Download International School of Advanced BioMedicine

Catania [ Acitrezza ] - Italy 2007
The International School of Advanced BioMedicine and
Bioinformatics
Oncogenes and Tumor Suppressor Genes
Pietro BUFFA
Department of Biomedical Science
University of Catania
OVERVIEW
OVERVIEW
ONCOGENES & PROTO-ONCOGENES
History
Fundamental Concepts
Classes of Oncogenes and main activation mechanisms
Examples: ras, abl, myc
TUMOR SUPPRESSOR GENES
Fundamental Concepts
Principal differences between Oncogenes and Tumor Suppressor Genes
Examples: Rb, p53
Final Considerations and Conclusions
Slides are available on www.biocode.it [Download section]
ONCOGENES
-ONCOGENES: History
ONCOGENES and
and PROTO
PROTO-ONCOGENES:
History
1909 - Peyton Rous was the first to describe the existence of a virus which seems to cause a
trasmissible form of tumor in chickens (Fibrosarcoma). The Rous Sarcoma Virus (RSV), a retrovirus
very well studied many years after, could effectively cause trasformation of the infected cells. It was the
first virus “cancer promoting” to be identifyed and for this reason it played a very important role in the
development of the actual knowledge on cancer.
About ‘70 years, the gene responsible for the neoplastic transformation was identifyed in RSV genome:
v-src.
EXPERIMENTAL HYPOTHESIS
Cancer is caused by viral infections which
transfer a cancer promoting viral gene, called
ONCOGENE, in the human DNA
ONCOGENES
-ONCOGENES: History
ONCOGENES and
and PROTO
PROTO-ONCOGENES:
History
Curiously, about the end of ’70 years, the v-src sequence was found, in highly conserved form, even in the chicken genome …
… and in other vertebrates, human comprised.
Why our DNA should contain genes
which cause cancer ?
ONCOGENES
-ONCOGENES: History
ONCOGENES and
and PROTO
PROTO-ONCOGENES:
History
1989 - Michael Bishop and Harold Varmus, on the basis of the previous discovery,
demonstrated that the viral v-src ONCOGENE derives from a normal gene already present in
the vertebrates genome, the c-src PROTO-ONCOGENE, integrated in the virus genome
consequently to a recombination event during its replication in eucariotic cells.
The v-src and the c-src genes, are not perfectly identical, but point mutations exist at the level
of the entire coding sequence, beyond to the deletion of the c-terminal region.
Michael Bishop
Harold Varmus
It is thought that the cellular gene initially
integrated in the RSV genome, underwent
several modifications that transformed it
in oncogene, rendering the protein
codified constitutively active
ONCOGENES
-ONCOGENES: Fundamental
ONCOGENES and
and PROTO
PROTO-ONCOGENES:
Fundamental Concepts
Concepts
CONCLUSION
The discovery of a relationship between the cellular PROTO-ONCOGENE (c-src) and the viral ONCOGENE
(v-src) has represented a fundamental point in the history of the cancer research, demonstrating that
cancer can be induced by modification of normal genes
All genes can promote cancer… Or does it exist a particular
fraction more involved ?
Of approximately 30.000 genes present in the Human genome, only hundreds are involved in the regulation of the delicate balance
between cellular proliferation/death. Therefore it is corrected to think that, only specific events which
which alter the function of
PROTOPROTO-ONCOGENES / TUMOR SUPPRESSOR GENES
[positive and negative regulatory genes of cell proliferation]
proliferation]
PROPRO-APOPTOTIC GENES / ANTIANTI-APOPTOTIC GENES
[positive and negative regulatory genes of the programmed cell death
death]]
are responsible of the origin of numerous and very well known Human
Human tumors
ONCOGENES
-ONCOGENES: Fundamental
ONCOGENES and
and PROTO
PROTO-ONCOGENES:
Fundamental Concepts
Concepts
Today, more than 50 oncogenes has been identifyed in Human tumors
The reserches carryed out to the identification of several oncogenes involved in the neoplastic transformation, were followed
from those undertaken to identify and characterize the structure of the proteins codified
Numerous oncogenic proteins seem to be structurally different respect to those codified by corresponding proto-oncogenes
GAIN OF FUNCTION
CLASSE 1
GROWTH FACTORS
CLASSE 2
GROWTH FACTORS
RECEPTORS
CLASSE 3
CYTOPL. REGULATION
PROTEINS
CLASSE 4
TRANSCRIPTION
FACTORS
Sis (Sarcoma)
Hst (Sarcoma)
EGFR (erb-B)(Carcinoma)
ras(Carcinoma/Leucemia)
myc(Linfoma/Leucemia)
abl (Leucemia)
fos & jun (Sarcoma)
ONCOGENES:
ONCOGENES: Activation
Activation Mechanisms
Mechanisms
ACTIVATION MECHANISMS
STRUCTURAL ALTERATIONS
INCREASED EXPRESSION
1
3
Point mutation Activation
Gene amplification or over-expression Activation
Anomalous gene product
costitutively active
Overproduction of normal gene product
2
Chromosomic translocation Activation
Transfer of one portion of chromosome to an other
Anomalous gene product
costitutively active
ACTIVATION
ACTIVATION FOR
FOR MUTATION:
MUTATION: Oncogene
Oncogene ras
ras
1
ONCOGENE RAS
The ras family genes ( HH-ras,
ras, KK-ras,
ras, NN-ras ) are all functionally
active and codify for proteins belonging to a circuit that,
that, in
response to specific stimuli,
stimuli, mediates the transmission of
messages able to address the cell towards the proliferation
and differenziation.
differenziation.
Adattatore
RAS is a “switch protein” of about 170aa and with a
GTPasic activity
RAS Inactive
RAS Active
ACTIVATION
ACTIVATION FOR
FOR MUTATION:
MUTATION: Oncogene
Oncogene ras
ras
1
ONCOGENE RAS & CANCER
The first oncogene identified in a human tumor [bladder carcinoma]
carcinoma]
The discovery of constitutively active ras proteins in bladder tumor cells
has been the first dimostration of an oncogene involved in a neoplasia.
The 35% of Human tumor present at least one of the mutated ras
genes,
genes, with synthesis of a structurally alterated protein
The most observed mutations occur in critical regions which cause decrement
of ras ability to bind GAP (GTPase Activating Protein) and activate the intrinsic
ras GTPase function [codons: 12, 13 e 61]. The constitutively active protein
primes several metabolic pathways even in absence of apstream specific
signals.
Structurally
alterated RAS
protein
Persistence of the active state of RAS
ACTIVATION
ACTIVATION FOR
FOR CHROMOSOMIC
CHROMOSOMIC TRASLOCATION:
TRASLOCATION: Oncogene
Oncogene abl
abl
2
ONCOGENE ABL & HUMAN CRONIC MYELOID LEUKEMIA [CML]
The 20% of the Leukemias
The CML is the result of the fast (anomalous) proliferation of the myeloid cellular line (granulociti). The disease
has initially slow passing (chronic), with little symptoms, after a variable period of some years the disease
becomes unavoidablly acute leukaemia
In 95% of the cases exists a reciprocal translocation t(9;22), an exchange of genetic material, between
chromosomes 9 and 22 and the presence of this translocation is a highly sensitive test for CML
Cromosoma
Cromosoma
9
9+
Cromosoma
Cromosoma
22
Ph
Midollo osseo normale
BCR
BCR-ABL
ABL
Leucemia Mieloide Cronica
Ph chromosome finds in 95% of the patients
affected from Cronic Myeloid Leukemia
Proteina di fusione
con attività
tirosino-chinasica
leucemogena
ACTIVATION
ACTIVATION FOR
FOR CHROMOSOMIC
CHROMOSOMIC TRASLOCATION:
TRASLOCATION: Oncogene
Oncogene abl
abl
2
P210
BCR-ABL
BCR
ABL
NLS
Oligomerization
Domain
SH3 SH2
YY-Kinase
BreakPoint
NES
DNA
ACTIN
BD
BD
The Ph chromosome codify for a
very big protein called BCR-ABL
which shows one Kinase activity
exalted and refractory to the
normal cellular control.
