[go: up one dir, main page]
Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Systemic depletion of L-cyst(e)ine with cyst(e)inase increases reactive oxygen species and suppresses tumor growth

Nature Medicine volume 23, pages 120–127 (2017)Cite this article

Subjects

Abstract

Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than do non-malignant cells because of genetic alterations and abnormal growth; as a result, maintenance of the antioxidant glutathione (GSH) is essential for their survival and proliferation1,2,3. Under conditions of elevated ROS, endogenous L-cysteine (L-Cys) production is insufficient for GSH synthesis. This necessitates uptake of L-Cys that is predominantly in its disulfide form, L-cystine (CSSC), via the xCT(−) transporter. We show that administration of an engineered and pharmacologically optimized human cyst(e)inase enzyme mediates sustained depletion of the extracellular L-Cys and CSSC pool in mice and non-human primates. Treatment with this enzyme selectively causes cell cycle arrest and death in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS; yet this treatment results in no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts, reduced tumor growth in both prostate and breast cancer xenografts and doubled the median survival time of TCL1-Tg:p53−/− mice, which develop disease resembling human chronic lymphocytic leukemia. It was observed that enzyme-mediated depletion of the serum L-Cys and CSSC pool suppresses the growth of multiple tumors, yet is very well tolerated for prolonged periods, suggesting that cyst(e)inase represents a safe and effective therapeutic modality for inactivating antioxidant cellular responses in a wide range of malignancies4,5.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Engineered human cyst(e)inase for cancer treatment.
Figure 2: Effect of cyst(e)inase in PCa cells.
Figure 3: Pharmacokinetics, pharmacodynamics and efficacy of cyst(e)inase administration in mice.
Figure 4: In vitro and in vivo efficacy of cyst(e)inase in the TCL1-Tg:p53−/− mouse model and primary CLL cells.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Trachootham, D., Alexandre, J. & Huang, P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat. Rev. Drug Discov. 8, 579–591 (2009).

    CAS  PubMed  Google Scholar 

  2. Dixon, S.J. & Stockwell, B.R. The role of iron and reactive oxygen species in cell death. Nat. Chem. Biol. 10, 9–17 (2014).

    CAS  PubMed  Google Scholar 

  3. Harris, I.S. et al. Glutathione and thioredoxin antioxidant pathways synergize to drive cancer initiation and progression. Cancer Cell 27, 211–222 (2015).

    CAS  PubMed  Google Scholar 

  4. Liu, H., Zhi, Y., Geng, G., Yu, Z. & Xu, H. [Effect of phenethyl isothiocyanate given at different duration of gestation on the outcome of pregnancy in rats]. J. Hyg. Res. 40, 283–286 (2011).

    CAS  Google Scholar 

  5. Reliene, R. & Schiestl, R.H. Glutathione depletion by buthionine sulfoximine induces DNA deletions in mice. Carcinogenesis 27, 240–244 (2006).

    CAS  PubMed  Google Scholar 

  6. Belalcázar, A.D., Ball, J.G., Frost, L.M., Valentovic, M.A. & Wilkinson, J. IV. Transsulfuration is a significant source of sulfur for glutathione production in human mammary epithelial cells. ISRN Biochem. 2013, 637897 (2013).

    PubMed Central  Google Scholar 

  7. Persa, C., Osmotherly, K., Chao-Wei Chen, K., Moon, S. & Lou, M.F. The distribution of cystathionine β-synthase (CBS) in the eye: implication of the presence of a trans-sulfuration pathway for oxidative stress defense. Exp. Eye Res. 83, 817–823 (2006).

    CAS  PubMed  Google Scholar 

  8. Vitvitsky, V., Thomas, M., Ghorpade, A., Gendelman, H.E. & Banerjee, R. A functional transsulfuration pathway in the brain links to glutathione homeostasis. J. Biol. Chem. 281, 35785–35793 (2006).

    CAS  PubMed  Google Scholar 

  9. You, X.-J. et al. Expression of cystathionine β-synthase and cystathionine γ-lyase in human pregnant myometrium and their roles in the control of uterine contractility. PLoS One 6, e23788 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Zhao, H. et al. Frequent epigenetic silencing of the folate-metabolising gene cystathionine-beta-synthase in gastrointestinal cancer. PLoS One 7, e49683 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Kim, J. et al. Expression of cystathionine β-synthase is downregulated in hepatocellular carcinoma and associated with poor prognosis. Oncol. Rep. 21, 1449–1454 (2009).

    CAS  PubMed  Google Scholar 

  12. Timmerman, L.A. et al. Glutamine sensitivity analysis identifies the xCT antiporter as a common triple-negative breast tumor therapeutic target. Cancer Cell 24, 853–863 (2013).

