Oncogenes-basic science for students.pptx

In this lecture, we will describe Oncogenes and Tumor virus
Proto-oncogene, Oncogene,
Oncogenic activation
Other Examples
Understand the mechansims of important oncogenes
Understand how oncogene induce cell transformation.

1. 1 Oncogenes MING-DERG LAI, Distinguished Professor TZU-YANG WENG, PhD. Department of Biochemistry and Molecular Biology College of Medicine National Cheng Kung University 2020.03.10

2. 2 Outline History: Discovery of Oncogenes Oncogenes Tumor virus Proto-oncogene  Oncogene Oncogenic activation Other Examples  Understand the mechansims of important oncogenes  Understand how oncogene induce cell transformation.

3. 3 The First Documented Case of Cancer The world's oldest documented case of cancer hails from ancient Egypt, in 1500 b.c . The details were recorded on a papyrus, documenting 8 cases of tumors occurring on the breast . It was treated by cauterization, a method to destroy tissue with a hot instrument called "the fire drill." It was also recorded that there was no treatment for the disease, only palliative treatment. 

4. 4 Tumor 「腫瘤」一詞，自古有之，在中國殷墟的 甲骨文 中，已有「瘤」字的出現，而到了距今兩千多年前的春秋時代，在 周禮 一書中對周代有關腫瘤的事跡就有所記載，據周禮所言，周代已有專治腫瘍的醫生，稱為「瘍醫」。以後中國歷代都有關於腫瘤的記載，當時所採用的病名很多，但都是和腫瘤有密不可分的關係，不但論及內因病邪，還特別強調「 邪之所湊，其氣必虛 」，即認為體內氣血虧虛，運行失常，以及五臟六腑的 蓄毒 等 體內失調 ，才導致腫瘤的發生。

5. Tumor Formation to Cancer Inducer of tumor formation 1. Carcinogen 2. Viruses Tumor formation 1. Oncogene 2. Tumor suppressor gene 3. Immune surveillance 5

6. Discovery of Oncogenes 1908- Ellerman and Bang showed that avian leukemia could be transmitted by filtered extracts. 1911- Peyton Rous demonstrated that sarcomas in chickens had a viral etiology. 1933- Richard Shope discovered 1 st DNA tumor virus (Papilloma in cottontail rabbits) 6

7. Discovery of Oncogenes 7 Figure 3.2 The Biology of Cancer (© Garland Science 2007)

8. Retroviruses & Oncogenes-History (Rous Sarcoma Virus) Early 1970s RSV  Retrovirus containing v- Src as an oncogene for tumorigenesis In 1975 scientists found a normal version of v- Src in healthy cells. This normal version of v- Src , called c- Src , was defined a proto-oncogene, playing an essential role in cell growth. RSV picked up c- Src  mutation  RSV w/v- Src  tumor-inducing virus 8

9. 9

10. Identification of Retroviral Oncogene 10 ALV DNA LTR LTR gag pol env RSV DNA LTR gag pol env src LTR RSV ALV 1-2 weeks Several months Sarcomas Lymphomas Transformation No transformation

11. 11

12. 12 Src - Tyrosine kinase Src operates as a protein kinase - an enzyme that removes a high energy phosphate group from ATP and transfers it to a suitable protein substrate . Src was itself a phosphoprotein, that is, it carried phosphate groups attached covalently to one or more of its amino acid side chains.

13. 13 Src - Tyrosine kinase After transformation of cells by the v- src oncogene , the level of phospho -tyrosine was found to rise dramatically, becoming as much as 1% of the total phospho - amino acids in these cells

14. Where does src come from? 14 Southern hybridization of the uninfected cells revealed the presence of src gene  c-src.

15. 15

16. Structure of Src protein 16

17. The vertebrate genome carries a large group of proto-oncogenes 17

18. Virus containing DNA molecules are also able to induce cancer 18

19. What about viral infections in human cancer: carrying oncogenes, inflammation, apoptosis and regeneration 19

20. Do viral oncogenes play a major role in human cancer? 20 Virus oncogenes only play a role in a potion of human cancer.

21. 21 Non-viral carcinogenesis Carcinogens function as mutagens. Whether physical (X-ray) or chemical (tobacco tar), these agents induce cancer through their ability to mutate critical growth-controlling genes in the genomes of susceptible cells. Once there genes were mutated, the resulting mutant alleles might function as active oncogenes, driving the cancerous growth of the cells that carried them.

