AIM2, an IFN-Inducible Cytosolic DNA Sensor, in the Development of Benign Prostate Hyperplasia and Prostate Cancer

Published OnlineFirst July 17, 2013; DOI: 10.1158/1541-7786.MCR-13-0145

Molecular Cancer Cell Death and Survival Research

Larissa Ponomareva1,2, Hongzhu Liu1,2, Xin Duan1,2, Eric Dickerson1,2, Hui Shen1,2, Ravichandran Panchanathan2, and Divaker Choubey1,2

Abstract Close links have been noted between chronic inflammation of the prostate and the development of human prostatic diseases such as benign prostate hyperplasia (BPH) and prostate cancer. However, the molecular mechanisms that contribute to prostatic inflammation remain largely unexplored. Recent studies have indicated that the IFN-inducible AIM2 protein is a cytosolic DNA sensor in macrophages and keratinocytes. Upon sensing DNA, AIM2 recruits the adaptor ASC and pro-CASP1 to assemble the AIM2 inflammasome. Activation of the AIM2 inflammasome cleaves pro-interleukin (IL)-1b and pro-IL-18 and promotes the secretion of IL-1b and IL-18 proinflammatory cytokines. Given that human prostatic infections are associated with chronic inflammation, the development of BPH is associated with an accumulation of senescent cells with a proinflammatory phenotype, and the development of prostate cancer is associated with the loss of IFN signaling, the role of AIM2 in mediating the formation of prostatic diseases was investigated. It was determined that IFNs (a, b,org) induced AIM2 expression in human prostate epithelial cells and cytosolic DNA activated the AIM2 inflammasome. Steady-state levels of the AIM2 mRNA were higher in BPH than in normal prostate tissue. However, the levels of AIM2 mRNA were significantly lower in clinical tumor specimens. Accordingly, constitutive levels of AIM2 mRNA and protein were lower in a subset of prostate cancer cells as compared with BPH cells. Further, the cytosolic DNA activated the AIM2 inflammasome in the androgen receptor–negative PC3 prostate cancer cell line, suggesting that AIM2- mediated events are independent of androgen receptor status.

Implications: The AIM2 inﬂammasome has a fundamental role in the generation of human prostatic diseases. Mol Cancer Res; 11(10); 1193–202. 2013 AACR.

Introduction history of certain sexually transmitted infections or prosta- fl The etiologic agents that contribute to the development of titis, which are often associated with intraprostatic in ammation (6, 7). Furthermore, studies have indicated possible human prostatic diseases such as benign prostate hyperplasia fl (BPH) and prostate cancer remain largely unknown (1–4). links between certain prostatic infections and in ammation Studies have suggested close links among certain infections (6, 7, 11, 12). of the prostate, chronic inflammation, and the development Prostatic infections, which can induce the production of of inflammation-associated prostatic diseases (5–10). It has type-I IFN-a/b) and the expression of the IFN-inducible been proposed that certain infections of the prostate con- genes (ISG; refs. 13, 14), reduce telomere length (15), tribute to the development of chronic inflammation and resulting in an accumulation of senescent prostate epithelial regenerative "risk factor" lesions, referred to as proliferative cells (PrEC) in BPH (16). Accordingly, expression of a set of inflammatory atrophy, contribute to the development of ISGs is upregulated during the onset of prostate epithelial – cellular senescence (17–19). Furthermore, levels of the IFN- prostate cancer (3 5). Accordingly, studies have found an fi increased relative risk of prostate cancer in men with a prior b increase in human diploid broblasts (HDF) when HDFs approach cellular senescence (20) and the expression of IFN- inducible proteins is upregulated in senescent PrECs (21) Authors' Affiliations: 1Cincinnati VA Medical Center; and 2Department of Environmental Health, University of Cincinnati, Cincinnati, Ohio and HDFs (22). Senescent HDFs (and other cell types) are known to secret proinflammatory cytokines (23). This Corresponding Author: Divaker Choubey, Department of Environmental Health, University of Cincinnati, 3223 Eden Avenue, PO Box 670056, phenotype of senescent cells has been termed senescence- Cincinnati, OH 45267. Phone: 513-558-1014; Fax: 513-558-0925; E-mail: associated secretory phenotype (SASP). The SASP, which [email protected] depends on activation of NF-kB (24), activates the immune doi: 10.1158/1541-7786.MCR-13-0145 surveillance and exhibits protumorigenic properties (23). 2013 American Association for Cancer Research. Furthermore, the cultures of senescent HDFs contain dead

