Dysregulation of the mTOR pathway in p53-deficient mice

Abstract

Mammalian or mechanistic target of rapamycin (mTOR) is involved in growth, aging, and age-related diseases including cancer. There is an extensive cross talk between p53 and mTOR. In cell culture, p53 inhibits the mTOR pathway in a cell type-dependent manner. p53-deficient mice develop pro-inflammation and cancer. We have shown that rapamycin delayed cancer and extended lifespan, thus partially substituting for p53. Here we show that a marker of mTOR activity, phosphorylated S6 (p-S6), is increased in the hearts of p53-deficient mice. Furthermore, cardiac p-S6 correlated with body weight. Also, p53^−/− mice were slightly hyperinsulinemic with a tendency to elevated IGF-1. Radiation exacerbated the difference between IGF-1 levels in normal and p53^−/− mice. Noteworthy, radiation induced Thr-308 Akt phosphorylation in the livers (but not in the hearts) of both p53^+/+ and p53^−/− mice. Simultaneously, radiation decreased p-S6 in the livers of normal mice, consistent with the negative effect of p53 on mTOR. Our data indicate that the activity of mTOR is increased in some but not all tissues of p53^−/− mice, associated with the tendency to increased insulin and IGF-1 levels. Therefore, the absence of p53 may create oncophilic microenvironment, favoring cancer.

Keywords: :

• rapamycin
• mTOR
• rapalog
• cancer
• aging
• senescence
• p53
• inflammation

Introduction

The p53 tumor suppressor can cause cell cycle arrest, apoptosis, and cellular senescence depending on cellular context.^1-5 Although this is sufficient to explain its anti-cancer effect, p53 can suppress cancer independently of these activities.^6,7 Noteworthy, p53 suppresses anabolic metabolism^5-11 expression of repeats and noncoding RNAs,¹² NFκB,¹³ the mTOR pathway,^9,14-20 and conversion from quiescence to senescence.^21,22 p53 may promote and suppress senescence.^23-31 While arresting cell cycle, p53 itself does not promote cellular senescence. It is growth-promoting pathways such as PI3K/mTOR that convert p53-induced cell cycle arrest into senescence.^21,32-35 By inhibiting the mTOR pathway, p53 can suppress conversion from arrest to senescence.^21,22,36 Senescent microenvironment favors cancer.^37-46 Noteworthy, overexpression of p53 under physiological regulation increases lifespan and decreases cancer incidence.⁴⁷ Cancer is an age-related disease and its incidence increases exponentially with age. Aging is the most important “risk factor” of cancer and any interventions that slow aging also delay cancer.^44,48-52 Rapamycin slows down aging in numerous organisms, including those that do not die from cancer.^53-59 As an anti-cancer and anti-aging agent, rapamycin can delay cancer not only directly but also indirectly: by slowing down aging. Mice lacking p53 die from cancer early in life.^60-62 Rapamycin extends lifespan and delays cancer in p53^+/− and p53^−/− mice.^63-65 In p53^−/− mice both direct (anti-cancer) and indirect (anti-aging) models identically predict that rapamycin will delay cancer and extend life span. There is a hint for indirect effects: rapamycin is more effective when given early in life.⁶³ (In contrast, classic anti-cancer agents such as radiation promote cancer, especially when given early in life.) Do p53^−/− mice have metabolic and tissue alterations that may favor both aging and cancer? It was already shown that p53^−/− mice are characterized by pro-inflammatory syndrome^66,67 and accelerated atherosclerosis.^68-70 Here we investigated the insulin/Akt/mTOR pathway in p53^−/− mice. Conventionally, we performed measurements after overnight fasting. We also included a second condition: radiation. In cell culture, in order to affect mTOR pathway, p53 should be induced. Therefore, we expected that the difference between normal and p53^−/− mice would be exacerbated by radiation. For consistency, all mice were fasted overnight before examination.

