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%


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%


4%

Esophagus 4%

Liver 3%

Bladder 3%

Kidney 3%

All other 21%

Lung 32%

Breast 10%

Colon 10%

Ovary 5%

Pancreas 5%

Leukemia 4%


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