1. Biochemistry and Chemical Biology
  2. Cancer Biology
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Cysteine dioxygenase 1 is a metabolic liability for non-small cell lung cancer

  1. Yun Pyo Kang
  2. Laura Torrente
  3. Aimee Falzone
  4. Cody M Elkins
  5. Min Liu
  6. John M Asara
  7. Christian C Dibble
  8. Gina M DeNicola  Is a corresponding author
  1. H Lee Moffitt Cancer Center and Research Institute, United States
  2. Beth Israel Deaconess Medical Center, United States
  3. Harvard Medical School, United States
Research Article
Cite this article as: eLife 2019;8:e45572 doi: 10.7554/eLife.45572
9 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Nrf2 promotes the accumulation of intracellular cysteine and sulfur-containing metabolites.

(a) Schematic of the murine Keap1R554Q allele. The Keap1R554Q allele was created by inserting a loxP-flanked wild-type Keap1 cDNA containing exons 3–5 upstream of a R554Q mutation in the endogenous exon 4 of the Keap1 gene. Prior to recombination, wild-type Keap1 protein is expressed due to splicing into the cDNA containing exons 3–5, and is referred to as Keap1WT. Following Cre-mediated excision of the loxP-flanked cargo, Keap1R554Q is expressed at physiological levels and is referred to as Keap1 R554Q. PA, poly A signal. (b) Western blot analysis of Nrf2, Nqo1, and β-Actin levels following Nrf2 stabilization in wild-type (WT) vs. homozygous Keap1R554Q/R554Q (R554Q) MEFs. (c) LC-HRMS metabolomics profiling of Keap1R554Q/R554Q MEFs compared to Keap1WT/WT MEFs. GSH, glutathione. CSA, cysteine sulfinic acid. HTAU, hypotaurine. CTH, cystathionine. NAC, N-acetyl cysteine. SAH, S-adenosyl homocysteine. aKG, α-ketoglutarate. PYR, pyruvate. N = 3, representative of 2 individual MEF lines. (d–i) Quantitation of cystine ([CYS]2, d), cysteine (CYS, e), glutathione (GSH, f), cysteine sulfinic acid (CSA, g), hypotaurine (HTAU, h) and taurine (TAU, i) levels in Keap1R554Q/R554Q MEFs compared to Keap1WT/WT MEFs. Cysteine and glutathione were derivatized with N-ethylmaleamide (NEM) to prevent oxidation during extraction here and for all quantification experiments. TXN, thioredoxin. TXN-ox, oxidized thioredoxin. GSH-ox, oxidized glutathione. GLU, glutamate. GLY, glycine. N = 3.

https://doi.org/10.7554/eLife.45572.003
Figure 1—source data 1

Nrf2 promotes the accumulation of intracellular cysteine and sulfur-containing metabolites.

https://doi.org/10.7554/eLife.45572.005
Figure 1—figure supplement 1
Cre infection does not induce taurine pathway activity in MEFs.

(a) LC-HRMS metabolomics profiling of wild-type MEFs infected with adenoviral-cre compared to MEFs infected with empty adenovirus. N = 3 replicates/group. (b) Verification of stable-isotope labeled CSA and cystine internal standards for absolute quantification. Stable isotope labeled CSA and cystine standards were synthesized from [13C3, 15N]-cysteine and analyzed by LC-MS/MS based MRM against authentic standards. RT, retention time.

https://doi.org/10.7554/eLife.45572.004
Figure 1—figure supplement 1—source data 1

Cre infection does not induce taurine pathway activity in MEFs.

https://doi.org/10.7554/eLife.45572.006
Nrf2 promotes the accumulation of cysteine dioxygenase (Cdo1) to promote entry of cysteine into the taurine synthesis pathway.

(a) Western blot analysis of Cdo1 and β-Actin levels following Nrf2 stabilization in wild-type (WT) vs. homozygous Keap1R554Q/R554Q (R554Q) MEFs. (b) Real-time PCR analysis of Cdo1 and Nqo1 mRNA levels following Nrf2 stabilization in wild-type (WT) vs. homozygous Keap1R554Q/R554Q (R554Q) MEFs. mRNA expression was normalized to β-Actin expression, followed by normalization to WT. N = 3. (c) Western blot analysis of Nrf2, Cdo1, Nqo1 and β-Actin levels following expression of sgControl (sgCON) or Cdo1 deletion (sgCdo1 #2 and #3) with CRISPR/Cas9 in primary wild-type (WT) and homozygous Keap1R554Q/R554Q (R554Q) MEFs. (d–i) Quantitation of cysteine (CYS, (d), glutathione (GSH, (e,f), cystine ([CYS]2,(g), and hypotaurine (HTAU, (h,i) total levels and 13C-labeling from 13C-cystine in cells from (c). 13C-label is shown in red, while 12C-label is gray. Cells were labeled for 4 hr. N = 3.

https://doi.org/10.7554/eLife.45572.007
Figure 2—source data 1

Nrf2 promotes the accumulation of cysteine dioxygenase (Cdo1) to promote entry of cysteine into the taurine synthesis pathway.

https://doi.org/10.7554/eLife.45572.008
Figure 3 with 3 supplements
CDO1 is preferentially silenced in KEAP1 mutant NSCLC and antagonizes proliferation.

