Tobias Haber

Bone Metastasis in Renal Cell Carcinoma is Preprogrammed in the Primary Tumor and Caused by AKT and Integrin α5 Signaling

PURPOSE Bone metastasis develops in 30% of all patients with renal cell carcinoma. We elucidated the mechanisms that lead to and predict bone metastasis of renal cell carcinoma. MATERIALS AND METHODS Nine renal cell carcinoma primary cell lines and 30 renal cell carcinoma tissue specimens (normal and tumor tissue) were collected from 3 patients with no metastasis and 10 with lung or bone metastasis within 5 years after nephrectomy. Cell migration was analyzed in a Boyden chamber and proliferation was assessed by bromodeoxyuridine incorporation. Adhesion to fibronectin, and collagen I and IV was determined after cell staining. The expression and/or activity of cellular signaling molecules was quantified by Western blot. RESULTS Compared to renal cell carcinoma cells from patients without metastasis, the migration of cells from patients with bone metastasis was enhanced 13.5-fold (p = 0.034), and adhesion to fibronectin and collagen I was enhanced 5.8-fold and 6.1-fold (p = 0.002 and 0.014, respectively). In general proliferation was decreased in metastasizing cells. In accordance with these results we detected higher activity of AKT (p = 0.011) and FAK (p = 0.054), higher integrin α5 expression (p = 0.052) and lower PTEN expression in primary cells from patients with bone metastasis compared to nonmetastasizing cells. An almost similarly altered expression pattern was also observed in the renal cell carcinoma tissue specimens and the normal renal tissue of patients with bone metastasis. CONCLUSIONS 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.

Overriding TKI resistance of renal cell carcinoma by combination therapy with IL-6 receptor blockade

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.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.

Calcium-sensing receptor (CaSR) promotes development of bone metastasis in renal cell carcinoma

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.

MP60-07 THE CALCIUM-SENSING RECEPTOR (CASR) IS RESPONSIBLE FOR THE DEVELOPMENT OF BONE METASTASIS IN RENAL CANCER

INTRODUCTION AND OBJECTIVES: We previously reported that high MET and matriptase expression in RCC cells in bone metastasis indicates their importance in bone metastasis (Mukai et al. Hum Cell, 2015). MET is a high-affinity receptor tyrosine kinase of hepatocyte growth factor (HGF). HGF is secreted as an inactive singlechain precursor, which requires proteolytic activation for conversion to an active form. Matriptase is the most efficient known cellular activator of pro-HGF. Furthermore, activation of matriptase is regulated by HGF activator inhibitor (HAI). In this study, we employed a previously reported mouse model of bone metastasis (Strube et al. Clin Exp Metastasis, 2010) to clarify the significance of the matriptase-induced HGF/MET signaling axis in RCC bone metastasis. METHODS: Luciferase-transfected 786-O cells were injected into the left cardiac ventricle of female nude mice (5 weeks old). After 6 weeks, we confirmed the formation of bone metastasis by whole-body bioluminescent imaging, and extracted specimens. Expression of matriptase, MET and HAI was analyzed by PCR, immunohistochemistry (IHC) and immunoblots. Phosphorylation of MET was also investigated. Based on the result, we produced HAI-2 (specific inhibitor of matriptase) stable knock down (KD) 786-O cells, and analyzed the difference of expression in each molecule, cell-migration assay and invasion assay. RESULTS: Expression of matriptase was increased significantly in bone metastasis compared with parent cell line, and we confirmed increased phosphorylation of MET in bone metastasis. On the other hand, decreased expression of HAI-2 was observed in bone metastasis. Interestingly, increased matriptase expression was observed by HAI-2 KD in 786-O cells. In addition, invasive activity was increased significantly by knock down of HAI-2. CONCLUSIONS: These results have suggested that matriptase contributes to the HGF-dependent MET activation in the pericellular microenvironment of bone metastasis in RCC. In addition, upregulation of matriptase and downregulation of HAI-2 may have important roles in their progression.

