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Medical Oncology (1998) 15, 20-26
9 1998 Stockton Press All rights reserved 0736-0118/98 $12.00
REVIEW
How do mutated oncogenes and tumor
suppressor genes cause cancer?
Dan Grand6r
Research Laboratory of Radiumhemmet, Karolinska Hospital, Stockholm, Sweden
In recent decades we have been given insight into the process that transforms a normal cell
into a malignant cancer cell. It has been recognised that malignant transformation occurs
through successive mutations in specific cellular genes, leading to the activation of
oncogenes and inactivation of tumor suppressor genes. The further study of these genes has
generated much of its excitement from the convergence of experiments addressing the
genetic basis of cancer, together with cellular pathways that normally control important
cellular regulatory programmes. In the present review the context in which oncogenes and
tumor suppressor genes normally function as key regulators of physiological processes such
as proliferation, cell death/apoptosis, differentiation and senescence will be described, as well
as how these cellular programmes become deregulated in cancer due to mutations.
Keywords: oncogenes; tumor suppressor genes; proliferation; apoptosis; differentiation;
senescence
Introduction
It is today clear that cancer is caused by specific
mutations in specific key regulatory genes. Since the
discovery of the first such genes some 30 years ago, we
have seen remarkable progress in the definition of
genetic lesions in malignancy, and furthermore our
general understanding of cell biology has made us able
to comprehend how these cancer genes normally
function in the control of the non-malignant cell, and
how this relates to the ability of these genes to cause
malignant transformation when mutated.
In normal tissues of :aaulticellular organisms the
function of the single cell is tightly controlled due to
inner and surrounding constraints, to maintain tissue
homeostasis and function. During malignant transformation the cancer cell will gradually become more
autonomous, and develop into an 'asocial citizen' of its
tissue, growing in an uncontrolled manner at the
Correspondence: Dr G Grand6r, Research Laboratory of
Radiumhemmet, CCK, Karolinska Hospital, S-171 76 Stockholm, Sweden.
Received 23 December 1997; accepted 2 January 1998
expense of the function of the normal tissue. More
specifically, the cancer cell has obtained relaxed control
of several normal cell regulatory mechanisms. The most
obvious such function is proliferative control. In many
types of malignancies (but not all), the tumor cells
divide at a higher rate, often irrespective of the
extracellular growth signals that usually control cell
growth. 1 Although obvious today, it was not until
recently that it was realised that tissue growth is not
only regulated by the rate of cell division, but that the
rate of cell death or apoptosis is another key factor in
tissue homeostasis9 With this came the understanding
that malignancy may also develop through reduced cell
death rate. Furthermore, a common feature of cancer
cells is their inability to differentiate terminally, which
in normal tissues would lead to cessation of proliferation. Cell cultures derived from human tumors often
divide indefinitely; furthermore, the number of population doublings a cell has to go through in order to
become a clinically detectable tumor is very large. This
is in sharp contrast to non-transformed cells which, with
few exceptions, have a limited lifespan and will, after a
defined number of population doublings, enter a non-
Consequences of mutations in oncogenes and TSG
D Grand6r
21
replicative but metabolically active state termed senescence. 3 This has led to the hypothesis that cellular
senescence is a tumor suppressor mechanism which is
abrogated in malignancy.4
As previously mentioned a number of genes that are
specifically mutated in malignant cell have been defined
to date. They can generally be divided into two groups:
oncogenes and tumor suppressor genes. Oncogenes
take part in malignant transformation due to activating
mutations such as amplification, small mutations or
translocations. Due to their activating nature, mutations in these genes are usually dominant and only one
allele of the gene needs to be affected. The function of
tumor suppressor genes on the other hand seems to be
to protect the normal cell from developing into a cancer
cell, and a loss of their function leads to malignant
transformation. As the majority of human genes are
present in two copies in the genome, both alleles
usually have to be inactivated for their tumor-suppressing activity to be lost. Recently a third category of
cancer-related genes has been defined; namely the
DNA repair genes. These are a group of genes that take
part in the normal repair of DNA damage, and somatic
or inherited loss of their function leads to an increased
frequency of secondary mutations in oncogenes and
tumor suppressor genes. 5 With the definition of the
molecular background of a number of different inherited cancer syndromes, it has been shown that the
increased cancer risk in these families may often be due
to the inheritance of a mutated tumor suppressor
gene.
