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. 2019 Nov 5;30(5):865-876.e5.
doi: 10.1016/j.cmet.2019.09.009. Epub 2019 Oct 10.

Transsulfuration Activity Can Support Cell Growth upon Extracellular Cysteine Limitation

Jiajun Zhu  1 , Mirela Berisa  2 , Simon Schwörer  1 , Weige Qin  2 , Justin R Cross  2 , Craig B Thompson  3
Affiliations

Transsulfuration Activity Can Support Cell Growth upon Extracellular Cysteine Limitation

Jiajun Zhu et al. Cell Metab. .

Abstract

Cysteine acts both as a building unit for protein translation and as the limiting substrate for glutathione synthesis to support the cellular antioxidant system. In addition to transporter-mediated uptake, cellular cysteine can also be synthesized from methionine through the transsulfuration pathway. Here, we investigate the regulation of transsulfuration and its role in sustaining cell proliferation upon extracellular cysteine limitation, a condition reported to occur in human tumors as they grow in size. We observed constitutive expression of transsulfuration enzymes in a subset of cancer cell lines, while in other cells, these enzymes are induced following cysteine deprivation. We show that both constitutive and inducible transsulfuration activities contribute to the cellular cysteine pool and redox homeostasis. The rate of transsulfuration is determined by the cellular capacity to conduct methylation reactions that convert S-adenosylmethionine to S-adenosylhomocysteine. Finally, our results demonstrate that transsulfuration-mediated cysteine synthesis is critical in promoting tumor growth in vivo.

Keywords: cancer; cysteine; glutathione; metabolism; methylation; redox homeostasis; transsulfuration; xCT.

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Conflict of interest statement

DECLARATION OF INTERESTS

C.B.T. is a founder of Agios Pharmaceuticals and a member of its scientific advisory board. He is also a former member of the Board of Directors and stockholder of Merck and Charles River Laboratories. He holds patents related to cellular metabolism.

