Download molecular mechanisms of malignant transformation

Oncogene (2004) 23, 6484–6491
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Epidemiology and molecular pathology at crossroads to establish causation:
molecular mechanisms of malignant transformation
Maurizio Bocchetta*,1 and Michele Carbone1
1
Cardinal Bernardin Cancer Center, Loyola University Chicago, 2160 South First Ave, Maywood IL 60153, USA
Epidemiology is a very reliable science for the
identification of carcinogens. Epidemiological studies
require that the effect, cancer in this case, has already
occurred, when of course it would be more desirable to
identify potential carcinogenic substances at an earlier
stage before they have caused a large number of
malignancies and thus become identifiable by epidemiological studies. In the past 30 years, molecular
pathology (which includes chemistry, biochemistry,
molecular biology, molecular virology, molecular genetics, epigenetics, genomics, proteomics, and other molecular-based approaches) has identified some key
alterations that are required for cellular transformation
and malignancy. Agents that specifically interfere with
some of these mechanisms are suspected human
carcinogens. It can be stated that tumor formation
requires the following steps: (1) inactivation of Rb and
p53 cellular pathways; (2) activation of Ras and/or
other growth promoting pathways; (3) inactivation of
phosphatase 2A that causes changes in the phosphorylation and activity of several cellular proteins; (4) evasion
of apoptosis; (5) telomerase activation or alternative
mechanisms of cellular immortalization; (6) angiogenic
activity; and (7) the ability to invade surrounding tissues
and to metastasize. Here, we review the molecular
mechanisms of cellular transformation. The integration
of this knowledge with classical epidemiology and
animal studies should permit a more rapid and accurate
identification of human carcinogens.
Oncogene (2004) 23, 6484–6491. doi:10.1038/sj.onc.1207855
Keywords: tumor suppressor genes; senoscence; cell
cycle checkpoints; cell immortalization; inflammation
Introduction
A carcinogen, or initiator, is a factor that causes the
molecular damage that leads to cancer. A cocarcinogen
is an agent that usually is not carcinogenic by itself but
that enhances the activity of a carcinogen (it can also
become carcinogenic at high doses or in the presence of
*Correspondence: M Bocchetta; E-mail: [email protected]
other cocarcinogens or predisposing factors). A tumor
promoter is an agent that is not carcinogenic by itself
but enhances the activity of an initiator when administered after the initiator. Epidemiology allows the
determination of the overall effect of a given carcinogen,
cocarcinogen, or promoter in the human population (for
example, hepatitits B virus, HBV and hepatocellular
carcinoma, HCC), but cannot prove causality in the
individual tumor patient. Molecular pathology cannot
determine the overall impact of a carcinogen in the
population, but can at times prove causality in the
individual tumor patient (such as the detection of highrisk human papillomavirus (HPV) in a cervical carcinoma biopsy). This is possible when molecular techniques
have shown that the agent is required for transformation
or malignant growth of human cells (such as antisense
HPV strategies showing the requirement for the expression of HPV proteins for tumor cell growth), and when
there is supportive experimental animal evidence.
Ideally, epidemiology and molecular pathology information together with experimental evidence in animals
should be available for the most reliable identification of
human carcinogens. Here, we review the key molecular
mechanisms of malignant transformation. Agents that
specifically alter some of these mechanisms are those
that pose a higher risk for tumor formation (Carbone
et al., in press).
In higher organisms, individual cells follow a strictly
regulated life cycle, so that the architecture and
functions of the various tissues can cooperate in
harmony during ontogenesis and postnatal life. When
cells are artificially removed from their natural context
and cultured in vitro, they still behave as members of a
regulated population. For example, mammalian fibroblasts stop proliferating when in contact with each other
to form a monolayer of cells (this property is termed
contact inhibition). Therefore, cells maintain their
‘social’ identity even when they are put in a context
that is profoundly different from the body, and they do
not proliferate in an unregulated fashion. Cells maintain
also a limitation of their proliferative potential. For
example, primary human fibroblasts can sustain up to
50 cell divisions before entering the so-called ‘senescence’ (Hayflick and Moorhead, 1961). Introduction
into such cells of certain DNA viruses oncoproteins
(such as the Simian Virus 40 –(SV40) large T antigen, or
Tag) can extend the number of cell doublings up to 20
more cell division, then cells undergo the so-called
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‘crisis’, characterized by cell growth arrest and apoptosis
(Shay et al., 1991). This emphasizes the fact that cells
can tolerate a limited number of cell divisions even in the
presence of gene products whose main function is to
induce cells into proliferation. Only occasional cells
escape crisis and develop into an immortal cell line
(Bryan and Reddel, 1994).
