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MDH1 and MPP7 Regulate Autophagy in Pancreatic Ductal Adenocarcinoma Maria New1,Tim Van Acker1, Jun-Ichi Sakamaki2, Ming Jiang3, Rebecca E

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MDH1 and MPP7 Regulate Autophagy in Pancreatic Ductal Adenocarcinoma Maria New1,Tim Van Acker1, Jun-Ichi Sakamaki2, Ming Jiang3, Rebecca E Published OnlineFirst February 14, 2019; DOI: 10.1158/0008-5472.CAN-18-2553 Cancer Molecular Cell Biology Research MDH1 and MPP7 Regulate Autophagy in Pancreatic Ductal Adenocarcinoma Maria New1,Tim Van Acker1, Jun-Ichi Sakamaki2, Ming Jiang3, Rebecca E. Saunders3, Jaclyn Long2, Victoria M.-Y. Wang4, Axel Behrens4, Joana Cerveira5, Padhmanand Sudhakar6,7,8, Tamas Korcsmaros6,7, Harold B.J. Jefferies1, Kevin M. Ryan2, Michael Howell3, and Sharon A. Tooze1 Abstract Pancreatic ductal adenocarcinoma (PDAC) is driven by genase 1), revealed their role in early stages of autophagy metabolic changes in pancreatic cells caused by oncogenic during autophagosome formation. MPP7 was involved in mutations and dysregulation of p53. PDAC cell lines and the activation of YAP1 (a transcriptional coactivator in the PDAC-derived xenografts grow as a result of altered meta- Hippo pathway), which in turn promoted autophagy, whereas bolic pathways, changes in stroma, and autophagy. Selective MDH1 was required for maintenance of the levels of the targeting and inhibition of one of these may open avenues essential autophagy initiator serine–threonine kinase ULK1, for the development of new therapeutic strategies. In this and increased in the activity upon induction of autophagy. study, we performed a genome-wide siRNA screen in a Our results provide a possible explanation for how autophagy PDAC cell line using endogenous autophagy as a readout is regulated by MPP7 and MDH1, which adds to our under- and identified several regulators of autophagy that were standing of autophagy regulation in PDAC. required for autophagy-dependent PDAC cell survival. Validation of two promising candidates, MPP7 (MAGUK Significance: This study identifies and characterizes MPP7 p55 subfamily member 7, a scaffolding protein involved in and MDH1 as novel regulators of autophagy, which is thought cell–cell contacts) and MDH1 (cytosolic Malate dehydro- to be responsible for pancreatic cancer cell survival. Introduction autophagy inhibition resulting in loss of viability in cell models and PDAC xenografts (4). Despite current advances, PDACs Pancreatic ductal adenocarcinoma (PDAC) is a cancer of become resistant to most therapies, and new treatment avenues unmet need with a median patient survival of only 6–9 are urgently needed. months (1). Key PDAC features include a high rate of activating Macroautophagy (hereafter autophagy) is an essential, KRAS mutations, a hypervascular and hypoxic microenviron- evolutionarily conserved membrane-mediated process that ment, and reprogramming of cellular metabolism (2). delivers cytoplasmic constituents in double-membrane autop- A number of studies have linked autophagy to PDAC survival hagosome vesicles to lysosomes for degradation, energy and progression. Autophagy is constitutively activated in PDAC release, and component recycling (5). Mammalian autophagy cell lines and tumors (3). This is required for tumor cell is mediated by at least 18 autophagy (ATG) proteins acting survival, as demonstrated by either pharmacologic or genetic in a concerted hierarchy (6). The mammalian ATG8s, which include LC3B, are used to monitor autophagosome formation fl 1Molecular Cell Biology of Autophagy Laboratory, The Francis Crick Institute, and autophagic ux (7). Autophagy has a homeostatic role London, United Kingdom. 2Tumour Cell Death Laboratory, Cancer Research UK under basal conditions, eliminating damaged organelles and Beatson Institute, Glasgow, United Kingdom. 3High Throughput Screening, The misfolded proteins that may otherwise diminish cellular fit- Francis Crick Institute, London, United Kingdom. 4Adult Stem Cell Laboratory, ness and integrity. Autophagy is also induced in response 5 The Francis Crick Institute, London, United Kingdom. Flow Cytometry, The to stimuli, including starvation, hypoxia, and other stresses, 6 Francis Crick Institute, London, United Kingdom. Korcsmaros Group, Earlham to ensure cell survival. Institute, Norwich, United Kingdom. 7Korcsmaros Group, Quadram Institute, Norwich, United Kingdom. 