Glutathione biosynthesis is a metabolic vulnerability in PI(3)K/Akt-driven breast cancer

  • Nature Cell Biology volume 18, pages 572578 (2016)
  • doi:10.1038/ncb3341
  • Download Citation


Cancer cells often select for mutations that enhance signalling through pathways that promote anabolic metabolism1. Although the PI(3)K/Akt signalling pathway, which is frequently dysregulated in breast cancer2, is a well-established regulator of central glucose metabolism and aerobic glycolysis3,4, its regulation of other metabolic processes required for tumour growth is not well defined. Here we report that in mammary epithelial cells, oncogenic PI(3)K/Akt stimulates glutathione (GSH) biosynthesis by stabilizing and activating NRF2 to upregulate the GSH biosynthetic genes. Increased NRF2 stability is dependent on the Akt-mediated accumulation of p21Cip1/WAF1 and GSK-3β inhibition. Consistently, in human breast tumours, upregulation of NRF2 targets is associated with PI(3)K pathway mutation status and oncogenic Akt activation. Elevated GSH biosynthesis is required for PI(3)K/Akt-driven resistance to oxidative stress, initiation of tumour spheroids, and anchorage-independent growth. Furthermore, inhibition of GSH biosynthesis with buthionine sulfoximine synergizes with cisplatin to selectively induce tumour regression in PI(3)K pathway mutant breast cancer cells, both in vitro and in vivo. Our findings provide insight into GSH biosynthesis as a metabolic vulnerability associated with PI(3)K pathway mutant breast cancers.

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  1. 1.

    , & Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324, 1029–1033 (2009).

  2. 2.

    Targeting PI3K signalling in cancer: opportunities, challenges and limitations. Nat. Rev. Cancer 9, 550–562 (2009).

  3. 3.

    et al. Akt stimulates aerobic glycolysis in cancer cells. Cancer Res. 64, 3892–3899 (2004).

  4. 4.

    & Is Akt the “Warburg kinase”?—Akt-energy metabolism interactions and oncogenesis. Semin. Cancer Biol. 19, 25–31 (2009).

  5. 5.

    et al. A transforming mutation in the pleckstrin homology domain of AKT1 in cancer. Nature 448, 439–444 (2007).

  6. 6.

    et al. The landscape of cancer genes and mutational processes in breast cancer. Nature 486, 400–404 (2012).

  7. 7.

    et al. Knock in of the AKT1 E17K mutation in human breast epithelial cells does not recapitulate oncogenic PIK3CA mutations. Oncogene 29, 2337–2345 (2010).

  8. 8.

    et al. Differential effects of AKT1(p.E17K) expression on human mammary luminal epithelial and myoepithelial cells. Hum. Mutat. 33, 1216–1227 (2012).

  9. 9.

    , , & A positive/negative ion-switching, targeted mass spectrometry-based metabolomics platform for bodily fluids, cells, and fresh and fixed tissue. Nat. Protoc. 7, 872–881 (2012).

  10. 10.

    et al. Activation of a metabolic gene regulatory network downstream of mTOR complex 1. Mol. Cell 39, 171–183 (2010).

  11. 11.

    , , & Stimulation of de novo pyrimidine synthesis by growth signaling through mTOR and S6K1. Science 339, 1323–1328 (2013).

  12. 12.

    et al. Glutamine supports pancreatic cancer growth through a KRAS-regulated metabolic pathway. Nature 496, 101–105 (2013).

  13. 13.

    , , , & SOD2 to SOD1 switch in breast cancer. J. Biol. Chem. 289, 5412–5416 (2014).

  14. 14.

    et al. Superoxide dismutase 1 (SOD1) is a target for a small molecule identified in a screen for inhibitors of the growth of lung adenocarcinoma cell lines. Proc. Natl Acad. Sci. USA 108, 16375–16380 (2011).

  15. 15.

    & NRF2 and cancer: the good, the bad and the importance of context. Nat. Rev. Cancer 12, 564–571 (2012).

  16. 16.

    & The emerging role of the Nrf2-Keap1 signaling pathway in cancer. Genes Dev. 27, 2179–2191 (2013).

  17. 17.

    et al. Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis. Nature 475, 106–109 (2011).

  18. 18.

    et al. Direct interaction between Nrf2 and p21(Cip1/WAF1) upregulates the Nrf2-mediated antioxidant response. Mol. Cell 34, 663–673 (2009).

  19. 19.

    , & AKT/PKB phosphorylation of p21Cip/WAF1 enhances protein stability of p21Cip/WAF1 and promotes cell survival. J. Biol. Chem. 277, 11352–11361 (2002).

  20. 20.

    et al. SCF/β-TrCP promotes glycogen synthase kinase 3-dependent degradation of the Nrf2 transcription factor in a Keap1-independent manner. Mol. Cell Biol. 31, 1121–1133 (2011).