(a) CDO1 mRNA expression of normal lung (Normal) or or KEAP1 wild-type (WT) and mutant (MUT) lung adenocarcinoma patient tumor samples. Normal, N = 45. MUT, N = 39, WT, N = 189. (b) Methylation of the CDO1 promoter in samples from normal lung (Normal) or KEAP1 wild-type (WT) and mutant (MUT) lung adenocarcinoma patient tumor samples. Normal, N = 28. MUT, N = 29, WT, N = 154. (c) Western blot analysis of NRF2, CDO1, xCT and α-Tubulin levels in KEAP1 wild-type and KEAP1 mutant NSCLC cell lines. Arrowhead denotes specific band. (d) (Top) Western blot analysis of NRF2, CDO1 and α-Tubulin expression in NRF2LOW and NRF2HIGH (KEAP1 mutant: H1792, HCC15, A549, H322, and H460) NSCLC cell lines expressing CDO1Y157F. (Bottom) Intracellular cysteine concentration of the parental cell lines (minus doxycycline). (Right) Correlation between CDO1 protein levels and intracellular cysteine concentrations. CDO1 protein was normalized to α-Tubulin. N = 13. (e) (Top) Western blot analysis of CDO1 and β-ACTIN expression in H1581 cells following expression of control (sgCON) or CDO1-targeting sgRNAs (sgCDO1 #1 and #2) and Cas9. (Bottom) Intracellular cysteine concentration of the same cells. N = 4 replicates/group. (f–h) Western blot analysis of NRF2, CDO1, xCT, TXN1, α-Tubulin and β-ACTIN levels following re-expression of CDO1WT (WT), CDO1Y157F (Y157F), or GFP in (f) NRF2 knockout A549 cells reconstituted with either pLX317 empty (-) or pLX317-NRF2 (+), (g) H1975 and H1299 cells expressing either pLX317 empty (-) or pLX317-NRF2T80K (+), or (h) H1944 cells reconstituted with inactive pLenti-KEAP1 (C273S), super repressor KEAP1 (C151S) or wild-type KEAP1 (WT). (i) Analysis of cysteine levels in the cells from (e–g). N = 3 replicates/group. (j) Analysis of the proliferation of cells from (f). Cells were collected on the indicated days, stained with crystal violet and their absorbance at 600 nm determined. N = 3 replicates/group. (k) Analysis of proliferation of NSCLC cells expressing CDO1WT and correlation with CDO1 protein expression. Cells were collected after 3 days, and CDO1 dependent growth inhibition was determined by taking the ratio of CDO1WT / CDO1Y157F cell quantity. For individual lines (N = 13), see Figure 3—figure supplement 3D. For (d-j), cells were treated with 0.25 μg/ml doxycycline for 2 days prior to and during the assay and fresh medium was added 4 hr prior to sample collection.

https://doi.org/10.7554/eLife.45572.009
Figure 3—source data 1

CDO1 is preferentially silenced in KEAP1 mutant NSCLC and antagonizes proliferation.

https://doi.org/10.7554/eLife.45572.016
Figure 3—figure supplement 1
CDO1 is epigenetically silenced in NSCLC.

(a) Association of CDO1 mRNA expression with NSCLC patient outcome. (b) Correlation between CDO1 promoter methylation and mRNA expression in lung adenocarcinoma patient samples from the TCGA. N = 185. (c) Methylation of the CDO1 promoter in samples from normal lung (Normal) or lung adenocarcinoma patient tumor samples based on NRF2 activity. Normal, N = 28. NRF2 high group = KEAP1 mutant patients + KEAP1 wild-type patients with NRF2 activity score >lowest KEAP1 mutant tumor (N = 51). NRF2 low group = KEAP1 wild-type patients with NRF2 activity score <lowest KEAP1 mutant tumor (N = 134). (d) Real-time PCR analysis of CDO1 mRNA expression following 5 uM decitabine treatment for 3 days with daily medium change. CDO1 expression was normalized to β-ACTIN expression, and relative expression compared to H1581 DMSO is shown. H1581 demonstrates high basal expression, in agreement with the western blot from Figure 3C. N = 3 replicates/group. Cells were treated with 0.25 µg/ml doxycycline for 2 days prior to and during the assay and fresh medium was added 4 hr prior to sample collection.

https://doi.org/10.7554/eLife.45572.010
Figure 3—figure supplement 1—source data 1

CDO1 is epigenetically silenced in NSCLC.

https://doi.org/10.7554/eLife.45572.011
Figure 3—figure supplement 2
CDO1 protein does not correlate with mRNA expression.

(a) Correlation between CDO1 protein levels (from Figure 3d) and CDO1 mRNA expression in the same cell lines. N = 13. (b) Western blot analysis of NRF2, xCT, CDO1 and HSP90 expression in NSCLC cell lines expressing CDO1WT with mouse liver and lung lysates. Liver expresses high physiological levels of CDO1, while lung expresses low. Cells were treated with 0.25 µg/ml doxycycline for 2 days prior to and during the assay and fresh medium was added 4 hr prior to sample collection.

https://doi.org/10.7554/eLife.45572.012
Figure 3—figure supplement 2—source data 1

CDO1 protein does not correlate with mRNA expression.

https://doi.org/10.7554/eLife.45572.013
Figure 3—figure supplement 3
NRF2 promotes CDO1 expression in NSCLC.

(a) Western blot analysis of NRF2, xCT, CDO1 and β-Actin protein expression in H460 control cells, or CDO1WT-expressing H1299 and H1975 cells following treatment with 2 µM β-lapachone (βLap). Cells were treated with βLap for 8 hr (8 hr) or 4 hr followed by a 24 hr recovery period (24 hr). (b) Analysis of intracellular cysteine levels in the 24 hr group from (d). N = 3 replicates/group. (c) Western blot analysis of CDO1 and β-ACTIN levels in NRF2 knockout A549 cells reconstituted with either pLX317 empty (NRF2 -) or pLX317-NRF2 (NRF2 +), followed by re-expression of CDO1WT (WT), CDO1Y157F (Y157F), or GFP. Cells were grown in medium containing 200 μM or 10 μM CYS for 24 hr prior to lysate collection. (d) Individual proliferation data for Figure 3k. Cells were collected on the indicated days, stained with crystal violet and their absorbance at 600 nm determined. Proliferation of each line expressing CDO1WT was normalized to the proliferation of cells expressing CDO1Y157F. N = 3 replicates/group. (e) Correlation of CDO1 mRNA expression with cell growth inhibition (%). N = 13. For (a,b,e), cells were treated with 0.25 µg/ml doxycycline for 2 days prior to and during the assay and fresh medium was added 4 hr prior to sample collection.

https://doi.org/10.7554/eLife.45572.014
Figure 3—figure supplement 3—source data 1

NRF2 promotes CDO1 expression in NSCLC.

https://doi.org/10.7554/eLife.45572.015
Figure 4 with 1 supplement
CDO1 depletes cyst(e)ine, leading to its export as CSA.