The present review will not give a detailed list of
known cancer-related genes, but will rather describe the
normal context in which these genes regulate physiological processes such as proliferation, cell death/
apoptosis, differentiation and senescence, and how they
become deregulated in the malignant cell due to
mutations in oncogenes and tumor suppressor genes.
The review will not deal to any major extent with other
factors that may be of importance in the malignant
process, such as the mechanism behind the acquisition
of metastasising potential or the interaction of tumor
cells with surrounding stroma and blood vessels.
Proliferation
Our understanding of the regulation of eukaryotic cell
division took a quantum leap some ten years ago, when
it was realised that cell cycle progression is governed by
sequential formation and activation of enzyme complexes consisting of a family of related cyclin-depend-
ent serine-threonine kinases (cdks) and their cyclin
partners (for a review see Ref. 6). At the various cell
cycle phases, waves of kinase activity corresponding to
different family members of the calks will propel cells
through the periodic events of mitotic division by
phosphorylation of key substrates. The activity of these
kinases is positively and negatively regulated by various
growth regulatory signals. Positive external mitogenic
signals will come from stimulation of the cell by various
growth factors through binding to their specific cell
surface receptors. Growth factor stimulation will lead
to activation of intracellular signalling systems such as
membrane associated enzymes and transcription factors. These events will affect the cdks active in the G~
phase by increasing cyclin levels as well as changing the
phosphorylative status of the cdks themselves, leading
to full activation of these kinases and permitting further
progress in the cell cycle (see Figure 1). One of the
major substrates for the activated kinases in the G1
phase is the retinoblastoma protein (pRb). When pRb
becomes phosphorylated by the kinases this will lead to
release of a group of transcription factors of the E:F
family that activate genes essential for S phase such as
DNA polymerase alpha, and c-myc (see Figure 1).
Several observations suggest that the phosphorylation
of pRb by the cyclin-cdk complexes is a rate-limiting
step in Gj progression. After this point cells become
committed to cell division irrespective of whether
growth factors are present or not. 7 This event has
previously been termed the 'restriction point'.
The cdks can also be negatively regulated by the
newly discovered cdk inhibitors (ckis). These latter
inhibitory proteins have been found to be important
effector molecules for internal and external growth
inhibitory signals such as DNA damage, contact inhibition and the cell growth inhibitory cytokines TGFI3 and
intcrferons.6
As previously mentioned, uncontrolled growth is a
common feature of malignant cells. Genes that control
the decision to initiate a new round of DNA synthesis
are attractive candidates for oncogenes and tumor
suppressor genes, depending on whether they have a
stimulatory or inhibitory role in the process. Indeed,
several oncogenes and tumor suppressor genes have
been found to participate directly in cell cycle regulation. 1"6 For example the malignant cell may have
mutations in genes leading to an autonomous production of growth factors, constitutive activation of growth
factor rcceptors or membrane-bound signalling proteins, leading to more or less autonomous growth. It has
also become clear that malignant cells commonly carry
Consequences of mutations in oncogenes and TSG
D Grand6r
22
mutations in the very key cell cycle regulatory proteins
such as overproduction of cyclin D1 or cdk 4 or
inactivation of the cki p16. The inactivation of p16
seems to occur at high frequencies in a wide variety of
malignancies such as leukaemias, melanomas, pancreatic cancer and bladder cancer. 8 Malignant cells may
also have mutations in the more downstream effector
molecules, such as inactivation of the retinoblastoma
gene or overproduction of E2F responsive genes such
as c-myc. As delineated above, cancer cells commonly
have mutations at some level of the molecular machin-
growthfactor
receptorg
signa_.!jng
proteins
1
1
-nucleus
S . p h,,mu~mmlimm,,,~,n~,,~
ase\
x ' ~ . hinery
DNA/
Figure 1 Outline of cell cycle regulation in the Gz phase.
Binding of growth factors to their specific receptors will lead to
activation of membrane associated signalling enzymes such as
ras, increase the levels of D-tcpe cyclins and activation of the
cdk complex. Gt cdks will phosphorylate the retinoblastoma
protein leading to the release of E2F transcription factors. E2F
induces the transcription of S phase genes such as DNA
polymerase-a and c-myc The cdk complexes can be negatively
regulated by the cyclin kinase inhibitors as exemplified by p16.
