Oncogene

and
Tumor
Suppressor Gene
Oncogene
b- B2
er
- r a s N- N-myc
Ki r as
erb-B C-M
yc yc
L-m
 Central Dogma
Gene Genome

Transcription

RNA Transcriptome

Translation

Protein Proteome
 Cellular signal transduction

A process by which signals from outside the cell are transferred inside Regulate by protein phosphorylation.
the cell through cascade of proteins interaction. Involved in all cellular
activities (death, growth, differentiation, etc.)
What is cancer ?
Cancer : · abnormal cell growth in a tissue
· escape from normal control
· invasive, metastases

How does cancer occur ?

· Proliferation Apoptosis
 
Tumor Degenerative disease
Malignancy Autoimmune disease
· Multiple genetic abnormalities
· Multi step
 Cells premalignant

PROMOTION PROMOTION

Cells still Expansion of

have normal premalignant
phenotype clone
Multistage
INITIATION Progression to PROGRESSION

Malignancy
Cells
malignant

Further genetic
changes Expansion of
malignant clone

METASTASES
 Multiple Steps of Carcinogenesis

initiator promoter

CANCER

CANCER

NO CANCER

NO CANCER

CANCER
time
What is the cause of cancer ?
External factors Internal factors

 Chemicals  Repair failure

 Radiations  Error
 Ultraviolet rearrangement
 Carcinogens

PERSISTENT GENE ABNORMALITY

· In the cell nucleus : > 100 protooncogenes

many tumor suppressor genes
· Identified in different chromosomes
(chromosome 1-22, X and Y)
Viruses and Cancer

Viruses Associated with Human Cancers

Virus Associated tumors

DNA viruses
Epstein-Barr Burkitt’s lymphoma
Nasopharyngeal cancer
Hepatitis B Liver cancer
Papilloma virus Benign warts
Cervical cancer
RNA viruses
Human immunodeficiency virus (HIV-1) Kaposi’s sarcoma
Human T-cell leukemia virus Type I Adult T-cell leukemia
(HTLV-1)
HTLV-2 Hairy cell leukemia
HTLV-5 Cutaneous T-cell leukemia
Viruses and Cancer

Retroviral Oncogenes
Oncogene Virus Tumor

v-ABL Abelson leukemia virus Leukemia

v-ERBA Avian erythroblastosis virus Helps v-ERBB
v-ERBB Avian erythroblastosis virus Erythroleukemia
v-FMS Feline sarcoma virus Sarcoma
v-HRAS Rat sarcoma virus (Harvey strain) Sarcoma
v-KRAS Rat sarcoma virus (Kirsten strain) Sarcoma
v-JUN Avian sarcoma virus Fibrosarcoma
v-MYB Avian myeloblastosis virus Myeloblastosis
v-MYC Avian myelocytomatosis virus Leukemia
v-SIS Simian sarcoma virus Sarcoma
v-SRC Rous sarcoma virus Sarcoma
 Nutritionally Linked Cancers
Carcinogen Promoter
Site Inhibitors
From From Mechanism

Esophagus Pickled, salted ? Yellow-green

foods vegetables
Stomach Pickled, smoked Salt Atrophic Yellow-green
foods, nitrate gastritis vegetables
Liver Mycotoxins, Hepatitis Cytotoxicity ?
nitrosamines antigen
Senecio alkaloids, Alcohol Cytotoxicity
hepatitis antigen
Colon Fried foods Fats Bile acids Bran fiber,
calcium ion,
vegetables
Breast Fried foods Fats Hormonal Same as
imbalances above
Prostate Fried foods? Fats Hormonal ?
imbalances
Endometrium ? Fats Obesity, ?
and ovary estrogen
 Irreversibility or Reversibility of Carcinogenesis
as a Function of Action Mechanism

Type of Type and Mode of

Reversibility Evidence
Cancer Action of Carcinogen

Glandular, Genotoxic from salted Poor Migrant studies :