(a) Schematic depicting intracellular cysteine metabolism. Following uptake of cystine via xCT, it is reduced to cysteine via the cellular antioxidant systems. Cysteine then enters glutathione synthesis mediated by GCL and GSS, or undergoes irreversible metabolism by CDO1 to CSA, and subsequently to HTAU by CSAD. (b) Time-dependent quantification (0, 1, 2, 4, 8, 24 hr) of intracellular cysteine (CYS) and cysteine sulfinic acid (CSA) concentrations following medium replenishment of CDO1WT-expressing (red) and CDO1Y157F-expressing (gray) A549 cells. *= CDO1WT vs. CDO1Y157F CYS, #= CDO1WT vs. CDO1Y157F CSA. N = 3 replicates/group, except 8 hr CYS for which N = 6 replicates/group. (c) Time-dependent quantification of GSH, HTAU, and TAU in the extracts from (b). N = 3 replicates/group. (d) Time-dependent quantification of the levels of (CYS)2 and CSA in the medium from the cells from (b). N = 3 replicates/group. (e) Intracellular CSA concentration of H1581 cells following expression of control (sgCON) or CDO1-targeting sgRNAs (sgCDO1 #1 and #2) and Cas9. N = 4 replicates/group. (f) Quantification of (CYS)2 consumption from the medium of the cells from (e). N = 4 replicates/group. (g) CSA production rate of NRF2 KO A549 cells reconstituted with either pLX317 empty (NRF2 -) or pLX317-NRF2 (NRF2 +), followed by expression of CDO1Y157F (Y157F) or CDO1WT (WT). N = 6 replicates/group. (h) Quantification of (CYS)2 consumption from the medium of the cells from (g). (i) Intracellular CSA concentration in H1975 and H1299 cells expressing either pLX317 empty (-) or pLX317-NRF2T80K (NRF2T80K), followed by expression of CDO1Y157F (Y157F) or CDO1WT (WT). N = 3 replicates/group. (j) Quantification of (CYS)2 consumption from the medium of the cells from (i). (k–m) CSA production rate of A549 (k), H1944 (l) and H322 (m) cells following expression of CDO1Y157F (Y157F) or CDO1WT (WT), and reconstituted with inactive KEAP1 (C273S), super repressor KEAP1 (C151S) or wild-type KEAP1 (WT). N = 3 replicates/group. For (g–m) cells were treated with 0.25 μg/ml doxycycline for 2 days prior to and during the assay and fresh medium was added 4 hr prior to sample collection.

https://doi.org/10.7554/eLife.45572.017
Figure 4—source data 1

CDO1 depletes cyst(e)ine, leading to its export as CSA.

https://doi.org/10.7554/eLife.45572.019
Figure 4—figure supplement 1
CSA only accounts for a fraction of CDO1-dependent (CYS)2 consumption.

(a) Time-dependent western blot analysis of CDO1 and β-Actin protein expression at 0, 1, 2, 4, 8, or 24 hr following media replenishment of CDO1WT-expressing and CDO1Y157F-expressing A549 cells. (b) Quantification of CDO1-dependent (CYS)2 consumption and CSA production as cysteine molar equivalents. Because (CYS)2 contains 2 molecules of cysteine, it counts as two molar equivalents. CDO1-dependent changes were determined by subtracting the rates of change in CDO1Y157F cells from CDO1WT cells. CSA accounts for 2–25% of CDO1-dependent (CYS)2 consumption. N = 3 replicates/group, except NRF2 KO A549 (N = 6 replicates). Cells were treated with 0.25 µg/ml doxycycline for 2 days prior to and during the assay and fresh medium was added 4 hr prior to sample collection (b) or prior the indicated time points (a).

https://doi.org/10.7554/eLife.45572.018
Figure 4—figure supplement 1—source data 1

CSA only accounts for a fraction of CDO1-dependent (CYS)2 consumption.

https://doi.org/10.7554/eLife.45572.020
Figure 5 with 4 supplements
CDO1 expressing cells produce SO32-, which further depletes (CYS)2 via sulfitolysis.

(a) Relative abundance of cysteine-related metabolites in cell extracts following CDO1WT or CDO1Y157F expression in A549 cells. Fresh medium was added 4 hr prior to extraction. Metabolites were analyzed by untargeted LC-MS. N.B. - cysteine and GSH were not derivatized with NEM for this analysis. N = 3 replicates/group. (b) GOT1 mediates the transamination of CSA to produce β-sulfinyl pyruvate, which decomposes to pyruvate and sulfite (SO32-). Sulfite hydrolyzes (CYS)2 to produce cysteine-S-sulfate (CYS-SO3-) in a process known as sulfitolysis. (c,d) Time-dependent quantification of the medium concentrations of and SO32- (c) and CYS-SO3- (d) concentrations from Figure 4d. (e) Time-dependent quantification of (CYS)2 and CYS-SO3- following the addition of 1 mM Na2SO3 to RPMI +10% FBS in the absence of cells. N = 3 replicates/group. (f) CYS-SO3- production rate of H1581 cells following expression of control (sgCON) or CDO1-targeting sgRNAs (sgCDO1 #1 and #2) and Cas9. N = 4 replicates/group. (g) CYS-SO3- production rate of NRF2 KO A549 cells reconstituted with either pLX317 empty (NRF2 -) or pLX317-NRF2 (NRF2 +), followed by expression of CDO1Y157F (Y157F) or CDO1WT (WT). N = 6 replicates/group. (h) CYS-SO3- production rate of H1975 and H1299 cells expressing either pLX317 empty (-) or pLX317-NRF2T80K (NRF2T80K), followed by expression of CDO1Y157F (Y157F) or CDO1WT (WT). N = 3 replicates/group. (i) CYS-SO3- production rate of A549 cells following expression of CDO1Y157F (Y157F) or CDO1WT (WT), and reconstituted with inactive KEAP1 (C273S), super repressor KEAP1 (C151S) or wild-type KEAP1 (WT). N = 3 replicates/group. (j) Intracellular SO32- concentration in the cells from (i). (k) CYS-SO3- production rate of H1944 cells following expression of CDO1Y157F (Y157F) or CDO1WT (WT), and reconstituted with inactive KEAP1 (C273S), super repressor KEAP1 (C151S) or wild-type KEAP1 (WT). N = 3 replicates/group. For a, c, d, g-k, cells were treated with 0.25 μg/ml doxycycline for 2 days prior to and during the assay.

https://doi.org/10.7554/eLife.45572.021
Figure 5—source data 1

CDO1 expressing cells produce SO32-, which further depletes (CYS)2 via sulfitolysis.

https://doi.org/10.7554/eLife.45572.030
Figure 5—figure supplement 1
Sulfite reacts with cystine and oxidized glutathione in the absence of cells.

