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