Figures

Figure 1.
Figure 1.. The transsulfuration pathway contributes to de novo cysteine synthesis in cancer cells
(A) Schematic of the cellular cysteine acquisition strategies, including the transsulfuration pathway and the system Xc- amino acid transporter. (B) Protein levels of cystathionine β-synthase (CBS), cystathionine γ-lyase (CTH) and xCT by Western blot analysis across the indicated cancer cell lines. (C) Western blot analysis in SHSY5Y cells expressing control small guide RNA (sgCtrl) or two independent sgRNA sequences targeting the CBS gene (sgCBS-1 and sgCBS-2). (D)-(F) Growth curves of SHSY5Y cells expressing sgRNA targeting control region or the CBS gene, cultured in (D) full medium (FM) containing 100 μM cysteine, (E) cysteine-deficient medium (FM-Cys), or (F) cysteine-deficient medium supplemented with 50 μM βME (FM-Cys+βME). (G) Schematic of [3-13C] serine isotope tracing. Grey circles indicate 13C carbon atoms. Clear circles indicate unlabeled carbon atoms. (H)-(J) Labeled and unlabeled metabolite levels in SHSY5Y cells expressing sgCtrl, sgCBS-1 or sgCBS-2, cultured in FM-Cys+βME containing [3-13C] serine for 72 hours. Natural isotope corrected isotopologue abundances normalized to biomass are shown. (K)-(M) Growth curves of SHSY5Y cells expressing (K) sgCtrl, (L) sgCBS-1, or (M) sgCBS-2, cultured under the indicated medium conditions. FM, full medium containing 100 μM cysteine. FM-Cys, cysteine-deficient medium. FM-Cys+Hcy, cysteine-deficient medium supplemented with 100 μM homocysteine. All error bars in this figure represent mean±SD, n=3. *p<0.05, two-sided Student’s t-Test of total metabolite levels between the indicated groups.
Figure 2.
Figure 2.. The transsulfuration pathway responds to cysteine limitations through GCN2-ATF4 signaling
(A) and (B) Growth curves of (A) SKMEL30 cells and (B) LN229 cells cultured under the indicated medium conditions. FM, full medium containing 100 μM cysteine. FM-Cys, cysteine-deficient medium. FM-Cys+Hcy, cysteine-deficient medium supplemented with 100 μM homocysteine. FM-Cys (Extra Met and Ser), cysteine-deficient medium supplemented with additional 100 μM methionine and 250 μM serine. (C) Western blot analysis of cancer cells cultured either in FM or in FM-Cys. SE, short exposure. LE, long exposure. (D) and (E) ATF4 or IgG ChIP followed by quantitative PCR (ChIP-qPCR) in (D) SKMEL30 cells and (E) LN229 cells, at the indicated gene regions. Prom., gene promoter region. Neg., negative control region 1.5 kb upstream of the gene promoter. ChIP results are normalized to the corresponding input signals. (F) and (G) Western blot analysis of (F) SKMEL30 cells and (G) LN229 cells expressing sgRNA targeting control region or the ATF4 gene, cultured in FM or FM-Cys for 8 hours. All error bars in this figure represent mean±SD, n=3. *p<0.05, two-sided Student’s t-Test between the indicated groups.
Figure 3.
Figure 3.. Transsulfuration activity is limited by the cellular capacity to convert SAM to SAH
(A) Schematic of the transsulfuration pathway and its connection to the methionine cycle. MAT2A, methionine adenosyltransferase 2A. MTs, methyltransferases, such as GNMT (glycine N-methyltransferase). AHCY, adenosyl-homocysteinase. MTR, 5-methyltetrahydrofolate-homocysteine methyltransferase. (B) Western blot analysis of SHSY5Y cells ectopically expressing enzymes denoted in (A). (C) Cell number fold change (Day 3/Day 0) of SHSY5Y cells ectopically expressing enzymes denoted in (A), cultured in FM or FM-Cys. (D) Cell number fold change (Day 3/Day 0) of SHSY5Y cells expressing sgRNA targeting control region or the CBS gene, and ectopically expressing vector control or GNMT, cultured in FM, FM-Cys, or FM-Cys+Hcy. (E) Cell number fold change (Day 3/Day 0) of SHSY5Y cells ectopically expressing different methyltransferases, cultured in FM or FM-Cys. (F) Relative SAH to SAM ratio analyzed by LC-MS in SHSY5Y cells ectopically expressing different methyltransferases, cultured in FM or FM-Cys. Ratio was calculated from raw peak areas measured by LC-MS/MS in MRM mode. (G) Western blot analysis of SHSY5Y cells ectopically expressing vector control or GNMT, cultured in FM, FM-Cys, or FM-Cys+Hcy. (H) Relative ROS levels in SHSY5Y cells ectopically expressing vector control or GNMT, cultured in FM-Cys. All error bars in this figure represent mean±SD, n=3. *p<0.05, two-sided Student’s t-Test between the indicated groups.
Figure 4.
Figure 4.. Enhanced transsulfuration activity restores GSH level and promotes cell growth upon cysteine limitations
(A) Relative GSH levels in SHSY5Y cells ectopically expressing different methyltransferases, cultured in FM or FM-Cys, measured by LC-MS/MS in MRM mode. (B)-(D) Labeled and unlabeled metabolite levels in SHSY5Y cells ectopically expressing vector control or GNMT, cultured in FM or FM-Cys containing [3-13C] serine for 48 hours. Natural isotope corrected isotopologue abundances normalized to biomass are shown. (E)-(G) Growth curves of SHSY5Y cells ectopically expressing vector control or GNMT, cultured in medium containing (E) 100 μM, (F) 30 μM or (G) 10 μM cysteine. (H)-(J) Growth curves of LN229 cells ectopically expressing vector control or GNMT, cultured in medium containing (H) 100 μM, (I) 30 μM or (J) 10 μM cysteine. All error bars in (A)-(J) represent mean±SD, n=3. *p<0.05, two-sided Student’s t-Test of total metabolite levels between the indicated groups. (K) Xenograft tumor growth of SHSY5Y cells expressing control vector or GNMT. Error bars represent mean±SEM, n=8. p-value is obtained by performing two-way ANOVA and corrected for multiple comparisons.
Figure 5.
Figure 5.. Endogenous transsulfuration activity supports cancer cell growth as extracellular cysteine levels decrease
(A) and (B) Growth curves of SHSY5Y cells expressing sgRNA targeting control region or the CBS gene, cultured in medium containing (A) 30 μM or (B) 10 μM cysteine. Error bars represent mean±SD, n=3. (C) Xenograft tumor growth of SHSY5Y cells expressing sgRNA targeting control region (sgCtrl) or the CBS gene (sgCBS-1). Error bars represent mean±SEM, n=7. p-value is obtained by performing two-way ANOVA and corrected for multiple comparisons. (D) Cystathionine and (E) GSH levels in xenograft tumors derived from SHSY5Y cells expressing sgCtrl or sgCBS-1, measured by LC-MS/MS in MRM mode. *p<0.05, two-sided Student’s t-Test.
Figure 6.
Figure 6.. GNMT expression supports prostate cancer cell growth upon cysteine limitation
(A) GNMT mRNA expression levels across different types of cancer by analyzing The Cancer Genome Atlas (TCGA) datasets in the cBioportal collection. (B) and (C) Growth curves of LNCaP cells expressing sgRNA targeting control region or the CBS gene, cultured in medium containing (B) 100 μM, or (C) 10 μM cysteine. (D) Western blot analysis of LNCaP cells expressing sgRNA targeting control region or the GNMT gene. (E) and (F) Growth curves of LNCaP cells expressing sgRNA targeting control region or the GNMT gene, cultured in medium containing (E) 100 μM, or (F) 10 μM cysteine. (G)-(I) Growth curves of LNCaP cells ectopically expressing vector control or GNMT, cultured in medium containing (G) 100 μM, (H) 30 μM or (I) 10 μM cysteine. All error bars in (B)-(I) represent mean±SD, n=3. (J) Xenograft tumor volumes of LNCaP cells expressing vector control or GNMT, and grown in immune-compromised mice for a period of 7 weeks. p-value is obtained by performing Mann-Whitney U test.
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