Behavioral stress accelerates prostate cancer development in mice

Prostate cancer patients have increased levels of stress and anxiety. Conversely, men who take beta blockers, which interfere with signaling from the stress hormones adrenaline and noradrenaline, have a lower incidence of prostate cancer; however, the mechanisms underlying stress–prostate cancer interactions are unknown. Here, we report that stress promotes prostate carcinogenesis in mice in an adrenaline-dependent manner. Behavioral stress inhibited apoptosis and delayed prostate tumor involution both in phosphatase and tensin homolog–deficient (PTEN-deficient) prostate cancer xenografts treated with PI3K inhibitor and in prostate tumors of mice with prostate-restricted expression of c-MYC (Hi-Myc mice) subjected to androgen ablation therapy with bicalutamide. Additionally, stress accelerated prostate cancer development in Hi-Myc mice. The effects of stress were prevented by treatment with the selective β₂-adrenergic receptor (ADRB2) antagonist ICI118,551 or by inducible expression of PKA inhibitor (PKI) or of BCL2-associated death promoter (BAD) with a mutated PKA phosphorylation site (BAD^S112A) in xenograft tumors. Effects of stress were also blocked in Hi-Myc mice expressing phosphorylation-deficient BAD (BAD^3SA). These results demonstrate interactions between prostate tumors and the psychosocial environment mediated by activation of an adrenaline/ADRB2/PKA/BAD antiapoptotic signaling pathway. Our findings could be used to identify prostate cancer patients who could benefit from stress reduction or from pharmacological inhibition of stress-induced signaling.

Substantial geographical variations in prostate cancer incidence in men with similar genetic backgrounds suggest that environmental factors are important in prostate cancer development (1). Several studies in prostate cancer patients have shown that changing to a low-fat, plant-based diet combined with stress management modulates gene expression and slows the rate at which levels of prostate-specific antigen (PSA) increase (2, 3). Curiously, although these studies included a stress reduction component to alleviate the stress of dietary changes, they did not examine the effects of the stress reduction per se on prostate cancer. Indeed, most attention so far has been focused on diet as the dominant environmental factor that influences cancer. Yet for higher organisms, psychosocial interactions could have a substantial effect on hormonal status and well being, as evidenced by reports that chronic stress and depression predict cancer progression and mortality (4, 5).

A cancer diagnosis itself is known to be a major distress factor, causing anxiety and depression (4), and patients with prostate cancer reportedly show higher levels of anxiety compared with other cancer patients (6). Furthermore, higher levels of PSA have been observed in patients under behavioral stress (7, 8), and increased stress levels have been connected with inflammatory prostatitis (9). Conversely, an 18% reduction of prostate cancer risk has been reported in patients who take beta blockers, which interfere with signaling of the stress hormones adrenaline and noradrenaline (10). Still, information is limited concerning mechanisms by which stress influences prostate cancer. Experiments in tissue culture models have shown that β₂-adrenergic receptor (ADRB2) activation inhibits apoptosis and stimulates migration, whereas glucocorticoid receptor activation inhibits proliferation, in prostate cancer cells (11–13). In vivo experiments showed that implanting nude mice with a noradrenaline-releasing micropump increased metastasis of PC3 xenografts by 1.6-fold (12). However, to date, there is no definitive experimental evidence on the mechanisms by which behavioral stress may influence prostate cancer development and therapy resistance.

Considering that increased stress and anxiety are common comorbidities for prostate cancer, we decided to test the effects of behavioral stress in vivo in mouse models of prostate cancer. We examined the effects of immobilization stress on therapeutic sensitivity of prostate cancer C42 xenografts in nude mice and on prostate tumors in mice with prostate-restricted expression of c-MYC (referred to herein as Hi-Myc mice). We report that the PI3K inhibitor ZSTK474 induced apoptosis in C42 prostate cancer xenografts, whereas subjecting mice to immobilization stress or to injection with adrenaline prevented ZSTK474-induced apoptosis and sustained tumor growth. Experiments with (a) ICI118,551, a selective antagonist of ADRB2; (b) inducible expression of the PKA inhibitor PKI-GFP; and (c) BCL2-associated death promoter (BAD) with a mutated PKA phosphorylation site at S112 delineated a dominant role of the ADRB2/PKA/BAD signaling pathway in apoptosis inhibition. The generality of this signaling mechanism was demonstrated by experiments with Hi-Myc mice. Immobilization stress induced BAD phosphorylation in prostates of these mice, inhibited apoptosis, and increased size of mouse prostatic intraepithelial neoplasia (PIN) and overall prostate weight compared with nonstressed mice. Stress also delayed prostate involution in mice subjected to androgen ablation therapy with bicalutamide (Casodex). Consistent with the role of the ADRB2/PKA/BAD signaling pathway, the effects of stress were blocked by providing Hi-Myc mice with ICI118,551 as well as in Hi-MycBAD^3SA/WT mice, in which endogenous BAD was replaced with phosphorylation-deficient mutant BAD^3SA (14).

Immobilization stress protects prostate cancer xenografts from apoptosis via adrenaline/ADRB2 signaling. To address the role of stress in therapeutic resistance of prostate cancer, we examined effects of immobilization stress on the responses of C42LucBAD xenografts to the PI3K inhibitor ZSTK474. Immobilization and exposure to predator scent is a well-established method of inducing behavioral stress in mice (15, 16). In our experiments, this technique increased adrenaline blood levels on average to 12 nM in stressed groups for at least 12 hours, whereas in nonstressed intact (or calm) mice, adrenaline was typically less than 1 nM (Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI63324DS1). Analysis of tumor tissue extracts also showed increased catecholamine levels in stressed mice (Supplemental Table 1).

