In this study, we examined the effects of social isolation on development and tumorigenesis of the mammary gland. Using this established model for chronic stress combined with p53 heterozygosity, mimicking the Li Fraumeni syndrome in women, we demonstrated that social experience alters mammary gene expression, development and tumorigenesis. Females housed individually beginning at puberty, regardless of genotype, displayed underdeveloped mammary ductal trees, which correlated with reduced estrous cycle length, significantly elevated kiss1r transcripts in the MPOA of the hypothalamus, and reduced mammary transcripts for the epigenetic regulators, Mecp2 and DNMT3b. Despite morphologic similarity to mammary glands of wildtype female controls, p53 heterozygotes, regardless of housing status, displayed a very different profile of gene expression, characterized by reduced mammary transcripts for ERα, PR, Kiss1r, and all 4 epigenetic regulators. Contrary to our hypothesis, IH reduced the incidence of endstage mammary tumors in p53+/- females. Our findings indicate that social isolation initiated at puberty inhibits mammary development and alters its epigenome, which may confound studies of mammary tumorigenesis.
Both wildtype and p53+/-
IH females displayed a significant and unpredicted lack of mammary development. Although ductal elongation was complete, glands of IH young adult females of both genotypes exhibited reduced numbers of pre-terminal end buds. This phenotype suggests altered progesterone responsiveness (29
), but we found no significant difference in either ERα or PR mRNA levels in IH animals. However, differences in estrous cycling in IH and GH mice suggest altered function of the hypothalamic-pituitary-gonadal axis, which may lead to these differences in morphology.
p53 is a powerful tumor suppressor, and exerts its actions via multiple pathways, including regulation of the cell cycle and apoptosis (33
). Genetic defects in one allele result in the Li Fraumeni syndrome in humans (16
). Afflicted individuals are at high risk for multiple cancers, including a 55% chance of breast cancer in women by age 45 (35
). Here we employed a mouse model heterozygous for a well-studied germline mutation of this gene (18
). We backcrossed this mutation, originally developed in 129/SV × C57BL/6 mice, into the FVB/N genetic background, in order to study its behavior in the context of a mouse strain that is extensively used for transgenic models of cancer. This change in strain background increased overall tumor incidence (>90%), and reduced latency, compared to the original report (18
). Interestingly the distribution of tumor types in group-housed p53+/-
FVB/N females also shifted toward a predominance of mammary tumors, more similar to the human syndrome.
Social isolation, conferred by individual housing of normally social species, is a stress paradigm which is relevant to human disease. Indeed, use of this stressor increases progression of ovarian tumors in mice (11
), and two recent studies also linked this stress to progression of mammary tumors in rat (10
) and mouse (25
) models. In the latter study, Williams and colleagues used a very similar experimental paradigm and the same mouse strain (FVB/N) as we employed in our study. In seeming contrast to our observed reduced incidence of invasive mammary carcinomas in IH p53+/-
females, they demonstrated that isolation increased mammary lesions and tumor burden induced by the C3(1
)/SV40 T-antigen (Tag) transgene (25
)/SV40 Tag model differs from p53
heterozygotes in several important respects. Transformation induced by SV40-Tag also involves loss of p53 function, but retinoblastoma protein is also inactivated (36
). Moreover, C3(1
) targets Tag expression to only epithelial cells, in contrast to the germline p53+/-
model employed in our study. The consequences of these differences on tumor incidence and progression in the context of social isolation merit additional investigation. However, the impact of IH on mammary development in both wildtype and p53+/-
females found in our study re-emphasizes the importance of a long understood consequence of isolation: IH exerts complex effects on estrous cycling (37
), and consequently steroid hormone action. Particularly when housing treatments are initiated at puberty, this can disrupt normal mammary development, which may impede tumorigenic processes (e.g., 39). Furthermore, mammary epithelial populations and potential tumor precursors expand rapidly at this time (40
); manipulations that interfere with these events may also inhibit oncogenesis. In addition, ovarian steroids are potent mitogenic stimuli for hormonally responsive mammary lesions, which include those induced by the absence of p53 in other strain backgrounds (41
). By reducing mammary development and ovarian steroid action, individual housing may mask possible effects of stress on tumor incidence from stimuli, such as p53
heterozygosity, which may be relatively weak compared to viral oncogenes. Thus experimental social isolation, particularly in the vulnerable peri-pubertal period, may confer additional complications in efforts to tease out any relationship between psycho-social factors and breast tumor incidence, as opposed to cancer progression. Techniques that reliably induce stress without depriving subjects of pheromonal cues that drive estrous cycling may be more appropriate for studies of mammary cancer.
Contrary to our expectations, IH also did not alter the overall survival of p53+/-
females in our study, despite behavioral and physiologic evidence of increased stress. Larger studies examining cell specific loss of p53
alleles may reveal effects of isolation on individual tumor types. However, in the initial months after weaning, IH animals died at a more rapid rate. The convergence of the survival rates with time suggests that some, but not all, mice could “adapt” to the higher stress level. This is consistent with a growing body of literature suggesting that individual temperaments play a role in the experience of stress and risk for disease (42
Kisspeptin was originally identified as an anti-metastatic agent in breast and other cancers (44
). This ligand and its cognate receptor, Kiss1r, also play critical roles in regulating maturation and function of the HPG axis in humans, non-human primates and rodents (31
transcripts in the hypothalami and MPOA of adults have been shown to be modulated by several stressors (32
), supporting the hypothesis that kisspeptin mediates the effect of stress on the estrous cycle by modulation of gonadotropin secretion. The altered cycling and higher kiss1r
mRNA levels in the MPOA of IH females in our study, regardless of genotype, suggest that IH may be exerting its effects on mammary development in part via kisspeptin action on the HPG. A similar mechanism may also mediate the delayed mammary development reported in isolated Sprague Dawley rats (10
Studies exploring the impact of maternal care have demonstrated that social experience in infancy can alter methylation of genes in the brain (47
). Similarly, studies in adult animals show that injury to the central nervous system induces changes in the epigenome (49
). Here we showed that postpubertal social isolation can also result in lower expression of genes regulating methylation in the mammary gland. The reduced DMNT3b and Mecp2 mRNA that we observed may reflect the relatively underdeveloped glands; DNA methylation and Mecp2 both play a role in some (50
), but not all (51
), aspects of mammary development. Alternatively, IH may alter this system regardless of mammary maturation. Examination of the effect of IH on fully mature glands would further illuminate this relationship.
Mammary glands of p53+/-
females exhibited reduced transcripts for all four epigenetic regulators. This is surprising in light of the association of heterozygosity of p53
with regional hypermethylation of CpG islands, repression of DNMT1 transcription by p53, and corepression of some promoters by p53 and DNMTs (52
). However, global hypomethylation as well as localized hypermethylation are hallmarks of cancer (54
). DNA hypomethylation and reduced DMNT3b, in particular, increase chromosomal instability (54
), and global 5-methylcytosine levels are reduced in breast as well as prostatic disease (56
). Hypomethylation may be particularly important for early neoplastic events (54
). Together, these observations indicate a need for more study of the role of epigenetic changes in tumorigenesis secondary to mutations in p53
Our study demonstrates that social isolation initiated at puberty exerts complex physiological effects, including dysregulation of mammary development and expression of epigenetic modulators. In the context of p53 heterozygosity, this experimental paradigm unexpectedly reduces the incidence of mammary tumors, highlighting the neglected interplay between social experience, mammary development and tumor biology. These studies in the context of the literature underscore the need for further investigation of the impact of social experience on the risk and progression of mammary cancer.