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Transcript of ONCOGENES AND CANCER MCB 720 Susan Evans John Kopchick.
ONCOGENES AND CANCER
MCB 720
Susan Evans
John Kopchick
ONCOGENES AND CANCER
MCB 720
1/07
• Statistics
• Introduction to cancer and oncogenes
• Compare tumor suppressors and oncogenes
• Tumor progression
• Mechanisms of oncogenes
• Examples of mutations in oncogenes
Mortality for Leading Causes of Death, United States-2001
Cause of deathNumber 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 2001National Center for Health StatisticsCenters for Disease Control and Prevention, 2003
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
Change in US Death Rates
0
100
200
300
400
500
600
700
Heart diseases Cerebrovasculardiseases
Pneumonia/influenza Cance
Ra
te p
er
10
0,0
00
1950
2001
Cancer
Prostate 33%
Lung 13%
Colon 11%
Bladder 6%
Melanoma 4%
Non-Hodgkin Lymphoma
4%
Kidney 3%
Oral cavity 3%
Leukemia 3%
Pancreas 2%
All other 18%
Breast 32%
Lung 12%
Colon 10%
Uterus 6%
Ovary 4%
Non-Hodgkin Lymphoma
4%
Melanoma 4%
Thyroid 3%
Pancreas 2%
Bladder 2%
All other 20%
2004 Estimated US Cancer Causes
Male Female
Lung 32%
Prostate 10%
Colon 10%
Pancreas 5%
Leukemia 5%
Non-Hodgkin Lymphoma
4%
Esophagus 4%
Liver 3%
Bladder 3%
Kidney 3%
All other 21%
Lung 32%
Breast 10%
Colon 10%
Ovary 5%
Pancreas 5%
Leukemia 4%
Non-Hodgkin Lymphoma
4%
Uterus 3%
Multiple myeloma 3%
Brain 3%
All other 21%
Male Female2004 Estimated US Cancer Deaths
0
50
100
150
200
250
300
350
400
White African American Asian American Indian Hispanic
Rat
e p
er 1
00,0
00
Men
Women
Comparison between men and women of different ethnicities
Introduction to cancer
Proliferation
Survival
Undifferentiated
Arrest
Apoptosis
Differentiated
Cellular homeostasis
CANCER
(oncogenes) (tumor suppressors)
inactiveoveractive
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
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
Tumor suppressors
• Normally function to suppress the formation of cancer– Growth arrest– Apoptosis– DNA repair– Differentiation– Anti-angiogenesis
Tumor suppressors are recessive – require mutation of both alleles
Oncogenes are dominant – mutation of 1 allele is sufficient
Oncogenes
Normal genes (regulate cell
growth)
1st mutation(leads to
accelerated cell division)
1 mutation is sufficient for a role in cancer development.
Tumor Suppressor Genes
Normal genes Normal genes (prevent (prevent cancer)cancer)
1st mutation1st mutation(susceptible carrier)(susceptible carrier)
2nd mutation or 2nd mutation or loss (leads to loss (leads to
cancer)cancer)
2 mutations are necessary for a role in cancer development.
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 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
Comparison of Proto-oncogenes and tumor suppressors
Normal cells
• Anchorage dependence
• Growth factor dependent
• Contact inhibition
• Cytoskeletal organization
• Monolayer
Transformed cell
• Unregulated growth properties
• Serum independence
• Anchorage independent
• No contact inhibition (form foci)
• May induce tumors in vivo
Tumorigenesis is aMultistep Pathway
• Mutation of proto-oncogenes and tumor suppressor genes
• Special combination
• Particular order
-Yes
-Yes
Evidence for multistep cancer pathogenesis
Oncogene Cooperation
myc
ras
Myc + ras
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
Multistep tumorigenesis
• Initiation– 1st mutation– Increased proliferation of a single cell
• Progression– Additional mutations– Selection for more aggressive cells
Clonal selection!
