Chronic restraint stress attenuates function and promotes tumorigenesis

Zhaohui Fenga,1, Lianxin Liub,1, Cen Zhanga, Tongsen Zhengc, Jiabei Wangc, Meihua Lina, Yuhan Zhaoc, Xiaowen Wanga, Arnold J. Levined,2, and Wenwei Huc,2

aDepartment of Radiation Oncology and cDepartment of Pediatrics, Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey, New Brunswick, NJ 08903; bKey Laboratory of Hepatosplenic Surgery, First Affiliated Hospital of Harbin Medical University, Harbin 150086, China; and dSimons Center for Systems Biology, Institute for Advanced Study, Princeton, NJ 08540

Contributed by Arnold J. Levine, March 8, 2012 (sent for review January 18, 2012) Epidemiological studies strongly suggest that chronic psychologi- manner; chronic restraint enhanced growth of p53+/+ xenograft − − cal stress promotes tumorigenesis. However, its direct link in vivo tumors to a much greater extent than p53 / tumors. This report and the underlying mechanisms that cause this remain unclear. further demonstrates that elevated during chronic This study provides direct evidence that chronic stress promotes restraint decreased p53 function through the induction of serum- tumorigenesis in vivo; chronic restraint, a well-established mouse and -induced protein kinase (SGK1), a negative model to induce chronic stress, greatly promotes ionizing radiation regulator of p53. These data strongly suggest that glucocorticoids − (IR)-induced tumorigenesis in p53+/ mice. The tumor suppressor mediate the inhibitory effect of chronic restraint on p53 function, protein p53 plays a central role in tumor prevention. Loss or at- which in turn promotes tumorigenesis. tenuation of p53 function contriubutes greatly to tumorigenesis. We found that chronic restraint decreases the levels and function Results of p53 in mice, and furthermore, promotes the growth of human Chronic Restraint Promotes IR-Induced Tumorigenesis in Mice. To xenograft tumors in a largely p53-dependent manner. Our results investigate the impact of chronic stress upon tumorigenesis show that glucocorticoids elevated during chronic restraint medi- in vivo, a mouse model that combines chronic restraint and IR- ate the effect of chronic restraint on p53 through the induction of induced tumorigenesis was used. p53 prevents IR-induced tu- serum- and glucocorticoid-induced protein kinase (SGK1), which in morigenesis (11). Loss of p53 sensitizes IR-induced tumorigen- turn increases MDM2 activity and decreases p53 function. Taken esis in mice; 4 Gy IR significantly promotes tumor development, − together, this study demonstrates that chronic stress promotes mainly lymphomas, in p53+/ mice, but not p53+/+ mice (11). In − tumorigenesis in mice, and the attenuation of p53 function is an this study, 7-wk-old p53+/ C57BL6/J male mice were subjected important part of the underlying mechanism, which can be medi- to periodic physical restraint by immobilization in a mouse re- ated by glucocortcoids elevated during chronic restraint. straint apparatus (6 h/day) for 1 wk and then treated with 4 Gy IR, followed by periodic restraint for another 3 wk. This treat- he tumor suppressor p53 plays a crucial role in preventing ment induced high corticosterone levels in serum (Fig. 1A), Ttumor formation (1, 2). p53 responds to stress signals and a characteristic of chronic stress. Unrestrained mice were treated transcriptionally regulates its target to start various cellular with 4 Gy IR as controls. Consistent with previous reports, IR +/− responses, including cell cycle arrest and , thus pre- significantly promoted tumor formation in p53 mice; the tu- venting tumor initiation and/or progression. p53 is the most mor latency was significantly reduced from ∼77 to ∼49 wk of frequently mutated in tumors; ∼50% of human tumors median survival age (P = 0.006, Fig. 1B). Although chronic re- +/− harbor inactivating mutations in p53 (3). The p53 protein is straint had no significant effect on the survival of p53 mice under tight regulation in cells. Loss or the attenuation of p53 (P = 0.92), it significantly reduced tumor latency induced by IR ∼ ∼ P function (even a twofold modest change) affects cancer risk and (from 49 to 38 wk of median survival age) ( = 0.004, Fig. B tumor progression. For example, loss of one p53 allele in mice 1 ). The IR-induced tumor spectrum was similar between mice − (p53+/ ) leads to the early development of tumors (4). with and without chronic restraint; both groups of mice de- Epidemiological studies strongly suggest that chronic psycho- veloped predominantly lymphomas and sarcomas, suggesting logical stress has negative influences on the onset, progression, that the reduced tumor latency in mice with chronic restraint is C and mortality of cancers (5–8). A meta-analysis of 165 longitu- not due to the development of new types of tumors (Fig. 1 ). dinal studies demonstrated that psychosocial factors and stressful These results demonstrate that chronic restraint stress promotes

