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HSPA5 Regulates Ferroptotic Cell Death in Cancer Cells Shan Zhu1,2,3, Qiuhong Zhang4, Xiaofan Sun1, Herbert J

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HSPA5 Regulates Ferroptotic Cell Death in Cancer Cells Shan Zhu1,2,3, Qiuhong Zhang4, Xiaofan Sun1, Herbert J Published OnlineFirst January 27, 2017; DOI: 10.1158/0008-5472.CAN-16-1979 Cancer Therapeutics, Targets, and Chemical Biology Research HSPA5 Regulates Ferroptotic Cell Death in Cancer Cells Shan Zhu1,2,3, Qiuhong Zhang4, Xiaofan Sun1, Herbert J. Zeh III4, Michael T. Lotze4, Rui Kang4, and Daolin Tang1,2,3,4 Abstract Ferroptosis is a form of regulated cell death driven by oxidation. Importantly, the HSPA5–GPX4 pathway mediated oxidative injury promoting lipid peroxidation, although ferroptosis resistance, limiting the anticancer activity of gemci- detailed molecular regulators are largely unknown. Here, we tabine. Genetic or pharmacologic inhibition of the HSPA5– show that heatshock 70-kDa protein 5 (HSPA5) negatively GPX4 pathway enhanced gemcitabine sensitivity by disinhibit- regulates ferroptosis in human pancreatic ductal adenocarci- ing ferroptosis in vitro and in both subcutaneous and ortho- noma (PDAC) cells. Mechanistically, activating transcription topic animal models of PDAC. Collectively, these findings factor 4 (ATF4) resulted in the induction of HSPA5, which in identify a novel role of HSPA5 in ferroptosis and suggest a turn bound glutathione peroxidase 4 (GPX4) and protected potential therapeutic strategy for overcoming gemcitabine against GPX4 protein degradation and subsequent lipid per- resistance. Cancer Res; 77(8); 2064–77. Ó2017 AACR. Introduction contributes to ferroptotic cancer death (4). Conditional knockout of GPX4 in mice enhances ferroptosis in vivo (5–7). In addition to The therapeutic goal of all cancer treatment has been to trigger inhibition of GPX4 activity, increased GPX4 degradation also tumor-selective cell death and spare normal tissues. Acquisition of contributes to ferroptotic cancer cell death (8, 9). various genetic or epigenetic alterations in cancer cells may result The heatshock 70 kDa protein 5 (HSPA5, also termed GRP78 or in drug resistance by activating stress-adaptation responses such BIP) is a member of the molecular chaperones expressed primarily as the heat shock response or the unfolded protein response. in the endoplasmic reticulum (ER; ref. 10). As an important Understanding the molecular mechanisms of these protective component of the unfolded protein response, HSPA5 promotes responses in the setting of cell injury and death may help over- cell survival under conditions of ER stress (11). The role of HSPA5 come drug resistance in cancer cells. in ferroptosis has not been explored, although ferroptosis itself is Ferroptosis is a form of regulated cell death identified by Dr. associated with ER stress (12). Brent Stockwell's laboratory in 2012 (1). Different from apoptosis Pancreatic ductal adenocarcinoma (PDAC) is an extremely and necroptosis, activation of caspase and receptor-interacting lethal cancer with limited treatment options. In this study, we protein kinase is not required for induction of ferroptosis (1). In provide the first evidence that increased HSPA5 expression sup- contrast, lipid peroxidation plays a key role in mediating ferrop- presses ferroptosis by direct inhibition of GPX4 protein degrada- tosis (2). Recently, the discovery and characterization of ferrop- tion in human PDAC cells. Furthermore, we demonstrate that the tosis regulators has begun to unravel the molecular mechanisms HSPA5–GPX4 pathway mediates ferroptosis resistance, limiting of ferroptosis (3). Among them, glutathione peroxidase 4 (GPX4, the anticancer activity of gemcitabine (the first-line drug used an antioxidant enzyme) inhibits ferroptosis (4). Genetic or phar- alone or in combination for the treatment of patients with macologic inhibition of GPX4 increases lipid peroxidation, which advanced PDAC). These findings not only identify a novel role of HSPA5 in ferroptosis, but also suggest a potential therapeutic strategy for overcoming gemcitabine resistance in PDAC cells by 1 fi The Third Af liated Hospital, Key Laboratory for Major Obstetric Diseases of triggering ferroptosis. Guangdong Province, Key Laboratory of Reproduction and Genetics of Guang- dong Higher Education Institutes, Guangzhou Medical University, Guangzhou, Guangdong, China. 2Center for DAMP Biology, Guangzhou Medical University, Materials and Methods Guangzhou, Guangdong, China. 3Protein Modification and Degradation Labo- ratory, Guangzhou Medical University, Guangzhou, Guangdong, China. 