Early Social Enrichment Modulates Tumor Progression and p53 Expression in Adult Mice

Abstract

Epidemiological evidence indicates that stress and aversive psychological conditions can affect cancer progression, while well-being protects against it. Although a large set of studies have addressed the impact of stress on cancer, not much is known about the mechanisms that protect from cancer in healthy psychological conditions. C57BL/6J mouse pups were exposed to an environmental enrichment condition consisting of being raised until weaning by the biological lactating mother plus a non-lactating virgin female (LnL = Lactating and non-Lactating mothers). The Control group consisted of mice raised by a single lactating mother (L = Lactating). Four months after weaning, mice from LnL and L conditions were exposed to intramuscular injection of 3-methylcolantrene (3MCA), a potent tumorigenic drug, and onset and progression of 3MCA-induced fibrosarcomas were monitored over time. Pups from the LnL compared to the L group received more parental care and were more resilient to stressful events during the first week of life. In association, the onset of tumors in LnL adults was significantly delayed. At the molecular level, we observed increased levels of wild-type p53 protein in tumor samples of LnL compared to L adults and higher levels of its target p21 in healthy muscles of LnL mice compared to the L group, supporting the hypothesis of potential involvement of p53 in tumor development. Our study sustains the model that early life care protects against tumor susceptibility.

Keywords: 3-methylcolantrene (3MCA); DNA-damage; fibrosarcoma; p21; p53; social enrichment; tumorigenesis.

Figure 1

Experimental design and groups. Top panels: experimental manipulations are shown in blue and include: 1. grouping pregnant females in L and LnL cages four days before offspring birth; 2. weaning at postnatal day 28 (PND28) during which male mice were caged in groups of four made of two pairs of siblings from two distinct L or LnL home-cages; 3. 3-methylcolantrene injection in the right rear leg of 8 L and 8 LnL mice (the same number of mice from the two groups were left undisturbed and used as controls). Middle panels: temporal timeline for the experiments, with gray boxes indicating key time points. Bottom panels: measured variables are reported in green boxes and include: 1. assessment of maternal behavior during the first week of pups’ life; 2. pups’ ultrasonic vocalizations and anxiety behavior measured on biological mothers on the elevated plus maze on PND8; 3. assessment of T lymphocytes basal state; 4. tumor monitoring from their onset until sacrifice; 5. molecular analysis on tumor and muscle samples.

Figure 2

Increased maternal care from LnL mothers during the first postnatal week: (A) number of sampling points (mean ± SEM) for active maternal care (nursing posture, grooming, licking, nest building) during daily monitoring (*** p <0.0001; two-way Anova for repeated measures with between factors being L and LnL groups and days being within measures); (B) number of sampling points (mean ± SEM) for litter left alone during daily monitoring. (*** p <0.0001; two-way Anova for repeated measures with between factors being L and LnL groups and days being within measures). Daily monitoring consisted of 32 recordings collected during 30-min sessions repeated twice over 7 days. PND = post-natal day.

Figure 3

LnL condition enhances resilience in 8-day-old pups and reduces anxiety in biological mothers. (A) Duration (mean ± SEM) of ultrasonic vocalizations (USVs) emitted from L and LnL pups during 5 min isolation. (* p = 0.05, one-way Anova for repeated measures, with time as within factor); (B) body temperature (mean ± SEM) of L and LnL pups after 20 min isolation (* p < 0.05, two-tailed unpaired t-test). (C) body weight (mean ± SEM) of L and LnL pups (n.s.; two-tailed unpaired t-test) (D) percentage of time spent in open arms (mean ± SEM) of an elevated plus maze by biological mothers from L and LnL groups (* p < 0.05; two-tailed unpaired t-test).

Figure 4

Development of fibrosarcomas is delayed in mice from the LnL group. (A) Curves reporting the appearance of palpable tumors from L and LnL mice. (p < 0.01; χ² test); (B) relative tumor volume (mean ± SEM) over the interval of time between tumor appearance and mouse sacrifice (n.s., Mann–Whitney U test for each day).

Figure 5

P53 activity is increased in LnL group. (A) WB analysis of indicated protein levels in muscles derived from animals of L and LnL groups, GAPDH was used as loading control. (B) Densitometric analysis of p53 relative to GAPDH levels (mean ± SD) in indicated groups (n.s., two-tailed unpaired t-test, n = 7/group). (C) Densitometric analysis of p21 relative to GAPDH levels (mean ± SD) in indicated groups (* p < 0.05, two-tailed unpaired t-test, n = 7/group). (D) RT-PCR analysis of p21 mRNA levels in indicated groups (** p < 0.01, two-tailed unpaired t-test, n = 7/group).

Figure 6

Lower Mdm2 levels in LnL mice. (Top) WB analysis of indicated protein levels in muscles derived from animals of L and LnL groups, GAPDH was used as loading control. (Bottom) Graphs reporting densitometric analysis of Mdm2 relative to GAPDH levels (mean ± SD) in indicated groups (n.s., two-tailed unpaired t-test, n = 7/group).

Figure 7

P53 levels are increased in fibrosarcoma of LnL animals. (A) WB analysis of indicated protein levels in fibrosarcoma derived from animals of L and LnL groups, GAPDH was used as loading control. Numbers under the lanes report the densitometric value of p53/GAPDH ratio. * marks an outlier value (by ROUT method, Q = 0.1%) not shown in the graph. (B) Densitometric analysis of wild type p53 relative to GAPDH levels (mean ± SD) in indicated groups (* p < 0.05, two-tailed unpaired t-test, n = 4 L, 3 LnL). (C) Densitometric analysis of p21 (from samples expressing wild type p53) relative to GAPDH levels (mean ± SD) in indicated groups (n.s., two-tailed unpaired t-test, n = 5 L, 3 LnL).