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
Number of deaths Percent of total
Cause of death
Heart disease 700,142 29.0
Cancer 553,768 22.9
Cerebrovascular 163,538 6.8
disease
Lung disease 123,013 5.1
Accidents 101,537 4,2
Diabetes mellitus 71,372 3.0
Pneumonia and 62,034 2.6
influenza
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
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
700
600
500
Rate per 100,000
400
1950
2001
300
200
100
0
Heart diseases Cerebrovascular Pneumonia/influenza Cance
Cancer
diseases
2004 Estimated US Cancer Causes
Male Female
Prostate 33% Breast 32%
Lung 13% Lung 12%
Colon 11% Colon 10%
Bladder 6% Uterus 6%
Melanoma 4% Ovary 4%
Non-Hodgkin 4% Non-Hodgkin 4%
Lymphoma Lymphoma
Kidney 3% Melanoma 4%
Oral cavity 3% Thyroid 3%
Leukemia 3% Pancreas 2%
Pancreas 2% Bladder 2%
All other 18% All other 20%
2004 Estimated US Cancer Deaths
Male Female
Lung 32% Lung 32%
Prostate 10% Breast 10%
Colon 10% Colon 10%
Pancreas 5% Ovary 5%
Leukemia 5% Pancreas 5%
Non-Hodgkin 4% Leukemia 4%
Lymphoma Non-Hodgkin 4%
Esophagus 4% Lymphoma
Liver 3% Uterus 3%
Bladder 3% Multiple myeloma 3%
Kidney 3% Brain 3%
All other 21% All other 21%
Comparison between men and women of different ethnicities
400
350
300
250
Rate per 100,000
Men
200
Women
150
100
50
0
White African American Asian American Indian Hispanic
Introduction to cancer
Cellular homeostasis
Proliferation Arrest
Survival Apoptosis
Undifferentiated Differentiated
(oncogenes) (tumor suppressors)
overactive inactive
CANCER
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
(prevent
cancer)
1st mutation
(susceptible carrier)
2nd mutation or
loss (leads to
cancer)
2 mutations are necessary for a role in cancer development.
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 frequent Rare (1 example)
mutant allele
Somatic mutations yes yes
Function of mutant allele Loss of function (recessive Gain of function
allele) (dominant allele)
Effects on cell growth Inhibit cell growth Promote cell growth
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 a
Multistep Pathway
• Mutation of proto-oncogenes and tumor
suppressor genes
• Special combination -Yes
• Particular order -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
Human bladder tumor Isolate human DNA
cell line Alu probe (Alu PCR)
Isolate DNA
Result: A single human gene is
transfect responsible for transforming
capability
3T3 cells Transformed
cells sequence
Result: Human Ras
Isolate DNA Compare to normal gene
>99% mouse
Result: Ras activation is due to a
single point mutation
Oncogenes by location
c-sis
Secreted
wnt1
int1
c-erbB Transmembrane
gsp neu
gip kit
Membrane associated
ras mas
src
abl
fps
Cytoplasmic raf
mos
Vav
myc
AKT
myb
fos
Nuclear jun
rel
erbA
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
Basic HLH leucine
• Increased myc
Myc
synthesis drives Max
Max Basic HLH leucine to partner with Myc
• Myc has short half life
Binds DNA Dimerization
• Max is stable
Myc-Max Max-Max Mad-Max
Increases trx Represses trx
Oncogenic mutation of myc
1 2 3 • Chromosome
Proto-oncogene translocation puts
myc under control of
Translocation to
Ig locus strong promoter and
enhancer
2 3
Ig promoter and enhancer
• Increases the
Oncogene concentration of Myc-
Transcription Max heterodimers
Splicing
thus increasing cell
mRNA 2 3 proliferation
Translation • Burkitt’s lymphoma
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 bcr kinase DBl-H P Rho-GAP
kinase
• Bcr promotes
abl
oligomerization SH3 SH2 Kinase Tail
• Bcr-abl fusion promotes
activation of abl by P210
kinase Dbl-H P SH3 SH2 Kinase Tail
oligomerization induced bcr-abl
autophosphorylation
• Philadelphia chromosome P185 kinase SH3 SH2 Kinase Tail
– translocation of chr 9 bcr-abl
and 22
Signaling by secreted molecules
Endocrine
Paracrine
Y
Autocrine
Y
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)
Regulation of Ras
Inactive state
GTP
Ras GDP
P
GEF
GTPase
GDP
GAP
•Cycle is unidirectional
•GTP bound is active form Ras GTP
•Regulated by 2 classes of proteins
• + regulator Active state
• -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
mSos1
mSos2
Ras-GRF
INACTIVE GEF ACTIVE
Interaction
Ras-GDP Ras-GTP with target
GAP proteins
P120 gap
Neurofibromin
Gap 1m
X
Constitutively
Mutant Ras active
Point mutation of PTK
Induces dimerization in absence of ligand
Hormone
Adenylyl cyclase
Receptor
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
•G dissociates from complex
•Stimulates production of cAMP by adenylyl cyclase
•cAMP is a second messenger
Regulation of G proteins
Inactive state
GDP
GTP hydrolysis
Hormone binding induces
interaction of receptor
with G protein
GTP
GDP
GTP
Stimulates the
Target proteins GDP
exchange of
GTP for GDP
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
Number of deaths Percent of total
Cause of death
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, 1978
Modified 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
Cellular homeostasis
Proliferation Arrest
Survival Apoptosis
Undifferentiated Differentiated
(oncogenes) (tumor suppressors)
CANCER
Disruption of homeostasis
Activation of oncogenes
Inhibition of tumor suppressors
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
(prevent
cancer)
1st mutation
(susceptible carrier)
2nd mutation or
loss (leads to
cancer)
2 mutations are necessary for a role in cancer development.
