e105605902082b0b65afec7dc74b4a9e pmc_35158994.pdf 85244033d6e2976563cfbbd2d507318346840a5d pmc_35158994.pdf 7012c54fc4e5369f57939d7addc547de7e551fe164e2e37f2d06b6e3ae1ad8ef pmc_35158994.pdf Title: Glioblastoma Cells Counteract PARP Inhibition through Pro-Survival Induction of Lipid Droplets Synthesis and Utilization Subject: Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. Poly (ADP-ribose) polymerase inhibitors (PARPi) represent a new class of anti-neoplastic drugs. In the current study, we have characterized the mechanism by which glioblastoma cells evade the effect of PARPi as anti-tumor agents. We have found that suppression of PARP activity exerts an anti-stemness effect and has a dual impact on autophagy, inducing its activation in the first 24 h (together with down-regulation of the pro-survival mTOR pathway) and preventing autophagosomes fusion to lysosomes at later time-points, in primary glioma cells. In parallel, PARPi triggered the synthesis of lipid droplets (LDs) through ACC-dependent activation of de novo fatty acids (FA) synthesis. Notably, inhibiting -oxidation and blocking FA utilization, increased PARPi-induced glioma cell death while treatment with oleic acid (OA) prevented the anti-glioma effect of PARPi. Moreover, LDs fuel glioma cells by inducing pro-survival lipid consumption as confirmed by quantitation of oxygen consumption rates using Seahorse respirometry in presence or absence of OA. In summary, we uncover a novel mechanism by which glioblastoma escapes to anti-tumor agents through metabolic reprogramming, inducing the synthesis and utilization of LDs as a pro-survival strategy in response to PARP inhibition. Keywords: glioblastoma stem cells; PARP inhibitors; lipophagy; lipid droplets; Acyl-coA-carboxylase; metabolic adaptation Author: Jara Majuelos-Melguizo, José Manuel Rodríguez-Vargas, Nuria Martínez-López, Daniel Delgado-Bellido, Ángel García-Díaz, Víctor J. Yuste, Marina García-Macía, Laura M. López, Rajat Singh and F. J. 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no 280 0 NUUXUW+VnURWPalladioL-Italic Type 1 Custom yes yes no 288 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-03-23 02:23:24 CET RepresentationInformation: pmc_35158994.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-02-27 18:44:27 CET Size: 3311368 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 483 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: Cell Culture and Reagents Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Western Blot Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Transfection of Small Interfering (si) RNA (siRNA) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Transfection of mCherry-GFP-LC3 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Autophagy Flux Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: RT-qPCR Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: GICs Immunofluorescence Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: LAMP1-Bodipy Immunofluorescence Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Sudan-Red Staining Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: ATP Determination Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Short-Term Proliferation Assay: MTT Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: SubG1 Analysis: Propidium Iodide Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Seahorse Respirometry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Transmission Electron Microscopy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Children: Item: Title: PARP Inhibition Down-Regulates Pro-Survival Pathways in Glioblastoma Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: PARP Inhibition Induces Lipid Droplets (LDs) Formation and Utilization Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: PARPi-Mediated Suppression of AMPK Modulates LDs Formation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Lipids from LDs as Metabolic Fuel for Survival of Glioma Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Info: Title: Glioblastoma Cells Counteract PARP Inhibition through Pro-Survival Induction of Lipid Droplets Synthesis and Utilization Author: Jara Majuelos-Melguizo, José Manuel Rodríguez-Vargas, Nuria Martínez-López, Daniel Delgado-Bellido, Ángel García-Díaz, Víctor J. Yuste, Marina García-Macía, Laura M. López, Rajat Singh and F. J. Oliver Subject: Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. Poly (ADP-ribose) polymerase inhibitors (PARPi) represent a new class of anti-neoplastic drugs. In the current study, we have characterized the mechanism by which glioblastoma cells evade the effect of PARPi as anti-tumor agents. We have found that suppression of PARP activity exerts an anti-stemness effect and has a dual impact on autophagy, inducing its activation in the first 24 h (together with down-regulation of the pro-survival mTOR pathway) and preventing autophagosomes fusion to lysosomes at later time-points, in primary glioma cells. In parallel, PARPi triggered the synthesis of lipid droplets (LDs) through ACC-dependent activation of de novo fatty acids (FA) synthesis. Notably, inhibiting -oxidation and blocking FA utilization, increased PARPi-induced glioma cell death while treatment with oleic acid (OA) prevented the anti-glioma effect of PARPi. Moreover, LDs fuel glioma cells by inducing pro-survival lipid consumption as confirmed by quantitation of oxygen consumption rates using Seahorse respirometry in presence or absence of OA. In summary, we uncover a novel mechanism by which glioblastoma escapes to anti-tumor agents through metabolic reprogramming, inducing the synthesis and utilization of LDs as a pro-survival strategy in response to PARP inhibition. 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