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MCB 720 Susan Evans John Kopchick

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1 MCB 720 Susan Evans John Kopchick
ONCOGENES AND CANCER MCB 720 Susan Evans John Kopchick

2 ONCOGENES AND CANCER MCB 720 1/07

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
Cause of death Number of deaths Percent of total Heart disease 700,142 29.0 Cancer 553,768 22.9 Cerebrovascular disease 163,538 6.8 Lung disease 123,013 5.1 Accidents 101,537 4,2 Diabetes mellitus 71,372 3.0 Pneumonia and influenza 62,034 2.6 Alzheimer’s disease 53,852 2.2 Nephritis 39,480 1.6 Septicemia 32,238 1.3 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
Male Female Prostate 33% Lung 13% Colon 11% Bladder 6% Melanoma 4% Non-Hodgkin Lymphoma Kidney 3% Oral cavity Leukemia Pancreas 2% All other 18% Breast 32% Lung 12% Colon 10% Uterus 6% Ovary 4% Non-Hodgkin Lymphoma Melanoma Thyroid 3% Pancreas 2% Bladder All other 20%

8 2004 Estimated US Cancer Deaths Male Female
Lung 32% Prostate 10% Colon Pancreas 5% Leukemia Non-Hodgkin Lymphoma 4% Esophagus Liver 3% Bladder Kidney All other 21% Lung 32% Breast 10% Colon Ovary 5% Pancreas Leukemia 4% Non-Hodgkin Lymphoma Uterus 3% Multiple myeloma Brain All other 21%

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 Property Tumor suppressor genes Proto-oncogenes Alleles mutated in cancer Both alleles One allele Germ line transmission of mutant allele frequent Rare (1 example) Somatic mutations yes Function of mutant allele Loss of function (recessive allele) Gain of function (dominant allele) Effects on cell growth Inhibit cell growth Promote cell growth

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

32 Oncogenes by location c-sis Secreted wnt1 int1 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 Increases trx Represses 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 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 kinase DBl-H P Rho-GAP abl SH3 SH2 Kinase Tail P210 bcr-abl SH3 SH2 Kinase Tail kinase Dbl-H P P185 bcr-abl kinase SH3 SH2 Kinase Tail

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 Amplification Too much protein

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 Regulation of Ras Inactive state GTP Ras GDP 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

51 X ACTIVE INACTIVE Mutant Ras GEF GAP mSos1 mSos2 Ras-GRF Interaction
Ras-GDP Ras-GTP GEF GAP P120 gap Neurofibromin Gap 1m mSos1 mSos2 Ras-GRF Interaction with target proteins X Constitutively active Mutant Ras

52 Point mutation of PTK Induces dimerization in absence of ligand

53 Hormone Adenylyl cyclase Receptor bg a a a G protein Activated G protein ATP cAMP G protein associated with inner surface of PM Hormone bound receptor interacts with G protein Stimulates release of GDP and exchange for GTP Ga dissociates from complex Stimulates production of cAMP by adenylyl cyclase cAMP is a second messenger

54 Regulation of G proteins
Inactive state bg a GDP GTP hydrolysis Hormone binding induces interaction of receptor with G protein bg a GTP GDP GTP bg a Target proteins Stimulates the exchange of GTP for GDP GDP

55 Oncogenic mutations Locking a subunit in an active state
Pituitary tumors Gsp – encodes a mutated a subunit that blocks GTPase activity Constitutive production of cAMP Thyroid tumors Thyroid receptor mutated

56 Therapeutic implications
High doses of retinoic acid can induce differentiation Block growth factor/receptor interaction with antagonist Tyrosine kinase inhibitors Block protein interactions in signaling cascade (SH2 domain) Block membrane localization of Ras with farnesylation inhibitors

57 Mortality for Leading Causes of Death, United States - 1978
Cause of death Number of deaths Percent of total Heart disease 729510 37.8 Cancer 396992 20.6 Cerebrovascular disease 175629 9.1 Accidents 105561 5.5 Pneumonia and influenza 58319 3.0 Lung disease 50488 2.6 Diabetes mellitus 33841 1.8 Cirrhosis of liver 30066 1.6 Arteriosclerosis 28940 1.5 Suicide 27294 1.4 Disease of infancy 22033 1.1 Homicide 20432 All others 248543 12.9 Source: Vital statistics of the United States, 1978 Modified from: CA – A Journal for Clinicians, Vol 33, 1983.

58 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

59

60 Cellular homeostasis Proliferation Survival Undifferentiated Arrest
Apoptosis Differentiated (tumor suppressors) (oncogenes) CANCER

61 Activation of oncogenes Inhibition of tumor suppressors
Disruption of homeostasis Activation of oncogenes Inhibition of tumor suppressors

62 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

63 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 Angiogenic

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

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

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

67 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.

