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Role of Autophagy in Histone Deacetylase Inhibitor-Induced Apoptotic and Nonapoptotic Cell Death

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Role of Autophagy in Histone Deacetylase Inhibitor-Induced Apoptotic and Nonapoptotic Cell Death Role of autophagy in histone deacetylase inhibitor-induced apoptotic and nonapoptotic cell death Noor Gammoha, Du Lama,1, Cindy Puentea, Ian Ganleyb, Paul A. Marksa,2, and Xuejun Jianga,2 aCell Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065; and bMedical Research Council Protein Phosphorylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom Contributed by Paul A. Marks, March 16, 2012 (sent for review February 13, 2012) Autophagy is a cellular catabolic pathway by which long-lived membrane vesicle. Nutrient and energy sensing can directly proteins and damaged organelles are targeted for degradation. regulate autophagy by affecting the ULK1 complex, which is Activation of autophagy enhances cellular tolerance to various comprised of the protein kinase ULK1 and its regulators, stresses. Recent studies indicate that a class of anticancer agents, ATG13 and FIP200 (10–12). Under nutrient-rich conditions, histone deacetylase (HDAC) inhibitors, can induce autophagy. One mammalian target of rapamycin (mTOR) directly phosphor- of the HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA), is ylates ULK1 and ATG13 to inhibit the autophagy function of the currently being used for treating cutaneous T-cell lymphoma and ULK1 complex. However, amino acid deprivation inactivates under clinical trials for multiple other cancer types, including mTOR and therefore releases ULK1 from its inhibition. glioblastoma. Here, we show that SAHA increases the expression Downstream of the ULK1 complex, in the heart of the auto- of the autophagic factor LC3, and inhibits the nutrient-sensing phagosome nucleation and elongation, lie two ubiquitin-like kinase mammalian target of rapamycin (mTOR). The inactivation conjugation systems: the ATG12-ATG5 and the LC3-phospha- of mTOR results in the dephosphorylation, and thus activation, of tidylethanolamide (PE) conjugates (13). During autophagy, free the autophagic protein kinase ULK1, which is essential for cytosolic LC3 (termed LC3-I) becomes conjugated to PE autophagy activation during SAHA treatment. Furthermore, we (termed LC3-II). LC3-II is then incorporated to the growing CELL BIOLOGY show that the inhibition of autophagy by RNAi in glioblastoma autophagosome structure that, upon maturation, fuses with the cells results in an increase in SAHA-induced apoptosis. Importantly, lysosome compartment, leading to the degradation of the when apoptosis is pharmacologically blocked, SAHA-induced non- autophagosome content. apoptotic cell death can also be potentiated by autophagy in- The encapsulation and degradation of cytosolic materials by hibition. Overall, our findings indicate that SAHA activates autophagy aids in the clearance of damaged organelles and autophagy via inhibiting mTOR and up-regulating LC3 expression; misfolded proteins, and thereby plays an important role in the autophagy functions as a prosurvival mechanism to mitigate recycling of macromolecules and energy within the cells. In this SAHA-induced apoptotic and nonapoptotic cell death, suggesting context, autophagy may be regarded as a prosurvival mechanism that targeting autophagy might improve the therapeutic effects (14). Hence, autophagy is frequently activated during nutrient of SAHA. deprivation, hypoxia, and a wide range of anticancer therapy. The activation of autophagy has been frequently shown to inhibit transcription | ATG7 | necrosis the onset of apoptotic and necrotic cell death (15). However, in cases where autophagy may have an additive role in the death istone deacetylase (HDAC) inhibitors emerge as a new class process, autophagy may be regarded as a cell-death mechanism “ ” Hof therapeutic agents with promising outcomes during the (16). Here, excessive self-eating through autophagy may con- treatment of a wide range of cancer types (1). Hematological tribute to cell death by a yet unknown mechanism. Therefore, malignancies appear to be particularly sensitive to HDAC assessing the role of autophagy in a context-dependent manner is inhibitors; however, a number of additional cancer types are crucial, especially when considering whether autophagy-targeting currently being tested for their response to HDAC inhibition can be used during anticancer therapy. therapy. For an example, suberoylanilide hydroxamic acid We have previously shown that SAHA treatment induces (SAHA, vorinostat), which inhibits HDACs 1, 2, 3, and 6, has potent autophagy (8). In this study, we provide insights into the been approved for treatment against cutaneous T-cell lymphoma mechanism by which SAHA induces autophagy. We also show and also has modest effects as a single agent on