life experiences are associated with higher cancer incidence and IR-induced tumorigenesis in mice. GENETICS poorer cancer survival (8). Studies using chronic restraint or Chronic Restraint Decreases p53 Protein Levels and Function in social isolation in mice, two well-established chronic stress mod- Response to IR in Mice. In response to IR, p53 is activated and els, have shown that chronic stress enhances tumor growth and its protein levels increase, initiating cellular responses to prevent metastasis in xenograft tumor assays (9, 10). However, the direct tumorigenesis. Loss of p53 sensitizes IR-induced tumorigenesis evidence that chronic stress promotes tumorigenesis in vivo is still (11). The observation that chronic restraint promotes IR-induced lacking, and furthermore, the molecular mechanisms by which chronic stress promotes tumorigenesis remain unclear. To investigate the impact of chronic stress upon tumorigenesis Author contributions: Z.F., L.L., A.J.L., and W.H. designed research; Z.F., L.L., C.Z., T.Z., J.W., in vivo, we established a mouse model that combines chronic M.L., Y.Z., X.W., and W.H. performed research; Z.F., L.L., A.J.L., and W.H. analyzed data; restraint and ionizing radiation (IR)-induced tumorigenesis. IR and Z.F., L.L., A.J.L., and W.H. wrote the paper. induces tumorigenesis in both mice and humans. A single dose of The authors declare no conflict of interest. p53+/− 4 Gy IR results in tumor development in mice (11). This Freely available online through the PNAS open access option. report demonstrates that chronic restraint promoted IR-induced 1Z.F. and L.L. contributed equally to this work. p53+/− tumorigenesis in mice, providing direct evidence that 2To whom correspondence may be addressed. E-mail: [email protected] or huw1@umdnj. chronic stress enhances tumorigenesis in vivo. Furthermore, edu. chronic restraint decreased p53 function and promoted the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. growth of HCT116 xenograft tumors in a largely p53-dependent 1073/pnas.1203930109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1203930109 PNAS | May 1, 2012 | vol. 109 | no. 18 | 7013–7018 Downloaded by guest on September 23, 2021 AB C Median Survival 8 100 Tumor types p =0.0001 Control (n=12) 541 days (77 weeks) 6 80 Restraint (n=10) 522 days (75 weeks) Splenic Thymic Sarcoma Total 60 Lymphoma Lymphoma 4 IR (4 Gy) (n=15) 340 days (49 weeks) %(n) %(n) %(n) %(n) 40 Control (p=0.006 con vs. IR) 2 Restraint Restraint+IR (4 Gy) 267 days (38 weeks) IR 44 (8) 34 (6) 22 (4) 20 IR 100 (18) Relative serum (p=0.004 IR vs. Restraint+IR) 0 Percent survival Restriant+IR (n=28)

Corticosterone levels p Control Chronic 0 ( <0.0001 Restraint Restraint+IR 49 (14) 33 (10) 18 (5) 100 (29) Restraint 0 200 400 600 vs. Restraint+IR) days

− − Fig. 1. Chronic restraint promotes IR-induced tumorigenesis in p53+/ mice. Seven-week-old p53+/ C57BL6/J male mice were subjected to periodic restraint (6 h/day) for 1 wk and then treated with 4 Gy IR followed by periodic restraint for another 3 wk. (A) Chronic restraint increased serum corticosterone levels in mice as measured by ELISAs. (B) Chronic restraint promotes IR-induced tumorigenesis as demonstrated by the Kaplan-Meier curve. IR significantly reduced median survival age of mice. A significantly shorter median survival age was observed in mice with chronic restraint compared with mice without restraint. (C) Similar IR-induced tumor spectrum between mice with and without chronic restraint.

tumorigenesis in mice suggested that chronic restraint may re- mechanism by which chronic restraint promotes tumorigenesis −/− duce p53 function. To test this possibility, 7-wk-old p53+/+ in response to IR. In p53 mice, due to rapid development of C57BL6/J male mice were subjected to periodic restraint for spontaneous tumors (at ∼4 mo of age), the same 4-Gy IR 1 wk, followed by 4 Gy IR. The phosphorylation of p53 at Ser18 treatment does not accelerate tumor development (11). There- (p-p53 Ser18, equivalent to human p53 Ser15), which is the fore, it is difficult to compare the impact of chronic restraint initial step of p53 activation in response to IR, and the accu- mulation of p53 protein were measured in the spleen after IR. Spleen is a radiosensitive tissue that displays strong IR-induced A B p53 responses. The increase of p-p53 Ser18 levels and the ac- Spleen Thymus Small Intestine IR (4 Gy) - - - - + ++ + + + cumulation of p53 protein in response to IR were significantly Chronic 25 15 10 - - + + - - - + + + p=0.006 p=0.027 p=0.008 lower in spleen from mice with chronic restraint as determined at restraint 20 12 8 ∼ p-p53 different time points after IR (decreased by 50% at 6 h after Ser18 15 9 6 IR, Fig. 2 A and B and Fig. S1A for 12 and 24 h after IR). Similar p53 10 6 4 3 2 Induction fold results were observed in other tissues, including thymus and Actin of p53 protein 5 − small intestine (Fig. 2B) and in spleen from p53+/ mice where 1 2 3 4 5 6 7 8 9 10 0 0 0