Stomach pickled foods maintenance of
risk in migrants

Lung Small amounts of Good People who stop

genotoxic carcinogens, smoking have
polycyclic aromatic progressively
hydrocarbons and lesser risk
nicotine derived
nitrosamines, large
amounts of phenols,
catechols, terpenes,
epigenetic cytotoxic
enhancing agents
Oncogenes in Human Tumors

Tumor Associated oncogene

Bladder HRAS, KRAS

Brain ERBB1, SIS
Breast ERBB2, HRAS, MYC
Cervical MYC
Colorectal HRAS, KRAS, MYB, MYC
Gastric ERBB1, HST, MYB, MYC, NRAS, YES
Lung ERBB1, HRAS, KRAS, MYC, LMYC, NMYC
Melanoma HRAS
Neuroblastoma NMYC
Ovarian ERBB2, KRAS
Pancreas KRAS, MYC
Prostate MYC
Testicular MYC
Leukemia ABL, MYC, BCR, BCL1, BCL2
 Cellular oncogene
play an important
role in controlling
cell proliferation and
differentiation

PROTO ONCOGENE

TUMOR SUPPRESSOR GENES

Control
growth arrest
and apoptosis
Genes involved in cancer

· Neoplastic transformation involves :

 oncogene
 tumor suppressor genes
 mutator genes
 apoptotic

· Activation of oncogenes :
play a predominant role in the formation of
cancer development

· Inactivation of tumor suppressor genes :

multiple site of loss of heterozygosity are
most prevalent forms of cancer
How does cancer occur ?
External factors Internal factors

Protooncogene Oncogene
Tumor Suppressor Gene Inactive

TUMOR

When ???
1. Deletion / Point Mutation
2. Gene Amplification
3. Chromosome Rearrangement
 Three Ways in Which A Proto-Oncogene Can Be
Converted into An Oncogene

Deletion or
Point Mutation
Gene Amplification Chromosome Rearrangement
in Coding
Sequence

DNA or DNA

RNA RNA

hyperactive normal protein

protein made in greatly
normal amounts overproduced nearby strong fusion to actively
enhancer causes transcribed gene
normal protein to greatly
be overproduced overproduces
fusion protein; or
fusion protein is
hyperactive
 Five types of
proteins encoded by
proto-oncogenes
participate in control
of cell growth:
 Class I: Growth Factors
Class II: Receptors for Growth F
actors and Hormones

Class III: Intracellular Signal Tr

ansducers

Class IV: Nuclear Transcription

Factors

Class V: Cell-Cycle Control Protei

Examples of Cellular Oncogenes
erb-B codes for a receptor for epidermal growth
factor; involved in glioblastoma, a brain cancer, and
breast cancer
erb-B2 also called HER-2 or neu; involved in
breast, ovarian, and salivary gland cancers
Ki-ras codes for a protein that relays a stimulatory
signal; involved in lung, ovarian, colon, and pancreatic
cancers
N-ras involved in leukemias
c-Myc, N-myc, L-myc all genes for transcription
factors that activate growth promoting genes;
involved in leukemias, breast, stomach, and lung
cancers (c-Myc, L-myc); neuroblastoma (N-myc)
Bcl-1 codes for cyclin D1, a component of the cell
cycle clock; involved in breast, head and neck
cancers
Products of Proto-Oncogenes
Growth Factors Growth Factor Receptors

PDGF ERBB1 ERBB2 HER2/NEU FMS ROS

EGF, FGF TRK IGF-1R RET EEK
IGF 1 and 2 BEK MET SEA KIT
TGF and  FGFR1-4 FIG EPH ELK

Signal Transducers

Membrane associated/guanine nucleotide

RAS GSP GIP
binding proteins

Membrane associated/cytoplasmic protein ABL SRC FES

tyrosine kinases FGR SYN LCK

Cytoplasmic protein serine-threonine

RAF MOS COT
kinases
 Nuclear Transcription Factors

MYC FOS JUN MYB SKI EVI1 REL

Roles of Proto-Oncogenes
· regulate gene expression
· regulate cell proliferation and differentiation
· apoptosis
· protein activator

