Molecular Mechanisms of Malignant Transformation

Home , Linear chromosome, Malignant transformation, Tumor promotion

Epidemiology and molecular pathology at crossroads to establish causation: molecular mechanisms of malignant transformation

Maurizio Bocchetta*,1 and Michele Carbone1

1Cardinal Bernardin Cancer Center, Loyola University Chicago, 2160 South First Ave, Maywood IL 60153, USA

Epidemiology is a very reliable science for the other cocarcinogens or predisposing factors). A tumor identification of carcinogens. Epidemiological studies promoter is an agent that is not carcinogenic by itself require that the effect, cancer in this case, has already but enhances the activity of an initiator when adminis- occurred, when of course it would be more desirable to tered after the initiator. Epidemiology allows the identify potential carcinogenic substances at an earlier determination of the overall effect of a given carcinogen, stage before they have caused a large number of cocarcinogen, or promoter in the human population (for malignancies and thus become identifiable by epidemio- example, hepatitits B virus, HBV and hepatocellular logical studies. In the past 30 years, molecular carcinoma, HCC), but cannot prove causality in the pathology (which includes chemistry, biochemistry, individual tumor patient. Molecular pathology cannot molecular biology, molecular virology, molecular genet- determine the overall impact of a carcinogen in the ics, epigenetics, genomics, proteomics, and other mole- population, but can at times prove causality in the cular-based approaches) has identified some key individual tumor patient (such as the detection of high- alterations that are required for cellular transformation risk human papillomavirus (HPV) in a cervical carcino- and malignancy. Agents that specifically interfere with ma biopsy). This is possible when molecular techniques some of these mechanisms are suspected human have shown that the agent is required for transformation carcinogens. It can be stated that tumor formation or malignant growth of human cells (such as antisense requires the following steps: (1) inactivation of Rb and HPV strategies showing the requirement for the expres- p53 cellular pathways; (2) activation of Ras and/or sion of HPV proteins for tumor cell growth), and when other growth promoting pathways; (3) inactivation of there is supportive experimental animal evidence. phosphatase 2A that causes changes in the phosphoryla- Ideally, epidemiology and molecular pathology infor- tion and activity of several cellular proteins; (4) evasion mation together with experimental evidence in animals of apoptosis; (5) telomerase activation or alternative should be available for the most reliable identification of mechanisms of cellular immortalization; (6) angiogenic human carcinogens. Here, we review the key molecular activity; and (7) the ability to invade surrounding tissues mechanisms of malignant transformation. Agents that and to metastasize. Here, we review the molecular specifically alter some of these mechanisms are those mechanisms of cellular transformation. The integration that pose a higher risk for tumor formation (Carbone of this knowledge with classical epidemiology and et al., in press). animal studies should permit a more rapid and accurate In higher organisms, individual cells follow a strictly identification of human carcinogens. regulated life cycle, so that the architecture and Oncogene (2004) 23, 6484–6491. doi:10.1038/sj.onc.1207855 functions of the various tissues can cooperate in harmony during ontogenesis and postnatal life. When Keywords: tumor suppressor genes; senoscence; cell cells are artificially removed from their natural context cycle checkpoints; cell immortalization; inflammation 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 Introduction ‘social’ identity even when they are put in a context that is profoundly different from the body, and they do A carcinogen, or initiator, is a factor that causes the not proliferate in an unregulated fashion. Cells maintain molecular damage that leads to cancer. A cocarcinogen also a limitation of their proliferative potential. For is an agent that usually is not carcinogenic by itself but example, primary human fibroblasts can sustain up to that enhances the activity of a carcinogen (it can also 50 cell divisions before entering the so-called ‘senes- become carcinogenic at high doses or in the presence of cence’ (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 *Correspondence: M Bocchetta; E-mail: [email protected] more cell division, then cells undergo the so-called Molecular mechanisms of malignant transformation M Bocchetta and M Carbone 6485 ‘crisis’, characterized by cell growth arrest and apoptosis tion, or transcription factors). The oncogene v-erb (Shay et al., 1991). This emphasizes the fact that cells (encoded by the Avian leukosis virus) is a truncated can tolerate a limited number of cell divisions even in the version of the human EGF receptor. This truncated presence of gene products whose main function is to EGR receptor is constitutively active, and therefore cells