4Depart- Antibodies and reagents ment of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania. The antibodies to HSPA5 (#3177), CANX (#2679), EIF2AK3 (#3192), ACTB (#3700), glyceraldehyde 3-phosphate dehydro- Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). genase (GAPDH, #5174), ATF4 (#11815), HSP90 (#4877), SLC7A11 (#12691), and Myc-tag (#2278) were obtained from S. Zhu and Q. Zhang contributed equally to this article. Cell Signaling Technology. The antibody to GPX4 (#ab125066), Corresponding Author: Daolin Tang, Guangzhou Medical University, Guang- p-EIF2AK3 at T982 (#ab192591), and p-ERN1 at S724 zhou, Guangdong 510150. Phone: 020-81292013; Fax: 020-81292013; E-mail: (#ab124945) were obtained from Abcam. Z-VAD-FMK (#V116); [email protected]) staurosporine (#S4400), rapamycin (#R0395), H2O2 (#216763), doi: 10.1158/0008-5472.CAN-16-1979 EGCG (#E4143), cycloheximide (#C7698), sulfasalazine Ó2017 American Association for Cancer Research. (#S0883), and gemcitabine (#G6423) were obtained from Sigma. 2064 Cancer Res; 77(8) April 15, 2017 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst January 27, 2017; DOI: 10.1158/0008-5472.CAN-16-1979 Ferroptosis in Pancreatic Cancer Necrosulfonamide (#480073) was obtained from EMD Millipore thione reductase and NADPH. The reaction rate was proportional Corporation. TNFa (#210-TA-005) was obtained from R&D to the concentration of glutathione up to 2 mmol/L. The yellow Systems. Erastin (#E7781), ferrostatin-1 (#S7243), and liproxsta- product (5-thio-2-nitrobenzoic acid) was measured spectropho- tin-1 (#S7699) were obtained from Selleck Chemicals. tometrically at 412 nm. Cell culture GSSG assay PANC1, CFPAC1, MiaPaCa2, Panc2.03, and Panc02 cells were The relative GSSG concentration in cell lysates was assessed obtained from the ATCC or the National Cancer Institute (NCI, using a kit from Cayman (#703002) according to the manufac- À À USA). GPX4 / cells were a gift from Dr. Marcus Conrad (Institute turer's instructions. The quantification of GSSG, exclusive of GSH, of Developmental Genetics; Helmholtz Zentrum Munchen;€ was accomplished by first derivatizing GSH with 2-vinylpyridine. Munchen,€ Germany; ref. 7). These cells were grown in DMEM or In brief, 1 mL of 1 mol/L 2-vinylpyridine in absolute ethanol were RPMI1640 medium with 10% FBS, 2 mmol/L L-glutamine, and 100 added to 99 mL of cell homogenate. This suspension was incu- U/mL of penicillin and streptomycin. All cells were Mycoplasma free bated at room temperature for 60 minutes to block the thiol group and authenticated by short tandem repeat DNA profiling analysis. of the GSH already present. NADPH (95 mL of 2 mg/mL) in nanopure water and 5 mL of 2 U/mL glutathione reductase were Cell viability assay added to reduce GSSG. Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8; #96992, Sigma) according to the manufacturer's Western blot analysis instructions. The assay is based on using the highly water- Proteins in the cell lysate or supernatants were resolved on 4% soluble tetrazolium salt WST-8 [2-(2-methoxy-4-nitrophe- to 12% Criterion XT Bis-Tris gels (Bio-Rad) and transferred to a nitrocellulose membrane. After blocking with 5% milk, the mem- nyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] to produce a water-soluble formazan dye brane was incubated for two hours at 25 C or overnight at 4 C upon reduction in the presence of an electron carrier. Absor- with various primary antibodies. After incubation with peroxi- bance at 450 nm is proportional to the number of living cells dase-conjugated secondary antibodies for one hour at routine in the culture. temperature, the signals were visualized via using enhanced or super chemiluminescence (Pierce) and by exposure to X-ray films. Clonogenic cell survival assay The relative band intensity was quantified using the Gel-pro Long-term cell survival was monitored in a colony formation Analyzer software (Media Cybernetics). assay. In brief, 1,000 cells were plated into 24-well plates after treatment with indicated drugs for 24 hours. The cells were allowed Immunoprecipitation analysis to grow for the next 10 to 12 days to allow colony formation and Cells were lysed at 4C in ice-cold radioimmunoprecipitation the colonies were visualized using crystal violet staining. assay buffer (Millipore), and cell lysates were cleared by a brief centrifugation (12,000 Â g, 10 minutes). Concentrations of GPX4 activity assay proteins in the supernatant were determined by BCA assay. Before The activity of GPX4 was determined using l-a-phosphatidyl- immunoprecipitation, samples containing equal amount of pro- choline hydroperoxide substrate and a coupled enzymatic assay as teins were pre-cleared with protein A or protein G agarose/ previously described (13, 14). It was based on the oxidation of sepharose (Millipore; 4 C, 3 hours), and subsequently incubated glutathione (GSH) to oxidized glutathione disulfide (GSSG) with various irrelevant IgG or specific antibodies (5 mg/mL) in the catalyzed by GPX4, which was then coupled to
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