Malignant neoplasia is a group of different diseases
that share some common characteristics: tumor cells are
immortal and can proliferate indefinitely. Cancer cells
do not stop proliferating when in contact with other
cells, but rather infiltrate surrounding tissues. Malignant
transformation also involves the capacity of cells to
disseminate throughout the organism and form secondary colonies. Cancer cells arise from the malignant
transformation of their normal counterparts. This
implies that the latter have undergone a number
of modifications leading to partial or complete loss of
tissue identity. Malignant cells have become members of
a different ‘tissue’ that has lost control of proliferation,
an event ultimately leading to the death of the entire
organism.
In the last three decades a wealth of experimental data
have revealed many of the molecular pathways that
control cell proliferation and differentiation. The
analysis of human cancers using genomic analysis has
evidenced that the malignant phenotype can be achieved
through different combinations of gene mutations. In
other words, different combinations of mutations can
cause the same type of tumor, although some genetic
alterations are either characteristic or predominantly
found in certain tumor types. In this section, we will not
cover all the pathways that can eventually lead to the
malignant transformation of the cell. We will rather
discuss some central issues common to most (or all)
tumors. In essence, a tumor cell requires gaining one or
more chronic proliferative stimuli, evading senescence,
acquiring immortality, and interacting with the surrounding tissues, and often the immune system.
Gain of function in the pathway of malignant
transformation: oncogenes
Oncogenes were traditionally recognized as fragments of
genetic material carried by viruses (both DNA viruses
and retroviruses). Several retroviruses can induce certain
types of tumors (mainly sarcomas) in birds, monkeys,
mice, and cats. Some examples are represented by the
Rous sarcoma virus, the Kirsten murine sarcoma virus,
and the Avian erythoblastosis virus. These viruses can
induce tumors because they express upon infection
proteins that induce the host cell into proliferation. The
identification of these viral oncogenes led to the
discovery that normal cells contain homologue genes
to these viral oncogenes. For this reason, the cellular
counterparts of the viral oncogenes where termed protooncogenes. The latter encode cellular proteins that are
generally related to the control of proliferation (growth
factor receptors, proteins involved in signal transduc-
tion, or transcription factors). The oncogene v-erb
(encoded by the Avian leukosis virus) is a truncated
version of the human EGF receptor. This truncated
EGR receptor is constitutively active, and therefore cells
expressing v-erb undergo a chronic EGF stimulation
(Lax et al., 1985). An example of a viral oncogene
coding for a critical component of growth signal
transduction pathways is represented by Ha-MuSV
(homologue to the cellular gene Ha-ras) (DeFeo et al.,
1981), encoded by the Harvey murine sarcoma virus. An
example of a viral oncogene encoding a transcription
factor is provided by REV-T, homologous to a member
of the NFkB protein family (Wilhelmsen et al., 1984;
Lee et al., 1991), encoded by the Reticuloendotheliosis
virus. The link between viral oncogenes and cellular
proto-oncogenes is provided by the fact that mutations
in the cellular gene can give rise to constitutively active
version of the proto-oncogene that in turn behaves
similarly to a viral oncogene. For example, substitution
of the glycine at codon 12 of the Ki-ras gene produces a
constitutively active, oncogenic ras allele, which is
commonly found in human malignancies (Bos et al.,
1987).