the promoting effect of chronic restraint on IR-induced tumor- Control Control Control B C Restraint Restraint Restraint igenesis was observed (Fig. S1 ). These results demonstrate that p21 Noxa Puma chronic restraint decreases the accumulation of p53 protein in 70 10 10 p=0.041 p=0.0042 p=0.023 D response to IR. 60 8 8 IR (4 Gy) - - + + - - + + Restraint To investigate whether chronic restraint decreases p53 func- 40 6 6 - - - - + + + + 4 tion, p53 transcriptional activity was determined. The mRNA 4 p21 levels of a group of well-known p53 target genes, including p21, 20 2 2 Actin Puma, and Noxa, were examined in spleen at 6 h after IR. These Induction fold RNA 0 0 0 1 2 3 4 5 6 7 8 genes were induced by IR in a p53-dependent manner; they were Control Control +/+ Control Restraint induced at high levels in p53 mice (Fig. 2C), but to a signifi- Restraint Restraint p53−/− A cantly lesser extent in mice (Fig. S2 ). Notably, the in- E 60 p53+/+ F fi p IR (4 G) - + + duction of these mRNA levels was signi cantly lower in mice 50 =0.017 Control restraint - + - with chronic restraint versus mice without restraint by up to 50% p53+/+ 40 p=0.013 (Fig. 2C). The decrease of p21 induction was confirmed by Restraint 30 p53-/- Western blot assays (Fig. 2D). Similar results were observed in p53+/+ − spleen from p53+/ mice (Fig. S2B). These results demonstrate 20 that chronic restraint decreases p53 transcriptional activity. (%) Apoptosis 10 0 One major p53 function in tumor suppression is to induce p53-/- 12h 24h apoptosis in response to stress. To investigate whether p53-me- +/+ diated apoptosis is reduced by chronic restraint, p53 and Fig. 2. Chronic restraint decreases p53 protein levels and function in tran- − − p53 / C57BL6/J mice with or without chronic restraint were scriptional activity and apoptosis in response to IR in mice. Seven-week-old irradiated with IR (4 Gy). IR-induced apoptosis was measured p53+/+ C57BL6/J male mice with or without chronic periodic restraint (6 h/day in splenocytes using Annexin V staining in a flow cytometer. for 1 wk) were treated with 4 Gy IR, and various tissues were collected at Splenocytes underwent p53-dependent apoptosis in response to different time points after IR (n = 5 per group). (A) Chronic restraint de- creased the phosphorylation of p53 Ser18 (p-p53 Ser18) and accumulation of IR in p53+/+ mice without chronic restraint (∼40 and ∼25% E p53 protein in response to IR in the spleen. Levels of p-p53 Ser18 and p53 apoptotic cells at 12 and 24 h after IR, respectively) (Fig. 2 ). As protein were determined at 6 h after IR by Western blot assays. (B) Chronic −/− a control, very few splenocytes (<5%) died of apoptosis in p53 restraint decreased the accumulation of p53 protein induced by IR in dif- +/+ mice treated similarly. Compared with p53 mice without re- ferent tissues. The induction fold of p53 protein in the spleen, thymus, and straint, IR induced significantly less apoptosis in p53+/+ mice with small intestine was determined at 6 h after IR by ELISAs. (C) Chronic restraint chronic restraint (∼20 and ∼15% apoptotic cells at 12 and 24 h decreased the induction of p53 target genes. mRNA levels of p21, Noxa,and after IR, respectively) (Fig. 2E). These results were confirmed by Puma were determined at 6 h after IR by Taqman real-time PCR and nor- TUNEL assays in the spleen (Fig. 2F), demonstrating that chronic malized with actin. (D) Chronic restraint decreased the protein levels of p21. Protein levels of p21 were determined at 6 h after IR by Western blot assays. restraint reduces p53-dependent apoptosis induced by IR. (E and F) Chronic restraint decreased p53-mediated apoptosis in the spleen in response to IR. Splenocytes were isolated from the mice at 12 and 24 h Attenuation of p53 Function by Chronic Restraint Promotes Tumori- after IR, stained with annexin V, and analyzed in a flow cytometer to detect genesis in Xenograft Tumors. Considering the role of p53 in tumor apoptotic cells (E). TUNEL assay was used to detect apoptotic cells in spleen prevention, the attenuation of p53 function could be an important tissues at 12 h after IR (F).

