Stress alters the expression of cancer-related genes in the prostate

Stress is a highly complex process that disrupts homeostasis and involves environmental and psychosocial factors acting as stimuli (stressors) to induce a series of responses by the peripheral and central nervous systems [1]. One of the physiological systems that becomes activated during stress is the hypothalamic-pituitary-adrenal (HPA) axis. Activation of the HPA axis triggers the release of corticotrophin-releasing hormone (CRH) from the paraventricular nucleus of the hypothalamus (PVH) which, in turn, induces the anterior pituitary to secrete adrenocorticotropic hormone (ACTH), ultimately resulting in the production of cortisol by the adrenal cortex. The entire axis is then regulated via negative feedback provided by cortisol at the hypothalamic and pituitary levels [2]. Chronic stress can adversely alter hormone levels, thus affecting the regulation of the stress response which can ultimately have a negative effect on the overall physiology of the body [3, 4]. Therefore, stress can have deleterious effects on health, contributing to the incidence and progression of diseases such as cancer.

Chronic stress is, in fact, believed to be a significant factor in the development and progression of cancer, and a growing body of evidence suggests that the neuroendocrine stress response machinery is an important mediator during tumorigenesis and metastasis [5‐9]. Impaired T cell-mediated immunity, enhanced tumor proliferation, and decreased survival have been shown in response to chronic stress in a mouse lymphoma model [10]. Suppression of T cell function by chronic stress is also known to increase susceptibility to skin cancer [11], and β-adrenergic signaling has been shown to be linked to tumor growth and invasion in pancreatic and ovarian cancers [12, 13]. Stress-related hormones have been shown to be involved in accelerating cell proliferation and tumor growth in breast and prostate cancer [14, 15], while inhibiting cell growth in other tumors such as neuroblastoma [16], thus indicating that cell type and specific hormones are critical factors in mediating the stress-cancer relationship.

In addition to altering the regulation of the HPA axis, acute and chronic stress can also affect the hypothalamic-pituitary-gonadal (HPG) axis, which can in turn disrupt reproductive function since the hormones within this axis are responsible for orchestrating mammalian reproduction. The first step of the HPG axis is the secretion of gonadotropin-releasing hormone (GnRH) by the preoptic area of the hypothalamus, which then stimulates the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary in a fashion that is dependent on the frequency and amplitude of GnRH pulses; the gonadotropic hormones LH and FSH then regulate steroidogenesis and gametogenesis in the gonads [17, 18]. Stress can suppress the HPG axis through inhibiting GnRH secretion which, in turn, suppresses pituitary release of LH and FSH [19, 20]. Chronic restraint stress in rats also reduces GnRH pulsatility and lowers plasma LH and FSH [21]. It should be noted, however, that rebound GnRH secretion can occur following earlier suppression of its release [22‐24]. GnRH is strongly implicated as having a role cancer, but conflicting data on the effects of GnRH on cellular function have been published. GnRH analogs inhibit growth and proliferation in reproductive cancer cell lines in vitro [21, 25‐28], and also decrease growth in melanoma cells [29], suggesting that GnRH may have broad anti-cancer functions. Furthermore, GnRH receptor agonists are currently being evaluated for potential use in the treatment of prostate, breast, endometrial and ovarian cancers as well as glioblastomas and melanomas, due to their ability to halt cellular proliferation [30]. However, GnRH receptor antagonists may alternatively be used to knock down the effects of endogenous GnRH and inhibit the release of steroids that stimulate tumor growth in certain cancers [31], demonstrating that GnRH does not always produce desired effects. While accumulating evidence seems to support a clear link between stress and cancer, a direct, mechanistic relationship through which stress can alter reproductive function and subsequently contribute to cancer has not been established.

