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. 2024 Jun 7;25(12):6328.
doi: 10.3390/ijms25126328.

Metabolomics Reveals Tyrosine Kinase Inhibitor Resistance-Associated Metabolic Events in Human Metastatic Renal Cancer Cells

Filipa Amaro  1   2 , Márcia Carvalho  1   2   3 , Maria de Lourdes Bastos  1   2 , Paula Guedes de Pinho  1   2 , Joana Pinto  1   2
Affiliations

Metabolomics Reveals Tyrosine Kinase Inhibitor Resistance-Associated Metabolic Events in Human Metastatic Renal Cancer Cells

Filipa Amaro et al. Int J Mol Sci. .

Abstract

The development of resistance to tyrosine kinase inhibitors (TKIs) is a major cause of treatment failure in metastatic renal cell carcinoma (mRCC). A deeper understanding of the metabolic mechanisms associated with TKI resistance is critical for refining therapeutic strategies. In this study, we established resistance to sunitinib and pazopanib by exposing a parental Caki-1 cell line to increasing concentrations of sunitinib and pazopanib. The intracellular and extracellular metabolome of sunitinib- and pazopanib-resistant mRCC cells were investigated using a nuclear magnetic resonance (NMR)-based metabolomics approach. Data analysis included multivariate and univariate methods, as well as pathway and network analyses. Distinct metabolic signatures in sunitinib- and pazopanib-resistant RCC cells were found for the first time in this study. A common metabolic reprogramming pattern was observed in amino acid, glycerophospholipid, and nicotinate and nicotinamide metabolism. Sunitinib-resistant cells exhibited marked alterations in metabolites involved in antioxidant defence mechanisms, while pazopanib-resistant cells showed alterations in metabolites associated with energy pathways. Sunitinib-resistant RCC cells demonstrated an increased ability to proliferate, whereas pazopanib-resistant cells appeared to restructure their energy metabolism and undergo alterations in pathways associated with cell death. These findings provide potential targets for novel therapeutic strategies to overcome TKI resistance in mRCC through metabolic regulation.

Keywords: drug resistance; metabolic reprogramming; metabolomics; renal cell carcinoma; tyrosine kinase inhibitors.

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Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
(a,b) Non-linear regression models represented with mean values and 95% confidence intervals obtained for sunitinib- and pazopanib-induced cell death in parental (green line), sunitinib-resistant (red line) and pazopanib-resistant (blue line) Caki-1 cell lines as assessed by the MTT after 48 h exposure. Statistical significance assessed between models using the Extra Sum of Squares F test revealing a p < 0.0001. (c,d) Effects on cell proliferation of parental (green bars), sunitinib-resistant (red bars) and pazopanib-resistant (blue bars) Caki-1 cells exposed to 2 µM sunitinib or 24 µM pazopanib. (e) Representative phase contrast microscopy images of parental and resistant Caki-1 cell lines. Scale bar: 100 µm. Original magnification 10×. Results were obtained from three independent experiments, performed in triplicate, and are presented as mean ± standard error of the mean. **** p < 0.0001.
Figure 2
Figure 2
(a,b) PLS-DA score scatter and loading plots of intracellular polar metabolic profiles from sunitinib-resistant (green circles, n = 8) vs. parental (red circles, n = 8) Caki-1 cells. (c,d) PLS-DA score scatter and loading plots of intracellular lipid profiles from sunitinib-resistant (green circles, n = 8) vs. parental (red circles, n = 8) Caki-1 cells. (e,f) PLS-DA score scatter and loading plots of extracellular metabolic profiles from sunitinib-resistant (green circles, n = 8) vs. parental (red circles, n = 8) Caki-1 cell lines. Acc, R2 and Q2 values were obtained with two components. Abbreviations: Ala: alanine; Asp: aspartate; CEs: cholesteryl esters; ChoP: phosphocholine; ETA: ethanolamine; FC: free cholesterol; Gly: glycine; Gln: glutamine; GSH: glutathione; GPC: glycerophosphocholine; Iso: isoleucine; Leu: leucine; MGs: monoglycerides; NAD+: nicotinamide adenine dinucleotide; PEs: phosphatidylethanolamines; PUFAs: polyunsaturated fatty acids; Val: valine.
Figure 3
Figure 3
(a,b) PLS-DA score scatter and loading plots of intracellular polar metabolic profiles from pazopanib-resistant (green circles, n = 8) vs. parental (red circles, n = 8) Caki-1 cell lines. (c,d) PLS-DA scores scatter and loading plots of intracellular lipid profiles from pazopanib-resistant (green circles, n = 8) vs. parental (red circles, n = 8) Caki-1 cell lines. (e,f) PLS-DA scores scatter and loading plots of extracellular metabolic profiles from pazopanib-resistant (green circles, n = 8) vs. parental (red circles, n = 8) Caki-1 cell lines. Acc, R2 and Q2 values were obtained with two components. Abbreviations: Ala: alanine; Asn: asparagine; Asp: aspartate; CEs: cholesteryl esters; ChoP: phosphocholine; FAs: fatty acids: Gly: glycine; Gln: glutamine; Glu: glucose; GPC: glycerophosphocholine; Iso: isoleucine; Leu: leucine; MGs: monoglycerides; MNA: 1-methylnicotinamide; NAD+: nicotinamide adenine dinucleotide; UFAs: unsaturated fatty acids; Val: valine.
Figure 4
Figure 4
(a,b) Heatmaps illustrating the mean levels of intracellular and extracellular metabolites altered in sunitinib- and pazopanib-resistant Caki-1 cell lines and the putatively altered metabolic pathways. Columns represent each sample group, and rows correspond to the mean normalised peak area of each metabolite coloured from minimum value (dark blue) to maximum value (dark red). Statistical significance was assessed by comparison with the parental Caki-1 cell line (first column in each heatmap) * p < 0.05; ** p < 0.01; *** p < 0.001. (c,d) Pathway analysis was performed on the list of metabolites found to be altered in sunitinib- and pazopanib-resistant Caki-1 cell lines, respectively. The annotated pathways were considered statistically significant (p < 0.05). Abbreviations: ChoP: phosphocholine; FAs: fatty acids; PEs: phosphatidylethanolamine; NAD+: nicotinamide adenine dinucleotide; UFAs: unsaturated fatty acids.
Figure 5
Figure 5
(a,b) Putative metabolic changes in sunitinib- and pazopanib-resistant Caki-1 cells. Red and green squares indicate increased and decreased metabolites, respectively. Blue squares represent extracellular changes. Arrows indicate metabolites consumed (orientation from outside to inside) and excreted (orientation from inside to outside). The consumption and excretion of metabolites can be interpreted from the boxplots shown in Figure S4. Dashed lines represent multiple-step reactions. Abbreviations: Ala: alanine; Arg: arginine; Asn: asparagine; Asp: aspartate; C: cholesterol; CEs: cholesteryl esters; ChoP: phosphocholine; ETA: ethanolamine; FAs: fatty acids; Gly: glycine; Gln: glutamine; Glu: glutamate; GSH: glutathione; GPC: glycerophosphocholine; Iso: isoleucine; Leu: leucine; Lys: lysine; Met: methionine; MGs: monoglycerides; NAD+: nicotinamide adenine dinucleotide; PEs: phosphatidylethanolamines; TCA: tricarboxylic acid cycle; Tyr: tyrosine; UFAs: unsaturated fatty acids; Val: valine.
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