(a) Verification of the stable-isotope labeled CYS-SO3- internal standard for absolute quantification. Stable isotope labeled CYS-SO3- was synthesized from [13C3, 15N]-cysteine and analyzed by LC-MS/MS based SRM against an authentic standard. RT, retention time. (b) Time-dependent quantification of (CYS)2 and CYS-SO3- following the addition of 1 mM CSA to RPMI +10% FBS in the absence of cells. (c) Relative quantification of (CYS)2 and CYS-SO3- following the addition of 1 mM SO32- to cystine-free RPMI +200 µM (Cys)2 + 10% dFBS in the absence of cells. (d) Relative quantification of GSSG and GS-SO3- following the addition of 1 mM SO32- to cystine-free RPMI +200 µM GSSG +10% dFBS in the absence of cells. (e–f) Quantitation of the levels of (Cys)2 and GSSG in blank (blank) or the conditioned medium (e) or cellular extracts (f) from H460 cells expressing CDO1WT (WT) or CDO1Y157F (Y157F). Cells were treated with 0.25 µg/ml doxycycline for 2 days prior to and during the assay, and fresh medium was added 6 hr (e) or 4 hr (f) prior to sample collection.

https://doi.org/10.7554/eLife.45572.022
Figure 5—figure supplement 1—source data 1

Sulfite reacts with cystine and oxidized glutathione in the absence of cells.

https://doi.org/10.7554/eLife.45572.023
Figure 5—figure supplement 2
MEFs preferentially use the CSA decarboxylation pathway.

(a) Western blot analysis of NRF2, CDO1, GOT1, CSAD and β-ACTIN levels following expression of GFP, CDO1WT, or CDO1Y157F in Keap1WT/WT (WT) and Keap1R554Q/R554Q (R554Q) MEFs. A549s expressing CDO1WT are included for comparison. (b–g) Quantitation of cystine (CYS2, (b), cysteine (CYS, (c), glutathione (GSH, (d), cysteine sulfinic acid (CSA, (e), hypotaurine (HTAU, (f) and taurine (TAU, (g) levels in Keap1R554Q/R554Q MEFs (red) compared to Keap1WT/WT MEFs (black) following expression of CDO1Y157F (Y157F) or CDO1WT (WT). N = 3 replicates/group. Cells were treated with 0.25 µg/ml doxycycline for 2 days prior to and during the assay, and fresh medium was added 4 hr prior to sample collection.

https://doi.org/10.7554/eLife.45572.024
Figure 5—figure supplement 2—source data 1

MEFs preferentially use the CSA decarboxylation pathway.

https://doi.org/10.7554/eLife.45572.025
Figure 5—figure supplement 3
NRF2 promotes the CDO1-dependent production of sulfite in NSCLC cell lines.

(a) Western blot analysis of GOT1, CSAD and HSP90 levels in NRF2LOW and NRF2HIGH NSCLC cell lines. Mouse liver is included as a positive control for GOT1 and CSAD expression. Cells were fed with fresh RPMI +10% FBS 4 hr prior to lysate collection. (b) Intracellular SO32- concentration of NRF2 KO A549 cells reconstituted with either pLX317 empty (NRF2 -) or pLX317-NRF2 (NRF2 +), followed by re-expression of CDO1Y157F (Y157F) or CDO1WT (WT). N = 3 replicates/group. (c) SO32- production rate of cells from (b). (d–f) Intracellular CSA concentration of A549 (d), H1944 (e) and H322 (f) cells following re-expression of CDO1Y157F (Y157F) or CDO1WT (WT), and reconstituted with inactive KEAP1 (C273S), super repressor KEAP1 (C151S) or wild-type KEAP1 (WT). N = 3 replicates/group. (g) CYS-SO3- production rate of H322 cells following expression of CDO1Y157F (Y157F) or CDO1WT (WT), and reconstituted with inactive KEAP1 (C273S), super repressor KEAP1 (C151S) or wild-type KEAP1 (WT). N = 3 replicates/group. (h–j) Analysis of CDO1-dependent CSA production (h), Cys-SO3- production (i) and cystine consumption (j) in the 24 hr group from Figure 3—figure supplement 3B. N = 3 replicates/group. For a–j), cells were treated with 0.25 µg/ml doxycycline for 2 days prior to and during the assay, and fresh medium was added 4 hr prior to sample collection.

https://doi.org/10.7554/eLife.45572.026
Figure 5—figure supplement 3—source data 1

NRF2 promotes the CDO1-dependent production of sulfite in NSCLC cell lines.

https://doi.org/10.7554/eLife.45572.027
Figure 5—figure supplement 4
CSA and sulfite are toxic to NSCLC cells.

