Cellular Oncogenes The Story of Ras Last week, we made it to the 70s Viral Oncogenes Pave the Way c-src proto-oncogene and h-ras, v-myc , etc. v-src oncogene At the time, no known viral tumors in humans. Carcinogenes can cause cancer. Maybe oncogenes can rise independently of viruses. Bob Weinberg infect with DNA from a cell? Viral DNA can be transfected into cells using Calcium Phosphate Adenovirus Viral DNA can be transfected into cells and induce tumors Late 50s Late 70s Chiaho Shih Bob Weinberg Figure 4.2 (part 2 of 2) The Biology of Cancer (© Garland Science 2007) DNA from chemically treated Mouse Cells can Transform Normal Mouse Cells foci are formed and cells in foci show tumor characteristics Without a Virus: Cells can be Transformed It’s time to move on- the 80s Could This Work for Human Tumors? transfection of DNA from human cancer cell lines Human Cancer Cell Line Chiaho Shih Figure 4.8 The Biology of Cancer (© Garland Science 2007) Bob Weinberg DNA from Human Cancer Cell Lines can Transform Mouse Cells a focus after transfection cells from the focus cells near the focus Next Challenge: How to Isolate the Human DNA? Figure 4.3 The Biology of Cancer (© Garland Science 2007) How to Find a Needle in a Haystack mouse sequence Figure 4.8 The Biology of Cancer (© Garland Science 2007) How to Find a Needle in a Haystack bacteriophage genomic library Lodish et al. Fig. 24-4 The Race is ON Wigler lab December 1981 Barbacid lab Weinberg lab An Oncogene is Cloned From a Human Tumor !! Independent of any viral infection the cloned gene is ~25,000 bp no PCR available cutting, ligating, sequencing what about a negative control ? In the mean time, why not verify that the cellular oncogene is not a viral oncogene? Why to expect a negative answer? 14 v-oncogenes, ~50,000 human genes no evidence for a human tumor virus we don’t want the results to be positive Bob Weinberg Luis Parada Southern Blot Analysis what probe should we use ? v-src, v-ras (h-ras), v-myc, etc. Figure 4.4 The Biology of Cancer (© Garland Science 2007) The Transforming Oncogene is Ras human RAS mouse RAS Der et al. PNAS 82 Channing Der, UNC Ras Acts As a Molecular Switch Lodish et al. Fig. 20-5 How is oncogenic ras different from the normal proto-oncogene? A new race starts Figure 4.9 The Biology of Cancer (© Garland Science 2007) Table 4.2 The Biology of Cancer (© Garland Science 2007) G12V Mutation Prevents Endogenous and GAP-Stimulated GTPase Activity The Ras Oncogene is constitutively Active G12V Mutation Prevents Endogenous and GAP-Stimulated GTPase Activity The Ras Oncogene is constitutively Active G12V Lodish et al. Fig. 20-5 Why would constitutively active Ras lead to Cancer ? How does Ras act in our body, in vivo ? From Cell Culture to Model Organisms Model Organisms Teach Us About Us Mus musculus Xenopus laevis Californias governorus Caenorhabditis elegans Drosophila melanogaster Signal Transduction Pathways Important for development and homeostasis. All involved in Cancer. RTK Wnt TGF-β Hedgehog Notch Signal Transduction Pathways RTK Receptor Tyrosine Kinases Src RTKs Ras is anchored to the membrane by a lipid modification. MAPK/ERK enters nucleus Figure 15-60 Molecular Biology of the Cell (© Garland Science 2008) The RTK Pathway How Do We Know All of This? Model Organisms and Genetics The Model: The Compound Eye of the Fly The compound eye of insects is composed of 750 individual units: ommatidia. Each ommatidium is composed of 33 cells, including pigment cells, cone cells and in the middle: 8 photoreceptor neurons (the rods and cones of flies). The R8 cell differentiates first. The R7 cell differentiates last. The Sequential Induction Model differentiation is established through cell-cell interactions Figure 15-57 Molecular Biology of the Cell (© Garland Science 2008) How Do We find The Molecules Involved? Mutants Genetic Approach Reverse genetics Knockdown a specific candidate gene and test the consequences. Forward genetics Mutagenize animals randomly and select for mutations that affect eye differentiation. Genetic Screens treat flies with a mutagen (e.g. EMS) screen for mutants in eye differentiation assays rough vs. smooth eyes behavioral reaction to different wave lengths Genetic Screens mutants cloning sequence comparison suggested function antibodies and GFP constructs expression and localization sevenless (sev) encodes a TM protein with a cytoplasmic kinase domain (RTK?) bride of sevenless (boss) encodes a TM protein (ligand?) Mosaics analysis shows the molecular interactions between Boss in R8 and Sev in R7. No identified downstream genes to Sev and Boss in these screens. Why? Sev a downstream gene eye differentiation viability -/- Other developmental events How can we find mutants in the Sevenless pathway w/o affecting viability? Michael Simon and Gerald Rubin, Cell 1991 We made it to the 90s 10 years-old Britney Spears Travolta is still dancing A new graduate from Harvard Kurt Cobain Boss Sev protein X phosphorylated protein X Partially active Sev 50% of Protein X How can we find mutants in the Sevenless pathway w/o affecting viability? In a sensitized genetic background, where