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BIOL 445 Cancer Biology Spring 2015 Mark Peifer and Bob Duronio BIOL 445 In Bio 445 we combine the approaches of Bio 202 + Bio 205 with the current scientific literature to study the family of diseases known as cancer You may not believe it but by the end of the semester This will make sense! Hanahan and Weinberg, Cell 100:57-70 (2000) Virtually everything you’ll need is found at: http://www.bio.unc.edu/Courses/2017Spring/Biol445/ It’s a REALLY good idea to look through this carefully THIS WEEK AND Check back frequently for updates or changes BIOL 445 Textbooks -The Biology of Cancer by Robert Weinberg 2nd Edition What are you responsible for? - Classes (attendance AND participation) - Papers and assigned textbook reading(on website) -Your project- literature search and presentation Grading - Exams (20% X 2 midterms + 25% final) = 75 - Presentations = 15 - Classroom discussion & in-class evaluations = 10 Your Project - Choose a topic - Read the posted review paper - Do a thorough literature research, including primary data on the gene and the disease - Make a poster and present it to your peers - The final exam will cover ALL posters As you learned in the Online Introduction and the assigned readings for today, Cancer is a family of diseases caused by our own cells gone wrong Cancer is number 2 and rising relative to heart disease! CDC But as we’ll see, Cancer is not one disease, its many Source: American Cancer Society In the Online Introduction You learned about - Properties of cancer cells - Tumor progression - What causes cancer? - Accumulation of mutations - Molecular genetics of cancer Cancer often starts with a single mutation However One mutation is not enough !! Heard of natural selection? What types of genes are mutated in cancers? What types of genes are mutated in cancers? Two broad categories Oncogenes Mutational activation of proteins that normally Promote cell proliferation What types of genes are mutated in cancers? Two broad categories Oncogenes Mutational activation of proteins that normally Promote cell proliferation Tumor suppressor genes Mutational inactivation of proteins that normally inhibit cell proliferation Oncogenes proto-oncogene oncogene A proto-oncogene: a normal cellular gene that can become an oncogene, upon mutational change As you know from Bio 205, cells instruct one another via cell-cell signals Be Posterior! Turn on new genes! Signal transduction moves information from the cell surface to the nucleus & other cellular targets Cell surface Yes Ma'am! Nucleus Turn on new genes; pass it on Turn on new genes; pass it on Turn on new genes; pass it on Cell surface Signal transduction occurs in a series of steps Turn on new genes; pass it on Yes Ma'am! Nucleus Proliferate! Proliferate! Cell surface Cell-cell signals can regulate cell proliferation Proliferate! Proliferate! Yes Ma'am! Nucleus Cell-cell signals can regulate cell proliferation Cancer at the Cellular Level Signal transduction drives information from the cell surface to the nucleus & other cellular targets It’s time for a Bio 202 review How many copies of each gene do we have? Are most mutations dominant or recessive? Why? Oncogenes result from rare dominant mutations That lock signaling machinery in the ON state Genes did not evolve to cause cancer! Src in its normal context Normal skin cell tightly adherent to ECM Alberts et al. Signaling is OFF Wounding->platelet recruitment-> cell migration and proliferation Alberts et al. Signaling is ON Oncogenes Cellular signaling machinery is stuck ON One mutated copy = over active protein = over proliferation, oversurvival, etc. What types of genes are mutated in cancers? Two broad categories Oncogenes Tumor suppressor genes Mutational inactivation of proteins that normally inhibit cell proliferation Oncogenes The good guys become bad guys Tumor suppressor genes We lose the good guys Rb puts the brakes on cell proliferation by acting as A transcriptional co-repressor Rb E2F DP DO NOT transcribe genes required for DNA replication Rb phosphorylation frees E2F/DP to turn on genes required for proliferation P P E2F DP Transcribe gene required for DNA replication Retinal tumor in patient that inherited a mutant copy of Rb Lodish et al. Fig. 24-11 Retinoblastoma is inherited in a dominant fashion Lodish et al. Fig. 24-12 However, at a cellular level Retinoblastoma is Recessive! Normal individual with two functional copies of Rb gene Rb Rb Rb Rb Rb Rb Rb Rb Rb In rare cell one copy mutated, but second copy remains The remaining Rb still puts the brakes on cell proliferation Rb DP E2F DO NOT transcribe genes required for DNA replication It’s only when both copies of Rb are knocked out in a single cell that a tumor develops In person