Elevated Frequency of Loss of Heterozygosity in Mammary Tumors Arising in Mouse Mammary Tumor Virus/Neu Transgenic Mice1

[CANCER RESEARCH 59, 2438–2444, May 15, 1999] Elevated Frequency of Loss of Heterozygosity in Mammary Tumors Arising in Mouse Mammary Tumor Virus/neu Transgenic Mice1

Marc Cool and Paul Jolicoeur2 Laboratory of Molecular Biology, Clinical Research Institute of Montreal, Montreal, Quebec, H2W 1R7 Canada [M. C., P. J.]; Department of Microbiology and Immunology, University of Montreal, Montreal, Quebec, H3C 3J7 Canada [P. J.]; and Division of Experimental Medicine, McGill University, Montreal, Quebec, H3G 1A4 Canada [P. J.]

ABSTRACT LOH. The number and identity of most tumor suppressor genes involved in human breast cancers are unknown, and only a few of Loss of heterozygosity (LOH) analysis was performed on 62 mammary them have been identified; p53 (16), RB1 (17, 18), PTEN/MMAC1 tumors that were induced in (BALB/c ؋ C57BL/6)F mouse mammary 1 (19, 20), E-cadherin (21, 22), FHIT (23, 24), TSG101 (25), and tumor virus/neu transgenic mice. Eighty-six simple sequence length poly- INK4a morphism markers were used to cover all of the somatic chromosomes. p16 (26, 27) have all been found to be inactivated at various Frequency of LOH was observed to be significant for chromosomes 4 frequencies in sporadic breast carcinoma. In familial breast cancer, (50%), 19 (32%), and 8 (21%). On chromosome 4, at least three distinct inactivation of BRCA1 and BRCA2 is a relatively frequent event regions of allelic deletions could be identified: one proximal to 22 cM; the (28, 29). second close to the p16INK4a/p15INK4b locus, which is commonly deleted in To study the molecular nature of the erbB-2/neu transformation various tumors; and the third one in the proximity of Mom1. The fre- event of the mammary glands, we (30) and others (31) have generated quency of LOH on chromosome 19 was the same for the four markers Tg mouse animal models (MMTV/neu), which are more amenable to used. Our data suggested the presence of two distinct LOH loci, one experimentation. These mice harbor a transgene consisting of a full- proximal to 47 cM and the other at the distal region. On chromosome 8, length neu cDNA, with a Val-6643Glu mutation located in the possibly two distinct LOH loci could be recognized, one around 52 cM and transmembrane domain, expressed under the regulatory sequences of the other one at 67 cM or distal to it. These regions map close to E-cadherin (Cdh1) and M-cadherin (Cdh15) loci, respectively. Because the MMTV promoter (30). These MMTV/neu Tg mice developed LOH sites are thought to harbor tumor suppressor genes, this allelotype mammary carcinoma that was pathologically very similar to the screening has allowed the mapping of putative tumor suppressor genes human tumors. Although, mammary tumors have been reported to that may be implicated, in collaboration with the erbB-2/neu oncogene, in arise after a short latent period in some MMTV/neu Tg mice (31), the development of mammary tumors in these transgenic mice. most of these MMTV/neu Tg mice develop tumors stochastically and after a long latency (30, 32), strongly suggesting that the erbB-2/neu INTRODUCTION oncogene is not by itself sufficient for tumor formation and that other genetic events (such as activation of other oncogenes and inactivation The erbB-2/neu gene encodes a growth factor receptor transmem- of recessive oncogenes) are likely to contribute to the development of brane tyrosine kinase (1, 2). This gene is frequently amplified and these malignant tumors. overexpressed in human breast cancers, and this change is thought to In an attempt to identify novel tumor suppressor genes that may be contribute at least partially to malignancy (reviewed in Ref. 3). Other involved, in collaboration with the activated erbB-2/neu oncogene, in genetic modifications, such