    Google Scholar 

  13. Takeuchi, S. et al. Increased xCT expression correlates with tumor invasion and outcome in patients with glioblastomas. Neurosurgery 72, 33–41 discussion 41, (2013).

    PubMed  Google Scholar 

  14. Shiozaki, A. et al. xCT, component of cysteine/glutamate transporter, as an independent prognostic factor in human esophageal squamous cell carcinoma. J. Gastroenterol. bok49, 1–11 (2014).

    Google Scholar 

  15. Doxsee, D.W. et al. Sulfasalazine-induced cystine starvation: potential use for prostate cancer therapy. Prostate 67, 162–171 (2007).

    CAS  PubMed  Google Scholar 

  16. Zhang, W. et al. Stromal control of cystine metabolism promotes cancer cell survival in chronic lymphocytic leukaemia. Nat. Cell Biol. 14, 276–286 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Stone, E. et al. De novo engineering of a human cystathionine-γ-lyase for systemic L-methionine depletion cancer therapy. ACS Chem. Biol. 7, 1822–1829 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Huang, S. et al. Site-directed mutagenesis on human cystathionine-γ-lyase reveals insights into the modulation of H2S production. J. Mol. Biol. 396, 708–718 (2010).

    CAS  PubMed  Google Scholar 

  19. Kumar, B., Koul, S., Khandrika, L., Meacham, R.B. & Koul, H.K. Oxidative stress is inherent in prostate cancer cells and is required for aggressive phenotype. Cancer Res. 68, 1777–1785 (2008).

    CAS  PubMed  Google Scholar 

  20. Afanas'ev, I. Reactive oxygen species signaling in cancer: comparison with aging. Aging Dis. 2, 219–230 (2011).

    PubMed  Google Scholar 

  21. Höll, M. et al. ROS signaling by NADPH oxidase 5 modulates the proliferation and survival of prostate carcinoma cells. Mol. Carcinog. 55, 27–39 (2016).

    PubMed  Google Scholar 

  22. Saha, A., Blando, J., Fernandez, I., Kiguchi, K. & DiGiovanni, J. Linneg Sca-1high CD49fhigh prostate cancer cells derived from the Hi-Myc mouse model are tumor-initiating cells with basal-epithelial characteristics and differentiation potential in vitro and in vivo. Oncotarget 7, 25194–25207 (2016).

    PubMed  PubMed Central  Google Scholar 

  23. Ellwood-Yen, K. et al. Myc-driven murine prostate cancer shares molecular features with human prostate tumors. Cancer Cell 4, 223–238 (2003).

    CAS  PubMed  Google Scholar 

  24. Cardaci, S., Filomeni, G. & Ciriolo, M.R. Redox implications of AMPK-mediated signal transduction beyond energetic clues. J. Cell Sci. 125, 2115–2125 (2012).

    CAS  PubMed  Google Scholar 

  25. Ghislat, G., Patron, M., Rizzuto, R. & Knecht, E. Withdrawal of essential amino acids increases autophagy by a pathway involving Ca2+/calmodulin-dependent kinase kinase-β (CaMKK-β). J. Biol. Chem. 287, 38625–38636 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Mungai, P.T. et al. Hypoxia triggers AMPK activation through reactive oxygen species-mediated activation of calcium release-activated calcium channels. Mol. Cell. Biol. 31, 3531–3545 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Liang, J. et al. The energy sensing LKB1-AMPK pathway regulates p27(kip1) phosphorylation mediating the decision to enter autophagy or apoptosis. Nat. Cell Biol. 9, 218–224 (2007).

    CAS  PubMed  Google Scholar 

  28. Jones, R.G. et al. AMP-activated protein kinase induces a p53-dependent metabolic checkpoint. Mol. Cell 18, 283–293 (2005).

    CAS  PubMed  Google Scholar 

  29. Foye, W., Lemke, T. & Williams, D. Foye's Principles of Medicinal Chemistry, 2007 (Lippincott Williams & Wilkins, 2007).

  30. West, G.B. & Brown, J.H. The origin of allometric scaling laws in biology from genomes to ecosystems: towards a quantitative unifying theory of biological structure and organization. J. Exp. Biol. 208, 1575–1592 (2005).

    PubMed  Google Scholar 

  31. Lewerenz, J. et al. The cystine/glutamate antiporter system x(c)(-) in health and disease: from molecular mechanisms to novel therapeutic opportunities. Antioxid. Redox Signal. 18, 522–555 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Kato, S., Ishita, S., Sugawara, K. & Mawatari, K. Cystine/glutamate antiporter expression in retinal Müller glial cells: implications for DL-alpha-aminoadipate toxicity. Neuroscience 57, 473–482 (1993).

