Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB4) as a Novel Autophagy Regulator by High Content shRNA Screening

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 shRNA 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, 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 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms Corresponding Author is Eileen, White [email protected], [email protected], FAX (732) 235-5795; VOICE: (732) 235-5329. *Drs. Strohecker and Joshi contributed equally to this work Potential Conflicts of Interest 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. Supplementary Information accompanying this paper is archived on the Oncogene website (http://www.nature.com/onc) HHS Public Access Author manuscript Oncogene. Author manuscript; available in PMC 2016 May 05. Published in final edited form as: Oncogene. 2015 November 5; 34(45): 5662–5676. doi:10.1038/onc.2015.23. A uhor M anscript