with hereditary Retinoblastoma, all cells start with one mutant copy Rb Rb Rb Rb If somatic mutation occurs, cell has no functional Rb and tumor develops Loss of functional Rb frees E2F/DP to turn on genes required for proliferation continuously E2F DP Transcribe gene required for DNA replication Thus oncogenes and tumor suppressors differ in both cellular function and genetics dominant recessive Figure 20-27 Molecular Biology of the Cell (© Garland Science 2008) The story of Src What Viruses and Nobel Laureates Taught Us About Cancer No thank you! Peyton Rous discovered a virus that causes cancer in chickens The Rous Sarcoma Virus (RSV) A virus can transform a normal cell into a tumor The Rous Sarcoma Virus (RSV) A virus can transform a normal cell into a tumor Nobel Prize in Physiology or Medicine 1966 The Rous Sarcoma Virus (RSV) A virus can transform a normal cell into a tumor But what’s a virus??? However the field then deviated-->Carcinogens Chemicals can directly induce cancer 1920s Viral Infection Out Yamagiwa Chemical Induction In 30 Years Later There was a Rebirth of RSV research RSV can transform cells in culture RSV stock Howard Temin Harry Rubin Immortality This allowed us to study cancer at the cellular level RSV infection Changed cells No contact inhibition on cell division No contact inhibition of cell division and significant alterations in cell adhesion and behavior Normal Normal RSV infected = Cancer RSV infected = Cancer But how??? Normal Normal RSV infected = Cancer RSV infected = Cancer I HOPE you remember the central dogma This is one of those biology facts That you need to have permanently stored The central dogma DNA Transcription mRNA Translation Protein RSV is a retrovirus These viruses reverse the central dogma, making a DNA copy of their RNA genome and inserting it into your DNA Alberts et al. Fig. 24-23 This earned the Nobel Prize in Physiology and Medicine in 1975!!! Howard Temin and David Baltimore Alberts et al. Fig. 24-23 Your genome is a retrovirus graveyard: Living and dead retroviruses make up 8% of your genome, with ~100,000 whole or partial copies! Alberts et al. Fig. 24-23 NEXT Breakthrough discovery Retroviruses can cause cancer by picking up mutated versions of normal cellular genes Alberts et al. Fig. 24-23 The paper that created two more Nobel laureates and founded the modern field of Cancer biology Let’s take a very short detour Retroviruses can also cause cancer by inserting next to and thus activating the expression of proto-oncogenes Retroviral insertion sites in different tumors Transcribe to mRNA 5 kilobases exons wnt-1 gene Alberts et al. Fig. 22-24 This turned out to be a very valuable discovery (for science and for Roel ) There are two mechanisms of gene activation by retroviral insertion Lodish et al. Fig. 24-10 OK—Back to src You know mis-expressing this gene Can initiate cancer What do you want to know now?? So, what job does the protein encoded by src do within the cell? The first BIG step: using antibodies to immunoprecipitate the v-Src protein Who can tell us what “immunoprecipitation” means? The first BIG step: using antibodies to immunoprecipitate the v-Src protein This led to the discovery that Src is post-translationally modified This led to the discovery that Src is post-translationally modified What’s translation?? Protein kinases and protein phosphatases add and remove phosphate groups from target proteins Lodish et al. Fig. 20-5 Adding labeled ATP to immunoprecipitated Src showed that Src can phophorylate a substrate Src is a kinase! in the presence of P32-ATP A substrate is phosphorylated Which amino acids can be phosphorylated? And Why those amino acids?? Src is a Tyrosine Kinase As a kinase, it can affect many cellular events Figure 15-18a Molecular Biology of the Cell (© Garland Science 2008) Normally, Src kinase intrinsic activity is low What makes Src so active in transformed cells? Western Blot with antibody that recognizes Tyr phosphorylated proteins What is a Western blot? Western Blot with antibody that recognizes Tyr phosphorylated proteins The structures of c-src and v-src provided an important clue! Lodish et al. Fig. 24-17 Src contains three domains that are shared with other proteins Binds polyproline motifs Phosphorylates other proteins Binds peptides phosphorylated on Tyr Scientists have determined the precise 3-dimensional structure of Src Xu et al. Nature. 