Breast cancer is the most frequent malignant tumor affecting women in the western world, and it accounts for more deaths than any other single cancer in women ...
Comparative Molecular Carcinogenesis, @ 1992 Wiley.Liss, Inc.
pages 137-153
CHROMOSOME ALTERATIONS IN RAT MAMMARY TUMOR PROGRESSION C. Marcelo Aldaz, Lauren S. Gollahon and Aaron Chen Department of Carcinogenesis, The University of Texas M. D. Anderson Cancer Center, Science Park Research Division, Smithville, Texas 78957 Breast cancer is the most frequent malignant tumor affecting women in the western world, and it accounts for more deaths than any other single cancer in women. Research efforts led to the development of numerous in vivo and in vitro models for the study of mammary carcinogenesis. Among these, the rat mammary carcinogenesis system is one of the most widely used. Since the pioneering work of Huggins in the early sixties (Huggins and Yang, 1962), much has been investigated using this model. Although most of the research has been conducted with dimethylbenz(a)anthracene as the carcinogenic agent, in 1975, Gullino et a1. demonstrated that the direct alkylating agent nitrosomethylurea (NMU) was also a potent mammary carcinogen. Later it was shown that NMU-induced tumors are responsive to specific ovarian and pituitary hormones and have receptors for estrogen, progesterone, prolactin, androgen, and glucocorticoids (reviewed by Welsch, 1985). Molecular studies on NMUinduced tumors provided evidence that the Harvey-IWi (H-ma) oncogene was directly involved in carcinogenesis (Sukumar et aI., 1983). It was shown that most tumors induced by this carcinogen had a point mutation (G-to-A transition) in the second nucleotide of codon 12 of the H-x:.a.agene; it was postulated that this mutation could be the initiation event. Very little is known about further steps required to achieve the full malignant phenotype.
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Much evidence from this model and other models has supported the concept of a multistage carcinogenesis. It is now clear that activation of oncogenes, inactivation of tumor suppressor genes, and gross chromosomal abnormalities play key roles in the complexity of the neoplastic progression. Practically no information is available on the role of gross chromosomal abnormalities in the rat mammary carcinogenesis system, despite widespread use of the model. Most previous cytogenetic information was obtained before the development of chromosomal banding (Majumdar and Rees, 1970;Takahashi et al., 1977). The first goal of our studies was to fill that gap in the understanding of the physiopathologyof the rat mammary tumor model. It is known that most rat mammary adenocarcinomas are well-differentiated lesions with little invasion and low metastic potential (Russo et al., 1990). Thus in our study we analyzed the cytogenetic profile of primary NMU-induced tumors and to study more advanced stages of progression, we follow two approaches: 1) we analyze the cytogenetic progression of tumors successively transplanted into younger syngeneic hosts (Fig. lA); 2) we also analyze cytogenetically ovary-independent tumors, defined as those lesions that did not regress or that developed after the hosts were subjected to ovariectomy(Fig. IB). CYTOGENETIC FINDINGS, CHROMOSOME 1 AND 15 INVOLVEMENT The chromosomal analysis was performed on shortterm tumor cultures, 2-4 days after plating. The culture technique has been described elsewhere (Aldaz et al., submitted for publication). Tumor organoids were plated using a low-serum (2%), low-calcium medium. This culture condition does not favor the attachment and growth of contaminating fibroblasts (Fig. 2). As can be observed from representative samples shown in Table 1, every primary tumor, including the ovaryindependent tumors, showed a diploid chromosomemode of 42 chromosomes.
Cyto~.n.t1cs of Rat Mammary Tumor. I 139
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q42. Previously, two other members of the same linkage group had been mapped by in situ hybridization, the hemoglobin beta gene cluster (HBB)and the insulin 2 gene (lNS2) (Soares et al., 1985; Levan et aI., 1991). Interestingly, both genes were mapped far apart from each other within chromosome 1: HBB was mapped to band 1q22 and INS2 to region 1q41-->q42.Our mapping study locates the H-ull.1 gene in the same region as INS2. However, all three genes in humans map to the same band llp15.5 (Fig.7). Numerous studies on different tumor types have shown LOH for genes located in llp15.5, in many cases such LOH involved the H-r..a.a.1and HBB loci. If indeed a tumor suppressor gene is in that region (Newsham et al., 1991), the rat may be a good model for discerning association of that putative gene with either HBB or H-maJ because of their distant chromosomal location (Fig. 6). Interestingly, the area of eytogenetic deletion that we observed in our rat mammary tumors involved the same band (lq22) where the HBB locus had been mapped. Thus, the occurrence of deletion and breakpoint for translocation in 1q22 may be pointing to a putative tumor suppressor locus in that region. The observed cytogenetic overrepresentation of chromosome 1 material - either through the direct duplication 1q22q43or through trisomy 1 observedin several tumors (Fig. 4C and D) - may be a mechanism used by tumor cells to amplify the mutated H-Illll.allele. In fact, all six tumors that showedthis type of abnormality also showedthe codon 12 G-toA transition. Furthermore our mapping of H-Illll.in 1q41-->q42 located this gene within the area contained by the direct duplication (Figs.4 and 6).
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Figure 6. Left: rat chromosome 1 diagram according to Levan et al.(1991). Bars represent regional sublocalization of the indicated genes. RIght: representative chromosomes 1 demonstrating hybridization of the H-ras probeto region lq41-->q42.
