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Allele-specific Hras Mutations and Genetic Alterations at Tumor Susceptibility Loci in Skin Carcinomas from Interspecific Hybrid Mice¹

The Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263 [H. N.], and University of California San Francisco Cancer Center and Cancer Research Institute, University of California San Francisco, San Francisco, California 94105 [H. N., J-H. M., A. B.]

	ABSTRACT

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We have investigated the effects of germ-line variants that influence skin tumor susceptibility loci on the patterns of somatic genetic alterations in mouse skin cancers. Using a two-stage skin carcinogenesis model, we previously identified at least 13 skin tumor susceptibility (Skts) loci in a large interspecific F1 backcross [(NIH/Ola x M. spretus) x NIH/Ola] study. In this report, we describe the analysis of allele-specific alterations at these loci in skin tumors from the same backcross animals. The mouse Hras gene, located close to Skts2 on chromosome 7, had specific activating mutations in the Mus musculus allele in 23 of 26 carcinomas. In all cases, tumors with Hras mutations also showed specific imbalance of chromosome 7 markers that favored the chromosome carrying the mutant allele. Allele-specific quantitative microsatellite analysis was also carried out, using DNA from 62 carcinomas from (NIH/Ola x M. spretus) x NIH/Ola mice. Frequent allelic imbalance was detected at five additional tumor-susceptibility loci on chromosomes 4, 6, 7, 9, and 16 (Skts7, Skts12, Skts1, Skts6, and Skts9, respectively). At all except Skts7, we found loss of the allele inherited from the resistant strain or amplification of the allele from the susceptible strain. We conclude that polymorphisms in some low-penetrance tumor modifier genes are reflected in the pattern of somatic alterations in tumors. Analysis of such allele-specific changes in tumors may facilitate the identification of functional germ-line variants that control tumor susceptibility.

	INTRODUCTION

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The major determinant of cancer predisposition in hereditary cancer syndromes is a recessive germ-line mutation. Subsequent loss or mutation of the remaining wild-type allele in a somatic cell gives rise to cancer, usually after the development of multiple additional genetic alterations (1) . Predisposition to sporadic cancers in the normal human population is also controlled in part by "low-penetrance" genetic variants that have weak effects but are relatively common and could account for a substantial proportion of the human cancer burden (2) . This model is supported by studies using mouse models, which have clearly indicated that tumor predisposition is controlled by multiple genes, each with a relatively small effect, that can interact additively or synergistically to confer high risk (3 , 4) .

The relationship between the genetic variants that control germ-line predisposition to cancer and the multiple somatic events that take place during tumor development has not been rigorously explored. It is possible, by analogy with the classical two-hit process seen in familial cancer syndromes (1) , that low-penetrance susceptibility genes with tumor suppressor or oncogenic potential may be altered in an allele-specific manner in sporadic cancers. One example is the Pas1 pulmonary adenoma susceptibility region that is located close to the Kras proto-oncogene. Activating mutations in Kras are detected in a large proportion of pulmonary adenomas, and these occur exclusively in the Kras allele inherited from the sensitive parent in hybrid mice (5 , 6) . It has been suggested that Kras is therefore likely to be the germ-line susceptibility gene, but others have proposed that an adjacent gene is responsible for germ-line predisposition (7) . In some other mouse model systems, allele-specific changes have been found at high frequency in tumors (8) , but in these cases the relationship to susceptibility genes has not been explored. In other cases where this question has been addressed, no correlation was found (9) .

We have collected skin cancers at various stages of progression, including high-grade carcinomas from interspecific backcross animals that had been used previously for linkage analysis to identify tumor susceptibility regions (10 , 11) . Our results show that some of these regions undergo allele-specific changes in tumors that in most cases are consistent with the conclusions from the linkage studies, thus demonstrating that low-penetrance genes with modest effects can influence patterns of somatic alterations during tumor development.