Results

AKT/mTOR pathway in p53-knockout mice

First we measured phosphorylated AKT and S6 in the hearts (Fig. 1). We previously found that the heart muscle is suitable to measure these markers.⁷¹ Levels of Akt phosphorylation at Thr-308 were not significantly different in p53-knockout mice (Fig. 1). Phospho-S6 (p-S6), the most validated target of the mTOR pathway, was significantly higher in p53-knockout mice (P = 0.0108). Radiation tended to decrease p-S6 in normal mice but not in p53-knockout mice (Fig. 1).

Figure 1. Levels of p-S6 and p-AKT in the hearts of normal (p53^+/+) and p53-knockout (p53^−/−) mice. (A) Immunoblot analysis of protein lysates from the hearts of 3.5-mo-old normal (p53^+/+) and p53-knockout (p53^−/−) mice, which were untreated (control) or irradiated with 10 gray (Rad), fasted overnight, and sacrificed. Numbers indicate individual mice. (B and C) Quantitave analysis of the data shown in (A). Quantified intensities of phosphorylated S6 (p-S6) signal (left panel) and signal of AKT phosphorylated at Thr308 (right panel) presented as mean ± SE. P values indicate statistically significant difference in p-S6 levels between p53^+/+ vs p53^−/− mice.

We next measured p-Akt-Thr-308 and p-S6 in the liver. Basal levels of p-Akt (Thr-308) and p-S6 were not changed in the livers of p53-knockout mice (Fig. 2). Radiation dramatically increased hepatic p-AKT (Thr-308) in both types of mice (Fig. 2A and C). However, this was not translated into phosphorylation of S6 in normal mice (Figs. 2 and 3). In normal mice, there was a dose-dependent decrease in p-S6 by radiation, which reached statistical significance at 10 Gray (Fig. 3B). While activating Akt (Thr-308) in a p53-independent manner (Fig. 2C), radiation decreased p-S6 in a p53-dependent manner (Fig. 3B). Phosphorylation of Akt at Ser-473, a site phosphorylated by mTORC2, was not significantly affected by radiation in the livers of normal mice (Fig. 3).

Figure 2. Levels of p-S6 and p-AKT in the livers of normal (p53^+/+) and p53-knockout (p53^−/−) mice. (A) Immunoblot analysis of protein lysates from the livers of 3.5-mo-old normal (p53^+/+) and p53-knockout (p53^−/−) mice, which were untreated (control) or irradiated for 1 h (Rad), fasted overnight and sacrificed. Note: p53^−/− mice in Rad group received 10 gray radiation; p53^+/+ Rad group received 5 gray (mice 18 and 19) or 10 gray (mice 23 and 24) radiation. Numbers indicate individual mice. (B and C) Quantitative analysis of data shown in (A). Quantified intensities of phosphorylated S6 (p-S6) signal (left panel) and signal of AKT phosphorylated at Thr308 (right panel) presented as mean ± SE. P < 0.0001 values indicate statistically significant difference between respective control and irradiated groups using two-tailed t test analysis.

Figure 3. p-S6 and p-AKT (S473) in the livers of normal (p53^+/+) mice: control and irradiated. (A) Immunoblot analysis of protein lysates from the livers of 3.5-mo-old normal (p53^+/+) mice, which were untreated (control) or irradiated for 1 h with 5 gray (Rad [5]) or 10 gray (Rad [10]), fasted overnight, and sacrificed. (B and C) Quantitative analysis of data shown in (A). Quantified intensities of phosphorylated S6 (p-S6) signal (left panel) and signal of AKT phosphorylated at Ser473 (right panel) presented as mean ± SE. P = 0.0115 value indicates statistically significant difference between control and irradiated with 10 gray groups using two-tailed t test analysis.