(a) Analysis of cell viability following treatment of A549 cells with 0–10 mM HTAU, CSA or Na2SO3 for 5 days. Viable cells were analyzed with CellTiter-Glo and normalized to untreated cells. N = 3 replicates/group. (b) Analysis of cell viability following starvation of NRF2LOW (gray) and NRF2HIGH (red) NSCLC cell lines of cystine for 3 days. Cell numbers were analyzed with crystal violet and normalized to cells grown in cystine-containing media. N = 3 replicates/group. (c) Analysis of cell viability following treatment of NRF2LOW (gray) and NRF2HIGH (red) NSCLC cell lines with 0–10 mM Na2SO3 for 3 days. Viable cells were analyzed with CellTiter-Glo and normalized to untreated cells. N = 3 replicates/group. (d) Analysis of cell viability following treatment of A549 cells expressing GFP, CDO1Y157F or CDO1WT with 0–100 µM cumene hydroperoxide (CuH2O2) for 24 hr. A549s were treated with 0.25 µg/mL doxycycline for 3 days prior to CuH2O2 treatment to induce cDNA expression. Cell numbers were analyzed with crystal violet and normalized to untreated cells. *CDO1WT vs. CDO1Y157F. #CDO1WT vs. GFP. N = 3 replicates/group. (e) Analysis of cell viability following treatment of A549 cells with 2.5 mM CSA, 2.5 mM Na2SO3 or control for 3 days, followed by treatment with 0–100 µM CuH2O2 for 24 hr. Cell numbers were analyzed with crystal violet and normalized to untreated cells. *Control vs. CSA. #Control vs. Na2SO3. N = 3 replicates/group.

https://doi.org/10.7554/eLife.45572.028
Figure 5—figure supplement 4—source data 1

CSA and sulfite are toxic to NSCLC cells.

https://doi.org/10.7554/eLife.45572.029
Sulfitolysis is not required for the inhibition of proliferation by CDO1.

(a) Western blot analysis of GOT1 and β-ACTIN expression in parental (P) A549 and H460 cells, and GOT1 KO clones #1 and #2 for each cell line expressing empty pMXS (GOT1 -) and reconstituted with pMXS-GOT1 (GOT1 +). (b) Contribution of sulfitolysis to (CYS)2 consumption by CDO1. CDO1-dependent (CYS)2 consumption, and CSA, SO32-, and CYS-SO3- production were determined as cysteine molar equivalents. N = 3 replicates/group. (c) Analysis of the proliferation of GOT1 KO A549 cells from (a) expressing CDO1Y157F (Y157F) or CDO1WT (WT). Cells were collected on the indicated days, stained with crystal violet and their absorbance at 600 nm determined. N = 3 replicates/group. (d,e) Quantitation of glutathione (GSH) total (12C + 13C) levels (d) and 13C-labeling only (e) from 13C-cystine in cells from (A) following expression of CDO1Y157F (Y157F) or CDO1WT (WT). 13C-label is shown in red, while 12C-label is gray. Cells were labeled for 1 hr. N = 3 replicates/group. (f) Analysis of Coenzyme A (CoA) labeling from 13C, 15N-cystine in cells from (a) expressing CDO1Y157F (Y157F) or CDO1WT (WT). 13C,15N-label (M + 3) is shown in red, while 12C, 14N-label is gray. Cells were labeled for 4 hr. N = 3 replicates/group. (g) Analysis of protein synthesis rates with azidohomoalanine labeling in cells from (a) expressing CDO1Y157F (Y157F) or CDO1WT (WT). Cells treated with 50 μg/mL cyclohexamide (C) were used as a positive control for translation inhibition. N = 4 replicates/group. For b–g,) cells were treated with 0.25 μg/ml doxycycline for 2 days prior to and during the assay.

https://doi.org/10.7554/eLife.45572.031
Figure 6—source data 1

Sulfitolysis is not required for the inhibition of proliferation by CDO1.

https://doi.org/10.7554/eLife.45572.032
Figure 7 with 1 supplement
CDO1-dependent cystine reduction limits NADPH availability for cellular processes.

(a) The NADPH/NADP + ratio was assayed following expression of CDO1Y157F (Y157F) or CDO1WT (WT) in A549 and H460 GOT1 KO cells expressing empty pMXS (GOT1 -) or reconstituted with pMXS-GOT1 (GOT1 +). N = 3 replicates/group. (b) Quantitation of reduced glutathione (GSH) and oxidized glutathione (GSSG) in KEAP1C273S-expressing cells from Figure 4k,l following expression of CDO1Y157F (Y157F) or CDO1WT (WT). N = 3 replicates/group. (c) A549 and H460 GOT1 KO cells expressing empty pMXS (dashed line, open circle) or reconstituted with pMXS-GOT1 (solid line, solid circle), followed by expression of CDO1Y157F (gray) or CDO1WT (red), were treated with 0–25 μM cumene hydroperoxide (CuH2O2) for 24 hr. Cell numbers were analyzed using crystal violet and normalized to untreated cells. *=GOT1+ CDO1WT vs. CDO1Y157F, #=GOT1- CDO1WT vs. CDO1Y157F. N = 3 replicates/group. (d) Analysis of proline (M + 5) labeling from L-[U]-13C-glutamine in GOT1 KO A549 and H460 cells expressing empty pMXS (Control) or reconstituted with pMXS-GOT1 (GOT1), followed by expression of CDO1Y157F or CDO1WT. Proline M + 5 abundance was normalized to the abundance of its precursor glutamate M + 5, and then CDO1WT levels were normalized to CDO1Y157F. Cells were labeled for 1 hr in proline-free media. N = 3 replicates/group. (e,f) Mass isotopomer analysis of citrate labeling in GOT1 KO A549 (d) and H460 (e) cells expressing empty pMXS (Control) or reconstituted with pMXS-GOT1 (GOT1) following expression of CDO1Y157F or CDO1WT cultured with L-[U]-13C-glutamine for 4 hr. N = 3 replicates/group. For (a–f), cells were treated with 0.25 µg/ml doxycycline for 2 days prior to and during the assay.

https://doi.org/10.7554/eLife.45572.033
Figure 7—source data 1

CDO1-dependent cystine reduction limits NADPH availability for cellular processes.

https://doi.org/10.7554/eLife.45572.035
Figure 7—figure supplement 1
Cystine uptake and reduction depletes NADPH.