C42 prostate cancer cells, which were used for xenograft studies, are among the best-characterized models of androgen-independent prostate cancer (17). The PI3K/AKT pathway is constitutively active in these cells due to the loss of tumor suppressor phosphatase and tensin homolog (PTEN), as is true for up to 70% of advanced androgen-independent prostate cancers (18). C42LucBAD cells were engineered to express firefly luciferase and HA-BAD. Expression of BAD provides a more uniform response of xenograft tumors to PI3K inhibitors, whereas luciferase expression allows noninvasive monitoring of xenograft tumors in mice in vivo by luminescent imaging.

Injection of the PI3K inhibitor ZSTK474 into C42LucBAD xenografts substantially reduced luminescence, as well as tumor volume (as measured by magnetic resonance imaging; Supplemental Figure 2), whereas subjecting mice to immobilization stress prevented this reduction of luminescence in C42LucBAD xenografts (Figure 1, A and C). Immunohistochemical analysis of tumor xenografts with antibodies that recognize active caspase-3 showed increased staining of prostate cancer cells in ZSTK474-injected xenografts; staining was decreased in xenografts from mice subjected to immobilization stress (Figure 1B). The validity of staining for active caspase-3 to identify apoptosis in C42LucBAD cells was confirmed by TUNEL; in most cases, staining for TUNEL and active caspase-3 overlapped (Supplemental Figure 3). Analysis of proteins extracted from tumor xenografts by Western blotting confirmed the appearance of the active cleaved form of caspase-3 and cleavage of the caspase substrate PARP in tumors with reduced luminescence (Figure 1D), whereas no changes in cleaved caspase-3 and PARP were observed in xenografts excised from stressed mice not injected with ZSTK474 (Supplemental Figure 4). Therefore, diminished luminescence of xenograft tumors reflects increased apoptosis of C42LucBAD cells.

Figure 1

Stress or adrenaline prevents apoptosis induced by PI3K inhibitor in prostate cancer xenografts. (A) Representative images of mice with C42LucBAD xenografts. Mice were injected with DMSO, ZSTK474 (ZSTK), or ZSTK474 followed by immobilization stress. Luminescence of xenograft tumors was measured before injection of PI3K inhibitors (0 hour) and after injections at 24 and 72 hours. (B) Immunohistochemical analysis of cleaved caspase-3 in C42LucBAD xenograft tumors. Mice were treated as in A, and xenografts were excised 6 hours after injections. Scale bars: 50 μm. (C) Dynamics of luminescence in C42LucBAD xenograft tumors. Mice were injected with DMSO (n = 3), ZSTK474 (n = 3), ZSTK474 followed by immobilization stress (n = 3), ZSTK474 and adrenaline (adren; n = 3), ZSTK474 and ICI118,551 (ICI) followed by immobilization stress (n = 3), or ZSTK474, ICI118,551, and adrenaline (n = 4). Error bars show SD from the average of measurements in at least 3 mice. Comparisons between pairs of groups were performed using t tests derived from the overall ANOVA model. Significant between-group differences over time were as follows: P < 0.02, ZSTK vs. DMSO; P < 0.01, ZSTK+stress vs. ZSTK; P < 0.03, ZSTK+adren vs. ZSTK; P < 0.003, ZSTK+ICI+stress vs. ZSTK+stress; P < 0.006, ZSTK+ICI+adren vs. ZSTK+adren. (D) Western blot analysis of C42LucBAD xenograft tumor tissues excised 6 hours after injection of ZSTK474 and/or ICI118,551 (ICI) into mice that were then subjected to immobilization stress, left intact, or injected with epinephrine. At the time of xenograft excision, blood was collected for adrenaline measurements (shown above blots).

Because beta blockers were reported to reduce prostate cancer incidence and mortality (10, 19), and ADRB2 is the predominant adrenergic receptor in LNCaP/C42 prostate cancer cells (20), we examined the effects of stress in the presence of the selective ADRB2 antagonist ICI118,551 (21). Effects of stress on xenograft luminescence and apoptosis (i.e., PARP and caspase-3 cleavage) were completely blocked by ICI118,551 (Figure 1, C and D). Conversely, injections of adrenaline mimicked the effects of stress on xenografts’ luminescence and apoptosis. As was the case with stress, effects of adrenaline injections were blocked by ICI118,551 (Figure 1C). Taken together, our results suggest that adrenaline signaling via ADRB2 is necessary and sufficient to inhibit apoptosis in prostate cancer xenografts of mice subjected to immobilization stress.

Stress protects C42Luc xenografts from apoptosis via PKA/BAD^S112 signaling. To examine signaling pathways that control apoptosis in prostate cancer xenografts, we targeted phosphorylation of BAD, a BH3-only proapoptotic member of the Bcl2 protein family. Earlier tissue culture experiments identified BAD as a convergence node for several survival signaling pathways in prostate cancer and breast cancer cells (22, 23). However, in vivo evidence that BAD phosphorylation plays a critical role in antiapoptotic signaling in prostate tumors has been lacking. BAD is continuously phosphorylated at S112, S136, and S155 in PTEN-deficient prostate cancer cells. Phosphorylation at either BAD^S112 or BAD^S136 facilitates binding to 14-3-3 proteins and neutralizes the proapoptotic properties of BAD (24). In prostate cancer cells, inhibition of PI3K led to dephosphorylation of BAD^S112 and BAD^S136 and increased apoptosis, while antiapoptotic signaling by vasoactive intestinal peptide, adrenaline, or the RAF/MAPK pathway restored phosphorylation of BAD^S112 and inhibited apoptosis (11, 22).