  21. 21.

    The Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature 490, 61–70 (2012).

  22. 22.

    et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149, 1060–1072 (2012).

  23. 23.

    et al. Regulation of ferroptotic cancer cell death by GPX4. Cell 156, 317–331 (2014).

  24. 24.

    , & Buthionine sulfoximine induced growth inhibition in human lung carcinoma cells does not correlate with glutathione depletion. Cell Biol. Toxicol. 7, 249–261 (1991).

  25. 25.

    Cell signaling. H2O2, a necessary evil for cell signaling. Science 312, 1882–1883 (2006).

  26. 26.

    , & Modulation of oxidative stress as an anticancer strategy. Nat. Rev. Drug Discov. 12, 931–947 (2013).

  27. 27.

    , , & PTEN-deficient tumors depend on AKT2 for maintenance and survival. Cancer Discov. 4, 942–955 (2014).

  28. 28.

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

  29. 29.

    et al. Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment. Nature 461, 109–113 (2009).

  30. 30.

    & Cisplatin in cancer therapy: molecular mechanisms of action. Eur. J. Pharmacol. 740, 364–378 (2014).

  31. 31.

    & Role of glutathione in the regulation of Cisplatin resistance in cancer chemotherapy. Met. Based Drugs 2010, 430939 (2010).

  32. 32.

    & Tumor-infiltrating lymphocytes and response to platinum in triple-negative breast cancer. J. Clin. Oncol. 33, 969–971 (2015).

  33. 33.

    et al. Oncogenic PI3K mutations lead to NF-κB-dependent cytokine expression following growth factor deprivation. Cancer Res. 72, 3260–3269 (2012).

  34. 34.

    & Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat. Protoc. 1, 1112–1116 (2006).

  35. 35.

    et al. Quantitative determination of apoptotic death in cultured human pancreatic cancer cells by propidium iodide and digitonin. Cancer Lett. 142, 129–137 (1999).

  36. 36.

    Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal. Biochem. 27, 502–522 (1969).

  37. 37.

    & NFAT induces breast cancer cell invasion by promoting the induction of cyclooxygenase-2. J. Biol. Chem. 281, 12210–12217 (2006).

  38. 38.

    , & Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30, 256–268 (2003).

  39. 39.

    , , & Methods for long-term 17β-estradiol administration to mice. Gen. Comp. Endocrinol. 175, 188–193 (2012).

  40. 40.

    et al. A novel model of continuous depletion of glutathione in mice treated with L-buthionine (S, R)-sulfoximine. J. Toxicol. Sci. 28, 455–469 (2003).

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We thank J. Brugge, B. Manning, J. Blenis, A. Beck, I. Harris and members of the Toker and Cantley laboratories for suggestions; A. Baldwin (Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, USA), Y. R. Chin (Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, USA) and G. DeNicola (Department of Medicine, Weill Cornell Medical College, USA) for critical reagents; and M. Yuan and S. Breitkopf for technical assistance with mass spectrometry. Research support was derived in part from the National Institutes of Health (R01CA177910 (A.T.), P01CA120964 (J.M.A.), P30CA006516 (J.M.A.), R01GM041890 (L.C.C.)). E.C.L. is a pre-doctoral fellow of the NSF graduate research fellowship programme (NSF DGE1144152). C.A.L. is financially supported in part by the Pancreatic Cancer Action Network as a Pathway to Leadership Fellow and through a Dale F. Frey Breakthrough award from the Damon Runyon Cancer Research Foundation.

Author information


  1. Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA

    • Evan C. Lien
    •  & Alex Toker
  2. Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48109, USA

    • Costas A. Lyssiotis
  3. Department of Internal Medicine, Division of Gastroenterology, University of Michigan, Ann Arbor, Michigan 48109, USA

    • Costas A. Lyssiotis
  4. Division of Hematology and Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA

    • Ashish Juvekar
    •  & Hai Hu
  5. Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA

    • John M. Asara
  6. Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA

    • John M. Asara
    •  & Alex Toker
  7. Department of Medicine, Weill Cornell Medical College, New York, New York 10065, USA

    • Lewis C. Cantley


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E.C.L., C.A.L., L.C.C. and A.T. designed the study and interpreted the results. E.C.L. and A.T. wrote the manuscript. E.C.L. performed the experiments. J.M.A. and C.A.L. assisted with the LC–MS/MS metabolomic studies and data interpretation. A.J. and H.H. assisted with the in vivo xenograft studies.

Competing interests

L.C.C. owns equity in, receives compensation from, and serves on the Board of Directors and Scientific Advisory Board of Agios Pharmaceuticals. Agios Pharmaceuticals is identifying metabolic pathways of cancer cells and developing drugs to inhibit such enzymes to disrupt tumour cell growth and survival.

Corresponding author

Correspondence to Alex Toker.

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