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Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel autophagy regulator by high content shRNA screening

Abstract

Deregulation of autophagy has been linked to multiple degenerative diseases and cancer, thus the identification of novel autophagy regulators for potential therapeutic intervention is important. To meet this need, we developed a high content image-based short hairpin RNA screen monitoring levels of the autophagy substrate p62/SQSTM1. We identified 186 genes whose loss caused p62 accumulation indicative of autophagy blockade, and 67 genes whose loss enhanced p62 elimination indicative of autophagy stimulation. One putative autophagy stimulator, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), drives flux through pentose phosphate pathway. Knockdown of PFKFB4 in prostate cancer cells increased p62 and reactive oxygen species (ROS), but surprisingly increased autophagic flux. Addition of the ROS scavenger N-acetyl cysteine prevented p62 accumulation in PFKFB4-depleted cells, suggesting that the upregulation of p62 and autophagy was a response to oxidative stress caused by PFKFB4 elimination. Thus, PFKFB4 suppresses oxidative stress and p62 accumulation, without which autophagy is stimulated likely as a ROS detoxification response.

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References

  1. Rabinowitz JD, White E . Autophagy and metabolism. Science 2010; 330: 1344–1348.

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Strohecker AM, Guo JY, Karsli-Uzunbas G, Price SM, Chen GJ, Mathew R et al. Autophagy sustains mitochondrial glutamine metabolism and growth of BRAFV600E-driven lung tumors. Cancer Disc 2013; 3: 1272–1285.

    CAS  Google Scholar 

  3. Guo JY, Karsli-Uzunbas G, Mathew R, Aisner SC, Kamphorst JJ, Strohecker AM et al. Autophagy suppresses progression of K-ras-induced lung tumors to oncocytomas and maintains lipid homeostasis. Genes Dev 2013; 27: 1447–1461.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Rao S, Tortola L, Perlot T, Wirnsberger G, Novatchkova M, Nitsch R et al. A dual role for autophagy in a murine model of lung cancer. Nat Commun 2014; 5: 3056.

    PubMed  Google Scholar 

  5. Rosenfeldt MT, O'Prey J, Morton JP, Nixon C, MacKay G, Mrowinska A et al. p53 status determines the role of autophagy in pancreatic tumour development. Nature 2013; 504: 296–300.

    CAS  PubMed  Google Scholar 

  6. Wei H, Wei S, Gan B, Peng X, Zou W, Guan JL . Suppression of autophagy by FIP200 deletion inhibits mammary tumorigenesis. Genes Dev 2011; 25: 1510–1527.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Huo Y, Cai H, Teplova I, Bowman-Colin C, Chen G, Price S et al. Autophagy opposes p53-mediated tumor barrier to facilitate tumorigenesis in a model of PALB2-associated hereditary breast cancer. Cancer Disc 2013; 3: 894–907.

    CAS  Google Scholar 

  8. Guo JY, Chen HY, Mathew R, Fan J, Strohecker AM, Karsli-Uzunbas G et al. Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes Dev 2011; 25: 460–470.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Yang A, Rajeshkumar NV, Wang X, Yabuuchi S, Alexander BM, Chu GC et al. Autophagy is critical for pancreatic tumor growth and progression in tumors with p53 alterations. Cancer Disc 2014; 4: 905–913.

    CAS  Google Scholar 

  10. Yang S, Wang X, Contino G, Liesa M, Sahin E, Ying H et al. Pancreatic cancers require autophagy for tumor growth. Genes Dev 2011; 25: 717–729.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Karsli-Uzunbas G, Guo JY, Price S, Teng X, Laddha SV, Khor S et al. Autophagy is required for glucose homeostasis and lung tumor maintenance. Cancer Disc 2014; 4: 914–927.