ACTIVE Proliferation, Differentiation, Apoptosis

CELL
INACTIVE Interphase
Function of the Proto-Oncogenes

Cellular Locations of Oncogene Products

CELL
 SIS MEMBRANE
ERBB FMS
ABL
 RAS  MYC
 JUN
 YES  MYB
 SKI
 SRC  POS
NUCLEUS
 ERBA
 MOS
 Activation of the Transmembrane
Tyrosine Kinase Receptors

Recruitment of
Phosphorylation
Ligand
andbinding
Dimerization
SH2 SH3
and activation
domain proteins

Activation of Cytoplasmic tyrosine

phospholipase C kinase phosphorylation
RAS Activation of
signaling phosphatidylinositol 3-kinase
 Signal Transduction from Receptor
to Nucleus Via RAS p21
Growth factor

P P Ras Ras
14-3-3 14-3-3
Grb2 GDP GTP GTP
Sos 1 2 1 2
Active
P120-GAP 14-3-3 3 3 Raf
Neurofibromin 1 2
3
MEK

P13-K
14-3-3 ERK1 ERK1 ?
1 2
3
Rac and Rho
Inactive Raf Transcription
pathway
factors etc.
Nucleus

Morphological DNA
change synthesis
Apoptosis

· Program Cell Death (PCD)

· Genetically controlled unwanted cell
- during morphogenesis
- during proliferation and differentiation
· Differs from necrosis / accidental cell death
- active process
- no surrounding tissue damage or induction of
inflammatory responses
Apoptosis

· Characteristic morphology :
- condensation of nuclear heterochromatin
- cell shrinkage
- loss of positional organization of organellas in
cytoplasm
- ladder phenomen
· Controlled by protooncogenes & tumor suppressor
genes
 Protein Controlling Apoptosis

Promoting Inhibiting

Intrinsic  p53  BCL2/BCLXL

 MYC  A20
 Interleukin-1
converting enzyme
 BAX/BCLXS

Extrinsic  TNF Many, for example :

 TGF  erythropoietin
 PDGF/IGF1
 sex hormones
 -4 RB WT1

Tumor
C
DP
NF-2 p53 NF-1
BRCA2
CA1
BR

Suppressor
Gene
Tumor Suppressor Genes

· Negative regulator of cell cycle & involve in

growth arrest
· Knudson’s “ Two-Hit ” hypothesis (1975) the
development of several human cancer is thought
to involveloss of heterozygosity (LOH) of tumor
suppressor gene
· Rb gene, p53, HNPCC, PAC, DCC
Definition
· genes involved in the control of abnormal cell
proliferation
· genes loss or inactivation malignancy

Evidence
· the supression of malignancy in somatic cell hybrids
· a consistent loss of chromosomal regions
- initially seen in hereditary cancers
- subsequently also shown in sporadic cancers
· suppression of malignancy when malignant cells were
fused with normal diploid cells (dependent on
retention of a specific chromosome)
Suppression of Malignancy by Cell Fusion

Normal cell Loss of specific

chromosome(s)

Nontransformed Transformed cell

hybrid
Transformed cell
Example :
The presence of chromosome 11 from the normal
partner is necessary to maintain the supression of
the malignant phenotype when HeLa cervical
carcinoma cells are fused with normal fibroblasts.

Example :
Reversion of malignant phenotype can also be
demonstrated in the presence of an activated oncogene.

