Polyi:C–Induced, TLR3/RIP3-Dependent Necroptosis

Published OnlineFirst April 21, 2015; DOI: 10.1158/2326-6066.CIR-14-0219

Research Article Cancer Immunology Research PolyI:C–Induced, TLR3/RIP3-Dependent Necroptosis Backs Up Immune Effector–Mediated Tumor Elimination In Vivo Ryo Takemura1,2, Hiromi Takaki1, Seiji Okada3, Hiroaki Shime1, Takashi Akazawa4, Hiroyuki Oshiumi1, Misako Matsumoto1, Takanori Teshima2, and Tsukasa Seya1

Abstract

Double-stranded RNA directly acts on fibroblast and myeloid species, but not on MDA5, MAVS, or the caspases/inflammasome lineages to induce necroptosis as in TNFa. Here, we investigated activation. However, the RNA-derived necroptosis was barely whether this type of cell death occurred in cancer cells in response reproduced in vivo in a CT26 tumor–implanted Balb/c mouse to polyinosinic–polycytidylic acid (polyI:C) and the pan-caspase model with administration of polyI:C þ zVAD. Significant shrink- inhibitor z-Val-Ala-Asp fluromethyl ketone (zVAD). We found age of CT26 tumors was revealed only when polyI:C (100 mg) was that the colon cancer cell line CT26 is highly susceptible to injected intraperitoneally and zVAD (1 mg) subcutaneously into necroptosis, as revealed by staining with annexin V/propidium tumor-bearing mice that were depleted of cytotoxic T lympho- iodide. CT26 cells possess RNA sensors, TLR3 and MDA5, which cytes and natural killer cells. The results were confirmed with are upregulated by interferon (IFN)-inducing pathways and immune-compromised mice with no lymphocytes. Although linked to receptor-interacting protein kinase (RIP) 1/3 activation necroptosis-induced tumor growth retardation appears mecha- via TICAM-1 or MAVS adaptor, respectively. Although exogenous- nistically complicated and dependent on the injection routes of ly added polyI:C alone marginally induced necroptosis in CT26 polyI:C and zVAD, anti-caspase reagent directed to tumor cells cells, a combined regimen of polyI:C and zVAD induced approx- will make RNA adjuvant immunotherapy more effective by mod- imately 50% CT26 necroptosis in vitro without secondary effects ulating the formation of the tumoricidal microenvironment and of TNFa or type I IFNs. CT26 necroptosis depended on the TLR3– dendritic cell–inducing antitumor immune system. Cancer Immunol TICAM-1–RIP3 axis in the tumor cells to produce reactive oxygen Res; 3(8); 902–14. 2015 AACR.

Introduction categorized as apoptosis, pyroptosis, and necroptosis, based on the difference of caspases involved (4). In general, apoptosis is A cell death response frequently occurs in malignantly trans- induced by activation of caspase-8 or caspase-9. Pyroptosis is a formed cells accompanied with infection or inflammation. Viral form of caspase-dependent cell death initiated by the activation of infection also induces programmed cell death, which is believed caspase-1 or caspase-11 (4). Although necroptosis is usually to be a host defense to restrict viral spread (1). However, recent triggered by death receptors, including Fas receptor (FasR), tumor advance in cell death studies may offer an alternative interpreta- necrosis factor (TNF) receptor, and TNF-related apoptosis-induction of cell death in tumor cell biology: The products of dead cells ing ligand receptor, Toll-like receptors (TLR) can also induce profoundly modulate the immune system and microenviron- necroptosis when caspase-8 is inhibited by gene depletion or ment around the established tumor, which is largely relied on pharmacologic inhibitors (4). Caspase inhibitors, such as z-Val- the mode of cell death (2, 3). Programmed cell death was Ala-Asp fluoromethyl ketone (zVAD), block TNF-induced apoptosis in many cell lines, whereas some cell lines respond to 1Department of Microbiology and Immunology, Graduate School of TNFþzVAD by activating necroptosis pathways (5). Necroptosis, 2 Medicine, Hokkaido University, Sapporo, Japan. Department of but not apoptosis, is believed to cause modulation of the tumor Hematology, Graduate School of Medicine, Hokkaido University, Sap- poro, Japan. 3Division of Hematopoiesis, Center for AIDS Research, microenvironment (TME) that affects tumor progression and Kumamoto University, Kumamoto, Japan. 4Department of Tumor invasion (6). Exosomes, proteins, and nucleic acids, released Immunology, Research Institute, Osaka Medical Center for Cancer and from dying cells, may be an important extracellular source of Cardiovascular Diseases, Osaka, Japan. the environmental effectors (7). Double-stranded (ds) and Note: Supplementary data for this article are available at Cancer Immunology stem-structured RNAs representing host and viral patterns of Research Online (http://cancerimmunolres.aacrjournals.org/). innate immunity also serve as modifiers for inflammatory R. Takemura and H. Takaki contributed equally to this article. environment and host immune response (8–10). Corresponding Author: Tsukasa Seya, Department of Microbiology and Immu- Polyinosinic–polycytidylic acid (polyI:C), a synthetic analogue nology, Graduate School of Medicine, Hokkaido University, Kita 15, Nishi 7, Kita- of dsRNA, has been known to have direct cytolytic activity ku, Sapporo 060-8638 Japan. Phone: 81-11-706-5073; Fax: 81-11-706-7866; on fibroblast and macrophage lineages. Myeloid cells in tumor E-mail: [email protected] are highly susceptible to dsRNA compared with normal cells doi: 10.1158/2326-6066.CIR-14-0219 (7, 11), whereas antigen-presenting dendritic cells (DC) mature 2015 American Association for Cancer Research. in response to dsRNA followed by immune activation (8, 10),