    CAS  Google Scholar 

  12. White E . Deconvoluting the context-dependent role for autophagy in cancer. Nat Rev Cancer. 2012; 12: 401–410.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson D, Chen G et al. Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell 2006; 10: 51–64.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Karantza-Wadsworth V, Patel S, Kravchuk O, Chen G, Mathew R, Jin S et al. Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis. Genes Dev 2007; 21: 1621–1635.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Mathew R, Karp CM, Beaudoin B, Vuong N, Chen G, Chen HY et al. Autophagy suppresses tumorigenesis through elimination of p62. Cell 2009; 137: 1062–1075.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Mathew R, Karantza-Wadsworth V, White E . Role of autophagy in cancer. Nat Rev Cancer. 2007; 7: 961–967.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Takamura A, Komatsu M, Hara T, Sakamoto A, Kishi C, Waguri S et al. Autophagy-deficient mice develop multiple liver tumors. Genes Dev 2011; 25: 795–800.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Komatsu M, Wang QJ, Holstein GR, Friedrich VL Jr., Iwata J, Kominami E et al. Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration. Proc Natl Acad Sci USA 2007; 104: 14489–14494.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Rubinsztein DC, Marino G, Kroemer G . Autophagy and aging. Cell 2011; 146: 682–695.

    CAS  PubMed  Google Scholar 

  20. Masiero E, Agatea L, Mammucari C, Blaauw B, Loro E, Komatsu M et al. Autophagy is required to maintain muscle mass. Cell Metab 2009; 10: 507–515.

    CAS  PubMed  Google Scholar 

  21. Sandri M, Coletto L, Grumati P, Bonaldo P . Misregulation of autophagy and protein degradation systems in myopathies and muscular dystrophies. J Cell Sci 2013; 126: 5325–5333.

    CAS  PubMed  Google Scholar 

  22. Grumati P, Coletto L, Sabatelli P, Cescon M, Angelin A, Bertaggia E et al. Autophagy is defective in collagen VI muscular dystrophies, and its reactivation rescues myofiber degeneration. Nat Med 2010; 16: 1313–1320.

    CAS  PubMed  Google Scholar 

  23. Bernardi P, Bonaldo P . Mitochondrial dysfunction and defective autophagy in the pathogenesis of collagen VI muscular dystrophies. Cold Spring Harbor Perspect Biol 2013; 5: a011387.

    Google Scholar 

  24. Shanmugam M, McBrayer SK, Qian J, Raikoff K, Avram MJ, Singhal S et al. Targeting glucose consumption and autophagy in myeloma with the novel nucleoside analogue 8-aminoadenosine. J Biol Chem 2009; 284: 26816–26830.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Hoang B, Benavides A, Shi Y, Frost P, Lichtenstein A . Effect of autophagy on multiple myeloma cell viability. Mol Cancer Ther 2009; 8: 1974–1984.

    CAS  PubMed  Google Scholar 

  26. Ding WX, Ni HM, Gao W, Chen X, Kang JH, Stolz DB et al. Oncogenic transformation confers a selective susceptibility to the combined suppression of the proteasome and autophagy. Mol Cancer Ther 2009; 8: 2036–2045.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Bellodi C, Lidonnici MR, Hamilton A, Helgason GV, Soliera AR, Ronchetti M et al. Targeting autophagy potentiates tyrosine kinase inhibitor-induced cell death in Philadelphia chromosome-positive cells, including primary CML stem cells. J Clin Invest 2009; 119: 1109–1123.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Maclean KH, Dorsey FC, Cleveland JL, Kastan MB . Targeting lysosomal degradation induces p53-dependent cell death and prevents cancer in mouse models of lymphomagenesis. J Clin Invest 2008; 118: 79–88.