ZSTK474 inhibited phosphorylated AKT^S473 (pAKT^S473) and pBAD^S112 in xenograft tumors of intact mice. However, in mice subjected to immobilization stress or injected with adrenaline, BAD phosphorylation was restored, despite continuous inhibition of the PI3K/AKT pathway (Figure 1D). Injection of ICI118,551 prevented BAD phosphorylation by either stress or adrenaline. Since C42Luc xenografts are characterized by constitutive activation of the PI3K pathway, BAD was constitutively phosphorylated. Its phosphorylation status was not changed significantly by stress in the absence of ZSTK474, although CREB phosphorylation was increased (Supplemental Figure 4). These results demonstrated that immobilization stress triggers PI3K/AKT-independent phosphorylation of BAD via the adrenaline/ADRB2 axis in prostate cancer xenografts.

PKA has been identified as one of the downstream effectors of ADRB2 signaling and as a BAD^S112 kinase (25, 26). We thus analyzed phosphorylation of CREB^S133, an established PKA substrate (27), in prostate cancer xenografts; similar to BAD^S112, both stress and adrenaline stimulated pCREB^S133, and these effects were reversed by ICI118,551 (Figure 1D).

To directly test the role of PKA in antiapoptotic signaling by stress or adrenaline in vivo, we generated C42LucPKI cells that inducibly express a chimera of the PKA inhibitor peptide PKI and GFP (PKI-GFP; ref. 28). Induction of PKI-GFP expression in C42LucPKI prostate cancer cells prevented adrenaline-induced activation of PKA to a degree similar to pretreatment with ICI118,551, as shown by lack of CREB phosphorylation (Supplemental Figure 5). Likewise, in subcutaneously implanted C42LucPKI xenografts, doxycycline-induced PKI-GFP expression prevented phosphorylation of the PKA substrates CREB^S133 and BAD^S112 in response to stress or adrenaline injection (Figure 2A). Furthermore, doxycycline-induced expression of GFP-PKI in C42Luc xenografts blocked the antiapoptotic effects of stress or adrenaline, as evidenced by the increased levels of cleaved caspase-3 and cleaved PARP.

Figure 2

Activation of PKA is necessary for stress- or adrenaline-induced protection from apoptosis in prostate cancer xenografts. (A) Analysis of C42LucPKI xenograft tumors by Western blotting. ZSTK474 inhibited pAkt^S473 and pBAD^S112 and induced cleavage of PARP and caspase-3. Stress or adrenaline induced pBAD^S112 and pCREB^S133 and inhibited cleavage of PARP and caspase-3. These effects of stress or adrenaline were blocked by doxycycline-induced (Dox) PKI-GFP expression. At the time of tumor excision, blood was collected for adrenaline measurements (shown above blots). (B and C) Effects of stress and adrenaline on tumor luminescence depend on PKA activity. Mice were injected with DMSO as a control or with ZSTK474, with or without adrenaline or subsequent immobilization stress. Comparisons between groups were performed using t tests derived from the overall ANOVA model. (B) In mice that did not receive doxycycline, significant differences in luminescence across time were found in ZSTK+stress (P < 0.0002) and ZSTK+adren (P < 0.0002) groups versus ZSTK. (C) These differences were eliminated by doxycycline-induced PKI-GFP expression (P > 0.63 and P > 0.13, respectively). Error bars in B and C show the SD from the average of measurements in at least 4 mice.

Consistent with increased apoptosis, decreased luminescence was observed in C42LucPKI xenografts injected with ZSTK474 in mice that received doxycycline before stress or adrenaline injection, whereas in the absence of doxycycline, stress and adrenaline prevented the loss of luminescence in ZSTK474-injected xenograft tumors (Figure 2, B and C). Thus, inhibition of PKA rendered cancer cells insensitive to antiapoptotic signaling activated by adrenaline or by immobilization stress.

A similar strategy was used to test the role of pBAD^S112 in antiapoptotic signaling by stress. We generated C42LucBAD^1SA cells that express BAD^S122A under a doxycycline-inducible promoter. In the absence of doxycycline, these xenograft tumors responded to injection of ZSTK474 by decreased luminescence; immobilization stress or injection of adrenaline reversed the effect of ZSTK474. Yet in xenografts with doxycycline-induced expression of mutant BAD^S112A, neither stress nor adrenaline restored luminescence in tumors injected with ZSTK474 (Figure 3). Consistent with these data, doxycycline-induced expression of BAD^S112A prevented inhibition of caspase-3 cleavage and PARP cleavage, despite increased phosphorylation of CREB, in response to stress or adrenaline. These results suggest that expression of the phosphorylation-deficient BAD mutant inhibits the antiapoptotic effects of stress or adrenaline downstream from PKA. In contrast, C42LucBAD xenografts that ectopically express WT BAD were protected from apoptosis by stress and by adrenaline (Figure 1). In summary, these results provide evidence that PKA-dependent BAD^S112 phosphorylation plays a decisive role in antiapoptotic effects of stress in prostate xenograft tumors.

Figure 3

Activation of pBAD^S112 is necessary for stress- or adrenaline-induced protection from apoptosis in prostate cancer xenografts. (A) Analysis of C42LucBAD^1SA xenograft tumors by Western blotting. ZSTK474 inhibited pAkt^S473 and pBAD^S112 and induced cleavage of PARP and caspase-3. Stress or adrenaline induced pBAD^S112 and pCREB^S133 and inhibited cleavage of PARP and caspase-3. Effects of stress or adrenaline on apoptosis (cleavage of caspase-3 and PARP) were blocked by doxycycline-induced expression of mutant HA-BAD^S112A. Arrowheads denote HA-BAD^1SA (top) and endogenous BAD (bottom). The inset at right from a pBAD blot shows lysates of C42LucBAD cells that served as positive control for phosphorylated HA-BAD. Mutant HA-BAD^S112A (lanes 5–7) could not be phosphorylated and was not recognized by pBAD^S112-specific antibodies. (B and C) Effects of stress or adrenaline on tumor luminescence depend on pBAD^S112. (B) In mice that did not receive doxycycline, luminescence in ZSTK+stress and ZSTK+adren groups was highly significantly different compared with the ZSTK group (P < 0.0001 for both). (C) These differences were completely eliminated by doxycycline-induced expression of pBAD^S112-deficient HA-BAD^1SA (P > 0.65 and P > 0.52, respectively). Error bars in B and C show the SD from the average of measurements in at least 4 mice.

Behavioral stress activates adrenaline/ADRB2 signaling, inhibits apoptosis, and accelerates cancer development in mouse prostates. Experiments with subcutaneously implanted C42 xenografts demonstrated that psychosocial stress could activate the adrenaline/ADRB2/PKA/BAD antiapoptotic signaling pathway in prostate tumors that grow outside of prostate gland and boost their therapeutic resistance. However, these experiments did not elucidate whether stress could activate antiapoptotic mechanisms in the prostate. To address this question, we examined phosphorylation of the PKA substrates CREB and BAD in prostates of mice subjected to immobilization stress. pBAD^S112 and pCREB^S133 increased in prostates of stressed FVB or BALB/c nude mice (Figure 4A and Supplemental Figure 6A). Statistically significant positive correlations were observed between adrenaline levels and pBAD^S112 and pCREB^S133 in prostates of stressed mice (Supplemental Figure 6B).

Figure 4

Stress induces BAD phosphorylation and inhibits cleavage of PARP and caspase-3 in Hi-Myc mouse prostate glands. WT and Hi-Myc mice were subjected to recurrent 1-hour immobilization stress at 12-hour intervals for 7 consecutive days; blood and prostates were collected immediately after the last stress procedure. ICI118,551 was given 30 minutes before stress. (A) Western blot analysis of DLP glands excised from intact (denoted “calm” or “C”) and stressed (“stress” or “S”) mice was conducted with antibodies to pCREB^S133, pBAD^S112, cleaved caspase-3, cleaved PARP, α-tubulin, and c-Myc. 4 representative samples from each treatment group are shown. (B) Densitometric analysis of Western blots revealed statistically significant increases of BAD and CREB phosphorylation in stressed versus intact mice (pBAD^S112, P = 0.005 between WT groups; P = 0.02 between Hi-Myc groups; pCREB^S133, P = 0.02 between WT groups; P = 0.0019 between Hi-Myc groups) and reduced cleavage of caspase-3 (P = 0.005) and PARP (P = 0.01) in stressed versus intact Hi-Myc mice. These effects of stress were completely eliminated by ICI118,551. Each experimental group contained at least 5 mice. Error bars represent SD from the average of at least 5 samples.

These results suggest that an adrenaline/ADRB2/PKA/BAD antiapoptotic signaling pathway activated in response to behavioral stress could contribute to the progression of primary prostate tumors in mice. To test this hypothesis, we chose Hi-Myc mice, a model of prostate cancer driven by prostate-restricted expression of c-MYC under a probasin promoter (29). Ectopic expression of c-MYC increases both proliferation and apoptosis in various cell types, including prostate epithelial cells (29, 30); however, it did not increase BAD phosphorylation (Figure 4A). At the same time, subjecting mice to immobilization stress increased BAD phosphorylation in prostates of WT and Hi-Myc mice and decreased cleavage of caspase-3 and PARP in Hi-Myc prostates (Figure 4, A and B). Furthermore, immobilization stress administered for 7 consecutive days led to increased prostate weight in Hi-Myc mice, but not WT mice (Figure 5A). Histological analysis of prostate tissue sections revealed reduced apoptosis, based on increased numbers of cells with active caspase-3 or with positive TUNEL staining (Figure 5B and Supplemental Figure 7), and larger PIN area (Figure 5C), in Hi-Myc mice subjected to stress. Consistent with our results in C42 xenografts, stress-induced phosphorylation of BAD, inhibition of apoptosis, more extensive PIN, and increased prostate weight were all blocked in stressed mice by the ADRB2 antagonist ICI118,551 (Figures 4 and 5).

