in cellular genes but may also result from introduction of viral genes which are typically mutant version of what were once cellular genes Oncogenes are cellular gene in which gain offunction mutant alleles contribute to cancer Tumor Suppressor genes are cellular genes in which loss of function mutant alleles contribute to cancer Growth Factor EGF TKR EGFR Grb2 SOS Ras Raf Given what you know about the role of Ras in cancer can you think of other possible mutations that might lead to cancer Why might mutations in Ras be more common than other mutations MEK MAPK Transcription Entry into cell cycle proliferation Growth Factor EGF TKR EGFR Chemo therapy Drugs that Target Ras MAPK pathway Grb2 SOS Ras Raf MEK MAPK Transcription Entry into cell cycle proliferation Topic 4 Basics of Genetics Some Cancer Genetics Initial Cell Biology of Ras Identification of Pathway Components in Fruit Flies and Yeast Ordering genes and protein on a pathway How Did Scientists Figure Out Ras was part of a Growth Factor Signaling Pathway Fibroblast tissue culture cells require EGF and PDGF to grow in culture and Receptors Inject cells with Ras blocking Abs cell stop dividing Ras required for response to GFs Transform with RasD allele and now divide in the absence of growth factors Ras acts downstream of GFs and Receptor Topic 4 Basics of Genetics Some Cancer Genetics Initial Cell Biology of Ras Identification of Pathway Components in Fruit Flies and Yeast Ordering genes and protein on a pathway How were the other players in the Ras and MAP kinase pathway identified Genetic Screens in Drosophila fruit flies the round worm C elegans and for the MAPK part of the pathway yeast Genetics Mutant Hunts need a phenotype in this case look for mutants that lack the R7 photoreceptor In a genetic screen we mutagenize an organism and look among a collection of mutagenized organisms for those that display the particular phenotype we are looking for This screen was designed to understand development and differentiation Looked for mutants that lacked R7 photorecptor cell that could not see UV light Identified a LOF mutant that did not make R7 Sevenless gene RTK Looked for other mutations that resulted in loss of R7 Bride of Sevenless gene signaling molecule on surface of R8 Son of Sevenless SOS Ras GEX a factor and a factor are pheromones Yeast Mating To understand the events involved in mating look for mutants defective in mating Identified the ste mutants the STE genes LOF alleles ste mutants cannot mate they are sterile Yeast Mating Signaling Pathway MAPK Mating factors Receptor Ste11 MEKK Raf MAPKKK Ste7 MEK MAPKK Fus3 MAPK MAPK Events involved in mating txn cell fusion Scaffold Proteins Prevent Cross talk between Pathways Topic 4 Basics of Genetics Some Cancer Genetics Initial Cell Biology of Ras Identification of Pathway Components in Fruit Flies and Yeast Ordering genes and protein on a pathway Upstream and Downstream Components in Pathway Genetic Analysis of Pathways Growth Factor EGF TKR EGFR Grb2 SOS Ras Raf MEK MAPK Transcription RasD Constitutively active Ras Is not dependent on upstream events it bypasses upstream components RasD phenotype is dependent on downstream components and events Double mutant analysis allows ordering of component along a pathway Entry into cell cycle proliferation Ras MAPK pathway What you need to know Human proteins What they do and how regulated Role of drugs and possible drug targets Genetic analysis reasoning Scaffold protein concept RAS MAP Kinase Pathway Cast of Characters EGF epidermal growth factor EGFR EGF receptor a receptor tyrosine kinase GRB2 G protein Receptor Binding SOS Son Of Sevenless RAS Rat Sarcoma RAF Rapidly Accelerated Fibrosarcoma MEK Map and Erk Kinase MAPK Mitogen Activated Protein Kinase 14 3 3 Was