Other cellular genes may become oncogenes as a
result of chromosomal deletions or rearrangements,
whether they have viral homologues or not. The
deletion mutant TAN-1, coding for a truncated version
of the Notch-1 gene (Aster et al., 1994), produces a
constitutively active Notch-1 protein that is implicated
in about 10% of human T-cell acute lymphoblastic
leukemias (Ellisen et al., 1991). A hallmark of human
chronic myeloid leukaemia is a 9;22 chromosome
translocation that generates the so-called ‘Phyladelphia
chromosome’. The junction of chromosome 9 and 22
produces a fusion of two genes, with the consequent
creation of the bcr-abl oncogene (Hariharan and
Adams, 1987). Another mechanism for the transformation of a normal cellular gene into an oncogene can be
either overexpression or gene amplification. The best
example of this phenomenon is provided by myc. Myc
is a transcription factor that regulates the transcription
of many downstream genes involved in cell cycle entry
and DNA synthesis. Amplification of myc expression
levels (due to different reasons, such as chromosomal
instability or deregulated transcription) can result in
aberrant myc-dependent transcription and consequent
induction of the transformed phenotype (Collins and
Groudine, 1982). In conclusion, cellular proto-oncogenes are molecules that normally regulate the cell’s
response to growth factors and hormones (either as
receptors, signal transduction molecules, or transcription factors). Upon gene mutations, which can be
caused by environmental carcinogens, these molecules
can be transformed into constitutively active factors
thus mimicking a chronic stimulation by a growth
signal.
Although all cancer cells contain one or more active
oncogenes, the expression of such molecules into normal
cells does not usually lead to increased proliferation. For
example, expression of an oncogenic ras allele in both
human and mouse primary cells causes complete cell
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growth arrest and senescence (Serrano et al., 1997).
Same effects are obtained when constitutively active
MEK, a critical component of the mytogen-activated
protein-kinase (MAP-K) cascade, is expressed in these
cells (MAP-K is one of the major downstream effectors
of ras signaling, see Figure 1) (Lin et al., 1998). This
indicates that cells have mechanisms in place to respond
to the deregulated expression of an oncogene by
eliminating that cell. This is just one of the many
mechanisms that prevent deregulated cell growth, which
can lead to tumor formation. When the same oncogenes
are aberrantly expressed in premalignant (or malignant)
cells, because those cells have lost the ability to arrest
cell growth, the oncogene is able to further drive the
expansion of the aberrant cell clone.
Oncogenes encoded by DNA tumor viruses are a
different class of oncogenes. DNA tumor viruses include
members of very different families of viruses. Their
genome can be large and encode numerous viral
proteins, as in the case of Herpes viruses, or their
chromosome can be very small and code for a limited
number of proteins, as in the case of HPV and SV40
(Butel, 2000). One common requirement of DNA
viruses is to induce the host cell to enter S phase,
because these viruses rely on the cellular machinery of
DNA replication to different extents for the amplifica-
tion of their genome. Different viral proteins that target
central functions controlling the G1–S checkpoint
achieve this task. DNA viruses’ oncogenes do not have
cellular homologues, and are often multifunctional. The
SV40 Tag achieves the highest degree of multifunctionality. SV40 is a small virus encoding three capsid
proteins, a small ancillary protein of uncertain biological function, and three oncoproteins, namely Tag, the
small t antigen (or tag), and the 17 kDa protein (Rundell
and Parakati, 2001). The Tag is the major SV40
oncoprotein, because it targets a number of cellular
proteins and transactivates a number of cellular
promoters. The net result of these activities is the
simultaneous inactivation of the two major cellular G1–
S checkpoints and the induction of a number of cellular
pathways that induce proliferation (Ali and DeCaprio,
2001). Overall, the common characteristic of DNA
tumor viruses’ oncogenes is that they do not require
mutations to exert their transforming functions, they do
not have cellular homologues, and that they are often
multifunctional. The latter is the reason why the
introduction, for instance, of the Tag into a primary
cell does not cause senescence or apoptosis, as often
caused by deregulated expression of cellular protooncogenes. This is because Tag targets numerous cell
pathways simultaneously.
The induction of cellular senescence and cell cycle
checkpoints: tumor suppressors
Figure 1 Representative schematic of the principal mediators of
the ras signaling pathway. The pathway has been divided into four
major branches, but these branches should not be considered as
independent; rather they are activated in concert, and overlap
extensively. For instance, MEK-K1 is involved in the phosphorylation of Jun N-terminal kinase-kinase (JNK-K), and of inhibitors
of NFkB (IkB), and is also involved in the activation of
extracellular regulated kinase (MEK and ERK). The Raf-1
serine–threonine protein kinase associates with ras upon ras
activation and phosphorylates a number of downstream mediators.