	MATERIALS AND METHODS

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DNA Samples from Normal Tissues and Tumors.
The mice used for this study have been described previously (11) . Briefly, female interspecific F1 hybrid mice between NIH/Ola and M. spretus were crossed with male NIH/Ola to generate the backcross mice (NIH/Ola x M. spretus) x NIH/Ola. NIH/Ola is an inbred strain, and M. spretus is from an outbred stock. The (NIH/Ola x M. spretus) x NIH/Ola mice were treated with DMBA³ and 12-O-tetradecanoylphorbol-13-acetate according to the standard two-stage carcinogenesis protocol (11) . A total of 62 carcinomas from the same animals used for linkage analyses (4 , 11) were investigated, using microsatellite markers to detect chromosomal imbalances at skin tumor susceptibility loci. After taking a thin slice of the center portion for histological evaluation, we dissected macroscopically normal tissue away from the tumor mass, and the carcinoma with a corresponding normal tail tip were immediately frozen in liquid nitrogen. DNAs were directly purified from frozen cancers and corresponding normal tail tip by standard methods. Histological sections were evaluated as described previously (12) , and a total of 18 stage I, 19 stage II, 9 stage III, and 16 stage IV tumors were used for this study.

Allele-specific Hras Mutation Detection System.
To clarify the mutation status of the Hras gene, we developed a simple allele-specific Hras mutation detection system to distinguish between mutations in the NIH/Ola and M. Spretus Hras alleles (Fig. 1A) . Exon 2 of the Hras gene was amplified using the primers 5'-AAGCCTGTTGTTTTGCAGGA-3' and 5'-GGTGGCTCACCTGTACTGATG-3'. Amplified 207-bp fragments were digested by a combination of restriction enzymes XbaI and AvaII at 37°C for 2 h and electrophoresed in a 4% Nusieve 3:1 agarose gel. A M. spretus polymorphism at codon 68 destroys the AvaII restriction site. Nearly 100% of tumors induced by DMBA have a specific CAA-to-CTA Hras gene mutation at codon 61, creating a new XbaI site. Therefore, the Hras mutation status of tumors can be detected by the restriction fragment length differences. The presence of a mutation in the M. spretus allele is detected by the diagnostic 116-bp band after digestion of tumor DNA with both XbaI and AvaII (Fig. 1) .

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Allele-specific Hras mutations are detected by restriction enzyme digestions after PCR. A, representation of restriction fragments expected from each allele. XbaI is used to detect the specific point mutation at codon 61 of the Hras gene induced by DMBA. After amplification of Hras exon 2 by primers EJ2A and EJ2B, PCR products from the mutated Hras allele create 116- and 91-bp fragments after XbaI single digestion. At codon 68, a single nucleotide polymorphism between M. spretus and M. musculus creates an AvaII site in the M. musculus allele. Double digestion with added AvaII creates 96- and 20-bp bands from the 116-bp fragments in the M. musculus allele of Hras mutated tumors. B, electrophoretic pattern of the double-digestion product in a 4% Nusieve 3:1 agarose gel. Lane 1, X174DNA/HaeIII molecular weight marker (Promega); Lane 2, Hras-mutated NIH allele papilloma; Lane 3, Hras-mutated M. spretus allele papilloma; Lanes 4–10, skin carcinoma samples (identification of tumor is indicated above each lane). The 116-bp band is retained after double XbaI/AvaII digestion as a diagnostic marker of the Hras-mutated M. spretus allele tumor. C, examples of allele-specific chromosomal alterations of three carcinomas. All DNA samples have a 116-bp band representing Hras codon 61 mutations as seen in lanes indicated by X (XbaI single digestion). The 116-bp band is retained after double XbaI/AvaII digestion (X-A) of the DNA in Lane 5, from the C44 carcinoma sample, which has a mutation in the M. spretus allele. Shown on the right is allele-specific quantitation of genomic DNA at the D7Mit12 locus. PCR products created by amplification using end-labeled fluorescent primers for microsatellite markers were electrophoresed on the ABI 377 sequencer. Peaks of fluorescently labeled PCR fragments were detected by ABI prism software. Carcinoma samples (top) and corresponding normal tail DNA (bottom) were amplified by quantitative PCR using the D7Mit12 primers. In the C8 carcinoma sample, the D7Mit12 M. musculus allele is amplified and the M. musculus allele of the Hras gene is mutated (left panel, Lanes 2 and 3). A similar situation is seen for the C10 carcinoma, which also carries a mutant M. musculus Hras allele, but here the normal M. spretus allele is lost (left panel, Lanes 6 and 7) In the C44 carcinoma sample, the M. spretus allele is mutated and also increased in copy number (left panel, Lanes 4 and 5).

where R is the ratio of AI in a tumor, Ts is a peak area from tumor DNA corresponding to a M. Spretus allele, Tn is a peak area from tumor DNA corresponding to a NIH/Ola allele, Ns is a peak area from normal DNA corresponding to a M. Spretus allele, and Nn is a peak area from normal DNA corresponding to a NIH/Ola allele. A positive imbalance was scored when R was >1.5 or <1/1.5. Each pair of DNA samples was analyzed by quadruplicate PCR analysis.