Figure 5

Stress accelerates PIN growth and inhibits apoptosis in DLP glands of Hi-Myc mice. (A) Stress increased prostate weight. Mouse prostates (AP, DLP, and VP lobes) were dissected and weighed, and the total prostate wet weight was expressed as mg/25 g body weight. Statistically significant differences were observed between intact and stressed Hi-Myc mice (P = 0.002), but not between WT groups (P = 1.00). This difference in Hi-Myc mice was eliminated by ICI118,551 injection prior to stress (P = 0.49). (B) Stress reduced apoptosis in DLP glands. Percent cleaved caspase-3–labeled cells in immunostained sections was determined relative to the total number of glandular epithelial cells in whole sections of DLP. Representative images of cleaved caspase-3 IHC-stained sections from DLP of intact or stressed Hi-Myc mice are also shown. Scale bars: 50 μm. Insets show the original images (×40 objective) enlarged ×5. (C) Stress increased PIN in DLP glands of Hi-Myc mice. Percent PIN area was determined as area of PIN divided by total DLP glandular area. Representative microphotographs of H&E-stained sections from DLP glands of intact and stressed Hi-Myc mice are also shown. Scale bars: 50 μm. See Supplemental Figure 8D for morphology of mouse PIN. Error bars in A–C show SD from the average of at least 5 samples.

Tumor-promoting effects of behavioral stress depend on BAD phosphorylation. To test whether BAD phosphorylation is necessary for the effects of stress on prostate tumors, we used mice with BAD^3SA knockin and c-Myc expression (referred to herein as Hi-MycBAD^3SA/WT mice). BAD^3SA/3SA knockin mice are generated by replacing endogenous BAD with mutant BAD in which the major phosphorylation sites S112, S136, and S155 are substituted by alanines (14). Because homozygous knockin Hi-MycBAD^3SA/3SA mice were born at lower than expected frequency and often had smaller body weights and smaller prostates, we decided to use heterozygous knockin Hi-MycBAD^3SA/WT mice and WTBAD^3SA/WT littermates that showed similar phenotypes.

Analysis of BAD phosphorylation and of apoptosis markers (cleaved PARP and cleaved caspase-3) revealed no difference between prostates from intact or stressed Hi-MycBAD^3SA/WT mice; however, pCREB^S1133 in prostates of stressed mice was significantly increased (Figure 6, A and B, and Supplemental Figure 8, A and B), which confirmed that stress activated the PKA pathway. Analysis of prostate tissue sections showed that, similar to WTBAD^3SA/WT littermates, Hi-MycBAD^3SA/WT mice were characterized by PIN, predominantly in the dorsolateral prostate (DLP) lobe. At the same time, there were no significant differences in prostate weight between Hi-MycBAD^3SA/WT mice subjected to repeated immobilization stress or left intact (Figure 6, C and D), in striking contrast to Hi-MycBAD^WT/WT mice, which express WT BAD (Figure 5A). Comparable results were obtained in 12- and 24-week-old Hi-MycBAD^3SA/WT mice (Figure 6 and Supplemental Figure 8). Thus, similar to the C42 prostate cancer xenograft model, BAD phosphorylation was necessary for behavioral stress to manifest tumor-promoting effects in mouse prostate glands.

Figure 6

Antiapoptotic and tumor-promoting effects of stress are eliminated in BAD^3SA/WT knockin mice. (A) Western blot analysis of DLP glands excised from 12-week-old intact and stressed Hi-MycBAD^3SA/WT mice was conducted with antibodies to pCREB^S133, pBAD^S112, cleaved caspase-3, cleaved PARP, and β-actin. (B) Densitometric analysis of Western blots revealed statistically significant increases of pCREB^S133 in stressed versus intact Hi-MycBAD^3SA/WT mice (P = 0.003); however, no significant differences between intact and stressed mice were found in pBAD^S112 (P = 0.29), cleaved caspase-3 (P = 0.47), or cleaved PARP (P = 0.25). Lysates of 4 intact mice were used for densitometry (2 mice with adrenalin levels greater than 1 nM were excluded as erroneously stressed); the stressed group contained 6 mice. (C) Prostates of intact and stressed Hi-MycBAD^3SA/WT (n = 5 per group) and WTBAD^3SA/WT (n = 3 per group) mice were analyzed as in Figure 3B. (D) Microphotographs of dissected prostate glands from Hi-MycBAD^3SA/WT or WTBAD^3SA/WT mice either subjected to repeated immobilization stress for 7 days or left intact. (E) Prostates of intact and stressed Hi-MycBAD^3SA/WT mice were analyzed as in Figure 5B (n = 5 per group). Bars in B, C, and E show SD. (F) Representative images of H&E-stained sections of DLP glands of intact or stressed Hi-MycBAD^3SA/WT mice. Scale bars: 50 μm.

Behavioral stress delays bicalutamide-induced involution and apoptosis in prostates of Hi-Myc mice. Androgen ablation therapy, introduced by Huggins in 1941, remains the most effective systemic treatment for prostate cancer (reviewed in ref. 31). Bicalutamide, an androgen receptor (AR) antagonist that, upon binding, inhibits transcriptional activity of AR, is widely used to treat prostate cancer. Since AR activity is necessary for survival of prostate cells, bicalutamide induces apoptosis and involution of human and rodent prostates (32–34).