    CAS  PubMed  Google Scholar 

  29. Lu Z, Luo RZ, Lu Y, Zhang X, Yu Q, Khare S et al. The tumor suppressor gene ARHI regulates autophagy and tumor dormancy in human ovarian cancer cells. J Clin Invest 2008; 118: 3917–3929.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Degtyarev M, De Maziere A, Orr C, Lin J, Lee BB, Tien JY et al. Akt inhibition promotes autophagy and sensitizes PTEN-null tumors to lysosomotropic agents. J Cell Biol 2008; 183: 101–116.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Amaravadi RK, Yu D, Lum JJ, Bui T, Christophorou MA, Evan GI et al. Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest 2007; 117: 326–336.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Carew JS, Nawrocki ST, Kahue CN, Zhang H, Yang C, Chung L et al. Targeting autophagy augments the anticancer activity of the histone deacetylase inhibitor SAHA to overcome Bcr-Abl-mediated drug resistance. Blood 2007; 110: 313–322.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Amaravadi RK, Lippincott-Schwartz J, Yin XM, Weiss WA, Takebe N, Timmer W et al. Principles and current strategies for targeting autophagy for cancer treatment. Clin Cancer Res 2011; 17: 654–666.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. White E, DiPaola RS . The double-edged sword of autophagy modulation in cancer. Clin Cancer Res 2009; 15: 5308–5316.

    PubMed  PubMed Central  Google Scholar 

  35. Barnard RA, Wittenburg LA, Amaravadi RK, Gustafson DL, Thorburn A, Thamm DH . Phase I clinical trial and pharmacodynamic evaluation of combination hydroxychloroquine and doxorubicin treatment in pet dogs treated for spontaneously occurring lymphoma. Autophagy 2014; 10: 1415–1425.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Rangwala R, Chang YC, Hu J, Algazy K, Evans T, Fecher L et al. Combined MTOR and autophagy inhibition: phase I trial of hydroxychloroquine and temsirolimus in patients with advanced solid tumors and melanoma. Autophagy 2014; 10: 1391–1402.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Rangwala R, Leone R, Chang YC, Fecher L, Schuchter L, Kramer A et al. Phase I trial of hydroxychloroquine with dose-intense temozolomide in patients with advanced solid tumors and melanoma. Autophagy 2014; 10: 1369–1379.

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Rosenfeld MR, Ye X, Supko JG, Desideri S, Grossman SA, Brem S et al. A phase I/II trial of hydroxychloroquine in conjunction with radiation therapy and concurrent and adjuvant temozolomide in patients with newly diagnosed glioblastoma multiforme. Autophagy 2014; 10: 1359–1368.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Vogl DT, Stadtmauer EA, Tan KS, Heitjan DF, Davis LE, Pontiggia L et al. Combined autophagy and proteasome inhibition: a phase 1 trial of hydroxychloroquine and bortezomib in patients with relapsed/refractory myeloma. Autophagy 2014; 10: 1380–1390.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Mahalingam D, Mita M, Sarantopoulos J, Wood L, Amaravadi R, Davis LE et al. Combined autophagy and HDAC inhibition: A phase I safety, tolerability, pharmacokinetic, and pharmacodynamic analysis of hydroxychloroquine in combination with the HDAC inhibitor vorinostat in patients with advanced solid tumors. Autophagy 2014; 10: 1403–1414.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Cheong H, Lu C, Lindsten T, Thompson CB . Therapeutic targets in cancer cell metabolism and autophagy. Nat Biotechnol 2012; 30: 671–678.

    CAS  PubMed  Google Scholar 

  42. Bjorkoy G, Lamark T, Brech A, Outzen H, Perander M, Overvatn A et al. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol 2005; 171: 603–614.

    PubMed  PubMed Central  Google Scholar 

  43. Lamark T, Kirkin V, Dikic I, Johansen T . NBR1 and p62 as cargo receptors for selective autophagy of ubiquitinated targets. Cell Cycle 2009; 8: 1986–1990.

    CAS  PubMed  Google Scholar 

  44. Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem 2007; 282: 24131–24145.