Metabolic alterations in p53-knockout mice

Given that mTOR drives cellular mass growth, hypertrophy of p53^−/− mice was expected. In agreement, p53^−/− mice were heavier than normal (p53^+/+) mice (Fig. 4A). The difference was not dramatic but still statistically significant (P = 0.0355). Noteworthy, fasting leptin levels showed the opposite tendency (Fig. 4B), in contrast to a strong positive correlation between weight and leptin in mice of the same genotype shown previously.^71,72 Since leptin is secreted by fat cells and reflects fat content, p53-knockout mice seem to be more hypertrophic rather than fatter. Importantly, there was a positive correlation between body weight and p-S6 levels in the heart tissue (Fig. 4C). Fasting levels of glucose did not differ between p53-deficient and normal mice (Fig. 4D). Fasting insulin levels were increased in p53^−/− mice (Fig. 4E). Noteworthy, levels of insulin and IGF-1 were not altered in younger p53^−/− mice, when measured without fasting (not shown). This indicates that hyperinsulinemia is detectable in certain conditions, depending on the age and fasting. We next irradiated mice to induce p53. Radiation did not affect glucose levels in both types of mice and increased insulin levels only in normal mice (Fig. 4). Levels of IGF-1, a marker associated with accelerated aging,^73-75 tended to be increased in p53^−/− mice, reaching a statistical significance after radiation (Fig. 4F).

Figure 4. Metabolic profile of 3.5-mo-old normal (p53^+/+) and p53^−/− mice. (A) Weight (g) of normal (n = 15) and p53-knockout (n = 9) mice. Data present mean ± SE. (B) Fasting leptin levels of control normal (n = 5) and p53-knockout (n = 4) mice presented as mean ± SE. (C) Correlation between weight and intensity of p-S6 signal in the heart of control normal and p53-knockout mice. r, Pearson coefficient. (D) Glucose levels (fasting) in normal and p53-knockout mice: control and irradiated—presented as mean ± SE. (E) Insulin levels (fasting) in normal and p53-knockout mice: control and irradiated—presented as mean ± SE. P = 0.0392 and P = 0.0442 values indicate statistically significant difference between control and combined irradiated groups of p53^+/+ mice and control and irradiated groups of p53^−/− mice, respectively. (F) IGF1 levels in plasma of normal and p53-knockout mice: control and irradiated—presented as mean ± SE. P = 0.0115 value indicated statistically significant difference between control and combined irradiated groups of p53^+/+ mice.

Discussion

We showed that nutrient- and insulin-sensing pathways are slightly dysregulated in p53^−/− mice. Our study provides a glimpse into the complexity of p53-dependent regulation of mTOR in the organism. There were tissue-specific (the liver vs. the heart) differences in phosphorylation of S6 and Akt. P-S6 levels were increased in the heart muscle of p53^−/− mice. Increased levels of p-S6 predict systemic alterations: increased body growth and insulin levels. In fact, p53^−/− mice were slightly heavier than normal mice, consistent with the role of mTOR in growth. Also, activated mTOR can cause insulin resistance by feedback inhibition of insulin signaling.^76,77 In fact, basal levels of fasting insulin were increased in p53^−/− mice, consistent with resistance to insulin, given that levels of glucose were unchanged. The mTOR/S6K pathway is involved in aging.⁷⁸ Hyperinsulinemia due to insulin resistance may accelerate aging and cancer.⁷⁹ In cell culture, in order to inhibit the mTOR pathway, p53 should be induced.²¹ Therefore, mice were irradiated to imitate the cell culture conditions and to exacerbate p53-dependent trends. Indeed, in irradiated p53^−/− mice, levels of IGF-1 were significantly increased compared with irradiated normal mice. Higher levels of IGF-1 are associated with accelerated aging in mice and humans.^80-84 The most striking alteration caused by radiation was phosphorylation of Akt at Thr-308 in the livers of both types of mice, thus in a p53-independent manner (Fig. 5). In response to insulin and IGF-1, Thr-308 is normally phosphorylated, which leads to activation of Akt (Fig. 5). In normal mice, radiation induced Thr-308 phosphorylation and decreased p-S6 (Fig. 5).

