Sunitinib activates Axl signaling in renal cell cancer

Int J Cancer. 2016 Jun 15;138(12):3002-10. doi: 10.1002/ijc.30022. Epub 2016 Mar 1.

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.

Keywords: AXL; FAK; phosphoproteomics; renal cell cancer; signaling; sunitinib.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents / pharmacology*
  • Carcinoma, Renal Cell / drug therapy
  • Carcinoma, Renal Cell / metabolism*
  • Cell Line, Tumor
  • Cell Survival
  • Gene Ontology
  • Human Umbilical Vein Endothelial Cells / metabolism
  • Humans
  • Indoles / pharmacology*
  • Inhibitory Concentration 50
  • Kidney Neoplasms / drug therapy
  • Kidney Neoplasms / metabolism*
  • Phosphorylation
  • Protein Processing, Post-Translational / drug effects
  • Proto-Oncogene Proteins / metabolism*
  • Pyrroles / pharmacology*
  • Receptor Protein-Tyrosine Kinases / metabolism*
  • Signal Transduction
  • Sunitinib

Substances

  • Antineoplastic Agents
  • Indoles
  • Proto-Oncogene Proteins
  • Pyrroles
  • Receptor Protein-Tyrosine Kinases
  • axl receptor tyrosine kinase
  • Sunitinib