    CAS  PubMed  Google Scholar 

  45. Sanchez P, De Carcer G, Sandoval IV, Moscat J, Diaz-Meco MT . Localization of atypical protein kinase C isoforms into lysosome-targeted endosomes through interaction with p62. Mol Cell Biol 1998; 18: 3069–3080.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Duran A, Linares JF, Galvez AS, Wikenheiser K, Flores JM, Diaz-Meco MT et al. The signaling adaptor p62 is an important NF-kappaB mediator in tumorigenesis. Cancer Cell 2008; 13: 343–354.

    CAS  PubMed  Google Scholar 

  47. Wooten MW, Geetha T, Seibenhener ML, Babu JR, Diaz-Meco MT, Moscat J . The p62 scaffold regulates nerve growth factor-induced NF-kappaB activation by influencing TRAF6 polyubiquitination. J Biol Chem 2005; 280: 35625–35629.

    CAS  PubMed  Google Scholar 

  48. Komatsu M, Waguri S, Koike M, Sou YS, Ueno T, Hara T et al. Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell 2007; 131: 1149–1163.

    CAS  PubMed  Google Scholar 

  49. Li L, Shen C, Nakamura E, Ando K, Signoretti S, Beroukhim R et al. SQSTM1 is a pathogenic target of 5q copy number gains in kidney cancer. Cancer Cell 2013; 24: 738–750.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Rodriguez A, Duran A, Selloum M, Champy MF, Diez-Guerra FJ, Flores JM et al. Mature-onset obesity and insulin resistance in mice deficient in the signaling adapter p62. Cell Metab 2006; 3: 211–222.

    CAS  PubMed  Google Scholar 

  51. Duran A, Amanchy R, Linares JF, Joshi J, Abu-Baker S, Porollo A et al. p62 is a key regulator of nutrient sensing in the mTORC1 pathway. Mol Cell 2011; 44: 134–146.

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Linares JF, Duran A, Yajima T, Pasparakis M, Moscat J, Diaz-Meco MT . K63 polyubiquitination and activation of mTOR by the p62-TRAF6 complex in nutrient-activated cells. Mol Cell 2013; 51: 283–296.

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Yalcin A, Telang S, Clem B, Chesney J . Regulation of glucose metabolism by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases in cancer. Exp Mol Pathol 2009; 86: 174–179.

    CAS  PubMed  Google Scholar 

  54. Okar DA, Lange AJ . Fructose-2,6-bisphosphate and control of carbohydrate metabolism in eukaryotes. BioFactors 1999; 10: 1–14.

    CAS  PubMed  Google Scholar 

  55. Okar DA, Manzano A, Navarro-Sabate A, Riera L, Bartrons R, Lange AJ . PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6-bisphosphate. Trends Biochem Sci 2001; 26: 30–35.

    CAS  PubMed  Google Scholar 

  56. Lipinski MM, Hoffman G, Ng A, Zhou W, Py BF, Hsu E et al. A genome-wide siRNA screen reveals multiple mTORC1 independent signaling pathways regulating autophagy under normal nutritional conditions. Dev Cell 2010; 18: 1041–1052.

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Chan EY, Kir S, Tooze SA . siRNA screening of the kinome identifies ULK1 as a multidomain modulator of autophagy. J Biol Chem 2007; 282: 25464–25474.

    CAS  PubMed  Google Scholar 

  58. Orvedahl A, Sumpter R Jr., Xiao G, Ng A, Zou Z, Tang Y et al. Image-based genome-wide siRNA screen identifies selective autophagy factors. Nature 2011; 480: 113–117.

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Szyniarowski P, Corcelle-Termeau E, Farkas T, Hoyer-Hansen M, Nylandsted J, Kallunki T et al. A comprehensive siRNA screen for kinases that suppress macroautophagy in optimal growth conditions. Autophagy 2011; 7: 892–903.