induce cells into proliferation. Only occasional cells expressing v-erb undergo a chronic EGF stimulation escape crisis and develop into an immortal cell line (Lax et al., 1985). An example of a viral oncogene (Bryan and Reddel, 1994). coding for a critical component of growth signal Malignant neoplasia is a group of different diseases transduction pathways is represented by Ha-MuSV that share some common characteristics: tumor cells are (homologue to the cellular gene Ha-ras) (DeFeo et al., immortal and can proliferate indefinitely. Cancer cells 1981), encoded by the Harvey murine sarcoma virus. An do not stop proliferating when in contact with other example of a viral oncogene encoding a transcription cells, but rather infiltrate surrounding tissues. Malignant factor is provided by REV-T, homologous to a member transformation also involves the capacity of cells to of the NFkB protein family (Wilhelmsen et al., 1984; disseminate throughout the organism and form second- Lee et al., 1991), encoded by the Reticuloendotheliosis ary colonies. Cancer cells arise from the malignant virus. The link between viral oncogenes and cellular transformation of their normal counterparts. This proto-oncogenes is provided by the fact that mutations implies that the latter have undergone a number in the cellular gene can give rise to constitutively active of modifications leading to partial or complete loss of version of the proto-oncogene that in turn behaves tissue identity. Malignant cells have become members of similarly to a viral oncogene. For example, substitution a different ‘tissue’ that has lost control of proliferation, of the glycine at codon 12 of the Ki-ras gene produces a an event ultimately leading to the death of the entire constitutively active, oncogenic ras allele, which is organism. commonly found in human malignancies (Bos et al., In the last three decades a wealth of experimental data 1987). have revealed many of the molecular pathways that Other cellular genes may become oncogenes as a control cell proliferation and differentiation. The result of chromosomal deletions or rearrangements, analysis of human cancers using genomic analysis has whether they have viral homologues or not. The evidenced that the malignant phenotype can be achieved deletion mutant TAN-1, coding for a truncated version through different combinations of gene mutations. In of the Notch-1 gene (Aster et al., 1994), produces a other words, different combinations of mutations can constitutively active Notch-1 protein that is implicated cause the same type of tumor, although some genetic in about 10% of human T-cell acute lymphoblastic alterations are either characteristic or predominantly leukemias (Ellisen et al., 1991). A hallmark of human found in certain tumor types. In this section, we will not chronic myeloid leukaemia is a 9;22 chromosome cover all the pathways that can eventually lead to the translocation that generates the so-called ‘Phyladelphia malignant transformation of the cell. We will rather chromosome’. The junction of chromosome 9 and 22 discuss some central issues common to most (or all) produces a fusion of two genes, with the consequent tumors. In essence, a tumor cell requires gaining one or creation of the bcr-abl oncogene (Hariharan and more chronic proliferative stimuli, evading senescence, Adams, 1987). Another mechanism for the transforma- acquiring immortality, and interacting with the sur- tion of a normal cellular gene into an oncogene can be rounding tissues, and often the immune system. 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 Gain of function in the pathway of malignant and DNA synthesis. Amplification of myc expression transformation: oncogenes levels (due to different reasons, such as chromosomal instability or deregulated transcription) can result in Oncogenes were traditionally recognized as fragments of aberrant myc-dependent transcription and consequent genetic material carried by viruses (both DNA viruses induction of the transformed phenotype (Collins and and retroviruses). Several retroviruses can induce certain Groudine, 1982). In conclusion, cellular proto-onco- types of tumors (mainly sarcomas) in birds, monkeys, genes are molecules that normally regulate the cell’s mice, and cats. Some examples are represented by the response to growth factors and hormones (either as Rous sarcoma virus, the Kirsten murine sarcoma virus, receptors, signal transduction molecules, or transcrip- and the Avian erythoblastosis virus. These viruses can tion factors). Upon gene mutations, which can be induce tumors because they express upon infection caused by environmental carcinogens, these molecules proteins that induce the host cell into proliferation. The can be transformed into constitutively active factors identification of these viral oncogenes led to the thus mimicking a chronic stimulation by a growth discovery that normal cells contain homologue genes signal. to these viral oncogenes. For this reason, the cellular Although all cancer cells contain one or more active counterparts of the viral oncogenes where termed proto- oncogenes, the expression of such molecules into normal oncogenes. The latter encode cellular proteins that are cells does not usually lead to increased proliferation. For generally related to the control of proliferation (growth example, expression of an oncogenic ras allele in both factor receptors, proteins involved in signal transduc- human and mouse primary cells causes complete cell