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suggesting the presence of a cell type–specific RNA-sensing mach- Japan); G1 (hepatocellular carcinoma) and G5 (hepatocellular inery that determines the life or death fate in the cell. PolyI:C carcinoma) from Dr. Y. Saeki (Osaka Medical Center for Cancer appear to trigger both apoptosis and necroptosis (8, 10). In vitro and Cardiovascular Diseases, Osaka, Japan); Renca (renal ade- studies on innate immunity suggested that several signaling path- nocarcinoma) from Dr. Y. Matsushita (Iwate Medical University ways could be involved in polyI:C–derived cell death, although School of Medicine, Iwate, Japan); and MC38 (colon adenocar- there are cell type–dependent variations in the resultant cell death cinoma) from Dr. H. Tahara (University of Pittsburgh Medical (12, 13). However, in tumor cells, the signal that induces cell Center, Pittsburgh, PA). A B16 subline, B16D8, was characterized death by polyI:C remains largely undetermined in in vivo models. as NK sensitive in our laboratory (21). All cell lines were con- PolyI:C is a ligand for both endosomal TLR3 and cytoplasmic firmed as Mycoplasma free. melanoma differentiation-associated protein-5 (MDA5) and YAC-1, EL4, B16F1, B16F10, B16D8, Renca, G1, G5, 3LL, L929, induces the activation of NF-kB and interferon regulatory factor EG-7, C1498, and CT26 cells were maintained in RPMI-1640 (IRF) 3 transcription factors followed by production of inflam- supplemented with 10% heat-inactivated fetal bovine serum and matory cytokines and type I/III interferons (IFN; refs. 8, 14). TLR3 antibiotics. MC38 cells were maintained in RPMI-1640 supple- and MDA5 are upregulated by polyI:C or IFN stimulation, sug- mented with 10% heat-inactivated fetal bovine serum, antibio- gesting that they are IRF3- and IFN-inducible factors (14). MDA5 tics, 2 mmol/L glutamine, 50 mmol/L 2-mercaptoethanol, 1 and TLR3 recruit different adaptors, mitochondrial antiviral sig- mmol/L sodium pyruvate, and nonessential amino acid. Necrop- naling protein (MAVS), or Toll-interleukin 1 receptor domain tosis-resistant CT26 cells were cells that survived after stimulation (TIR)–containing adaptor molecule (TICAM)-1, respectively (15, with 25 mg/mL polyI:C and 25 mmol/L zVAD, and maintained in 16), which confers distinct functional properties on the two RPMI-1640 supplemented with 10% heat-inactivated fetal bovine pathways. In response to polyI:C, TLR3/TICAM-1 activates IRF3 serum and antibiotics. For induction of bone marrow–derived as well as receptor-interacting protein kinase (RIP) 1/3 in a cell dendritic cells (BMDC), bone marrow cells from C57B6/J WT type–dependent manner (17). Upon malignant transformation, mice were cultured in RPMI-1640 with 10% heat-inactivated fetal cells usually express high levels of TLR3 and MDA5, which sense bovine serum and antibiotics containing J558 supernatant for 7 polyI:C and initiate RNA-sensing signals that are sometimes days with medium replenished every other day. linked to cell death or live output, including IFN/cytokine pro- Antibodies used were: anti-RIP1 (BD Biosciences), anti-RIP3 duction (13, 18). What factors discriminate between the death (QED Bioscience), anti-FLAG monoclonal (Sigma), anti-FLAG and live signal is yet unknown, but both IRF3-derived and IFN polyclonal (Sigma), anti-tubulin (BioLegend), anti-HA monoclo- receptor (IFNAR)–derived cell death have been reported (13, 18). nal (Covance), anti-b-actin (Sigma), allophycocyanin (APC) anti- Here, we found that polyI:C and zVAD induced cell death in the TLR3 (BioLegend), and fluorescein isothiocyanate (FITC) anti- mouse colon carcinoma cell line CT26. Because the dead cells TLR4 (MBL) antibodies. PolyI:C was purchased from Amersham were stained propidium iodide (PI)/annexin V–double positive Biosciences, zVAD, butylated hydroxyanisole (BHA), and nec-1 and a necroptosis inhibitor (necrostatin, nec-1) blocked the cell were from Sigma. Anti-IFNAR and anti-TNFa antibodies were death (12), we concluded that the polyI:C/zVAD–induced CT26 from BioLegend. cell death was necroptosis. This form of cell death was abrogated in CT26 cells after depletion of Ticam-1 or Ripk3 or treatment with Mice nec-1. In contrast, Mavs knockdown barely affected tumor cell Balb/c AJcl and C57B6/J WT female mice were purchased from death. Notably, blocking IFNAR or TNFa hardly affected the CLEA Japan. Rag-2/Jak3 double-KO mice in Balb/c background degree of polyI:C–induced CT26 necroptosis. Thus, necroptosis (22) were housed and monitored in our animal research facility was induced in CT26 colon cancer cells in vitro directly by polyI:C according to institutional guidelines. All mice were maintained and zVAD through the TLR3–TICAM-1–RIP3 pathway, indepen- under specific pathogen-free conditions in the Animal Facility in dent of IFN or TNFa. Neither the cytoplasmic RNA-sensor path- Hokkaido University Graduate School of Medicine (Sapporo, way (15) nor the TICAM-1–mediated inflammasome-caspase Japan) and used when they were 7 to 9 weeks of age. This study activation (19) participates in this type of tumor necroptosis. was carried out in strict accordance with the recommendations in In wild-type mice, polyI:C adjuvancy promotes cross-priming the Guide for the Care and Use of Laboratory Animals of the NIH þ of CD8 T lymphocytes, activation of natural killer (NK) cells, (Bethesda, MD). The protocol was approved by the Committee on and IFN/cytokine production by DCs (14, 20). We detected NK/ the Ethics of Animal Experiments in the Animal Safety Center, cytotoxic T lymphocyte (CTL)/cytokine-independent tumor- Hokkaido University (Hokkaido, Japan). All mice were used necroptotic shrinkage in CT26-bearing immune-compromised according to the guidelines of the Institutional Animal Care and Balb/c mice by injection of polyI:C and zVAD in vivo. Use Committee of Hokkaido University, approval no. 13-0043. All efforts were made to minimize suffering.

