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Cellular Oncogenes
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We made it to the 70s
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v-src c-src proto-oncogeneoncogene Viral oncogenes paved the way
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v-src c-src proto-oncogeneoncogene Viral oncogenes paved the way The concept: -Viruses kidnap a normal proto-oncogene -During the “kidnapping”, the mutated proto-oncogene became an oncogene -A new viral infection inserted an oncogene into the recipient, leading to cancer
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At the time, there were no known viral tumors in humans However….
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We ultimately want to know the cause of human diseases like colon cancer, rather than curing chicken sarcomas http://www.clevelandclinic.org/registries/inherited/fap.htm
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One oncogene mutated in the progression to malignancy is K-ras Lodish et al. Fig. 24-6
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How did we get there?
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Bob Weinberg long before he wrote our textbook Onto the stage stepped a bright and ambitious young assistant professor with a crazy idea
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But how can we identify oncogenes that are not viral oncogenes?
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But how can we find oncogenes that are not viral oncogenes? Add DNA from carcinogen-treated cell?
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First you need a way to get DNA into cells How would you do that?
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DNA can be “transfected” into cells using Calcium Phosphate
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As you know, Oncogenes relieve contact inhibition. This can be visualized in culture by “ focus formation ” Transfect with oncogene J Virol 2000 74:1008-13 Yoshioka et al. J Biol Chem 2002 277:10813-23 Fiordalisi et al.
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One can then test if the cells induce tumors
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But it’s a crazy idea 1. you need to find the right needle in a very large haystack 2.What if you need to find two or more needles??
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Bob Weinberg Chiaho Shih
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Bob Weinberg Chiaho Shih Maybe it wasn’t So crazy after all! Soon everybody was trying it!
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DNA from chemically treated mouse cells can transform normal mouse cells Without a virus: cells can be transformed a focus after transfection cells from the focus cells near the focus
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It’s time to move on- the 80s
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Transfect DNA from human cancer cell lines Human Cancer Cell Line Could this work for human tumors? Bob Weinberg Chiaho Shih
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Human Cancer Cell Line Somewhere here, among the normal mouse genes, we have a human oncogene Low transformation rates suggest that we are dealing with a single oncogene
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Figure 4.8 The Biology of Cancer (© Garland Science 2007) How to find a needle in a haystack
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Lodish et al. Fig. 24-4 How to find a needle in a haystack? Generate a bacteriophage genomic library Searching for the one human gene among many mouse genes
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An Oncogene is Cloned From a Human Tumor !! The Race is ON December 1981 Wigler lab Barbacid lab Weinberg lab But what does it encode?
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The cloned gene is ~25,000 bp In 1981 there was no way to sequence 25 kilobases
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The cloned gene is ~25,000 bp In 1981 there was no way to sequence 25 kilobases Luis Parada Bob Weinberg Let’s try a long-shot shortcut--what if the cellular oncogene is one of the known viral oncogenes? Its very unlikely - 14 v-oncogenes, ~30,000 human genes
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Use Southern blot analysis to look for one human gene in the otherwise mouse genome What probe should we use ?
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Der et al. PNAS 82 Channing Der, now at UNC mouse RAS human RAS human cellsmouse cells Just go through the viral oncogenes one by one
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Der et al. PNAS 82 Channing Der, UNC mouse RAS human RAS human cellsmouse cells Mouse cells transformed by a human oncogene The transforming oncogene is Ras Cellular oncogenes = Viral oncogenes
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v-rasc-ras (cellular ) (viral) Proto-oncogeneOncogene Viruses Carcinogens Random mutations
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But what does ras do in the cell??
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But what does ras do in the cell?? What do we want to know?
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But what does ras do in the cell?? Where does it do its work?
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Ras is postranslationally modified by addition of a lipid --where will that put it?
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Lipid modification targets Ras to the plasma membrane
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Farnesyltransferase inhibitors offer a way of reducing Ras activity
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Clinicians then tried them in a variety of tumors With activated Ras involvement Examples of Phase II trials: Leukemias (esp. AML/CML): Metastatic breast cancer(with capecitabine): Pancreatic cancer (with gemcitabine): Ovarian cancer (with current 2 drug combo): Neuroblastoma and Small cell lung cancer (with Taxol): Tipifarnib Or Lonafarnib
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Sadly, it was largely an epic fail Examples of Phase II trials: Leukemias (esp. AML/CML)May have some efficacy Metastatic breast cancer(with capecitabine): no significant improvement Pancreatic cancer (with gemcitabine): no improvement Ovarian cancer (with current 2 drug combo): no effect Neuroblastoma and Small cell lung cancer (with Taxol): Discontinued. Tipifarnib Or Lonafarnib
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What else could we ask about Ras?
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Lodish et al. Fig. 20-5 Scientists found Ras binds GTP Is it a new type of kinase?
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Lodish et al. Fig. 20-5 Ras is an enzyme and hydolyzes GTP
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Lodish et al. Fig. 20-5 Does this remind you of another well studied signaling pathway that was one of the first identified?
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Nobel Prize.org 2012 Nobel Prize in Chemistry!
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Lodish et al. Fig. 20-5 This allows Ras to act as a molecular switch
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The activity of Ras is regulated by GEFs and GAPs
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How is oncogenic ras different from the normal proto-oncogene? A new race starts
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How is oncogenic ras different from the normal proto-oncogene?
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Table 4.2 The Biology of Cancer (© Garland Science 2007) Ras is a key player in >50% of human tumors!
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Table 4.2 The Biology of Cancer (© Garland Science 2007) A note for the future—Ras is mutated in <5% of human breast cancers—we’ll see why later
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The G12V mutation prevents endogenous and GAP-stimulated GTPase activity
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Lodish et al. Fig. 20-5 G12V Thus the Ras oncogene is constitutively active i.e. stuck in the ON state!
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How does Ras act in our body, in vivo ? Why would constitutively active Ras lead to cancer ? From cell culture to model organisms
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