    CAS  PubMed  Google Scholar 

  60. He P, Peng Z, Luo Y, Wang L, Yu P, Deng W et al. High-throughput functional screening for autophagy-related genes and identification of TM9SF1 as an autophagosome-inducing gene. Autophagy 2009; 5: 52–60.

    CAS  PubMed  Google Scholar 

  61. Sarkar S, Perlstein EO, Imarisio S, Pineau S, Cordenier A, Maglathlin RL et al. Small molecules enhance autophagy and reduce toxicity in Huntington's disease models. Nat Chem Biol 2007; 3: 331–338.

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Williams A, Sarkar S, Cuddon P, Ttofi EK, Saiki S, Siddiqi FH et al. Novel targets for Huntington's disease in an mTOR-independent autophagy pathway. Nat Chem Biol 2008; 4: 295–305.

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Criollo A, Senovilla L, Authier H, Maiuri MC, Morselli E, Vitale I et al. The IKK complex contributes to the induction of autophagy. EMBO J 2010; 29: 619–631.

    CAS  PubMed  Google Scholar 

  64. McKnight NC, Jefferies HB, Alemu EA, Saunders RE, Howell M, Johansen T et al. Genome-wide siRNA screen reveals amino acid starvation-induced autophagy requires SCOC and WAC. EMBO J 2012; 31: 1931–1946.

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Luo B, Cheung HW, Subramanian A, Sharifnia T, Okamoto M, Yang X et al. Highly parallel identification of essential genes in cancer cells. Proc Natl Acad Sci USA 2008; 105: 20380–20385.

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Mi H, Muruganujan A, Casagrande JT, Thomas PD . Large-scale gene function analysis with the PANTHER classification system. Nat Protocols 2013; 8: 1551–1566.

    PubMed  Google Scholar 

  67. Hoyer-Hansen M, Bastholm L, Szyniarowski P, Campanella M, Szabadkai G, Farkas T et al. Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2. Mol Cell 2007; 25: 193–205.

    PubMed  Google Scholar 

  68. Law BY, Wang M, Ma DL, Al-Mousa F, Michelangeli F, Cheng SH et al. Alisol B, a novel inhibitor of the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase pump, induces autophagy, endoplasmic reticulum stress, and apoptosis. Mol Cancer Ther 2010; 9: 718–730.

    CAS  PubMed  Google Scholar 

  69. Rusten TE, Stenmark H . How do ESCRT proteins control autophagy? J Cell Sci 2009; 122: 2179–2183.

    CAS  PubMed  Google Scholar 

  70. Rusten TE, Vaccari T, Stenmark H . Shaping development with ESCRTs. Nat Cell Biol 2012; 14: 38–45.

    CAS  Google Scholar 

  71. Tamai K, Tanaka N, Nara A, Yamamoto A, Nakagawa I, Yoshimori T et al. Role of Hrs in maturation of autophagosomes in mammalian cells. Biochem Biophys Res Commun 2007; 360: 721–727.

    CAS  PubMed  Google Scholar 

  72. Li L, Guan KL . Microtubule-associated protein/microtubule affinity-regulating kinase 4 (MARK4) is a negative regulator of the mammalian target of rapamycin complex 1 (mTORC1). J Biol Chem 2013; 288: 703–708.

    CAS  PubMed  Google Scholar 

  73. Qin Q, Inatome R, Hotta A, Kojima M, Yamamura H, Hirai H et al. A novel GTPase, CRAG, mediates promyelocytic leukemia protein-associated nuclear body formation and degradation of expanded polyglutamine protein. J Cell Biol 2006; 172: 497–504.

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Fallon L, Moreau F, Croft BG, Labib N, Gu WJ, Fon EA . Parkin and CASK/LIN-2 associate via a PDZ-mediated interaction and are co-localized in lipid rafts and postsynaptic densities in brain. J Biol Chem 2002; 277: 486–491.