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 frequent Rare (1 example)
mutant allele
Somatic mutations yes yes
Function of mutant allele Loss of function (recessive Gain of function
allele) (dominant allele)
Effects on cell growth Inhibit cell growth Promote cell growth
Definitions
• Tumor – abnormal growth
– Benign
– Remain localized to tissue where they
originated
• Cancer – malignant
– Invades surrounding tissue
– Has the ability to metastasize
Transport
Primary neoplasm neovascularization Invasion
Arrest in organ
extravasation Adherence
Angiogenesis
and
proliferation
Establishment of
microenvironment metastases
Angiogenesis
Definition: Process where tumor cells encourage the in-
growth of capillaries and vessels from adjacent normal
tissue
Tumor releases Endothelial cells proliferate
angiogenic factors Tumor 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 a
Multistep 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
Human bladder tumor Isolate human DNA
cell line Alu probe (Alu PCR)
Isolate DNA
Result: A single human gene is
transfect responsible for transforming
capability
3T3 cells Transformed
cells sequence
Result: Human Ras
Isolate DNA Compare to normal gene
>99% mouse
Result: Ras activation is due to a
single point mutation
Oncogenes by location
c-sis
Secreted
wnt1
int1
c-erbB Transmembrane
gsp neu
gip kit
Membrane associated
ras mas
src
abl
fps
Cytoplasmic raf
mos
Vav
myc
AKT
myb
fos
Nuclear jun
rel
erbA
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
Basic HLH leucine
• Increased myc
Myc
synthesis drives Max
Max Basic HLH leucine to partner with Myc
• Myc has short half life
Binds DNA Dimerization
• Max is stable
Myc-Max Max-Max Mad-Max
Increases trx Represses trx
Oncogenic mutation of myc
1 2 3 • Chromosome
Proto-oncogene translocation puts
myc under control of
Translocation to
Ig locus strong promoter and
enhancer
2 3
Ig promoter and enhancer
• Increases the
Oncogene concentration of Myc-
Transcription Max heterodimers
Splicing
thus increasing cell
mRNA 2 3 proliferation
Translation • Burkitt’s lymphoma
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 bcr kinase DBl-H P Rho-GAP
kinase
• Bcr promotes
abl
oligomerization SH3 SH2 Kinase Tail
• Bcr-abl fusion promotes
activation of abl by P210
kinase Dbl-H P SH3 SH2 Kinase Tail
oligomerization induced bcr-abl
autophosphorylation
• Philadelphia chromosome P185 kinase SH3 SH2 Kinase Tail
– translocation of chr 9 bcr-abl
and 22
Signaling by secreted molecules
Endocrine
Paracrine
Y
Autocrine
Y
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 EGF TGF
Growth factor receptor EGFR TGFR
p27
cyclinD1/cdk4 cyclinE/cdk2
P
Rb-E2F1 Rb + E2F1
CyclinD1 – amplification S phase genes
associated with cancer
proliferation
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)
Regulation of Ras
Inactive state
GTP
Ras GDP
P
GEF
GTPase
GDP
GAP
•Cycle is unidirectional
•GTP bound is active form Ras GTP
•Regulated by 2 classes of proteins
• + regulator Active state
• -regulator
mSos1
mSos2
Ras-GRF
INACTIVE GEF ACTIVE
Interaction
Ras-GDP Ras-GTP with target
GAP proteins
P120 gap
Neurofibromin
Gap 1m
X
Constitutively
Mutant Ras 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
Hormone
Adenylyl cyclase
Receptor
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
•G dissociates from complex
•Stimulates production of cAMP by adenylyl cyclase
•cAMP is a second messenger
Regulation of G proteins
Inactive state
GDP
GTP hydrolysis
Hormone binding induces
interaction of receptor
with G protein
GTP
GDP
GTP
Stimulates the
Target proteins GDP
exchange of
GTP for GDP
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