(a) The NADPH/NADP+ ratio was assayed following growth of A549 cells in medium containing 200 µM or 0 µM cystine for 24 hr. The ratio was normalized to cells grown in 200 µM cystine. N = 3 replicates/group. (b) H1581 cells were infected with lentivirus encoding Cas9 and non-targeting (sgCON) or CDO1-targeting (#1, #2) sgRNAs to delete CDO1 in the bulk population. Cells were treated with the indicated doses of CuH2O2 for 24 hr and cell number normalized to EtOH (Vehicle) treated cells. N = 3 replicates/group. (c) Soft agar growth of NSCLC cell lines expressing CDO1Y157F (Y157F) or CDO1WT (WT). Cells were treated with 0.25 µg/ml doxycycline for 2 days prior to and during the assay. N = 3 replicates/group. (d) Western blot analysis of CDO1 and HSP90 levels in GOT1 KO A549 and H460 cells expressing empty pMXS (Cntl) or reconstituted with pMXS-GOT1 (GOT1), followed by expression of CDO1Y157F or CDO1WT. Cells were cultured in either 200 µM or 10 µM (Cys)2, or 200 µM (Cys)2 + 0.5µM Erastin. (e) Quantification of medium (Cys)2 levels following Erastin treatment or growth in 10 µM (Cys)2. Control medium contains 200 µM (Cys)2. (f) Analysis of the proliferation of the cells from (d). Cells were fed with fresh media daily, collected on the indicated days, stained with crystal violet and their absorbance at 600 nm determined. N = 3 replicates/group. For (d,f), cells were treated with 0.25 µg/ml doxycycline for 2 days prior to and during the assay, and fresh medium was added 5 hr prior to sample collection (d) or during the assay (f).

https://doi.org/10.7554/eLife.45572.034
Figure 7—figure supplement 1—source data 1

Cystine uptake and reduction depletes NADPH.

https://doi.org/10.7554/eLife.45572.036
Nrf2 activation promotes Cdo1 expression in murine lung tumors in vivo.

(a) Overall survival of Trp53flox/flox and KrasG12D; Trp53flox/flox mice expressing homozygous Keap1WT (WT/WT), heterozygous for Keap1R554Q (R554Q/WT) or homozygous for Keap1R554Q (R554Q/R554Q). (b) Representative hematoxylin and eosin (H&E) stained section depicting lung hemorrhage in KrasG12D; Trp53flox/flox; Keap1R554Q/R554Q mice compared to KrasG12D; Trp53flox/flox; Keap1WT/WT mice. (c) (Left) Representative H&E image of lung tumor burden in KrasG12D; Trp53flox/flox mice expressing Keap1WT/WT, Keap1R554Q/WT or homozygous for Keap1R554Q/R554Q. (Right) Quantification of average lung tumor size per mouse. WT/WT (N = 10 mice), R554Q/WT (N = 8 mice), R554Q/R554Q (N = 10 mice). (d) (Left) Representative Nqo1 and Cdo1 immunohistochemistry of KrasG12D; Trp53flox/flox mouse tumors expressing Keap1WT/WT, Keap1R554Q/WT or homozygous for Keap1R554Q/R554Q. B, Bronchiole; T, Tumor. (Right) Average score (scale = 0–4) for Nqo1 staining across mice. WT/WT (N = 10 mice), R554Q/WT (N = 6 mice), R554Q/R554Q (N = 10 mice).

https://doi.org/10.7554/eLife.45572.037
Figure 8—source data 1

Nrf2 activation promotes Cdo1 expression in murine lung tumors.

https://doi.org/10.7554/eLife.45572.038
Model: CDO1 antagonizes the growth and survival of KEAP1MUT cells by producing toxic products and depleting NADPH.

(Left) Elevated intracellular cysteine (CYS) stabilizes CDO1, leading to the production of CSA and SO32-. In turn, SO32- depletes cystine via sulfitolysis to produce CYS-SO3-, further limiting cysteine availability and utilization. SO2 (sulfur dioxide) exists in equilibrium with SO32- and is a gas that may diffuse in and out of cells. The continual reduction of (CYS)2 to CYS depletes cellular NADPH, limiting its availability for cellular processes. (Right) CDO1 silencing promotes the accumulation of intracellular CYS and NADPH, and prevents wasting of CYS as CSA and toxic SO32-. GSR, glutathione reductase. TXNRD1, thioredoxin reductase 1. Fe-S, iron sulfur cluster.

