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

Abstract

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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Figure 1: Oncogenic signalling through AKT2(E17K) stimulates GSH biosynthesis.
Figure 2: Enhanced GSH biosynthesis confers resistance to oxidative stress.
Figure 3: AKT2(E17K) activates NRF2 to upregulate the GSH biosynthetic genes.
Figure 4: GSH biosynthesis is required for the PI(3)K/Akt-driven initiation of tumour spheroids.
Figure 5: BSO synergizes with CDDP to selectively induce cell death and tumour regression in PI(3)K pathway mutant breast cancer cells.

References

  1. Vander Heiden, M. G., Cantley, L. C. & Thompson, C. B. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324, 1029–1033 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Lauring, J. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Yuan, M., Breitkopf, S. B., Yang, X. & Asara, J. M. 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).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  11. Ben-Sahra, I., Howell, J. J., Asara, J. M. & Manning, B. D. Stimulation of de novo pyrimidine synthesis by growth signaling through mTOR and S6K1. Science 339, 1323–1328 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. Papa, L., Hahn, M., Marsh, E. L., Evans, B. S. & Germain, D. SOD2 to SOD1 switch in breast cancer. J. Biol. Chem. 289, 5412–5416 (2014).

    Article  CAS  Google Scholar 

  14. Somwar, R. 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).

    Article  CAS  Google Scholar 

  15. Sporn, M. B. & Liby, K. T. NRF2 and cancer: the good, the bad and the importance of context. Nat. Rev. Cancer 12, 564–571 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. Li, Y., Dowbenko, D. & Lasky, L. A. AKT/PKB phosphorylation of p21Cip/WAF1 enhances protein stability of p21Cip/WAF1 and promotes cell survival. J. Biol. Chem. 277, 11352–11361 (2002).

    Article  CAS  Google Scholar 

  20. Rada, P. 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).

    Article  CAS  Google Scholar 

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

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  24. Kang, Y. J., Emery, D. & Enger, M. D. Buthionine sulfoximine induced growth inhibition in human lung carcinoma cells does not correlate with glutathione depletion. Cell Biol. Toxicol. 7, 249–261 (1991).

    CAS  PubMed  Google Scholar 

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

    Article  Google Scholar 

  26. Gorrini, C., Harris, I. S. & Mak, T. W. Modulation of oxidative stress as an anticancer strategy. Nat. Rev. Drug Discov. 12, 931–947 (2013).

    Article  CAS  Google Scholar 

  27. Chin, Y. R., Yuan, X., Balk, S. P. & Toker, A. PTEN-deficient tumors depend on AKT2 for maintenance and survival. Cancer Discov. 4, 942–955 (2014).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  30. Dasari, S. & Tchounwou, P. B. Cisplatin in cancer therapy: molecular mechanisms of action. Eur. J. Pharmacol. 740, 364–378 (2014).

    Article  CAS  Google Scholar 

  31. Chen, H. H. W. & Kuo, M. T. Role of glutathione in the regulation of Cisplatin resistance in cancer chemotherapy. Met. Based Drugs 2010, 430939 (2010).

    Article  Google Scholar 

  32. Tung, N. M. & Winer, E. P. Tumor-infiltrating lymphocytes and response to platinum in triple-negative breast cancer. J. Clin. Oncol. 33, 969–971 (2015).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  34. Vichai, V. & Kirtikara, K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat. Protoc. 1, 1112–1116 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  36. Tietze, F. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  39. Ingberg, E., Theodorsson, A., Theodorsson, E. & Strom, J. O. Methods for long-term 17β-estradiol administration to mice. Gen. Comp. Endocrinol. 175, 188–193 (2012).

    Article  CAS  Google Scholar 

  40. Watanabe, T. 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).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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.

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Contributions

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.

Corresponding author

Correspondence to Alex Toker.

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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.

Integrated supplementary information

Supplementary Figure 1 Characterization of AKT1(E17K) and AKT2(E17K).

(a) Anti-HA immunoprecipitates from serum-starved cells were assayed for their ability to phosphorylate a GSK-3β fusion peptide in vitro (data is representative of two independent experiments). (b) Proliferation of cells grown in the presence of serum and growth factors was determined using the WST-1 assay (data are from one experiment that was independently repeated two times with similar results (Supplementary Table 1)). Unprocessed original scans of blots are shown in Supplementary Fig. 6.