The end result of the activation of the ras signaling pathway is the
activation of several transcription factors that promote cell growth,
survival, angiogenesis
Oncogene
The activation of a cellular oncogene represents a gain
of functions. The mutation of one allele of a protooncogene is almost invariably dominant over its wildtype counterparts. The genetic analysis of human cancer
has revealed that loss of function is also a common
characteristic in the process of malignant transition.
Since the loss of both copies of certain genes is often
required for tumor formation, these mutations are
considered to be recessive (the remaining wild-type
allele is dominant over the mutated one), and these
genes were named tumor suppressor genes. The reason
for the denomination resides in the need for the
complete loss of them for tumors to arise; one copy of
these genes is still able to suppress malignant transformation. In reality, this is an oversimplification, since
haploinsufficiency of certain tumor suppressor genes is
compatible with tumor formation (Kwabi-Addo et al.,
2001; McLaughlin and Jacks, 2002; Magee et al., 2003).
Tumor suppressors include proteins with very different
activities. Some are transcription factors, such as p53
(Chene, 2003). Others are proteins that through their
interaction with other proteins can either inhibit the
activity of kinases involved in cell cycle progression,
such as p16INK4A and p21WAF-1 (Malumbres and
Barbacid, 2001), or affect the cellular localization of
other proteins, such as the members of the 14-3-3
protein family (Hermeking, 2003). Some tumor suppressors are kinases that activate a number of other
kinases and transcription factors, such as members of
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the PI3K-like protein kinases (PIKKs) family (Shiloh,
2003). These include ATM (ataxia-telangiectasia mutated), ATR (ataxia telangiectasia-mutated and Rad3related), and other members. These kinases are critically
involved in the signaling of DNA damage and can
trigger cell cycle arrest throughout G1 to the G2/M
checkpoint. The list of all known tumor suppressor gene
products and the description of the intricate interconnections among them is beyond the scope of this section,
and the reader is referred to a number of excellent
reviews, some of which have been mentioned above. One
general feature of tumor suppressors is the participation
in the establishment of critical checkpoints during G1/S
and G2/M transitions. Tumor suppressors become
activated and halt the cell cycle in the presence of
aberrant proliferative stimuli, DNA damage, and other
‘out-of context’ biological situations. The cell cycle is
arrested to allow the cell to ‘fix’ the problem. If such
problem cannot be corrected, the cell enters into
senescence or initiates apoptosis. This is the reason
why all tumor cells contain mutations in a number of
tumor suppressor genes that ultimately disrupt the two
major tumor suppressor pathways of the cell: the p53
and Retinoblastoma pathways (Hahn and Weinberg,
2002). p53 activates the G1/S checkpoint mainly by
inducing the cyclin-dependent kinase (CDK) inhibitor
p21WAF. Alternatively, p53 can induce apoptosis because
it regulates the transcription of proapoptotic proteins.
Retinoblastoma family member activate the G1/S
checkpoint by binding to and inhibiting the E2F
transcription factors (the latter positively regulate the
transcription of a wide number of genes responsible for
cell cycle progression). Retinoblastoma proteins can be
inactivated through phosphorylation by CDK (mainly
CDK4 and 6) bound to G1 cyclins (cyclins D and E
members). The tumor suppressor p16INK4A (similarly to
p21WAF) can inhibit CDK–cyclin complexes, therefore
re-establishing the G1/S checkpoint (see Figure 2).