Oncogene Molecular mechanisms of malignant transformation M Bocchetta and M Carbone 6486 growth arrest and senescence (Serrano et al., 1997). tion of their genome. Different viral proteins that target Same effects are obtained when constitutively active central functions controlling the G1–S checkpoint MEK, a critical component of the mytogen-activated achieve this task. DNA viruses’ oncogenes do not have protein-kinase (MAP-K) cascade, is expressed in these cellular homologues, and are often multifunctional. The cells (MAP-K is one of the major downstream effectors SV40 Tag achieves the highest degree of multifunction- of ras signaling, see Figure 1) (Lin et al., 1998). This ality. SV40 is a small virus encoding three capsid indicates that cells have mechanisms in place to respond proteins, a small ancillary protein of uncertain biologi- to the deregulated expression of an oncogene by cal function, and three oncoproteins, namely Tag, the eliminating that cell. This is just one of the many small t antigen (or tag), and the 17 kDa protein (Rundell mechanisms that prevent deregulated cell growth, which and Parakati, 2001). The Tag is the major SV40 can lead to tumor formation. When the same oncogenes oncoprotein, because it targets a number of cellular are aberrantly expressed in premalignant (or malignant) proteins and transactivates a number of cellular cells, because those cells have lost the ability to arrest promoters. The net result of these activities is the cell growth, the oncogene is able to further drive the simultaneous inactivation of the two major cellular G1– expansion of the aberrant cell clone. S checkpoints and the induction of a number of cellular Oncogenes encoded by DNA tumor viruses are a pathways that induce proliferation (Ali and DeCaprio, different class of oncogenes. DNA tumor viruses include 2001). Overall, the common characteristic of DNA members of very different families of viruses. Their tumor viruses’ oncogenes is that they do not require genome can be large and encode numerous viral mutations to exert their transforming functions, they do proteins, as in the case of Herpes viruses, or their not have cellular homologues, and that they are often chromosome can be very small and code for a limited multifunctional. The latter is the reason why the number of proteins, as in the case of HPV and SV40 introduction, for instance, of the Tag into a primary (Butel, 2000). One common requirement of DNA cell does not cause senescence or apoptosis, as often viruses is to induce the host cell to enter S phase, caused by deregulated expression of cellular proto- because these viruses rely on the cellular machinery of oncogenes. This is because Tag targets numerous cell DNA replication to different extents for the ampliﬁca- pathways simultaneously.

The induction of cellular senescence and cell cycle checkpoints: tumor suppressors

The activation of a cellular oncogene represents a gain of functions. The mutation of one allele of a proto- oncogene is almost invariably dominant over its wild- type 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). Figure 1 Representative schematic of the principal mediators of Tumor suppressors include proteins with very different the ras signaling pathway. The pathway has been divided into four activities. Some are transcription factors, such as p53 major branches, but these branches should not be considered as (Chene, 2003). Others are proteins that through their independent; rather they are activated in concert, and overlap interaction with other proteins can either inhibit the extensively. For instance, MEK-K1 is involved in the phosphorylation of Jun N-terminal kinase-kinase (JNK-K), and of inhibitors activity of kinases involved in cell cycle progression, of NFkB(IkB), and is also involved in the activation of such as p16INK4A and p21WAF-1 (Malumbres and extracellular regulated kinase (MEK and ERK). The Raf-1 Barbacid, 2001), or affect the cellular localization of serine–threonine protein kinase associates with ras upon ras other proteins, such as the members of the 14-3-3 activation and phosphorylates a number of downstream mediators. The end result of the activation of the ras signaling pathway is the protein family (Hermeking, 2003). Some tumor sup- activation of several transcription factors that promote cell growth, pressors are kinases that activate a number of other survival, angiogenesis kinases and transcription factors, such as members of