7014 | www.pnas.org/cgi/doi/10.1073/pnas.1203930109 Feng et al. Downloaded by guest on September 23, 2021 upon IR-induced tumorigenesis between mice with or without tisol on p53 protein accumulation was abolished in cells treated p53. Here, the xenograft tumor assays were used to investigate with RU486 (Fig. 4B). Consistently, knockdown of GR by siRNA whether the attenuation of p53 contributes to the promoting abolished the inhibitory effect of cortisol on p53 accumulation in effect of chronic restraint on tumorigenesis. A pair of isogenic response to IR in HCT116 p53+/+ cells (Fig. 4C). Similar results human colorectal cancer cell lines with or without p53 (HCT116 were observed for two different siRNA oligos against GR. These − − p53+/+ and HCT116 p53 / ) were used to measure the impact of results suggest that cortisol down-regulates p53 levels through GR chronic stress upon the maintenance and propagation of tumor signaling. In addition to IR, cortisol greatly decreased the levels of growth. BALB/c athymic nude mice with periodic restraint for p-p53 Ser15 and p53 protein accumulation induced by etoposide, − − +/+ 1 wk were s.c. inoculated with HCT116 p53+/+ or HCT116 p53 / a chemotherapeutic agent, in HCT116 p53 cells (Fig. S3A). cells, followed by periodic restraint for another 3 wk. Consistent Similar results were observed in other human cells, such as breast with previous studies showing that p53 deficiency promotes xe- cancer cell MCF7 (Fig. S3B). Similarly, corticosterone, a major − − nograft tumor growth, the growth rate of HCT116 p53 / tumors glucocorticoid synthesized in mice, greatly decreased the levels of was faster than that of HCT116 p53+/+ tumors in mice without p-p53 Ser18 and the accumulation of p53 protein in response to fi D restraint (Fig. 3 A and B). Notably, chronic restraint significantly IR in mouse embryonic broblasts (MEFs) (Fig. 4 ), which can E promoted the growth rate of HCT116 p53+/+ tumors in mice; be abolished in RU486-treated cells (Fig. 4 ). The impact of cortisol upon p53-mediated transcriptional ac- the average tumor size was increased by 3.77-fold in mice with +/+ chronic restraint versus mice without restraint (Fig. 3 A and C), tivity and apoptosis was determined in HCT116 p53 cells with which demonstrates that chronic restraint promotes the main- or without cortisol treatment followed by IR (4 Gy). Cortisol significantly decreased the induction of p53 target genes, in- tenance and propagation of xenograft tumors. Furthermore, the p21 Puma F promoting effect of chronic restraint on the growth of HCT116 cluding and (Fig. 4 ). The decrease of p21 induction − − fi A p53 / tumors was significantly less pronounced (increased by was con rmed at the protein level (Fig. 4 ). Similar results were 1.52-fold) compared with HCT116 p53+/+ tumors (P < 0.0001) also observed in MEFs treated with corticosterone (Fig. S4). (Fig. 3 A and C–E). These results demonstrate that chronic re- Cortisol clearly decreased p53-mediated apoptosis induced by IR (Fig. 4G). The inhibitory effect of cortisol on the p53-mediated straint promotes xenograft tumor growth in a largely p53-de- transcriptional activity and apoptosis was abolished in RU486- pendent manner, suggesting that attenuation of p53 function is treated cells (Fig. 4 F and G). These results demonstrate that an important mechanism by which chronic stress promotes tu- glucocorticoids reduce p53 protein levels and functions, in- morigenesis. This experiment also demonstrates that the psycho- cluding transcriptional activity and apoptosis. logical state and its physiological consequence upon the host can affect tumor growth and that p53 plays a critical role in medi- Glucocorticoids Decrease p53 Protein Levels and Function in Vivo. ating this effect. Repeated s.c. injection of corticosterone can mimic stress-in- duced elevation of corticosterone levels and induces Glucocorticoids Decrease p53 Protein Levels and Function in Cultured in animal models (15). To investigate the effect of glucocorti- +/+ Cells. Glucocorticoids are among the hormones elevated during coids on p53 in vivo, p53 C57BL6/J mice were injected with chronic stress and affect the systemic physiology of the host (12). corticosterone (20 mg/kg) for 1 wk followed by IR (4 Gy). Cor- Prolonged elevation of glucocorticoids could have a direct impact ticosterone decreased p53 protein accumulation in response to upon tumorigenesis. It has been reported that glucocorticoids IRinthespleen(Fig.5A and B), thymus, and small intestine (Fig. promote survival and proliferation of tumor cells and the che- 5B). Furthermore, corticosterone injection decreased p53 function motherapeutic resistance of tumor cells (13). However, the un- in transcriptional induction of p21 and Puma by IR as demon- derlying mechanisms are largely unknown. To investigate whether C +/+ strated in the spleen (Fig. 5 ). Corticosterone injection also re- the elevated glucocorticoids reduce p53 function, HCT116 p53 duced IR-induced apoptosis as determined by the TUNEL assays cells were treated with cortisol for 12 h, a major glucocorticoid in the spleen (Fig. 5D) and small intestine (Fig. S5). elevated during chronic stress in humans, followed by 4 Gy IR. Cortisol greatly decreased the levels of p-p53 Ser15 and p53 SGK1 Mediates the Inhibitory Effect of Glucocorticoids on p53 Protein. protein at 6 h after IR in cells (Fig. 4A). Glucocorticoids exert most of their functions through binding to (GR), which is ubiquitously expressed in many types of cells, GR, which functions as a transcription factor to regulate a group mediates most of the effects of glucocorticoids (14). RU486, a GR of genes (16). SGK1, a ubiquitously expressed serine-threonine antagonist, was used to determine whether cortisol decreases p53 kinase, can be transcriptionally induced by glucocorticoids (17). protein levels through GR signaling. The inhibitory effect of cor- Consistent with the previous report (17), corticosterone clearly GENETICS