Figure 5. Relationship between p53 and the insulin/AKT/MTOR/S6 pathway.

Metabolic alterations in normal tissues of p53^−/− mice can be termed oncophilic. The main cause of cancer in p53^−/− mice is the loss of cell cycle control in cells that give rise to tumors. Also, loss of p53 may activate mTOR in tumor cells, given that activation of the PI-3K/mTOR pathway is the most common alteration in cancer.^85,86 In addition, our data suggest that there may be also organism-dependent factors of accelerated tumorogenesis. This can explain why calorie restriction, which is known to inhibit mTOR and decelerate organismal aging, delays cancer, and prolongs life span in p53^−/− mice.⁸⁷ Similarly, rapamycin delayed cancer in p53^+/− and p53^−/− mice.^63,64 Noteworthy, the effect of rapamycin was blunted when it was used later in life, consistent with its indirect anti-cancer effect.⁶³ In conclusion, mice lacking p53 is characterized by oncophilic metabolism, an additional factor fostering carcinogenesis.

Materials and Methods

Mice

Animal studies were conducted in accordance with the regulations of the Committee of Animal Care and Use at Roswell Park Cancer Institute. In the study 15 normal (p53^+/+) 3.5-mo-old C57B1/6 male and 9 p53-knockout 3.5-mo-old male mice were used. The colony of p53-knockout mice on a C57B1/6 background (obtained from Jackson Laboratories) was maintained by crossing p53^+/− females with p53^−/− males followed by genotyping of the progeny by PCR as described previously.⁸⁸ Animals were randomly assigned to control and irradiated groups. Normal (p53^+/+) mice were divided into 3 groups: control (untreated) (n = 5), irradiated with 5 gray (n = 5), and irradiated with 10 gray (n = 5). p53^−/− mice were divided into 2 groups: control (n = 4) and irradiated with 10 gray (n = 5). All mice were fasted overnight and sacrificed. Fasted blood and organs were collected. Plasma was prepared and used for biochemical analyses.

Immunoblot analysis

Tissues were homogenized in Bullet blender using stainless steel 0.5 mm diameter beads (Next Advantage, Inc.) and RIPA lysis buffer supplemented with protease and phosphatase inhibitors tablets (Roche Diagnostics). Lysates were cleared by centrifugation at 4 °C at 13 000 rpm. Equal amounts of protein were separated on gradient Criterion gels (BioRad). Primary antibodies used: rabbit anti-phospho S6(Ser 240/244), anti-phospho AKT(Ser473), and phospho AKT(Thr308) (Cell Signaling Biotechnology); monoclonal anti-β-actin-peroxidase (Sigma-Aldrich).

Glucose levels in blood plasma

Glucose levels in blood plasma were measured using Accu-Chek Aviva strips (McKesson).

Insulin concentration in blood plasma

Insulin concentration in blood plasma was measured using Insulin (Mouse) Ultrsensitive ELISA kit (ALPCO Diagnostics) according to manufacturer’s protocol. Data were analyzed using range of insulin standards and four parameter logistic fit.

IGF1 concentration in blood plasma

IGF1 concentration in blood plasma was determined using IGF1 (Mouse/Rat) ELISA kit (ALPCO Diagnostics) according to manufacturer’s protocol. Data were analyzed using range of IGF1 standards and four parameter logistic fit.

Leptin concentration in blood plasma

Leptin concentration in blood plasma was determined using Mouse Leptin ELISA kit (Crystal Chem Inc.) using according to manufacturer’s protocol. Data were analyzed using range of leptin standards and four parameter logistic fit.

Statistical analyses

Statistical analyses were performed using GraphOad Prizm 5.00 for Windows, GraphPad Software, http://www.graphpad.com.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

10.4161/cbt.26947