Initiation
Progression
Aggressive, rapidly growing tumor
With increase in histopathological abnormalities, there is an increase in the number of mutations at defined genetic loci
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
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
Oncogenes
Activated oncogenes from DNA transfection
3T3 cells
Human bladder tumorcell line
Isolate DNA
Transformedcells
Isolate DNA>99% mouse
Isolate human DNAAlu 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
Oncogenes by locationSecreted
Transmembrane
Cytoplasmic
Nuclear
c-siswnt1int1
c-erbBneukitmas
Membrane associated
gspgiprassrc
ablfpsrafmosVavAKT
mycmybfosjunrelerbA
Oncogenes by function
• Growth factors
• Growth factor receptors
• G proteins
• Intracellular kinases
• Transcription factors
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
Mechanisms of conversion
• Insertional mutagenesis
• Translocation and inversion
• Amplification
• Point mutations
Chromosomal translocation
Myc genes
• Increased myc synthesis drives Max to partner with Myc
• Myc has short half life• Max is stable
Basic HLH leucine
Basic HLH leucine
Myc
Max
Binds DNA Dimerization
Myc-Max Max-Max Mad-Max
Increases trx Represses trx
Oncogenic mutation of myc
• 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
1 2 3
Proto-oncogene
Translocation to Ig locus
Ig promoter and enhancer 2 3
Oncogene
2 3
TranscriptionSplicing
mRNA
Translation
Increased expression of normal myc protein
Translocation resulting in fusion of 2 genes
Alters structure of normal c-abl protein
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
kinase DBl-H P Rho-GAPbcr
SH3 SH2 Kinase Tailabl
SH3 SH2 Kinase Tailkinase Dbl-H PP210
bcr-abl
SH3 SH2 Kinase TailkinaseP185
bcr-abl
YY
Y
Endocrine
Paracrine
Autocrine
Signaling by secreted molecules
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
Receptor activation
Rearrangement
N terminal domain is replaced by a transcription factor that
can associate with itself
Amplification
Too much protein
Amplification
Increased density induces dimer formation, autophosphorylation -thus constitutively active
Point mutation
Constitutively active
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)
Ras GDP
GEF
GDP
GTP
Ras GTP
GAP
P
Inactive state
Active state
GTPase
Regulation of Ras
•Cycle is unidirectional•GTP bound is active form•Regulated by 2 classes of proteins
• + regulator• -regulator
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
INACTIVE ACTIVE
Ras-GDP Ras-GTP
GEF
GAP
P120 gapNeurofibromin
Gap 1m
mSos1mSos2
Ras-GRF
Interactionwith target
proteins
Mutant RasX Constitutively active
Point mutation of PTK
Induces dimerization in absence of ligand
Adenylyl cyclase
ATPcAMP
G protein ActivatedG protein
Hormone
Receptor
•G protein associated with inner surface of PM•Hormone bound receptor interacts with G protein•Stimulates release of GDP and exchange for GTP•G dissociates from complex•Stimulates production of cAMP by adenylyl cyclase•cAMP is a second messenger
GDP
Inactive state
Hormone binding induces interaction of receptor with G protein
Stimulates the exchange of GTP for GDP
GDP
GTP
GDP
GTP
Target proteins
GTP hydrolysis
Regulation of G proteins
Oncogenic mutations
• Locking subunit in an active state
• Pituitary tumors– Gsp – encodes a mutated subunit that
blocks GTPase activity– Constitutive production of cAMP
• Thyroid tumors– Thyroid receptor mutated– Constitutive production of cAMP
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
Mortality for Leading Causes of Death, United States - 1978
Cause of deathNumber 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 1.1
All others 248543 12.9
Source: Vital statistics of the United States, 1978Modified from: CA – A Journal for Clinicians, Vol 33, 1983.
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
Proliferation
Survival
Undifferentiated
Arrest
Apoptosis
Differentiated
Cellular homeostasis
CANCER
(oncogenes) (tumor suppressors)
Activation of oncogenes
Inhibition of tumor suppressors
Disruption of homeostasis
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
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
Tumor suppressors
• Normally function to suppress the formation of cancer– Growth arrest– Apoptosis– DNA repair– Differentiation– Anti-angiogenesis
Tumor suppressors are recessive – require mutation of both alleles
Oncogenes are dominant – mutation of 1 allele is sufficient
Oncogenes
Normal genes (regulate cell
growth)
1st mutation(leads to
accelerated cell division)
1 mutation is sufficient for a role in cancer development.
Tumor Suppressor Genes
Normal genes Normal genes (prevent (prevent cancer)cancer)
1st mutation1st mutation(susceptible carrier)(susceptible carrier)
2nd mutation or 2nd mutation or loss (leads to loss (leads to
cancer)cancer)
2 mutations are necessary for a role in cancer development.