Materials and Methods Tumor challenge and antibody treatment Cell culture and reagents Mice were shaved at the back and injected 200 mLof1 105 or Cell lines EG7 (lymphoma) and C1498 (acute myeloid leuke- 5 105 CT26 cells in PBS. Tumor size was measured using caliper. mia) were obtained from the American Type Culture Collection; Tumor volume was calculated using the following formula: tumor 3LL (Lewis lung carcinoma), YAC-1 (lymphoma), and colon26 volume (cm3) ¼ (long diameter) (short diameter)2 0.4. PolyI: (CT26, colon carcinoma) from Summit Pharmaceuticals Interna- C (usually 100 mg) was injected into mice intraperitoneally (i.p.) tional Corporation; L929 (ﬁbroblast) from RIKEN Cell Bank; EL4 or subcutaneously (s.c.), whereas zVAD (1 mg) was s.c. injected þ (lymphoma) from Dr. N. Sato (Sapporo Medical University per mouse. For depletion of CD8 T cells and NK cells, anti- School, Sapporo, Japan); B16F1 (melanoma) and B16F10 (mel- murine CD8b antibody prepared from H35.17.2 hybridoma that anoma) from Dr. O. Hazeki (Hiroshima University, Hiroshima, was kindly provided by Dr. Toshitada Takahashi (Aichi Cancer

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Center, Aichi, Japan; ref. 23) and anti-asialo-GM1 antibody FACS analysis of dead cells (WAKO) were i.p. injected before polyI:C treatment (21). Opti- For the detection of dead cells, 1 105 CT26 cells were plated in mal doses of the antibodies were determined in preliminary 24-well plates. The following day, cells were stimulated with 25 or studies (anti–asialo-GM1 30 mL/body), and the same lot of 50 mg/mL of polyI:C, 25 mmol/L of zVAD, and 50 mmol/L of nec-1. anti-CD8b antibody was used in the experiment. After 24 hours, cells were stained with PI and annexin V–FLOUS staining kit (Roche) following the manufacturer's instructions. Water-soluble tetrazolium salts-1 assay Cells were stained by intracellular staining with APC anti-TLR3 A water-soluble tetrazolium (WST)-1 Cell Counting kit (11F8), FITC anti-TLR4 (UT49), or isotype control antibody with (Dojindo) was used following the manufacturer's instructions. or without permeabilization. Stained cells were analyzed by flow Cells (2 104) were plated in a 96-well plate. The following day, cytometry. cells were stimulated with 25 or 50 mg/mL of polyI:C, 25 mmol/L of zVAD, and 50 mmol/L of nec-1. After 24 hours, 10 mL of WST-1 reagent was added to each well and incubated at 37C for 1 to 4 Results hours. The absorbance at 450 nm was measured by a microplate Necroptosis induced by polyI:C and zVAD reader. Necoptosis is induced in macrophages by TLR stimulation when caspase-8 is inhibited by caspase inhibitors, such as Detection of reactive oxygen species zVAD (4). To determine whether polyI:C or combination of CT26 cells (2 104) were plated in a 96-well plate. The polyI:C/zVAD could induce cell death in tumor cell lines, we following day, cells were stimulated with 50 mg/mL of polyI:C, added these reagents to the culture of mouse tumor cell lines, 25 mmol/L of zVAD, 100 mmol/L of BHA, and 50 mmol/L of nec-1. including YAC-1, EL-4, B16F1, B16F10, B16D8, Renca, G1, G5, After 6 hours, CT26 cells were incubated with 5 mmol/L CM- 3LL, MC38, and CT26. The L929 fibroblast cell line was used as H2DCFDA (Invitrogen) at 37 C for 15 minutes. Cells were washed a positive control for cell death (24). Cell viability was mea- with culture medium and incubated at 37C for 15 minutes. Cells sured by the WST-1 assay (Supplementary Table S2). Cell were washed with FACS buffer and analyzed by flow cytometry. death was detected after polyI:C and zVAD treatment only in the CT26 cell line of all the tumor cell lines tested. A negligible Measurement of high-mobility group protein B1 (HMGB1) level of cell death was detected by treatment with either polyI: An HMGB1 ELISA kit (Shino test) was used as per the manu- C or zVAD alone (Fig. 1A and B). PolyI:C/zVAD–dependent facturer's instructions. CT26 cells (2 104) were plated in a 96-well cell death was observed in the CT26 as well as the control L929 plate. The following day, cells were stimulated with 25 mg/mL of cell line (Supplementary Table S2), suggesting that polyI:C– polyI:C, 25 mg/mL of zVAD, and 50 mmol/L of nec-1. After 24 hours, induced factors other than caspases are involved in polyI:C– HMGB1 in culture supernatants was quantified with the HMGB1 derived cell death in most tumor lines. Although polyI:C ELISA kit (Shino test) following the manufacturer's instructions. activates NALP3 inflammasome via TICAM-1 and caspase-11 in bacterial infections (19), no zVAD-affecting factor partici- Cytometric bead array assay and ELISA pates in caspase-11 or IL1b levels in CT26 cells (Supplemen- The production of cytokines was measured by a cytometric tary Fig. S1). bead array (CBA) assay (BD Biosciences). Culture supernatants or Comparing CT26 cells treated with polyI:C and zVAD with sera were incubated with capture beads for 1 hour at room CT26 cells treated with polyI:C alone, the population of cells temperature following incubation with phycoerythrin (PE)- stained annexin V–positive (10.5% vs. 0.3%) and double-positive labeled detection reagents. The intensity of beads bound to for annexin V and PI (23.4% vs. 3.7%) were increased by the cytokines was detected by flow cytometry. Data analysis was addition of zVAD to polyI:C (Fig. 1C). Although dead cells were performed by FCAP Array Software. Culture supernatants of CT26 stained with PI (Fig. 1D), the rate of single positive for annexin V cells or concanavalin A–stimulated splenocytes (5 105) were was increased after polyI:C and zVAD treatment (Fig. 1C), indi- analyzed for IFNg levels using ELISA. An IFNg ELISA kit was cating that polyI:C/zVAD–induced cell death was involved, in purchased from eBiosciences. The assay was performed according part, in apoptosis. HMGB1, a necrosis marker, was produced by to the manufacturer's instructions. IFNb levels in sera were mea- polyI:C/zVAD stimulation (Fig. 1E). Hence, both necrosis and sured by a mouse IFN beta ELSA kit (PBL) following the manu- apoptosis are induced in CT26 cells by treatment with polyI:C facturer's instructions. independent of caspases.

TUNEL staining The properties of CT26 necroptosis induced by polyI:C and Tumors isolated from CT26-bearing Rag2 / /Jak3 / mice zVAD were fixed with 4% paraformaldehyde/PBS for 30 minutes at To further analyze the features of cell death after polyI:C and 4 C. Fixed tissues were impregnated with 15% sucrose/PBS for 2 zVAD stimulation, we examined whether cell death induced by hours following 30% sucrose/PBS for overnight at 4 C. Tissues polyI:C and zVAD could be prevented by treatment with nec-1, an were then embedded in O.T.C. compound (Sakura Finetek inhibitor of RIP1 kinase in the necroptosis pathway. Consistent Japan), and the frozen tissue blocks were sectioned by using a with previous observations (25), when 50% of cell death was cryotome (LEICA CM1850). Terminal deoxynucleotidyl transfer- induced by polyI:C and zVAD, nec-1 treatment fully restored cell ase–mediated dUTP nick end labeling (TUNEL) staining of frozen viability (Fig. 2A and B). The population of PI-positive cells was sections was performed using an in situ cell death detection kit reduced with nec-1 treatment (Fig. 2C). Moreover, treatment with (Roche) following the manufacturer's instructions. Stained sec- nec-1 reduced the population of cells that were double positive for tions were monitored at 20 or 40 magnification using LSM510 annexin V and PI (Fig. 2D). By imaging analysis with PI, we META microscopy (Zeiss). confirmed that nec-1 inhibited cell death (Fig. 2E). Initiation of

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Figure 1. PolyI:C and zVAD induce necroptosis in CT26 cells. A–E, CT26 cells were stimulated with 25 mg/mL of polyI:C or 25 mmol/L of zVAD for 24 hours. A, cell viability was measured by the WST-1 assay. Data, mean SD of three independent samples. , P < 0.01. B, morphology was analyzed by microscopy. Arrowheads show cells with cytoplasmic swelling. C, cells were stained with PI and Annexin V and analyzed by flow cytometry. The number shows percentages of the gated population. One of two experiments is shown. D, cells were stained with PI and Hoecst33342 and analyzed by fluorescent microscopy. One representative of three experiments is shown. E, HMGB1 in the culture supernatants was quantified with an HMGB1 ELISA kit.

pyroptosis is mediated by caspase-11 expression that is dependent tion is crucial for necroptosis triggered by polyI:C and zVAD in on TLR4–TICAM-1 (26), but neither expression of caspase-11, tumor cells. production of IL1b (Supplementary Fig. S1), nor TLR4 protein Type I IFN, IFNg, and TNFa are inducers of necroptosis (27, was induced by treatment with polyI:C and zVAD in CT26 cells 28). To examine the participation of type I IFN and TNFa in (the latter compared with control JAWSII cells; Supplementary necroptosis induced by polyI:C and zVAD, CT26 cells were pre- Fig. S2). No evidence thus far endorses that cell death induced by treated with anti-IFNAR antibody or anti-TNFa antibody (Fig. polyI:C and zVAD involves NALP3-inflammasome- or caspase- 3A). Blocking IFNAR or TNFa signaling by specific antibody did mediated pyroptosis. not affect cell viability (Fig. 3A). The levels of TNFa protein in the Because reactive oxygen species (ROS) generation is a major culture supernatant of stimulated CT26 cells were below the inducer of necroptosis, we examined the possibility that ROS detection limit (Fig. 3B). CT26 cells were resistant to IFNg- being involved in polyI:C/zVAD–derived necroptosis. ROS gen- induced cell death (Fig. 3C) and they barely produced IFNg eration was induced in CT26 cells by polyI:C and zVAD treatment following polyI:C treatment (Fig. 3D). (Fig. 2F). Nec-1 treatment suppressed the ROS generation induced by polyI:C and zVAD (Fig. 2F). To investigate the effect of ROS The signaling pathway for necroptosis in CT26 cells generation on cell death induced by polyI:C and zVAD, CT26 cells To determine the pathway involved in polyI:C– and zVAD- were pretreated with BHA, a ROS scavenger. Both cell death and induced necroptosis, genes encoding molecules that might par- ROS generation induced by polyI:C and zVAD were suppressed ticipate in polyI:C recognition were silenced by siRNA in CT26 with BHA (Fig. 2F and G). These data indicate that ROS produc- cells with knockdown efficiencies higher than 50% (Fig. 4A). Tlr3