	RESULTS

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Allele-specific Hras Mutations.
The initiating event for mouse skin tumors induced by treatment with DMBA involves mutation of the Hras gene at codon 61 (13) . This mutation is detected in >90% of papillomas and carcinomas and is followed by trisomy of chromosome 7, on which Hras is located, leading to preferential duplication of the mutant allele (14) . The location of Hras on distal chromosome 7 is very close to Skts2, a significant M. spretus papilloma resistance locus (10) . We therefore investigated the possibility that Skts2 might influence the allele specificity of the mutation and/or selection of the Hras-initiated cell. Sequence analysis of Hras exons 1 and 2 was carried out, and we detected no amino acid differences between M. spretus and NIH/Ola. This analysis identified an AvaII restriction enzyme site polymorphism in exon 2 (Fig. 1A) that enabled us to develop an allele-specific mutation assay. The somatic codon 61 mutation introduces a new restriction site for the enzyme XbaI, and mutations in the M. musculus or M. Spretus alleles could be distinguished by further digestion with AvaII (Fig. 1B) . If the mutation occurs in the M. spretus allele, this can be detected by the presence of the diagnostic 116-bp band in tumor DNA that has been subjected to double digestion with both XbaI and AvaII (Fig. 1B) .

Forty-one of 42 (97.6%) carcinomas had the same CAA-to-CTA transversion at codon 61 in the Hras gene. Twenty-six tumors were from mice heterozygous at the Hras allele, but only 3 carried a mutation in the M. spretus allele, whereas 23 of 26 (P < 0.001) had a mutant NIH/Ola allele. Because the initial Hras mutation is followed by trisomy of chromosome 7, we investigated the allele specificity of this imbalance, using markers close to the Hras gene on distal chromosome 7. Fig. 1C shows examples of AI at D7Mit12, which is close to the Hras gene and within the region containing the Skts2 locus. As can be seen, tumors C8 and C10, which carry the mutant M. musculus Hras allele, show preferential imbalance in favor of the M. musculus marker at D7Mit12, whereas the opposite is the case for tumor C44, which carries the mutant M. spretus allele. Similar results were observed with other tumors, showing concordance between the presence of a particular mutant parental Hras allele and the direction of imbalance on distal chromosome 7. All three carcinomas with Hras mutations in the M. spretus allele had duplicated this allele and/or lost the NIH/Ola allele, whereas 15 of 23 carcinomas with Hras mutations in the NIH/Ola allele had preferential imbalance in favor of the M. musculus alleles at the flanking markers D7Mit12 and D7Mit14 (P = 0.038). The results with the remaining eight carcinomas were not clearly interpretable because of discrepancies between the imbalance patterns at the two markers, suggesting that complex genetic alterations may have taken place in these tumors (data not shown). We conclude that the M. spretus resistance locus Skts2 influences the allelic preference for somatic events at or near the initiating Hras gene on distal chromosome 7.

Allelotype Analysis of Carcinomas from Backcross Mice.
Our results with Skts2 prompted us to carry out a genome-wide study of preferential AI in mouse skin carcinomas from backcross mice. A series of 62 carcinomas were genotyped at fluorescently labeled simple sequence repeat polymorphism markers close to the known Skts loci. Fig. 2 shows specific examples of chromosomal imbalances on chromosomes 4 and 16, demonstrating a clear imbalance in favor of one parental allele. It should be noted that no quantitative conclusions could be drawn from AI using microsatellite markers, and for this reason, we refer to preferential imbalance (>1.5-fold difference) without concluding that this is attributable to loss of one allele or gain of the other.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Allele-specific LOH on chromosomes 4 and 16. Two markers on chromosome 4 were simultaneously loaded, and two peaks at each end-labeled marker are seen, representing the two parental M. musculus and M. spretus alleles Peaks from C05 carcinoma (top) and peaks from corresponding normal tail DNA (bottom) are similar at D4Mit17, but the peak representing the M. musculus allele at D4Mit166 in carcinoma C05 is significantly reduced in intensity. The same carcinoma sample was tested, using the D16Mit2 marker on chromosome 16. The peak representing the M. spretus allele is also significantly reduced in the C05 carcinoma sample.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Allele-specific chromosomal imbalance in 62 carcinomas from backcross mice at Skts loci. The percentage of chromosome imbalance is plotted at each microsatellite marker on a bar graph. A total of 34 microsatellite markers indicated on the X axis and in Table 1 were analyzed. represents M. spretus allele loss or NIH/Ola allele amplification; represents NIH/Ola allele loss or M. spretus amplification.

