Ellen S. Dickinson

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

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.

A mammary-specific model demonstrates the role of the p53 tumor suppressor gene in tumor development

Although alterations in the p53 tumor suppressor gene are detected frequently in human breast cancers, mammary tumors are observed infrequently in p53null mice. This has led to the suggestion that absence of p53 alone is not sufficient for induction of mammary tumors. However, early death of p53null mice from thymic lymphomas may obscure tumor phenotypes that would develop later. Therefore, p53null mammary epithelium was transplanted into cleared mammary fat pads of wild type p53 BALB/c hosts to allow long-term analysis of mammary tumor phenotypes. Five treatments were compared for their effects on tumor incidence in hosts bearing transplants of p53null and p53wt mammary epithelium. The treatment groups were: (1) untreated; (2) continuous hormone stimulation with pituitary isografts; (3) multiple pregnancies; (4) DMBA alone; and (5) DMBA+pituitary isografts. The tumor incidences in p53null vs p53wt mammary transplants for each treatment group were 62% vs 0%, 100% vs 0%, 68% vs 0%, 60% vs 4% and 91% vs 14%, respectively. The mammary tumors that developed in the p53null mammary epithelium were all adenocarcinomas and were frequently aneuploid. These data demonstrate that the absence of p53 is sufficient to cause development of mammary tumors and that hormonal stimulation enhances the tumorigenicity of p53null mammary epithelium to a greater extent than DMBA exposure alone. This model provides an in situ approach to examine the molecular basis for the role of p53 in the regulation of mammary tumorigenesis.

Delayed involution of the mammary epithelium in BALB/c-p53null mice

In mammals, weaning of neonates and subsequent milk stasis initiates removal of the secretory epithelium of the mammary gland by apoptosis. The p53 tumor suppressor gene is induced rapidly following weaning of neonates, but its role in the process of involution has not been defined. Therefore, experiments were performed to identify the cell types in which the p53 gene is expressed during involution and determine the consequences of its absence in BALB/c-p53null mice. Both p53 mRNA and protein were detected in the mammary epithelium within 48 h following weaning and resulted in an eightfold increase in levels of p21WAF1 mRNA. Induction of p21WAF1 mRNA was absent in BALB/c-p53null mice, and therefore, was shown to be p53-dependent. The BALB/c-p53null mice exhibited delayed involution of the mammary epithelium, as measured by 60% greater epithelial area compared to BALB/c-p53wt mice through 5 days post-weaning. The delay was transient with no differences being apparent at 7 days post-weaning. Expression of the stromal protease stromelysin-1 was unaffected by the absence of p53 suggesting that stromal responses were intact. These data demonstrate that p53 participates in the first stage of involution initiated by the epithelium itself, but does not affect the second phase during which stromal proteases are induced.

Estrogen and progesterone regulate radiation-induced p53 activity in mammary epithelium through TGF-beta-dependent pathways

DNA damage normally induces p53 activity, but responses to ionizing radiation in the mammary epithelium vary among developmental stages. The following studies examined the hormones and growth factors that regulate radiation-responsiveness of p53 in mouse mammary epithelium. Immunoreactive p21/WAF1 and TUNEL staining were used as indicators of p53 activity following exposure to ionizing radiation. In ovariectomized mice, radiation-induced accumulation of p21/WAF1 was minimal in the mammary epithelial cells (<1%). Systemic injections of estrogen and progesterone (E+P) for 72 h were necessary to recover maximal expression of p21/WAF1 following ionizing radiation (55%). The effects of E+P on radiation-induced p21/WAF1 were p53-dependent as responses were absent in Trp53−/− mice. Though hormonal treatments stimulated increases in the proportion of cycling cells (PCNA-positive), this was not directly correlated with p53 activity. Whole organ cultures were used to determine whether E+P act directly upon the mammary gland. Treatment with E+P was sufficient to render p53 responsive to radiation, but TGF-β-neutralizing antibodies blocked responsiveness. In the absence of E+P, TGF-β1 alone did not alter p53 activity. These results demonstrate that estrogen and progesterone together with TGF-β signaling are necessary for maintenance of p53 activity in the mammary epithelium.

Plasma-derived serum as a selective agent to obtain endothelial cultures from swine aorta

SummaryEndothelial cell and smooth muscle cell cultures from artery wall provide a potential model system for studying cellular processes involved in atherogenesis. To prepare serial subcultures of swine arterial endothelial cells that are free of smooth muscle cells without either selecting a small population or subjecting the cells to cytotoxic conditions, we used swine plasma-derived serum (SPDS) to establish conditions in which endothelial cells have a growth advantage. Endothelial cells were collected by collagenase digestion and smooth muscle cell cultures were prepared by outgrowth from explants of arterial medial segments. Growth rates were compared when each cell type was maintained on SPDS, or fetal bovine serum (FBS), or swine whole serum (SWS). When 20% FBS or SWS were used the doubling times were <30 h for both endothelial cells and smooth muscle cells. On 20% SPDS the doubling time for endothelial cells was 32 h, but for smooth muscle cells it was at least 168 h. Using SPDS, we prepare endothelial subcultures from swine aorta that express principally polygonal morphology at confluence. Endothelial cell cultures grown on SPDS have higher angiotensin-converting enzyme than those grown on FBS.