As mentioned above, introduction of oncogenic ras
into primary human cells causes senescence. This
phenomenon is accompanied by a dramatic increase of
the p16INK4A CDK inhibitor (Wei et al., 2001). The CDK
inhibitor p16INK4A binds to CDK4 and CDK6 complexed to D-type cyclins displacing the latter from their
association with CDKs. The result of this action is the
inactivation of CDKs, which in turn are not able to
phosphorylate Retinoblastoma family members and, as
a consequence, the cell remains blocked in G1
(Malumbres and Barbacid, 2001). These data underscore
the importance of the Retinoblastoma tumor suppressor
pathway in the establishment of senescence in human
cells. However, p53 is also considered to contribute to
senescence in human cells (Macip et al., 2003; Horner
et al., 2004), especially in the process of replicative
senescence (see below). Loss of p53 is important
not only to evade senescence. Tumor cells are characterized by a high degree of chromosomal instability
(Rangarajan and Weinberg, 2003). In such environment,
an intact p53 pathway would invariably cause G1 arrest
or p53-dependent apoptosis. Therefore, it is not surprising
that p53 itself is lost in about 60% of all human cancers
Figure 2 Representative schematic of the two major checkpoint of
the cell cycle and involvement of some of the major tumor
suppressor gene products. The ATM and ATR kinases are involved
in the signaling of DNA damage. These kinases phosphorylate a
number of downstream mediators, including p53 and Chk
(Checkpoint kinases 1 and 2). Phosphorylation of p53 activates
this transcription factor that in turn promotes synthesis of p21WAF,
which inhibits the cyclin/CDK complexes, thus activating the G1/S
checkpoint. If DNA damage cannot be repaired, p53 promotes
apoptosis. p53 is also involved in the establishment of the G2/M
checkpoint. The tumor suppressor p16INK4A is involved in inhibition
of CDKs, thus contributing to the G1/S checkpoint. The
checkpoint kinases halt the cell cycle by phosphorylating (thus
inactivating) cdc25 protein family members. Cdc25 are phosphatases that activated cdc2 complexed with cyclin B. The latter
complex is the mytosis-promoting factor (MPF), which is the
complex that regulates cell entry into the M phase
(Levine et al., 1994). More recent data indicated that
tumor suppressor genes are not exclusively proteins
involved in checkpoint control, but that different
proteins having functions apparently unrelated to the
control of the cell cycle can nevertheless be important
for cancer prevention. One good example is provided by
dyskerin, a protein involved in the modification of
uridine residues of ribosomal RNA into pseudouridine
and therefore important for ribosomal biogenesis.
Dyskerin-deficient mice display signs of human dyskeratosis congenita, a disease characterized by premature
aging and cancer susceptibility, and are markedly
susceptible to cancer development (Ruggero et al.,
2003).
Mutations (both point mutations and deletions) are
not the only way through which a tumor suppressor
gene can be inactivated. In recent years, the importance
of epigenetic changes in the establishment of the
malignant phenotype has been underscored. Some
tumor suppressor genes are not transcribed because of
methylation of their promoter. Common examples of
tumor suppressor genes that are inactivated because
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of hypermethylation of their promoter are represented
by those encoded by the INK4A locus (Myohanen et al.,
1998) and RASSF1A (Agathanggelou et al., 2001;
Dammann et al., 2001).
The expression of tumor suppressors can be lost
because of differential splicing. One example is
provided by IG20, a splice variant of MADD (MAPK activating death domain-containing protein). IG20 is
a protein interacting with the tumor necrosis factor
alpha (TNFa) receptor (Al-Zoubi et al., 2001) and
TNF-related apoptosis-inducing ligand) (TRAIL) receptor (Efimova et al., 2003). Through its activities,
IG20 sensitizes the cell to apoptotic stimuli and induces
the secretion of interleukin (IL)-6, thus suppressing
tumor cell growth (BS Prabhakar, personal communication). In certain tumors, IG20 expression could be
lost because of perturbed splicing of the IG20 gene
primary transcript leading to overexpression of an
IG20 splice variant (DENN-SV) that induces cell
proliferation and resistance to apoptosis (Efimova
et al., 2004).
