Anti-proliferative effects of sorafenib in clear cell renal cell carcinoma (CCRCC) cell lines: Relationship to von Hippel Lindau protein (pVHL) expression and hypoxia

Abstract

4601 Background: Sorafenib, a multikinase inhibitor targeting raf kinase and several receptor tyrosine kinases including VEGFR-2 and PDGFR, has demonstrated clinical activity in the treatment of metastatic CCRCC. To further characterize the molecular basis of response to this agent, we tested the functional role of pVHL and hypoxia in the anti-tumor activity of sorafenib in a panel of CCRCC cell lines. Methods: CCRCC cell lines CAKI-1 (wild-type pVHL), CAKI-2 (mutant VHL) and the isogenic pair, 786–0-neo (vector control) and 786–0-VHL (pVHL expressing) cells were treated with sorafenib (2.5 - 20 μM) for 24 - 96 hours at 37°C in a atmosphere of either normoxia (21% oxygen) or hypoxia (1% oxygen), 5% carbon dioxide and remainder nitrogen. Gene expression analysis of control and sorafenib treated CCRCC cell lines was carried out using a custom cancer cDNA array containing probes to 9240 cDNA clones corresponding to 4900 Unigenes or unclustered ESTs relevant to cancer or kidney development. Results: Anti-proliferative effects of sorafenib were both concentration and time of treatment dependent; the IC50 of sorafenib (inhibitory concentration for 50% cell kill) ranged from 7.5-10 μM and required continuous treatment for at least 48-72 hours for maximal cell kill. In contrast to CAKI-2 and 786–0-neo cells harboring mutant VHL, both CAKI-1 and 786–0-VHL cells expressing wild type VHL were ∼2-fold more resistant to the anti-proliferative effects of sorfaenib when treated under hypoxic conditions. No difference in resistance was observed under normoxic conditions. In contrast to 786–0-neo cells, there were ∼40 genes overexpressed (>5-fold) under hypoxia in control 786–0-VHL cells, that were predominantly related to anti-apoptosis (e.g. bcl2-associated athanogene) or angiogenesis (e.g. PDGF beta). Bcl2-associated athanogene and PDGF beta were down regulated >2-fold in hypoxia with sorafenib treatment. Conclusions: CCRCC with wild type VHL under hypoxic conditions can promote overexpression of anti-apoptotic and angiogenesis genes that attenuate the anti-proliferative effects of sorafenib. pVHL and hypoxia related genes may be important biomarkers for assessing the clinical efficacy of sorafenib in CCRCC. [Table: see text]