Comprehensive Metabolic Tracing Reveals the Origin and Catabolism of Cysteine in Mammalian Tissues and Tumors

Abstract

Cysteine plays critical roles in cellular biosynthesis, enzyme catalysis, and redox metabolism. The intracellular cysteine pool can be sustained by cystine uptake or de novo synthesis from serine and homocysteine. Demand for cysteine is increased during tumorigenesis for generating glutathione to deal with oxidative stress. While cultured cells have been shown to be highly dependent on exogenous cystine for proliferation and survival, how diverse tissues obtain and use cysteine in vivo has not been characterized. We comprehensively interrogated cysteine metabolism in normal murine tissues and cancers that arise from them using stable isotope 13C1-serine and 13C6-cystine tracing. De novo cysteine synthesis was highest in normal liver and pancreas and absent in lung tissue, while cysteine synthesis was either inactive or downregulated during tumorigenesis. In contrast, cystine uptake and metabolism to downstream metabolites was a universal feature of normal tissues and tumors. However, differences in glutathione labeling from cysteine were evident across tumor types. Thus, cystine is a major contributor to the cysteine pool in tumors, and glutathione metabolism is differentially active across tumor types.

Significance: Stable isotope 13C1-serine and 13C6-cystine tracing characterizes cysteine metabolism in normal murine tissues and its rewiring in tumors using genetically engineered mouse models of liver, pancreas, and lung cancers.

Graphical abstract

Figure 1.

Cultured cancer cell lines lack de novo cysteine synthesis capacity. A–C, Analysis of de novo cysteine synthesis in cultured NSCLC, SCLC, PDAC, and HCC cell lines with ¹³C₃-serine tracing. Cell lines were incubated with ¹³C₃-serine containing media for 4 hours, followed by analysis of the fraction labeling in serine (A), cystathionine (B), and cysteine (C). Data are presented as mean ± SD and N = 3 biological replicates for each cell line. D, Immunoblotting for the transsulfuration enzymes CBS and CSE. HSP90 was used for the loading control and HepG2 was used for relative comparison between different membranes. Cth, cystathionine; Cys, cysteine; Ser, serine.

Figure 2.

Contribution of de novo cysteine synthesis to the cysteine pool varies across healthy mouse tissues. A, Schematic depicting 1-[¹³C₁]-serine infusion and its metabolism via the transsulfuration and glutathione synthesis pathways. B–G, Healthy C57BL/6J mice were infused with 1-[¹³C₁]-serine, followed by analysis of the fraction labeling in serine (B), glycine (C), cystathionine (D), glutathione (E), cysteine (F), and γ-glutamylcysteine (G). For B–G, data are presented as mean ± SD and N = 10 mice (5 male, 5 female). N.D., not detected. H, Fractional contribution of serine to intracellular cysteine synthesis in each tissue from B–G. Cysteine labeling was normalized to the fraction labeling of serine in each tissue. I, Immunoblots of CBS and CSE for each tissue. HSP90 was used for the loading control. αKB, α-ketobutyrate; Cth, cystathionine; Cys, cysteine; Gly, glycine; Glut, glutamate; GSH, glutathione; γ-Glu-Cys, γ-glutamylcysteine; Ser, serine. (A, Created with BioRender.com.)

Figure 3.

Cyst(e)ine supplies the cysteine pool in all tissues. A, Schematic depicting ¹³C₆-cystine infusion and its metabolism to glutathione and taurine. B–G, Healthy C57BL/6J mice were infused with ¹³C₆-cystine, followed by analysis of the fraction labeling in cysteine (B), γ-glutamylcysteine (C), cysteine (D), glutathione (E), hypotaurine (F) and taurine (G). For B–G, data are presented as mean ± SD and N = 5 mice. N.D., not detected. H, Immunoblots of xCT, CDO1, CSAD, ADO, FMO1, GCLC, GCLM, and GSS for each tissue. HSP90 was used for the loading control. αKB, α-ketobutyrate; Cth, cystathionine; Cys, cysteine; Cys₂, cystine; γ-Glu-Cys, γ-glutamylcysteine; Glut, glutamate; Gly, glycine; GSH, glutathione; Hcy, homocysteine; Htau, hypotaurine; Tau, taurine. (A, Created with BioRender.com.)

Figure 4.