Shaded proteins are commonly mutated in malignant cells
leading to relaxed control of ,Tell growth
ery that regulates G1 progression into S phase, explaining increased and uncontrolled proliferation in malignancy (Figure 1).
Apoptosis
It is not until recently that the major importance o f
tightly regulated cell death in tissue homeostasis has
been duly recognised. Apoptosis is the descriptive
name given to the process of physiological programmed
cell death in vertebrates (for a review see Ref. 2).
During apoptosis a cell activates an intrinsic suicide
machinery that results in cell death: its surface membrane begins to bleb, the cell shrinks, the chromatin
becomes condensed and cleaved, and eventually the
whole cell fragments into membrane-bound vesicles
that are rapidly ingested by neighbouring cells. Apoptosis has been shown to be important in a number of
physiological processes such as embryonic development, immune regulation and tissue homeostasis.
Recently we have gained important knowledge in the
molecular regulation of the apoptotic process.
A number of stimuli have the capability of activating
the apoptotic programme. These stimuli include both
extracellular factors, such as the FAS antigen, TNE
interferons and matrix attachments as well as intracellular events, as for example DNA damage
(Figure 2). 2
However, not every cell that is exposed to an
apoptotic signal undergoes apoptosis, which demonstrates the importance of intracellular modulators
regulating the sensitivity of the cell. The Bcl2 family of
proteins are the best known rheostats in regulating the
cellular sensitivity to apoptosis. 9 The family members
either promote cell survival (eg Bcl2) or augment
programmed cell death (eg Bax). It is believed that the
agonistic and antagonistic proteins of the Bcl2 family
interact through hetero- or homodimerisation, and the
relative protein amounts of pro- and anti-apoptotic
Bcl2 family members will determined whether an
apoptotic signal will result in cell death or not
(Figure 2).
Recently, an emerging family of cysteine proteases
have been found to be essential downstream effectors
in the actual killing of the apoptotic cell. In this family
the nematode C. elegans protease Ced3 and its vertebrate homologue interleukin-l[3 converting enzyme
(ICE) are the prototype cysteine proteases. 1~ All the
members in the ICE/Ced3 protease family share some
features:
(1) They all induce apoptosis when overexpressed.
Consequences of mutations in oncogenes and TSG
D Grand6r
23
TNF
FasL ( ~
cell death
inducers
imlmnmnm..................
DNA damage
/
~1r ?
Bcl-2
protein
family
rheostat
ICE/CED-3
protease
cascade
cell death
Figure 2 A model o f the molecular regulation o f apoptosis.
The cell receives apoptotic signals from exogenous stimuli such
as TNF and FasL or internal stimuli such as DNA damage. The
relative ratios o f pro- and anti-apoptotic Bcl-2 family members
will determine whether an apoptotic signal will result in cell
death or not. Apoptotic signals result in the activation o f the
ICE/CED-3 cystein protease cascade, presumably cleaving
essential cellular substrates leading to cell death. Shaded
proteins such as Bcl-2 and p53 are commonly mutated in
malignant cells leading to a decreased cellular sensitivity to
apoptosis
(2) They are all produced as proenzymes and have
substrate specificity for cleavage after an aspartate residue.
Several different classes of substrate have been identified including the pro-proteascs themselves. Poly
(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK) are two proteins
involved in DNA repair, which are cleaved by the ICE
proteases, but also structural proteins such as lamins
and actin are substrates for these enzymes. However,
other unknown important targets probably exist.