1997 385:595-602 Scientists have determined the precise 3-dimensional structure of Src This is the bit missing in v-src We now know that tyrosine phosphorylation of the C-terminus creates an intramolecular and inhibitory interaction Lodish et al. Fig. 24-17 Thus Src is normally inactive due to intramolecular inhibition Lodish et al. Fig. 24-17 Recent work provided a more detailed model of Src activation Closed = OFF Open = ON Cowen-Jacob et al. Structure 13, 861-871 (2005) v-src lacks the C-terminal Tyr and thus cannot be inactivated! Lodish et al. Fig. 24-17 Activation of Src has multiple consequences From Schwartzenberg, Oncogene 17, 1463-1468 (1 Scientists next asked “Where is Src within cells?” This is a covalently attached lipid what might that mean? Experiments revealed that Myristylation of Src is essential for transformation This was built into an even more detailed model of Src activation Cowen-Jacob et al. Structure 13, 861-871 (200 OK-- c- Src is a tyrosine kinase What does it do in the cell? What are its targets? Remember, we are still in the late 70s Bishop and Varmus To Identify The KEY Targets of Src, let’s look for Proteins ONLY modified by biologically active Src Western blotting with antiphosphotyrosine antibodies V = v-Src transfected cells 2A/V = non-myristylated v-Src transfected cells Reynolds et al. MCB (1989) Identifying The Targets of Src-look for Proteins ONLY modified by biologically active Src Western blotting with antiphosphotyrosine antibodies V = v-Src transfected cells 2A/V = non-myristylated v-Src transfected cells p120 catenin: modulates cellcell adhesion Reynolds et al. MCB (1989) Here are a sampling of the targets of Src - p120 catenin: modulates cell-cell adhesion - Cortactin A: regulates actin polymerization - Focal Adhesion Kinase: involved in cell-matrix interactions Mike Schaller, ex-UNC Professor Src modulates both cell-cell and cell matrix adhesion: The basics Cell-cell junctions Cell-matrix junctions Basal lamina Src modulates both cell-cell and cell matrix adhesion: The basics Lodish et al. Fig. 22-2 Epithelial cells secrete a special ECM called the basal lamina Epithelial cells Basal Lamina Alberts et al. Fig. 19-54 Cells interact with the ECM via Focal adhesions, which also Anchor the actin cytoskeleton Focal Adhesions (orange) Actin: Green Alberts et al. Fig. 17-42 Focal adhesions are linked to the actin cytoskeleton Alberts et al. Fig. 16-75 A complex network of proteins links the focal adhesion to actin and regulates actin polymerization Alberts et al. Fig. 16-75 Focal adhesions are sites of intense protein tyrosine phosphorylatio Focal adhesions Actin: Green Phosphotyrosine: Red An oversimplified model of Src function Normal skin cell tightly adherent to ECM Wounding->platelet recruitment-> cell migration and proliferation Alberts et al. A less oversimplified model Migratory growth factors Extracellular matrix e.g., EGF, PDGF RTKs Src Integrins FAK PI-3kinase Adaptors Actin Remodel cell-matrix junctions -> cell motility From Jones et al. Eur J. Cancer 36, 1595-1606 (2000) FAK is recruited by integrins to the membrane and is autophosphorylated - Src binds to phosphorylated FAK - Src changes conformation and becomes active - Src further phosphorylates FAK - Src and FAK phosphorylate target proteins Src and FAK act together to regulate other focal adhesion proteins They regulate focal adhesion turnover Src-FAK active = less adhesion, more migration If Src is a critical regulator of cell adhesion, what happens to an animal without any Src? If Src is a critical regulator of cell adhesion, what happens to an animal without any Src? Cell 1991 64:693-702 Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Soriano P, Montgomery C, Geske R, Bradley A. Why is this phenotype so modest? Redundancy!! Why is this phenotype so modest? Redundancy!! Src has two very close relatives: Fyn and Yes Different Src family kinases work downstream of different receptors Alberts et al. Fig. 23-54 Fyn mutant mice are viable but have defects in myelination of brain neurons Yes mutant mice are viable but have subtle changes in B-cell function Src; Fyn; Yes triple mutant mice die at embryonic day 9.5 with multiple defects Triple mutant Wild-type That’s more like it! Triple mutant Wild-type However, triple mutant cells still make focal adhesions But src; fyn; yes (SYF) triple mutant cells fail to migrate! Scratch Wound assay Now let’s take all this basic science and put it to work to discover new drugs to treat cancer Scientists have determined the precise 3-dimensional structure of Src Active site This aided identification of kinase inhibitors that block Src action Active site SU6656 In leukemia, adding Src inhibition to inhibition of the related kinase Abl improved prognosis in phase II trials and was FDA-approved to help get around drug resistance in CML dasatinib Ottmann et al. Blood 110, 2309 (2007) This same Src inhibitor is in Phase II trials for advanced breast and Lung cancer dasatinib Ottmann et al. Blood 110, 2309 (2007) Another Src inhibitor failed in Phase I/II trials for metastatic pancreatic, breast, ovarian, and prostate cancers Active site AZD0530