Target identification by phosphoproteomics: RIN1 modulation of sorafenib-induced cytotoxicity in renal cell carcinoma

Abstract

e14543 Background: Sorafenib (SFB) is a multi-tyrosine kinase inhibitor clinically useful in treatment of metastatic renal cancer. While the inhibition of angiogenesis is considered a major mechanism of action, identification of targets regulating growth inhibitory effects of SFB is necessary to further improve its efficacy and reduce toxicity. Methods: In this study we used targeted phosphoproteomics to identify tyrosine phosphorylated proteins that are differentially affected in control and SFB-treated human CAKI-1 renal cell carcinoma cells. The strategy involved immunoaffinity isolation of phosphotyrosine containing proteins and liquid chromatography - tandem mass spectrometry (MS) for identification of candidate proteins. Results: Among identified proteins, signal transducer and activator of transcription 1 (STAT1) and Ras and Rab interactor 1 (RIN1) were found to be hypophosphorylated in SFB-treated compared to untreated CAKI-1 cells based on quantitative MS analysis, by peptide counts and native peptide reference method. A ∼4-fold decrease in expression and phosphorylation of STAT1 was observed in cells treated with 10 μM SFB for 48h. Up to 8-fold SFB dose-dependent (5–15 μM) decrease in phosphorylation of RIN1 at tyrosine 36, but not in total RIN1 expression, was observed. Similar effects on decreased phosphorylation of STAT1 and RIN1 were also observed in 786-O renal cell carcinoma treated with SFB. Hypophosphorylation of RIN1 at tyrosine 36 was observed in CAKI-1 cells treated with 5 μM sunitinib but not with imatinib (≤ 10 μM). Treatment of CAKI-1 cells with RIN1 targeted, but not control si-RNA led to down-regulation of RIN1 expression and attenuation of antiproliferative effects of SFB. Notably, ∼2-fold higher expression of RIN1 protein (total and phosphorylated) was observed in CAKI-1 cells selected for resistance following continuous exposure to 7.5 μM SFB. However, unlike parent CAKI-1 cells, prolonged exposure of these SFB-resistant CAKI-1 cells to 7.5 μM SFB did not completely abrogate phosphorylation of RIN1 at tyrosine 36. Conclusions: These results demonstrate that RIN1, a Ras effector protein with multiple biochemical functions, is a target for the anti-tumor effects of SFB in kidney cancer cells. [Table: see text]