cancers of the that autophagy-targeting can enhance SAHA-induced apoptotic prostate, ovaries, breast, colorectal, and glioblastoma (2, 3). and nonapoptotic cell death in glioblastoma cells. Although their precise mode of action remains uncertain, Results a number of recent data suggest that HDAC inhibitors may in- duce apoptotic cell death through both chromatin-dependent SAHA Induces Autophagy and LC3 Transcription in Mouse Embryonic and -independent mechanisms. Fibroblast Cells. To determine the mechanism by which the Treatment with HDAC inhibitors most frequently induces HDAC inhibitor SAHA induces autophagy, we treated mouse apoptosis via the programmed activation of a series of proteases, called caspases (4–6). More recently, HDAC inhibition has been also shown to induce autophagy (7, 8). Unlike apoptosis, the Author contributions: N.G., P.A.M., and X.J. designed research; N.G., D.L., C.P., and I.G. performed research; N.G., P.A.M., and X.J. analyzed data; and N.G., P.A.M., and X.J. wrote contribution of autophagy to cell death remains controversial the paper. and, most likely, context-dependent. Autophagy is a catabolic The authors declare no conflict of interest. process by which cytosolic material is targeted for lysosomal 1Present address: Novartis Oncology Global Development, Novartis Pharmaceuticals, Flor- degradation by means of double-membrane cytosolic vesicles, ham Park, NJ 07932. termed autophagosomes (9). The formation of autophagosomes 2To whom correspondence may be addressed. E-mail: [email protected] or marksp@ is orchestrated by upstream signaling molecules, including the mskcc.org. ULK1 and PI3K complexes, which signal to downstream com- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. plexes involved in the nucleation and maturation of the double- 1073/pnas.1204429109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1204429109 PNAS Early Edition | 1of5 Downloaded by guest on September 26, 2021 Fig. 1. SAHA induces autophagy and LC3 up-reg- ulation in MEF cells. (A) MEF cells seeded in six-well dishes were treated with the indicated concentra- tion of SAHA for 8 h. Where indicated, 20 nM Baf A1 was added 2 h before harvesting cells. Cell extracts were analyzed by Western blot using antibodies against the indicated proteins. The ac- cumulation of LC3-II (faster migrating form) rela- tive to LC3-I (slower migrating form) is indicative of the induction of autophagy. (B) MEF cells stably expressing GFP-LC3 grown on glass cover-slips were either left untreated or treated with 5 μMSAHAfor 24 h. Cells were then fixed with 3.7% PFA, pro- cessed for imaging, and visualized under the con- focal microscope using the 60× magnification objective. (C) MEF cells were seeded in six-well dishes and treated with 20 μM of SAHA for 20 h followed by 20 nM Baf A1 for a further 4 h. Cell extracts were − − analyzed by Western blot analysis using the indicated antibodies. (D) Wild-type or ATG3 / MEF cells were treated with the indicated concentrations of SAHA for 24 h. (E) A semiquantitative RT-PCR detecting LC3 expression was performed using RNA extracted from wt MEFs either treated with 10 μM SAHA or left untreated. GAPDH RT-PCR was used as a loading control. embryonic fibroblast (MEF) cells with various concentrations of induction of autophagy by monitoring LC3 conversion. As shown SAHA and assayed the expression of the autophagy marker, in Fig. 2A, unlike wild-type MEFs that contained an intact LC3, by both Western blot and microscopy. As shown in Fig. 1A, autophagy response (Fig. 2A, Upper), LC3 conversion was de- treatment of MEF cells with SAHA resulted in the accumulation fective in ULK1/2 DKO MEFs (Fig. 2A, Lower), indicating that of LC3-II (faster migrating form); additional treatment of cells SAHA induces autophagy in a ULK1-dependent mechanism. with the lysosomal inhibitor Bafilomycin A1 (Baf A1) caused Previous reports suggest that the autophagy function of the a further increase of LC3-II level, demonstrating that SAHA ULK1 complex is suppressed by the nutrient-sensing kinase induces a full flux of autophagy, resulting in the lysosomal deg- mTOR (10, 12) and mTOR does so by directly phosphorylating radation of LC3-II. Induction of autophagy by SAHA was fur- ULK1 and its regulator ATG13. This finding prompted us to ther confirmed by imaging GFP-tagged LC3 expressed in MEF examine whether SAHA treatment can cause the inactivation of cells (Fig. 1B). In untreated cells, which mostly express LC3-I, mTOR and thereby activation of the ULK1 complex. Indeed, as GFP-LC3 showed a cytosolic, diffused localization (Fig. 1B, shown in Fig. 2B, two well-known mTOR substrates, p70S6K and Left). However, SAHA treatment resulted in the relocalization 4EBP, are dephosphorylated upon SAHA treatment in a similar of GFP-LC3 into punctate structures corresponding to auto- manner to that upon amino acid starvation.
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