https://doi.org/10.7554/eLife.45572.039

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional information
Chemical
compound, drug
CycloheximideSigma Aldrich01810–1G
Chemical
compound, drug
PyridineSigma Aldrich270970–1L
Chemical
compound, drug
CuCl2Sigma Aldrich203149–10
Chemical
compound, drug
Methoxylamine
hydrochloride
Sigma Aldrich226904–5G
Chemical
compound, drug
Ethyl acetate
(LC-MS grade)
Sigma Aldrich34972–1 L-R
Chemical
compound, drug
Methanol
(HPLC grade)
Sigma Aldrich34860–1 L-R
Chemical
compound, drug
CysteineSigma AldrichC6852-25G
Chemical
compound, drug
CystineSigma AldrichC6727-25G
Chemical
compound, drug
L-Cysteine S-sulfateSigma AldrichC2196-25MG
Chemical
compound, drug
L-Cysteine
sulfinic acid
Sigma Aldrich270881–1G
Chemical
compound, drug
HypotaurineSigma AldrichH1384-100MG
Chemical
compound, drug
Na2SO3Sigma Aldrich71988–250G
Chemical
compound, drug
[34S]-Na2SO3,Sigma Aldrich753572
Chemical
compound, drug
DoxycyclineSigma AldrichD9891-1G
Chemical
compound, drug
[13C2,15N]-Glutathione
trifluoroacetate salt,
Sigma Aldrich683620
Chemical
compound, drug
GSSGSigma AldrichG4376-250MG
Chemical
compound, drug
Decitabine
(5-Aza-2’-deoxycytidine)
Sigma AldrichA3656-5MG
Chemical
compound, drug
Cumene
hydroperoxide
Invitrogencomponent
of C10445
Chemical
compound, drug
N-EthylmaleimideChem-Impex International00142
Chemical
compound, drug
[13C5]-glutamine,Cambridge Isotope LabsCLM-1822-H-0.25
Chemical
compound, drug
[13C3, 15N]-CysteineCambridge Isotope LabsCNLM-3871-H-0.25
Chemical
compound, drug
[13C3]-Cysteine,Cambridge Isotope LabsCLM-4320-H-0.1
Chemical
compound, drug
[D4]-Cystine,Cambridge Isotope LabsDLM-1000–1
Chemical
compound, drug
[13C2]-TaurineCambridge Isotope LabsCLM-6622
Chemical
compound, drug
[D5]-GlutathioneSanta Cruz Biotechnologysc-489493
Chemical
compound, drug
[D4]-HypotaurineCDN IsotopesH1384-100MG
Chemical
compound, drug
Formic acidFisher ChemicalMFX04405
Chemical
compound, drug
NaOHFisher ChemicalSS256500
Chemical
compound, drug
MSTFA + 1% TMCS
solution
Fisher ChemicalTS-48915
Chemical
compound, drug
HPLC-grade
water
Fisher ChemicalW5-1
Chemical
compound, drug
Acetonitrile
(HPLC grade)
Honeywell34967
Chemical
compound, drug
ErastinCayman Chemical17754
Chemical
compound, drug
β-lapachoneDr. David Boothman
Chemical
compound, drug
puromycinInvivogenant-pr-1
Chemical
compound, drug
blasticidinInvivogenant-bl-1
Strain, strain
background (mouse)
LSL-KrasG12D(Jackson et al., 2001)
Strain, strain
background (mouse)
Trp53flox(Marino et al., 2000)
Strain, strain
background (mouse)
Keap1R554Qthis paperA minigene containing
a cDNA encoding wild
-type exons 3–5, followed
by a SV40 polyA signal,
was inserted upstream
of endogenous exon 3
of the Keap1 gene. Codon
554 in endogenous
exon four was mutated
from arginine to glutamine
Recombinant
DNA reagent
Keap1R554Q
genotyping primers
this paperCommon (R) 5’-GCCACC
CTATTCACAGACCA-3’
Mutant (F) 5’-ATGGCCA
CACTTTTCTGGAC 3’
WT (F) 5’-GGGGGTAGA
GGGAGGAGAAT-3’
WT PCR product = 326 bp
Mutant PCR product
= 584 bp
Recombinant
DNA reagent
adenoviral-CreUniversity of IowaVVC-U of Iowa-5
Recombinant
DNA reagent
pRRL-CDO1this paperThe LT3GEPIR vector
backbone (Fellmann et al., 2013)
was obtained from
Johannes Zuber and the
MiR-E cassette was excised
to generate pRRL-GFP. GFP
was excised and replaced
with CDO1 using pQTEV-CDO1
(addgene# 31292) as a PCR
template.
Recombinant
DNA reagent
pRRL-CDO1 Y157Fthis paperThe enzyme inactive
mutant (CDO1Y157F) was
generated from pRRL-CDO1
by site-directed mutagenesis
of the wild-type protein.
Recombinant
DNA reagent
pQTEV-CDO1Addgene31292
Recombinant
DNA reagent
lentiCRISPR-V2(Shalem et al., 2014)
Recombinant
DNA reagent
lentiCRISPR-V2
mCdo1 #2
this paperprogenitor: lentiCRISPR-V2;
oligonucleotides for sgRNAs
targeting mCDO1 (#2F 5’-
caccgCGAGAGCAATCCCGCCGAGT-
3’, #2R 5’-aaacACTCGGCG
GGATTGCTCTCGc-3’)
Recombinant
DNA reagent
lentiCRISPR-V2
mCdo1 #3
this paperprogenitor: lentiCRISPR-V2;
oligonucleotides for sgRNAs
targeting mCDO1 (#3F
5’-caccgCGAAGAGCTCATGTAA
GATG-3’, #3R 5’-aaacCATCTT
ACATGAGCTCTTCGc-3’)
Recombinant
DNA reagent
lentiCRISPR-V2
hCDO1 #1
this paperprogenitor: lentiCRISPR-V2;
oligonucleotides for sgRNAs
targeting hCDO1 (F - 5’-caccgGAT
GCGGATCAGATCAGCCA-3’,
R - 5’-aaacTGGCTGATCT
GATCCGCATCc-3’)
Recombinant
DNA reagent
lentiCRISPR-V2
hCDO1 #2
this paperprogenitor: lentiCRISPR-V2;
oligonucleotides for
sgRNAs targeting hCDO1
(R - 5’-caccgCGAGAGCGACCCC
ACCGAGT-3’,R - 5’-aaacACTCG
GTGGGGTCGCTCTCGc-3’)
Recombinant
DNA reagent
pLX317-NRF2Dr. Alice Berger,
(Berger et al., 2016)
Recombinant
DNA reagent
pLX317-NRF2T80KDr. Alice Berger,
(Berger et al., 2016)
Recombinant
DNA reagent
pLX317-emptythis paperpLX317-empty was
generated from pLX317
-NRF2 by site
directed mutagenesis.
Recombinant
DNA reagent
pLenti KEAP1WTthis paperThe KEAP1WT cDNA was
provided by Dr. Christian
Metallo (Zhao et al., 2018);
pLenti-GFP-blast was
generated from pLenti-
GFP-puro (addgene #17448)
Recombinant
DNA reagent
pLenti KEAP1C151Sthis paperThe KEAP1C151S cDNA
was provided by Dr. Christian
Metallo (Zhao et al., 2018);
pLenti-GFP-blast was
generated from pLenti-GFP