Natively Unfolded Region
Recentemente, il gruppo di G. S. Furga
ha risolto la struttura tridimensionale del
dominio legante l’actina di ABL
Circa 6 anni fa, il gruppo di John Kuriyan ha
risolto la struttura tridimensionale del dominio
catalitico (SH3-SH2-YK) di ABL, complessato con
una variante di IM
ACTIVATION
-EXPRESSION: Oncogene
ACTIVATION FOR
FOR OVER
OVER-EXPRESSION:
Oncogene myc
myc
3
ONCOGENE MYC & BURKITT LYMPHOMA
The 50% of infantile lymphomas
Burkitt Lynphoma is a tumor of the linfatic system (in particular, B lymphocytes)
Exist in two forms: Endemic (Africa equatoriale) and sporadic
In 75% of the cases exists a reciprocal translocation t(8;14) that insert the oncogene c-myc
(8q24) to the to the place of the gene codifying for the heavy chains of the immunoglobuline
(14q32)
As result of the translocation, c-myc it is found, therefore, under
the transcritional control of elements (promoters, enhancer) that,
in B lymphocytes, are permanently active, with the consequence
that it will codify continuously for an excess of its product.
Linfonodo Normale
Linfonodo Patologico
TUMOR
TUMOR SUPPRESSOR
SUPPRESSOR GENES:
GENES: The
The fusion
fusion of
of one
one hypothesis
hypothesis
THE CANCER IS A GENETIC DISEASE
The tumor cells present one or more alterations in their
PROTO-ONCOGENES that, constitutively activated,
become ONCOGENES
EXPERIMENTAL HYPOTHESIS
NORMAL Cells
TUMOR Cells
’70 years – Cellular fusion experiments
Normal cells prevail on the tumor cells
The healthy cells express genes able to
suppress the transformation potential of
the oncogenes and for this reason
called TUMOR SUPPRESSOR GENES
The biological effect of the Tumor Suppressor Genes is
dominant respect to the oncogenes
TUMOR
TUMOR SUPPRESSOR
SUPPRESSOR GENES
GENES
BIOLOGICAL FUNCTION OF TUMOR SUPPRESSOR GENES
CELL CYCLE
DNA REPAIR
CELL ADESION
REGULATORS
REGULATORS
REGULATORS
IN TUMOR CELLS
THE TUMOR SUPPRESSOR GENES ARE GENERALLY
LOST
REPRESSED
ALTERED
[Chromosom or Gene Deletion]
[Ipermetilazion Events]
[Mutational Events]
Not codified Products
LOSS OF FUNCTION
Product structurally
incapable to carry out its
physiological function
TUMOR
TUMOR SUPPRESSOR
SUPPRESSOR GENES
GENES
GENE
RB
WT1
P53
APC
VHL
P16
BRCA1
BRCA2
...
NEOPLASIA
(Retinoblastoma)
(Tumore di Wilms)
(Osteosarcoma)
(Colon)
(Renal carcinoma)
(Melanoma)
(Mammella e ovaio)
(Mammella e ovaio)
CROMOSOMA
Crom.
Crom.
Crom.
Crom.
Crom.
Crom.
Crom.
Crom.
13q14
11p13
17p13
5q21
3p25
9p21
17q21
13q12
ANNO
(1986)
(1990)
(1990)
(1991)
(1993)
(1994)
(1994)
(1995)
TUMOR
TUMOR SUPPRESSOR
SUPPRESSOR GENES
GENES
GENE
RB
WT1
P53
APC
VHL
P16
BRCA1
BRCA2
...
NEOPLASIA
(Retinoblastoma
)
(Retinoblastoma)
(Tumore di Wilms)
(Osteosarcoma
)
(Osteosarcoma)
(Colon)
(Renal carcinoma)
(Melanoma)
(Mammella e ovaio)
(Mammella e ovaio)
CROMOSOMA
Crom.
Crom.
Crom.
Crom.
Crom.
Crom.
Crom.
Crom.
Crom.
Crom.
13q14
11p13
17p13
5q21
3p25
9p21
17q21
13q12
ANNO
(1986)
(1990)
(1990)
(1991)
(1993)
(1994)
(1994)
(1995)
Two of these genes have been mainly study and the absence
or the inattivation of their products is involved in the
development of various malignant tumors.
TUMOR
Rb)
TUMOR SUPPRESSOR
SUPPRESSOR GENES:
GENES: Retinoblastoma
Retinoblastoma ((Rb)
First human tumor whose origin is
attributable to the inactivation of a
specific tumor suppressor gene
Retinoblastoma is a cancer of the retina. It accounts for about 3% of the
cancers occurring in children younger than 15 years. Retinoblastoma is
usually confined to the eye but can spread to the brain via the optic nerve.