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or Ticam-1 knockdown resulted in a recovery of cells from necroptosis induced by polyI:C and zVAD (Fig. 4B). RIPK3 is the key molecule that interacts with mixed lineage kinase domain-like protein (MLKL) and RIP1 to transmit necroptosis signal (25, 28). Cell viability was recovered in Ripk3 knockdown cells (Fig. 4B). Mavs knockdown did not affect cell viability in polyI:C/zVAD– induced cell death (Fig. 4B). Collectively, necroptosis initiated by polyI:C and zVAD critically depends on the TLR3–TICAM-1–RIP3 pathway. In conﬁrmation experiments, CT26 cells expressed TLR3 protein (Fig. 4C) and Tlr3 knockdown cells did not induce Ifn-b mRNA in response to polyI:C (Fig. 4D), suggesting that TLR3 mainly signals the presence of polyI:C through the TICAM-1 pathway in CT26 cells. To identify the molecular mechanism of necroptosis induced by polyI:C and zVAD, we examined the physical interaction among TICAM-1, RIP1, and RIP3 in cells stimulated with polyI:C. CT26 cells were transfected with HA-tagged TICAM-1 and FLAG-tagged RIP3. Immunoprecipitation assays using anti- FLAG antibody after 24 hours of transfection showed that RIP3 interacted with TICAM-1 but not RIP1 in steady state conditions (Fig. 5A). Upon polyI:C stimulation, RIP3 interacted with both TICAM-1 and RIP1 in CT26 cells (Fig. 5A). However, in necroptosis-resistant B16D8 cells, RIP3 did not bind TICAM-1 after polyI:C stimulation, and little necroptosis was induced after treatment with polyI:C and zVAD (Fig. 5B and Supplementary Table S1). We sometimes found a minute amount of TICAM-1– RIP3 interaction in the absence of polyI:C stimulation in B16D8 cells (Fig. 5B), although the reason is as yet unknown. Even when the RIP3 protein was overexpressed, extrinsic RIP3 did not interact with TICAM-1 in B16D8 cells stimulated with polyI:C (Fig. 5B), and cell death was not induced by polyI:C and zVAD in RIP3- overexpressing cells (Supplementary Fig. S3). To identify the molecules that deﬁne sensitivity to cell death induced by polyI:C and zVAD, we established necroptosis-resistant CT26 cells by culturing in medium with polyI:C and zVAD (Fig. 5C). Expression of RIP3 was decreased in necroptosis- resistant CT26 cells (Fig. 5D and E). The expression levels of negative regulatory molecules of necroptosis, including cellular FLICE (FADD-like IL1b-converting enzyme)–inhibitory protein (cFILP)s, cFLIPL, and inhibitor of apoptosis (cIAP; ref. 4), remain unchanged between parent and death-resistant CT26 cells (Sup- plementary Fig. S4). Expression of 5-azacytidine–induced 2 (Azi2), dynamin-related protein (Dnm1l), Mlkl and phosphoglyc- erate mutase family member 5 (Pgam5), which are critical necroptosis factors downstream of RIP1 and RIP3 (4), was unaltered in necroptosis-resistant CT26 cells (Fig. 5D). RIP3-mediated cell death was observed with mouse bone marrow–derived

WST-1 assay. Data, mean SD of three independent samples. , P < 0.01. B, morphology was analyzed by microscopy. C, cells were stained with PI and analyzed by flow cytometry. The graph shows percentages of the PI-positive cells. Data, mean SD of three independent samples. , P < 0.01. D, cells were stained with PI/Annexin V and analyzed by flow cytometry. The number shows percentages of the gated population. One of two experiments is shown. E, cells were stained with PI and Hoecst33342 and analyzed by fluorescent microscopy. One of two experiments is shown. F and G, CT26 cells were stimulated with 50 mg/mL of polyI:C and 25 mmol/L of zVAD with or without 50 mmol/L of nec-1 or 100 mmol/L of BHA for 6 hours. F, cells were Figure 2. stained with CM-H2DCFDA and analyzed by flow cytometry. One Necrostatin-1 inhibits the necroptosis in CT26 cells. A–E, CT26 cells were representative of three experiments is shown. G, cell viability was measured stimulated with 25 mg/mL polyI:C and 25 mmol/L zVAD with or without 50 by the WST-1 assay. Data, mean SD of three independent samples. mmol/L necrostatin (nec)-1 for 24 hours. A, cell viability was measured by the , P < 0.01.

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Figure 3. Type I IFN, IFNg, and TNFa signaling barely participate in the CT26 necroptosis. CT26 cells were stimulated with 25 mg/mL of polyI:C and 25 mmol/L of zVAD in the presence of 10 mg/mL anti-IFNAR antibody or 10 mg/mL anti-TNFa antibody for 24 hours. A, cell viability was measured by the WST-1 assay. Data, mean SD of three independent samples. , P < 0.01. B, TNFa protein in culture supernatants of CT26 cells or 100 ng/mL LPS-stimulated BMDCs was measured by the CBA assay. Data, mean SD of two independent samples. C, cell viability of CT26 cells treated with polyI:C or 100 U/mL IFNg for 24 hours was measured by the WST-1 assay. Data, mean SD of three independent samples. D, IFNg protein in culture supernatants of CT26 cells or 5 mg/mL concanavalin A (ConA)-stimulated splenocytes was measured by ELISA. N.D., not detected. Data, mean SD of two independent samples. N.S., not statistically signiﬁcant.

macrophages (BMDM; ref. 25) and L929 (24), but not with EL4 the effect of zVAD on polyI:C–treated, NK/CD8b-depleted, CT26- (Supplementary Table S2). Tlr3 mRNA and Ifn-b mRNA expres- bearing mice. Mice were s.c. inoculated with 1 mg zVAD, which sions were not induced in EL4 cells in response to polyI:C (Takaki, suppresses death receptor–mediated liver injury in vivo (29). Unpublished Data), suggesting unresponsiveness of our EL4 cells Injection with s.c. zVAD and i.p. polyI:C resulted in tumor to polyI:C, the mechanism of which remains to be determined. regression in immune effector–depleted mice (Fig. 6C). In the Ripk3 mRNA was hardly expressed in B16D8, B16F10, 3LL, MC38, presence of CD8 T and NK cells, the zVAD effect was less than that C1498, and Renca cell lines that were resistant to cell death by expected from the in vitro results. Therapeutic use of polyI:C polyI:C and zVAD (Fig. 5F and Supplementary Table S2). Thus, promotes DC maturation but combination with zVAD tends to the interaction between TICAM-1 and RIP3 is a regulatory step in decrease the viability of BMDMs (25). Subcutaneous administra- polyI:C–induced necroptosis in some types of tumor cells. tion of polyI:C alone effectively induced growth retardation of CT26 tumors, but s.c. administration of polyI:C together with Tumor shrinkage induced by polyI:C/zVAD in CT26-implanted zVAD hampered the antitumor activity in NK/CD8 T cell–deplet- mice ed tumor-bearing mice (Fig. 6D). In our setting (Fig. 6D), NK and To investigate whether polyI:C/zVAD–induced necroptosis was CD8 T cells preferentially kill the tumor cell population that can involved in tumor retraction in vivo, CT26 cells were implanted in be targeted by polyI:C/zVAD. Although the reason why the polyI: Balb/c mice. Constant tumor growth was observed, as expected C/zVAD tumor suppression is abrogated by s.c. administration is (Fig. 6A). PolyI:C i.p. injection effectively and dose dependently unknown, these reagents might target epidermal myeloid and suppressed tumor growth (Fig. 6A). Injection of 100 mg polyI:C fibroblastic cells in this route, which alters the TME to barely resulted in >80% regression by day 20 of CT26-implanted tumors promote tumor retardation (Fig. 6D). compared with injection of a PBS control, and tumoricidal action To confirm the antitumor effect of zVAD and polyI:C in the of 1 mg zVAD only was negligible (data not shown). Tumor absence of immune cells, CT26-bearing Rag2 / /Jak3 / mice, regression by polyI:C treatment was partially abrogated when NK which lacks T, B, and NK cells (22), were injected with i.p. polyI:C þ cells or CD8 T cells were depleted by anti-asialo GM1 or CD8b- and s.c. zVAD (Fig. 7A). Although injection of polyI:C alone did specific antibodies (Fig. 6B), suggesting that tumor growth retar- not decrease tumor volume, additional s.c. zVAD treatment þ dation by polyI:C was mediated by NK cells and CD8 T cells. Of significantly suppressed tumor growth (Fig. 7A). Serum IFNb and note, DCs did not directly induce cytotoxicity in CT26 cells TNFa levels in polyI:C/zVAD–treated mice were comparable with (Supplementary Fig. S5). On the basis of these results, we assessed those treated with polyI:C alone (Fig. 7B). IFNg was not detected

