Abstract 140: Oncogenic signaling by MET and other cabozantinib targets in cells derived from urothelial carcinoma of the bladder
Authors: Lee Young H. , Wong Tiffany K. , Apolo Andrea B. , Agarwal Piyush K. , Bottaro Donald P. 
Published: August 2015

Abstract
Background: Mounting evidence supports MET as a target in urothelial carcinoma (UC) of the bladder. Activated MET can promote tumor growth and metastasis, as well as tumor angiogenesis by upregulating VEGF, and may play a role in UC pathogenesis. The effects of MET inhibitor cabozantinib on MET-driven UC cell growth, invasion and tumorigenicity were previously analyzed in biochemical, cell-based and animal models. Current known cabozantinib targets (MET, KDR, FLT3, KIT, RET, AXL and ROS) and possible targets based on structural similarity (MERTK, TYRO3, RON, RYK, CSF1R, ROR1, ROR2, ALK and LTK) were characterized in 13 UC-derived cell lines by determining the frequency and absolute level of mRNA expression, and ultimately, for targets where mRNA was present, encoded protein abundance, ligand expression and cabozantinib sensitivity.
Methods: The effects and the targets of cabozantinib on human UC-derived cell lines were studied in vitro. Cells at 80% confluence were serum deprived 16h, then left untreated or treated with hepatocyte growth factor (HGF) and/or cabozantinib prior to analysis of MET, phospho- (p)MET, pAKT1, total AKT1, pMAPK1/2 and total MAPK1/2 by two-site immunoassay and/or immunoblotting. Cabozantinib effects on basal and HGF-induced UC cell invasion and proliferation were measured. Gene mRNA abundance (absolute copy number) of cabozantinib targets was measured using real time PCR from the extracted RNAs of 13 UC cell lines.
Results: Basal pMET content was uniformly low, and was increased significantly by HGF; the latter was reversed by cabozantinib treatment. HGF-driven increases in pAKT1:AKT1 and pMAPK1/2:MAPK1/2 ratios in all cell lines were reversed by cabozantinib treatment, as were HGF-enhanced UC cell invasion, proliferation and tumorigenicity. UC cell lines displayed uniformly robust mRNA expression of MET, AXL, RYK, RON, CSF1R, ROR1 and ROR2, and frequent modest expression of RET and ROS, whereas KDR, ALK, KIT, LYK, TYRO3, MERTK and FLT3 showed little or no expression. MET mRNA and protein abundance were significantly positively correlated among these 13 UC cell lines.
Conclusions: UC cell MET content was higher with higher UC grade. HGF was not expressed in any UC cell line tested, suggesting that this pathway is predominantly paracrine in vivo. Added HGF stimulated activation of MET and known effectors, and enhanced invasion, growth rate and anchorage-independent growth; cabozantinib effectively reversed these HGF-driven effects. Based on receptor expression, cabozantinib is most likely acting through MET, AXL and ROS, and potentially through RYK, ROR1, ROR2, and/or RET, in UC cells, all of which have been implicated in oncogenesis in other human malignancies.
Note: This abstract was not presented at the meeting.
Citation Format: Young H. Lee, Tiffany K. Wong, Andrea B. Apolo, Piyush K. Agarwal, Donald P. Bottaro. Oncogenic signaling by MET and other cabozantinib targets in cells derived from urothelial carcinoma of the bladder. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 140. doi:10.1158/1538-7445.AM2015-140