68 Comparison of Proto-oncogenes and tumor suppressors
Property Tumor suppressor genes Proto-oncogenes Alleles mutated in cancer Both alleles One allele Germ line transmission of mutant allele frequent Rare (1 example) Somatic mutations yes Function of mutant allele Loss of function (recessive allele) Gain of function (dominant allele) Effects on cell growth Inhibit cell growth Promote cell growth

69 Definitions Tumor – abnormal growth Cancer – malignant Benign
Remain localized to tissue where they originated Cancer – malignant Invades surrounding tissue Has the ability to metastasize

70 Primary neoplasm neovascularization Invasion Transport Arrest in organ extravasation Adherence Angiogenesis and proliferation Establishment of microenvironment metastases

71 Endothelial cells proliferate
Angiogenesis Definition: Process where tumor cells encourage the in-growth of capillaries and vessels from adjacent normal tissue Tumor releases angiogenic factors Endothelial cells proliferate Tumor grows

72 How does a normal cell become a tumor cell?
Immortalization Normal cells have fixed amount of doublings (~50) Senescence Telomerase activity decreases Shorter telomeres Crisis Increase telomerase Transformation

73 Immortalized/established cell line
Anchorage dependence Growth factor dependent Contact inhibition Cytoskeletal organization Monolayer

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

75 Focus Forming Assay Lacks contact inhibition

76

77 Tumorigenesis is a Multistep Pathway
Mutation of proto-oncogenes and tumor suppressor genes Special combination - yes Particular order - yes

78 Evidence for multistep cancer pathogenesis

79 Cooperation between genes
Myc – few mice with tumors Ras – more mice with tumors myc + ras – all mice with tumors Conclusion: Complementary activities of 2 distinct oncogenes function collaboratively to create fully tumorigenic cells

80 Oncogene Cooperation myc ras Myc + ras

81 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

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

83

84 Initiation Progression Aggressive, rapidly growing tumor

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

86 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

87 How does damage affect function?
Quantitative model- increased and sustained activity on a gene or its protein product Altered gene expression Change in protein structure Qualitative model- change in the specificity or function of the protein Substrate specificity Transactivation of different genes

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

89 Oncogenes by location c-sis Secreted wnt1 int1 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

90 Oncogenes by function Growth factors Growth factor receptors
G proteins Intracellular kinases Transcription factors

91 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 so that it is locked in the active state Interpreted by cell as a continuous and unrestricted growth inducing signal

92 Mechanisms of conversion
Induced by virus Transduction Insertional mutagenesis May or may not be virus induced Chromosomal translocation and inversion Amplification Point mutations

93 Chromosomal translocation

94 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 Increases trx Represses trx

95 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

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

97 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 kinase DBl-H P Rho-GAP abl SH3 SH2 Kinase Tail P210 bcr-abl SH3 SH2 Kinase Tail kinase Dbl-H P P185 bcr-abl kinase SH3 SH2 Kinase Tail

98 Signaling by secreted molecules
Endocrine Y Paracrine Y Autocrine Y

99 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

100 Receptor activation

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

102 Amplification Too much protein

103 Amplification Increased density induces dimer formation, autophosphorylation thus constitutively active

104 Growth factor EGF TGFb Growth factor receptor EGFR TGFR p27 cyclinD1/cdk4 cyclinE/cdk2 P Rb-E2F1 Rb + E2F1 CyclinD1 – amplification associated with cancer S phase genes proliferation

105 Point mutation Constitutively active

106 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)

107 Regulation of Ras Inactive state GTP Ras GDP 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

108 X ACTIVE INACTIVE Mutant Ras GEF GAP mSos1 mSos2 Ras-GRF Interaction
Ras-GDP Ras-GTP GEF GAP P120 gap Neurofibromin Gap 1m mSos1 mSos2 Ras-GRF Interaction with target proteins X Constitutively active Mutant Ras

109 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

110 Point mutation of PTK Induces dimerization in absence of ligand

111 Hormone Adenylyl cyclase Receptor bg a a a G protein Activated G protein ATP cAMP G protein associated with inner surface of PM Hormone bound receptor interacts with G protein Stimulates release of GDP and exchange for GTP Ga dissociates from complex Stimulates production of cAMP by adenylyl cyclase cAMP is a second messenger

112 Regulation of G proteins
Inactive state bg a GDP GTP hydrolysis Hormone binding induces interaction of receptor with G protein bg a GTP GDP GTP bg a Target proteins Stimulates the exchange of GTP for GDP GDP

113 Oncogenic mutations Locking a subunit in an active state
Pituitary tumors Gsp – encodes a mutated a subunit that blocks GTPase activity Constitutive production of cAMP Thyroid tumors Thyroid receptor mutated

114 Therapeutic implications
High doses of retinoic acid can induce differentiation Block growth factor/receptor interaction with antagonist Tyrosine kinase inhibitors Block protein interactions in signaling cascade (SH2 domain) Block membrane localization of Ras with farnesylation inhibitors


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