Oncogenes and Tumor-Suppressor Genesin Mesothelioma A SynopsisJohn F. Lechner,l Johannes Tesfaigzi,l andBrenda 1. Gerwin2'Lovelace Respiratory Research Institute, Albuquerque, New Mexico;2Laboratory of Human Carcinogenesis, National Cancer Institute,National Institutes of Health, Bethesda, Maryland

Invariably mesothelioma is diagnosed late in the development of the disease when treatment isno longer effective. Therefore, a key to reducing the mortality rate of this neoplasm is knowledgeof the general sequence of genetic events between initiation of mesothelial cells and theemergence of the metastatic tumor cells. Unfortunately, relatively little is known about the earlychanges in the genesis of this disease. Of the known changes, the most frequent are in thetumor-suppressor genes p 161NK4 and NF2 and possibly the SV40 virus large T-antigen oncogene.The molecular nature of the changes in these genes as well as other alterations are addressed inthis overview. Environ Health Perspect 105(Suppl 5):1061-1067 (1997)

Key words: oncogenes, tumor-suppressor genes, PDGF, p1&NKa, SV40 T-antigen, NF2,mesothelioma, human, fibers, cancer

Delineating the genetic changes thatproduce a cell with uncontrolled and oftenunlimited growth potential is important tothe understanding of carcinogenesis mech-anisms. Knowledge of these molecularprocesses also enhances the design of earlydetection and therapeutic protocols.Unfortunately, relatively minimal informa-tion is available on cancers associated withfiber exposure. This overview summarizesthese observations, with primary emphasison the alterations that have been describedin human mesotheliomas.

This paper is based on a presentation at The SixthInternational Meeting on the Toxicology of Naturaland Man-Made Fibrous and Non-Fibrous Particlesheld 15-18 September 1996 in Lake Placid, NewYork. Manuscript received at EHP 27 March 1997;accepted 11 July 1997.

This work was supported in part by the U.S.Department of Energy/Office of Health EffectsResearch (contract DE-AC04-76EV01013).

Address correspondence to Dr. J.F. Lechner,Building 9200, Area Y Kirtland AFB East Albuquerque,NM 87115. Telephone: (505) 845-1121. Fax: (505)845-1193. E-mail: jlechner@lm.org

Abbreviations used: CDK, cyclin-dependent kinase;CKI, cyclin-dependent kinase inhibitor; EGFR, epider-mal growth factor receptor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-monocytecolony-stimulating factor; IGF-1, insulinlike growth fac-tor 1; IL, interleukin; LIF, leukemia inhibitory factor;MCP-1, monocyte chemotatic protein-1; M-CSF,macrophage colony-stimulating factor; NF2, neurofi-bromatosis type 2; PCR, polymerase chain reaction;PDGF, platelet-derived growth factor; Rb, retinoblas-toma; T-ag, SV40 virus T-antigen; TGF-P, transforminggrowth factor beta; TNF-a, tumor necrosis factor alpha;WT1, VWilms' tumor.

Molecular Pathwaysof Growth Control

Progression of eukaryotic cells through theircell cyde is regulated by the sequential forma-tion, activation, and inactivation ofa series ofcyclin/cydlin-dependent kinase (CDK) com-

plexes. In addition to positive regulation bythe activation of cyclin/CDK complexes,negative regulation of the cell cycle occurs

through cyclin-dependent kinase inhibitors(CKIs) to prevent premature entry into thenext phase of the cyde before completion ofnecesay macromolecular reactions (1-3).

Cell replication begins at the restrictionpoint (R Figure 1), where growth and arrestsignals from the extracellular environment are

integrated to determine whether the celldivides or remains quiescent (1-3). Forexample, elevated expression of platelet-derived growth factor (PDGF), which is a

hallmark of mesothelioma, can elevate theconcentration of c-myc in the nudeus. c-myc

then induces the expression of cyclin Ds, andthe balance between cycin D/CDK4 and itsCKI is tipped toward more rapid cell division(3). Once a growth-positive balance of thesefactors has developed, the cell is committedto traverse the cell cyde.