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 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
Comparison of Proto-oncogenes and tumor suppressors
Definitions
• Tumor – abnormal growth– Benign– Remain localized to tissue where they
originated
• Cancer – malignant– Invades surrounding tissue– Has the ability to metastasize
Primary neoplasm neovascularization Invasion
Transport
Arrest in organAdherenceextravasation
Establishment of microenvironment
Angiogenesisand
proliferation
metastases
Angiogenesis
Definition: Process where tumor cells encourage the in-growth of capillaries and vessels from adjacent normal tissue
Tumor releases angiogenic factors
Endothelial cells proliferateTumor grows
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
Immortalized/established cell line
• Anchorage dependence
• Growth factor dependent
• Contact inhibition
• Cytoskeletal organization
• Monolayer
Transformed cell
• Unregulated growth properties
• Serum independence
• Anchorage independent
• No contact inhibition (form foci)
• May induce tumors in vivo
Focus Forming Assay
Lacks contact inhibition
Tumorigenesis is aMultistep Pathway
• Mutation of proto-oncogenes and tumor suppressor genes
• Special combination - yes
• Particular order - yes
Evidence for multistep cancer pathogenesis
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
Oncogene Cooperation
myc
ras
Myc + ras
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
Multistep tumorigenesis
• Initiation– 1st mutation– Increased proliferation of a single cell
• Progression– Additional mutations– Selection for more aggressive cells
Clonal selection!
Initiation
Progression
Aggressive, rapidly growing tumor
With increase in histopathological abnormalities, there is an increase in the number of mutations at defined genetic loci
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
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
Activated oncogenes from DNA transfection
3T3 cells
Human bladder tumorcell line
Isolate DNA
Transformedcells
Isolate DNA>99% mouse
Isolate human DNAAlu 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
Oncogenes by locationSecreted
Transmembrane
Cytoplasmic
Nuclear
c-siswnt1int1
c-erbBneukitmas
Membrane associated
gspgiprassrc
ablfpsrafmosVavAKT
mycmybfosjunrelerbA
Oncogenes by function
• Growth factors
• Growth factor receptors
• G proteins
• Intracellular kinases
• Transcription factors
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
Mechanisms of conversion
• Induced by virus
• Transduction
• Insertional mutagenesis
• May or may not be virus induced
• Chromosomal translocation and inversion
• Amplification
• Point mutations
Chromosomal translocation
Myc genes
• Increased myc synthesis drives Max to partner with Myc
• Myc has short half life• Max is stable
Basic HLH leucine
Basic HLH leucine
Myc
Max
Binds DNA Dimerization
Myc-Max Max-Max Mad-Max
Increases trx Represses trx
Oncogenic mutation of myc
• 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
1 2 3
Proto-oncogene
Translocation to Ig locus
Ig promoter and enhancer 2 3
Oncogene
2 3
TranscriptionSplicing
mRNA
Translation
Increased expression of normal myc protein
Translocation resulting in fusion of 2 genes
Alters structure of normal c-abl protein
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
kinase DBl-H P Rho-GAPbcr
SH3 SH2 Kinase Tailabl
SH3 SH2 Kinase Tailkinase Dbl-H PP210
bcr-abl
SH3 SH2 Kinase TailkinaseP185
bcr-abl
YY
Y
Endocrine
Paracrine
Autocrine
Signaling by secreted molecules
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
Receptor activation
Rearrangement
N terminal domain is replaced by a transcription factor that can associate with itself
Amplification
Too much protein
Amplification
Increased density induces dimer formation, autophosphorylation thus constitutively active
Growth factor
Growth factor receptor
EGF TGF
EGFR TGFR
cyclinD1/cdk4
p27
Rb-E2F1 Rb E2F1
proliferation
S phase genes
P
cyclinE/cdk2
+
CyclinD1 – amplification associated with cancer
Point mutation
Constitutively active
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)
Ras GDP
GEF
GDP
GTP
Ras GTP
GAP
P
Inactive state
Active state
GTPase
Regulation of Ras
•Cycle is unidirectional•GTP bound is active form•Regulated by 2 classes of proteins
• + regulator• -regulator
INACTIVE ACTIVE
Ras-GDP Ras-GTP
GEF
GAP
P120 gapNeurofibromin
Gap 1m
mSos1mSos2
Ras-GRF
Interactionwith target
proteins
Mutant RasX Constitutively active
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
Point mutation of PTK
Induces dimerization in absence of ligand
Adenylyl cyclase
ATPcAMP
G protein ActivatedG protein
Hormone
Receptor
•G protein associated with inner surface of PM•Hormone bound receptor interacts with G protein•Stimulates release of GDP and exchange for GTP•G dissociates from complex•Stimulates production of cAMP by adenylyl cyclase•cAMP is a second messenger
GDP
Inactive state
Hormone binding induces interaction of receptor with G protein
Stimulates the exchange of GTP for GDP
GDP
GTP
GDP
GTP
Target proteins
GTP hydrolysis
Regulation of G proteins
Oncogenic mutations
• Locking subunit in an active state
• Pituitary tumors– Gsp – encodes a mutated subunit that
blocks GTPase activity– Constitutive production of cAMP
• Thyroid tumors– Thyroid receptor mutated– Constitutive production of cAMP
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