22. 22 Transfection of DNA provides a strategy for detecting nonviral oncogenes 1972

23. Transformation of mouse cells by human DNA 23 Figure 4.3 The Biology of Cancer (© Garland Science 2007) Foci Foci cells Surrounding normal cells

24. 24 Transfection of DNA provides a strategy for detecting nonviral oncogenes Oncogenes exist in transformed cells. However, it is necessary to identify a single gene. 1972

25. 25 Southern and Northern blotting procedures DNA probe specific for retrovirus-associated oncogene probe

26. 26 Oncogene: H- ras Nature 297: 474-478, 1982 H- ras oncogene probe was cloned from Harvey murine sarcoma virus. Genomic DNA from NIH 3T3 were transfected with the DNA extracted from human bladder sarcoma cell line (a-j).

27. Is the cellular oncogene important in carcinogenesis? 27

28. Kaplan-Meir plot, in which the percentage of patients surviving is plotted on the ordinate as the function of time after initial diagnosis or treatment, which is plotted on abscissa 28

29. Oncogenes 29 Cellular Proto-Oncogenes Cell cycle, cell proliferation, differentiation, survival, and apoptosis … Proteins

30. Mechanism of oncogene perturbation Retroviral transduction Mutations ： H- ras mutation Cytogenetic mechanism of oncogene activation Translocation- myc Amplification- erbB2/ neu 30

31. Mechanism of oncogene perturbation Retroviral transduction Mutations ： H- ras mutation Cytogenetic mechanism of oncogene activation Translocation- myc Amplification- erbB2/ neu 31

32. The life cycle of an RNA tumor virus like RSV 32

33. Mechanisms of cell transformation by retroviruses Retroviral transduction of oncogene (transducing retrovirus) Oncogene activation by retroviral insertion ( cis- acting) Oncogenesis mediated by essential retrovirus proteins ( trans- activating) 33

34. Mechanisms of cell transformation by retroviruses 1. Retroviral transduction of oncogene (transducing retrovirus) 34

35. Mechanisms of cell transformation by retroviruses 2. Oncogene activation by retroviral insertion ( cis- acting) 35

36. 36 Retrovirus-associated oncogenes

37. Mechanism of oncogene perturbation Retroviral transduction Mutations ： H- ras mutation Cytogenetic mechanism of oncogene activation Translocation- myc Amplification- erbB2/ neu 37

38. 38 The Regulation of Ras Proto-oncoprotein

39. Localization of an oncogene-activating mutation 39 Nature 300:143-149, 1982

40. Mutation responsible for H- ras mutation 40

41. 41

42. Mechanism of oncogene perturbation Retroviral transduction Mutations ： H- ras mutation Cytogenetic mechanism of oncogene activation Translocation- myc Amplification- erbB2/ neu 42

43. Burkitt’s lymphoma incidence in Africa 43

44. Chromosome translocations in Burkitt’s lymphoma 44

45. 45

46. Mechanism of oncogene perturbation Retroviral transduction Mutations ： H- ras mutation Cytogenetic mechanism of oncogene activation Translocation- myc Amplification- erbB2/ neu 46

47. Amplification of oncogene erbB2/ neu in breast caners 47

48. 48

49. 49

50. Summary I. Proto-oncogene  Oncogene Mechanism of oncogene perturbation Viral transduction (DNA/RNA) Mutations Chromosome Translocation Amplification 50

51. Mutant p53: an oncogenic transcription factor 51 Oncogene  (2007)  26,  2212–2219

52. 52 Outline History: Discovery of Oncogenes Oncogenes Tumor virus Proto-oncogene  Oncogene Oncogenic activation Other Examples  Understand the mechansims of important oncogenes  Understand how oncogene induce cell transformation.

53. 53 Oncogenes Growth factors Growth factors receptors Cytoplasmic kinase Intracellular tyrosine kinases ： Non-receptor tyrosine kinase ; e.g. src signaling G protein/signal transduction Serine/threonine kinases; e.g. raf-1 signal transduction Transcription factors Proteins involve in cell cycle & apoptosis

54. 54 Oncogenes Oncogenes are mutated in ways that render gene constitutively active or active under conditions in which the wild-type gene is not . Examples: Cell Surface protein Cytoplasmic protein

55. 55 Growth Factors The normal versions of oncogene-encoded proteins often serve as components of the machinery that enables cells to receive and process biochemical signals regulating cell proliferation . Growth Factors These are relatively small proteins that are released by some cells , make their way through intercellular space, and eventually impinge on yet other cells, carrying with them specific biological messages.