We examined effects of systemically injected bicalutamide on prostates of Hi-Myc mice. The most significant reduction in prostate weight (from 45 ± 8.3 mg to 29 ± 8.4 mg) was observed after 2 daily injections of bicalutamide, with little further decrease (to 28 ± 3.7 mg) after third bicalutamide injection. Effects of behavioral stress on prostate weight were also most evident after 2 days of bicalutamide injections; therefore, this time point was selected for subsequent experiments. As shown in Figure 7, behavioral stress delayed Hi-Myc prostate involution and apoptosis induced by bicalutamide and increased prostate weight in bicalutamide-treated stressed versus intact Hi-Myc mice. Consistent with the effects on prostate weight, stress caused a significant 3.6-fold reduction in apoptosis, based on the percentage of cells with cleaved caspase-3 (Figure 7B). Analysis of Hi-Myc prostates by Western blotting confirmed quantitative analysis of apoptosis: cleaved caspase-3 and cleaved PARP were increased in prostates of mice that received bicalutamide (Figure 8A). Behavioral stress induced pCREB^S133 and pBAD^S112 and inhibited apoptosis, as shown by lower levels of cleaved caspase-3 and cleaved PARP, in prostates of stressed mice (Figure 8, B and C). Expression of probasin, a known androgen-responsive gene, was significantly reduced in prostates of bicalutamide-treated mice, yet no significant increase in probasin expression was detected in stressed mice (Figure 8, A and D), which indicates that stress does not transactivate AR signaling or activate transcriptional targets of AR.

Figure 7

Stress delays bicalutamide-induced involution and apoptosis in Hi-Myc prostates via the ADRB2/BAD pathway. Hi-Myc mice were subjected to subcutaneous injection of bicalutamide (bicalut.; 50 mg/kg, once daily) and recurrent 1-hour immobilization stress at 12-hour intervals for 3 consecutive days; blood and prostates were collected immediately after the last stress procedure. ICI118,551 was given 30 minutes before stress. (A) Stress delayed bicalutamide-induced prostate involution in Hi-Myc mice. Mouse prostates (AP, DLP, and VP lobes) were dissected and weighed, and the total prostate wet weight was expressed as mg/25 g body weight. Statistically significant differences were observed between intact and bicalutamide-treated intact Hi-Myc mice (P = 0.01) and between bicalutamide-treated intact and stressed Hi-Myc mice (P = 0.002). The effect of bicalutamide on prostate weight was completely eliminated with ICI118,551 (P = 0.54) and significantly reduced from 2- to 0.2-fold in Hi-MycBAD^3SA/WT mice (P = 0.049). Representative images of prostates of Hi-Myc intact and stressed mice treated with bicalutamide are also shown. (B) Stress delayed bicalutamide-induced apoptosis in DLP glands of Hi-Myc mice (P = 0.002). The effect of stress on bicalutamide-induced apoptosis was eliminated in Hi-Myc mice injected with ICI118,551 (P = 0.84) and in compound transgenic Hi-MycBAD^3SA/WT mice (P = 0.47). Representative images of cleaved caspase-3 IHC-stained sections from DLP of intact and stressed Hi-Myc mice treated with bicalutamide are also shown. Scale bars: 50 μm. Insets show the original images (×40 objective) enlarged ×2.33.

Figure 8

Stress inhibits bicalutamide-induced cleavage of PARP and caspase-3 through ADRB2 and BAD phosphorylation in prostates of Hi-Myc mice. (A) Western blot analysis of prostate glands excised from intact mice and bicalutamide-injected intact or stressed mice was conducted with antibodies to pCREB^S133, pBAD^S112, cleaved caspase-3, cleaved PARP, β-actin, and probasin. 3 representative samples for each treatment group are shown. (B) Densitometric analysis of Western blots showed significant increases of BAD and CREB phosphorylation in stressed versus intact Hi-Myc mice (pCREB^S133, P = 0.01; pBAD^S112, P = 0.008), which was blocked by ICI118,551. No significant differences were found in pCREB^S133 (P = 0.35) or pBAD^S112 (P = 0.34) between the intact and stressed bicalutamide treatment groups. In Hi-MycBAD^3SA/WT mice, stress significantly increased pCREB^S133 (P = 0.003), but not pBAD^S112 (P = 0.15). (C) Stress decreased cleavage of caspase-3 and PARP induced by bicalutamide (P = 0.003, intact vs. bicalutamide intact; P = 0.004, bicalutamide intact vs. bicalutamide stress). These effects were eliminated in mice treated with ICI118,551 and in Hi-MycBAD^3SA/WT mice (P > 0.5). P values were similar for cleaved caspase-3 and PARP. (D) Bicalutamide treatment significantly decreased probasin expression (P = 0.01), and stress did not affect probasin expression in prostates of bicalutamide-treated mice (P = 0.58). Each experimental group contained at least 5 mice. Error bars in B–D show SD from the average of at least 4 samples.

The effect of stress on prostate weight was completely blocked by the ADRB2 antagonist ICI118,551 and was decreased from 2-fold to 0.2-fold in Hi-MycBAD^3SA/WT prostates (Figure 7). Conversely, no significant decrease of apoptosis was observed in prostates of Hi-MycBAD^3SA/WT mice or of mice that received ICI118,551 (Figure 7). Western blot analysis showed that BAD phosphorylation was completely blocked in mice that received ICI118,551 as well as in compound transgenic Hi-MycBAD^3SA/WT mice (Figure 8, A and B). Consistent with lack of BAD phosphorylation, stress did not significantly decrease cleaved PARP and cleaved caspase-3 in prostates of Hi-MycBAD^3SA/WT mice or mice treated with ICI118,551 (Figure 8, A and C). Taken together, results from experiments with Hi-Myc mice support our conclusion drawn from the xenograft experiments: antiapoptotic signaling via the adrenaline/ADRB2/PKA/BAD pathway in prostate cells plays a decisive role in the effects of behavioral stress on prostate cancer.

