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
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
Cause of death
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
Rate per 100,000
500
400
1950
2001
300
200
100
0
Heart diseases
Cerebrovascular
diseases
Pneumonia/influenza
Cance
Cancer
2004 Estimated US Cancer Causes
Female
Male
Prostate
Lung
Colon
33%
13%
11%
Bladder
Melanoma
Non-Hodgkin
Lymphoma
Kidney
Oral cavity
Leukemia
6%
4%
4%
Pancreas
All other
2%
18%
3%
3%
3%
Breast
Lung
32%
12%
Colon
Uterus
Ovary
Non-Hodgkin
Lymphoma
Melanoma
Thyroid
Pancreas
10%
6%
4%
4%
Bladder
2%
All other
20%
4%
3%
2%
2004 Estimated US Cancer Deaths
Female
Male
Lung
Prostate
32%
10%
Lung
Breast
32%
10%
Colon
Pancreas
Leukemia
Non-Hodgkin
Lymphoma
Esophagus
Liver
10%
5%
5%
4%
Colon
Ovary
Pancreas
Leukemia
10%
5%
5%
4%
4%
4%
3%
Non-Hodgkin
Lymphoma
Uterus
Bladder
Kidney
All other
3%
3%
21%
Multiple myeloma
Brain
All other
3%
3%
21%
3%
Comparison between men and women of different ethnicities
400
350
300
Rate per 100,000
250
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
(tumor suppressors)
(oncogenes)
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
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
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
cell line
Isolate human DNA
Alu probe (Alu PCR)
Isolate DNA
Result: A single human gene is
responsible for transforming
capability
transfect
3T3 cells
Transformed
cells
sequence
Result: Human Ras
Isolate DNA
>99% mouse
Compare to normal gene
Result: Ras activation is due to a
single point mutation
Oncogenes by location
Secreted
Membrane
associated
c-sis
wnt1
int1
gsp
gip
ras
src
Cytoplasmic
Nuclear
c-erbB
neu
kit
mas
myc
myb
fos
jun
rel
erbA
abl
fps
raf
mos
Vav
AKT
Transmembrane
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
Myc
Max
Basic
HLH
leucine
Basic
HLH
leucine
Binds DNA
Dimerization
Myc-Max
Increases trx
Max-Max
• Increased myc
synthesis drives Max
to partner with Myc
• Myc has short half life
• Max is stable
Mad-Max
Represses trx
Oncogenic mutation of myc
1
2
3
Proto-oncogene
Translocation to
Ig locus
2
Ig promoter and enhancer
Oncogene
Transcription
Splicing
mRNA
2
3
Translation
3
• Chromosome
translocation puts
myc under control of
strong promoter and
enhancer
• Increases the
concentration of MycMax heterodimers
thus increasing cell
proliferation
• 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
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
abl
SH3
P210
kinase
bcr-abl
P185 kinase
bcr-abl
SH2
Dbl-H
SH3
P
Rho-GAP
Kinase
Tail
P
SH2
SH3
Kinase
SH2
Tail
Kinase
Tail
Signaling by secreted molecules
Endocrine
Y
Paracrine
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
•Regulated by 2 classes of proteins
• + regulator
• -regulator
Ras GTP
Active state
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
Ras-GDP
ACTIVE
Ras-GTP
Interaction
with target
proteins
Mutant Ras
Constitutively
active
GAP
P120 gap
Neurofibromin
Gap 1m
X
Point mutation of PTK
Induces dimerization in absence of ligand
Hormone
Adenylyl cyclase
Receptor
bg
a
G protein
a
a
Activated
G protein
ATP
•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
cAMP
Regulation of G proteins
Inactive state
bg
GTP hydrolysis
bg
a
a
GDP
Hormone binding induces
interaction of receptor
with G protein
GTP
GDP
GTP
Target proteins
Stimulates the
exchange of
GTP for GDP
bg
a
GDP
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
– 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
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
Cause of death
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
(tumor suppressors)
(oncogenes)
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
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
Definitions
• Tumor – abnormal growth
– Benign
– Remain localized to tissue where they
originated
• Cancer – malignant
– Invades surrounding tissue
– Has the ability to metastasize
neovascularization
Invasion
Arrest in organ
extravasation
Adherence
Angiogenesis
and
proliferation
Establishment of
microenvironment
metastases
Transport
Primary neoplasm
Angiogenesis
Definition: Process where tumor cells encourage the ingrowth of capillaries and vessels from adjacent normal
tissue
Tumor releases
angiogenic factors
Endothelial cells proliferate
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
cell line
Isolate human DNA
Alu probe (Alu PCR)
Isolate DNA
Result: A single human gene is
responsible for transforming
capability
transfect
3T3 cells
Transformed
cells
sequence
Result: Human Ras
Isolate DNA
>99% mouse
Compare to normal gene
Result: Ras activation is due to a
single point mutation
Oncogenes by location
Secreted
Membrane
associated
c-sis
wnt1
int1
gsp
gip
ras
src
Cytoplasmic
Nuclear
c-erbB
neu
kit
mas
myc
myb
fos
jun
rel
erbA
abl
fps
raf
mos
Vav
AKT
Transmembrane
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
Myc
Max
Basic
HLH
leucine
Basic
HLH
leucine
Binds DNA
Dimerization
Myc-Max
Increases trx
Max-Max
• Increased myc
synthesis drives Max
to partner with Myc
• Myc has short half life
• Max is stable
Mad-Max
Represses trx
Oncogenic mutation of myc
1
2
3
Proto-oncogene
Translocation to
Ig locus
2
Ig promoter and enhancer
Oncogene
Transcription
Splicing
mRNA
2
3
Translation
3
• Chromosome
translocation puts
myc under control of
strong promoter and
enhancer
• Increases the
concentration of MycMax heterodimers
thus increasing cell
proliferation
• 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
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
abl
SH3
P210
kinase
bcr-abl
P185 kinase
bcr-abl
SH2
Dbl-H
SH3
P
Rho-GAP
Kinase
Tail
P
SH2
SH3
Kinase
SH2
Tail
Kinase
Tail
Signaling by secreted molecules
Endocrine
Y
Paracrine
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
Growth factor receptor
EGF
TGFb
EGFR
TGFR
p27
cyclinD1/cdk4
Rb-E2F1
CyclinD1 – amplification
associated with cancer
cyclinE/cdk2
P
Rb
+
E2F1
S phase genes
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
•Regulated by 2 classes of proteins
• + regulator
• -regulator
Ras GTP
Active state
mSos1
mSos2
Ras-GRF
INACTIVE
GEF
Ras-GDP
ACTIVE
Ras-GTP
Interaction
with target
proteins
Mutant Ras
Constitutively
active
GAP
P120 gap
Neurofibromin
Gap 1m
X
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
bg
a
G protein
a
a
Activated
G protein
ATP
•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
cAMP
Regulation of G proteins
Inactive state
bg
GTP hydrolysis
bg
a
a
GDP
Hormone binding induces
interaction of receptor
with G protein
GTP
GDP
GTP
Target proteins
Stimulates the
exchange of
GTP for GDP
bg
a
GDP
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
– 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