Transcript Molecular-Pathology2010

Oncogenes and
tumour suppressor
genes
Prof Orla Sheils,
Department of Histopathology.
2nd year Pathology 2010
Introduction
 cell division process is dependent on a
tightly controlled sequence of events.
 dependent on the proper levels of
transcription and translation of certain genes.
 When this process does not occur properly,
unregulated cell growth may be the end
result.
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Mutation
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Introduction
 Of the 30,000 or so genes that are currently
thought to exist in the human genome, there is
a small subset that seems to be particularly
important in the prevention, development, and
progression of cancer.
 These genes have been found to be either
malfunctioning or non-functioning in many
different kinds of cancer.
 The genes that have been identified to date
have been categorized into two broad
categories, depending on their normal functions
in the cell.
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 Genes whose protein products stimulate
or enhance the division and viability of
cells. This first category also includes
genes that contribute to tumour growth
by inhibiting cell death.
 Genes whose protein products can
directly or indirectly prevent cell division
or lead to cell death.
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 The normal versions of genes in the first
group (whose protein products stimulate or enhance the division
and viability of cells )are called proto-oncogenes.
 The mutated or otherwise damaged
versions of these genes are called
oncogenes.
 The genes in the second group (whose protein
products can directly or indirectly prevent cell division or lead to cell
death
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) are called tumour suppressors.
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Definition and discovery
 Cancer is caused by an accumulation of genetic
alterations that confer a survival advantage to
the neoplastic cell.
 Genetic Changes affect multiple facets:
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Cell proliferation
Apoptosis
Tissue invasiveness
Production of growth and angiogenic factors
Ability to escape immune surveillance
Genetic Basis for Cancer
 Reflected in the clonal nature of neoplastic cells
 Polyclonal growth/ hyperplasia
 Response to an
 Extrinsic growth factor or
 Internal genetic mutation shared by
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All cells - MEN2a –germline ret mutation
Some cells – McCune Albright, postzygotic somatic
Gsa mutation.
 Hyperplastic cells may subsequently acquire
one or more somatic mutations and develop
clonal derivatives.
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Genetic Basis for Cancer
 Reflected in the clonal nature of
neoplastic cells
 Monoclonal growth reflects the
acquisition of somatic mutations that
confer survival advantage.
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 tumour suppressors function in many
key cellular processes including the
regulation of transcription, DNA repair
and cell:cell communication.
 The loss of function of these genes leads
to abnormal cellular behavior.
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Oncogenes
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Cancer – a genetic disease
 Intensive effort to characterise genetic alterations
associated with different forms of cancer
 Landmark advances:
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Recognition that some cancers ‘run in families’
1900s- Rous sarcoma virus (RSV)
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sarcoma transmission in chickens
Retrovirus
Harboured a abnormal variant of a normal cellular gene
src
Viral gene product referred to as an oncogene
 Greek onkos = mass or tumour
Many viral oncogenes correspond to altered versions of
normal cellular genes (proto-oncogenes) [src, ras, raf, kit,
jun, fos…].
DNA tumour viruses
 Important role in current understanding of
neoplasia.
 Viruses produce proteins - target key cellular
regulatory proteins
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Rb, p53
SV40 large T Ag associates with and inactivates Rb.
Adenovirus E1A also targets Rb
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Hereditary Cancers
 Transmitted in autosomal dominant manner.
 Based on age dependent appearance of
retinoblastoma –Knudson postulated a “two-hit”
model for the disease.
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Hit 1 = germline inherited mutation in Allele 1
Hit 2 = somatic event involving remaining normal
allele.
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Tumour Suppressor Genes
 Historically –suspected based on several lines
of evidence:
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Malignant phenotype suppressed by fusion with
normal cells (presence of tumour suppressor in
normal implied).
Chromosomal losses in hybrids caused reversion
to malignant phenotype.
Introduction of single chromosomes into malignant
cells:
 e.g. insertion of chromosome 11( WT-1
gene) could suppress tumourigenicity in
Wilm’s tumour cell line.
tumour Suppressor Genes
 Some genes suppress tumour formation.