Tumorigenesis of liver and pancreas induces downregulation of de novo cysteine synthesis. A, Schematic for the generation of Myc; p53^−/− HCC and Kras^G12D; p53^+/− PDAC GEMM tumors. B, Analysis of the fraction labeling in serine, glycine, cystathionine, glutathione, cysteine, and γ-glutamylcysteine in liver tissues (N = 8) compared with HCC tumors (N = 8) and their matched serum normal (N = 8) and HCC (N = 5) following infusion with 1-[¹³C₁]-serine. C, Analysis of the fraction labeling in serine, glycine, cystathionine, glutathione, cysteine, and γ-glutamylcysteine in pancreas tissues (N = 5) compared with PDAC tumors (N = 5) and their matched serum from normal (N = 5) and PDAC (N = 5) following infusion with 1-[¹³C₁]-serine. D, Fractional contribution of serine to intracellular cysteine synthesis in HCC and PDAC. Cysteine labeling was normalized to the fraction labeling of serine in each tissue. One healthy pancreas sample was excluded because of a division error. For B–D, data are presented as mean ± SD. N.D., not detected. E, Immunoblots of CBS and CSE for each tissue. HSP90 was used for the loading control. *, P < 0.05; **, P < 0.01. Cth, cystathionine; Cys, cysteine; Gly, glycine; GSH, glutathione; γ-Glu-Cys, γ-glutamylcysteine; Ser, serine. (A, Created with BioRender.com.)

Figure 5.

De novo cysteine synthesis does not contribute to the cysteine pool of lung tumors. A, Schematic for the generation of Kras^G12D; p53^−/− and Kras^G12D; p53^−/−; Nrf2^D29H LUAD, and Rb1^−/−; p53^−/−; Myc^T58A/+ or Rb1^−/−; p53^−/−; Myc^T58A/T58A SCLC GEMM tumors. B, Analysis of the fraction labeling in serine, glycine, cystathionine, glutathione, cysteine, and γ-glutamylcysteine in normal lung tissues (N = 8) compared with Nrf2^WT LUAD and (N = 10), Nrf2^D29H LUAD tumors (N = 10) and their matched serum from normal (N = 8), Nrf2^WT (N = 5), and Nrf2^D29H (N = 5) following infusion with 1-[¹³C₁]-serine. C, Analysis of the fraction labeling in serine, glycine, cystathionine, glutathione, cysteine, and γ-glutamylcysteine in normal lung tissues (N = 8) compared with SCLC tumors (N = 9), and their matched serum normal (N = 8) and SCLC (N = 9) following infusion with 1-[¹³C₁]-serine. The control lung samples in C are the same as in B. D, Fractional contribution of serine to intracellular cysteine synthesis in LUAD and SCLC. Cysteine labeling was normalized to the fraction labeling of serine in each tissue. For B–D, data are presented as mean ± SD. N.D., not detected. E, Immunoblots of CBS and CSE for each tissue. HSP90 was used for the loading control. *, P < 0.05; ****, P < 0.0001. Cth, cystathionine; Cys, cysteine; Gly, glycine; GSH, glutathione; γ-Glu-Cys, γ-glutamylcysteine; Ser, serine. (A, Created with BioRender.com.)

Figure 6.

Cystine is a major contributor to the cysteine pool in tumors. A, Analysis of the fraction labeling in cysteine, γ-glutamylcysteine, and glutathione in liver tissues (N = 9), HCC tumors (N = 9), lung tissues (N = 10), Nrf2^WT LUAD tumors (N = 16), Nrf2^D29H LUAD tumors (N = 10), pancreas tissues (N = 3), PDAC tumors (N = 12), and their matched serum from normal control mice for HCC (N = 6), HCC (N = 7), normal control mice for PDAC (N = 3), PDAC (N = 6), normal control mice for LUAD (N = 5), Nrf2^WT LUAD (N = 8), and Nrf2^D29H LUAD serum (N = 5) following infusion with ¹³C₆-cystine. B, Total signal of glutathione in the tissues from A. C, Total signal of cysteine in the tissues from A. For A–C, data are presented as mean ± SD. N.D., not detected. D, Immunoblots of xCT, GCLC, GCLM, and GSS for each tissue. HSP90 was used for the loading control. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Cys, cysteine; GSH, glutathione; γ-Glu-Cys, γ-glutamylcysteine.