In the first phase (Gi), different speciesofcydin D (DI, D2, D3) are expressed andform complexes with CDK4 and CDK6(1,2,4). The activated cyclin/CDK com-

plexes phosphorylate the retinoblastoma(Rb) gene product and its related proteins

(pI07, p130). When these proteins arehypophosphorylated, they are complexeswith transcription factors, e.g., E2F; hyper-phosphorylation of the Rb protein releasesthese transcription factors (1,2,4-6), whichthen stimulate the transcription of mRNAsthat encode proteins required for the cell toprogress further through the cell cycle(1,2,4,5). The next complex, cyclinE/CDK2, further phosphorylates Rb familyproteins, and the cell begins to synthesizeDNA. Once the cell enters S phase, i.e.,begins to synthesize DNA, a complexbetween cyclin A and CDK2 kinase forms.This complex, whose role is unclear, isdegraded as the cell enters the G2 phase.Finally, the cyclin B/CDC2 complex phos-phorylates proteins involved in chromo-some condensation and the progression ofthe cell through mitosis, e.g., HI histone,nuclear lamins, nudeolin, and intermediatefilaments (1,2).

Two checkpoints in the cell cyde providethe cell with opportunities to govern its rateof cycling. The first, which is at the G1/Sborder, employs proteins denoted as CKIs.Currently, two structurally defined classesare known. The first, exemplified bypl6INK4a and pi5INK4b and induding p19and p18, primarily regulates CDK4 andCDK6 (7,8) by binding to the associatedcydins (9). The second family, characterizedby 21cip1, and including p27KIPl andp57KP2, regulates the activities of the CDK2and CDK4/6 complexes (1,2,4). As will bediscussed later, aberrations in the G1/Scheckpoint function are associated withmesothelioma. Inhibitor proteins that pre-vent the activation of the cyclin B/CDC2complex at the G2/M checkpoint also havebeen described (1,2), but no correlationsbetween these proteins and fiber-caused car-cinogenesis have been reported. However,because disruption of mitotic processes is asalient feature of cell-fiber interactions(10-14), examining mesothelioma cells foraberrations of the G2/M checkpoint genesmay identify one or more that are highlycorrelated with mesothelioma.

Expression of CKI genes is stimulatedby stress factors. For example, damagedDNA initiates events that cause the p53gene product to accumulate within the cellnucleus (15,16). This protein induces thetranscription of certain genes whileinhibiting others. Two major regulatoryproteins induced by the accumulation ofp53 are the kinase inhibitor p2lCiPl and theprotooncogene mdm2 (15). The ability of

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LECHNER ET AL

Figure 1. Relationships of genes involved in mesothelial cell carcinogenesis to the cell cycle.

p53 to arrest cell growth can be explainedin part by its ability to induce the kinaseinhibitor p21CiPl, which inhibits the actionof the G 1 phase cyclin-CDK complexes(1). During the arrest period, the DNAdamage is repaired. p53 also initiates tran-scription of the protooncogene mdm2,which then inactivates the p53 protein.This decrease in p53 activity leads to thefall in concentration of p21CiP1 protein, andthe cell resumes its progress through the cellcycle (17).

The cell cycle is permanently stoppedby programmed cell death, e.g., apoptosis.Overabundance of the Bax protein pro-motes apoptosis; on the other hand, excessBcl-2 protein can extend cell survival bypreventing apoptosis. Excessive damage toDNA elevates the concentration of the p53gene product, which transactivates expres-sion of the Bax protein (18). This mecha-nism is responsible for apoptotic death of apotentially mutant cell. However, if p53 isdysfunctional or is inactivated by bindingwith excess mdm2 protein, Bax levels remainlow and the mutated cell can survive.Alternatively, inappropriate expression ofvarious growth factors or growth factorintermediates can cause overexpression ofBcl-2, which prevents programmed celldeath (18), in part by inhibiting expressionof p21ClPl (19). Consequently, mutatedcells continue to replicate.