-puro (addgene #17448)
Recombinant
DNA reagent
pLenti KEAP1C273Sthis paperThe KEAP1C273S cDNA
was provided by Dr. Christian
Metallo (Zhao et al., 2018);
pLenti-GFP-blast was
generated from
pLenti-GFP-puro
(addgene #17448)
Recombinant
DNA reagent
plentiCRISPR-sgGOT1addgene72874
Recombinant
DNA reagent
pMXS-GOT1addgene72872
Recombinant
DNA reagent
pMXS-emptythis paperpMXS-empty was generated
from pMXS-GOT1 by
site-directed mutagenesis.
Recombinant
DNA reagent
pCMV-dR8.2 dvpraddgene8455
Recombinant
DNA reagent
pCMV-VSV-Gaddgene8454
Cell line
(Homo-sapiens)
Lenti-X 293TTakara632180
Cell line
(Homo-sapiens)
Phoenix-AMPHOATCCCRL-3213RRID:CVCL_H716
Cell line
(Homo-sapiens)
Calu3Dr John Minna,
Hamon Cancer Center
Collection (University
of Texas-Southwestern
Medical Center)
(DeNicola et al., 2015)RRID:CVCL_0609
Cell line
(Homo-sapiens)
H1581Dr John Minna,
Hamon Cancer Center
Collection (University
of Texas-Southwestern
Medical Center)
(DeNicola et al., 2015)RRID:CVCL_1479
Cell line
(Homo-sapiens)
H1975Dr John Minna,
Hamon Cancer Center
Collection (University of
Texas-Southwestern
Medical Center)
(DeNicola et al., 2015)RRID:CVCL_1511
Cell line
(Homo-sapiens)
H2087Dr John Minna,
Hamon Cancer Center
Collection (University
of Texas-Southwestern
Medical Center)
(DeNicola et al., 2015)RRID:CVCL_1524
Cell line
(Homo-sapiens)
H2347Dr John Minna,
Hamon Cancer Center
Collection (University
of Texas-Southwestern
Medical Center)
(DeNicola et al., 2015)RRID:CVCL_1550
Cell line
(Homo-sapiens)
H1792Dr John Minna,
Hamon Cancer Center
Collection (University
of Texas-Southwestern
Medical Center)
(DeNicola et al., 2015)RRID:CVCL_1495
Cell line
(Homo-sapiens)
H1944Dr John Minna,
Hamon Cancer Center
Collection (University
of Texas-Southwestern
Medical Center)
(DeNicola et al., 2015)RRID:CVCL_1508
Cell line
(Homo-sapiens)
H322Dr John Minna,
Hamon Cancer Center
Collection (University
of Texas-Southwestern
Medical Center)
(DeNicola et al., 2015)RRID:CVCL_1556
Cell line
(Homo-sapiens)
H460Dr John Minna,
Hamon Cancer Center
Collection (University
of Texas-Southwestern
Medical Center)
(DeNicola et al., 2015)RRID:CVCL_0459
Cell line
(Homo-sapiens)
HCC15Dr John Minna,
Hamon Cancer Center
Collection (University
of Texas-Southwestern
Medical Center)
(DeNicola et al., 2015)RRID:CVCL_2057
Cell line
(Homo-sapiens)
H2009ATCCCRL-5911RRID:CVCL_1514
Cell line
(Homo-sapiens)
H1299ATCCCRL-5803RRID:CVCL_0060
Cell line
(Homo-sapiens)
H1993ATCCCRL-5909RRID:CVCL_1512
Cell line
(Homo-sapiens)
H441ATCCHTB-174RRID:CVCL_1561
Cell line
(Homo-sapiens)
A549ATCCCCL-185RRID:CVCL_0023
Cell line
(Homo-sapiens)
NRF2 KO A549Dr. Laureano
de la Vega
(Torrente et al., 2017)
Cell line (Mus
musculus)
mouse embryonic
fibroblasts
this paperMEFs were isolated
from E13.5–14.5 day
old embryos
Commercial
assay or kit
JetPRIMEVWR89129–922
Commercial
assay or kit
ImmPRESS HRP
anti-rabbit kit
Vector LabsMP-7451
Commercial
assay or kit
E.Z.N.A. Total RNA Kit IOmega BiotekR6834-02
Commercial
assay or kit
PrimeScript
RT Master Mix
TakaraRR036A
Commercial
assay or kit
Taqman gene
expression assays
- mCdo1
Thermo Fisher4448892,
Mm00473573_m1
Commercial
assay or kit
Taqman gene
expression assays
- hCDO1
Thermo Fisher4448892,
Hs01039954_m1
Commercial
assay or kit
Taqman gene
expression assays
- mActb
Thermo Fisher4448892,
Mm02619580_g1
Commercial
assay or kit
Taqman gene
expression assays
- hACTB
Thermo Fisher#4333762F
Commercial
assay or kit
CellTiter-GloPromegaG7571
Commercial
assay or kit
DC protein assayBiorad500112
AntibodyNRF2 (Rabbit mAb)Cell Signaling
Technologies
12721RRID:AB_2715528;
1:1000 WB
AntibodyHSP90 (Rabbit pAb)Cell Signaling
Technologies
4874 sRRID:AB_2121214;
1:5000 WB
AntibodyTXN1 (Rabbit mAb)Cell Signaling
Technologies
2429SRRID:AB_2272594;
1:1000 WB
Antibodyα-tubulin (Mouse mAb)Santa Cruzsc-8035, clone TU-02RRID:AB_628408;
1:500 WB
Antibodyβ-actin (Mouse mAb)Thermo FisherAM4302, clone AC-15RRID:AB_2536382;
1:100,000 WB
AntibodyCDO1 (Rabbit pAb)Abcamab53436RRID:AB_940958;
1:1000 WB, discontinued,
verified with Sigma
CDO1 antibody
AntibodyxCT (Rabbit pAb)Abcamab37185RRID:AB_778944;
1:1000 WB
AntibodyGOT1 (Rabbit pAb)BiovisionA1272RRID:AB_2801348;
1:1000 WB
AntibodyCSAD (Rabbit pAb)LSBioC375526RRID:AB_2801349;
1:1000 WB
AntibodyAnti-NQO1
antibody
(Rabbit pAb)
Sigma AldrichHPA007308RRID:AB_1079501;
1:100, IHC; 1:1000 WB
AntibodyAnti-CDO1
antibody
(Rabbit pAb)
Sigma AldrichHPA057503RRID:AB_2683451;
1:100, IHC
Otherdialyzed FBSSigma AldrichF0392
OtherCysteine/cystine,
methionine and
glutamine free RPMI
MP Biomedicals91646454
OtherCysteine/cystine,
methionine, pyruvate
and glutamine free
DMEM
Gibco21013024
Software,
algorithm
MZmine 2(Pluskal et al., 2010)Version 2.30
Software,
algorithm
Thermo Xcaliber
Qual Browser
Thermo FisherVersion 4.0.27.19
Software,
algorithm
EI Mavenhttps://elucidatainc.github.io/ElMavenVersion 0.3.1
Software,
algorithm
Agilent Mass Hunter
Workstation Software
- Qualitative Analysis
AgilentVersion B.07.00
Software,
algorithm
Image Scope softwareAperio

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all figures.

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