 Their protein product inhibits mitosis.
 When mutated, the mutant allele behaves as a
recessive; that is, as long as the cell contains
one normal allele, tumour suppression
continues.
 (Oncogenes, by contrast, behave as dominants;
one mutant, or overly-active, allele can
predispose the cell to tumour formation).
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Example 1: RB - the retinoblastoma gene
 Retinoblastoma is a cancerous tumour of the
retina. It occurs in two forms:
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Familial retinoblastoma
 Multiple tumours in the retinas of both eyes
occurring in the first weeks of infancy.
Sporadic retinoblastoma
 A single tumour appears in one eye sometime in
early childhood before the retina is fully developed
and mitosis in it ceases.
Familial retinoblastoma
 Familial retinoblastoma occurs when the fetus
inherits from one of its parents a chromosome
(number 13) that has its RB locus deleted (or
otherwise mutated). The normal Rb protein
prevents mitosis.
 Mechanism. The Rb protein prevents cells from
entering S phase of the cell cycle. It does this
by binding to a transcription factor called E2F.
 This prevents E2F from binding to the
promoters of such proto-oncogenes as c-myc
and c-fos.
 Transcription of c-myc and c-fos is needed for
mitosis so blocking the transcription factor
needed to turn on these genes prevents cell
division.
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Retinoblastoma
 A random mutation of the
remaining RB locus in
any retinal cell
completely removes the
inhibition provided by the
Rb protein, and the
affected cell grows into a
tumour. So, in this form of
the disease, a germline
mutation plus a somatic
mutation of the second
allele leads to the
disease.
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Example 2: p53
 The product of the tumour suppressor gene p53
is a protein of 53 kilodaltons (hence the name).
 The p53 protein prevents a cell from completing
the cell cycle if
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its DNA is damaged or
the cell has suffered other types of damage.
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 the damage is minor, p53 halts the cell cycle —
hence cell division — until the damage is repaired.
 the damage is major and cannot be repaired, p53
triggers the cell to commit suicide by apoptosis.
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 These functions make p53 a key player in protecting
us against cancer; that is, an important tumour
suppressor gene.
 More than half of all human cancers do, in fact,
harbour p53 mutations and have no functioning p53
protein.
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Loss Of Heterozygosity (LOH)
 Because tumour suppressor genes are
recessive, cells that contain one normal and
one mutated gene — that is, are heterozygous
— still behave normally.
 However, there are several mechanisms which
can cause a cell to lose its normal gene and
thus be predisposed to develop into a tumour.
These may result in a "loss of heterozygosity"
or "LOH".
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Mechanisms of LOH:
1.
Deletion of
the normal allele;
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the chromosome arm containing the normal allele;
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the entire chromosome containing the normal allele
(resulting in aneuploidy).
Loss of the chromosome containing the normal allele
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followed by duplication of the chromosome containing
the mutated allele.
Mitotic recombination. The study of tumour suppressor
genes revealed (for the first time) that crossing over —
with genetic recombination — occasionally occurs in
mitosis (as it always does in meiosis).
In #2 and #3, the resulting cell now carries two copies of the
"bad" gene. This is called "reduction to homozygosity".
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Methylation
 Mutation is not the only way to inactivate
tumour suppressor genes.
 Their function can also be blocked by
methylation of their promoter.
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Mechanism of parental imprinting
 The process of imprinting start in the gametes where the
allele destined to be inactive in the new embryo (either the
father's or the mother's as the case may be) is "marked".
The mark appears to be methylation of the DNA in the
promoter(s) of the gene.
 Methyl groups are added to cytosines (Cs) in the DNA.
 occurs at stretches of alternating Cs and Gs called CpG
islands.
 Methylation of promoters prevents binding of transcription
factors to the promoter thus shutting down expression of the
gene.
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Methylation
 Cancer cells often contain a methylated
promoter on one tumour suppressor gene
accompanied by
 a similarly blocked promoter on the other allele
(producing the same effect as #2 above);
 a loss of that locus on the other chromosome
(like the LOH in #1 above);
 an inactivating mutation in the other allele.