    CAS  PubMed  Google Scholar 

  33. Narang, V.S., Pauletti, G.M., Gout, P.W., Buckley, D.J. & Buckley, A.R. Suppression of cystine uptake by sulfasalazine inhibits proliferation of human mammary carcinoma cells. Anticancer Res. 23, 4571–4579 (2003).

    CAS  PubMed  Google Scholar 

  34. Fang, J., Lu, J. & Holmgren, A. Thioredoxin reductase is irreversibly modified by curcumin: a novel molecular mechanism for its anticancer activity. J. Biol. Chem. 280, 25284–25290 (2005).

    CAS  PubMed  Google Scholar 

  35. Kanai, M. et al. A phase I study investigating the safety and pharmacokinetics of highly bioavailable curcumin (Theracurmin) in cancer patients. Cancer Chemother. Pharmacol. 71, 1521–1530 (2013).

    CAS  PubMed  Google Scholar 

  36. Carroll, R.E. et al. Phase IIa clinical trial of curcumin for the prevention of colorectal neoplasia. Cancer Prev. Res. (Phila.) 4, 354–364 (2011).

    CAS  Google Scholar 

  37. Kanai, M. et al. A phase I/II study of gemcitabine-based chemotherapy plus curcumin for patients with gemcitabine-resistant pancreatic cancer. Cancer Chemother. Pharmacol. 68, 157–164 (2011).

    CAS  PubMed  Google Scholar 

  38. Liu, J. et al. Loss of p53 and altered miR15-a/16-1→MCL-1 pathway in CLL: insights from TCL1-Tg:p53(−/−) mouse model and primary human leukemia cells. Leukemia 28, 118–128 (2014).

    CAS  PubMed  Google Scholar 

  39. Olsen, L.F., Issinger, O.-G. & Guerra, B. The yin and yang of redox regulation. Redox Rep. 18, 245–252 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Vousden, K.H. & Ryan, K.M. p53 and metabolism. Nat. Rev. Cancer 9, 691–700 (2009).

    CAS  PubMed  Google Scholar 

  41. Zhang, W. et al. Expression profiling of homocysteine junction enzymes in the NCI60 panel of human cancer cell lines. Cancer Res. 65, 1554–1560 (2005).

    CAS  PubMed  Google Scholar 

  42. Mandal, P.K. et al. System x(c)- and thioredoxin reductase 1 cooperatively rescue glutathione deficiency. J. Biol. Chem. 285, 22244–22253 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Pader, I. et al. Thioredoxin-related protein of 14 kDa is an efficient L-cystine reductase and S-denitrosylase. Proc. Natl. Acad. Sci. USA 111, 6964–6969 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Trachootham, D. et al. Effective elimination of fludarabine-resistant CLL cells by PEITC through a redox-mediated mechanism. Blood 112, 1912–1922 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Zhang, W. et al. Effective elimination of chronic lymphocytic leukemia cells in the stromal microenvironment by a novel drug combination strategy using redox-mediated mechanisms. Mol. Med. Rep. 12, 7374–7388 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Lee, W.-J. et al. Nonautophagic cytoplasmic vacuolation death induction in human PC-3M prostate cancer by curcumin through reactive oxygen species–mediated endoplasmic reticulum stress. Sci. Rep. 5, 10420 (2015).

    PubMed  PubMed Central  Google Scholar 

  47. Sánchez, Y., Simón, G.P., Calviño, E., de Blas, E. & Aller, P. Curcumin stimulates reactive oxygen species production and potentiates apoptosis induction by the antitumor drugs arsenic trioxide and lonidamine in human myeloid leukemia cell lines. J. Pharmacol. Exp. Ther. 335, 114–123 (2010).

    PubMed  Google Scholar 

  48. Chaiswing, L., Zhong, W., Liang, Y., Jones, D.P. & Oberley, T.D. Regulation of prostate cancer cell invasion by modulation of extra- and intracellular redox balance. Free Radic. Biol. Med. 52, 452–461 (2012).

    CAS  PubMed  Google Scholar 

  49. Minor, W. & Otwinowski, Z. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    PubMed  Google Scholar 

  50. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007).

    CAS  Google Scholar 

  51. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. DeLano, W.L. The PyMOL molecular graphics system (Schrödinger LLC, 2002).

  53. Cheson, B.D. et al. Guidelines for clinical protocols for chronic lymphocytic leukemia: recommendations of the National Cancer Institute-sponsored working group. Am. J. Hematol. 29, 152–163 (1988).

    CAS  PubMed  Google Scholar 

  54. Huang, P., Sandoval, A., Van Den Neste, E., Keating, M.J. & Plunkett, W. Inhibition of RNA transcription: a biochemical mechanism of action against chronic lymphocytic leukemia cells by fludarabine. Leukemia 14, 1405–1413 (2000).