Oncogene Molecular mechanisms of malignant transformation M Bocchetta and M Carbone 6487 the PI3K-like protein kinases (PIKKs) family (Shiloh, 2003). These include ATM (ataxia-telangiectasia mutated), ATR (ataxia telangiectasia-mutated and Rad3- related), 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 intercon- nections 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 Figure 2 Representative schematic of the two major checkpoint of major tumor suppressor pathways of the cell: the p53 the cell cycle and involvement of some of the major tumor and Retinoblastoma pathways (Hahn and Weinberg, suppressor gene products. The ATM and ATR kinases are involved 2002). p53activates the G1/S checkpoint mainly by in the signaling of DNA damage. These kinases phosphorylate a number of downstream mediators, including p53and Chk inducing the cyclin-dependent kinase (CDK) inhibitor (Checkpoint kinases 1 and 2). Phosphorylation of p53activates WAF p21 . Alternatively, p53can induce apoptosis because this transcription factor that in turn promotes synthesis of p21WAF, it regulates the transcription of proapoptotic proteins. which inhibits the cyclin/CDK complexes, thus activating the G1/S Retinoblastoma family member activate the G1/S checkpoint. If DNA damage cannot be repaired, p53promotes checkpoint by binding to and inhibiting the E2F apoptosis. p53is also involved in the establishment of the G2/M checkpoint. The tumor suppressor p16INK4A is involved in inhibition transcription factors (the latter positively regulate the of CDKs, thus contributing to the G1/S checkpoint. The transcription of a wide number of genes responsible for checkpoint kinases halt the cell cycle by phosphorylating (thus cell cycle progression). Retinoblastoma proteins can be inactivating) cdc25 protein family members. Cdc25 are phospha- inactivated through phosphorylation by CDK (mainly tases that activated cdc2 complexed with cyclin B. The latter complex is the mytosis-promoting factor (MPF), which is the CDK4 and 6) bound to G1 cyclins (cyclins D and E complex that regulates cell entry into the M phase 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 (Levine et al., 1994). More recent data indicated that phenomenon is accompanied by a dramatic increase of tumor suppressor genes are not exclusively proteins the p16INK4A CDK inhibitor (Wei et al., 2001). The CDK involved in checkpoint control, but that different inhibitor p16INK4A binds to CDK4 and CDK6 com- proteins having functions apparently unrelated to the plexed to D-type cyclins displacing the latter from their control of the cell cycle can nevertheless be important association with CDKs. The result of this action is the for cancer prevention. One good example is provided by inactivation of CDKs, which in turn are not able to dyskerin, a protein involved in the modification of phosphorylate Retinoblastoma family members and, as uridine residues of ribosomal RNA into pseudouridine a consequence, the cell remains blocked in G1 and therefore important for ribosomal biogenesis. (Malumbres and Barbacid, 2001). These data underscore Dyskerin-deficient mice display signs of human dysker- the importance of the Retinoblastoma tumor suppressor atosis congenita, a disease characterized by premature pathway in the establishment of senescence in human aging and cancer susceptibility, and are markedly cells. However, p53is also considered to contribute to susceptible to cancer development (Ruggero et al., senescence in human cells (Macip et al., 2003; Horner 2003). et al., 2004), especially in the process of replicative Mutations (both point mutations and deletions) are senescence (see below). Loss of p53is important not the only way through which a tumor suppressor not only to evade senescence. Tumor cells are char- gene can be inactivated. In recent years, the importance acterized by a high degree of chromosomal instability of epigenetic changes in the establishment of the (Rangarajan and Weinberg, 2003). In such environment, malignant phenotype has been underscored. Some an intact p53pathway would invariably cause G1 arrest tumor suppressor genes are not transcribed because of or p53-dependent apoptosis. Therefore, it is not surprising methylation of their promoter. Common examples of that p53itself is lost in about 60% of all human cancers tumor suppressor genes that are inactivated because

Oncogene Molecular mechanisms of malignant transformation M Bocchetta and M Carbone 6488 of hypermethylation of their promoter are represented crisis (Chin et al., 1999). Therefore, telomeres work as a by those encoded by the INK4A locus (Myohanen et al., cellular hour glass that determines how many replication 1998) and RASSF1A (Agathanggelou et al., 2001; rounds a cell can afford. Germinal cells and most of Dammann et al., 2001). tumor cells evade this phenomenon (and therefore The expression of tumor suppressors can be lost achieve immortality) through the activity of a ribo- because of differential splicing. One example is protein complex called telomerase. This enzyme is a provided by IG20, a splice variant of MADD (MAP- reverse transcriptase that adds TTAGGG repeats to the K activating death domain-containing protein). IG20 is telomeres, thus preventing their shortening and the a protein interacting with the tumor necrosis factor consequent induction of senescence and apoptosis. alpha (TNFa) receptor (Al-Zoubi et al., 2001) and Telomerase has an RNA component (TERC) that is TNF-related apoptosis-inducing ligand) (TRAIL) re- universally expressed in human cells, and functions as ceptor (Efimova et al., 2003). Through its activities, template for the protein component of telomerase to IG20 sensitizes the cell to apoptotic stimuli and induces synthesize TTAGGG tandem repeats (Goytisolo and the secretion of interleukin (IL)-6, thus suppressing Blasco, 2002). The protein component of telomerase tumor cell growth (BS Prabhakar, personal commu- (TERT, the reverse transcriptase) is not expressed (or nication). In certain tumors, IG20 expression could be expressed at insufficient levels) in most somatic cells. lost because of perturbed splicing of the IG20 gene For this reason, somatic cells cannot restore the length primary transcript leading to overexpression of an of telomeres during cell proliferation and undergo IG20 splice variant (DENN-SV) that induces cell replicative senescence and crisis. As a result of muta- proliferation and resistance to apoptosis (Efimova tions, or of viral infection in certain cells (Foddis et al., et al., 2004). 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 Immortality is acquired at chromosomes’ ends become clinically evident (Reddel, 2000). This issue was raised from the observation that breast cancer is a very The ends of all eucaryotes’ chromosome are organized fastidious tissue to obtain cell lines from, arguing into structures termed telomeres. Human telomeres are against the case that these tumors are truly made of a 5–15 Kbp long, and contain tandem repeats of the uniformly immortal cell population. However, more sequence 50-TTAGGG-30 (Moyzis et al., 1988). Telo- than 85% of human cancers (including breast cancer) meres end in a 30 overhang (about 300 nucleotides long) have readily measurable telomerase activity (Shay and that invades the double-stranded portion of telomeres to Bacchetti, 1997). Many of those cancers in which form a lariat structure also known as T loop (Griffith telomerase activity is not detected have an alternative et al., 1999). Several proteins are bound to the T loop process to preserve telomeres length called ALT (alter- and contribute to its stability and/or signal whether such native lengthening of telomeres; Bryan et al., 1997). structure is perturbed. Telomere’s end is structured this ALT cancer cells are characterized by unusually long way because a terminal DNA sequence of a putative telomeres, and the process underlying the ALT pheno- linear chromosome would be detected as a double- type is believed to be nonreciprocal recombination strand break in DNA and it would trigger DNA repair between telomeres of different chromosomes (reviewed response, including activation of a cell cycle checkpoint in Neumann and Reddel, 2002). All these evidences and/or apoptosis. Furthermore, unprotected chromoso- indicate that cell immortalization is a necessary event for mal ends would lead to end-to-end chromosomal fusion, malignant growth. thus leading to mitosis catastrophe (Goytisolo and Blasco, 2002). One characteristic of DNA replication is that the ends of linear DNA molecules are progres- sively shortened at each round of replication because Dual role in cancer pathogenesis: the immune system DNA polymerase uses RNA primers that are degraded after elongation, a phenomenon known as ‘end replica- Researchers are increasingly focusing on the interplay tion problem’ (Watson, 1972). Bacteria solve this between cancer cells and the surrounding stromal problem by having a circular genome, while bacter- tissues. It appears more and more evident that the iophages with a linear chromosome arrange it in tandem microenvironment surrounding cancer cells plays a concatemers, therefore limiting the importance of pivotal role in tumor promotion, vascularization. and terminal DNA sequences. In most normal somatic cells, metastatization (Coussens and Werb, 2002). The phy- instead chromosomes shorten at each round of cell siological rearrangements of connective tissue are replication (Harley et al., 1990). This progressive erosion largely under the control of the various members of of chromosomes ends ultimately results in progressive the immune system (Schreiber, in press). The fact that shortening of telomeres. When telomeres shorten the immune system could have played a major role in beyond a critical threshold, they start to become tumor containment and, at the same time, promotion structurally dysfunctional. This process eventually leads was long known. In the late 19th century, it was shown to activation of the p53signaling pathway with that a minority of terminally ill cancer patients showed consequent establishment of replicative senescence or partial or complete remission of their malignancy when