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tumour suppressor genes = anti-oncogenes
 Genes like RB and p53 are also called anti-
oncogenes. They were first given this name
because they reverse, at least in cell culture,
the action of known oncogenes.
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Tumour suppressor genes
 This image (courtesy of Moshe Oren, from
Cell 62:671, 1990)
shows petri dishes
which were seeded with the
same number of cells that had
been transformed by two
oncogenes: myc and ras.
 Many of those on the left have
grown into colonies of cells.
 However, the cells plated on the
right also contained the tumour
suppressor p53 gene. Only a few
have been able to grow into
colonies.
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Human Papillomavirus (HPV)
 The name anti-oncogene may be even more
appropriate than originally thought.
 Both the Rb protein and the p53 protein
complex directly in the cell with an oncogene
product of some human papilloma viruses.
 Once inside the cells of their host, human
papilloma viruses synthesise
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a protein designated E7 and
another designated E6.
Human Papillomavirus
 The E7 protein of one of these binds to the Rb
protein preventing it from binding to the host
transcription factor E2F.
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Result: E2F is now free to bind to the promoters of genes
(like c-myc) that cause the cell to enter the cell cycle .
Thus this version of E7 is an oncogene product.
 The E6 protein of human papilloma virus implicated
in cervical cancer binds the p53 protein targeting it
for destruction by proteasomes and thus removing
the block on the host cell's entering the cell cycle.
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Oncogenes
 Genes associated with the stimulation of cell
division.
 Cancers result from only one mutant allele of
gene.
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Oncogenes
Growth Factors or Receptors for
Growth Factors
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PDGF Platelet Derived Growth Factor
(brain and breast cancer)
erb-B receptor for epidermal growth
factor (brain and breast cancer)
erb-B2 receptor for growth factor (breast,
salivary, and ovarian cancers)
RET growth factor receptor (thyroid
cancer)
Oncogenes
2. Cytoplasmic Relays in Stimulatory
Signaling Pathways
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Ki-ras activated by active growth factor
receptor proteins (lung, ovarian, colon
and pancreatic cancer)
N-ras activated by active growth factor
receptor proteins (leukaemias)
c-src is a protein kinase that becomes
overactive in phosphorylation of target
proteins
Oncogenes
3. Transcription Factors that Activate
Growth Promoting Genes
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c-myc activates transcription of growth
stimulation genes (leukemia, breast,
stomach, and lung cancer)
N-myc (nerve and brain cancer)
L-myc (lung cancer)
c-jun and c-fos function as transcription
factors
Oncogenes
4. Other types of molecules
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Bcl-2 normal protein blocks cell suicide
(lymphoma)
Bcl-1 codes for cyclin D1, stimulatory
protein of the cell cycle (breast, neck,
head cancers)
MDM2 codes for antagonist of p53
(sarcomas)
RAS
 Ras gene products are involved in kinase
signalling pathways that control the
transcription of genes, which then regulate cell
growth and differentiation.
 To turn "on" the pathway, the ras protein must
bind to a particular molecule (GTP) in the cell.
 To turn the pathway "off," the ras protein must
break up the GTP molecule.
 Alterations in the ras gene can change the ras
protein so that it is no longer able to break up
and release the GTP.
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RAS Pathway
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 These changes can cause the pathway to be stuck in
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the "on" position.
The "on" signal leads to cell growth and proliferation.
ras overexpression and amplification can lead to
continuous cell proliferation, which is a major step in the
development of cancer.
Cell division is regulated by a balance of positive and
negative signals.
When ras transcription is increased, an excess of the
gene's protein is in the cell, and the positive signals for
cell division begin to outweigh the negative signals.
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RAS
 The conversion of ras from a proto-oncogene
into an oncogene usually occurs through a
point mutation in the gene.
 The altered function can affect the cell in
different ways because ras is involved in many
signaling pathways that control cell division and
cell death.