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cells, exhibit apoptosis, and the senescent HDFs release binant IFN-a, recombinant human IFN-b, recombinant "exosomes" (microvesicles), which when fused with other human IFN-g, and human TNF-a were purchased from cells, deliver dsDNA (a "danger signal") into the cytoplasm R&D Systems. Caspase inhibitor VI (Z-VAD-FMK) was of neighboring cells (25, 26). purchased from EMD Millipore. Poly(dA:dT)/LyoVec (syn- A complex of cytosolic proteins, which is termed "inflam- thetic double-stranded DNA in complex with LyoVec masome", is activated in innate immune cells (macrophages transfection agent) was purchased from InvivoGen. and dendritic cells) in response to sensing "danger signals" by receptors (27–29). These receptors include members of the Cell lines, culture conditions, and treatments nucleotide binding and oligomerization domain (NOD)-like Human normal PrECs prostate cancer cell lines were fi receptor (NLR) family proteins such as NLRP3 and certain cultured at 37 C in a humidi ed cell culture incubator members of the IFN-inducible p200-family proteins such as containing 5% CO2. Human normal PrECs in culture (at murine Aim2 and human AIM2 (29–31). The NLRP3 passage 2) were purchased from Lonza and were maintained receptor can sense cytoplasmic pathogens, self-derived mole- in culture as suggested by the supplier. Human prostate cules, and environmental materials (29). Upon sensing these cancer cell lines (RWPE-1, RWPE-2, DU-145, PC-3, and molecules, activated NLRP3 recruits an adaptor protein ASC LNCaP) were originally purchased from the American Type and pro-caspase-1 to form the NLRP3 inflammasome. Acti- Culture Collection (ATCC) and maintained in culture as vation of an inflammasome proteolytically cleaves pro-IL-1b suggested by the supplier. BPH-1 cell line was generously and pro-IL-18 to the mature forms and potentiates their provided by Dr. Simon Hayward (Vanderbilt University secretion from cells (27, 28). In addition, the activation of Medical Center, Nashville, TN; ref. 38). Cells were main- fi inflammasomes in macrophages induces a rapid highly tained in Dulbecco's modi ed Eagle medium (high glucose) inflammatory cell death called pyroptosis (29–31). culture medium (Invitrogen Life Technologies) supplemen- Activation of inflammasomes has been known in myeloid ted with 10% (v/v) FBS and antibiotics (Invitrogen). cells such as monocytes and macrophages (32). Interestingly, When indicated, cells were treated with either IFN-a activation of an inflammasome has also been reported in (1,000 units/ml), IFN-b (1,000 u/ml), or IFN-g (10 ng/ nonmyeloid cells (33, 34), which are the preferred host cells mL) for the indicated time (hours). When indicated, PrECs for many intracellular pathogens. For example, an infection of and PC-3 cells in culture were "primed" by incubating with cervical epithelial cells by Chlamydia trachomatis, an intra- IFN-g (10 ng/mL) and TNF-a (10 ng/mL) for the indicated cellular pathogen, leads to activation of caspase-1 through the time as described previously (34). NLRP3 inflammasome (33). The human AIM2 protein from the IFN-inducible p200- Antibodies protein family senses cytoplasmic dsDNA (hereafter referred Rabbit polyclonal antibodies that were raised against the as DNA) in myeloid cells (35–37) and "primed" (treated keyhole limpet hemocyanin-conjugated hAIM2 C-terminal with IFN-g and TNF-a) human keratinocytes (34). Upon peptide (GVHSTIKVIKAKKKT) have been described pre- fi sensing DNA, the AIM2 protein recruits an adaptor protein viously (20). Antibodies speci c to detect ASC (sc-22514) ASC to form the AIM2 inflammasome (31, 35–37). The and IL-18 (sc-7954) in immunoblotting were purchased activated AIM2 inflammasome in macrophages through from Santa Cruz Biotechnology. Antibodies for b-actin (cat activation of caspase-1 promotes the proteolytic cleavage #4967); IkBa (#9247); histone 3 (#9715); caspase 1 and secretion of proinflammatory cytokines (IL-1b and IL- (#3866); and IL-1b (#2022) were purchased from Cell 18). Given that human prostatic infections are associated Signaling Technology. Horseradish peroxidase-conjugated with chronic inflammation (6, 7), the development of BPH secondary anti-mouse (NXA-931) and anti-rabbit (NA-934) is associated with an accumulation of senescent cells with a antibodies were from GE Healthcare Biosciences. proinflammatory phenotype (16, 23), and the development of prostate cancer is associated with the loss of type I IFN Immunoblotting fl signaling (18, 21), we investigated the role of AIM2 protein Cells from subcon uent cultures were lysed with radio- in human PrECs and prostatic diseases. Here, we report that immunoprecipitation assay (RIPA) buffer (50 mmol/L Tris- cytosolic DNA can activate the AIM2 inflammasome in Cl (pH 7.4), 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% human PrECs. In addition, we found that the expression of sodium deoxycholate, 0.1% SDS). The buffer was supple- AIM2 mRNA was higher in BPH than normal prostate, mented with complete mini EDTA-free protease inhibitor fi cocktail. Lysates were centrifuged at 12,000 rpm for 5 whereas levels of AIM2 mRNA were signi cantly lower in prostate tumors and certain cancer cell lines that were tested. minutes at 4 C. Cell lysates containing equal amounts of Our observations show a role for the AIM2 inflammasome in protein ( 50 mg) were separated by SDS-PAGE, transferred fl the development of human prostatic diseases. to a polyvinylidene di uoride membrane, and immuno- blotted according to the manufacturer's guidelines.