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Figure 4. The TLR3–TICAM-1–RIP3 pathway governs polyI:C–derived necroptosis. A and B, cells were knocked down for Tlr3, Ticam-1, Ripk3,andMavs by siRNA. A, total RNA was collected and the mRNA levels of Tlr3, Ticam-1, Ripk3,andMavs were determined by real-time PCR. mRNA expression levels are shown as relative expression to b-actin. Data, mean SD of two independent samples. B, cells were stimulated with 25 mg/mL of polyI:C and 25 mmol/L of zVAD for 24 hours. Cell viability was measured by the WST-1 assay. Data, mean SD of three independent samples. , P < 0.01; N.S., not statistically signiﬁcant. C, cells were stained with anti-TLR3 antibody with or without permeabilization and analyzed by ﬂow cytometry. One of two experiments is shown. D, Tlr3-silenced cells were stimulated with polyI:C for 1 hour. Ifn-b mRNA expression was measured by real-time PCR. mRNA expression levels are shown as relative expression to b-actin.

in sera of mice treated with polyI:C or polyI:C/zVAD (Fig. 7B), plementary Fig. S6). Taken together, polyI:C/zVAD treatment indicating that IFNb, TNFa, or IFNg is dispensable for tumor induces cell death in CT26 tumors in mice without participation retardation in vivo as is in vitro. Upregulation of Ripk3, Ripk1, Mlkl of immune cells, resulting in tumor retardation. mRNA, which is a marker of necroptosis in vivo (30), was observed in tumors prepared from polyI:C/zVAD–treated mice as compared with those of polyI:C alone (Fig. 7C). TUNEL-positive cells Discussion also increased in tumors in polyI:C/zVAD–treated mice as com- Here, we demonstrate that administration of a TLR3 agonist pared with those treated with polyI:C alone (Fig. 7D and Sup- and pan-caspase inhibitor, zVAD, results in tumor regression in

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Figure 5. PolyI:C stimulation increases the coupling of TICAM-1 with RIP1 and RIP3. A, 6 105 CT26 cells and (B) 5 105 B16D8 cells were transfected with 2 mg of pCMVRIP3-FLAG and 2 mgof pcDNA4TICAM-1-HA, following stimulation with 25 mmol/L of zVAD. Twenty-four hours after transfection, cells were stimulated with 50 mg/mL polyI:C for 30 minutes and then lysed with lysis buffer. Total cell lysate (TCL) was subjected to immunoprecipitation with anti-FLAG antibody, and the complexes were analyzed by immunoblot with anti- FLAG, anti-HA and anti-RIP1 antibodies. C, parent and resistant cells were stimulated with 25 mg/mL of polyI:C and 25 mmol/L of zVAD for 24 hours and cell viability was measured by the WST-1 assay. Data, mean SD of three independent samples. , P < 0.01. D, mRNA expression of the indicated genes was determined by real-time PCR. mRNA expression levels are shown as relative expression to b-actin.E,2 106 parental CT26 cells or necroptosis- resistant CT26 cells were lysed with lysis buffer, and then analyzed by immunoblot with anti-RIP3 and anti- tubulin antibodies. F, level of Ripk3 mRNA in various cell lines. Total RNA was collected from CT26, EL4, EG7, C1498, Renca, L929, 3LL, B16D8, MC38, and B16F10 cells and expression of Ripk3 were determined by real-time PCR. mRNA expression is shown as relative expression to b-actin. Data, mean SD of two independent samples. N.D., not detected.

mice secondary to tumor cytolysis. Notably, direct action of these observed in fibroblasts and macrophages is mainly induced reagents on tumor cells induces a tumoricidal event. In the through the TICAM-1–RIP3 pathway that involves no caspases literature, activation of caspases usually accelerates programmed (13): This pathway is activated in the absence of caspase-8 activity. cell death, and prior polyI:C–mediated priming of TICAM-1 is This cell death process fits the definition of necroptosis, in which crucial in promoting caspase-mediated inflammasome activation cell death is RIR1/3-dependent and can be inhibited by nec-1, and in lipopolysaccharide (LPS) signaling (19, 26). In contrast with based on the production of ROS (4). We found this type of tumor the TICAM-1–inflammasome axis, which involves caspase-11 and death evidenced in the CT26 colon cancer cell line. Because effector caspases-3 and -7 (19, 31), the necrotic cell death similar RIP1/3-mediated necroptosis has been reported with