Oncogene Molecular mechanisms of malignant transformation M Bocchetta and M Carbone 6489 injected with a mixture of bacterial toxins (the so-called et al., 2000). IFN-g is a multifactorial cytokine that Coley extract) in order to cause a potent inflammatory probably mediates a wide variety of cell effects, but one response (Nauts et al., 1953). These data indicated that a possible mechanism to explain these data is that IFN-g massive inflammatory stimulus can trigger a generalized can upregulate MHC class I molecules and therefore reorganization of the immune systems capable of render cancer cells easier targets for T cell-mediated activating a response against the tumor cells (even if targeting (Shankaran et al., 2001). Other experiments the primary stimulus was not specific for the tumor conducted in immunocompromized mice showed a itself). On the other hand, it is also known that cancer protective role for the immune system against cancer, arises preferentially within a context of chronic inflam- although other mice models, especially those deficient in mation. Currently, about 15% of all human tumors are the production of certain cytokines yielded opposite attributable to infectious agents, and inflammation is a effects. These apparently paradoxical results indicate critical component of these chronic infections (Parkin that in cancer biology it is impossible to envision the role et al., 1999). of the immune system in just one direction. Despite a Experimental evidences have underscored that in the wide variety of possible mechanisms of self-defense, the early stages of cancer, the presence of consistent immune system has obviously lost its battle against intratumoral lymphocyte infiltrations are strongly cor- cancer when the disease finally becomes clinically related with reduced metastasis formation and pro- manifest. The principle of boosting this compromised longed patient survival (Clark et al., 1989; Naito et al., immune system following a wide variety of strategies in 1998). Indeed, both innate and adaptive immunity can order to fight clinically evident cancer has been the fight cancer development. Innate immunity cells deci- central hypothesis of Cancer Immunology. A number of pher pattern-recognition of receptors and other trans- studies are presently in progress to produce therapeutic membrane proteins expressed by cancer cells and cancer vaccines, or to infuse genetically modified directly target them. The main cellular players in this immune cells into cancer patients to trigger a cancer- mechanism of recognition are natural killer cells (NK). specific immune response. However, immune depressed The latter recognize stress-related proteins on the patients (e.g. AIDS patients, patients who are immuno- surface of cancer cells (Diefenbach and Raulet, 2003). compromised following organ transplant, etc.) do not NK can also recognize cells with decreased expression of develop an excess of malignancies other than those that HMC class I, a common feature in cancer cells (Karre, are virally induced (e.g. EBV-related lymphomas, 2002). Dendritic cells (DC) also play an essential role. Kaposi sarcoma, etc.). This indicates that the immune They can phagocytose apoptotic cancer cells, a process system has a much more limited role in cancer eventually leading to activation of adaptive immunity prevention than originally hypothesized and that this against cancer cell-specific antigens. DC also phagocy- role is mainly limited to malignancies that express tose heat-shock proteins (HSP) released by necrotic unique antigens, such as viral proteins. Moreover, also cancer cells. HSP released in the extracellular space for virally induced cancers, the immune system can activate inflammation (Basu et al., 2001). Adaptive promote cancer growth, such as in the case of Hepatitis immune system targets tumor cells indirectly. Tumor C infection that leads to chronic inflammation, which in cells potentially express a number of tumor-specific turn can result in the development of HCC (Carbone antigens (deriving from mutations of wild-type pro- et al., in press). teins), and therefore should be easy targets for CD4 þ As stated above, cancer and inflammation have been and CD8 þ T cells. However, cancer cells decrease the correlated. A number of pathologies characterized by level of expression of major histocompatibility complex chronic inflammation are strikingly associated with a (MHC) class I molecules and that of critical immuno higher cancer incidence. Individuals who have taken costimulatory proteins such as the member of the B7 aspirin and other cyclooxygenases inhibitors over a protein family, which are required for efficient T- period of several years have a 40–50% reduced risk of dependent response (Sharpe and Freeman, 2002). This developing colon cancer (Garcia-Rodriguez and Huerta- problem is overcame by DC that, after phagocytosis of Alvarez, 2001). There are a number of reasons why tumor cells, debris migrate to lymph nodes and activate inflammation could play a landscaping role for cancer a tumor-specific T response (Banchereau and Steinman, development, mainly due to the action of proinflam- 1998). A limitation of this process (termed ‘cross matory cytokines secreted by various component of the priming’) is that DC infiltrating cancer tissues are often innate immune system, including macrophages, mast immature, a process mediated by a quite complicated cells, neutrophils, eosinophils, and other members. interplay between tumor cells and immune