BackgroundThe prognosis for renal cell carcinoma (RCC) is related to a high rate of metastasis, including 30% of bone metastasis. Characteristic for bone tissue is a high concentration of calcium ions. In this study, we show a promoting effect of an enhanced extracellular calcium concentration on mechanisms of bone metastasis via the calcium-sensing receptor (CaSR) and its downstream signaling molecules.MethodsOur analyses were performed using 33 (11/category) matched specimens of normal and tumor tissue and 9 (3/category) primary cells derived from RCC patients of the 3 categories: non-metastasized, metastasized into the lung and metastasized into bones during a five-year period after nephrectomy. Expression of CaSR was determined by RT-PCR, Western blot analyses and flow cytometry, respectively. Cells were treated by calcium and the CaSR inhibitor NPS 2143. Cell migration was measured in a Boyden chamber with calcium (10 μM) as chemotaxin and proliferation by BrdU incorporation. The activity of intracellular signaling mediators was quantified by a phospho-kinase array and Western blot.ResultsThe expression of CaSR was highest in specimens and cells of patients with bone metastases. Calcium treatment induced an increased migration (19-fold) and proliferation (2.3-fold) exclusively in RCC cells from patients with bone metastases. The CaSR inhibitor NPS 2143 elucidated the role of CaSR on the calcium-dependent effects. After treatment with calcium, the activity of AKT, PLCγ-1, p38α and JNK was clearly enhanced and PTEN expression was almost completely abolished in bone metastasizing RCC cells.ConclusionsOur results indicate a promoting effect of extracellular calcium on cell migration and proliferation of bone metastasizing RCC cells via highly expressed CaSR and its downstream signaling pathways. Consequently, CaSR may be regarded as a new prognostic marker predicting RCC bone metastasis.

Metastatic renal cell carcinoma (RCC) is a tumor entity with poor prognosis due to limited therapy options. Tyrosine kinase inhibitors (TKI) represent the standard of care for RCCs, however a significant proportion of RCC patients develop resistance to this therapy. Interleukin-6 (IL-6) is considered to be associated with poor prognosis in RCCs. We therefore hypothesized that TKI resistance and IL-6 secretion are causally connected. We first analyzed IL-6 expression after TKI treatment in RCC cells and RCC tumor specimens. Cell proliferation and signal transduction activity were then quantified after co-treatment with tocilizumab, an IL-6R inhibitor, in vitro and in vivo. 786-O RCC cells secrete high IL-6 levels after low dose stimulation with the TKIs sorafenib, sunitinib and pazopanib, inducing activation of AKT-mTOR pathway, NFκB, HIF-2α and VEGF expression. Tocilizumab neutralizes the AKT-mTOR pathway activation and results in reduced proliferation. Using a mouse xenograft model we can show that a combination therapy with tocilizumab and low dosage of sorafenib suppresses 786-O tumor growth, reduces AKT-mTOR pathway and inhibits angiogenesis in vivo more efficient than sorafenib alone. Furthermore FDG-PET imaging detected early decrease of maximum standardized uptake values prior to extended central necrosis.Our findings suggest that a combination therapy of IL-6R inhibitors and TKIs may represent a novel therapeutic approach for RCC treatment.

We describe evidence that molecular predispositions determine the potential for bone metastasis to develop in renal cell carcinoma, which may serve as prognostic markers after initial tumor detection.

Bone metastasis is an important prognostic factor in renal cell carcinoma (RCC). The calcium-sensing receptor (CaSR) has been associated with bone metastasis in several different malignancies. We analyzed the impact of CaSR in bone metastasis in RCC in vitro and in vivo. The RCC cell line 786-O was stably transfected with the CaSR gene and treated with calcium alone or in combination with the CaSR antagonist NPS2143. Afterwards migration, adhesion, proliferation and prominent signaling molecules were analyzed. Calcium treated CaSR-transfected 768-O cells showed an increased adhesion to endothelial cells and the extracellular matrix components fibronectin and collagen I, but not to collagen IV. The chemotactic cell migration and proliferation was also induced by calcium. The activity of SHC, AKT, ERK, P90RSK and JNK were enhanced after calcium treatment of CaSR-transfected cells. These effects were abolished by NPS2143. Development of bone metastasis was evaluated in vivo in a mouse model. Intracardiac injection of CaSR-transfected 768-O cells showed an increased rate of bone metastasis. The results indicate CaSR as an important component in the mechanism of bone metastasis in RCC. Therefore, targeting CaSR might be beneficial in patients with bone metastatic RCC with a high CaSR expression.