A B C D E Control Restraint HCT116 ) 1000 HCT116 p53+/+ HCT116 p53-/- Fold increase of tumor volume

p53+/+ p53-/- 3 1000 HCT116 p53+/+ 1400 promoted by chronic restraint Control Restraint Control Restraint Control Control 800 HCT116 p53-/- 1200 800 Restraint Restraint 1000 _ 600 p53+/+ 3.77+ 0.69 n=10 600 800 p53-/- 1.52 +_ 0.45 n=10 400 400 600 p <0.01 p 400 <0.01 p<0.0001 200 200 p53+/+ p53-/-p53+/+ p53-/- 200 HCT116 (mmTumor volume 0 0 0 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 day

Fig. 3. Chronic restraint promotes the growth of HCT116 xenograft tumors in a largely p53-dependent manner. Seven-week-old BALB/c athymic nude mice with or without chronic restraint were inoculated (s.c.) with HCT116 p53+/+ or HCT116 p53−/− cells (n = 10 per group). (A) Representative images of mice at day 21 after inoculation of tumor cells (Left) and tumors harvested from these mice (Right) are presented. (B–D) Tumor volume is presented as mean ± SD. (B) Loss − − of p53 promotes HCT116 tumor growth in mice without restraint; growth rate of HCT116 p53 / tumors was much faster than HCT116 p53+/+ tumors in mice − − without restraint. (C and D) Chronic restraint preferentially promoted the growth of HCT116 p53+/+ xenograft tumors (C) compared with HCT116 p53 / tumors (D). (E) Fold increase of tumor volume promoted by chronic restraint at day 21 after inoculation of tumor cells was much higher in HCT116 p53+/+ − − xenograft tumors than HCT116 p53 / tumors.

Feng et al. PNAS | May 1, 2012 | vol. 109 | no. 18 | 7015 Downloaded by guest on September 23, 2021 A +/+35p611TCH B +/+35p611TCH Cortisol (nM) Cortisol (nM) 0 50 200 0 200 200 RU486 (100 nM) - - + IR (4 Gy) - + - + - + IR (4 Gy) - + - + - + p-p53 ser15 p-p53- ser15 p53 p53

