Published OnlineFirst March 28, 2011; DOI: 10.1158/0008-5472.CAN-10-4475 Cancer Therapeutics, Targets, and Chemical Biology Research Following Cytochrome c Release, Autophagy Is Inhibited during Chemotherapy-Induced Apoptosis by Caspase 8–Mediated Cleavage of Beclin 1 Hua Li1, Peng Wang1, Quanhong Sun2, Wen-Xing Ding2, Xiao-Ming Yin2, Robert W. Sobol1,4, Donna B. Stolz3, Jian Yu2, and Lin Zhang1 Abstract Autophagy is an evolutionarily conserved stress response mechanism that often occurs in apoptosis-defective cancer cells and can protect against cell death. In this study, we investigated how apoptosis and autophagy affect each other in cancer cells in response to chemotherapeutic treatment. We found that specific ablation of the proapoptotic function of cytochrome c, a key regulator of mitochondria-mediated apoptosis, enhanced autophagy following chemotherapeutic treatment. Induction of autophagy required Beclin 1 and was associated with blockage of Beclin 1 cleavage by caspase 8 at two sites. To investigate the role of Beclin 1 cleavage in the suppression of autophagy and cell survival, a caspase-resistant mutant of Beclin 1 was knocked into HCT116 colon cancer cells. Beclin 1 mutant knockin resulted in markedly increased autophagy and improved long-term cell survival after chemotherapeutic treatment but without affecting apoptosis and caspase activation. Furthermore, Beclin 1 mutant tumors were significantly less responsive to chemotherapeutic treatment than were wild-type tumors. These results show that chemotherapy-induced apoptosis inhibits autophagy at the execution stage subsequent to cytochrome c release through caspase 8–mediated cleavage of Beclin 1. If apoptosis fails to execute, autophagy is unleashed due to lack of Beclin 1 cleavage by caspases and can contribute to cancer cell survival and therapeutic resistance. Therefore, Beclin 1 may be a useful target for inhibiting autophagy to sensitize chemotherapy. Cancer Res; 71(10); 3625–34. Ó2011 AACR. Introduction apoptogenic mitochondrial proteins such as cytochrome c, and activation of the caspase cascade (3, 4). Apoptosis is a major cytotoxic mechanism of chemother- Autophagy, a catabolic degradation process, has recently apy, and its deregulation contributes to therapeutic resistance emerged as an important stress response and cell death (1, 2). Stress-induced apoptosis in mammalian cells often regulatory mechanism (5). It is characterized by the formation proceeds through the intrinsic pathway mediated by mito- of double membrane autophagosomes, which enclose cyto- chondria and the Bcl-2 family proteins. Once cells are com- solic materials and organelles, and then fuse with lysosomes, mitted to apoptosis, a number of downstream events are leading to degradation of their luminal content by lysosomal triggered to execute cell death, including permeabilization proteases (6). Autophagy is controlled by a group of evolu- of the mitochondrial outer membrane, release of several tionarily conserved autophagy-related genes (ATG), among which Bcl-2–interacting protein 1 (Beclin 1) is a key regulator Authors' Affiliations: Departments of 1Pharmacology and Chemical of autophagy (7). Beclin 1 binds to VPS34, a class III phos- Biology, 2Pathology, 3Cell Biology and Physiology, and 4Human Genetics, phoinositide 3-kinase, to drive autophagosome formation (6). University of Pittsburgh Cancer Institute, University of Pittsburgh, Pitts- A number of studies indicate a functional role of Beclin 1 in burgh, Pennsylvania cancer (7–9). Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Apoptosis interconnects with autophagy in a number of ways (10). Morphologic features of autophagy are often H. Li and P. Wang contributed equally to this work. observed in dying cells in which caspases are not sufficiently Current address for W.-X. Ding: Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, activated (11). Autophagy can be inhibited by antiapoptotic Kansas Bcl-2 and Bcl-XL (12), which interact with Beclin 1 through its Bcl-2 homology 3 (BH3) domain (13). Several ATG proteins, Current address for X.