Pattern of secondary genomic changes in pancreatic tumors of Tgfα/Trp53+/− transgenic mice

Pattern of Secondary Genomic Changes inPancreatic Tumors of Tgf�/Trp53�/� Transgenic Mice

Bettina Schreiner,1 Dorothee M. Baur,2 Alexander A. Fingerle,2 Ulrich Zechner,2,3 Florian R. Greten,4,5

Guido Adler,2 Bence Sipos,6 Gunter Kloppel,6 Horst Hameister,1* and Roland M. Schmid2,4

1Department of Human Genetics, University of Ulm, Ulm, Germany2Department of Internal Medicine I, University of Ulm, Ulm, Germany3Institute of Human Genetics, Johannes Gutenberg-University Mainz, Mainz, Germany4Department of Internal Medicine II, Technical University of Munich, Munich, Germany5Department of Pharmacology, University of California–San Diego, La Jolla, California6Department of Pathology, Christian-Albrechts-University, Kiel, Germany

Trp53�/� mice overexpressing Tgf� in a pancreas-specific manner represent a well-established animal model for pancreaticcancer. In this study we analyzed 38 pancreatic adenocarcinomas of these mice for secondary genomic changes by comparativegenomic hybridization (CGH), loss of heterozygosity (LOH) analysis, real-time PCR, and methylation-specific analysis. CGHscreening of the tumors revealed a recurrent pattern of genomic changes. In more than 50% of the tumors, chromosome 11was affected. The gain of the proximal part spans about 16 cM, including the genes for Egfr, Rel, and Stk10. The distal part ofchromosome 11, which contains the Trp53 locus, was deleted. LOH analysis proved that almost all tumors segregate thewild-type Trp53 allele. The Cdkn2a locus on chromosome 4 was inactivated by hypermethylation in 55% of all tumors. Inaddition, two other changes were detected in a mutually exclusive manner: overrepresentation of part of chromosome 15,or more rarely, loss of the distal part of chromosome 14. Together these data suggest the induction of a uniform pattern ofsecondary genomic changes in this transgenic tumor model for pancreatic cancer. © 2003 Wiley-Liss, Inc.

INTRODUCTION

Cancer is a multistep disease involving the pro-tracted acquisition of a series of mutations thathave differing properties. The nature and se-quence of these mutations vary from tissue to tis-sue because of the vastly different architecture andfunctions possessed by differing organs. These ge-netic changes have been investigated by use ofallelotyping and comparative genomic hybridiza-tion (CGH). Allelotype studies of human pancre-atic cancer have demonstrated recurrent loss ofheterozygosity (LOH) at multiple chromosome loci(Seymour et al., 1994; Hahn et al., 1995; Kimura etal., 1996; Shridhar et al., 1996). CGH analyses haveconfirmed these losses and have also revealed fre-quent chromosome gains (Solinas-Toldo et al.,1996; Fukushige et al., 1997; Mahlamaki et al.,1997; Schleger et al., 2000; Harada et al., 2002).Among these, frequent changes were gains of 3q,5p, 8q, 12p, 17q, and 20q, and losses of 3p, 6q, 8p,9p, 17p, 18q, and 22q. Despite the identification ofnumerous chromosomal regions that are consis-tently altered, only some tumor suppressors and afew oncogenes have been identified to be fre-quently affected in pancreatic cancer. These in-clude deletion or inactivation of CDKN2A, TP53,and MADH4, which have already been detected in

precancerous lesions (Moskaluk et al., 1997;Wilentz et al., 1998; Apple et al., 1999; Luttges etal., 1999; Wilentz et al., 2000). Among the domi-nantly acting oncogenes, KRAS2 mutations aremost common and have been reported in nearly90% of all cases analyzed (Rozenblum et al., 1997).Overexpression of the receptor tyrosine kinaseERBB2 is a further common finding (Day et al.,1996).

