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  1. ONCOGENES AND CANCER MCB 720 Susan Evans John Kopchick


  3. Statistics • Introduction to cancer and oncogenes • Compare tumor suppressors and oncogenes • Tumor progression • Mechanisms of oncogenes • Examples of mutations in oncogenes

  4. Mortality for Leading Causes of Death, United States-2001 Source: US Mortality Public Use Data Tape 2001 National Center for Health Statistics Centers for Disease Control and Prevention, 2003

  5. Who gets cancer? • Over 1 million people a year • 1 out of 2 men • 1 out of 3 women • ~80% of cancers occur in people over 55

  6. Cancer

  7. 2004 Estimated US Cancer Causes Female Male

  8. 2004 Estimated US Cancer Deaths Female Male

  9. Comparison between men and women of different ethnicities

  10. Introduction to cancer

  11. Cellular homeostasis Proliferation Survival Undifferentiated Arrest Apoptosis Differentiated (tumor suppressors) (oncogenes) overactive inactive CANCER

  12. Definitions • Oncogene – a gene that when mutated or expressed at abnormally high levels contributes to converting a normal cell into a cancer cell • Proto-oncogene – the “normal” cellular progenitors of oncogenes that function to promote the normal growth and division of cells

  13. Proto-oncogene to oncogene • An alteration occurs in a normal cellular gene (proto-oncogene) that makes the protein hyper-functional (oncogene) • Proteins involved in the cell signaling pathways are products of proto-oncogenes • Proliferative • Anti-apoptotic (survival) • Angiogenic

  14. Tumor suppressors • Normally function to suppress the formation of cancer • Growth arrest • Apoptosis • DNA repair • Differentiation • Anti-angiogenesis

  15. Tumor suppressors are recessive – require mutation of both alleles Oncogenes are dominant – mutation of 1 allele is sufficient

  16. Oncogenes Normal genes (regulate cell growth) 1st mutation (leads to accelerated cell division) 1 mutation is sufficient for a role in cancer development.

  17. Tumor Suppressor Genes Normal genes (prevent cancer) 1st mutation (susceptible carrier) 2nd mutation or loss (leads to cancer) 2 mutations are necessary for a role in cancer development.

  18. Comparison of Proto-oncogenes and tumor suppressors

  19. Normal cells • Anchorage dependence • Growth factor dependent • Contact inhibition • Cytoskeletal organization • Monolayer

  20. Transformed cell • Unregulated growth properties • Serum independence • Anchorage independent • No contact inhibition (form foci) • May induce tumors in vivo

  21. Tumorigenesis is aMultistep Pathway • Mutation of proto-oncogenes and tumor suppressor genes • Special combination • Particular order -Yes -Yes

  22. Evidence for multistep cancer pathogenesis

  23. Oncogene Cooperation myc ras Myc + ras

  24. Mechanisms of collaboration • Multiple mutated genes disrupt multiple control points of anti-cancer mechanism • Synergistic/complementary activities • Cell tries to apoptose but selects for more aggressive cell with increased proliferative abilities

  25. Multistep tumorigenesis • Initiation • 1st mutation • Increased proliferation of a single cell • Progression • Additional mutations • Selection for more aggressive cells Clonal selection!

  26. Initiation Progression Aggressive, rapidly growing tumor

  27. With increase in histopathological abnormalities, there is an increase in the number of mutations at defined genetic loci

  28. What causes the mutations that lead to cancer? • Anything that damages DNA • Physical agents (radiation) • Chemical agents (carcinogens) • Anything that stimulates the rate of mitosis • Viruses • Oncogenes • Tumor suppressor genes

  29. How does damage affect function? • Increased and sustained activity on a gene or its protein product • Altered gene expression • Change in protein structure • Change in the specificity or function of the protein • Substrate specificity • Transactivation of different genes

  30. Oncogenes

  31. Isolate human DNA Alu probe (Alu PCR) Human bladder tumor cell line Isolate DNA Result: A single human gene is responsible for transforming capability transfect Transformed cells sequence Result: Human Ras Isolate DNA >99% mouse Compare to normal gene Result: Ras activation is due to a single point mutation Activated oncogenes from DNA transfection 3T3 cells

  32. Oncogenes by location c-sis wnt1 int1 Secreted Transmembrane c-erbB neu kit mas gsp gip ras src Membrane associated abl fps raf mos Vav AKT Cytoplasmic myc myb fos jun rel erbA Nuclear

  33. Oncogenes by function • Growth factors • Growth factor receptors • G proteins • Intracellular kinases • Transcription factors

  34. Oncogenic mutations • GF receptors and signaling proteins can exist in active and inactive state • Active state is rapidly turned over • Dephosphorylation of kinases • Hydrolysis of GTP to GDP • Protein degradation • Oncogenic mutation alters protein product -locked in the active state • Interpreted by cell as a continuous and unrestricted growth inducing signal

  35. Mechanisms of conversion • Insertional mutagenesis • Translocation and inversion • Amplification • Point mutations

  36. Chromosomal translocation

  37. Myc genes • Increased myc synthesis drives Max to partner with Myc • Myc has short half life • Max is stable Myc Basic HLH leucine Max Basic HLH leucine Binds DNA Dimerization Myc-Max Max-Max Mad-Max Represses trx Increases trx

  38. Oncogenic mutation of myc 1 2 3 • Chromosome translocation puts myc under control of strong promoter and enhancer • Increases the concentration of Myc-Max heterodimers thus increasing cell proliferation • Burkitt’s lymphoma Proto-oncogene Translocation to Ig locus Ig promoter and enhancer 2 3 Oncogene Transcription Splicing mRNA 2 3 Translation Increased expression of normal myc protein

  39. Translocation resulting in fusion of 2 genes Alters structure of normal c-abl protein

  40. SH3 SH3 SH3 SH2 SH2 SH2 Kinase Kinase Kinase Tail Tail Tail Cytoplasmic tyrosine kinase • C-abl encodes a cytoplasmic tyrosine kinase • Bcr promotes oligomerization • Bcr-abl fusion promotes activation of abl by oligomerization induced autophosphorylation • Philadelphia chromosome – translocation of chr 9 and 22 bcr P kinase DBl-H Rho-GAP abl P210 bcr-abl kinase Dbl-H P P185 bcr-abl kinase

  41. Signaling by secreted molecules Endocrine Y Paracrine Y Autocrine Y

  42. Growth factor expression • Controlled at the level of gene expression • Autocrine • Cell produces a growth factor to which it also responds • Sis – encodes a variant form of PDGF • Astrocytomas • Increases cell growth • Paracrine • VEGF • Increases growth of endothelial cells • Secreted by tumor

  43. Receptor activation

  44. Rearrangement N terminal domain is replaced by a transcription factor that can associate with itself

  45. Too much protein Amplification

  46. Amplification Increased density induces dimer formation, autophosphorylation -thus constitutively active

  47. Point mutation Constitutively active

  48. Ras proteins • Activation of PTK receptor by ligand binding • Receptor associates with adaptor protein (grb2) • Grb2 SH3 domain binds guanine exchange factor (Sos) • Sos activates Ras • Activated Ras interacts with protein kinase (Raf)

  49. Ras GDP Regulation of Ras Inactive state GTP GEF P GTPase GDP GAP • Cycle is unidirectional • GTP bound is active form • Regulated by 2 classes of proteins • + regulator • -regulator Ras GTP Active state

  50. Oncogenic mutations that constitutively activate Ras • Constitutive activation of GEFs (positive regulator) • Reduction of GAP activity (negative regulator) • Mutation of Ras gene • Cannot hydrolyze GTP