Development of spontaneous mammary tumors in BALB/c p53 heterozygous mice. A model for Li-Fraumeni syndrome

Abstract

Breast cancer is the most frequent tumor type among women in the United States and in individuals with Li-Fraumeni syndrome. The p53 tumor suppressor gene is altered in a large proportion of both spontaneous breast malignancies and Li-Fraumeni breast cancers. This suggests that loss of p53 can accelerate breast tumorigenesis, yet p53-deficient mice rarely develop mammary tumors. To evaluate the effect of p53 loss on mammary tumor formation, the p53(null) allele was back-crossed onto the BALB/c genetic background. Median survival was 15.4 weeks for BALB/c-p53(-/-) mice compared to 54 weeks for BALB/c-p53(+/-) mice. Sarcomas and lymphomas were the most frequent tumor types in BALB/c-p53(-/-) mice, whereas 55% of the female BALB/c-p53(+/-) mice developed mammary carcinomas. The mammary tumors were highly aneuploid, frequently lost the remaining wild-type p53 allele, but rarely lost BRCA1. Although mammary tumors were rarely detected in BALB/c-p53(-/-) female mice, when glands from BALB/c-p53(-/-) mice were transplanted into wild-type BALB/c hosts, 75% developed mammary tumors. The high rate of mammary tumor development in the BALB/c background, but not C57Bl/6 or 129/Sv, suggests a genetic predisposition toward mammary tumorigenesis. Therefore, the BALB/c-p53(+/-) mice provide a unique model for the study of breast cancer in Li-Fraumeni syndrome. These results demonstrate the critical role that the p53 tumor suppressor gene plays in preventing tumorigenesis in the mammary gland.

Figure 1.

Survival curves of BALB/c p53-deficient mice. The probability of tumor-free survival of BALB/c-p53^−/− mice (n = 85; closed circles), and BALB/c-p53^+/− mice (n = 56; open circles) was monitored for 80 weeks. No wild-type animals died during this study (n = 5). Dashed lines represent the median time at which 50% of the animals had developed tumors.

Figure 2.

Tumor distribution and mammary abnormalities in BALB/c-p53^−/− mice. A: Tumor spectrum from BALB/c-p53^−/− mice. Frequency histogram of tumor types observed in both male and female mice (n = 44). B: Mammary phenotype of female BALB/c-p53^−/− mice. Relative frequency of abnormalities detected in the inguinal or thoracic gland from nulliparous and breeder females (n = 36). C: H&E-stained sections of mammary tissue from BALB/c-p53^−/− females. The normal murine mammary gland architecture is characterized by large adipocytes in the stroma surrounding a sparse population of ducts (i, ×40 objective). Typical stromal changes observed in BALB/c-p53^−/− females (ii and iii). Stroma characterized by microvesicular adipocytes and proliferation of mammary ducts (ii, ×40 objective). Hypercellular stroma and dilated ducts with attenuation of the mammary epithelium (iii, ×20 objective).

Figure 3.

Tumor distribution and mammary abnormalities in BALB/c-p53^+/− mice. A: Tumor spectrum from BALB/c-p53^+/− mice. Frequency histogram of tumor types observed in both male and female mice (n = 45). B: Mammary phenotype of female BALB/c-p53^+/− mice. Relative frequency of abnormalities detected in the inguinal or thoracic mammary glands from nulliparous and breeder females (n = 38). Mammary hyperplasia was observed either alone or in association with tumors in all glands analyzed. C: H&E-stained sections of mammary tumor tissue from BALB/c-p53^+/− females (×20 objective). A typical adenoacanthoma characterized by keratin formation (i) and an adenocarcinoma with small acinar structures (ii). H&E section of a mammary carcinoma infiltrating adjacent stroma. (iii, ×20 objective). Ductal hyperplasia commonly seen in BALB/c-p53^+/− female mice (iv, ×40 objective).

Figure 4.

Analysis of DNA from BALB/c-p53^+/− mammary carcinomas. A: Representative DNA histograms determined by FACS analysis from mammary tumors (n = 14). Seventy-one percent of the tumors were aneuploid with DNA content >40 (i), and the remaining 29% of the tumor were diploid (ii). The solid line represents the distribution of cells. Subpopulations of cells were calculated using the ModFit LT software and are represented by the shaded peaks. B: Representative Geimsa-stained (original magnification, ×100) metaphase spreads of mammary carcinomas from BALB/c-p53^+/− females. Typical abnormalities such as quadriradial chromosomes (i) and chromosome breaks commonly found in aneuploid karyotypes (ii) marked by arrows, respectively. C: Southern blot analysis of p53 and BRCA1 in BALB/c-p53^+/− mammary tumors. Genomic DNA from mammary tumors (MT), a prepucial adenoma (PA), a salivary gland carcinoma (SC), and tails (tail) were digested with EcoRI and StuI. Loss of heterozygosity was determined using a genomic DNA clone spanning exon 7 to exon 9 of the p53 gene. Three bands were detected in tail DNAs from BALB/c-p53^+/− mice (tail +/−). These fragments represent the wild-type allele (wt p53), the mutant allele (mut p53), and the pseudogene (ψ p53). The blot was reprobed with exon 11 of BRCA1 to determine whether there was loss of BRCA1 in the mammary tumors from BALB/c-p53^+/− mammary tumors.

Figure 5.

Histology of mammary carcinomas derived from transplants. A: The transplantation of a BALB/c-p53^−/− whole gland into a wild-type recipient yielded mammary tumors as early as 7 months. Typical adenocarcinoma with structures reminiscent of mammary ducts and several mitotic figures (H&E, ×40 objective). B: Adenocarcinomas also developed from reconstituted glands consisting of p53-deficient epithelium in a wild-type fat pad. A poorly differentiated mammary carcinoma invading the adjacent skeletal muscle (H&E, ×20 objective). C: An adenocarcinoma arising from a p53^null/wt transplant that has both acinar structures (white arrow) and a spindle cell component (×40 objective). D: Lung metastasis from a mammary carcinoma derived from a reconstituted gland consisting of p53^−/− epithelium in a wild-type fat pad (H&E, ×10 objective).