On chromosome 9, where we found linkage to the M. spretus Skts6 papilloma resistance allele with a log of the odds score of 3.71, AI occurred in 16 of 23 carcinomas at D9Mit9, 1 cM centromeric from Skts6. Thirteen alterations in 16 carcinomas were M. spretus allele losses or NIH/Ola allele gains (P = 0.011). Similarly, at the Skts9 locus (LOD score of 5.90) on chromosome 16, M. spretus allele losses and/or NIH/Ola allele gains were seen at the marker D16Mit2 in all 10 chromosomal imbalances (P = 0.001) detected in a total of 29 informative carcinomas.

On chromosome 1, the Skts8 locus showed no AI in any of the 31 informative cases. The Skts3 locus on chromosome 5 conferred significant resistance to both papilloma and carcinoma development, but only 2 of 23 carcinomas were found to have AI in this region. A similar lack of obvious genetic alterations was found at other susceptibility loci on chromosomes 12 and 17 (Fig. 3 ; Table 1 ).

We investigated a subset of 42 carcinomas, using an additional panel of markers located in other chromosomal regions (Fig. 4) . Chromosomal imbalances were detected in >30% of informative carcinomas at several chromosome locations in addition to those described above, including chromosomes 8, 10, 18, and 19. All chromosomal imbalances except that on chromosome 8 were confirmed by use of flanking markers, whereas on chromosome 8 only one marker at D8Mit112 showed consistent imbalance. The flanking markers D8Mit211, D8Mit121, and D8Mit13 were imbalanced in <10% of cases. It is possible that a small, localized genetic event may be responsible for the pattern of imbalances seen around D8Mit112.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Allele-specific chromosomal imbalances in 42 carcinomas from backcross mice at non-Skts loci. The percentage of chromosome imbalance is plotted for each microsatellite marker on a bar graph. A total of 38 microsatellite markers are shown. represents M. spretus allele loss or NIH/Ola allele amplification; represents NIH/Ola allele loss or M. spretus amplification.

	DISCUSSION

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The identification of human low-penetrance tumor susceptibility genes is complicated by several factors including their multiplicity, weak effects, gene-gene and gene-environment interactions, and population heterogeneity (2) . On the other hand, mouse models of human cancer have proven to be powerful tools for the detection of quantitative trait loci that influence tumor susceptibility (15) . Nevertheless, even with mouse model systems, the definitive identification of these weak susceptibility genes is hampered by the lack of sufficient resolution because of the small number of informative meiotic recombinations in crosses between sensitive and resistant strains. We propose that more detailed investigations of somatic recombinations, deletions, and amplifications that take place during tumor development can provide important clues that will help to fine map and eventually identify an important subset of these genes. On the basis of the observation that in most cases of high-penetrance familial cancer the wild-type allele of the predisposing gene is lost by a somatic event during tumor development, we carried out an investigation of allele-specific somatic changes that involve known Skts loci. The mouse offers some distinct advantages for this kind of study because the parental origin of each allele is known and the number of tumors potentially available from informative, genetically uniform animals is unlimited. The observation that approximately half of the mapped Skts loci show allele-specific alterations in carcinomas from informative mice suggests that this approach could be a fruitful route to the identification of at least some of the polymorphic variants in the mouse genome that confer increased cancer risk. Considering the overall similarity between mechanisms of carcinogenesis in mouse and human cells and the current explosion of information on comparative mouse-human genetics and genomics (16) , this information should also prove to be useful for the discovery of human low-penetrance tumor susceptibility genes.

