Introduction
The corticotropin-releasing hormone (CRH) pathway, primarily described as the major neuroendocrine pathway modulating stress-related response through the hypothalamic–pituitary–adrenal (HPA) axis, consists of CRH, urocortin (UCN or UCN1), UCN2, and UCN3. These peptides act through activation of two G-protein-coupled receptors with different specificities and effects—CRHR1 and CRHR2. The corresponding binding protein CRHBP to which a regulatory function is attributed binds CRH analogues and serves as a ligand trap in the CRH system [
1‐
7]. It has been shown that CRH analogues inhibit tumor growth via effects on angiogenesis by reduction of vascular endothelial growth factor (VEGF) synthesis and cell proliferation through activation of CRH receptors in different tumor entities, especially by activation of CRHR2 [
8‐
11]. Expression and pathophysiological implication of the CRH pathway in several human cancers have been reviewed lately [
11,
12]. Recently, we reported the translocation of UCN and reduction of CRHR2 in clear cell renal cell carcinoma (ccRCC) [
5]. UCN3 in human kidney and its presence in serum and urine have also been described, but with approximately 5-fold higher urine levels than that of plasma and higher urine levels compared to other vasoactive peptides found in urine. This supports the hypothesis of renal tubular cells as the origin of UCN3 [
2,
4]. Up to now, there are no comparable data available about the gene expression of
UCN3 in RCC and its role in renal cancer biology, although alterations of other members of the CRH peptide family such as UCN, CRHBP, or CRHR2 have already been reported. Kaprara et al. reviewed literature showing that different tumor entities, e.g., prostate, thyroid, lung, breast, and gastrointestinal tumors, exhibit different expression patterns of members of the CRH system depending on tumor grading and staging [
12]. Therefore, and in order to extend the understanding of the CRH peptide family and its influence on the tumor biology of RCC, another member of the CRH system, UCN3, was investigated in this study. We examined the expression of
UCN3 in 106 human kidney specimens and available corresponding benign peritumoral specimens by qPCR analysis. Protein signals were examined by western blot analysis. Tissue localization was investigated by immunohistochemistry. Moreover, a statistical correlation analysis between mRNA expression of
UCN3 in kidney cancer with clinicopathological parameters was performed.
Discussion
Here and for the first time a kidney cancer-specific downregulation of UCN3, which is a selective CRHR2 agonist [
2], was demonstrated using the example of the two most common histological subtypes—ccRCC and pRCC.
UCN3 mRNA was significantly downregulated in comparison to adjacent normal renal tissue. Expression loss of
UCN3 showed no correlation with clinicopathological parameters of this cohort. The UCN3 protein was located in renal tubular cells of normal kidneys, particularly in distal tubules, whereas no immunohistochemical signals were detected in ccRCC.
Various studies have shown that members of the CRH peptide family are regulated and presented differently in a large number of human cancer entities [
5,
12,
14]. Recently, we demonstrated that CRHR2, which is the only receptor known to match UCN3 [
2], is not available in the vasculature of ccRCC [
5]. Those findings were in accordance with recent knowledge from in vivo and in vitro studies showing that activation of CRHR2 suppresses the neovascularization through reduction of vascular endothelial growth factor (VEGF) production in an upstream regulatory way [
8,
10,
11]. Excessive expression of VEGF and VEGF receptors in ccRCC has been reported frequently, which is in accordance with its histological architecture. Furthermore, a significant positive correlation between higher expression of VEGF and advanced disease stages of RCC (pT3–4 and/or N+/M+) was observed [
18].
Hsu and Hsueh introduced UCN3 as a CRHR2-selective agonist with no affinity to CRHR1 and were the first to show its mRNA expression in human kidney [
2]. Later, Takahashi et al. succeeded in detecting the UCN3 protein in normal human kidney, plasma, and urine [
4]. Presence of UCN3 in plasma and its higher level in urine prove a regulatory role for this peptide in tubular reabsorption and renal circulation. It presumes that this peptide may have a role as a circulating factor in humans [
4].