Tumor-Suppressor Genesin MesotheliomasTumor-suppressor genes check and regulatecell division. In general, these genes canbecome aberrant by point mutation, partialdeletion, inappropriate expression, epige-netic silencing, gene amplification, gene

rearrangement, complete gene loss, orcombinations of these mechanisms. Allcould be involved in the genesis of amesothelioma from exposure to asbestos.However, a hallmark of human (10-14,20-34) and rodent (35-37) mesotheliomasis the large number of their nonrandomcytogenetic alterations. For human tumors,these include monosomy or partial mono-somy of chromosomes 1, 3, 4, 6, 9, 14, 15,18, 19, and 22, and trisomies and polysomiesof chromosomes 1, 5, 7, 11, 12, and 20.This array of nonrandom chromosomedeletions in human mesotheliomas suggeststhat many tumor-suppressor genes andoncogenes are probably involved in the gen-esis of the disease. The tumor-suppressorgenes located at bands lp21-22, 3p2l,6ql5-21, and 22q will be identified even-tually because these cytogenetic regions areinvolved in the genesis of other human can-cers (21,26,28,30-34,38). However, cur-rent information confirms the involvementof only the following five tumor suppressorgenes, i.e., pl6NK4, pl5INK4b, p53 NF2and WT1.

Cytogenetic analyses have shown thatchromosome bands 9pl3 to p22 are deletedin 50% of mesotheliomas. Two knowntumor suppressors have been mapped tothis region: the CDK4,6 inhibitorsp 16INK a and p 1 5INK4b (39). Cheng et al.(40) showed that 85% of the mesothelialcell lines they investigated had a homo-zygous deletion of p16INK4a. However, only22% of the primary tumors showed thisdeletion. In contrast, Xiao and co-workers(39) reported that both of these genes weredeleted in more than 70% of primar1mesotheliomas. A similar loss of p1 6INK awas recently reported by Kratzke et al. (41).

In addition, these authors showed thatwhen pI 6INK4a was transfected intomesothelioma cell lines, their growth rateswere inhibited 5- to 10-fold. In parallel,Spillare et al. (42) showed a 50% inhibi-tion of colony-forming efficiency whenp16INK4a was transfected into culturedmesothelioma cells. Several primarymesotheliomas exhibit cell heterogeneityfor expression of p16INK4a and p15INK4bimplying that the loss of these genes was alate event (41). On the other hand, a pri-mary consequence of asbestos exposure isextensive chromosome alterations (11,24).Thus, it is also possible that these geneswere deleted as a direct result of a fiber inter-acting with chromatin or spindle proteins(43). In human lung tumor cells, loss ofpl6INK4a is inversely related to retainingthe Rb tumor-suppressor gene, and viceversa (41). Mesotheliomas follow this ruleas well; three studies have shown that Rb isnot deleted in these tumors (44-46).

The p53 tumor-suppressor gene, whichresides in band p13 of chromosome 17,shows the most frequent rate of geneticalteration in human cancers, especially ofthe lung (15). In support of this statement,Cote et al. (47) found that three of fourmesothelioma cell lines exhibited an abnor-mal p53 gene. Specifically, two had pointmutations, and the third did not transcribep53 mRNA. One of the cell lines with apoint mutation, as well as the nontranscrib-ing line, also showed loss of heterozygosityfor 17p. However, Metcalf et al. (48) ana-lyzed 20 mesothelioma cell lines and foundthat only 2 had point mutations, and 1failed to transcribe p53. These latter data arein accord with the fact that mesotheliomaswith deletions of chromosome 17p havebeen reported only occasionally (22,24).These data on human mesothelioma con-trast with murine data where frequent(76%) rearrangement of the gene was foundand p53 mRNA expression was frequentlyreduced or absent (49).

p53 overexpression in the nucleus oftenreflects abnormal p53 function (15).Metcalf et al. (48) reported that three of thelines without mutations exhibited strongstaining of p53 protein, while most of theothers showed some positive cells. Further,Mayall et al. (50), Kafiri et al. (51), andSegers et al. (52) reported strong immuno-staining of p53 protein in tumor sections.As noted by Metcalf et al. (48), p53 expres-sion without gene mutation suggests upreg-ulation of p53 because of overexpression ofc-myc, or perhaps the stabilization of theprotein because of complexing with an

