Abstract A68: Tumor cell dedifferentiation into endothelial cells and resistance to sunitinib in renal carcinoma models

Abstract

Abstract Background: Acquired resistance to multikinase inhibitors stands as a major clinical challenge. Treatment with sunitinib was shown to strikingly reduce the tumor blood flow, which in turn induces cellular starvation, hypoxia, and necrosis. When angiogenesis inhibition is maintained, cancer cells and other cells in the stroma may adapt to recover from the primary stress to allow continued perfusion and tumor growth that is less reliant on VEGF/VEGFR signaling. Clinical evidence has suggested that resistance to VEGFR-targeted agents was mediated through changes in both cancer cells and the microenvironment. The aim of present study was to study in vitro and in vivo the molecular mechanisms of acquired resistance to sunitinib in RCC models. Materials and Methods: CAKI1 (VHL+) and 786-0 (VHL−) renal carcinoma cells were exposed to stepwise increasing doses of sunitinib for >6 months. Invasiveness of cells was determined by Matrigel invasion assay. qRT-PCR, western blot and IHC assays were used to assess a panel of genes and proteins. To compare the molecular mechanisms of sunitinib in vitro and in vivo, CAKI1 cells were engrafted in nude mice and treated continuously with a tumoristatic dose of 60 mg/kg oral sunitinib until progression. Results: CAKI-Suni, 786-Suni cells were 2-fold more resistant to sunitinib than parental cells. Several changes in gene expressions were detected in cells acquiring resistance to sunitinib. Protracted exposure to sunitinib led to a >3-fold increase of ErbB3 and CDH1 in CAKI-Suni and 786-Suni as compared to parental CAKI1 and 786-0 cells. Resistance to sunitinib was associated also with AKT and ERK activation. In addition, CAKI-Suni cells were at least 3-fold more invasive than their parental counterpart. In vivo, sunitinib induced significant tumor growth retardation with median PFS of 59 against 22 days for placebo (p<0.0001). After median of 35 days several tumors became progressive under sunitinib. IHC analysis of tumors revealed less necrosis and more blood vessels in sunitinib resistant as compared to sunitinib-sensitive tumors. Two-fold increased mRNA expression of VEGFA, CXCR4, CA9 and HIF1A and 4-fold decreased expression of E-cadherin were found in progressive tumors. CXCR4, vimentin and ErbB3 protein levels were higher in progressive tumors than in tumors responding to sunitinib. Using human and mouse primers for mRNA expression of CD31, we suggested that human cancer cells may differentiate into endothelial cells at progression. Tumor cells resistant to sunitinib situated in tumor vessels co-expressed both human renal cell carcinoma marker CD10 and CD31/Von Willebrand using immunostaining. Conclusions: Sustained exposure to sunitinib induces changes in gene expression, invasion properties and survival signaling pathways in RCC cells. In CAKI1 xenograft tumor cells that become insensitive to sunitinib may be found in tumor vessels and display markers of endothelial cells.