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

Oncogene volume 34, pages 5662–5676 (2015)Cite this article

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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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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

Author information

Author notes

  1. A M Strohecker and S Joshi: These authors contributed equally to this work.

Authors and Affiliations

  1. Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA

    A M Strohecker, S Joshi & E White

  2. Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ, USA

    A M Strohecker, S Joshi & E White

  3. Departments of Molecular Virology, Immunology, and Medical Genetics, and Surgery, The Ohio State University, Columbus, OH, USA

    A M Strohecker

  4. The Comprehensive Cancer Center and Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University, Columbus, OH, USA

    A M Strohecker

  5. Whitehead Institute for Biomedical Research, Cambridge, MA, USA

    R Possemato & D M Sabatini

  6. Broad Institute of Harvard and MIT, Cambridge, MA, USA

    R Possemato & D M Sabatini

  7. Department of Biology, MIT, Cambridge, MA, USA

    R Possemato & D M Sabatini

  8. Department of Pathology, NYU, New York, NY, USA

    R Possemato

  9. Pfizer, Oncology Research Unit, San Diego, CA, USA

    R T Abraham

Authors
  1. A M Strohecker
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  2. S Joshi
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  3. R Possemato
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  4. R T Abraham
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  5. D M Sabatini
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  6. E White
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Corresponding author

Correspondence to E White.

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Competing interests

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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Supplementary Information accompanies this paper on the Oncogene website

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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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  • DOI: https://doi.org/10.1038/onc.2015.23

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