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Figure 6. Antitumor effect of polyI:C against CT26 cells. A, 1 105 CT26 cells were subcutaneously inoculated into Balb/c mice. Ten days later, tumor-bearing mice were injected i.p. with the indicated doses of polyI:C. The polyI:C treatment was repeated at days 17 and 24. Results represent means SD of 3 mice. , P < 0.05; , P < 0.01. B, 10 days after inoculation, tumor-bearing Balb/c mice were injected i.p. with anti-asialo GM1 or anti-CD8b antibodies following injection i.p. of 100 mg polyI:C. These treatments were repeated on day 14. Results represent mean SD of 3 mice. , P < 0.05; , P < 0.01. C and D, schedule of treatment is shown on the bottom. Balb/c mice inoculated with CT26 cells (5 105) were injected i.p. with anti-asialo GM1 and anti-CD8b antibodies and then injected i.p. (C) or s.c. (D) with 100 mg polyI:C with or without s.c. injection of 1 mg zVAD. These treatments were repeated 4 days after the ﬁrst treatment (day 10). Results represent mean SD of 3 mice. , P < 0.01. N.S., not statistically signiﬁcant.

neuroblastoma cell lines, most of which are caspase-8 deﬁcient not only TLR3 but also MDA5, as shown by the IFNb reporter (32), the RIP1/3-mediated tumor shrinkage we observed is not an assay (33): Type I IFN represents an output of the live signal isolated phenomenon in tumors. induced by RNA sensors. Virtually, no upregulation of IL1b is In CT26 necroptosis induction, the TLR3–TICAM-1 pathway detected in CT26 cells by stimulation with polyI:C/zVAD (Sup- plays a pivotal role without the involvement of TNFa and IFNs in plementary Fig. S1B), suggesting that inﬂammasome activation is the process of RIP3 activation followed by cytolysis. Thus, exog- again dispensable for the CT26 cell death. Upon polyI:C stimu- enously added polyI:C acts on TLR3 and, together with zVAD, lation, on the other hand, TICAM-1 immediately and transiently induces necroptosis in tumor cells. Exogenous polyI:C activates interacts with RIP1 and RIP3 to initiate necroptosis signaling,

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Figure 7. polyI:C and zVAD treatment induces tumor regression in CT26-bearing Rag2//Jak3/ mice. A, Rag2//Jak3/ mice inoculated with CT26 cells (5 105) were injected with or without i.p. of 100 mg polyI:C and s.c. of 1 mg zVAD at days 12 and 16 after inoculation. Results represent mean SD of 3 mice. , P < 0.01. B and C, Rag2 / /Jak3 / mice inoculated with CT26 cells (5 105) were injected with or without i.p. of 100 mg polyI:C and s.c. of 1 mg zVAD at day 14. B, after 2 hours, IFNb,IFNg, and TNFa proteins in sera were measured by the CBA assay or ELISA. Results represent mean SD of 3 samples. N.D., not detected; N.S., not statistically signiﬁcant. C, after 6 hours, Ripk1, Ripk3,andMlkl mRNA expression in tumors was measured by real-time PCR. Results represent mean SD of three samples. , P < 0.01. D, Rag2 / /Jak3 / mice inoculated with CT26 cells (5 105) were injected with or without i.p. of 100 mg of polyI:C and s.c. of 1 mg of zVAD. After 24 hours, frozen sections of tumors were prepared. Frozen sections were subjected to TUNEL staining. Stained sections were monitored at 40 magniﬁcation using microscopy. One representative of two experiments is shown.