p21 p21

Actin Actin 1 2 3 4 5 6 1 2 3 4 5 6 C HCT116 p53+/+ 1.2 Cortisol (nM) 0 200 200 1 siRNA con con GR 0.8 IR (4 Gy) - + - + - + 0.6 p53 0.4 0.2 Actin 0 1 2 3 4 5 6 levels Relative GR RNA siRNA Con GR Fig. 4. Glucocorticoids decrease p53 levels and function in cultured cells. (A) Cortisol decreased p-p53 Ser15 levels, p53 D MEF E MEF accumulation, and induction of p21 protein in response to IR. CORT (n M ) 0 50 200 HCT116 p53+/+ cells were treated with cortisol for 12 h, fol- CORT (nM) 0 200 0 200 IR (4 Gy) - + - + - + lowed by IR, and analyzed at 6 h after IR. (B) RU486, a GR RU486 (100 nM) - - + + p-p53 antagonist, blocked the effect of cortisol on p-p53 Ser15 IR (4 Gy) - + - + - + - + levels, p53 accumulation, induction of p21 protein in re- Ser18 +/+ p53 sponse to IR in HCT116 p53 cells, as determined at 6 h after p53 IR. (C) Knockdown of GR by siRNA blocked the effect of cor- +/+ Actin Actin tisol on IR-induced p53 accumulation in HCT116 p53 cells (Left). Knockdown of GR mRNA levels was confirmed by 1 2 3 4 5 7 86 1 2 3 4 5 6 Taqman real-time PCR and normalized with actin (Right). (D) HCT116 Corticosterone (CORT) decreased p-p53 Ser18 levels and p53 F G HCT116 accumulation induced by IR in p53+/+ MEFs. Cells were treated p21 Puma with corticosterone for 12 h, followed by IR, and analyzed at 8 12 p p 50 p 6 h after IR. (E) RU486 blocked the effect of corticosterone on =0.01 =0.01 p=0.01 p=0.02 p=0.005 =0.006 6 40 IR-induced p53 accumulation in MEFs. (F) Cortisol (200 nM for 8 12 h) decreased p53 transcriptional induction of p21 and 30 4 Puma in response to IR, which can be blocked by RU486 in +/+ 4 20 HCT116 p53 cells. mRNA levels were determined at 6 h 2 after IR by Taqman real-time PCR and normalized with actin. 10 (G) Cortisol (200 nM for 12 h) decreased p53-mediated apo- 0 +/+ RNA Induction fold RNA 0 0 ptosis induced by IR (4 Gy) in HCT116 p53 cells, which can be Con Cortisol Cortisol Con % of apoptoic cells Con Cortisol Cortisol Con Con +RU486 Con Cortisol Cortisol blocked by RU486, as determined at 36 h after IR by annexin V +RU486 +RU486 − − staining. HCT116 p53 / cells were used as negative controls p53+/+ p53-/- p53+/+ p53-/- p53+/+ p53-/- in F and G. Con, control.

increased the expression of SGK1 at both RNA and protein restraint and corticosterone injection clearly induced the ex- levels in MEFs (Fig. 6A), whereas IR had no obvious effect on pression levels of SGK1 in mice (Fig. 6B). SGK1 shares high the expression of SGK1 (Fig. S6A). Furthermore, both chronic with AKT (∼50% through their catalytic

IR (4 Gy) ABSpleen C D - + CORT - - + + - - - + + + Spleen Thymus Small Intestine p21 Puma IR (4 Gy) - - - - + + + + + + 25 12 12 40 8 p=0.015 p=0.02 p=0.004 p=0.036 - + p-p53 20 p=0.01 30 6 Ser18 9 9 15 6 p53 10 6 20 4

Actin 5 3 3 10 2 CORT (200 nM) CORT

0 Induction fold RNA 1 2 3 4 5 6 7 8 9 10 0 0 0 0

Induction fold of p53 protein Control CORT Control CORT Control CORT Control CORT Control CORT

Fig. 5. Corticosterone decreases p53 protein levels and function in vivo. p53+/+ C57BL6/J male mice were s.c. injected with corticosterone (CORT; 20 mg/kg) daily for 1 wk. Mice with or without corticosterone injection were exposed to IR, and various tissues (spleen, thymus, and small intestine) were collected at different time points after IR (n = 5 per group). (A and B) Corticosterone injection decreased the accumulation of p53 protein in response to IR in vivo. Levels of p53 protein in various tissues were determined at 6 h after IR by Western blot (A) and ELISA (B) assays, respectively. (C) Corticosterone injection decreased the transcriptional induction of p53 targets in response to IR in vivo. mRNA levels of p21 and Puma were determined at 6 h after IR in the spleen by Taqman real-time PCR and normalized with actin. (D) Corticosterone injection greatly decreased p53-mediated apoptosis in spleen in response to IR. TUNEL assay was used to detect apoptotic cells in the spleen at 12 h after IR.