    CAS  PubMed  Google Scholar 

  55. Pelicano, H. et al. Inhibition of mitochondrial respiration: a novel strategy to enhance drug-induced apoptosis in human leukemia cells by a reactive oxygen species-mediated mechanism. J. Biol. Chem. 278, 37832–37839 (2003).

    CAS  PubMed  Google Scholar 

  56. Tiziani, S. et al. Optimized metabolite extraction from blood serum for 1H nuclear magnetic resonance spectroscopy. Anal. Biochem. 377, 16–23 (2008).

    CAS  PubMed  Google Scholar 

  57. Okuda, S. et al. KEGG Atlas mapping for global analysis of metabolic pathways. Nucleic Acids Res. 36, W423–W426 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Wishart, D.S. et al. HMDB: the human metabolome database. Nucleic Acids Res. 35, D521–D526 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Sana, T.R., Roark, J.C., Li, X., Waddell, K. & Fischer, S.M. Molecular formula and METLIN Personal Metabolite Database matching applied to the identification of compounds generated by LC/TOF-MS. J Biomol Tech 19, 258–266 (2008).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to D. Lowe (Aeglea Biotherapeutics), O. Paley, and M. Bonem for assistance with various aspects of this work and data interpretation and to L. Beltran and S. Carbajal for their expert technical assistance. Instrumentation and technical assistance for parts of this work were provided by the Macromolecular Crystallography Facility, with financial support from the College of Natural Sciences, the Office of the Executive Vice President and Provost, and the Institute for Cellular and Molecular Biology at the University of Texas at Austin. Diffraction data was collected at the Advanced Light Source at the Berkeley Center for Structural Biology which is supported in part by the National Institutes of Health, National Institute of General Medical Sciences, and the Howard Hughes Medical Institute. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under Contract No. DE-AC02-05CH11231. This work was supported by funding from: Welch Foundation F-1778 (Y.J.Z.), NIH 1 R01 GM104896 (Y.J.Z.), NIH 1 R01 CA172724 (P.H.), NIH 1 R01 CA154754 (G.G., E.S.), NIH 1 RO1 CA189623 (E.S., J.D. and G.G.), NCI P30 CA54174 (S. Tiziani) and Aeglea Biotherapeutics.

Author information

Author notes

  1. Shira L Cramer, Achinto Saha and Jinyun Liu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA

    Shira L Cramer, Kendra Triplett, Candice Lamb & George Georgiou

  2. Division of Pharmacology and Toxicology and Dell Pediatric Research Institute, University of Texas at Austin, Austin, Texas, USA

    Achinto Saha & John DiGiovanni

  3. Department of Translational Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston, Texas, USA

    Jinyun Liu & Peng Huang

  4. Department of Nutritional Sciences, University of Texas at Austin, Austin, Texas, USA

    Surendar Tadi & Stefano Tiziani

  5. Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA

    Wupeng Yan, Kendra Triplett, Candice Lamb, Yan Jessie Zhang, George Georgiou & Everett Stone

  6. Aeglea Biotherapeutics, Austin, Texas, USA,

    Susan E Alters & Scott Rowlinson

  7. Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston, Texas, USA

    Michael J Keating

Authors
  1. Shira L Cramer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Achinto Saha
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jinyun Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Surendar Tadi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Stefano Tiziani
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wupeng Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kendra Triplett
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Candice Lamb
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Susan E Alters
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Scott Rowlinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Yan Jessie Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Michael J Keating
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Peng Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. John DiGiovanni
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. George Georgiou
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Everett Stone
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.L.C., A.S., J.L., S. Tadi, W.Y., K.T., C.L., and Y.J.Z. performed key experiments; E.S., P.H., J.D. and G.G. conceived and designed the research; S.L.C., A.S., J.L., S. Tadi, S. Tiziani, W.Y., K.T., S.E.A., S.R., Y.J.Z., M.J.K, P.H., J.D., G.G., and E.S. analyzed data; M.J.K. provided critical materials (CLL blood samples); S.L.C., A.S., J.L., G.G., J.D. and E.S. wrote the manuscript.

Corresponding authors

Correspondence to George Georgiou or Everett Stone.

Ethics declarations

Competing interests

G. Georgiou and E. Stone are inventors on intellectual property related to this work, and G. Georgiou, E. Stone, S. Rowlinson and S. Alters have an equity interest in Aeglea Biotherapeutics, a company pursuing the commercial development of this technology.

Supplementary information

Supplementary Figures, Table & Text

Supplementary Figures 1–14, Supplementary Table 1, Supplementary Methods and Dataset (PDF 3637 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cramer, S., Saha, A., Liu, J. et al. Systemic depletion of L-cyst(e)ine with cyst(e)inase increases reactive oxygen species and suppresses tumor growth. Nat Med 23, 120–127 (2017). https://doi.org/10.1038/nm.4232

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.4232

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer