title: Sunitinib activates Axl signaling in renal cell cancer.
abstract: Mass spectrometry-based phosphoproteomics provides a unique unbiased approach to evaluate signaling network in cancer cells. The tyrosine kinase inhibitor sunitinib is registered as treatment for patients with renal cell cancer (RCC). We investigated the effect of sunitinib on tyrosine phosphorylation in RCC tumor cells to get more insight in its mechanism of action and thereby to find potential leads for combination treatment strategies. Sunitinib inhibitory concentrations of proliferation (IC50) of 786-O, 769-p and A498 RCC cells were determined by MTT-assays. Global tyrosine phosphorylation was measured by LC-MS/MS after immunoprecipitation with the antiphosphotyrosine antibody p-TYR-100. Phosphoproteomic profiling of 786-O cells yielded 1519 phosphopeptides, corresponding to 675 unique proteins including 57 different phosphorylated protein kinases. Compared to control, incubation with sunitinib at its IC50 of 2 µM resulted in downregulation of 86 phosphopeptides including CDK5, DYRK3, DYRK4, G6PD, PKM and LDH-A, while 94 phosphopeptides including Axl, FAK, EPHA2 and p38α were upregulated. Axl- (y702), FAK- (y576) and p38α (y182) upregulation was confirmed by Western Blot in 786-O and A498 cells. Subsequent proliferation assays revealed that inhibition of Axl with a small molecule inhibitor (R428) sensitized 786-O RCC cells and immortalized endothelial cells to sunitinib up to 3 fold. In conclusion, incubation with sunitinib of RCC cells causes significant upregulation of multiple phosphopeptides including Axl. Simultaneous inhibition of Axl improves the antitumor activity of sunitinib. We envision that evaluation of phosphoproteomic changes by TKI treatment enables identification of new targets for combination treatment strategies.
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.|Department of Hematology/Oncology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA.
authors.forename: Johannes C
authors.initials: JC
authors.lastname: van der Mijn
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Henk J
authors.initials: HJ
authors.lastname: Broxterman
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Jaco C
authors.initials: JC
authors.lastname: Knol
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Sander R
authors.initials: SR
authors.lastname: Piersma
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Richard R
authors.initials: RR
authors.lastname: De Haas
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Henk
authors.initials: H
authors.lastname: Dekker
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Thang V
authors.initials: TV
authors.lastname: Pham
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Victor W
authors.initials: VW
authors.lastname: Van Beusechem
authors.affiliation: Department of Hematology/Oncology, Columbia University Medical Center, New York, NY.
authors.forename: Balazs
authors.initials: B
authors.lastname: Halmos
authors.affiliation: Department of Hematology/Oncology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA.
authors.forename: James W
authors.initials: JW
authors.lastname: Mier
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Connie R
authors.initials: CR
authors.lastname: Jiménez
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Henk M W
authors.initials: HM
authors.lastname: Verheul
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D002292:Carcinoma, Renal Cell / Q000188:drug therapy / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival; D063990:Gene Ontology; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D007680:Kidney Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011758:Pyrroles / Q000494:pharmacology*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D000077210:Sunitinib; D000094963:Axl Receptor Tyrosine Kinase
chemical_list: D000970:Antineoplastic Agents; D007211:Indoles; D011518:Proto-Oncogene Proteins; D011758:Pyrroles; D020794:Receptor Protein-Tyrosine Kinases; D000077210:Sunitinib; D000094963:Axl Receptor Tyrosine Kinase; C000722289:AXL protein, human
keywords: AXL; FAK; phosphoproteomics; renal cell cancer; signaling; sunitinib