Recently a direct link between the FAS/FASL and
TNF signalling and the ICE protease cascade was
found. In this system, a limited number of proteins
transmit the signal from the plasma membrane receptors to the proteases in the cytoplasm through proteinprotein interactions, using the conserved motives 'death
domain' and 'death effector domain'. 11
The finding that the Bcl2 oncogene functions in
preventing apoptosis instead of promoting proliferation
not only established a new class of oncogenes, but also
revealed the fact that malignant cells often exhibit
decreased sensitivity to apoptotic signals leading to
prolonged survival. 2 Furthermore, it seems that most
chemotherapeutic agents and radiotherapy utilised in
anticancer treatment act through induction of apoptosis
in the malignant cells. 2 It has also been realised that the
p53 tumor suppressor gene is involved in the apoptotic
response to several types of DNA-damaging agents,
including chemotherapy and radiotherapy. The p53
gene is the hitherto most commonly mutated gene in
human cancer, as abberations in this gene is estimated
to occur in more than 50% of all tumors. Tumor cells
with p53 mutation thus have a reduced susceptibility to
apoptosis induced by several stimuli, including agents
used in anticancer therapy. 12 Several other traditional
oncogenes and tumor suppressor genes, such as c-myc
and the retinoblastoma gene, have also been implicated
in regulating cell death 2 even though the mechanism
behind this is less clear.
Differentiation
The ability of cells to differentiate in an orderly and
controlled manner is of major importance for multicellular organisms, where all specialised cells are
derived from a single totipotent cell. In comparison
with our comprehension of cellular proliferation and
apoptosis, our understanding of the differentiation
process is less clear, possibly reflecting a greater
complcxity in its regulation and a larger variability
between tissues. One process that has been studied in
some detail is haematopoiesis and will therefore be
further discussed as an example in this context.
All types of mature specialised blood cells are
believed to originate from a small subset of ancestor
cells. During haematopoiesis immature multipotent
stem cells undergo progressive restriction of lineage
potential to give rise to mature, terminally differentiated functional blood cells, which eventually
undergo programmed cell death (Figure 3). 13 This
process is regulated by the intricate cooperation
between exogenous and endogenous molecules. The
exogenous molecules include different cytokines,
Consequences of mutations in oncogenes and TSG
D Granddr
24
whose cellular response is activated by binding to
specific receptors. These receptors transform the cytokine message into cellular signals that ultimately result
in altered DNA transcription, thus leading to the
activation of different genetic programmes and progressive differentiation. At the centre of this transcriptional regulation induced by the external factors reside
endogenous cell-specific proteins known as transcription factors. These proteins bind to short stretches of
DNA in a sequence-specific manner and thus alter the
transcription of specific genes in a positive or negative
manner. Some of these transcription factors have been
found to coordinate the expression of many different
genes involved in lineage specific genetic programmes
of differentiation, and are thus thought of as master or
key transcriptional regulalors (Figure 3).
In leukaemia as well as other malignancies, the
malignant cells commonly have lost the capacity to
Lymfocytes
/
~6
stemcell
progenitor
tLus;
-
-
GM-CSF
Senescence
Macrophages
-
"O'S'AY"I
o
differentiate, and become frozen at a certain stage of
differentiation. The pathomolecular background to this
is not entirely known, but in acute leukaemias a
recurrent theme of genetic alteration is the occurrence
of translocations involving known differentiation regulating transcription factors (Figure 3). These translocations usually lead to the formation of chimeric proteins
which either abrogate the function of the transcription
factor or lead to activation at an inappropriate timepoint, 13 presumably perturbing the normal differentiation programme of the affected cell. It has
furthermore been suggested that abrogation of the
differentiation blockage in malignant cells could lead to
reversion into a non or less malignant state. 14 In
different in vitro systems, mainly utilising leukaemic
cell lines, a wide variety of compounds have indeed
demonstrated differentiating activity with the induction
of mature non-proliferating cells. 13A4 Despite the progress with in vitro systems, little has been achieved in
vivo. To date, only patients with acute promyelocytic
leukaemia have come to benefit from this approach.
This disease is characterised by a balanced translocation between chromosomes 15 and 17, producing a
chimeric protein fusing the retinoic acid receptor
RARer to the transcription factor PML. In the majority
of these patients treatment with retinoic acid leads to
complete remission with the appearance of mature cells
containing the t(15;17). However, retinoic acid treatment does not cure acute promyelocytic leukaemia, as
the disease usually relapses in the absence of consolidating therapy.