7016 | www.pnas.org/cgi/doi/10.1073/pnas.1203930109 Feng et al. Downloaded by guest on September 23, 2021 A B C MEF Spleen Plasmid con SGK1 3 3.5 p 3.5 IR (4 Gy) =0.005 p=0.002 - + - + 2 3 3 SGK1 1 p53 MEF RNA levels RNA 0 2 2 Relative SGK1 Actin Time (h) 0 2 8 15 SGK1 SGK1 1 1 p53 Actin 0 0 Actin HCT116 p53+/+ Time (h) 0 2 8 15 Con Cort Relative SGK1 RNA levels Relative SGK1 RNA Con Restraint 1 2 3 4

D MEF E MEF F MEF CORT - + p21 CORT - + 12 siRNA con con SGK1 p=0.01 p=0.01 GSK 650394 - - +- - + IR (4 Gy) - + - + - + IR (4 Gy) - + + - + + 8 p53 p53 Actin 4 Actin 1 2 3 4 5 6 1 2 3 4 5 6 0 8 Induction fold RNA p=0.001 p=0.001 Con CORT CORT 8 p=0.004 p=0.001 6 +siSGK1 PUMA 6 4 8 p=0.01 p=0.02 4 2 protein levels Relative p53 6 2 Relative p53 0 protein levels CORT - + 4 0 CORT siRNA con con SGK1 2 - + IR (4 Gy) - + - + - + GSK 650394 - - +- - +

RNA Induction fold RNA 0 IR (4 Gy) - + + - + + Con CORT CORT +siSGK1

G MEF HCT116 p53+/+ I Chronic stress p-MDM2 Ser166/186 Glucocorticoids levels MDM2 Actin GR Time (h) 0 2 8 15 0 2 8 15 MEF SGK1 H HCT116 p53+/+ con siRNA SGK1 siRNA con SGK1 MDM2 CORT - + - + Cortisol p-MDM2 - + - + p-MDM2 Ser166/186 Ser166/186 p53 MDM2 MDM2 Actin Actin 1 2 3 4 1 2 3 4 tumorigenesis

Fig. 6. SGK1 mediates the inhibitory effect of glucocorticoids on p53. (A and B) Corticosterone induced SGK1 in cultured cells and in vivo. (A) mRNA and protein levels of SGK1 were determined in p53+/+ MEFs treated with corticosterone (200 nM). (B) SGK1 mRNA levels were determined in the spleen of mice with corticosterone (CORT) injection (Left) or chronic restraint (Right). Con, control. (C) SGK1 overexpression by expression plasmid (pCMV-SGK1) decreased IR-induced p53 protein accumulation in MEFs (Upper) and HCT116 p53+/+ cells (Lower) as determined at 6 h after IR. (D–F) Blocking SGK1 function abolished

the inhibitory effect of corticosterone on p53. Blocking SGK1 by SGK1 siRNA (siSGK1) (D and E) or a SGK1 inhibitor, GSK650394 (200 nM) (F) abolished the GENETICS effect of corticosterone (CORT; 200 nM) on p53 accumulation (D and F), and p53 transcriptional activity (E) in response to IR in MEFs. Bar graphs in D and F represent the quantification of p53 levels in Western blot assays. (G) Glucocorticoids increased phosphorylation of MDM2 on Ser166/186 (p-MDM2 Ser166/ 186) in MEFs treated with corticosterone (200 nM) (Left) and HCT116 p53+/+ cells treated with cortisol (200 nM) (Right) for different hours. (H) Knockdown of SGK1 by siRNA blocked the increase of p-MDM2 Ser166/186 in MEFs treated with corticosterone (CORT; 200 nM) (Left) and HCT116 p53+/+ cells treated with cortisol (200 nM) (Right). (I) Schematic model depicting the negative regulation of p53 by chronic stress, which could be an important mechanism that contributes to tumor promoting effect of chronic stress. Positive and negative regulatory interactions are indicated.

domain) and a similar consensus phosphorylation site RXRXXS/ regulates p53 function. Furthermore, knockdown of SGK1 by T with AKT (17), which negatively regulates p53 function through siRNA (Fig. S6B) abolished the inhibitory effect of glucocorti- the activation of MDM2. A recent study reported that SGK1 coids on p53 accumulation (Fig. 6D) and transcriptional activity negatively regulates p53 in several human cancer cell lines (Fig. 6E) in response to IR in MEFs. Similar results were ob- through the activation of MDM2 (18). This raises the possibility served for two different siRNA oligos against SGK1. These that the induction of SGK1 under chronic restraint may mediate results were confirmed by blocking the function of SGK1 with the effect of glucocorticoids on p53. Indeed, SGK1 over- a SGK1-specific inhibitor, GSK650394 (19), in MEFs (Fig. 6F). expression in both MEFs and HCT116 p53+/+ cells by SGK1 Similarly, blocking SGK1 function by siRNA or GSK650394 expression plasmid decreased p53 accumulation in response to abolished the inhibitory effect of cortisol on p53 accumulation in IR (Fig. 6C), which clearly demonstrates that SGK1 negatively response to IR in HCT116 p53+/+ cells (Fig. S7). These results