Prostate cancer (PC) is the most commonly diagnosed non-skin malignancy and the second most prevalent cause of cancer death in the U.S., accounting for ~10% of newly diagnosed male cancer cases worldwide [32‐34]. Taking current screening habits into consideration, it is predicted that 1 in 6 men alive today in the U.S. will be diagnosed with the disease, and roughly 3% of those patients will die from it [35, 36]. GnRH analogs have been previously used as antineoplastic drugs against PC [37‐39]. The use of GnRH agonists is the current choice for androgen-deprivation therapy for advanced and metastatic PC. The rationale behind this treatment is to stimulate the release of LH to achieve feedback regulation of testosterone levels. This method initially leads to a testosterone surge that can worsen the disease, but continuous administration of the agonist eventually downregulates the pituitary GnRH-R, regulating LH release and decreasing testosterone production [40]. On the other hand, GnRH antagonists act by directly binding to GnRH-R, thus avoiding the testosterone “flare” caused by GnRH agonists [41, 42]. Even though it is clear that chronic stress leads to the suppression of GnRH, and GnRH constitutes a target for PC treatment, no study to date has directly examined the relationship between stress-induced modulation of the GnRH system and PC-related outcomes in a single in vivo model. Therefore, we aimed to determine if chronic stress modulates hypothalamic GnRH and consequently affects the expression of genes associated with prostate cell proliferation, and if recovery from stress could induce a rebound of GnRH release that subsequently contributes to an upregulation of genes that promote metastasis.

Accumulating evidence provided by epidemiological studies strongly suggests that chronic psychological stress plays an important role in the initiation and progression of cancer [45‐47]. Results from clinical studies also strongly support a link between stressful events in a patient’s life with reduced cancer survival [46, 48]. However, the precise mechanisms by which chronic stress influences tumorigenesis and carcinogenesis remain poorly understood. Although much progress has been made through the use of animal models, most of these studies have used mouse xenograft and genetic models to study the effects of chronic stress (such as restraint and social isolation) on cancer progression and metastasis [12, 49‐52].To the best of our knowledge, this is the first study designed to identify changes in the expression of cancer-associated genes as a response to chronic emotional stress in a specific organ of an otherwise healthy animal. This allowed us to determine changes in prostate gene expression in response to chronic stress compared to basal levels, which has provided us with insights as to which cellular pathways can be initially disrupted by stress that may lead to abnormal cell proliferation and tumor formation.

Our results demonstrate that restraint stress (both acute and chronic) is sufficient to alter the expression of genes associated with tumor proliferation and metastasis in the rat prostate. Previous studies using a rat model of chronic stress (by restraint water immersion) have shown that 14 days of stress can induce histological changes in the prostate, as well as proliferation of epithelial cells of the ventral lobe of the prostate [53, 54]. Although the expression of proliferation-associated genes was not measured in these studies, our data supports the notion that restraint stress can induce aberrant gene expression, which could potentially lead to increased cell proliferation. The genes included in this investigation have broad cellular functions and their up and downregulation may not necessarily indicate cancer development or progression in this model. However the sensitivity of these genes to stress provides evidence of potential mechanisms through which increased stress exposure might ultimately contribute to cancer.

Although PC is usually considered a disease of aged men (~70% of diagnosed patients are over 65), and age itself is one of the major risk factors in developing the disease [36], histopathological studies using prostate tissue samples from younger healthy patients (age 20–40) have demonstrated the presence of proliferative lesions, indicating that cancer initiation may take place at an early age and remain undetected for many years [55‐58]. Our data demonstrate aberrant expression of cancer-related genes in the tissues of adult animals in response to chronic stress. It is therefore reasonable to speculate that chronic stress may either trigger localized cell proliferation at an early age, which can then accumulate additional “hits” over time until progressing into full fledged cancer. Another interesting possibility is that chronic stress may serve to aggravate pre-existing neoplastic lesions, thus contributing to cancer progression.

Despite the lack of knowledge regarding specific mechanisms underlying tumorigenic processes induced by stress, previous studies have identified several physiological and molecular pathways that are influenced by stress and can contribute to cancer progression. Several studies have focused on immune function, since it is very well established that stress can downregulate immune function, which in turn can impair the immune response against tumor cells [59‐61]. Importantly, it was recently shown that chronic stress can induce a re-organization of lymphatic vessels to facilitate the dissemination of tumor cells in a mouse model of breast cancer, and this was proven to be regulated by the sympathetic nervous system [62]. However, decreased immune function alone cannot account for the multitude of processes that need to be altered in order for cancer to progress. One major contributor is the neuroendocrine response to stress, including the HPA and HPG axes, which trigger hormone signaling descending from the brain and the pituitary to the adrenal glands and reproductive tissues. These systems can have myriad effects on peripheral tissues, including neurotransmitter release and production of mitogenic factors that can promote tumor growth by acting directly on receptors present in cancer cells [6], as well as suppression of hormone pulses that can potentially promote cell proliferation, such as GnRH.