cells in which However, tumor-associated macrophages are increas- cytokines play a major role (see below). The importance ingly implicated as possible tumor-promoting cells. of the immune system in the protection against tumor These macrophages produce a wide array of cytokines, formation is underscored also by a number of studies proteases, angiogenic factors, all of which may play an conducted in experimental animals. Mice defective for important role in tumor progression and dissemination interferon g (IFN-g, a cytokine produced mainly by T (Schoppmann et al., 2002). Additionally, tumor-asso- cells and NK and, to a lesser extent, by DC and ciated macrophages secrete the proinflammatory che- macrophages), or for other cytokines important for mokine TNFa (Torisu et al., 2000). This cytokine, IFN-g production display an increased susceptibility to through induction of the NFkB signaling pathway, chemical carcinogenesis (Kaplan et al., 1998; Smyth 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 Conclusions reactive oxygen species (ROS) and other potentially mutagenic molecules produced during infection (Mae- Our knowledge about the molecular mechanisms da and Akaike, 1998). Another powerful cytokine required for tumor formation has tremendously im- produced by macrophages and T cells is macrophage proved during the past 40 years. Key molecular migration inhibitory factor (MIF). MIF inhibits p53 mechanisms required for malignancy have been identi- transcriptional activity (Hudson et al., 1999). There- fied (Carbone et al., in press). It can be easily predicted fore, this cytokine may promote extended lifespan, that new oncogenes will be identified in the future; accumulation of mutations due to impaired p53 however, it can be predicted that these new ‘cancer function, inhibition of p53-mediated apoptosis. This genes’ will affect those pathways required for tumor situation may explain why inflammation may be fertile formation described above. Thus, the apparent com- ground for cancer development. Inhibition of p53 plexity of tumor formation can be reduced to a much functions, paralleled by NFkB activation, provide cells simpler picture. Regardless of the specific mechanism by with survival and proliferative stimuli in the absence of which a given gene pathway is altered, carcinogenesis the major tumor suppressor gene product of the cell. requires the activation or inactivation of the pathways Potentially mutagenic substances produced during we have discussed in this review. infection may introduce mutations into cellular proto- Cancer is a multifactorial event in which numerous oncogenes, thus allowing the mutated cell to survive alterations contribute to the emergence of the malignant the activities of an active oncogene. Furthermore, as a cell. Tumor cells per se can be innocuous unless response to pro-inflammatory cytokines, mast cells additional factors such as tumor promoters stimulate release a number of proteases, including heparin, cell growth (hormones, chronic inflammation, etc.). heparanase, matrix metalloproteinases and serine pro- Moreover, tumor cells that express viral antigens or teases, all thought to play a key role in tissue dynamics some other type of unique antigen can be eliminated by and in metastatic dissemination of cancer cells the immune system. Therefore, malignant tumor growth (Balkwill and Mantovani, 2001). A role for certain is a dynamic process in which it is difficult to identify a proinflammatory cytokines in tumor formation has unique event that caused the process. Rather, it is been demonstrated in experimental animals. TNFa possible to identify numerous events that together knockout mice are resistant to skin cancer induced by contributed to malignancy. This information should different chemical carcinogens (Moore et al., 1999), allow scientists to identify those agents that are more while mice homozygous for the IL-6-null allele (À/À) likely to cause or to contribute to cancer before they are resistant to plasma cell tumor development (Hilbert have caused a substantial number of cancer deaths et al., 1995). Other proinflammatory cytokines, such as detectable by classical epidemiology. Thus, it is hoped colony-stimulating factor 1 (C-SF1) (Lin et al., 2001) that the integration of molecular pathology with and IL-1 (Voronov et al., 2003), proved to be required classical epidemiological and animal studies will lead for tumor formation in experimental animals. In to a more rapid and accurate identification of human conclusion, the immune system is one of the most carcinogens. important lines of defense for tumor formation for malignancies that express unique antigens, such as viral Acknowledgements proteins. The immune system can also promote tumor Dr Carbone’s work is supported by grants from the National growth through the stimulatory and mutagenic effects Cancer Institute, USA, RO1CA92657; the American Cancer of cytokines released by inflammatory cells (Wogan Society, RSG-04-029-01-CCE, and by the Cancer Research et al., in press; Schreiber, in press). Foundation of America.

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