In this study, we demonstrated that chronic stress profoundly accelerates the emergence and development of squamous cell carcinoma and their precursors while suppressing tumor regression in a mouse model. To our knowledge, this is the first study to elucidate the cellular and molecular mechanisms mediating a chronic stressinduced increase in susceptibility to skin cancer. Chronic stress administered during the middle 3 weeks of mild, non-blistering, UVB exposure caused more mice to develop tumors and to develop them faster than non-stressed mice (). These chronically stressed mice had an earlier onset of the first tumor and also reached 50 % and 100% incidence earlier than the non-stressed mice (). Although median time to first tumor and time of 50% incidence were the same for the chronically stressed group, the 4.5-week gap between median time to first tumor and time of 50% incidence in the non-stressed mice indicates greater spontaneous regression of tumors during this period in the non-stressed group. In addition, chronically stressed mice showed no tumor regression at week 34 whereas non-stressed mice did ().
Our results suggest that suppressed immune function and increased tumor promotion during chronic stress exposure have long lasting effects. Support for this also comes from studies that show that chronic stress suppresses skin cell-mediated immunity (3
) and that cell-mediated immunity is critical for elimination of squamous cell carcinomas that are known to be immunogenic tumors (8
). Stress has also been shown to suppress NK activity and enhance experimental tumor metastasis following intravenous injection of a tumor cell line (25
). Parker et al. (27
) have used an extended model of chronic stress that was initiated 2 weeks before, and administered during, a more prolonged period of UV exposure, to show accelerated formation of cutaneous neoplasms. It is important to appreciate that in the current study, chronic stress was administered for only 3 weeks during the period of UV-induced tumor promotion and yet had effects on critical molecular and cellular mediators of antitumor immunity that were observed 8 months later. This result suggests that a relatively moderate chronic stressor can be a potent immunosuppressor during critical periods of immune response development and can set the stage for suppression of anti-tumor immunity long after stress has ended.
Stressed mice had higher basal corticosterone levels nearly 28 weeks after the cessation of stress. A salubrious corticosterone rhythm is one that shows low levels of plasma corticosterone at the beginning of the inactive period of the diurnal cycle (morning for nocturnal rodents) and higher levels at the beginning of the active period (evening) (28
). The higher morning corticosterone levels observed in the stressed group indicate dysregulation of the circadian rhythm. Circadian rhythm dysregulation may negatively affect immune function and health (29
), and has been associated with increased cancer progression (30
) and mortality (33
). In addition, a dysregulated cortisol rhythm may induce immune dysregulation (28
) and accelerate tumor growth because the host circadian clock is an important control point in tumor progression (31
). Future studies using additional sampling time points will be required to elucidate the kinetics and magnitude of circadian rhythm dysregulation during skin cancer and to determine if the dysregulated rhythm was a cause or effect of increased tumor burden.
We used real-time PCR to quantify differences in gene expression between naïve, non-stressed and chronically stressed animals. IL-12p40 and IFNγ gene expression was suppressed in chronically stressed animals. IL-12 has been shown to promote Th1 responses that drive cell-mediated immunity (35
). IL-12 therapy has been shown to slow the growth of murine mammary tumors by preventing neovascularization and by increasing the number of infiltrating leukocytes (36
) and intra-tumoral administration of IL-12 has been shown to elicit a Th1 response profile in sentinel lymph nodes (37
). Although measurement of IL-12p40 gene expression may not be entirely indicative of the biologically active IL-12p70 heterodimer, IL- 12p40 does appear to be an important representation of IL-12 activity in vivo (38
). It has also been shown that the p19 subunit of IL-23 forms a heterodimer with p40 subunit of IL-12 to form IL-23 (40
). Therefore, it is possible that the level of IL- 12p40 gene expression may indicate IL-23 activity. Although further analyses of cytokine proteins are required to determine the exact cytokine involved, both IL-12 and IL-23 are important mediators of cellular immunity (41
), and IL-23 promotes anti-tumor immunity (42
). IFNγ promotes tumor recognition and elimination (43
), is a critical mediator of the anti-tumor effects of IL-12 (44
), and enables immune system suppression of tumors (45
). Therefore, stress-induced decreases in IL-12 and IFNγ gene expression suggest suppression of critical protective immune mechanisms. In contrast, IL-4 and IL-10 gene expression was not different between groups. Based on these studies, it appears that stress-induced immunosuppression of anti-squamous cell carcinoma responses may be mediated by suppression of Type 1 versus Type 2 cytokines that would in turn suppress anti-tumor immunity.