Tumor-prone transgenic mice offer powerfultools for investigating the molecular mechanisms ofoncogenesis (Jonkers and Berns, 2002). We re-cently characterized a transgenic mouse model thatspontaneously develops pancreatic cancer (Wagneret al., 1998). In these mice, the epidermal growthfactor receptor ligand Tgf� is targeted to mousepancreas by use of the acinar cell-specific elastase-1promoter/enhancer elements and promotes devel-

Supported by: Dr. Mildred Scheel Stiftung, Apoptoseschwer-punkt; Deutsche Forschungsgemeinschaft SFB 518.

*Correspondence to: Dr. Horst Hameister, Department of Hu-man Genetics, University of Ulm, Albert-Einstein Allee 11, 89070Ulm, Germany. E-mail: [email protected]

Received 5 May 2003; Accepted 1 August 2003DOI 10.1002/gcc.10285

GENES, CHROMOSOMES & CANCER 38:240–248 (2003)

© 2003 Wiley-Liss, Inc.

opment of acinar-to-ductal metaplasia (Sandgren etal., 1990; Song et al., 1999). Mice overexpressingTgf� develop pancreatic tumors with a long latency.When bred onto a Trp53�/� background, Tgf�transgenic mice develop invasive carcinoma char-acterized by LOH for the remaining Trp53 allele(Wagner et al., 2001). Tumor development is ac-celerated in these mice. However, it still takes 120days for tumors to develop in Tgf� transgenic micecompletely deficient for Trp53, suggesting that lossof Trp53 is not sufficient to overcome the G1/S-phase arrest (Wagner et al., 2001).

We therefore used these mice to facilitate thediscovery of genes that contribute to the pathogen-esis of pancreatic cancer in this model. We per-formed CGH on 38 pancreatic tumors fromTrp53�/� mice overexpressing Tgf�. We identifieda remarkably consistent set of genetic changes,such as loss of the distal part of chromosome 11(second allele of Trp53) and of chromosome 14(Rb1), and gains on chromosomes 11 (Egfr) and 15(Myc). Interestingly, alterations on chromosomes 14and 15 were mutually exclusive, suggesting thattwo distinct pathways can lead to G1/S-phase tran-sition in the mouse pancreas. Although it is sug-gested that both pathways affect cell cycle transi-tion, tumor progression is 2 months earlier withdeletion of chromosome 14 than in tumors withgain of chromosome 15.

MATERIALS AND METHODS

Tumor Specimens

The EL-Tgf�-hGH transgenic mice (line no.2261-3) and Trp53-deficient mice were previouslydescribed (Sandgren et al., 1990; Jacks et al., 1994).Two different series of animals were investigated.In the first series, progeny derived from matingsbetween C57Bl/6-EL-Tgf�-hGH transgenic miceand (C57Bl/6-Trp53�/� � BALB/c) � BALB/cbackcross mice (between backcross 3 to backcross6) were used. In the second series, C57Bl/6-EL-Tgf�-hGH transgenic mice and BALB/c-Trp53�/�

mice were crossed, resulting in F1 C57Bl/6-BALB/c progeny of Tgf�/Trp53�/� mice, whichwere completely informative for polymorphic ge-netic markers. After removal, tumor samples weredivided into three parts. One part was used forhistopathological evaluation. Areas of obvious puretumor tissue were identified, isolated by laser cap-ture microdissection, and DNA was prepared forLOH analysis. The second tumor sample was usedto establish a cell culture. From the third part,

genomic DNA was isolated for CGH and real-timePCR.

Histopathological Analysis

From each tumor sample, H&E-stained slideswere checked by two experienced pathologists(B.S. and G.K.). Immunohistochemical analysis forcytokeratin 8/18 and cytokeratin 19 was performedas previously described (Wagner et al., 2001).

Laser Capture Microdissection and LOH Analysis

Paraffin sections were used for laser capture mi-crodissection as described previously (Wagner etal., 2001). The genomic DNA was eluted and usedfor PCR amplification of three microsatellite mark-ers in close proximity to the following loci: Cdkn2aon chromosome 4, Trp53 on chromosome 11, Rb1on chromosome 14, and Madh4 on chromosome 18.The markers used were described earlier (Wagneret al., 2001).

Comparative Genomic Hybridization

CGH was performed as described by Kallioniemiand coworkers, with slight modifications as re-ported (Kallioniemi et al., 1992; Bruch et al., 1998).The tumor DNA was labeled with biotin-16-dUTPand the normal reference DNA with digoxigenin-11-dUTP, by use of standard nick translation.

Real-Time PCR Analysis

Genomic DNA amplification was quantified byuse of real-time PCR (TaqMan, ABI PRISM 7700Sequence Detection System; PE Applied Biosys-tems, Norwalk, CT). PCR reactions (denaturation95°C for 2 min followed by 40 cycles of 95°C for 15sec and 60°C for 1 min) were performed by use oftumor DNA as well as normal DNA as reference.The following target genes were tested: Egfr, Rel,Stk10, Flt4, Myc, Adcy8, Ptk2, and Junb, and Gcnt2 orAnt1 as endogene control genes. The followingprimers and concentrations were used:

Egfr-forward primer (FP) (300 nM) 5�-GCC CTCTTG CCC TGT GC-3�; Egfr-reverse primer (RP)(300 nM) 5�-GTC CGT AGT GTT AAC CTCTTG TGA AG-3�

Stk10-FP (300 nM) 5�-AGA TCG AGC CCCCAC ACC-3�; Stk10-RP (300 nM) 5�-CGA CTTGGC AAT CTT GAG CAG-3�

Rel-FP (900 nM) 5�-AAT AAT TGA ACA GCCAAG GCA GAG-3�; Rel-RP (900 nM) 5�-CCTGGG ATG GCT ACC TGC TGA-3�

241GENOMIC CHANGES IN MURINE PANCREATIC CARCINOMA

Flt4-FP (300 nM) 5�-GCC ACG CCA CCC TCAG-3�; Flt4-RP (300 nM) 5�-TTC ACA CAC GTAGTC ACC CTC G-3�

Myc-FP (300 nM) 5�-CAA GTT GGA CAGTGG CAG GGT-3�; Myc-RP (300 nM) 5�-TGTCCG CCT CTT GTC GTT T-3�

Adcy8-FP (300 nM) 5�-CCG GGC AAT ACTCTC TGG C-3�; Adcy8-RP (300 nM) 5�-GCCTTT GCC TGT TGA GAG AC-3�

Ptk2-FP (300 nM) 5�-CCT GGA GGA GGAGAA GGT GC-3�; Ptk2-RP (300 nM) 5�-ACA CGGTAG CCT GTC TTC TGG A-3�

Junb-FP (900 nM) 5�-CAC CGA GGA GCAGGA GGG-3�; Junb-RP (300 nM) 5�-GGC GTCACG TGG TTC ATC TT-3�

Gcnt2-FP (900 nM) 5�-GCC TGA ACT CTTAAT CCT CCA C-3�; Gcnt2-RP (300 nM) 5�-CAGACT GCC AGA GCC AGG TG-3�

Ant1-FP (300 nM) 5�-ATC CGG TAC TTCCCC ACT CAA-3�; Ant1-RP (300 nM) 5�-GGCGAT CCA CGC CTC C-3�

The relative quantitation of the target DNA levelwas calculated with the comparative CT methodaccording to the manufacturer’s protocol.

Cdkn2a Methylation Assay

Total genomic DNA (700 ng) was digested withEcoRI at 37°C for at least 6 hr. The bisulfite treat-ment was performed as described by Hajkova et al.(2002). The methylated sequence was amplifiedwith the primers p16-M1 (5�CGA TTG GGCGGG TAT TGA ATT TTC GC 3�) and p16-M2(5�CAC GTC ATA CAC ACG ACC CTA AAC CG3�) (Bardeesy et al., 2002). To amplify the unmod-ified sequence, the PCR reaction was performedwith primers Un1-p16 (5�G TGA TTG GGT GGGTAT TGA ATT TTT GTG 3�) and Un2-p16(5�CA CAC ATC ATA CAC ACA ACC CTC CACC/TA 3�).

RESULTS

Tumor Characteristics

In Table 1, the tumors derived from both seriesof animals analyzed during this study are listed.Tumors that developed in progeny derived frommatings between C57Bl/6-EL-Tgf�-hGH transgenicmice and (C57Bl/6-Trp53�/� � BALB/c) �BALB/c backcross mice (between backcross 3 tobackcross 6) are designated as “BC.” Tumors thatdeveloped in F1 C57Bl/6-BALB/c progeny of Tgf�/Trp53�/� mice are designated as “F1.”

Histological examination of the pancreatic tu-mors revealed a variety of morphologies, including

pronounced tubular and cystic transformation, cys-tic complexes, acinar hyperplasia, carcinoma withacinar differentiation, carcinoma with acinar differ-entiation and transition into undifferentiated pleo-morphic carcinoma, carcinoma with adenoid-tubu-lar differentiation, and carcinoma of anundifferentiated solid type. Four tumors thatshowed a histology incompatible with pancreaticorigin were not included in the analysis.

Immunohistochemical analyses with cytokeratin8/18 indicated an epithelial origin and analyseswith cytokeratin 19, a ductal origin of these pan-creatic tumors (Table 1). Several studies demon-strated that the adult pancreas exhibits remarkabledevelopmental plasticity, reflecting the origin ofacinar, ductal, and islet cell types from commonprecursor populations in embryonic pancreatic ep-ithelium. Therefore, it is not clear whether tumorcells arise from an undifferentiated precursor pop-ulation, from acinar cells, or from duct-like cellswithin tubular complexes.

In Table 1, the survival of the animals is alsolisted. There was considerable variation of the sur-vival period from 153 to 441 days, as previouslydescribed (Wagner et al., 2001).

Comparative Genomic Hybridization

For CGH analysis, tumor DNA was extractedfrom a tumor sample. The CGH profile of the Xchromosome was used as an internal control for thedynamic range of the hybridization. Therefore, im-balances of the X and Y chromosomes were notconsidered during the following analyses. The se-ries analyzed consists of 38 tumors, of which threetumors were completely uninformative. Overall,there were more losses (76) than gains (68). Weidentified gains with amplifications of various de-grees, in which the profile surpasses the ratio of 1.5.In general, the overall number of chromosomalimbalances per tumor specimen in these advancedtumor stages was low: 3.8 imbalances/tumor.

The results of the CGH analysis are summarizedin Figure 1. The most conspicuous aberration pat-tern was observed for chromosome 11, with gain oreven amplification of proximal chromosome 11 andloss of an even larger part of distal chromosome 11.This combination of gain and loss for DNA fromchromosome 11 in the same tumor was observed 11times. The second specific chromosomal imbalanceconcerns chromosome 15, which in some tumorsshows gain of the whole chromosome and in othertumors, amplification within its distal two thirds.Other regions of specific imbalance are deletions indistal chromosomes 14, 18, and 19, and various

242 SCHREINER ET AL.

regions of chromosome 10. For chromosome 1, gainof most of the whole chromosome was observed insix specimens (Fig. 1).

Alternative Patterns of Secondary GeneticChanges

In Figure 1, the loci most possibly involved inthe indicated chromosomal imbalance are alsolisted. The Egfr gene maps to proximal chromo-some 11 and the Trp53 locus is on distal chromo-some 11. The region of overlap of chromosome15 gains includes the Myc gene locus. Anotherconspicuous finding is the loss of distal chromo-some 14, where the tumor suppressor gene Rb1 islocated. It was very interesting to realize that

each type of these imbalances belongs to alter-native patterns of chromosomal aberration. Asshown in Figure 2b, gains of chromosome 15were observed in 14 tumors. Interestingly, thesegains coincide in some tumors with the specificaberration pattern of chromosome 11. In sevenother tumors, deletion of distal chromosome 14was apparent (Fig. 2a), which was combined inseveral tumors again with the specific aberrationpattern of chromosome 11. In none of the 38tumors analyzed were deletion of chromosome14 and gain of chromosome 15 observed togetherin the same tumor. This observation is not com-patible with the independent occurrence of thesetwo specific aberrations (P � 0.0274, Fisher’s

TABLE 1. Characteristics of Tumors Analyzed*

Mouseno.

Geneticbackground

Survivalperiod

(no. of days)Cytokeratin

8/18Cytokeratin

19LOH oftrp53

Cdkn2ahypermethylation

Gainchromosome11 proximal

Gainchromosome

15 distal

Losschromosome

14 distal

2 BC 317 nt � � � �5 BC 214 nt �� 20 BC ? nt � � � �23 F1 153 � nt � � �29 F1 336 � �� 31 BC 306 nt � � �41 F1 238 � � � � �45 BC 262 nt � � � �56 BC 223 nt �� 66 F1 335 � �� 67 BC 294 nt � � � �68 F1 368 �� 70A F1 220 � � � � �70B BC 230 � �� nt � � � �78A BC 243 nt � � � �78B BC 220 nt � � � �84 F1 367 �� 89 F1 427 �� 97 F1 422 �� 106 F1 229 � �� 111 F1 358 � �� 119 F1 441 � � � � � �125 F1 321 � �� 126 F1 381 � �� 129 F1 361 � �� 138 F1 431 �� 181 F1 247 �� 304 BC 274 �� nt �� 307 BC 324 nt � � � �316 BC 317 nt � � � �317 BC 278 nt � � � �321 BC 292 nt � � � �324 BC 292 �� nt nt � � �332 BC 347 nt � � � �340 BC 390 nt � � � �341 BC 374 nt � � � �346 BC 175 nt � � � �349 BC 278 nt � � � �

*F1, C57Bl/6-EL-Tgf�-hGH � Balb/c Trp53�/� F1-hybrids; BC, C57Bl/6-EL-Tgf�-hGh � backcross 3-6 [(C57Bl/6 Trp53�/� � Balb/c) � Balb/c];Cytokeratin 8/18 and cytokeratin 19 immunohistochemical staining: ��, most of the tumor tissue stains positive; �, part of the tumor tissue stainspositive; LOH of trp53; �, no loss; �, loss; nt, tumors were not tested; methylation-specific PCR for Cdkn2a: ��/�, hypermethylation was detected;�, no PCR product was obtained; nt, tumors were not tested; gain of chromosome 11 proximal and chromosome 15 distal, detected by CGH analysis:�, gain; �, no gain of this chromosomal region; loss of chromosome 14 distal: �, loss, �, no loss of this chromosomal region.


exact test). Thus, these analyses identify twoalternative aberration patterns, gain of chromo-some 15 or loss of chromosome 14, suggestingtwo different genetic pathways in pancreatic tu-mor development and/or progression. We did notfind a correlation between these two differentaberration patterns and the histological appear-ance. However, there was an obvious differencein the mean survival of the animals in relation tosecondary genetic changes (P � 0.046, two-sidedMann–Whitney U test). Animals that developedtumors with gains or an amplification of chromo-some 15 showed a mean survival of 320.4 � 63.4days, whereas animals that developed tumorswith deletion of chromosome 14 showed a meansurvival of 251.4 � 49.2 days (Fig. 2c).

Real-Time PCR

By CGHs it is not possible to determine theprecise number of gene copy gain, and the exact

genes involved in gain or amplification on therespective chromosome. Therefore, all tumorswere tested by real-time PCR for the extent ofamplification of candidate genes from proximalchromosome 11 and distal chromosome 15. Forproximal chromosome 11, the epidermal growthfactor receptor gene Egfr, reticuloendotheliosisoncogene Rel, serine/threonine kinase 10 Stk10,and FMS-like tyrosine kinase 4 Flt4, which mapfrom 9.0 to 25.0 cM on that chromosome, werechosen. Among the 38 tumors analyzed, 22 tu-mors showed a gain in gene copy number at leastfor one of these genes. In 20 tumors, gain/ampli-fication included the Egfr locus at 9 cM, whichshowed the highest amplification rate of all locitested, varying between 1.75- and 7.3-fold. Inseveral tumors, the amplification unit extendsover an enlarged chromosomal region, which in-cludes Rel in 10 tumors at 13 cM and is evenfurther enlarged in six tumors, including Stk10 at

Figure 1. Summary of DNA copy number changes detected byCGH analysis in 38 ductal pancreatic tumors. Green lines on the leftside of each chromosome ideogram represent losses; red lines on theright side indicate gains. Amplification sites are represented as red bars

with varying widths corresponding to the degree of amplification of therespective chromosomal region. Lines in the same vertical positionindicate changes within one and the same tumor. Candidate genes of themost affected chromosomal regions are added next to them.


16 cM. Flt4, mapping more distal at 25.0 cM,defines the distal part of chromosome 11, whichshows loss of chromosomal material in eight tu-mors. From distal chromosome 15, the myelocy-tomatosis oncogene Myc, adenylate cyclase 8Adcy8, and protein kinase 2 Ptk2 (formerly Fadk)were tested. These genes map from 32.0 cM(Myc) to 42.0 cM (Ptk2). In 15 tumors, a gain incopy number of at least one of the analyzedgenes was found. In several tumors an extendedchromosomal region covering all genes analyzedwas amplified, but the highest amplification val-ues were determined for Myc. In tumor 126, agreater than 77-fold amplification is evident (notshown). In six tumors, only gain or amplificationof Myc was found. The genes investigated areindicated in Figure 3, in which the real-timePCR results of the tumors with gain of chromo-some 15 or loss of distal chromosome 14 accord-ing to CGH results are summarized.

A further chromosomal amplification was ob-served once for chromosome 8. In this tumor, dis-tinct amplification of distal chromosome 8 waspresent. At this site, maps the Jun B proto-onco-gene Junb, which was determined to be amplified

3.8-fold in this tumor. Several other tumors with anambiguous CGH profile for chromosome 8 weretested for Junb amplification by real-time PCR, butdid not reveal any gene copy gain at this site (datanot shown).

LOH Analysis

For LOH studies, a different DNA sample wasused that was isolated from pure tumor tissue bylaser capture microdissection. The LOH analysiswas performed only for tumors removed from trueF1 animals, which were available in the secondseries of tumors studied. As described earlier (Wag-ner et al., 2001), loss of the wild-type Trp53 alleleis common to most tumors (Table 1), whereasLOH at the loci Cdkn2a, Madh4, and Rb1 was foundin only a variable minority of tumors (results notshown).

Cdkn2a Methylation Studies

CDKN2A is frequently inactivated in human pan-creatic tumors, mostly by LOH and/or promoterhypermethylation (Herman et al., 1996; Schutte etal., 1997). CGH and LOH analysis of these tumorsrevealed loss of this particular locus in only three

Figure 2. Overview of the CGH analysis of chromosomes 11, 14,and 15 of (A) all tumors with loss of distal chromosome 14 and (B) alltumors with gain of chromosome 15. Gains and losses are representedby red and green lines on the right or left side of each ideogram. C:

Survival period (in days) of animals with loss of distal chromosome 14and animals with gain of chromosome 15, according to CGH results.The mean age at death (in days) is 251.4 � 49.2 (loss 14) and 320.4 �63.4 (gain 15).

Figure 3. Real-time PCR results for tumors with gainof chromosome 15 (no. 2-340) and loss of distal chromo-some 14 (no. 56-346). Egfr, Rel, Stk10, and Flt4 were testedfrom proximal chromosome 11, and Myc, Adcy8, and Ptk2from distal chromosome 15. Gains (rate � 1.75) areindicated in gray, amplifications (rate � 3.00) in dark gray,no gain or loss in white, and * was not tested.


tumors. Therefore, the methylation status of CpGdinucleotides in the 5�-promoter region of theCdkn2a gene was determined. A PCR assay by useof primers specific for the methylated and unmeth-ylated sequence (Bardeesy et al., 2002) was done(Fig. 4). Methylated alleles were detected in morethan 58% of all tumors. The total conversion ofcytosine to uracil residues by bisulfite treatmentwas confirmed by an additional primer set specificfor the unmodified sequence. No PCR product wasamplified (data not shown).

DISCUSSION

The Tgf�/Trp53�/� transgenic mouse has provedto be an informative animal model for human pan-creatic cancer. Overexpression of Tgf�, driven bythe elastase promoter, induces constitutive activityof the Ras–Erk1/2 pathway and of the cyclin-D1–Cdk4 complex (Wagner et al., 2001). In a search forfurther, secondary genetic changes, a genome-widesurvey by CGH was performed.

By CGH, only large gene dosage differencesattributed to genomic imbalances become apparent(Kallioniemi et al., 1992). The resolution of thistechnique is limited. Deletions down to 2 Mb willbe detected and gains from 4 Mb upward (Bentz etal., 1998). Because of the inherent poor quality ofmouse chromosomes, the resolution in the mousewill be even less than that reported for man. Fur-ther, gains or losses by gene copy variation consti-tute only one of several possibilities by which geneactivity is regulated in tumors. It is thus notewor-thy that in the series of tumors described in thisstudy a relative uniform pattern of genomic imbal-ances becomes apparent.

A prediction from our earlier study was that Tgf�transgenic mice hemizygous for Trp53 deficiencysegregate the remaining wild-type Trp53 allele. Wefound frequent losses of the distal two thirds ofchromosome 11 where the tumor suppressor genesTrp53, Nf1, and Brca1 are located (Fig. 1), andLOH analysis confirmed that Trp53 is the targetgene in chromosome 11 in these tumors. In 15 of 17tumors, the wild-type allele of Trp53 was lost (Ta-

ble 1). Our previous study suggested homozygousloss of the Cdkn2a locus in more than 50% ofpancreatic tumors in Tgf�/Trp53�/� mice (Wagneret al., 2001). In the present study, losses at theCdkn2a locus or LOH were found in only a fewtumors. However, methylation or even hypermeth-ylation of the Cdkn2a promoter region was con-firmed in 58% of all tumors (Fig. 4). Thus, in mosttumors, both tumor suppressor genes Trp53 andCdkn2a are inactive.

Copy losses of the distal part of chromosome 11were associated with gains and even amplificationsof gene copies on the proximal part of that chro-mosome, including the Egfr gene locus, supportingthe central role for Tgf�-Egf-receptor signaling inthis model. In the precancerous period, overexpres-sion of Tgf� results in up-regulation of epidermalgrowth factor receptor in cells within tubular com-plexes, establishing an autocrine loop (Wagner etal., 1998). A similar regulation was previously de-scribed in human pancreatic tumors (Korc et al.,1992). Activation of epidermal growth factor recep-tor results in potent signals for cell proliferation,survival, invasion, and angiogenesis (Olayioye etal., 2000). Distal mouse chromosome 11 is homol-ogous to human chromosome 17. Interestingly, onmouse chromosome 11, in close proximity to theTrp53 locus, the Erbb2 gene locus maps. In hu-mans, overexpression of ERBB2 is an early event inpancreatic cancer development and is frequentlydetected in early lesions (Day et al., 1996; Apple etal., 1999). In contrast, in Tgf�/Trp53�/� mice, in-herent constraints force deletion of the remainingTrp53 allele on distal chromosome 11 and thereforein addition deletion of the Erbb2 gene. Conse-quently, enhanced Tgf� signaling accomplishesamplification of the Egfr gene on proximal chromo-some 11.

Our tumor model seems to favor up-regulation ofMyc or deletions of Rb1 as further genetic changes.Whereas the above-mentioned changes are rela-tively uniform in the Tgf�/Trp53�/� pancreatic can-cer model, it is interesting that the cancer cellsseem to use two alternative pathways to overcome

Figure 4. PCR analysis of the methylation status of the Cdkn2apromoter region of several bisulfite-treated tumor DNAs. Special prim-ers for the hypermethylated sequence were used. Additionally, DNA of

normal pancreatic tissue (p), DNA of non-bisulfite–treated normalpancreatic tissue (p nt), and DNA of the cell line TD2 as positivecontrol (p. c.) (Greten et al., 2002) were tested; negative control: n. c.


the G1/S arrest. In a minority of tumors, deletion ofdistal chromosome 14 was observed, whereas inother tumors gain or even amplification of distalchromosome 15 was present (Fig. 2). The domi-nant acting tumor gene on distal chromosome 15 isobviously Myc, which was shown by real-time PCRto be amplified up to 77-fold (Fig. 3). The cell lineTD2 has been established (Greten et al., 2002),which harbors the characteristic chromosomal ab-erration pattern for chromosomes 11 and 15. In thiscell line, overexpression of Myc has been confirmed(Schreiner et al., 2003). On distal chromosome 14,Rb1 was suggested to be the suspected tumor sup-pressor gene that is deleted. However, this couldnot be authenticated by independent LOH analy-sis because most tumors have uninformative Rb1alleles. Both genetic changes, Myc overrepresenta-tion/overexpression or Rb1 deletion/inactivation,are alternative mechanisms to overcome inhibitoryeffects at the G1/S-phase transition. A Myc-depen-dent dissociation of Cdkn1b from Cdk2 resulting ina rapid activity of the cyclinE/Cdk2 complex waspreviously reported (Bouchard et al., 1999; Elendand Eilers, 1999). We tested Cdk2 and also Cdc2kinase activity and found both kinase activitiesenhanced compared to non-cancerous pancreatictissue, although there was no difference betweentumors with Myc amplification or Rb1 deletion. In-terestingly, animals that develop tumors with Rb1deletion have a considerably shorter survival thananimals that develop tumors with Myc gains oramplifications (Fig. 2c).

In conclusion, this study has revealed a surpris-ingly uniform pattern of secondary genomicchanges in this transgenic tumor model of pancre-atic cancer, and it is tempting to arrange thesechanges into a cascade of consecutive events (Fig.5). In the Tgf�/Trp53�/� mouse tumor model, pre-

determined primary changes are active Tgf� ex-pression and heterozygosity for Trp53. This in-duces a uniform pattern of secondary changes:deletion of Trp53, inactivation of the Cdkn2a locus,and overrepresentation/overexpression of Egfr,which induces the Ras–Erk1/2 pathway. Thesechanges are relatively common to all tumors andare related to the promotion of acinar-to-ductalmetaplasia (Sandgren et al., 1990; Song et al.,1999). To overcome the G1/S-phase arrest, twoalternative pathways are followed, indicated by amutually exclusive pattern of tertiary changes.These are exemplified by Myc gene activity or Rb1gene inactivity, with a considerably faster tumorprogression in the Rb1-deleted specimens.

Here we present a first step toward the identifi-cation of tumor-suppressor networks that cooperatewith Tgf� in pancreatic cell transformation in aTrp53-deficient background. This model might beuseful for testing therapeutic strategies to counter-act the selective disruption of tumor-suppressorpathways in pancreatic carcinogenesis.

ACKNOWLEDGMENTS

We are indebted to Rosi Rittelmann, SabineSchirmer, and Antje Kollak for excellent technicalassistance, and Dr. J. Hogel for help with the sta-tistical evaluation.

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Figure 5. Cascade of genetic changes during pancreatic tumor in-duction in Tgf�/Trp53�/� mice. The activation of the Ras/Erk1/2 path-way is shown (Wagner et al., 2001).


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Pattern of secondary genomic changes in pancreatic tumors of Tgfα/Trp53+/− transgenic mice