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 mM resulted in downregulation of 86 phosphopeptides including CDK5, DYRK3, DYRK4, G6PD, PKM and LDH-A, while 94 phosphopeptides including Axl, FAK, EPHA2 and p38a were upregulated. Axl-(y702), FAK-(y576) and p38a (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.

Kinase Inhibitor Treatment of Patients with Advanced Cancer Results in High Tumor Drug Concentrations and in Specific Alterations of the Tumor Phosphoproteome

“…Figure 3 shows the marginal overlap between the SOR, ERL, DAS and VEM treatment groups of Fc > 1.5 up-or downregulated phosphopeptides in ≥ 3 of 5 patients within each cohort, confirming that, even at a much less stringent selection, these 39-74 up-and 4-135 downregulated phosphopeptides were drug-specific. These data support preclinical evidence that at the concentrations we have measured (Table 2), these drugs do affect multiple kinases [5,22]. Analysis of Pearson's correlations between reference samples measured along with the drug cohorts on subsequent days indicated that the differences in regulated peptides between the drug cohorts could not be attributed to day to day variation or batch effects ( Figure S2).…”

“…The observation of multiple inhibitory effects is considered beneficial in terms of potential anticancer activity, but the observed upregulation of phosphorylated peptides may reflect stimulatory and pro-survival events, as for example visible in the networks of the SOR and VEM cohorts ( Figure S3). PKI-induced upregulation of phosphorylated proteins has been reported in in vitro experiments by Zhang et al and by ourselves [22,37]. These potential pro-survival signals support the search for alternative combination treatment strategies in order to circumvent these potential resistant mechanisms [38].…”

RETRACTED ARTICLE: LncRNA HOTAIR regulates HIF-1α/AXL signaling through inhibition of miR-217 in renal cell carcinoma

“…25, 26 In addition, AXL mediates a resistance to sunitinib treatment in Rcc. 27 AXL is highly expressed in Rcc tissues and cells, and knockdown of AXL reduces cell viability. 28 To evaluate whether HOTAIR regulates Rcc activity through HIF-1 α /AXL signaling, we detected AXL expression after gain- and loss-of-function of HOTAIR in Rcc cells.…”

Identification of mutations associated with acquired resistance to sunitinib in renal cell cancer

“…25 Incubation of 786-O, 769-p, and A-498 RCC cell lines with sunitinib downregulated 86 phosphopeptides including G6PD, while upregulating Axl signaling. 26 Our analysis showed only modest or no difference in G6PD levels between parental and sunitinib-desensitized RCC cells. Consistently, treatment of parental RCC cells was not associated with a significant change in G6PD levels, in contrast to the five other proteins, and G6PD depletion had the least effect on sensitizing RCC cells to sunitinib.…”