Immortality is acquired at chromosomes’ ends
The ends of all eucaryotes’ chromosome are organized
into structures termed telomeres. Human telomeres are
5–15 Kbp long, and contain tandem repeats of the
sequence 50 -TTAGGG-30 (Moyzis et al., 1988). Telomeres end in a 30 overhang (about 300 nucleotides long)
that invades the double-stranded portion of telomeres to
form a lariat structure also known as T loop (Griffith
et al., 1999). Several proteins are bound to the T loop
and contribute to its stability and/or signal whether such
structure is perturbed. Telomere’s end is structured this
way because a terminal DNA sequence of a putative
linear chromosome would be detected as a doublestrand break in DNA and it would trigger DNA repair
response, including activation of a cell cycle checkpoint
and/or apoptosis. Furthermore, unprotected chromosomal ends would lead to end-to-end chromosomal fusion,
thus leading to mitosis catastrophe (Goytisolo and
Blasco, 2002). One characteristic of DNA replication
is that the ends of linear DNA molecules are progressively shortened at each round of replication because
DNA polymerase uses RNA primers that are degraded
after elongation, a phenomenon known as ‘end replication problem’ (Watson, 1972). Bacteria solve this
problem by having a circular genome, while bacteriophages with a linear chromosome arrange it in tandem
concatemers, therefore limiting the importance of
terminal DNA sequences. In most normal somatic cells,
instead chromosomes shorten at each round of cell
replication (Harley et al., 1990). This progressive erosion
of chromosomes ends ultimately results in progressive
shortening of telomeres. When telomeres shorten
beyond a critical threshold, they start to become
structurally dysfunctional. This process eventually leads
to activation of the p53 signaling pathway with
consequent establishment of replicative senescence or
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crisis (Chin et al., 1999). Therefore, telomeres work as a
cellular hour glass that determines how many replication
rounds a cell can afford. Germinal cells and most of
tumor cells evade this phenomenon (and therefore
achieve immortality) through the activity of a riboprotein complex called telomerase. This enzyme is a
reverse transcriptase that adds TTAGGG repeats to the
telomeres, thus preventing their shortening and the
consequent induction of senescence and apoptosis.
Telomerase has an RNA component (TERC) that is
universally expressed in human cells, and functions as
template for the protein component of telomerase to
synthesize TTAGGG tandem repeats (Goytisolo and
Blasco, 2002). The protein component of telomerase
(TERT, the reverse transcriptase) is not expressed (or
expressed at insufficient levels) in most somatic cells.
For this reason, somatic cells cannot restore the length
of telomeres during cell proliferation and undergo
replicative senescence and crisis. As a result of mutations, or of viral infection in certain cells (Foddis et al.,
2002), tumor cells regain sufficient amounts of TERT
expression and thus can proliferate indefinitely. Some
have questioned whether certain cancers (especially
breast cancer) need to become immortal in order to
become clinically evident (Reddel, 2000). This issue was
raised from the observation that breast cancer is a very
fastidious tissue to obtain cell lines from, arguing
against the case that these tumors are truly made of a
uniformly immortal cell population. However, more
than 85% of human cancers (including breast cancer)
have readily measurable telomerase activity (Shay and
Bacchetti, 1997). Many of those cancers in which
telomerase activity is not detected have an alternative
process to preserve telomeres length called ALT (alternative lengthening of telomeres; Bryan et al., 1997).
ALT cancer cells are characterized by unusually long
telomeres, and the process underlying the ALT phenotype is believed to be nonreciprocal recombination
between telomeres of different chromosomes (reviewed
in Neumann and Reddel, 2002). All these evidences
indicate that cell immortalization is a necessary event for
malignant growth.
Dual role in cancer pathogenesis: the immune system
Researchers are increasingly focusing on the interplay
between cancer cells and the surrounding stromal
tissues. It appears more and more evident that the
microenvironment surrounding cancer cells plays a
pivotal role in tumor promotion, vascularization. and
metastatization (Coussens and Werb, 2002). The physiological rearrangements of connective tissue are
largely under the control of the various members of
the immune system (Schreiber, in press). The fact that
the immune system could have played a major role in
tumor containment and, at the same time, promotion
was long known. In the late 19th century, it was shown
that a minority of terminally ill cancer patients showed
partial or complete remission of their malignancy when
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injected with a mixture of bacterial toxins (the so-called
Coley extract) in order to cause a potent inflammatory
response (Nauts et al., 1953). These data indicated that a
massive inflammatory stimulus can trigger a generalized
reorganization of the immune systems capable of
activating a response against the tumor cells (even if
the primary stimulus was not specific for the tumor
itself). On the other hand, it is also known that cancer
arises preferentially within a context of chronic inflammation. Currently, about 15% of all human tumors are
attributable to infectious agents, and inflammation is a
critical component of these chronic infections (Parkin
et al., 1999).
Experimental evidences have underscored that in the
early stages of cancer, the presence of consistent
intratumoral lymphocyte infiltrations are strongly correlated with reduced metastasis formation and prolonged patient survival (Clark et al., 1989; Naito et al.,
1998). Indeed, both innate and adaptive immunity can
fight cancer development. Innate immunity cells decipher pattern-recognition of receptors and other transmembrane proteins expressed by cancer cells and
directly target them. The main cellular players in this
mechanism of recognition are natural killer cells (NK).
