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 . 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 , 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 , 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) . 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.