The detection of frequent mutations specifically in the M. musculus allele of the Hras gene at Skts2 in skin tumors mimics the situation observed for the Kras gene at Pas1 in mouse lung tumors (5 , 6) . It has been proposed that the Pas1 gene is in fact the Kras gene (6 , 17) . The selection of M. musculus allele duplications in early skin tumors (14) and M. musculus Hras mutations in carcinomas could be attributable to the negative influence of a strong resistance allele distinct from Hras on the parental M. spretus chromosome. It is also possible that more than one gene may confer susceptibility in this region, as has been demonstrated for the Mom1 locus and intestinal neoplasia (18) . Our preliminary data have also shown that carcinomas obtained from mice that are homozygous for M. musculus alleles at Skts2 and heterozygous M. musculus/M. spretus at Skts1 also have preferential chromosome 7 imbalances. Because the M. spretus allele confers resistance when transmitted from either the mother or the father (data not shown), preferential AI at Skts1 is not a result of an imprinting effect. Therefore, at least two polymorphic Skts genes influence somatic alterations on chromosome 7. Further studies will be necessary to dissect the various possible contributions of ras genes and adjacent modifier genes to tumor susceptibility in these mouse models.

Some similarities exist between the regions of allele-specific LOH detected in this study and the results obtained with other model systems. Skts6 and Skts9 on chromosomes 9 and 16, respectively, overlap with Loh1 and Loh2, where LOH is found in pancreatic tumors from SV40 Tag transgenic mice (19) . Although allele-specific losses were not detected in pancreatic tumors, the common LOH region at Skts9 overlaps with Loh2 and the common deletion region at Skts6 includes Loh1. The Loh1 gene has been proposed to be a regulator of apoptosis and/or telomerase activity, and Loh2 is predicted to be a suppressor of tumor angiogenesis (19) . Polymorphic variants of these genes in appropriate crosses may therefore influence susceptibility to pancreatic cancer, but this question has not yet been addressed. On chromosome 4, the LOH in skin carcinomas showed no specificity for either parental allele. The critical region in this case includes the Cdk inhibitor p16^Ink4a, which has been implicated as a modifier of plasmacytoma susceptibility (20) . Further detailed analysis is necessary to investigate the possible role of this candidate gene in skin carcinogenesis.

Other cancer susceptibility loci, such as Skts3, Skts5, Skts8, Skts10, and Skts11 on chromosomes 5, 12, 1, 17, and 6, respectively, rarely show evidence of somatic imbalance. This may indicate that these loci act in a non-cell autonomous fashion, e.g., as secreted proteins, or that the polymorphisms induce haploinsufficiency that affects tumor growth without requiring additional changes at the "wild-type" allele. On the other hand, frequent allele-specific somatic alterations on chromosomes 8, 10, and 19 provide evidence for the existence of additional tumor predisposition loci on these chromosomes. Particularly at D10Mit180, AI was detected in four stage IV, two stage III, and one stage II carcinomas. It is possible that a locus on this chromosome may control tumor progression, but the numbers of animals in the study were not sufficiently large to permit linkage analysis for this phenotype (only 17 of 326 mice had stage IV carcinomas; Ref. 11 ). More definitive answers must await the cloning and identification of the specific genes involved. Nevertheless, our data demonstrate that the presence of tumor modifier genes strongly influences the patterns and allele specificity of somatic alterations in mouse skin tumors.

	ACKNOWLEDGMENTS

 
We would like to thank Frances Fee, Pi-Hsia Su, Sheila Bryson, Tomoe Minami, and Dr. Kenneth Brown for useful discussions and technical support. We are very grateful to Stephen Bell and the CRC Beatson Labs Animal House staff for excellent assistance with animal husbandry.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was funded by a grant from the Cancer Research Campaign (United Kingdom), and further support was provided by Onyx Pharmaceuticals (Richmond, CA) and by a grant from the NCI Mouse Models of Human Cancer Consortium. Dr. Jian-Hua Mao was supported by a grant to A. B. from the European Community. H. N. is also supported by the Roswell Park Alliance Foundation and by Roswell Park Cancer Institute’s NCI-funded Cancer Center Support Grant CA16056.

² To whom requests for reprints should be addressed, at Roswell Park Cancer Institute, Department of Cancer Genetics, Elm and Carlton Streets, Buffalo, NY 14263. Phone: (716) 845-1546; Fax: (716) 845-1698; E-mail: Hiroki.Nagase{at}RoswellPark.org

³ The abbreviations used are: DMBA, 7,12-dimethyl-benzanthracene; Skts, skin tumor susceptibility locus; AI, allelic imbalance; LOH, loss of heterozygosity.

Received 2/ 5/03. Revised 6/ 2/03. Accepted 6/ 9/03.

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