The latter recognize stress-related proteins on the
surface of cancer cells (Diefenbach and Raulet, 2003).
NK can also recognize cells with decreased expression of
HMC class I, a common feature in cancer cells (Karre,
2002). Dendritic cells (DC) also play an essential role.
They can phagocytose apoptotic cancer cells, a process
eventually leading to activation of adaptive immunity
against cancer cell-specific antigens. DC also phagocytose heat-shock proteins (HSP) released by necrotic
cancer cells. HSP released in the extracellular space
activate inflammation (Basu et al., 2001). Adaptive
immune system targets tumor cells indirectly. Tumor
cells potentially express a number of tumor-specific
antigens (deriving from mutations of wild-type proteins), and therefore should be easy targets for CD4 þ
and CD8 þ T cells. However, cancer cells decrease the
level of expression of major histocompatibility complex
(MHC) class I molecules and that of critical immuno
costimulatory proteins such as the member of the B7
protein family, which are required for efficient Tdependent response (Sharpe and Freeman, 2002). This
problem is overcame by DC that, after phagocytosis of
tumor cells, debris migrate to lymph nodes and activate
a tumor-specific T response (Banchereau and Steinman,
1998). A limitation of this process (termed ‘cross
priming’) is that DC infiltrating cancer tissues are often
immature, a process mediated by a quite complicated
interplay between tumor cells and immune cells in which
cytokines play a major role (see below). The importance
of the immune system in the protection against tumor
formation is underscored also by a number of studies
conducted in experimental animals. Mice defective for
interferon g (IFN-g, a cytokine produced mainly by T
cells and NK and, to a lesser extent, by DC and
macrophages), or for other cytokines important for
IFN-g production display an increased susceptibility to
chemical carcinogenesis (Kaplan et al., 1998; Smyth
et al., 2000). IFN-g is a multifactorial cytokine that
probably mediates a wide variety of cell effects, but one
possible mechanism to explain these data is that IFN-g
can upregulate MHC class I molecules and therefore
render cancer cells easier targets for T cell-mediated
targeting (Shankaran et al., 2001). Other experiments
conducted in immunocompromized mice showed a
protective role for the immune system against cancer,
although other mice models, especially those deficient in
the production of certain cytokines yielded opposite
effects. These apparently paradoxical results indicate
that in cancer biology it is impossible to envision the role
of the immune system in just one direction. Despite a
wide variety of possible mechanisms of self-defense, the
immune system has obviously lost its battle against
cancer when the disease finally becomes clinically
manifest. The principle of boosting this compromised
immune system following a wide variety of strategies in
order to fight clinically evident cancer has been the
central hypothesis of Cancer Immunology. A number of
studies are presently in progress to produce therapeutic
cancer vaccines, or to infuse genetically modified
immune cells into cancer patients to trigger a cancerspecific immune response. However, immune depressed
patients (e.g. AIDS patients, patients who are immunocompromised following organ transplant, etc.) do not
develop an excess of malignancies other than those that
are virally induced (e.g. EBV-related lymphomas,
Kaposi sarcoma, etc.). This indicates that the immune
system has a much more limited role in cancer
prevention than originally hypothesized and that this
role is mainly limited to malignancies that express
unique antigens, such as viral proteins. Moreover, also
for virally induced cancers, the immune system can
promote cancer growth, such as in the case of Hepatitis
C infection that leads to chronic inflammation, which in
turn can result in the development of HCC (Carbone
et al., in press).
As stated above, cancer and inflammation have been
correlated. A number of pathologies characterized by
chronic inflammation are strikingly associated with a
higher cancer incidence. Individuals who have taken
aspirin and other cyclooxygenases inhibitors over a
period of several years have a 40–50% reduced risk of
developing colon cancer (Garcia-Rodriguez and HuertaAlvarez, 2001). There are a number of reasons why
inflammation could play a landscaping role for cancer
development, mainly due to the action of proinflammatory cytokines secreted by various component of the
innate immune system, including macrophages, mast
cells, neutrophils, eosinophils, and other members.