~
Erythrocytes
Figure 3 Haematopoietic differentiation and some of its
important molecular regulators. Exogenous signals such as
cytokines (denoted below the arrows) and endogenous proteins
in the form of key transcription factors (denoted within the
arrows), will have positive or negative roles in the commitment
of cells to maturation along a specific lineage. Some of the
transcription factors known to be mutated during malignant
transformation are indicated by italics
As described by Hayflick more than 30 years ago,
human cells have a limited lifespan and will, after a
defined number of population doublings, enter a nonreplicative but metabolically active state termed senescence. 3 During replicative senescence, cells arrest in the
GI phase of the cell cycle. With the discovery of a
number of proteins normally regulating the cell cycle
and G1 progression, some of the molecular events
involved in replicative senescence have been identified. 4 It has recently been demonstrated that senescent
cells express increased levels of the previously mentioned ckis p16, p15, p21. 4"15The p16 and p15 proteins
accumulate as a consequence of the increasing number
of population doublings, eventually leading to senescence, t5 Another feature shared by senescent cells is
telomere shortening. The telomeres are repetitive
sequences at the ends of chromosomes, and their
shortening has been suggested as a candidate for a cell
division 'counting' mechanism in normal somatic cells
Consequences of mutations in oncogenes and TSG
D Grand6r
25
of higher organisms, 16 as the telomere length decreases
with increasing population doublings. It has been
proposed that during successive rounds of replication,
progressive loss of telomere length is sensed as DNA
damage and causes cells to exit the cell cycle.
Senescence has been suggested to be an important
tumor suppressive mechanism, and there is substantial
evidence to support this idea. 4 First, senescence prevents cells from acquiring the multiple mutations that
are needed for malignant transformation. Indeed,
many, if not most, malignant tumors contain cells that
have an extended or indefinite division potential.
During malignant transformation there seems to be a
selection for cells that can bypass senescence. Secondly,
several oncogenes, both cellular and viral, act at least in
part by extending the replicative lifespan. Furthermore,
the p16 and p15 ckis that have been shown to
accumulate during normal senescence, are commonly
inactivated in many human tumors, possibly allowing
the malignant cells to escape the senescence programme, s Malignant cells have often also acquired an
ability to maintain telomere length through the expression of the enzyme telomerase which adds essential
telomeric sequences to maintain the ends of
chromosomes. 16
Other Deregulated Processes in Malignant
Cells
Although it is not the scope of the present review to
cover other functions that may be altered in malignant
cells and their molecular background, one such area,
metastasising potential, will be mentioned briefly. The
majority of cancer-related deaths are not due to growth
of the primary tumor, but rather because of metastasising disease. The molecular background of metastasis is
still unclear, even though some mechanisms have been
proposed, such as changes in cell adhesion molecules
and expression of proteases. 17
Alterations in the above mentioned oncogenes and
tumor suppressor genes leading to transformation of
cells exhibiting undifferentiated features, autonomous
growth and decreased sensitivity to apoptotic signals,
probably also create cells with an ability to reside in
other environments than the original organ. This is also
exemplified by a host of animal experiments, in which
normal cells transfected with a limited number of
oncogenes gain the ability to form tumors at various
sites in the animal. Other important functions aiding in
the malignant transformation is the ability of the
malignant cells to interact with the surrounding stroma
and vasculature, but these functions will not be further
discussed.
Current Perspectives and Future
Directions
During recent decades we have seen remarkable
progress in our understanding of the events that
transform a normal cell into a malignant cell. It is now
clear that malignant transformation is caused by
specific mutations in oncogenes and tumor suppressor
genes. We are also starting to comprehend how these
mutations lead to the malignant phenotype, as we begin
to understand the way in which these genes interact
with cellular programmes such as control of proliferation, apoptosis, differentiation and senescence, and the
way that mutations in oncogenes and tumor suppressor
genes lead to a relaxed control of these processes. To
date, this increase in our basic knowledge of malignant
transformation has unfortunately been of little help to
the vast majority of patients with cancer. Hopefully, we
are now starting to obtain the tools to utilise this
knowledge in designing more efficient and specific
anticancer therapies. The current understanding of the
malignant process has, for example, formed the basis of
a number of current clinical pilot studies, based on
gene-therapeutic approaches. In the future we will
probably be able to design chemicals and peptides that
can specifically interfere with the functions that are
dysregulated in malignant cells. Although no clear data
concerning the beneficial effects of such novel treatments exist to date, some studies have shown promising
results, giving clear hope for the future.
Acknowledgements
Supported by grants from the Swedish Cancer Society and the
Cancer Society of Stockholm. The author is a recipient of a
fellowship from the Stockholm County Council.
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