56. 56 Effects of growth factors on cells PDGF and medium medium Wild type Fibroblast Fibroblast loss of PDGF receptor

57. 57 Structure of tyrosine kinase receptors (Receptor tyrosine kinase, RTKs)

58. 58 Signaling network of ErbB family

59. 59 Clinical and Applied Immunology Reviews

60. 60 EGF and EGFR (ErbB1) EGF was able to bind to the surfaces of the cells whose growth it stimulated. Involvement of a cell surface protein, an EGF receptor (EGF-R), which was able to specifically recognize EGF in the extracellular space. 621 amino acid 542 amino acid 23 amino acid

61. 61 EGFR has a Src -like kinase domain 621 amino acid 542 amino acid 23 amino acid EGF Phosphorylate tyrosine on certain protein (in cytoplasm) Cell proliferation

62. 62 Receptor dimerization following ligand binding

63. 63 Phosphotyrosine on the EGFR following ligand addition A fluorescent reagent that binds to a phosphotyrosine resides. Blue- above the basal level Red- below the basal level AG1478: EGFR kinase inhibitor

64. 64 Deregulation of receptor firing -> Ligand-independent

65. Example (1): EGFR mutations 65

66. Example (2): v- ErbB 66

67. 67 Oncogenic growth factor receptor

68. Molecular targets  68 Nature Reviews Clinical Oncology  8 , 492-503

69. 69 Deregulation of receptor firing -> Autocrine signaling loop

70. 70

71. 71 Cytokine receptor -IFN-R

72. TGF-beta receptor 72

73. 73 Chromosome Translocation -> Bcr-Abl fusion gene Bcr (breakpoint cluster region) gene - Abl (Abelson proto-oncogene The resulting fusion of Abl with Bcr amino acid sequences deregulates the normally well-controlled Abl protein, causing it to emit growth-promoting signals in a strong, deregulated fashion. The BCR-ABL fusion gene is found in most patients with chronic myelogenous leukemia (CML), and in some patients with acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML).

74. 74 Bcr-Abl

75. 75

76. 76 Signaling network of Ras oncogene

77. Example: Signaling network (EGFR  RAS ) Ligand binding to the EGFR induces dimerization through a receptor-mediated mechanism. EGFR stimulation results in activation of signalling cascades that include the RTK–GRB2–SOS–RAS–RAF–MEK–ERK. 77 GRB2 SOS (GEF)

78. 78

79. 79 Nat Rev Drug Discov. 2014 ;13(11):828-51

80. 80 Ras oncogenes Ras oncoproteins (H- ras , K- ras and N- ras ) were frequently found to carry amino acid substitutions in residues 12, 13 or 61, which resulted in constitutive activation of signaling. More than 20% of human tumors arising in a variety of tissues carry such point-mutated ras genes.

81. Targeting Mutant Kras ? Inhibit the posttranslational modification of Ras proteins by Ftase  Farnesyltransferase inhibitors (FTIs)  Geranylgeranyltransferase type1 (GGT1) Targeting its GTP binding pocket Targeting downstream Ras effector signaling pathways , including the Raf –MEK–ERK and PI3K–AKT– mTOR pathways 81 81

82. Targeting mutant Kras ? Inhibit the posttranslational modification of Ras proteins by Ftase  Farnesyltransferase inhibitors (FTIs)  Geranylgeranyltransferase type1 (GGT1) Targeting its GTP binding pocket Targeting downstream Ras effector signaling pathways , including the Raf –MEK–ERK and PI3K–AKT– mTOR pathways 82 82

83. RAS processing and association with the plasma membrane. 83 Nature Reviews Cancer  3 , 945-951

84. Targeting Mutant Kras ? Inhibit the posttranslational modification of Ras proteins by Ftase  Farnesyltransferase inhibitors (FTIs)  Geranylgeranyltransferase type1 (GGT1) Targeting its GTP binding pocket Targeting downstream Ras effector signaling pathways , including the Raf –MEK–ERK and PI3K–AKT– mTOR pathways 84 84

85. 85 The Regulation of Ras Proto-oncoprotein

86. Figure 6.14 The Biology of Cancer (© Garland Science 2007) 86

87. Targeting Mutant Kras ? Inhibit the posttranslational modification of Ras proteins by Ftase  Farnesyltransferase inhibitors (FTIs)  Geranylgeranyltransferase type1 (GGT1) Targeting its GTP binding pocket Targeting downstream Ras effector signaling pathways , including the Raf –MEK–ERK and PI3K–AKT– mTOR pathways 87 87

88. Figure 6.15 The Biology of Cancer (© Garland Science 2007) 88

89. 89 Dual PI3k/mTOR inhibition of the PAM pathway (stimulatory connections in green, negative feedback loops in red, pathways activated by suppression of negative feedback loops highlighted in yellow (from Rozengurt E et al, 2014). Complex Signal Pathways

90. 90 https://www.marycrowley.org/groundbreaking-research/cancer-pathways/ Multi Therapeutic Approaches