In the case of our study, the cell counts of activated neurons in the PVH, as identified by Fos immunostaining, provide evidence that acute restraint stress is activating the HPA axis. This response is well-established, and involves an increase in the neuroendocrine secretion of corticotropin-releasing factor and, ultimately, enhanced release of the glucocorticoid hormone cortisol. Of our genes displaying upregulated expression levels in the Acu condition, a number of them are regulated by cortisol or have related functions. Krt14, for example, is one of many glucocorticoid-regulated keratin genes that tend to be repressed in skin following cortisol-receptor monomers binding to glucocorticoid response elements in the presence of corticosteroid binding protein [63]. Our observed increase in Krt14 expression may be tissue-specific or a function of alternate binding strategies. Increases in Arnt and Fgfr4 may directly result from increased cortisol exposure. The aryl hydrocarbon receptor and glucocorticoid receptor have been shown previously to interact and participate in complex developmental and physiological processes [64]. Cortisol has been shown to increase Fgfr4 expression, and this molecule has been specifically associated with extracellular matrix degradation and tumor invasion in prostate and other cancers [65]. Metabolically, the glucoregulatory capacity of cortisol provides another link to Fgfr4 which also functions in glucose regulation [65], and to Slc2a1, a facilitated glucose transporter that showed upregulated expression after acute stress in our study. Lastly, prostate cancer patients have been shown to have increased circulating levels of cortisol [66], suggesting a possible bi-directional relationship between this stress hormone and cancer. Future experiments will focus on identifying the effects of our stress paradigm on different players within the HPG axis.

The broad molecular functions and biological processes affected by the genes that displayed significantly altered expression levels in our study underlie the observation that stress can affect a multitude of cellular functions that can ultimately lead to increased cell proliferation, including apoptosis, mitogenic signaling, extracellular matrix, DNA repair, and altered bioenergetics. This is not surprising, as cancer is a highly complex, multistep process that requires cells to acquire specific traits in order to proliferate and evolve into a malignant phenotype, and though it is unlikely that stress alone can provide cells with such specific characteristics, it is certainly possible that, given the progressive nature of cancer, stress can facilitate deregulation of certain pro- and anti-proliferative cellular processes [6, 67]. Interestingly, our results showed that restraint stress upregulated the expression of genes involved in the activation of apoptotic pathways (i.e. caspases 2, 7, and 9) and are thus associated with anti-proliferative activity. Recent studies have suggested that psychological stress can promote prostate carcinogenesis in mouse xenograft models via inhibition of apoptosis [68, 69]. While this finding appears to conflict with our gene expression data, it is also possible that our observed increase in the expression of pro-apoptotic genes could be a compensatory response by the prostate cells since we also observed an upregulation of genes that promote cell proliferation (i.e. cyclin D3, Src, MAPK). In fact, prostate tissue is known to be very sensitive to signals that influence tumorigenic growth, and therefore maintains a cell death/proliferation equilibrium [70, 71].

Of noteworthy importance is the upregulated expression of several genes identified as proto-oncogenes, including genes with very well characterized roles in cancer progression such as Map2k1 and Src. Importantly, the expression of tumor protein p53, one of the most characterized tumor suppressor genes, was found to be downregulated in accordance with its cellular role in cancer where inactivation of this gene is sufficient to predispose individuals to cancer [72, 73]. Moreover, chronic stress has been found to promote tumorigenesis in a p53 genetic mouse model of cancer, with tumor formation depending on glucocorticoid action [52].

The results of the present study illustrate the effects of emotional stress on prostate tissue gene expression. As shown by the functions and processes in which the affected genes participate, stress can influence a wide variety of cellular functions, and may be an early stimulus that promotes prostate cancer incidence or progression through modification by endocrine mediators. That recovery from stress is able to restore the expression of some genes, but not others, to normal levels provides support for future investigations into the importance of stress timing and duration in cancer diagnoses.