    CAS  PubMed  Google Scholar 

  75. Jager S, Bucci C, Tanida I, Ueno T, Kominami E, Saftig P et al. Role for Rab7 in maturation of late autophagic vacuoles. J Cell Sci 2004; 117: 4837–4848.

    PubMed  Google Scholar 

  76. Gutierrez MG, Munafo DB, Beron W, Colombo MI . Rab7 is required for the normal progression of the autophagic pathway in mammalian cells. J Cell Sci 2004; 117: 2687–2697.

    CAS  PubMed  Google Scholar 

  77. Ganley IG, Wong PM, Gammoh N, Jiang X . Distinct autophagosomal-lysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest. Mol Cell 2011; 42: 731–743.

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Ros S, Santos CR, Moco S, Baenke F, Kelly G, Howell M et al. Functional metabolic screen identifies 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 as an important regulator of prostate cancer cell survival. Cancer Disc 2012; 2: 328–343.

    CAS  Google Scholar 

  79. Ros S, Schulze A . Balancing glycolytic flux: the role of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatases in cancer metabolism. Cancer Metab 2013; 1: 8.

    PubMed  PubMed Central  Google Scholar 

  80. Minchenko OH, Ochiai A, Opentanova IL, Ogura T, Minchenko DO, Caro J et al. Overexpression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-4 in the human breast and colon malignant tumors. Biochimie 2005; 87: 1005–1010.

    CAS  PubMed  Google Scholar 

  81. Warmoes MO, Locasale JW . Heterogeneity of glycolysis in cancers and therapeutic opportunities. Biochem Pharmacol 2014; 92: 12–21.

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Jeon YK, Yoo DR, Jang YH, Jang SY, Nam MJ . Sulforaphane induces apoptosis in human hepatic cancer cells through inhibition of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase4, mediated by hypoxia inducible factor-1-dependent pathway. Biochimica et Biophysica Acta 2011; 1814: 1340–1348.

    CAS  PubMed  Google Scholar 

  83. Goidts V, Bageritz J, Puccio L, Nakata S, Zapatka M, Barbus S et al. RNAi screening in glioma stem-like cells identifies PFKFB4 as a key molecule important for cancer cell survival. Oncogene 2012; 31: 3235–3243.

    CAS  PubMed  Google Scholar 

  84. Kimura S, Noda T, Yoshimori T . Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 2007; 3: 452–460.

    CAS  PubMed  Google Scholar 

  85. Mizushima N, Yoshimori T, Levine B . Methods in mammalian autophagy research. Cell 2010; 140: 313–326.

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Yoshimori T, Yamamoto A, Moriyama Y, Futai M, Tashiro Y . Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J Biol Chem 1991; 266: 17707–17712.

    CAS  PubMed  Google Scholar 

  87. Zhou R, Yazdi AS, Menu P, Tschopp J . A role for mitochondria in NLRP3 inflammasome activation. Nature 2011; 469: 221–225.

    CAS  PubMed  Google Scholar 

  88. Scherz-Shouval R, Shvets E, Fass E, Shorer H, Gil L, Elazar Z . Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4. EMBO J 2007; 26: 1749–1760.

    CAS  PubMed  PubMed Central  Google Scholar 

  89. Chen Y, Azad MB, Gibson SB . Superoxide is the major reactive oxygen species regulating autophagy. Cell Death Differ 2009; 16: 1040–1052.

    CAS  PubMed  Google Scholar 

  90. Lee J, Giordano S, Zhang J . Autophagy, mitochondria and oxidative stress: cross-talk and redox signalling. Biochem J 2012; 441: 523–540.

    CAS  PubMed  Google Scholar 

  91. Jain A, Lamark T, Sjottem E, Larsen KB, Awuh JA, Overvatn A et al. p62/SQSTM1 is a target gene for transcription factor NRF2 and creates a positive feedback loop by inducing antioxidant response element-driven gene transcription. J Biol Chem 2010; 285: 22576–22591.

