Metabolomic Analysis to Elucidate Mechanisms of Sunitinib Resistance in Renal Cell Carcinoma

Abstract

Metabolomics analysis possibly identifies new therapeutic targets in treatment resistance by measuring changes in metabolites accompanying cancer progression. We previously conducted a global metabolomics (G-Met) study of renal cell carcinoma (RCC) and identified metabolites that may be involved in sunitinib resistance in RCC. Here, we aimed to elucidate possible mechanisms of sunitinib resistance in RCC through intracellular metabolites. We established sunitinib-resistant and control RCC cell lines from tumor tissues of RCC cell (786-O)-injected mice. We also quantified characteristic metabolites identified in our G-Met study to compare intracellular metabolism between the two cell lines using liquid chromatography-mass spectrometry. The established sunitinib-resistant RCC cell line demonstrated significantly desuppressed protein kinase B (Akt) and mesenchymal-to-epithelial transition (MET) phosphorylation compared with the control RCC cell line under sunitinib exposure. Among identified metabolites, glutamine, glutamic acid, and α-KG (involved in glutamine uptake into the tricarboxylic acid (TCA) cycle for energy metabolism); fructose 6-phosphate, D-sedoheptulose 7-phosphate, and glucose 1-phosphate (involved in increased glycolysis and its intermediate metabolites); and glutathione and myoinositol (antioxidant effects) were significantly increased in the sunitinib-resistant RCC cell line. Particularly, glutamine transporter (SLC1A5) expression was significantly increased in sunitinib-resistant RCC cells compared with control cells. In this study, we demonstrated energy metabolism with glutamine uptake and glycolysis upregulation, as well as antioxidant activity, was also associated with sunitinib resistance in RCC cells.

Molecular and Functional Analysis of Sunitinib-Resistance Induction in Human Renal Cell Carcinoma Cells

“…( Z )-sunitinib ( Figure 4 A, 2) is clinically relevant and active. The two stereoisomers could be detected at the same ratios in the supernatant, independent of the dose of sunitinib, with the percent of stereoisomeric excess (see Materials and Methods) being approximatively 85% ( Z/E ) at all concentrations ( Supplementary Figure S8B ), which is slightly lower than the values reported in the literature [ 31 , 33 ]. The increasing percentage of stereoisomeric excess at low concentrations can be attributed to the limit of detection of peak 1 (see Materials and Methods).…”

“…Investigation of the conversion of sunitinib in Caki-1-SR cells through LC-HRMS/MS was performed and showed two peaks corresponding to its ( E ) and ( Z )-isomer ( m/z = 399.2183; Figure 4 A and Supplementary Figure S8A ). Diverse studies showed the presence of both stereoisomers, ( E )-sunitinib and ( Z )-sunitinib [ 23 , 31 , 33 ]. Attribution was made by comparing the retention times and stereoisomeric ratios with the literature [ 32 ] and a standard solution in methanol ( Supplementary Figure S8A ).…”

“…Attribution was made by comparing the retention times and stereoisomeric ratios with the literature [ 32 ] and a standard solution in methanol ( Supplementary Figure S8A ). ( E )-sunitinib ( Figure 4 A, 1) appears to be clinically inactive, and its isomerization occurs through light exposure [ 31 , 33 ]. ( Z )-sunitinib ( Figure 4 A, 2) is clinically relevant and active.…”

“…When comparing the supernatant and the cellular extract, remarkable differences in the ratios of ( Z )-sunitinib and its stereoisomer were observed. While stereoisomeric excess of the ( Z )-form was observed (>96%) in both Caki-1-SR CTRL and supernatants of sunitinib-treated Caki-1-SR cells (Caki-1-SR Sun ) ( Supplementary Figure S8 ) [ 31 , 33 ], in the cell extract, the stereoisomeric excess decreased to 45% and 30%, respectively. The presence of ( Z )- N -desethyl sunitinib and ( E )- N -desethyl sunitinib in the cell extract of Caki-1-SR cells cultured for 24 h in the culture medium treated with 1 µM sunitinib was detected, whereas, in the supernatant, only the ( Z )-form could be detected ( Figure 4 B).…”

“…Evaluating the anti-cancer efficacy, as well as the pharmacokinetics of sunitinib and its active metabolite ( N -desethyl sunitinib), demonstrated high interpatient variability [ 31 ], potentially linked to the light-dependent isomerism of sunitinib [ 31 , 32 ]. The reversible isomerization from the ( Z )- to the ( E )-isomer occurs through light exposure, independent of the dose [ 31 , 32 , 33 ]. How isomerization and metabolism link to sunitinib resistance is not yet fully understood.…”