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oncogene. The latter mechanism is sup-ported by Segers et al. (52); 60% of the p53immunopositive but nonmutated tumorsthey evaluated also overexpressed the p53-inactivating oncogene mdm2. However,Ungar et al. (53) recently reported thatmdm2 was not overexpressed in the 17mesothelioma cell lines they analyzed.A second p53-inactivating mechanism

could be the expression of the T-antigen(T-ag) of the SV40 virus. In cells that havebeen infected, the p53 protein apparentlyis overexpressed because it is complexedwith large T-ag. This virus causes mesothe-liomas when injected into the pleural spaceof hamsters (54). Further, Carbone et al.(55) recovered fragments of SV40-likeDNA sequences from 60% of the meso-theliomas they examined by polymerasechain reaction (PCR). In addition, 79% ofthe tumors exhibited SV40 T-ag whenassessed using immunohistochemistry, andthere was a high correlation between T-agimmunoreactivity and the presence of SV40sequences. Subsequently, Cristaudo et al.(56) found the same SV40-like DNAsequences in 72% of the mesotheliomasthey investigated. In contrast, Metcalf et al.(48) found no evidence of SV40 T-ag inany of the 20 mesothelioma cell lines theyinvestigated using immunohistochemistry.Further conflicting information, however, isin the recent report by Galateau-Salle et al.(57). These authors showed that SV40-likesequences are found not only in somemesotheliomas but also in pleural plaques,lung tumors, parenchyma distal to thetumors, and in parenchyma of individualswithout cancer.

The NF2 autosomal dominant tumor-suppressor gene resides on chromosome 22(58). As noted above, this chromosomefrequently is abnormal in mesotheliomas(24). Consequently, this gene has beenevaluated in several cell lines. The resultsshow that frameshift and nonsense muta-tions, and deletions of small portions of theNF2 gene were often present in cell lines(58,59). These aberrations also have beenfound frequently in primary mesothe-liomas (59), but they are rare in humanlung carcinomas (59). NF2 codes for aprotein called merlin, which may play arole in cell surface dynamics and structureby linking the cytoskeleton to the plasmamembrane (58). Asbestos fibers are knownto be more efficient in deforming thecytoskeleton in mesothelial cells than inairway epithelial cells (10). Thus, loss ofstabilizing function supplied by a normalNF2 gene product may be involved in the

mechanism of fiber-caused transformationof the mesothelial cell.A small percentage of mesotheliomas

exhibit a cytogenic aberration-an intersti-tial deletion of bands 1ipl1 to l 1pl3 (24),where the transcription factor tumor sup-pressor gene WTI resides (60). For exam-ple, Park et al. (61) reported a homozygousmutation of this gene in a peritonealmesothelioma. Normal mesothelial tissue ofboth humans and rats abundantly expressesthis gene (60-63). Amin et al. (62) foundthat WTI mRNA was undetectable in 3 of19 mesothelioma cell lines and in 3 of 8mesothelioma tumors. In contrast, Walkeret al. (63) reported that the WTJ was ubiq-uitously expressed by the human and ratmesothelioma cell lines they studied,although there was a relatively lower expres-sion of the gene in the lines that gave rise tosarcomatous tumors in nude mice. Thislatter relationship could not be confirmedby Langerak et al. (60). It is possible, how-ever, that an aberration in WTI expressioncould stabilize and sequester p53 protein(64), thereby explaining the frequentimmunohistochemical detection of p53in mesotheliomas.

Oncogenes in MesotheliomasProtooncogenes are genes that promote celldivision, and alterations in protooncogenesmay contribute to uncontrolled cell growth.As noted above, nonrandom rearrangementsand polysomy of chromosomes 1, 7, and 22are also hallmarks of human mesotheliomas.These structural aberrations can generategrowth-promoting oncogenes by rearrange-ments and amplification. Using Southernblot analyses, Tiainen et al. (65) examined23 mesotheliomas and found no rearrange-ment and amplifications of several onco-genes located on these chromosomes, i.e.,N-ras, epidermal growth factor receptor(EGFR), Met, and PDGF-B chain. Further,Kishimoto (66) could find no immunohis-tochemical evidence of overexpression ofEGFR. On the other hand, Ramael et al.(67) detected amplifications or rearrange-ments of EGFR in all three of the mesothe-liomas they examined using a more sensitivePCR assay. Thus, reevaluation of the knownoncogenes on chromosomes 1, 7, and 22using PCR approaches may reveal addi-tional loci that are frequently rearranged oramplified in mesotheliomas.