    CAS  PubMed  PubMed Central  Google Scholar 

  92. Sahani MH, Itakura E, Mizushima N . Expression of the autophagy substrate SQSTM1/p62 is restored during prolonged starvation depending on transcriptional upregulation and autophagy-derived amino acids. Autophagy 2014; 10: 431–441.

    CAS  PubMed  PubMed Central  Google Scholar 

  93. Yang Z, Goronzy JJ, Weyand CM . The glycolytic enzyme PFKFB3/phosphofructokinase regulates autophagy. Autophagy 2014; 10: 382–383.

    CAS  PubMed  Google Scholar 

  94. Yang Z, Fujii H, Mohan SV, Goronzy JJ, Weyand CM . Phosphofructokinase deficiency impairs ATP generation, autophagy, and redox balance in rheumatoid arthritis T cells. J Exp Med 2013; 210: 2119–2134.

    CAS  PubMed  PubMed Central  Google Scholar 

  95. Klarer AC, O'Neal J, Imbert-Fernandez Y, Clem A, Ellis SR, Clark J et al. Inhibition of 6-phosphofructo-2-kinase (PFKFB3) induces autophagy as a survival mechanism. Cancer Metab 2014; 2: 2.

    PubMed  PubMed Central  Google Scholar 

  96. Bensaad K, Cheung EC, Vousden KH . Modulation of intracellular ROS levels by TIGAR controls autophagy. EMBO J 2009; 28: 3015–3026.

    CAS  PubMed  PubMed Central  Google Scholar 

  97. Bensaad K, Tsuruta A, Selak MA, Vidal MN, Nakano K, Bartrons R et al. TIGAR, a p53-inducible regulator of glycolysis and apoptosis. Cell 2006; 126: 107–120.

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank M Komatsu (conditional Atg7 mice) and Z Yue (Beclin1+/−) for mice and T Yoshimori for providing the ptf-LC3 plasmid. We also thank the staff of the Genetic Perturbation Platform of the Broad Institute (formerly the RNAi Platform) particularly S Silver and O Alkan for assistance with execution and analysis of screen data, The Genome Core of the Whitehead Institute for assistance with the Arrayscan, A Roberts for fluorescence-activated cell sorting (FACS) analysis, P Chin for technical assistance and members of the White and Sabatini laboratories for helpful discussions. This work was supported by NIH grants R37 CA53370, RC1 CA147961, R01 CA163591, R01 CA130893, the Rutgers Cancer Institute of New Jersey (P30 CA072720), Johnson & Johnson, and a gift to DMS and EW from Pfizer. AMS and SJ were supported by postdoctoral fellowships from the New Jersey Commission on Cancer Research (09-2406-CCR-E0 to AMS, DFHS13PPCO24 to SJ).

Abbreviations

DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; AMPK, 5′ adenosine monophosphate-activated protein kinase; BA1, Bafilomycin A1; ESCRT, endosomal sorting complexes required for transport; F6P, fructose-6-phosphate; F1,6BP, fructose-1,6-bisphosphate; F2,6BP, fructose-2,6-bisphosphate; LC3, microtubule-associated protein 1 light chain 3; LIR, LC3 interaction region; mTOR, mammalian/mechanistic target of rapamycin; NAC, N-acetyl-cysteine; NSCLC, non-small cell lung cancer; PFK-1, phosphofructokinase-1; PFKFB4, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4; PPP, pentose phosphate pathway; ROS, reactive oxygen species; TDCLs, tumor-derived cell lines; UBA, ubiquitin-binding domain

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EW is a member of the scientific advisory board of Forma Therapeutics. EW, DMS, RP and AMS are co-inventors on a related patent application. RTA is an employee and shareholder of Pfizer, Inc.

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Strohecker, A., Joshi, S., Possemato, R. et al. Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel autophagy regulator by high content shRNA screening. Oncogene 34, 5662–5676 (2015). https://doi.org/10.1038/onc.2015.23

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