The experiments presented above demonstrated that in 2 distinct in vivo models of prostate cancer, C42 xenografts and prostates of Hi-Myc mice, behavioral stress activated the adrenaline/ADRB2/PKA/BAD antiapoptotic signaling pathway, which in turn reduced therapeutic sensitivity and accelerated prostate cancer development. These data introduce behavioral stress as a new environmental component contributing to prostate cancer pathogenesis through a defined antiapoptotic signaling pathway that impinges on BAD phosphorylation. Considering that prostate cancer diagnosis increases stress and anxiety levels (6, 7), activation of a stress-induced antiapoptotic pathway may lead to a vicious cycle of stress and cancer progression (Figure 9).

Figure 9

A vicious circle of stress signaling in prostate cancer. Psychoemotional stress activates the hypothalamic-pituitary-adrenal axis (HPA); as a result, blood levels of adrenaline increase and activate ADRB2/PKA/BAD antiapoptotic signaling in prostate cells. Constitutive activation of this signaling pathway in stressed patients (Supplemental Figure 12) may trigger a vicious circle: stress and anxiety from a diagnosis of prostate cancer increase adrenaline levels that in turn reduce efficacy of anticancer treatments.

Numerous publications have demonstrated that BAD phosphorylation is necessary for inhibition of apoptosis by survival agonists in tissue culture models, including prostate cancer cell lines (11, 22, 35). However, the in vivo evidence supporting the role of BAD phosphorylation is circumstantial, and therefore cannot distinguish whether BAD phosphorylation plays a dominant or secondary role in translating survival signaling into tumor progression and therapy resistance. The results presented here are the first to establish the critical role of BAD phosphorylation in stress or adrenaline antiapoptotic signaling in prostate tumors in vivo.

In the context of prostate cancer, substantial attention is paid to the potential roles of ARs in regulation of apoptosis in prostate cells. Specifically, androgen independence is attributed to transactivation of AR in the absence of androgens by other signaling pathways or to activation of antiapoptotic and proliferative pathways that are entirely independent from AR (36). Several publications examined interactions between PKA signaling and AR and reported either transactivation (37, 38) or inhibition (39, 40) of AR.

We previously demonstrated that the antiapoptotic effect of activation of ADRB2s is modestly diminished in prostate cancer cells with knockdown of AR expression and thus is largely independent from AR (11). Conversely, unlike the AR agonist R1881, adrenaline did not induce increased PSA production by LNCaP and C42 cells that express AR (Supplemental Figure 9A). Thus, adrenaline cannot transactivate AR in prostate cancer cells. Consistent with this conclusion, stress did not increase PSA levels in mice with C42Luc xenografts (Supplemental Figure 9B), or probasin expression in prostates of mice treated with bicalutamide (Figure 8A).

Considering these recent data and earlier results of experiments with AR knockdown, we concluded that transactivation of AR plays a limited role in the antiapoptotic effects of adrenaline. Instead, adrenaline bypasses AR by activating the AR-independent ADRB2/PKA/BAD antiapoptotic signaling pathway and thus increases resistance of prostate cancer cells to androgen ablation therapy and other cytotoxic therapies that induce apoptosis.

A substantial body of literature describes the effects of stress on tumor development and metastases in various cancer models (5). Yet despite this abundant phenomenology, mechanisms of interaction between stress and cancer have not been well defined. The pioneering studies by Selye on stress-induced immunosuppression (41) stimulated significant interest in understanding how the modulation of immune responses by stress contributes to cancer initiation and progression. Thus, it was reported that catecholamine signaling accelerated primary tumor development by inhibiting natural killer cells and stimulated metastatic spread by attracting macrophages to tumor sites (42, 43).

Another line of research has focused on stress-regulated angiogenesis. Experiments in ovarian tumor xenografts have shown that ADRB2 activation leads to greater production of VEGF by ovarian cancer cells and neovascularization that supports tumor growth (16). These experiments established a new paradigm that connected the neuroendocrine system, cancer cells, and vascular growth into a stress-activated circuit that accelerated progression of ovarian cancer (44).

Since our xenograft experiments were conducted in immunocompromised mice, and the effects of stress on apoptosis were observed within 6 hours, contributions from the tumor microenvironment are unlikely to play a dominant role in inhibiting apoptosis in this model. Moreover, we did not find increased VEGF levels in plasma of stressed mice (Supplemental Figure 10A). Because C42Luc xenografts are already highly vascularized, this model may not permit detection of vascularity increases. Immunostaining of the prostates of intact and stressed Hi-Myc mice with the endothelial cell marker factor VIII–related antigen (FVIII-RA) did not show increased vascular density in stressed mice (Supplemental Figure 10B). Taken together, these experimental data suggest that, unlike in the models of ovarian cancer, increased vascularity is not a primary mediator of stress effects on prostate tumors.

Expression of functional ADRB2 was previously reported in normal prostate epithelium and in prostate cancer cells, including LNCaP and C42 human prostate cancer cells and rodent prostates (12, 20, 45, 46). We confirmed expression of ADRB2 in prostate cancer models (Hi-Myc mouse prostates and C42 prostate cancer cells) as well as in human prostates (Supplemental Figure 11). Our results showing inhibition of antiapoptotic effects of stress or adrenaline in C42Luc xenografts by inducible expression of PKI-GFP and of BAD^S112A in prostate cancer cells are consistent with the notion that activation of antiapoptotic signaling within prostate cancer cells (rather than systemic effects of adrenaline) are responsible for reduced therapeutic sensitivity and accelerated progression of prostate tumors. Results from experiments in Hi-Myc mice that express either WT BAD or BAD^3SA supported the conclusions from experiments in the xenograft model that antiapoptotic signaling via BAD phosphorylation mediates effects of stress on PIN extent and on prostate weight. These results suggest apoptosis inhibition by adrenaline as a new mechanism of the tumor-host interaction that, together with effects mediated via the immune and vascular systems, defines the effects of behavioral stress on tumor growth.