Metcalf et al. (48) found no activatingmutations of the K-ras oncogene in the20 mesothelioma cell lines they exam-ined. Further, Lee et al. (68) reported thatoverexpression of ras protein is a rare event

for mesotheliomas. More recently, how-ever, Kishimoto (66) did find frequentimmunostaining of N-ras, and Ramaelet al. (69) found that more than 50% ofthe cells within 78% of the mesotheliomasthey evaluated had immunoreactivity toN-ras antibody. Notably, N-ras is locatedon chromosome 1 which is often polysomicin mesotheliomas. Finally, sustainedexpressions of c-jun and c-fos is induced byfibers (70), and c-myc, along with c-neuwere found to be overexpressed in themajority of the mesotheliomas of Japanesepatients (66).

Many exogenous peptides increase thegrowth rate of mesothelial cells (71-74),and mesothelioma cells produce severalgrowth factors and cytokines (71,75-79).Oncogenes often cause inappropriateexpression of growth factors and/or theirreceptors. As a consequence, normal growthcontrol mechanisms are abrogated.Oncogenes causing the production ofgrowth factors that simulate autoreplica-tion are referred to as autocrines (80).Autocrine activity, shown not to be PDGF,TGF-,, or EGF, has been detected in theconditioned medium of a mesotheliomacell line; however, the actual factor has notbeen identified (81). Nonetheless, repeat-edly observed alterations in the expressionof PDGF proteins and the associatedPDGF receptors by mesotheliomas havesuggested that this ligand and receptor pro-duce an autocrine loop in many mesothe-liomas (77,82). Several laboratories haveexamined this possibility, with somewhatconflicting and puzzling results.

PDGF-A-protein and PDGF-B-proteinchains dimerize to form three species ofPDGF that are each mitogenic formesothelioma cells. One is A-chainhomodimers and one is B-chain homod-imers; the third is an A/B-chain het-erodimer. The PDGF-A chain andPDGF-B chains are coded by separategenes, as are the two receptor proteins, aand 3 (83). The A/B-chain heterodimerand the B-chain homodimer are recog-nized by the 3 receptor. All three forms ofPDGF bind the a receptor, although withdiffering affinities. Therefore, alterations inany of four different genes could affectgrowth of mesothelial cells. Normalmesothelial cells express the a and j recep-tors (82-89). Mesotheliomas often over-produce PDGF A-chain and B-chainproteins and low levels, if any, of the ,Breceptor. Therefore, the PDGF-B-chainprotein produced by the mesotheliomasmay serve as an autocrine for continuous

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replication of the tumor cells (82,85,89).However, mesothelioma cells also expressthe PDGF-A-chain growth factor, and thea receptor has been detected using RNaseprotection assays and recently by run-offanalyses (83). In addition, transfectionexperiments with the immortal but nontu-morigenic human mesothelial cell MeT-5Ahave shown that overexpression of thePDGF-A chain will transform these cells tothe tumorigenic phenotype (90,91).Further, it has been shown using antisenseoligonucleotides that the PDGF-A chain,but not the PDGF-B chain, will inhibit thegrowth of mesothelioma cell lines but notthe MeT-5A cells (84). Thus, even thoughthe PDGF growth factors and their recep-tors are differentially expressed in humanmesothelioma cells compared to their nor-mal cell counterparts, the role of thesechanges in the transformation of mesothe-lial cells is unclear. Parenthetically, ratmesotheliomas do not produce any form ofPDGF, which suggests that fiber exposurescan produce dissimilar results in differentspecies (92,93).