It is necessary that all the two copies (alleli)
of a tumor suppressor gene are lost or
changed, for the development of the disease
Hereditary Rb
Not Hereditary Rb
1/3 Germinal Mutation (Hereditary)
2/3 Not Germinal Mutation (Sporadic)
1971 Alfred Knudson, on the base of the observations about 48 cases of
familiar Retinoblastoma and not, assumed that this rare shape of cancer is
caused from 2 mutational events of the same gene.
1° Evento
Mutazionale
2° Evento
Mutazionale
“THE TWO KNOCK” KNUDSON’S HYPOTHESIS
Hereditary Rb una mutazione è
germinale (ereditata), mentre
l’altra avviene a livello delle
cellule somatiche in un secondo
momento
Not Hereditary Rb
[Sporadic] entrambe le
mutazioni sono
somatiche
TUMOR
Rb)
TUMOR SUPPRESSOR
SUPPRESSOR GENES:
GENES: Retinoblastoma
Retinoblastoma ((Rb)
MOLECULAR ASPECTS
The Rb gene codifies for the nuclear protein p105Rb that represents a critical check point of the cell cycle. Only the iperphosphorilated form, actives the transcription factor E2F, allowing the progression of the cell cycle towards the S phase.
Forma
Ipofosforilata
attiva
Forma
Iperfosforilata
inattiva
Inactivating mutations of Rb
allow the uncontrolled
cellular proliferation
TUMOR
TUMOR SUPPRESSOR
SUPPRESSOR GENES:
GENES: p53
p53
P53 & CANCER
The Tumor Suppressor Gene more involved in human tumors. TP53 is deleted or is mutated in about the 75% of human tumors. It maps in
Human chromosome 17p13 and codes for a multi domain protein p53 [53Kda - 393aa] which exibits sequences very well conserved during the
evolution.
1
Transactivation
Domain
2
DNA
Binding
Domain
Tetramerizzation
Domain
3
4
The p53 expression level is generaly low
In healthy individual, p53 is in balance between its production and degradation. Experimental data demostrate that there is an increase of p53
when the cell undergoes a damage to the DNA, showing an important role in the preservation of the genomic stability.
[Experiments on TP53 knockout rats]
TUMOR
TUMOR SUPPRESSOR
SUPPRESSOR GENES:
GENES: p53
p53
MOLECULAR ASPECTS
A FUNDAMENTAL TUMOR SUPPRESSOR GENE
DNA Damage
Sostanze genotossiche
Processi ossidativi
Radiazioni
SPORADIC MUTATION OF ONE TP53 ALLELE:
TP53 is able to induce neoplastic transformation also as result
of a single allele mutation.
P
ATM
GERMINAL MUTATION OF ONE TP53 ALLELE:
Li-Fraumeni Syndrome is a rareautosomal dominant
hereditary disorder that increases greatly the susceptibility to
cancer.
P53
Inattivo
mdm2
mdm2
P53
1
Attivo
ARRESTO CICLO
CELLULARE
Enzimes recruitment
involved to the DNA
reparation
Transactivation activity
towards genes involved in
the arrest of G1 fase
[Es. gene p21]
2
APOPTOSI
REPAIRED DNA
Restart of the cell
cycle
Start of cell death
mecchanisms
[Es. BAX gene]
GENOMIC STABILITY
FINAL
FINAL CONSIDERATIONS
CONSIDERATIONS
THERE ARE 2 WAYS TO RENDER FASTER ONE MACHINE
1] TO PRESS THE ACCELERATOR Æ ONCOGENES
2] TO ELIMINATE THE BRAKES Æ TUMOR SUPPRESSOR GENES
THE CANCER USES THEM BOTH
FINAL
FINAL CONSIDERATIONS
CONSIDERATIONS
Evolutionary stages of a epithelium tumor:
Colon Carcinoma
THE CANCER IS A COMPLEX GENETIC DISEASE
Cancer develops from the accumulation of lesions
localized on a limited number of genes that determine a
homeostatic imbalance between genes that stimulate cell
proliferation (ONCOGENES) and genes that stop cell
growth (TUMOR SUPPRESSOR GENES)
ACKNOWLEDGMENTS
ACKNOWLEDGMENTS
Director and Scientific Committee
of the
International School of Advanced BioMedicine and Bioinformatics