Cell lines carrying Hibrids :

either activated · phenotypically normal
NRAS/HRAS genes · tumorigenic revertants
fusion
· still express the product
Normal fibroblasts of respective oncogene
 Tumor Suppressor Genes in Hereditary
Cancers : The Retinoblastoma Model

The prototype for studies on hereditary cancer is :

RETINOBLASTOMA
· a childhood cancer
· occur in two forms
· affects the retina

Studies of the polymorphic enzyme, esterase D,

which had previously been mapped to 13q14,
supported the evidence for RB1 (retinoblastoma gene)
in this area.
Hereditary retinoblastoma :

· close linkage was demonstrated between esterase D

alleles and retinoblastoma
· suggesting that the two genes had to be close

Non-hereditary retinoblastoma :

· ± 20% of retinoblastomas but not the constitusional

DNA of the patient have an abnormality
· usually absence or a deletion in one copy of
chromosome 13
· reduced levels of esterase D in the tumor
Deletion of 13q14 in Retinoblastomas

Position of
RB1 gene

Chromosome 13 Chromosome 13
deleted for the
region containing
the RB1 gene
Detection :

1. The loss of heterozygosity test

· depends on differences in the lengths of DNA

fragments generated by digestion of genomic DNA
with restriction enzymes

2. Polymerize Chain Reaction

· use microsatellite markers which are distributed

throughout the genome
· more useful than RLFP analysis because of the
greater number of alleles present at any one locus
 RFLP Analysis as a Means of Detecting
Loss of Heterozygosity
Somatic cells Tumor cells
Part of one
Pair of chromosome
chromosomes deleted in
a b a tumor cell

a a No DNA from
DNA b region of deletion
containing allele b
Probe Probe

a a Allele b
RLFPs on
deleted in
Southern Blot b tumor
= restriction enzyme site
Regulation of RB Function
RB function is regulated by phosphorylation in a cell
cycle-specific manner
· hypophosphorylated in the G0/G1 phase of the cycle
· hyperphosphorylated prior to G1/S transition and
throughout S, in at least a two-step process
· dephosphorylated as the cell leaves mitosis and
goes back to G0/G1 phase

The phosphorylation state of the RB protein varies

throughout the cell cycle and correlates with its
capacity to interact with the transcription factors
and with viral oncoproteins.
 Mode of G0
RB
No transcription
E2F DP-1
Action of RB
RB
G1 E2F DP-1
No transcription
Cyclin D1/CDK4-6
Transcription
Cyclin E/CDK2 P P
RB E2F DP-1
S
TTTCGCGC
P
Cyclin A/CDK2 Phosphorylation
During G1, cyclinof RB
P P is maintainedand
D1/CDK4-6 by cyclin
cyclin
RB During G0 and G1 RB is
G2 A/CDK2 until mitosis
E/CDK2 phosphorylate
underphosphorylated
when
RB andit E2F-1
is is
P and is bound to
dephosphorylated the
released to interact
E2F-1
ready transcription
either to re-
with and promote
factor complexedgo with
Dephosphorylation M RB enter G1 or tofrom
transcription
DP-1.
into
genes
the stationary
necessaryphase.
for S
phase.
RB
G0 G1 E2F DP-1
No transcription
How Does RB Control Transcription ?
· RB does not act directly to control transcription
· act by interaction with transcription factor,such as:
E2F family (E2F 1-5)

· a family of transcription factors

· containing a basic helix-loop-helix (bHLH) motif
· interacts specifically with underphosphorylated RB protein in
the same region as that involved in binding viral oncoproteins
· once RB is phosphorylated, E2F-1 is released allowing it to
initiate transcription
· RB mutations result in truncated unstable proteins which leave
E2F-1 free to continually initiate transcription
· interaction of viral oncoproteins with RB also inactivate it,
similiarly leaving E2F-1 free and promoting cell growth
How Does RB Control Transcription ?
· RB can also positively regulate transcription as has
been demonstrated for the TGF2 gene
· RB regulates transcription in a cell type-dependent
manner :
 in specific cell lines such as lung epithelial cell
lines, the TGF1 gene is also activated by RB
 in lines from different origins, the TGF1 gene
is repressed
· RB1 has been found to be mutated in a wide range
of tumors
Hereditary Cancers
1. Wilms’ Tumor
· deletions of chromosome 11 centred around 11p13
· WT1 protein shows a high degree of homology with
the early growth response gene 1 (EGR1), a gene
which is involved in the control of cell proliferation