The other growth factors and cytokinesthat are produced by at least some humanmesothelioma cells include TGF-a,TGF-,B1, TGF-P2, IGF-1, IL-la, IL-1 p,G-CSF, GM-CSF, M-CSF, IL-8, MCP-1,LIF, TNF-a, IL-6, and IL-8 (71,75-78).Both normal mesothelial cells andmesotheliomas produce IGF-1 andexpress the receptor for this growth factor(94). Further, a hamster mesotheliomacell line that was transfected with anIGF-1 receptor antisense plasmid exhib-ited both decreased growth in vitro andtumorigenicity (95). These observationssuggest that IGF-1 may function as anautocrine for both the normal and trans-formed cells. Peritoneal mesothelial cellssecrete detectable amounts of IL-1 andexpress the IL-1 receptor. However, an invivo role for IL-I as an autocrine for thesecells is unclear because exogenously addedIL-1 is required for long-term growth ofthe cells in culture (79). IL-6 has beenshown to be an autocrine for normalhuman mesothelial cells (76,80); itremains to be determined if IL-6 is anautocrine factor for mesotheliomas, aswell. IL-8, on the other hand, is only pro-duced by mesothelioma cells (75); it isnot known whether the receptor for thisfactor resides on the surface of mesothe-lioma cells. However, because IL-8 is amajor angiogenesis factor (75), it mayplay an important role in the growth ofthe mesothelioma tumor.

ereditary ~Asbestos OtherHereditary f~iber burden carcinogen

Chromosome/DNA changes

bersGenomic Cromosome

ognekFI~~~~> nstablt rearrangements Mehlto Anignss

Normal Myc 1, 14, 21, 7, 5, 12, p16 Mlgat Metastasismesothelial fos --22, 16 30 20 p53 masthlignant 11-8

cell Loss Polysomy PDGF meohloa cytokines

0 20-40 Years

Figure 2. Possible steps in the genesis of a mesothelioma.

ConclusionsAlthough information is minimal on themolecular changes that produce fiber-associated malignant mesotheliomas, theavailable data do show that point muta-tions, partial deletions, inappropriateexpression, gene rearrangement and com-plete gene loss, and possibly epigeneticsilencing and gene amplification probablyare all involved. Many of these aberrationsoccur in the genes regulating the initiationof cell division, checking cell division rate,maintaining the cytoskeleton, regulatingtranscription, and inducing angiogenesis. Itis also known that these aberrations areattributed to carcinogens other thanasbestos fibers. Thus, the molecular biologyunderlying the genesis of these tumors isnot significantly different from other neo-plasms attributed to nonfibrous carcino-gens; only the actual battery of geneticalterations seems to differ. The obser-vations of Meloni et al. (33) and Knuutilaet al. (24) along with the recent reportfrom Hansteen et al. (23) do, however,permit a hypothesized ordering of geneticchanges in the genesis of human mesothe-lioma (Figure 2). Their models disagree,but in general, they propose that losses ofchromosomes 6q, lp, and 22q may beamong the earliest events. Other areas ofdata inconsistency include the specificform of PDGF and PDGF receptor, thestatus of p53 and WTI, and the possiblerole of SV40 viral infection. Plausible rea-sons for these discrepancies include inher-ent species differences, dissimilar routes offiber exposure, and the creation of newalterations that manifested themselves dur-ing the development of cell lines. Somediscrepancies might be resolved by the

exchange of tumor specimens and cell linesas well as protocols and reagents.

Mesothelioma invariably is diagnosedlate in the development of the diseasewhen treatment is no longer effective(24). Therefore, a key to reducing themortality rate of this disease is knowledgeof the general sequence of genetic eventsthat transpire between initiation ofmesothelial cells and the emergence of themetastatic tumor cells. As with all tumorsystems, but especially for mesothelioma,considerable work remains to be done toclarify which genetic changes are the mostimportant in transformation of the normalcell. The ultimate resolution of thesechanges will permit the designing of anti-tumor treatment approaches that couldreverse the effects of the altered genes.Future work will require confirmation ofthe proposed model (Figure 2), clarifyingthe role of the above discussed geneticchanges, and elucidating the importanceof as yet uninvestigated mechanisms, e.g.,the induction ofgenomic instability (96,97),reduced DNA repair capacity (96), dis-ruption of programmed cell death (98),and inherent susceptibility (99).

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