Bone tissue is one of the main locations of metastases in renal cell carcinoma (RCC). In bone tissue the concentration of calcium ions is very high. Cells recognize extracellular calcium by the calcium-sensing receptor (CaSR). To investigate the role of calcium in bone metastases, the CaSR was quantified in tumor tissue and primary tumor cells of patients who were free of metastases or developed bone or lung metastases during a time period of 5 years after nephrectomy. In tissue specimens and primary cells of patients developing bone metastases, CaSR expression was clearly enhanced. Functionally, analyses showed a higher sensitivity in bone metastasizing cells concerning proliferation and chemotactical migration. These effects were caused by enhanced activity of the downstream targets of CaSR, namely AKT, PLCg, JNK and p38, analyzed in a phospho-kinase array and western blot analysis. The extent to which CaSR is suitable as a new marker for bone-specific metastases from renal cancer must be examined further.

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INTRODUCTION AND OBJECTIVES: Abnormal chromosome segregation during mitosis causes aneuploidy, a hallmark of cancers associated with high risk for tumorigenesis. Mitotic checkpoint, or spindle assembly checkpoint prevents missegregation of chromosomes by arresting cells in metaphase until all the chromosomes are properly aligned. Mps1 kinase activity is essential for spindle checkpoint signaling. High levels of Mps1 serine/threonine kinase are found in colon cancer tissues and several cancer cell line. We found that Mps1 is over-expressed in ccRCC. We have further shown that Mps1 requires the molecular chaperone Hsp90 for its activity. Hsp90 protects an array of mutated, over-expressed and/or chimeric kinases that are vital for tumor growth and survival. The Hsp90 inhibitors are currently undergoing evaluation in human trials. We have previously shown that Hsp90 is a post-translationally modified protein and this regulates its chaperone activity. We hypothesize that Mps1 kinase targets and phosphorylates Hsp90 and therefore regulates its chaperone activity. This in turn impacts the tumor selectivity of Hsp90 inhibitors.METHODS: Functional genomics and quantitative proteomic (stable isotope labeling by amino acids in cell culture) was used to identify the Mps1 kinase that targets and phosphorylates Hsp90. ccRCC and adjacent normal tissue was obtained with written informed consent through the Upstate Department of Urology. 8-mm core of tissue was dissected into 3mm pieces and cultured in 24-well plates containing 1 mL RPMI-1640 with 10% FBS, antibiotic/antimycotic solution. Tissues were cultured at 37 C for 24 hours with or without Mps1 inhibitor NMS-P715 and for further 24 hours in the presence of fluorescently labeled Hsp90 inhibitor ganetespib (FL-ganetespib), then formalin-fixed and paraffin embedded. The tissues were immunostained for Hsp90.RESULTS: Mps1 kinase is over-expressed in ccRCC compared to normal adjacent tissue. Mps1 targets and phosphorylates T115 in the molecular chaperone Hsp90 and this regulates the chaperone activity. Ex vivo data showed that FL-ganetespib selectively accumulates in ccRCC and not in the normal tissue. Pharmacologic inhibition of Mps1 reverts this drug uptake by ccRCC. CONCLUSIONS: Mps1 protein kinase is an Hsp90 client and it is involved in post-translational regulation of the chaperone function. Over-expression of Mps1 in the ccRCC hyperphosphorylates Hsp90 and this is the molecular basis of the tumor selectivity of Hsp90 inhibitors.Source of Funding: This work was supported by SUNY INTRODUCTION AND OBJECTIVES: Tyrosine-kinase inhibitors (TKI) treatment targeted at the vascular endothelial growth factor (VEGF) pathways represents the standard of care in advanced renal cell carcinoma (RCC). Although the TKI treatment has promising effect against RCC, the development of resistance to TKIs should be solved. We have been reported that TKI treatment induces IL-6 on RCC cells. We investigated the effect of TKI in combination with IL-6 receptor antibody against RCC.METHODS: Human ren...