 Anti-cancer drugs are now being developed that
target ras dependent pathways. Much remains
to be discovered before these drugs can be put
into use
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RAS
 Mutant ras has been identified in cancers of
many different origins, including: pancreas
(90%), colon (50%), lung (30%), thyroid (50%),
bladder (6%), ovarian (15%), breast, skin, liver,
kidney, and some leukaemias.
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MYC
 The myc protein acts as a transcription
factor and it controls the expression of
several genes.
 Mutations in the myc gene have been
found in many different cancers,
including Burkitt's lymphoma, B-cell
leukemia, and lung cancer.
 The myc family of oncogenes may
become activated by gene
rearrangement or amplification.
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 Gene rearrangements involve the breakage and
re-sealing of chromosomes.
 This process can involve large amounts of DNA
and can affect many genes.
 The movement of a gene or group of genes to a
different location within the same chromosome
or to a different chromosome often leads to
altered gene expression and cell function.
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SRC
 The Src protein is a tyrosine kinase.
 Kinases are enzymes that transfer phosphate
groups onto target molecules.
 The important aspect of this process is that the
removal/addition of phosphates changes
biomolecules and is a key way by which the
activities of cells are regulated.
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SRC
 The phosphate addition/removal process acts
like an on/off switch to control the activity of the
target molecules.
 The src proteins alter several target molecules,
resulting in the transmission of signals to the
nucleus that help regulate the cell
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Tyrosine Kinases
 MAP kinase (MAPK) signaling is among central
signaling pathways that regulate cell
proliferation, cell differentiation and apoptosis.
 As MAPK should transmit extracellular signals
to proper regions or compartments in cells,
controlling subcellular localisation of MAPK is
important for regulating fidelity and specificity of
MAPK signaling.
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Tyrosine Kinases
 The ERK1/2-type of MAPK is the best
characterized member of the MAPK family. In
response to extracellular stimulus, ERK1/2
translocates from the cytoplasm to the nucleus
by passing through the nuclear pore by several
independent mechanisms.
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Tyrosine Kinases
 The MAP kinase (MAPK) pathway is a highly
conserved pathway involved in diverse cellular
functions, including cell proliferation, cell
differentiation and apoptosis.
 A wide variety of extracellular stimuli, such as
growth factors and environmental stresses,
induce sequential phosphorylation and
activation of three protein kinases, MAP kinase
kinase kinase (MAPKKK), MAP kinase kinase
(MAPKK) and MAPK.
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Tyrosine Kinases
 MAPK is a serine/threonine kinase activated by
MAPKK via phosphorylation on both threonine
and tyrosine residues in the TXY sequence
 The MAPK family consists of four members,
ERK1/2 (also known as classical MAPK),
JNK/SAPK, p38 and ERK5/BMK1.
 Each molecule is activated by distinct pathways
and transmits signals either independently or
co-ordinately
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Tyrosine Kinases
 MAPK plays an important role in
transmitting the signals from receptors
on cell membrane to cytoplasmic targets
such as cytoskeleton and downstream
kinases and nuclear targets such as
transcription factors.
 Thus, regulation of the subcellular
localisation of MAPK is important for
controlling MAPK signaling.
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TSG
 tumour Suppressor Genes
 Genes associated with inhibition of cell
division.
 Cancers require both alleles of the gene
to be altered.
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TSG
Cytoplasmic Proteins
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APC (colon and stomach cancers)
DPC4 codes for relay molecule in cell
division inhibitory pathway (pancreatic
cancer)
NF-1 codes for protein that inhibits a
stimulatory (Ras) protein (brain, nerve,
and leukemia)
NF-2 (brain and nerve cancers)
TSG
2.
Nuclear Proteins
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MTS1 codes for p16 protein, brake on cell cycle
clock (many cancers)
RB codes for pRB protein, master brake on cell
cycle (retinoblastoma, bone, bladder, lung, and
breast cancer)
p53 codes for p53 protein, halts cell cycle in G1 and
induces cell suicide (many cancers)
p16 inhibits cyclin D-dependent kinase activity
WT1 (Wilms tumour of the kidney)
BRCA1 functions in repair of damage to DNA
(breast and ovarian cancers)
BRCA2 functions in repair of damage to DNA
(breast cancer)
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