-M. Yin: Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana including ATG5 and Beclin 1, can be cleaved by calpain or Corresponding Author: Lin Zhang, the UPCI Research Pavilion, Room caspases that are activated during apoptosis (14, 15). Inhibi- 2.42a, Hillman Cancer Center, 5117 Centre Ave., Pittsburgh, PA 15213. tion of autophagy is known to enhance chemotherapy- Phone: 412 623-1009; Fax: 412 623-7778; E-mail: [email protected] induced apoptosis, and autophagy-modulating drugs are doi: 10.1158/0008-5472.CAN-10-4475 being tested in a number of combination chemotherapy trials Ó2011 American Association for Cancer Research. (16). However, it has remained unclear how apoptosis and www.aacrjournals.org 3625 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst March 28, 2011; DOI: 10.1158/0008-5472.CAN-10-4475 Li et al. autophagy affect each other in chemotherapy-induced apo- and pepstatin A (10 mg/mL). LC3II accumulation and p62 ptosis and whether autophagy is simply an innocent bystander degradation were analyzed by Western blotting. For analysis that is suppressed during the execution of apoptosis (17). of green fluorescent protein (GFP)-LC3 puncta formation, In this study, we used a genetic approach to dissect when cells were transfected with pEGFP-LC3 (19) prior to treatment and how autophagy is suppressed during chemotherapy- to induce autophagy. GFP-LC3 puncta signals were detected induced apoptosis. Our results revealed autophagy suppres- by confocal microscopy using a Zeiss LSM Pascal 5 and the sion through caspase 8–mediated cleavage of Beclin 1, follow- Plan-Apochromat 63Â/1.40 Oil DIC objective lenses. Images ing cytochrome c release. Specific ablation of Beclin 1 cleavage were acquired using the Zeiss LSM Image Browser Software. restored autophagy in apoptotic cells and led to enhanced GFP-LC3 puncta signals were quantified by counting at least cancer cell survival and therapeutic resistance. 300 cells for each sample and expressed as means Æ SD of 3 independent experiments. Autophagic vesicles, including Materials and Methods autophagosomes and autolysosomes, were analyzed by trans- mission electron microscopy as described (19). Electron Cell lines microscopy sections were imaged using a JEOL JEM 1011 Human colorectal cancer cell lines, including HCT116 (Peabody) at 80 V fitted with a bottom mount AMT 2k digital (obtained before 2002), Caco2 (obtained in 2008), RKO camera (Advanced Microscopy Techniques). (obtained in 2010), and LoVo (obtained before 2002), were from American Type Culture Collection. Cell lines were last Knockin of mutant Beclin 1 tested and authenticated for absence of Mycoplasma, geno- The Beclin 1 targeting vector was constructed using the types, drug response, and morphology in our laboratory in pUSER-rAAV (recombinant adenoassociated virus) System October 2010. (23). Briefly, 2 1-kb homologous arms flanking the fourth intron of Beclin 1 were inserted between 2 USER sites in Western blotting the AAV shuttle vector pTK-Neo-USER. The coding sequence Cells were lysed in the presence of a protease inhibitor for Beclin 1 double mutant (DM; D133A/D146A) was intro- cocktail (Roche Diagnostics) with RIPA radioimmunopreci- duced into the right arm using the QuickChange XL Site- pitation assay buffer (for probing LC3 and p62), a mixture of Directed Mutagenesis Kit (Agilent Technologies). For gene 0.25 mol/L sucrose, 10 mmol/L HEPES (pH 7.4), and 1 targeting, HCT116 cells were infected with the targeting rAAV mmol/L EGTA (for probing endogenous Beclin 1), or 2Â and selected by G418 (0.5 mg/mL; Mediatech) for 3 weeks. Laemmli sample buffer (for probing all other proteins). G418-resistant clones were pooled and screened by PCR for Western blotting was carried out as previously described targeting events using the primer pairs listed in Supplemen- (18). Antibodies included those against N-terminal and tary Table S1. To target the second allele, Neo flanked by 2 C-terminal Beclin 1 (Sigma), caspase 8 (Cell Signaling Tech- LoxP sites was excised from a heterozygous clone by infection nology), glutathione S-transferase (GST; GE Healthcare), V5 with an adenovirus expressing Cre recombinase (Ad-Cre). The (Invitrogen), cytochrome oxidase subunit IV (Cox IV; Invi- same targeting construct was used in the second round of trogen), a-tubulin (BD Biosciences), LC3 (19), and p62 gene targeting. After the second round, Neo was excised by Ad- (Novus Biologicals). Cre infection and targeting was verified by sequencing of genomic DNA and Western blotting. Analysis of apoptosis After treatment, attached and floating cells were har- Xenograft tumor experiments vested at different time points. Apoptosis was analyzed by All animal experiments were approved by the University of counting cells with condensed chromatin and micronuclea- Pittsburgh Institutional Animal Care and Use Committee. tion following nuclear staining with Hoechst 33258 (Invitro- Xenograft tumors were established by s.c. injecting 3.5 Â gen; ref. 20). A minimum of 300 cells were analyzed for each 106 wild-type (WT), or 7.0 Â 106 Beclin 1-KI HCT116 cells sample. Annexin V staining was carried out as previously into both flanks of 5- to 6-week-old female Nu/Nu mice