Supplementary Figure 2 Metabolomic profiling of serum-starved MCF10A cells expressing AKT2(E17K) vs. AKT2.

(a) Unbiased hierarchical clustering of relative metabolite abundances in serum-starved MCF10A cells expressing AKT2(E17K) versus AKT2. (b) Fold changes of metabolite abundances in serum-starved MCF10A cells expressing AKT2(E17K) versus AKT2 (data are from one metabolomics experiment that was independently repeated two times with similar results). (c) Relative abundances of glycolytic intermediates (n = 3 technical replicates from a single independent metabolomics experiment; the experiment was repeated twice with similar results (Supplementary Table 1)). (d) Relative abundances of metabolites in the pentose phosphate pathway (n = 3 technical replicates from a single independent metabolomics experiment; the experiment was repeated twice with similar results (Supplementary Table 1)). (e,f) Incorporation of U-13C5-glutamine into glutamine and GSSG over 1, 3, and 8 h in serum-starved cells (n = 3 technical replicates from a single metabolomics experiment (Supplementary Table 1)). All error bars represent s.e.m. P < 0.05, P < 0.01 by a two-sided Student’s t-test.

Supplementary Figure 3 Enhanced GSH biosynthesis confers resistance to oxidative stress.

(a, b) Cells were serum-starved for 20–24 h in the presence or absence of 1 μM GSK690693, followed by treatment with 1 mM H2O2 for 6 h. Cell death was assessed by 7-AAD staining followed by FACS (data are from one experiment that was independently repeated two times with similar results (Supplementary Table 1)). (c) Total glutathione levels in cells serum-starved for 20–24 h in the presence or absence of 50 μM BSO (n = 3 biologically independent replicates (Supplementary Table 1)). (df) Cells were serum-starved for 20–24 h in the presence or absence of 1 μM GSK690693 or 50 μM BSO, followed by treatment with 500 μM H2O2 for 4 h. Cells were immunoblotted for the indicated proteins (data is representative of three independent experiments). All error bars represent s.e.m. P < 0.01, P < 0.001 by a two-sided Student’s t-test. Unprocessed original scans of blots are shown in Supplementary Fig. 6.

Supplementary Figure 4 Nrf2 is necessary for AKT2(E17K)-mediated stimulation of GSH biosynthesis and resistance to oxidative stress.

NRF2 was knocked down over 72 h. (a) Nrf2 knock-down was confirmed by immunoblot analysis (data is representative of three independent experiments). (b) Total glutathione levels in serum-starved cells (data are from one experiment that was independently repeated two times with similar results (Supplementary Table 1)). (c) Serum-starved cells were treated with 500 μM H2O2 for 4 h (data is representative of three independent experiments). (d) Distribution of RPPA Z-scores for Akt S473 phosphorylation in patient tumors from TCGA BRCA data set. Breast tumors with Akt pS473 levels greater than 2 standard deviations from the mean were classified as ‘Akt pS473 high’, while tumors with Akt pS473 levels less than 1 standard deviation from the mean were classified as ‘Akt pS473 low’. Unprocessed original scans of blots are shown in Supplementary Fig. 6.

Supplementary Figure 5 Inhibition of GSH biosynthesis does not robustly inhibit the growth of MCF10A cells expressing AKT2 or PIK3CA variants in 2D culture conditions.

(a) mRNA levels were measured by qRT-PCR and are expressed as fold changes relative to MCF10A AKT2 cells (n = 3 biologically independent replicates (Supplementary Table 1)). (b,c) Proliferation of cells grown in the absence of serum and growth factors, and in the presence or absence of 50 μM BSO, was determined with the SRB assay (data are from one experiment (Supplementary Table 1)). (d,e) Viability of cells grown in full growth media for 48 h in the presence of increasing doses of BSO was measured using the WST-1 assay. Viability is expressed relative to the lowest BSO concentration (data are from one experiment (Supplementary Table 1)). All error bars represent s.e.m. P < 0.01 by a two-sided Student’s t-test.

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Lien, E., Lyssiotis, C., Juvekar, A. et al. Glutathione biosynthesis is a metabolic vulnerability in PI(3)K/Akt-driven breast cancer. Nat Cell Biol 18, 572–578 (2016). https://doi.org/10.1038/ncb3341

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