Pathways Contributing to Development of Spontaneous Mammary Tumors in BALB/c-Trp53+/− Mice

Mutation and loss of function in p53 are common features among human breast cancers. Here we use BALB/c-Trp53+/- mice as a model to examine the sequence of events leading to mammary tumors. Mammary gland proliferation rates were similar in both BALB/c-Trp53+/- mice and wild-type controls. In addition, sporadic mammary hyperplasias were rare in BALB/c-Trp53+/- mice and not detectably different from those of wild-type controls. Among the 28 mammary tumors collected from BALB/c-Trp53+/- mice, loss of heterozygosity for Trp53 was detected in more than 90% of invasive mammary tumors. Transplantation of Trp53+/- ductal hyperplasias also indicated an association between loss of the wild-type allele of Trp53 and progression to invasive carcinomas. Therefore, loss of p53 function seems to be a rate-limiting step in progression. Moreover, expression of biomarkers such as estrogen receptor alpha, progesterone receptor, Her2/Neu, and activated Notch1 varied among mammary tumors, suggesting that multiple oncogenic lesions collaborate with loss of p53 function. Expression of biomarkers was retained when tumor fragments were transplanted to syngeneic hosts. Tumors expressing solely luminal or basal keratins were also observed (27 and 11%, respectively), but the largest class of tumors expressed both luminal and basal keratins (62%). Overall, this panel of transplantable tumors provides a resource for detailed evaluation of the cell lineages undergoing transformation and preclinical testing of therapeutic agents targeting a variety of oncogenic pathways including cancer stem cells.

Regulation of p53 and Its Targets During Involution of the Mammary Gland

Post-lactational involution of the mammary glandprovides a system in which to study the expression andfunction of genes that regulate apoptosis in the contextof a normal tissue. The functions of the p53 tumor suppressor gene have been extensivelystudied as a mediator of apoptosis in response to DNAdamage, but its regulation in normal physiologicprocesses has been poorly characterized. Expression of p53 mRNA was shown to be among the firstgenes to be induced in mammary tissue following weaningof neonates. Although involution proceeds in the absenceof a functional p53 gene, it is delayed compared to normal individuals. Therefore, involutioncan be viewed as biphasic with initial responses beingsensitive to p53, whereas secondary responses beingp53-independent. These observations can be exploited to determine the subset of genes that arep53-responsive and that mediate the effects of p53 innormal mammary tissue.

Abstract B56: Identification of genes involved in mammary tumor susceptibility and resistance in two strains of mice

Loss of p53 can lead to a variety of cancers, including breast cancer. Mice heterozygous for the p53 gene (designated Trp53+/−) develop spontaneous mammary tumors, but this depends on the strain background and has been linked to a locus on chromosome 7 (designated SuprMam1). Mammary tumors are common in BALB/c‐Trp53+/−females, but are rare in C57BL/6‐Trp53+/− mice. Prevalence of genomic instability appears to contribute to the phenotype as loss of heterozygosity (LOH) through mitotic recombination is significantly more common among tumors arising in BALB/c‐Trp53+/− mice compared to C57BL/6J‐Trp53+/− mice. This increased LOH in BALB/c‐Trp53+/− tumors was shown to be due to recombination events. The Balb/c strain has been shown to have a deficiency in non‐homologous end joining (NHEJ) of DNA double strand breaks (dsb), however, this does not account for the increase of LOH events in tumors. Our hypothesis is that BALB/c‐Trp53+/− mice are more susceptible to mammary tumors due to impaired dsb repair leading to LOH. Using the COMET assay, we demonstrate that dsbs persist longer in BALB/c‐Trp53+/− mouse embryonic fibroblasts (MEFs) compared to C57BL/6J‐Trp53+/− MEFs. Similarly, co‐localization of H2AX and the homologous recombination protein RAD51 remain at dsb longer in BALB/c‐Trp53+/− MEFs compared to C57BL/6‐Trp53+/− MEFs suggesting a decreased efficiency of homologous recombination. Using a GFP reporter assay, homology‐directed repair was decreased by 36% in MEFs from BALB/c‐Trp53+/− compared to C57BL/6‐Trp53+/−. Palb2 is a candidate gene as it lies within the SuprMam1 interval and has been shown to contribute to heritable breast cancer. Although no coding SNPs were found within Palb2, preliminary results show the BALB/c‐Trp53+/− cells have a 30% decrease in Palb2 RNA compared to C57BL/6‐Trp53+/− cells as assayed by RT‐qPCR. These results identify defects in homology‐directed repair of dsb in BALB/c which are linked to susceptibility to mammary tumors in this strain. Experiments are in progress analyzing these features in congenic strains. The results will elucidate potential low penetrance modifiers that determine resistance to mammary tumors in both mice and humans which can be used as targets for therapy as well as breast cancer screening. Citation Information: Cancer Res 2009;69(23 Suppl):B56.