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Figure 7. Schematic representation of rat chomosome 1 and human chromosome 11. Both areas identified to be affected by cytogenetic or molecular abnormalities in rat chromosome 1 in mammary tumors map to human region llp15.5.
Cylo,enetica of Rat Mammary Tumor. I 149
Southern analysis of some of the tumor passages supported the idea that tumor cells tended to amplify the H-r.w> gene in more advanced stages of progression. Contrary to what was observed in the primary tumors, an increase in the H-rlll!1 signal was observed in the tumor passages (Aldaz et aI., submitted for publication). This coincided with the 'presence of trisomy 1 or the chromosome 1 direct duplication . Further these results are very similar to results in other models of epithelial carcinogenesis where H-m.a activation plays a relevant role in initiation and progression (Bianchi et a1.,1990). Although it has been demonstrated that H-ra.s.activation is unusual in human breast cancer, several reports have shown that overexpression of the H-nu. P21 protein is common. Nevertheless, the rat mammary system will constitute a good in vivo model to understand interactions between an activated oncogeneand tumor suppressor genes. Further studies will also be performed to elucidate the role of the chromosome 15 abnormalities. Several of the ovaryindependent tumors showed abnormalities affecting this chromosome. It is worth noting that at least three genes of interest in breast cancer have been mapped to rat chromosome 15 (Fig. 5). The retinoic acid receptor beta chain (RARB)and the thyroid hormone receptor beta (THRB)in humans map to chromosomeregions 3p24 and 3p24.1-22(Levan et aI., 1991). It has been shown that numerous human breast tumors present abnormalities affecting 3p (Trent, 1985) as well as LOH affecting region 3p21-p25 (Ali et aI., 1989; Callahan and Campbell, 1989). Also the retinoblastoma protein (RBI), that showed involvement in some breast tumors (Callahan and Campbell,1989) has been mapped to rat chromosome 15 (Fig. 5). In summary, additional studies on this model may identify specific similarites with human breast cancer and eventually allow the elucidation of the sequence and consequenceof each event.
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MODEL OF MAMMARY TUMOR PROGRESSION As previously mentioned, the lack of metastasis is a commonfeature of the rat mammary tumors. We generated a model that allows the expression of the metastatic phenotype. To date, we generated in vivo transplantable lines from 10 of 11 tumors from ovariectomized animals. This model is similar to that proposed by Dulbeccoand Armstrong (1988), but in our model the primary tumors are obtained from ovariectomized animals and the transplants are performed on ovariectomized hosts. As result of these studies we observed that forty percent of the primary tumors already showed metastases. Upon subsequent in vivo passages, 8 of the 10 tumor lineages showed metastatic phenotypes (Fig. 8). We hope that further cytogenetic and molecular analysis of ovary-independent tumor lineages will provide new insights into the mechanisms of .mamrna'rytumor progression. .
Figure ~. Spo'.'taneous lung (A) and .1iv~r (B) metastases that developed in an ovariectomized host from a third In VIVO passage level ovary independent rat mammary tumor with karyotype 45,xx,+1.+6,+12.
Cytogcoctlcs of Rat Mammary Tumors I IS 1
Acknowledgments: This work was supported by grant CA 48922from the United States National Institutes of Health. We are grateful to Dr. Jose Russo for the histopathologic evaluation of tumors. We wish to thank also Michelle Gardiner for excellent secretarial assistance, and Judy Ing and John Riley for the art work. REFERENCES Aldaz CM, Chen A, Gollahon LS, Russo J, Zappler K: Nonrandom abnormalities involving chromosome 1 and Harvey-ras-l alleles irrrat mammary tumor progression. Submitted for publication. Ali IU, Lidereau R, Theillet C, Callahan R (1987): Reduction to homozygosity of genes on chromosome 11 in human breast neoplasia. Science238:185-188. Ali IU, Lidereau R, Callahan R, (1989): Presence of two members of c-erbA receptor gene family (c-erbA~and cerbA2) in smallest region of somatic homozygosity on chromosome 3p21-25 in human breast carcinomas. J Natl Cancer Inst 81:1815-1820 Bianchi AB, Aldaz CM, Conti CJ (1990): Nonrandom duplication of the chromosomebearing a mutated Ha-ras1 allele in mouse skin tumors. Proc Nat! Acad Sci USA 87:6902-6906. Callahan R, Campbell G (1989): Mutations in human breast cancer an overview. J Nat! Cancer Inst 81:1780-1786. Dulbecco R, Armstrong B (1988): Stochastic development of invasive potential in rat mammary tumors induced by Nmethyl-N-nitrosourea. Proc Natl Acad Sci USA 85:86598663.Fallenius A, Skoog L, Svane G, Auer G (1984): Cytophotometrical and biochemical characterization of nonpalpable, mammographically detected mammary adenocarcinomas. Cytometry 5:426-429. Ferti-Passantonopoulou A, Panani AD, Raptis S (1991): Preferential involvement of llq23-24 and llp15 in breast cancer. Cancer Genet Cytogenet51:183-188. Freidlandler ML, Hedley DW, Taylor IW (1984): Clinical and biological significance of aneuploidy in human tumors. J Coo PathoI37:961-974.
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