8Department of Chronic Diseases, Metabolism and The role of autophagy in cancer is complex and context- Ageing, KU Leuven, Belgium. dependent with evidence for both tumor survival and tumor- suppressive roles. Autophagy is thought to be required for anti- Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). cancer immunosurveillance and inhibition of malignant trans- formation. However, in established tumors, autophagy can also M. New and T. Van Acker contributed equally to this article. provide a means for tumor cell survival and therapy resistance (8). Corresponding Author: Sharon A. Tooze, The Francis Crick Institute, 1 Midland In PDAC, autophagy is constitutively activated (9) and required Road, London NW1 1AT, United Kingdom. Phone: 203-796-1340; E-mail: for tumor development, metabolism, and growth (4, 10). [email protected] Although the importance of autophagy in cancer is established, doi: 10.1158/0008-5472.CAN-18-2553 the process is still not fully understood, and it remains unclear Ó2019 American Association for Cancer Research. how manipulation of autophagy in PDAC should be optimally www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst February 14, 2019; DOI: 10.1158/0008-5472.CAN-18-2553 New et al. deployed for clinical benefit. There is therefore a need for discov- 18.75 nmol/L, with 0.0075 mL/well of INTERFERin-HTS ery of novel autophagy regulators, as the enhanced understanding reagent (Polyplus), and 600 cells/well in a final volume of of autophagy may yield new therapeutic targets. 50 mL/well in black and clear-bottom 384-well plates (Greiner). To identify the novel targets, we performed a genome-wide siRNA libraries were aliquoted on a Beckman FX liquid loss-of-function screen to identify genes that regulate autophagy handling station and all other liquid addition steps performed in PDAC cells. Regulators identified through the screen will with a FluidX Xrd-384 plate dispenser. Following reverse siRNA elucidate molecular mechanisms governing tumor cell survival transfection, plates were incubated at 37 Cin5%CO2 for and autophagy. We report that levels of MPP7 and MDH1 72 hours, with BafA1 at a final concentration of 50 nmol/L influence autophagic flux under basal and hypoxic conditions. present for the final 6 hours of transfection. Cells were then Our ATG protein pathway mapping experiments show MPP7 and fixed in 4% paraformaldehyde (Sigma) for 30 minutes, fol- MDH1 influence the earliest stages of autophagy. Furthermore, lowed by incubation with methanol for 15 minutes and stain- our results suggest that YAP1 may contribute to the positive ing with primary [anti-LC3 5F10 antibody (nanoTools Anti- regulation of autophagy by MPP7, and reveal a functionally korpertechnik;€ used at 1:100 dilution in 1% BSA)] and sec- important role for MDH1 in maintaining cellular ULK1 levels. ondary antibody (anti-mouse Alexa Fluor 488 Life Technolo- Exploiting the apparent sensitivity of PDAC to autophagy inhi- gies at 1:1,000 dilution in 1% BSA). bition provides a rational basis for the consideration of these novel autophagy regulators as therapeutic targets. Image capture and data analysis To collect antibody labeling and nuclear staining, a Thermo Fisher Scientific Cellomics Arrayscan Vti (Â20 magnification, 0.4 Materials and Methods NA, BGRFR filter set, X1 camera, LED illumination) and accom- Cell culture and reagents panying image analysis software were used (8 fields scanned per Cell lines were ordered from ATCC (BxPc-3, Panc 02.03, Panc well). The parameters recorded were: number of nuclei (object 10.05, Panc-1, PL45), Riken (KLM-1, KP4-3, PK-1, PK-45H), count) from the DAPI channel; number of LC3 spots/cell; total JCRB (SUIT-2, KP-4, KP-2), and DSMZ (HUPT3, YAPC, PA-TU- spot count; total spot area; average spot area; total spot intensity; 8902, PA-TU-8999, DAN-G), expanded, and fluorescence-based and average spot intensity. More detailed descriptions of algo- Mycoplasma testing was performed, followed by agar culture confir- rithm parameters are available upon request. mation. A large batch of cells was frozen. Cells were passaged twice a Raw data for the individual parameters were normalized to week and used until 20 passages. All PDAC cell lines were grown in correct for plate, row, and column effects, then divided by the full medium: DMEM with 10% FCS and 4 mmol/L L-glutamine. individual plate median absolute deviation, thus creating a PK-1 cell lines stably expressing Tet-On–inducible myc-tagged Z-score. Replicates were summarized by taking the median MDH1 and Tet-On–inducible HA-tagged MPP7 were maintained Z-score for each siRNA pool and normalized scores for the three in DMEM with 10% tetracycline-free FCS, 4 mmol/L L-glutamine, parameters were then combined into a ranked list, thereby deter- and 1 mg/mL puromycin. mining the top 200 spot decreasers for the primary siGENOME Where indicated, cells were treated with 100 nmol/L screen. For the secondary deconvolution screen, results are Bafilomycin A1 (BafA1; Calbiochem), 100 nmol/L Torin1 expressed as percentage of control with respect to the RISC-free (Cayman Chemical), 100 nmol/L epoxomycin (Sigma) or for control (median of three replicates). 24 hours with 1 mg/mL doxycycline (Takara Bio). For autop- hagy induction by amino acid starvation, cells were washed Bioinformatic analysis three times in
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