2. Familial Adenomatous Polyposis (FAP)

· an autosomal dominantly inherited condition
· incidence around 1 in 10.000
· accounts for 1-2% of all colorectal cancer
· characterized by the presence of hundreds to
thousands of polyps throughout the colon and rectum
· the first gene identified as the gene for AFP was
the ‘mutated in colorectal cancer’ (MCC) gene
· APC (adenomatous polyposis coli) gene :
 localized to chromosome 5q21 by family linkage
 8,5 kb consisting of 15 exons and encoding
2834 amino acids
 mutations are found distributed throughout the
gene, which introduced premature stop codons
resulting in the production of a truncated
protein

3. Neurofibromatosis 1
· Von Recklinghausen neurofibromatosis (NF1) is an
autosomal dominant disorder
· incidence of 1 in 1000
· characterized by :
 café au lait spots, multiple neurofibromas
 increased risk of cancers such as
neurofibrosarcomas and phaeochromocytomas
· localized to chromosome 17 by linkage analysis and
also by the cytogenetic observation of translocations
involving 17q11
· 350 kb DNA, produces a transcript of 11-13 kb and
has 59 exons
· the protein product of NF1 is a 2818 amino acid
protein called neurofibromin
· neurofibromin shares sequence homology in the
central region with the mammalian GAP proteins which
play a role in regulating the RAS proto-oncogene
· reduced GAP activity increased levels of active
RAS p21 resulting in abnormal signaling
4. Neurofibromatosis 2
· much less common than NF1
· incidence of 1:35.000 - 1:40.000
· characterized by bilateral vestibular schwannomas
· predisposition to other tumors such as meningiomas,
astrocytomas and spinal schwannomas
· the gene was mapped to chromosome 22q

5. BRCA1 and BRCA2

· familial breast cancer was linked to a gene on
chromosome 17q, called BRCA1
· BRCA1 spans more than 80 kb of DNA and encodes
a 7.8 kb transcript composed of 24 exons
· the protein is an 1863 amino acid RING finger
protein
p53 Gene
· located on the short arm of chromosome 17
· involved in a wide range and number of tumors
· has important role in tumorigenesis
Tumors with deletions of 17p

Adrenal cortical tumors Lung tumors

Bladder cancer Neurofibrosarcomas
Brain tumors Osteosarcomas
Breast cancer Ovarian cancer
Cervical cancer Renal cell cancer
Colorectal tumors Testicular tumors
Hepatocellular cancer
· functions as a cell cycle checkpoint protein by
transactivation of genes which encode proteins
with growth suppressing activities
p53 Gene
· exerts it function during the G1 phase of the cycle
· however p53, like RB1, is not necessary for normal
cell growth and function
· major role of p53 : ‘ the guardian of the genome ‘
 a rapid increase in the level of p53 in response
to DNA damage
 cause arrest of the cell cycle during G1
 this give the cell time to repair its DNA
 if repair is not possible, p53 induces
programmed cell death (apoptosis)
 cells carrying mutations in p53 are able to
resist this process and become the predominant
cells in the tumor
Protein Product of the p53 Gene

· p53 is made up of 11 exons and encodes 2.8 kb

mRNA
· virtually undetectable in normal cells
· has a half-life of around 20 min
· located in the nucleus
· can be detected in the cytoplasm in G1 and also
following DNA synthesis
p53 as a Transcriptional Transactivator

· p53 inhibits cell growth by acting as a sequence-

specific transcription factor, promoting the
transcription of genes which encode proteins with
growth suppressive activity
· the gene which contains the consensus binding
motif, was called WAF1 and encodes the p21
protein
· overexpression of p21 can lead directly to growth
suppression
· WAF1 was shown to be identical to CIP1 gene,
isolated because of its ability to bind to the
cyclin-dependent kinase, CDK2
DNA lesion
Mode of Action of p53
p53 gene

? p53
?
STABILIZATION

WAF1/CIP1 gene

p21 APOPTOSIS

DNA REPAIR
CYC CYC
CDK CDK
STOP
Kinase No kinase GO
G1 S
activity activity
M G2
Inactivation of p53

Products of transforming viruses

(such as SV40 and HPV) interact
with the central domain

Inactivation of p53

Cell proliferation Viral multiplication

· p90 protein, the product of the cellular oncogene

MDM2 (mouse double minute 2), interacts with the
amino terminal end of p53 decreased
transcriptional transactivation
 Interactions and Differences between
Oncogenes and Tumor Suppressor Genes

Involvement of Oncogenes and Tumor

Suppressor Genes in Sporadic Colorectal Cancer

Percentage of adenomas Percentage

Gene Location
Early Intermediate Late of cancers

APC 5q21 60 60 60 60
DCC 18q 13 11 47 70
KRAS 12q 10 50 50 50
p53 17p 6 6 24 75
Model for the Interaction of Oncogenes and Tumor
Suppressor Genes in Colorectal Tumorigenesis

Normal epithelium 5q mutation or loss APC

Hyperproliferative gene
epithelium
DNA hypomethylation
Early adenoma
12p mutation KRAS
Intermediate
adenoma
18q loss DCC?
Late adenoma
17p loss p53
Carcinoma
Other alterations
Metastases
 Sites of possible tumor suppressor genes
in human cancers
Site Cancer Site Cancer

1p Melanoma 8p Colorectal carcinoma

MEN2 (8p23.2-8p22)
Medullary thyroid cancer (8p21.3-8p11.22)
Neuroblastoma Colorectal cancer
1q Breast cancer (8p22-p21.3)
3p Small cell lung cancer Urinary cancer
(3p14-23) (8p21-q11.2)
Lung cancer (3p21-3p22) 9p Head and neck
Cervical carcinoma Lung cancer (9p21-p22)
Renal cell carcinoma Melanoma, glioma, leukemia
Head and neck cancer 9q Bladder cancer
von Hippel-Lindau disease 10q Astrocytoma
Breast cancer MEN2 (10q11.2)
5q Familial adenomatous WT locus (11p13)
polyposis (5q21) 11p Wilms’ tumor
Colorectal carcinoma Rhabdomyosarcoma
 Sites of possible tumor suppressor genes
in human cancers
Site Cancer Site Cancer

11p Breast cancer Osteosarcoma

Hepatoblastoma Astrocytoma
Transitional cell bladder Lung cancer
carcinoma Head and neck cancer
Lung cancer (17p13.1-p11.1)
13q Rb (13q14) 17q NF type 1 (17q11.2)
Retinoblastoma Von Recklinghausen
Osteosarcoma neurofibromatosis
Small cell lung cancer BRAC1
Ductal breast cancer Breast cancer locus
Stomach cancer Ovarian cancer (17q12-21)
Bladder & colon cancer nm23-H1 (17q21)
16q E-cadherin gene (16q22) 18q DCC (18q21.3)
17p p53 (17p13) Colorectal carcinoma
Small cell cancer 22q NF type 2, Meningioma
Colorectal cancer Acoustic neuroma
Breast cancer Pheochromocytoma
 Pathway of signal transduction

PDGF receptor Ras proteins PDGF [ s/s ]

EGF receptor (erbB) [ H-ras ] Src protein EGF
M-CSF receptor (fms) [ N-ras ] kinase [src] M-CSF
[ K-ras ] Growth factors

Growth-factor
GTP-binding Membrane/cyto-
receptors acting
proteins skeleton-associated
via tyrosine-
specific protein- tyrosine-specific [ myc ]
kinase activity protein kinases [ fos ]
[ jun ]
Thyroid hormone
[ fes ] [ raf ]
receptor [ erbA ]

Nuclear protein

Cytoplasmic Steroid-type Serine/threonine-

tyrosine-specific growth-factor specific protein
protein kinases receptors kinases