Reduced mrna expression level of corticotropin-releasing hormone-binding protein is associated with aggressive human kidney cancer

The Corticotropin releasing factor (CRF) system in human includes all naturally occurring CRF peptide analogues namely Urocortin (Ucn) and Urocortin 3 (Ucn3), known as CRF counterparts in the periphery, the CRF receptors 1 and 2 (CRFR1 and CRFR2), and finally Corticotropin-releasing hormone-binding protein (CRHBP) [1‐4]. The existence and translation of Urocortin 2 (Ucn2) in human is still unclear. It has been shown that CRF analogues can inhibit tumor progression, can modulate proliferation and apoptosis, and can hinder angiogenesis by reduction of VEGF expression in vivo, through activation of CRF receptors, especially CRFR2 in different tumor entities [5‐8]. Expression and the pathophysiological relevance of the CRF system has been reviewed for different human cancers [9, 10]. Recently, we reported the expression of Ucn and CRFR2 in clear cell renal cell carcinoma (cc-RCC) [11]. In our study, a nuclear migration of Ucn and loss of expression of vascular CRFR2 in cc-RCC could be demonstrated [11]. Expression of CRHBP on mRNA level has been reported in human normal kidney [12] but there is still no data available about the expression of CRHBP in kidney cancer. Moreover, for other tumor entities it has been pointed out that expression patterns of the CRF system are related to grade and stage of tumors [10].

To assess a potential relevance of CRHBP expression alterations for cc-RCC we first compared the mRNA expression levels of CRHBP in cc-RCC fresh frozen specimens and paired normal appearing tissue samples using quantitative RT-PCR analysis. Moreover, relative mRNA expression levels were statistically evaluated for association with clinicopathological parameters of cc-RCC patients. Presence and localization of CRHBP protein expression in normal and malignant kidney tissues were investigated using immunohistochemistry and immunofluorescence.

Here we demonstrate that CRHBP expression is depleted both on mRNA and most likely also on protein level in cc-RCC. Moreover, we found that loss of CRHBP mRNA expression is correlated with advanced, metastasized and higher stages of disease. To our knowledge, tumor specific reduction of CRHBP expression has not been reported in any human malignancy so far, although results indicative of a decreased expression of corticotropin-releasing hormone-binding protein mRNA in pituitary adenomas have been described before [21]. On the other hand, various studies show that Ucn, CRFR1 and CRFR2 are differently regulated and expressed in human cancer [10, 11]. Moreover, several in vitro and in vivo studies reported that activation of CRFR2 suppresses the neovascularization through reduction of VEGF production. The loss of function, blocking or gene knock out of CRFR2 has been found to cause an up-regulation of VEGF in turn inducing neovascularization [5, 7, 8]. This is in good concordance with our previous finding that the CRFR2 protein is not expressed in vessels of cc-RCC [11], a cancer that is characterized by increased VEGF levels and substantial neovascularization. CRHBP is the only example of a neuro-peptide-binding protein that circulates in the plasma. CRHBP binds the Corticotropin –Releasing factor (CRF) and other CRF-family peptides, e.g. Urocortin (Ucn) with a high affinity but does not interact with Ucn3 [22, 23]. It has been hypothesized that extracellular CRHBP may serve as a ligand trap thus potentially be involved in regulation of the bound/free ligand equilibrium of locally required peptides and representing a potential pharmacological therapeutic target [24, 25]. Considering that ligand concentrations of locally required peptides have been shown to affect proliferation and angiogenesis of cancer cells, we suppose that the reduction of CRHBP mRNA and protein expression in tumors also contribute in those pivotal tumorigenic processes.

Immunopositivity was detected mainly in proximal tubular epithelial cells of normal tissue for both CRHBP itself in the present study as well as for the CRHBP ligand Ucn in a previous analysis [11], hence also supporting the hypothesis that CRHBP is involved in tumor specific alterations of the UCN system. Finally this point of view, is strengthened by our statistical findings revealing a significant relationship between reduced tumoral mRNA expression levels and the state of more aggressive tumors in cc-RCC. Considering that previous analyses support the relevance of the UCN system for human carcinogenesis including also renal cancer, we hypothesize that alteration of CRHBP as a putative modulator of UCN levels may also contribute to the development and course of cc-RCC.

Although we found reduction of both, mRNA levels and immunopositivity on protein levels in cc-RCC tissue compared to normal kidney, this study obviously does not allow functional implications about the role of CRHBP in cc-RCC. While a direct head to head association between mRNA and protein levels has not been shown in our study, it is known that epigenetic changes including altered expression of micro- or long non-coding RNA may effect protein expression after transcription. Such a candidate epigenetic effector is miR-200bc/429 which has already been annotated to the CRHBP gene (UCSC human genome browser) [26]. Therefore functional analyses such as targeted knock out of CRHBP gene in a cc-RCC tumor model or epigenetic characterization of the UCN system should provide an improved understanding of the CRHBP contribution to the pathogenesis of cc-RCC.

Interestingly, Ucn family peptides have also been detected in human circulation and urine [4, 27] while CRHBP expression beside those observed in normal tubules of the kidney is also found in podocytes of renal glomeruli. Whether Ucn and CRHBP levels in blood and urine become altered and possibly affect renal cancer progression or can serve as biomarker therefore appear as further relevant questions remaining to be clarified in future studies.

In conclusion our data give evidence that altered CRHBP expression as part of the UCN system is involved in kidney cancer. Our results underline the potential biological relevance of the CRF peptide family for renal cancer and may also be of clinical relevance for future diagnostic or molecular therapeutic approaches.