the pathway is only partially active, reducing another protein’s concentration by 50% might have an effect on eye development. We will look for dominant enhancers mutants that are hetrozygotes : m/+ Looking for other players in the pathway using genetics. Dominant Enhancer Screen R7 present Sev threshhold R7 absent wild type sev- sevts 22.7o C Lecture slide of Gian Garriga, UC Berkeley sevts 24.3o C sevts; enh*/+ 22.7o C The Final Ingredient Where do we find ts sev mutants? induced conserved mutations from src mutants in the sev gene: formed sev ts mutants Michael Simon and Gerald Rubin, Cell 1991 Looking for other players in the pathway using genetics. Dominant Enhancer Screen R7 present Sev threshhold R7 absent wild type sev- sevts 22.7o C Lecture slide of Gian Garriga, UC Berkeley sevts 24.3o C sevts; enh*/+ 22.7o C 7 genes were found that might function in the RTK pathway, including: 1) Son Of Sevenless (Sos) Sos sequence suggested it is a Ras GEF 2) Ras !!! Looking for other players in the pathway using genetics. ? R7 present Sev threshhold R7 absent wild type sev- sevts 22.7o C sevts 24.3o C sevts; m*/+ 22.7 24.3oo CC dominant suppressors genes involved in reducing RTK pathway activity Raf rapidly accelerated fibrosarcoma First, isolated as a viral oncogene. Sev a downstream gene eye differentiation viability -/- Other developmental events Figure 1 WT (slightly rough) raf(-) (dominant rough) sev(-); ectopic raf Sev Ras Raf normal eye sev(-); sev(-); ectopic ectopic ras raf (dominant rough) (dominant rough) sev(-); ectopic (suppresses ras; raf(-) dominant rough) Figure 1 (R7+) Sev WT (no R7) sev (-) (no R7) raf(-) (no R7) sev(-); normal raf Ras Raf Sina normal eye sev(-); ectopic sev(-); ectopic raf raf; sina(-) (R7 ++) (no R7) sev(-); ectopic sev(-); ras; raf(-) ectopic ras (R7 ++) no R7 Figure 2 western blot, verifying the presence of the proteins Ectopic Raf has increased kinase activity. Figure 3 early late sev(-) no R7 WT R7 (+) sev(-); ectopic Raf R7 (+) and even early Raf is necessary and sufficient for R7 differentiation Figure 4 Flies were bad enough, but worms ?? Caenorhabditis elegans exploratorium Nobel Prize lineage and programmed cell death Physiology and Medicine 2002 Bob Horvitz John Sulston Sydney Brenner Chemistry 2008 Physiology and Medicine 2006 GFP RNAi Marty Chalfie Andy Fire Craig Mello A simple model for organogenesis. Formation of the Vulva in C. elegans early oocytes embryos sperm vulva Only 22 cells! oocytes early oocytes embryos sperm vulva oocytes The Key Players one gonadal anchor cells (AC) 6 vulval precursor cells (VPCs) The anchor cell induces vulval fates. Sherwood and Sternberg (2003) Dev Cell How do we know this? killing the AC with a laser no vulva is formed vulvaless worm (bag of worms) Cell Induction The gonadal anchor cell (AC) signals vulval precursor cells (VPCs) to adopt vulval fates 3 VPCs will form the vulva, while the other 3 will fuse to the epidermis So, why call all of them VPCs ? All the VPCs are competent to form a vulva vulva A ligand gradient is secreted from the AC. The receptor for the secreted ligand is expressed in all the VPCs. Vulva Formation in C. elegans cell migration cell fusion ring formation How can we learn about the molecular mechanisms involved in vulva formation? Mutants wild type No vulva induction mutants with no vulval signaling vulvaless (Vul) mutants with too much vulval signaling multivulvae (Muv) A Screen for Vul and Muv Mutants some examples Bob Horvitz Nobel Prize physiology and medicine 2002 Vul Muv let-23 lin-15 lin-3 let-60 Next step: cloning of the genes Vul let-23 let-23 is an EGFR homologue = RTK. lin-3 lin-3 is an EGF homologue. Where do they function? Lin-3 is expressed in the AC. Let-23 is expressed in all the VPCs. Alberts et al. 21-44 Muv lin-15 lin-15 let-60 lin-15 was found to be an inhibitor of vulval induction (loss of function mutation) let-60 was found to promote vulval induction (gain of function mutation) Next step: clone the let-60 gene First evidence: Ras has an in vivo role as part of the RTK pathway. The gain-of-function mutation = Glycine 13 Glutamine Constitutively active Ras All the VPCs make vulvae. G13Q let-23 let = lethal Their good fortune: the screen yielded a weak mutant allele of let-23, affecting vulva formation, but not viability This suggests that other genes might be there, but those are essential for viability How can we find such genes? lin-15 Other mutations?of ras Suppressor Screens suppressors of lin-15 Muv mutants ? lin-15 suppressor of lin-15 Muv mutants Let-23 Let-60 GRB-2 Raf others Model Organisms Teach Us About Us Mus musculus Xenopus laevis Californias governorus Caenorhabditis elegans Drosophila melanogaster The RTK Pathways is Highly Conserved The RTK-Ras pathway offers drug targets for cancer treatment e.g., the Raf kinase inhibitor sorafenib (also inhibits the RTKs VEGFR, PDGFR, and Kit) In clinical trials for melanoma and lung cancer Approved for treatment of advanced renal cell carcinoma (Jan. 2006) Alberts et al. Fig. 21-44 Increased survival in Phase III liver cancer trial and approved for inoperable form (Nov. 2007)