CCL27/CTACK and CD3εgene expression was also suppressed in chronically stressed animals. CTACK is predominantly expressed in the skin and is critical for attracting skin homing T cells (46
). CD3ε gene expression can be considered an index of T-cell infiltration. Importantly, T cells have been implicated in both the regression (47
) and rejection (48
) of UV-induced squamous cell carcinoma. Therefore, downregulation of CTACK gene expression may contribute to suppression of T cell infiltration and T cell driven anti-tumor immune responses. With the exception of the effects of stress on CD3ε gene expression (which are confirmed by the observed effects on T cell infiltration), we did not examine changes in protein expression for the genes measured in this study. Further studies are required to confirm that the observed effects on gene expression translate to the level of protein expression.
Our observation that chronic stress decreased the numbers of infiltrating CD4 and CD8 T cells around tumors further supports the hypothesis that increased susceptibility to squamous cell carcinoma may be mediated by stress-induced suppression of protective T cell-mediated immunity. Both helper and cytolytic T cell responses are likely to be important in controlling tumor growth, and T cells have been shown to mediate regression of non-melanoma skin cancers (47
). CD4+ T cells have also been found to be crucial for controlling tumor growth by CTL-independent mechanisms (50
). Interestingly, IFN-γ, shown here to be suppressed by chronic stress, appears to be involved in CD4+ T cell mediated elimination of tumors (51
Regulatory/suppressor T cells are a subset of cells expressing both CD25 (the IL-2R-α chain) and CD4 (52
). High levels of these cells have been detected in human cancers (23
). CD25+ T cells impair effector cell function by suppressing IFNγ secretion (53
) and may suppress anti-tumor immune responses (23
). Given these findings, it is likely that CD25+ cell populations that we detected in skin tumors were of the T regulatory/suppressor subset although further studies using dual staining are required to specifically identify this leukocyte subset. Chronic stress increased the numbers of CD25+ cells within tumors () while decreasing the numbers of CD4+ and CD8+ cells around tumors (). It is possible that increased CD25+ cells within tumors of chronically stressed animals suppressed the recruitment of protective T cells and prevented them from mounting effective anti-tumor immune responses. The increased numbers of CD4+CD25+ cells in peripheral blood () show that chronic stress also increased regulatory/suppressor cell numbers in the circulation. Taken together, these results suggest that regulatory/suppressor T cells play an important role in chronic stress-induced immunosuppression, and increased susceptibility to squamous cell carcinoma.
The multi-factorial nature of cancer progression and the involvement of different physiologic systems (nervous, endocrine, and immune) described here, highlight the importance of conducting follow-up studies to further elucidate the molecular, cellular, and physiological mechanisms by which chronic stress increases susceptibility to skin cancer. Additional studies quantifying changes in protein levels need to be conducted to confirm the gene expression effects presented in the current study. The extent of the relative contribution of the individual cell types identified here to the tumor development process must be determined by selective depletion or via specific inactivation. Moreover, additional hormone analysis time points at different stages of tumor development are required to elucidate the kinetics and magnitude of circadian rhythm dysregulation and to determine whether the dysregulated rhythm is a cause or effect of increased tumor burden. Finally, future human studies elucidating mechanisms mediating the link between stress and skin tumor development are required to confirm and translate these findings from bench to bedside.
Our data suggest that chronic stress increases susceptibility to skin cancer and shifts the balance from protective to suppressive immune responses. On the one hand, chronic stress suppresses Type 1 cytokines and CCL27/CTACK gene expression, and CD4+ and CD8+ T cell infiltration at sites of tumor emergence and progression, while on the other, it increases the numbers of regulatory/suppressor cells at tumor sites and in circulation. These results are, to our knowledge, the first to show that chronic stressors increase susceptibility to disease by mobilizing endogenous immunosuppressive mechanisms like regulatory/suppressor T cells. Our results show that a moderate chronic stressor, one that does not have overall healthaversive effects (no change in body and organ weights), can significantly increase susceptibility to skin cancer. Moreover, the detrimental effects of stress on critical clinical, cellular, and molecular parameters, are observed months after the cessation of stress.
Stress pervades almost all aspects of life and is especially salient during diagnosis, treatment, and follow-up, for cancer and other diseases. Therefore, these findings may be relevant for conditions where chronic stress may increase susceptibility to other cancers, decrease effectiveness of tumor immunotherapy, or contribute to systemic immunosuppression during cancer treatment. It is hoped that knowledge gained from transdisciplinary studies such as these, which examine cancer in a holistic context, will increase the accuracy and timeliness of risk evaluation, improve preventative and therapeutic interventions, and help optimize a patient’s response to treatment.