Feng et al. PNAS | May 1, 2012 | vol. 109 | no. 18 | 7017 Downloaded by guest on September 23, 2021 indicate that SGK1 mediates the inhibitory effect of glucocorti- intact NK cell activity, suggesting that additional mechanisms coids on p53 function. contribute to tumor promoting effect of chronic stress. Indeed, AKT can negatively regulate p53 through phosphorylation of this study demonstrates that the attenuation of p53 could be an MDM2, a key negative regulator of p53, on Ser166/186 to in- important part of the mechanism by which chronic stress pro- crease MDM2 activity (20). Glucocorticoid treatment increased motes tumorigenesis. Our results demonstrated that chronic the phosphorylation of MDM2 on Ser166/186 in both MEFs and stress decreases p53 protein levels and function in mice. Fur- +/+ G HCT116 p53 cells (Fig. 6 ), which raised the possibility that thermore, chronic restraint of the host promotes the growth of SGK1 mediates the inhibitory effect of glucocorticoids on p53 a xenograft tumor in a largely p53-dependent manner. Consid- through increasing MDM2 activity. Indeed, the increase of ering the critical role of p53 in preventing the initiation and/or MDM2 phosphorylation by glucocorticoids largely disappeared progression of cancer, the attenuation of p53 function by chronic in cells when SGK1 was knocked down by siRNA (Fig. 6H). These results demonstrate that the induction of SGK1 by glu- stress could lead to the promoting effect of chronic stress on cocorticoids mediates the inhibitory effect of glucocorticoids on the initiation and/or progression of cancer. This study further p53 through increasing MDM2 activity. demonstrates that glucocortioids released during chronic stress decrease p53 protein levels and function through induction of Discussion SGK1, which in turn increases MDM2 activity to down-regulate Epidemiological studies have strongly suggested that chronic p53 (Fig. 6I). These results provide a mechanism by which psychological stress promotes tumor development. However, chronic restraint decreases p53 function. This study expands our the role of chronic stress in tumorigenesis remains controversial understanding of the biological pathways by which chronic stress and has not been well established in animal models. Chronic regulates cancer pathogenesis and demonstrates that the sys- restraint and social isolation in mice are two well-established temic communication between the circulation of neurohormones chronic stress models that mimic stress-induced neuroendocrine and the molecular pathways within the cells enables chronic responses in humans and induce depression- and anxiety-like psychological stress to promote the initiation and propagation of states in mice. Most studies used xenograft tumor assays to study tumorigenesis. These results suggest that the reactivation and/or the effect of chronic restraint or social isolation on tumor growth restoration of p53 functions may prevent tumor initiation, main- (9, 10, 21, 22). However, in xenograft tumor assays, the tumor tenance, and progression during or after chronic stress. growth could be greatly affected by the properties of the injected cancer cells in addition to the condition of the host. Here, the Materials and Methods impact of chronic restraint upon IR-induced tumorigenesis was +/+ +/− −/− p53+/− Mouse strains used in this study include the p53 , p53 , and p53 C57BL6/ directly investigated in vivo. In mice, chronic restraint J mice, BALB/c nu/nu athymic nude mice. Mouse restraint system (flat bottom greatly promoted IR-induced tumorigenesis. These results provide restrainers; Kent Scientific) was used for chronic periodic restraint. Details direct evidence that chronic stress promotes tumorigenesis in vivo. for mouse strains, chronic restraint, IR and corticosterone administration, The mechanisms by which chronic psychological stress pro- Western blot and ELISAs, Taqman real-time PCR, apoptosis analysis, xeno- motes tumorigenesis remain unknown. Many previous studies graft tumorigencity analysis, cell culture and treatments, and statistic anal- focused upon the down-regulation of the immune system. For ysis are given in SI Materials and Methods. instance, it has been reported that chronic stress decreases im- mune functions, including natural killer (NK) cell cytotoxicity ACKNOWLEDGMENTS. W.H. is supported by National Institutes of Health and T-cell responses to mitogen stimulation (23, 24). Previous (NIH) Grant 1P30CA147892-01, Department of Defense Grant W81XWH-10- 1-0435, CINJ new investigator award, and the Ellison Foundation. Z.F. is studies (10) and this study show that the promoting effect of supported by NIH Grant 1R01CA143204-01 and the New Jersey Commission chronic stress on the growth of xenograft tumors can be observed on Cancer Research (NJCCR). C.Z. is supported by a postdoctoral grant in immune-deficient nude mice with defective T-cell systems but from NJCCR.

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