as activation of other oncogenes or inac- the appearance of these tumors, we used the LOH analysis on tumors tivation of tumor suppressor genes, in addition to the increased ex- ϫ from (BALB/c C57BL/6)F1 MMTV/neu Tg mice. For this study, pression of the erbB-2/neu gene, are likely to be involved in the we used 62 mouse mammary tumors from MMTV/neu Tg animals development of these malignant tumors, as it has been elegantly 3 and performed a genome-wide analysis with 86 microsatellite SSLP demonstrated in other types of human tumors (4). In fact, LOH is the markers to search for LOH sites. A few specific regions of LOH on 3 most common type of mutation in human primary breast carcinoma chromosomes were identified. We are proposing that these regions (5). It affects at least 20 regions of the genome, encompassing the with allelic losses harbor tumor suppressor genes involved in the following human chromosome arms: 1p, 1q, 3p, 6q, 7q, 8p, 11p, 11q, development of these mammary tumors, in collaboration with the 13q, 16q, 17p, 17q, 18q, 22q (6–8), 2p (9), 7p (10), 8q, 9p, 15q (11), erbB-2/neu oncogene. 10q (9), 16p (12), Xp, and Xq (13). Some and maybe all of these regions of allelic loss are likely to harbor a tumor suppressor gene. This concept has now been validated by the cloning of several tumor MATERIALS AND METHODS suppressor genes in regions of LOH (14). This idea stems from the two-hit model of genetic inactivation of tumor suppressor genes by Mice. The MMTV/neu Tg mice line MN-10 has been described previously (30). They were initially produced in a (C57BL/6 ϫ C3H)F background and Knudson (15), stating that inactivation of a tumor suppressor gene 2 were subsequently maintained on a BALB/c background by crossing with male requires mutations affecting both alleles independently, the first event or female BALB/c mice (Charles River, St-Constant, Quebec, Canada) for 10 being a small germ-line mutation of a single allele, in the case of generations. At the 10th generation, the MMTV/neu Tg males were bred with familial cancer, and the second being a mutational event on the other C57BL/6 females (Charles River, St-Constant) to obtain (BALB/c ϫ C57BL/ allele, often consisting of a large chromosomal deletion scored as 6)F1 MMTV/neu Tg female mice, which developed spontaneous mammary tumors. The frequency and characterization of these tumors have been de- Received 10/5/98; accepted 3/18/99. scribed (30). The costs of publication of this article were defrayed in part by the payment of page DNA Preparation. Mammary tumors and kidneys were collected from F1 charges. This article must therefore be hereby marked advertisement in accordance with Tg females. Kidneys were used as source for normal DNA. DNA was extracted 18 U.S.C. Section 1734 solely to indicate this fact. as described previously (33) in 10 mM Tris (pH 7.5), 0.4 M NaCl, 2 mM EDTA, 1 This work was supported by a grant from the Canadian Breast Cancer Research Initiative (National Cancer Institute of Canada; to P. J.). 0.5% SDS, and 0.5 mg/ml Pronase buffer for overnight at 65°C, followed by 2 To whom requests for reprints should be addressed, at Clinical Research Institute of phenol extraction, then chloroform/isoamyl alcohol (24:1) extraction. DNA Montreal, 110 Pine Avenue West, Montreal, Quebec, H2W 1R7 Canada. Phone: was precipitated in 2 volumes of ethanol. (514) 987-5569; Fax: (514) 987-5794; E-mail: [email protected]. Microsatellites. The 86 microsatellite markers (SSLPs) used (Table 1) 3 The abbreviations used are: LOH, loss of heterozygosity; Tg, transgenic; MMTV, mouse mammary tumor virus; SSLP, simple sequence length polymorphism; AI, allelic have been described by Dietrich et al. (34, 35). They encompass all 20 somatic imbalance; Naad, neu-associated allelic deletion. chromosomes and exhibit polymorphism at the C57BL/6 and BALB/c alleles. 2438

Table 1 Characteristics of the 86 microsatellite markers used in the study of 62 background for 20 generations. In addition, in one region of chromosome 7, ϫ mammary tumors from (BALB/c-neu C57BL/6)F1, mice whose a large portion has remained heterozygote, the transgene may have cM position cM position cM position integrated in the C57BL/6 allele and been selected for. Strain bias was SSLP marker (MGD) SSLP marker (MGD) SSLP marker (MGD) observed to deviate from the expected 50% distribution and was confirmed by D1Mit70 17.8 D7Mit55 15.0 D13Mit16 10.0 a ␹2 statistical test. D1Mit132 43.1 D7Mit62 44.0 D13Mit20 35.0 D1Mit187 62.0 D7Mit68 60.0 D13Mit226 59.0 D1Mit106 85.0 D7Mit259 72.4 D13Mit151 71.0 RESULTS D1Mit113 92.3 D1Mit361 101.5 D8Mit4 14.0 D14Mit50 6.0 Characteristics of the Mammary Tumors. The MMTV/neu Tg D8Mit69 31.0 D14Mit60 15.0 D2Mit45 53.0 D8Mit86 52.0 D14Mit37 27.5 mice (line MN-10) described previously (30) and used in the present D2Mit340 68.9 D8Mit91 67.0 D14Mit34 40.0 study have been bred on a BALB/c background for 10 generations. D2Mit456 86.3 D8Mit280 72.0 D14Mit75 54.0 Both male and female MMTV/neu Tg mice were crossed with normal D2Mit230 107.0 D15Mit226 11.6 C57BL/6 mice to produce F1 mice. The F1 Tg female mice were bred D3Mit164 2.4 D9Mit247 17.0 D15Mit159 49.6 continuously to multiply the number of pregnancies and were allowed D3Mit182 23.3 D9Mit162 30.0 to age until they spontaneously develop mammary tumors between the D3Mit29 45.2 D9Mit269 43.0 D16Mit9 4.0 D3Mit189 49.7 D9Mit18 71.0 D16Mit4 27.3 ages of 10 and 26 months. Sixty-two independent mammary tumors D3Mit147 79.4 D16Mit5 38.0 (1–3 cm in diameter) were collected from these mice. Some mice D4Mit236 12.1 D10Nds1 6.0 D17Mit133 10.4 developed more than one tumor, and these were all collected and D4Mit111 21.9 D10Mit170 29.0 D17Mit221 56.7 treated as independent tumors. D4Mit178 35.5 D10Mit15 35.0 D4Mit12 57.6 D10Mit10 51.0 D18Mit64 2.0 LOH Analysis. The DNAs from these 62 MMTV/neu Tg mam- D4Mit54 66.0 D10Mit35 69.0 D18Mit63 16.0 mary tumors were analyzed by using 86 microsatellite markers D4Mit127 77.5 D18Mit52 32.0 D18Mit49 49.0 (SSLPs) distributed on each chromosome. These markers were chosen D5Mit346 1.0 D11Mit80 10.0 according to their polymorphism, and an effort was made to have D5Mit233 29.0 D11Mit20 20.0 D19Mit19 26.0 them evenly dispersed (ϳ20 cM; Table 1). Assuming that each marker D5Mit240 59.0 D11Mit5 37.0 D19Mit10 47.0 D11Mit41 49.0 D19Mit34 53.0 covers 10 cM on each of its sides, we estimated that 76% of all 20 D11Mit50 66.0 D19Mit71 54.0 chromosomes were covered by our 86 markers. LOH was a very D6Mit83 2.7 frequent event in these neu-induced tumors; as much as 56 of 62 D6Mit8 35.2 D12Mit37 1.0 DXMit73 19.0 D6Mit39 46.3 D12Mit110 19.0 DXMit64 45.0 tumors (90%) had at least one chromosome with LOH. The mean D6Mit374 63.9 D12Mit5 37.0 DXMit172 47.0 number of chromosomes having an LOH per tumor was 2.1, and the D6Mit14 74.0 D12Nds2 59.0 DXMit236 56.8 D6Mit15 74.0 DXMit249 70.8 median frequency was 2.4. We found that the highest number of The position given in cM is from Mouse Genome Database (MGD). chromosomes with LOH in individual tumors could go up to 9 (Fig. 1). The frequency of allelic loss on each chromosome is given in Fig. 2. The overall rate of LOH was 11%. On most chromosomes, the rate The linkage position for these markers was found in the Whitehead MIT database. These DNA marker oligonucleotides were purchased from Research of LOH was low, around 5% or below (chromosomes 2, 3, 5, 6, 10, Genetics (Hunstville, AL). 11, 12, 13, 15, 17, and 18) or close to the average rate of 11% PCR. Analysis for LOH was done by PCR amplification using one radio- (chromosomes 7, 9, 14, 16, and X). Chromosome 1 showed LOH at labeled primer, the “forward” primer. The labeling mix reaction consisted of a frequency of ϳ20%, but further analysis showed that this did not 0.05 ␮M “forward” primer, 1 ␮Ci/␮l (3000 Ci/mmol) [␥-32P]ATP (Amer- appear to be significantly different from the overall rate (see below). sham), and 0.125 unit/␮l T4 DNA polynucleotide kinase (Pharmacia) and was Interestingly, a much higher frequency of LOH was detected on incubated for 30 min at 37°C, then heated at 100°C for 30 s. The PCR mix chromosome 8 (21%), chromosome 4 (50%), and chromosome 19 reaction was made in a 10-␮l volume with 20 ng of genomic DNA, 0.05 ␮M (32%). The frequency of LOH was significantly different from the ␮ of labeled “forward” primer as well as 0.05 M of unlabeled “reverse” primer; overall rate of 11% for chromosomes 4 and 19 (P Ͻ 0.001) and for 200 ␮M of each four deoxynucleotides (dATP, dCTP, dGTP, and dTTP), 0.125 chromosome 8 (P ϭ 0.05). unit/␮l Taq polymerase (Pharmacia), and 1ϫ PCR buffer [10 mM Tris-HCl To have a better picture of the specific location of the LOH on each (pH 8.3), 50 mM KCl, and 1.5–2.5 mM MgCl2]. Amplification was performed in a Perkin-Elmer 9600 instrument with the following parameters; 94°C for 3 chromosome, the frequency of LOH detected with each marker was min, then 25 cycles of 94°C for 15 s, 55°-60°C for 2 min, 72°C for 2 min, and plotted (Fig. 3). The average mean frequency of the LOH background finally 72°C for 7 min. Analysis of SSLPs. Loading buffer (2ϫ; 0.08% bromphenol blue, 0.08% Xylene cyanol, and 10% glycerol) was added to amplified PCR mix reaction. Samples (5 ␮l) were loaded on a 6% nondenaturing PAGE and run in 1ϫ TBE buffer (0.09 M Tris, 0.09 M boric acid, 2 mM EDTA). Each sample was always compared with its normal DNA (kidney) extracted from the same animal. The assessment of allelic loss (LOH) was visually scored on an autoradiographic film. Only tumors showing 50% or more decrease in intensity of one of the allelic fragments were scored as positive for LOH. More than two assays were done to confirm LOH. In case of a moderate decrease (25%) in intensity for an allelic fragment, it was scored as an AI. Statistical analysis to determine the significance of the percentage values of tumors with LOH for one chromosome as compared with the overall rate of LOH for all chromosomes was done by a two-tailed Fisher exact test. The overall rate of LOH was calculated as the number of tumors with LOH over the number of all informative tumors. Noninformative tumors retained some C57BL/6 genomic regions present in the Fig. 1. Fraction of MMTV/neu Tg mammary tumors with LOH with any marker along ϫ a chromosome. Sixty-two tumors were used for this analysis. The average mean number (C3H C57BL/6)F2 embryo inoculated initially with the transgene. This may of chromosomes having LOH per tumor was 2.1, whereas the median number of reflect the fact that these Tg mice have not yet been bred on a BALB/c chromosomes with LOH per tumor was 2.4. 2439

observed with the D4Mit178 marker (35.5 cM). Interestingly, despite the fact that several tumors have lost apparently the entire chromosome 4, a small number of tumors showed selective LOH ϩ AI at each of the individual markers, except D4Mit127, independently of each other (Fig. 4B). Although the number of tumors exhibiting these characteristics is small, this observation suggests the presence of as many as four and possibly five independent distinct loci affected by allelic deletion along the chromosome 4. These regions have been designated Naad1 to Naad5 (Fig. 4B). Our results also show a strong strain bias on allelic loss on this chromosome 4, because deletions of these alleles significantly deviate from an expected 50% distribution between BALB/c and C57BL/6 alleles. Of 31 tumors with LOH, only 7 had lost the C57BL/6 allele, whereas 24 tumors lost the BALB/c allele. This represents a significant difference (P Ͻ 0.002). No such strain bias on all other chromosomes, except chromosome 19 (below), was found in this set of tumors. Fig. 2. Fraction of MMTV/neu Tg mammary tumors with LOH on each chromosome. The total number of informative tumors for each chromosome varies between 35 and 62. LOH on chromosome 19. Our analysis of chromosome 19 showed The overall rate of LOH in these tumors was 11%. The frequency of LOH on chromo- that the rate of LOH varies from 24 to 26%, along the chromosome ,P Ͻ 0.001; 32%, P Ͻ 0.001; 21% ,%50 :ء) somes 4, 19, and 8 was statistically significant P ϭ 0.05, respectively). from 26 to 54 cM (Fig. 5A). When the number of tumors with LOH ϩ AI are added, this enables us to distinguish from the relatively high rate of allelic loss for the marker D19Mit19 (44%), relative to the of all chromosomes was 8.7%. With most of the markers, the fre- three other markers at 39% (D19Mit10), 32% (D19Mit34), and 30% quency of LOH was 5% or below. A few markers scored LOH in (D19Mit71). Moreover, these data suggest that possibly two distinct ϳ 10% of the tumors, not significantly different than the overall rate. loci may be affected by allelic deletion, one (Naad6) at or proximal to Markers on chromosome 4 remained significantly different and could D19Mit10 and the other (Naad7)atD19Mit34 or distal to it (Fig. 5B). detect LOH in 28–48% of the tumors. Similarly, LOH on chromo- The fact that a high proportion of tumors exhibiting allelic loss some 19 was measured with four different markers in 24–26% of the (LOH ϩ AI) on this chromosome have lost most of the chromosome tumors. The frequency of LOH on chromosome 8 was lower. One also suggests the presence of more than one allelic deletion site (Fig. marker (D8Mit280) could detect LOH in only 3.2% of the tumors, 5A). Additional tumors will need to be analyzed to assess the number whereas another one (D8Mit91) could detect LOH in 21% of the tumors (three times the value of the background). Comparison of the and correct percentage of LOH sites on this chromosome. percentage of tumors with LOH detected with all four of chromosome As with chromosome 4, a strain bias for allelic loss was observed. 8 markers used showed a significant difference (14%) from the Fifteen tumors of 20 exhibited deletion of the C57BL/6 allele, whereas ϭ background mean of 8.7% (P ϭ 0.042). only five tumors lost the BALB/c allele (P 0.025). However, this Together these results indicate that LOH on chromosomes 4, 19, strain bias on allelic loss of chromosome 19 (C57BL/6 allele loss) was and 8 is a frequent event in mammary tumors arising in MMTV/neu different from the one observed on chromosome 4 (BALB/c allele Tg mice. loss). LOH on Chromosome 4. Of 62 tumors, 43 (69%) showed LOH or LOH on Chromosome 8. The highest percentage of tumors with AI on chromosome 4 (Fig. 4A). Nine, and possibly 12, tumors had LOH on this chromosome is 21% (Fig. 6A). The deleted region complete deletions of a region spanning from 12.1 to 77.5 cM, which includes the D8Mit86 and D8Mit91 markers and spans from 52 to 67 is almost the entire chromosome 4. This type of deletion accounts for cM between the D8Mit280 marker (72 cM) and the D8Mit69 marker 28% of the 43 tumors, and for 40% when considering the 31 tumors (31 cM). Possibly two distinct loci may be affected by allelic deletion with LOH only. The highest percentage of LOH (48%) was found on this chromosome: one (Naad8) proximal to D4Mit91 and the other with the D4Mit54 marker (66 cM). Another peak of LOH (42%) was one (Naad9) distal to D8Mit86. The analysis of additional tumors may

Fig. 3. Fraction of MMTV/neu Tg mammary tumors with LOH for each marker (SSLP) analyzed. The total number of informative tumors varies between 35 and 62. The overall rate of LOH in these tumors was 8.7%. The frequency of LOH for all markers used on chromosomes 4, 19, and 8 was statistically .(P Ͻ 0.001; P Ͻ 0.001; P ϭ 0.042, respectively ,ء) significant

2440

Fig. 4. Deletion mapping of chromosome 4 in MMTV/neu Tg mammary tumors. A, six microsatellite markers that cover from 12.1 to 77.5 cM were used. The two highest scores for LOH are shown at 35.5 and 66 cM along the chromosome box. f, tumors with LOH; u, tumors with a partial deletion of one allele (25% AI); Ⅺ, no change in the allelotyping (still heterozygotes for BALB/c and C57BL/6 alleles); E, noninformative (n.i.) samples (animals carrying homozygous C57BL/6 alleles). The number of tumors for each of the four types of data (LOH, AI, heterozygote, and n.i.; in parentheses are number of tumors from all 62 tumors) are presented at the bottom of the figure along with the calculated percentage of LOH for each marker. B, allelic deletions grouped in five putative regions (Naad1 to Naad5) representing tumors that share the same LOH or AI locus at each marker. Our data do not exclude the possibility that for example Naad1 and Naad2 represent a single locus mapping between D4Mit236 and D4Mit111. confirm the presence of these independent loci. No strain bias was is syntenic to human 16q22.1 where the E-cadherin (CDH1) gene has observed for this chromosome. been mapped. In human breast cancers, LOH has been observed in this 16q22.1 region (36), and E-cadherin has been found to be mutated DISCUSSION as a tumor suppressor gene (22). The second (Naad9) locus mapped distal to 67 cM and colocalized with the aprt (adenine phosphoribosyl The identification of additional genetic events, which might coop- transferase) and Cdh15 loci. The human syntenic 16q24.3, where erate with the activated neu/erbB-2 oncogene to generate mammary M-cadherin (CDH15) has been mapped, was reported to be deleted in carcinoma, appears to be important to understand the molecular basis human breast cancers (36, 37). Therefore, E- and M-cadherin (CDH1 of this neoplasia. To this end, we looked for recessive oncogenes that and CDH15) remain candidate tumor suppressor genes on the mouse may be selectively lost in these tumors. To map such putative tumor chromosome 8. suppressor genes, we used the allelic deletion or LOH approach on 62 The LOHs detected on chromosome 19 cover a large region of the mammary carcinoma DNAs from (BALB/c ϫ C575BL/6)F MMTV/ 1 chromosome, with a possibility that two distinct loci are affected by neu Tg mice with 86 microsatellite markers dispersed on all chromo- LOH. One of these regions, Naad6, may represent the Pas3 LOH somes. LOH was detected at a low frequency at several loci across the region proximal to D19Mit19 identified in other murine tumors, i.e., genome, with an overall rate of 8.7%. Although rare, these genetic liver and lung tumors (38–40). However, deletion of chromosome 19 events may not be insignificant for a given tumor but remain difficult to study experimentally. Interestingly, we also found that a few loci regions is an uncommon event in other mouse mammary tumors had sustained allelic deletion in a significant percentage (21–48%) of induced by other methods in different mouse strains (41–44). The these tumors, indicating that these genetic events tended to be specific human 11q region is syntenic to the proximal region of mouse and frequent in this set of tumors. The markers showing the highest chromosome 19 and is known to show LOH in breast carcinomas (45). score for LOH were D4Mit54 (48%), D4Mit178 (42%), D4Mit236 The other human syntenic region of this chromosome, 10q23–25, (28%), D4Mit111 (32%), D4Mit12 (37%), D4Mit127 (35%) on chro- harbors a known tumor suppressor gene, PTEN/MMAC1, deleted in a mosome 4 and D19Mit10 (26%), D19Mit34 (26%), D19Mit71 (26%) variety of human tumors, including breast cancers (19, 20). The allelic on chromosome 19. Another marker, D8Mit91, on chromosome 8 had deletion locus (Naad6) observed for the D19Mit19 (26 cM) marker is a lower score (21%). in the vicinity of the mouse PTEN gene, which maps at 24.5 cM. The Our analysis revealed that possibly up to two independent putative other putatively independent distal LOH region, Naad7, is syntenic to tumor suppressor loci are present on chromosome 8. One of them the human 10q25–26 region, which has not been reported to show (Naad8) mapped close to the E-cadherin (CDH1) gene (53.3 cM). It LOH in human breast cancers. However, LOH of this region has been 2441