Interactions between stress and cancer may not always be unidirectional. Thus, moderate stress from enriched environments reportedly causes inhibition of tumor growth and cancer remission in melanoma and colon cancer models (47). These antitumor effects of moderate stress are attributed to increased hypothalamic brain-derived neurotrophic factor that activates sympathetic β-adrenergic receptor signaling, which in turn reduces secretion of leptins by white adipose tissue (47). Unlike immobilization stress, enriched environments may not increase blood adrenaline sufficiently to activate antiapoptotic signaling in cancer cells. These bidirectional effects of behavioral stress evoke the distinction between “eustress” and “distress” made by Selye in his classic studies (41).

Several extrapolations relevant to human prostate cancer can be derived from our results. First, tumor-promoting effects of stress will be evident in patients who regularly experience increased adrenaline levels. We observed repeated increases greater than 1 nM in plasma adrenaline levels of 14 of 62 prostate cancer patients (22%; Supplemental Figure 12).

Second, antiapoptotic effects of stress will be restricted to cancer cells that express components of the ADRB2/PKA/BAD pathway. ADRB2, PKA, and BAD are ubiquitously expressed in normal prostate epithelial cells and in prostate cancer cells (20, 45, 48–50). Expression of ADRB2 is regulated by androgen and triiodothyronine and is reportedly increased in malignant prostate glands and in metastatic tumors (45). Yet another publication showed biphasic changes in ADRB2 expression characterized by increased levels of PIN and localized prostate cancer, followed by reduction of ADRB2 in metastatic cancer specimens due to transcriptional suppression by EZH2 (51). Still, despite apparent EZH2-dependent downregulation of ADRB2 in LNCaP cells, adrenaline in concentrations observed in stressed patients can protect these cells from apoptosis (11). There is also evidence that expression of βARK1, which desensitizes ADRB2, is lost in prostate tumors with high Gleason scores. This may lead to prolonged activation of adrenaline/ADRB2/PKA/BAD signaling (52).

Third, effects of behavioral stress would be most evident when apoptosis plays the leading role: for example, in tumors with deregulated oncogene expression. We have shown accelerated tumor progression in Hi-Myc mice subjected to repeated stress. Considering that increased c-MYC expression is reported in a substantial proportion of prostate cancers (53), stress-induced apoptosis suppression could synergize with c-MYC in prostate cancer development in men.

Fourth, considering the redundancy of the signaling pathways that control apoptosis, effects of stress in a given tumor will depend on activity of other pathways. For example, if the Ras/ERK or PI3K pathways that control phosphorylation of BAD^S112 (corresponding to human BAD^S75) are active, then stress signaling may have a lesser effect. Thus, in advanced prostate cancers that have lost PTEN, stress is not expected to have a significant effect unless patients are treated with PI3K inhibitors.

We are at the very beginning of understanding complex stress-cancer interactions, with multifaceted responses to stress that affect cancer cells, tumor microenvironment, and the organism overall. There is a vast body of literature on psychological disorders experienced by cancer patients (54), and yet very little is known about the changes in the “endocrinome” of cancer patients caused by behavioral stress. In addition, the limited mechanistic information on signaling by stress hormones in various tumor cells makes it difficult to design new epidemiological studies and to interpret the results of previous ones that have addressed the role of stress in cancer progression and the therapeutic potential of beta blockers as an adjuvant anticancer therapy. Together with recent reports on stress-induced VEGF and IL-8 secretion (16, 55), activation of FAK (56), and downregulation of p53 (57, 58) that stimulate tumor angiogenesis, migration, invasion, and survival (26), our data highlight the connection between activation of the ADRB2/PKA axis and tumor-promoting effects of stress and introduce BAD phosphorylation as a target of stress or adrenaline signaling. Future experiments will determine whether stress activates the adrenaline/ADRB2/PKA/BAD pathway in prostate tumors and other human cancers. We anticipate that components of this signaling pathway could be used as biomarkers to predict whether and how a given tumor will respond to stress and antistress therapies.

View Supplemental data

The authors are grateful to Anil Sood and Karen Klein for critical reading and helpful suggestions; to Dina Yamaleeva, Michael Mangiapani, and Michael Conlin for assistance with image analysis; to John Olson for MRI imaging; and to Michael Thomas for mass spectrometry detection of noradrenaline. This work was supported by federal grants PC073548 from the Department of Defense and R01CA118329 from the National Cancer Institute and by institutional grants from Wake Forest University Health Sciences to G. Kulik. D. Yancey was supported in part by Research Supplement to Promote Diversity in Health-Related Research.

Address correspondence to: George Kulik, Department of Cancer Biology, Wake Forest School of Medicine, Medical Center Blvd., Winston-Salem, North Carolina 27157, USA. Phone: 336.713.7650; Fax: 336.713.7661; E-mail: gkulik@wakehealth.edu.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2013;123(2):874–886. doi:10.1172/JCI63324.

See the related article at Why stress is BAD for cancer patients.

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