A concomitant loss at genetic, protein, and immunohistochemical levels, which were demonstrated in the present study, and the disruption of CRHR2 in ccRCC, which was observed by our group lately [
5], corresponds to the angiogenic nature of ccRCC known as a hypervascularized tumor—and to the inhibitory function of UCN3 in neovascularization through activation of CRHR2. Moreover, and as described before, activation of CRHR2 by its agonists inhibits the malignant cell proliferation in kidney cancer and other malignancies as well [
8‐
10]. Wang et al. inhibited growth of hepatocellular carcinoma by suppressing angiogenesis via UCN/CRHR2 activation in its vessels. Use of a CRHR2 antagonist reversed angiogenesis inhibition. Consequently, the role of CRHR2 in angiogenesis was strengthened and confirmed [
11]. However, in inflammatory vasculature Im et al. propagated diverging effects of CRHR1 and CRHR2 on intestinal angiogenesis. While CRH/CRHR1 promoted inflammatory vascular growth, UCN3/CRHR2 prevented these processes [
19,
20]. The extent to which these results can be directly transferred to the tumor biology of carcinomas still needs to be investigated. Several causal relationships have already been reported: Grivennikov et al. reviewed the interaction between inflammation and tumor and reported its critical role in carcinogenesis [
21]. Anand et al. confirmed that most tumors are caused or promoted mainly by lifestyle habits and environment; in the overall context, only a small proportion of tumors are linked to hereditary mutations [
22]. These lifestyle factors include smoking, nutrition [
23], obesity [
24], infection [
25], and environment [
26]. Aggarwal et al. identified chronic inflammation as a common characteristic of all these factors [
27]. Especially smoking and obesity, which can also maintain chronic inflammation, have been identified in numerous studies as risk factors for the development of renal cancer and have therefore been included as major risk factors in the German and European guidelines for renal cancer [
28‐
40]. A connection between inflammation and the development of cancer, also in the case of renal cancer, seems to be obvious, especially since the molecular mechanisms that apparently promote or prevent tumor development also maintain or prevent chronic inflammations, in this case various members of the CRH system. Recently, a significantly higher expression of
CRH and
CRHR1 mRNA was detected in human colon cancer cell lines compared to an immortalized colonic epithelial line [
41]. Fang et al. linked their results to the known involvement of CRH in inflammation by promoting VEGF and other cytokines via CRHR1 activation [
42‐
44] and proclaimed a critical role for the CRH/CRHR1 signaling pathway in cell proliferation and tumorigenesis of human colon cancer [
41]. Interestingly, there was a significantly lower mRNA expression of
UCN in all three tumor cell lines compared to the normal tissue tested, although immunohistochemically a higher staining of UCN in tumorous tissue compared to normal and peritumoral tissue was detected as well [
41]. It is known that CRH binds to CRHR1 with an affinity that is approximately 20-fold higher than that of CRHR2 [
41,
45,
46]. In contrast, UCN’s receptor affinity is distributed equally between CRHR1 and CRHR2 [
1,
47‐
49]. To what extent there is a difference, whether CRH or UCN binds to CRHR1, and what role CRHBP, which may act as a key regulator and is possibly involved in post-translational and post-secretional modifications or even subcellular translocation of these ligands, remains to be investigated [
5‐
7,
50,
51].
In carcinogenesis of ccRCC, a disturbance of the CRH system may occur at receptor and ligand levels, which has been shown in the present study by downregulation of
UCN3 mRNA and its corresponding protein signal loss in ccRCC—and by the disruption of its receptor CRHR2, which was shown in our previous studies [
5]. The point at which these changes contribute to the promotion of tumor metabolism is not yet known.
Members of the CRH peptide family have been detected in human blood and urine [
4]. A significant lower UCN3 level in urine or blood serum compared to physiological levels might serve as a potential biomarker for early diagnosis of RCC, especially when radiological differentiation to benign renal tissue is critical and biopsy takings, e.g., via surgical or interventional approach, due to patients’ health conditions are limited. So far, its involvement in tumor progression according to the correlation analysis in the present study, where there was no correlation between
UCN3 mRNA expression level and clinicopathological parameters, seems to be of lesser significance. This, in turn, could be an indication of its involvement in tumorigenesis and emphasize its use as a potential biomarker for screening and early diagnosis in contrast to our last results, where a negative correlation between
CRHBP mRNA levels in RCC and clinicopathological parameters—meaning tumor progression—such as advanced, metastasized, and higher stages of RCC was observed [
14]. Recently, a statistical association between hypermethylation of CRHBP in RCC and more aggressive tumors was detected [
52]. These epigenetic changes may influence the functional presence of CRHBP and could consecutively alter the receptor effects of CRHR1 and its corresponding ligands CRH and UCN. Thus, interactions on epigenetic levels, e.g., by epigenetic silencing of CRHBP, may also be involved in the carcinogenesis of RCC [
52]. It suggests that different members of the CRH system may act at specific points in the tumor biology of RCC. While some of the peptides are more involved in tumorigenesis, others seem to play remarkable roles in the progression of the disease. Of course, confirmatory studies are necessary to support this hypothesis. In the present study and for the first time, cancer-specific losses of
UCN3 mRNA levels and UCN3 protein signals in RCC were demonstrated. These findings support the hypothesis of the involvement of UCN3 in neovascularization and promotion of renal cancer. A re-induction of CRHR2—already mentioned in our previous studies [
5]—and an additional re-induction of the corresponding ligand UCN3, which—based on the now available results of the present study—is nearly lost in ccRCC, could be a possible therapeutic approach in the suppression of vascularization in ccRCC. In Lewis lung carcinoma (LLC) cells these tumor-suppressing effects have already been observed. UCN2/CRHR2 suppressed tumor vascularization and direct cell proliferation [
10]. Another translational link by comparison of UCN3 urine and blood levels of patients with questionable malignancy of renal tumors and confirmed histopathological diagnosis of RCC is of further interest and will be tested soon.
Acknowledgements
We gratefully thank Ms. M. Hepke for her technical support.