tumor suppressor genes. The Naad2 region is syntenic to the human 9q32–34, which harbors a candidate tumor suppressor gene known as DBCCR1, the promoter of which was found hypermethylated in bladder cancer (54). The Naad3 locus covers a region harboring two well-known genes, MTS1(p15INK4b) and MTS2(p16INK4a), which have been reported to be deleted in mouse hepatocellular carcinoma cell lines (55–57), in thymic lymphomas (49, 50, 58), and in lung tumors (44, 59–61). In the latter tumors, homozygous deletions of the genes p15INK4b and p16INK4a have been observed (62). Although we have not observed homozygous deletions of the D4Mit178 marker in the neu-induced mammary tumors studied here, p15INK4b and p16INK4a remain candidate tumor suppressor genes in these neu-induced mammary carcinomas. Naad3 corresponds to the TLSR1 locus of Santos et al. (49). This Naad3 locus is syntenic with human 9p21–23, which harbors the p15INK4b and p16INK4a (CDKN2a and CDKN2b) genes, in addition to being syntenic with the 1p31–35 region. The former region is frequently deleted in different types of human cancers (63), including breast cancers (26, 64). However, inactivation of p16/CDKN2a is a rare event in primary breast tumors and was found predominantly in immortal breast epithelial cell lines (26). More- over, it has been reported recently that p16INKa (CDKN2a) expression was increased in human breast carcinoma (65). In addition, Radany et al. (41) found that these two p15INK4b and p16INK4a genes were unlikely to be involved in ras-induced mammary carcinomas. Together, these results suggest that these two genes may not be implicated in neu-induced mammary tumors, and that a novel tumor suppressor gene may be inactivated.

Fig. 5. Deletion mapping of chromosome 19 in MMTV/neu Tg mammary tumors. A, four microsatellite markers that cover from 26 to 54 cM were used. Symbols are the same as those in Fig. 4. B, at least two putative regions of allelic deletions (Naad6 and Naad7) are shown by grouping tumors that share the same LOH or AI locus at each marker.

observed in other types of human cancers, and candidate tumor suppressor genes mutated on both alleles have been identified: MXI1 in prostate cancer (46), DMBT1 in malignant brain tumor cell lines (47), and h-neu (human homologue of Drosophila neuralized gene) in malignant astrocytomas (48). More than two and possibly five distinct loci (Naad1 to Naad5) exhibiting allelic deletion at relatively high frequency were found on chromosome 4. The simultaneous loss of several of these putative tumor suppressor genes may favor tumor growth, and this could explain the loss of the entire chromosome 4 observed in a significant proportion of the neu-induced mammary tumors (15%). A very similar pattern of LOH has been reported to occur in thymic lymphomas (49, 50), in ras-induced mammary carcinomas (41), and in lung tumors (51). These investigators have postulated the presence of at least two and up to three distinct loci of LOH. The Naad1 and Naad2 loci may correspond to the proximal LOH locus reported by Radany et al. (41). This Naad1 region is in synteny with the human regions Fig. 6. Deletion mapping of chromosome 8 in MMTV/neu Tg mammary tumors. A, four microsatellite markers that cover from 31 to 72 cM were used. The highest score for 8q11–q22, 6q14–21, and 9p13–q32, and the Naad2 region is syntenic to LOH is shown with the chromosome box at 67 cM. Symbols are the same as those in Fig. human 9p13-q34. The 6q14–15 region has been reported to exhibit LOH 4. B, two putative allelic deletion loci (Naad8 and Naad9) are shown by grouping tumors that share the same LOH or AI locus at each marker. No distinct allelic deletion locus was in breast cancer (52). LOH has been reported in the 8q22 region in adult found between the D8Mit69 and D8Mit86 markers. Our data do not exclude the possibility acute myeloid leukemia (53). This region harbors no known candidate that Naad8 and Naad9 represent a single locus mapping between D8Mit86 and D8Mit91. 2442

The presence of Naad4 (D4Mit12; 57.6) locus is suggested by AI in ACKNOWLEDGMENTS only two tumors. Additional tumors will have to be studied to confirm the presence of an allelic deletion site in this region. This region is We thank Michel Ste-Marie, Benoit Laganie`re, and Patrick Couture for excellent technical assistance. We are grateful to Alain Guimond and Ste´phane likely to correspond to the Lci LOH locus described in liver epithelial Sirois for initial work on this project. cell lines (66). Naad4 is syntenic to human 1p32–36, which harbors the mammary-derived growth inhibitor (MDGI) gene, reported to REFERENCES exhibit LOH in human breast tumors (67). The connexin 37 (GJA4) 1. Stern, D. F., Heffernan, P. A., and Weinberg, R. A. p185, a product of the neu gene (1p35.1; Ref. 68) is also present in this allelic deletion locus, proto-oncogene, is a receptorlike protein associated with tyrosine kinase activity. where it maps at 57.6 cM on this mouse chromosome. Mol. Cell. 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Marc Cool and Paul Jolicoeur

Cancer Res 1999;59:2438-2444.

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