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resulting in the production of ROS in CT26 cells. Although we cells and the parent CT26 cells (Fig. 5B). In L929 cells, which are cannot yet define what mechanism is responsible for the switch used for in vitro necroptosis studies (24), the Ripk3 mRNA level is from live to death signal in tumor cells by RNA stimulation, high as well as in CT26 and EL4 cells. Except for EL4, these RIP3 ROS production might reflect mitochondrial oxidative stress profiles imply that RIP3 expression correlates with sensitivity to induced by the TICAM-1 signal, leading to the PGAM5–DRP-1 polyI:C–induced necroptosis in most tumor lines. axis (4). However, simply overexpressing RIP3 is insufficient to trigger The function of the TICAM-1–RIP3 pathway has been shown to necroptosis: An additional phosphorylation or positive regulator be involved in the activation of the GTPase DRP1 that is translo- plays a key role in inducing cell death. Recently, sirtuin-2 (SIRT2), cated from cytosol to mitochondria to drive mitochondrial dam- MLKL, and PGAM5 were reported to positively regulate the TNF age in macrophages, which markedly modifies inflammation necroptotic pathway (4, 28, 42). MLKL interacts with RIP3 to (34). Because phosphorylation controls the RIP3–DRP1 activa- trigger necrosis in fibroblasts stimulated with TLR ligands (25). tion, the phosphatase–kinase balance in tumor cells needs to be The Pgam5 and Mlkl mRNA levels are unchanged between paren- further investigated. tal and resistant CT26 cells (Fig. 5D). Therefore, tumor necropto- In CT26 tumor–bearing mice, tumor growth is abrogated tic death represents an output produced by complex signaling with i.p. injection of polyI:C, which causes activation of anti- involving RNA sensors. þ tumor NK cells and CD8 CTLs (21, 23). These immune PolyI:C, a synthetic TLR3 agonist, is used as an effective effectors must be depleted in mice in order to detect an adjuvant for antitumor treatment and vaccines because of its þ alternative mode of tumor growth retardation by the direct prominent effects on DCs to induce CD8 T and NK cells action of polyI:C and zVAD on tumor cells. There appears no (21, 23). In addition, stem-structured RNA from viral replica- additive effect on immune activation and direct tumor killing tive intermediates can act as a TLR3-specific ligand (8, 9). A induced by polyI:C and zVAD in vivo, suggesting that the cell GpC-capped dsRNA named ARNAX, which exclusively targets þ death system supports the immune system in tumor clearance. TLR3, potently induces cross-presentation in CD8a DCs with- þ Notably, the direct tumoricidal activity by polyI:C/zVAD can be out a significant increase of serum cytokines (43). CD8a DCs, observed only when polyI:C is administered by the i.p. route. which have high expression of TLR3, are activated by i.p. or s.c. We surmised that s.c. injection of polyI:C and zVAD directly injection of these TLR3 ligands to promote cross-priming of T affected the viability of skin fibroblasts and Langerhans cells; cells in a TLR3/TICAM-1–dependent manner (23). This treat- the decreased viability of these lineages after polyI:C and zVAD ment also induces antitumor NK activation through induction treatment is consistent with that reported in the literature (25). of a polyI:C–inducible gene, INAM (IRF3-dependent NK-acti- Taken together, tumor necroptosis induced by polyI:C makes a vating molecule), in DCs (44, 45). Moreover, polyI:C injection small contribution to CT26 growth suppression in wild-type induces production of type I IFNs via the MAVS pathway in Balb/c mice with sufficient immune effectors in vivo.Yet,the stromal cells, which suppresses tumor growth (46). However, tumor necroptosis activity and tumor regression by polyI: the polyI:C/zVAD tumor regression was DC unrelated. Recent- C/zVAD become evident in Rag2 / /Jak3 / double-deficient ly, polyI:C was found to act on tumor-associated macrophages mice (Fig. 7). This study clarifies that the RNA-induced tumor to facilitate robust production of TNFa, resulting in hemor- clearance system works in tumors, which is engaged in necrop- rhagic necrosis of tumors (47). Tumors usually contain various tosis but is independent of the immune effectors or IFN/ types of macrophages concomitant with invasive properties cytokines. (48). The recruited macrophages are obliged to support tumor Although necrotic cells prepared by freeze–thaw cycles, form- progression, but often turn tumor suppressive in response to aldehyde fixation, or osmotic shock provoke no protective polyI:C via TNFa production (47). Like other myeloid species immune response in a tumor vaccination model (35–37), (48), ROS would be a macrophage-derived antitumor mod- heat-killed necrotic cells stimulate antigen-presenting cells to ulator induced via extracellular RNA stimulation. increase production of IL12 and TNFa (38). The inconsistent This study further emphasizes an alternative mode of RNA- results among studies might be explained by the differences in the mediated tumor suppression that is attributable to the direct composition and properties of damage-associated molecular effect of RNA on tumor cells independent of DC or macrophage patterns (DAMP)–containing RNA released from necrotic cells responses. Tumor regresses by direct action of RNA without that depends on type of stimulation, resulting in induction of participation of the products of macrophages or DAMPs. In diverse immune responses. In this context, self-RNA with incom- CT26 cells, tumor retardation by polyI:C depends on NK cells plete stems that activates TLR3 (39) would be involved in inflam- because of lower expression of class I MHC in CT26 cells (49). mation-mediated tumor cell death. Hence, in addition to the immune or macrophage activation by Although RIP1 and RIP3 are required to initiate necroptotic polyI:C, tumor cells can be direct targets of polyI:C for the signaling, necroptosis is induced in the absence of RIP1 (40, 41), induction of cell death. TLR3 is often expressed in murine and which has been reported to protect some tissue/organ cells from human tumor cells (50, 51). TLR3 levels in tumor cells would necroptosis, reflecting the complex arrays of necroptosis. Similar be a biomarker for the therapeutic efficacy of dsRNA therapy in to L929 cells, CT26 cells can be sensitized by polyI:C to induce renal cell carcinoma and breast cancer (52, 53). We found that necroptosis in the presence of zVAD (24). However, this result tumor necroptosis by TLR3 signaling occurred only under cannot always be generalized for the other tumor cell lines specific conditions, in line with the findings that caspase-8 (Supplementary Table S2 and Fig. 5F). The expression levels of deficiency, caspase inhibition by zVAD, or the presence of RIP3 is lower in B16D8 cells (resistant to the polyI:C–induced cell anticaspase viral proteins is required for necroptosis induced death) than in wild-type CT26 and L929 cells (Fig. 5F), in which by TNFa or death receptors (54, 55). Further elucidation of the necroptosis can be induced (24). The RIP3 protein expression molecular composition of RNA-mediated cell death and devel- levels are clearly different between necroptosis-resistant CT26 opment of a strategy to deliver an anti-caspase reagent to tumor

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cells will make RNA immunotherapy more potent in conjunc- Acknowledgments tion with dsRNA-mediated DC maturation. H. Takaki is a scholarship member in the Japan Society for the Promotion of Science. The authors thank laboratory members for valuable discussions. The Disclosure of Potential Conﬂicts of Interest authors are also grateful to Olivier Donze and George Chappuis for their kind gift of the RIR3 plasmid. No potential conﬂicts of interest were disclosed. Grant Support Authors' Contributions This work was supported, in part, by Grants-in-Aid from the Ministry of Conception and design: H. Takaki, M. Matsumoto, T. Seya Education, Science, and Culture (MEXT), "the Carcinogenic Spiral" a MEXT Development of methodology: R. Takemura, H. Shime, T. Seya Grant-in-Project, the Ministry of Health, Labor, and Welfare of Japan, the Takeda Acquisition of data (provided animals, acquired and managed patients, Foundation, the Yasuda Cancer Research Foundation, and the Kato Memorial provided facilities, etc.): R. Takemura, H. Takaki, S. Okada, H. Shime, Bioscience Foundation. T. Akazawa, T. Seya The costs of publication of this article were defrayed in part by the Analysis and interpretation of data (e.g., statistical analysis, biostatistics, payment of page charges. This article must therefore be hereby marked computational analysis): R. Takemura, H. Takaki, T. Seya advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Writing, review, and/or revision of the manuscript: H. Takaki, H. Oshiumi, this fact. M. Matsumoto, T. Seya Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Seya Received November 21, 2014; revised March 21, 2015; accepted April 7, 2015; Study supervision: T. Teshima, T. Seya published OnlineFirst April 21, 2015.

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914 Cancer Immunol Res; 3(8) August 2015 Cancer Immunology Research

PolyI:C−Induced, TLR3/RIP3-Dependent Necroptosis Backs Up Immune Effector−Mediated Tumor Elimination In Vivo

Ryo Takemura, Hiromi Takaki, Seiji Okada, et al.

Cancer Immunol Res 2015;3:902-914. Published OnlineFirst April 21, 2015.

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