Abstract 37: Bone metastases in renal cell carcinoma are predicted by characteristics of the primary tumor

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: The poor prognosis of renal cell carcinoma (RCC) is caused by a high risk of metastasis. 30 % of all RCC patients develop bone metastases during course of disease. The outcome of RCC patients with bone metastases is poor so that a prediction would be of advantage. The aim of this study was to elucidate the mechanisms that lead to an organ specific metastasis of RCC in bones and probably predict bone metastasis. Methods: In 30 RCC tissue specimens and 9 RCC primary cell lines collected from patients who had developed no metastases, lung or bone metastases within 5 years after nephrectomy (each 10 RCC tissue specimens, each 3 RCC cell lines) expression and/or activity of 46 cellular signalling molecules were quantified by a phospho kinase array and Western blot analyses. As steps of metastasis, migration of the primary RCC cells was analysed in a Boyden chamber with 10 µg/ml fibronectin as chemotaxin, adhesion to extracellular matrix compounds fibronectin, collagen I and IV was determined after cell staining with crystal violet. Results: In RCC tissue specimens and primary cells from patients who developed bone metastases a higher expression of alpha5 integrins, higher activity of Akt and FAK as well as a lower expression of PTEN was detected, compared to those from patients without or with lung metastases. According to these results we observed an enhanced chemotactic migration (13-fold) of bone metastatic RCC cells and adhesion to fibronectin (6-fold) or collagen type I (8-fold) - both components of the bone matrix. Conclusion: These results show that specific characteristics like the expression and activity of cell signalling mediators and the cellular behaviour of the primary tumor determine the location of the subsequent metastasis in RCC and could be used as prognostic marker for bone metastasis. This should be considered during follow-up care. Funded by the Wilhelm Sander Foundation Citation Format: Elke Schneider, Tobias Haber, Frederick C. Roos, Christian Hampel, Kerstin Junker, Joachim W. Thuroff, Walburgis Brenner. Bone metastases in renal cell carcinoma are predicted by characteristics of the primary tumor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 37. doi:10.1158/1538-7445.AM2014-37

616 CHARACTERISTICS OF PRIMARY TUMOR MAY PREDICT BONE METASTASIS IN RENAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVES: The poor prognosis of renal cell carcinoma (RCC) is caused by a high risk of metastasis. 30% of RCC patients develop bone metastases during the course of disease. A method of predicting bone metastasis would benefit the otherwise poor outcome of this patient group. The aim of this study was to elucidate the mechanisms that lead to organ-specific metastasis of RCC in bones and thereby predict bone metastasis. METHODS: In 30 RCC tissue specimens and 9 RCC primary cell lines collected from patients who had developed no metastases, lung or bone metastases within 5 years after nephrectomy (each 10 RCC tissue specimens, each 3 RCC cell lines) expression and/or activity of 46 cellular signaling molecules were quantified by phosphokinase array and Western blot analyses. To investigate metastatic behavior, migration of the primary RCC cells was analysed in a Boyden chamber with 10 g/ml fibronectin as chemotaxin, and adhesion to extracellular matrix compounds fibronectin, collagen I and IV was determined after cell staining with crystal violet. RESULTS: In RCC tissue specimens and primary cells from patients who developed bone metastases, a higher expression of 5 integrins, higher activity of Akt and FAK and a lower expression of PTEN were detected, compared to those from patients without or with lung metastases. These results show an enhanced chemotactic migration (13-fold) of bone metastatic RCC cells and adhesion to fibronectin (6-fold) or collagen type I (8-fold) both components of the bone matrix. CONCLUSIONS: Specific characteristics such as expression and activity of cell signaling mediators and cellular behavior of the primary tumor determine the location of subsequent metastasis in RCC and could be used as a prognostic marker for bone metastasis. This should be considered during follow-up care.