However, tumor-associated macrophages are increasingly implicated as possible tumor-promoting cells.
These macrophages produce a wide array of cytokines,
proteases, angiogenic factors, all of which may play an
important role in tumor progression and dissemination
(Schoppmann et al., 2002). Additionally, tumor-associated macrophages secrete the proinflammatory chemokine TNFa (Torisu et al., 2000). This cytokine,
through induction of the NFkB signaling pathway,
may provide surrounding cells with critical survival
Oncogene
Molecular mechanisms of malignant transformation
M Bocchetta and M Carbone
6490
stimuli in the presence of mutagenic molecules, such as
reactive oxygen species (ROS) and other potentially
mutagenic molecules produced during infection (Maeda and Akaike, 1998). Another powerful cytokine
produced by macrophages and T cells is macrophage
migration inhibitory factor (MIF). MIF inhibits p53
transcriptional activity (Hudson et al., 1999). Therefore, this cytokine may promote extended lifespan,
accumulation of mutations due to impaired p53
function, inhibition of p53-mediated apoptosis. This
situation may explain why inflammation may be fertile
ground for cancer development. Inhibition of p53
functions, paralleled by NFkB activation, provide cells
with survival and proliferative stimuli in the absence of
the major tumor suppressor gene product of the cell.
Potentially mutagenic substances produced during
infection may introduce mutations into cellular protooncogenes, thus allowing the mutated cell to survive
the activities of an active oncogene. Furthermore, as a
response to pro-inflammatory cytokines, mast cells
release a number of proteases, including heparin,
heparanase, matrix metalloproteinases and serine proteases, all thought to play a key role in tissue dynamics
and in metastatic dissemination of cancer cells
(Balkwill and Mantovani, 2001). A role for certain
proinflammatory cytokines in tumor formation has
been demonstrated in experimental animals. TNFa
knockout mice are resistant to skin cancer induced by
different chemical carcinogens (Moore et al., 1999),
while mice homozygous for the IL-6-null allele (/)
are resistant to plasma cell tumor development (Hilbert
et al., 1995). Other proinflammatory cytokines, such as
colony-stimulating factor 1 (C-SF1) (Lin et al., 2001)
and IL-1 (Voronov et al., 2003), proved to be required
for tumor formation in experimental animals. In
conclusion, the immune system is one of the most
important lines of defense for tumor formation for
malignancies that express unique antigens, such as viral
proteins. The immune system can also promote tumor
growth through the stimulatory and mutagenic effects
of cytokines released by inflammatory cells (Wogan
et al., in press; Schreiber, in press).
Conclusions
Our knowledge about the molecular mechanisms
required for tumor formation has tremendously improved during the past 40 years. Key molecular
mechanisms required for malignancy have been identified (Carbone et al., in press). It can be easily predicted
that new oncogenes will be identified in the future;
however, it can be predicted that these new ‘cancer
genes’ will affect those pathways required for tumor
formation described above. Thus, the apparent complexity of tumor formation can be reduced to a much
simpler picture. Regardless of the specific mechanism by
which a given gene pathway is altered, carcinogenesis
requires the activation or inactivation of the pathways
we have discussed in this review.
Cancer is a multifactorial event in which numerous
alterations contribute to the emergence of the malignant
cell. Tumor cells per se can be innocuous unless
additional factors such as tumor promoters stimulate
cell growth (hormones, chronic inflammation, etc.).
Moreover, tumor cells that express viral antigens or
some other type of unique antigen can be eliminated by
the immune system. Therefore, malignant tumor growth
is a dynamic process in which it is difficult to identify a
unique event that caused the process. Rather, it is
possible to identify numerous events that together
contributed to malignancy. This information should
allow scientists to identify those agents that are more
likely to cause or to contribute to cancer before they
have caused a substantial number of cancer deaths
detectable by classical epidemiology. Thus, it is hoped
that the integration of molecular pathology with
classical epidemiological and animal studies will lead
to a more rapid and accurate identification of human
carcinogens.
Acknowledgements
Dr Carbone’s work is supported by grants from the National
Cancer Institute, USA, RO1CA92657; the American Cancer
Society, RSG-04-029-01-CCE, and by the Cancer Research
Foundation of America.
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