Materials and Methods RNA isolation and PCR Reagents Total RNA was isolated from the cultured cells with Complete mini EDTA-free protease inhibitor cocktail was TRIzol reagent (Invitrogen). cDNA synthesis, reverse purchased from Roche Applied Science. Universal recom- transcription polymerase chain reaction (RT-PCR), and

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quantitative real-time TaqMan PCR were conducted to cultures of PC-3 cells in a 6-well plate were transfected with detect the expression of the AIM2 gene as described previ- the AIM2-luc reporter plasmid (1.8 mg) along with the pRL- ously (20). The following primers were used for RT-PCR: TK reporter plasmid (0.2 mg) as an internal control, using human AIM2 (forward: 50-ATGTG AAGCCGTCCAGA- FuGene6 Transfection Reagent (Roche). Thirty-six to forty 30; backward: 50-CATCATTTCTGA TGGCTGCA-30), hours after transfections, the firefly luciferase and Renilla human ASC (forward: 50-GCCTGCACTTTATAGAC- luciferase activities were assayed using dual-luciferase report- CAGC-30; backward: 50- GCTTCCGCATCTTGCT- er assay kit (Promega). The relative firefly luciferase activity is TGG-30), human CASP-1 (forward: 50-TCCAATAATG- expressed as the ratio of the firefly luciferase and Renilla GACAAGTC AAGCC-30; backward: 50-GCTGTACCC- luciferase. CAGATTTTGTAGCA-30), human IL-1b (forward: 50- CTCG CCAGTGAAATGATG GCT-30; backward: 50- Cell fractionations GTCGGAGATTCGTAGCTGGAT-30), and actin (for- Cytosolic and nuclear fractions from cultured cells were ward: 50-GCTCGTCGTCGACA ACGGCTC-30; back- prepared as described previously (20). Briefly, cell pellets ward: 50-CATGATCTGGGTCA TCTTCTC-30). The fol- were incubated on ice for 10 minutes in cell fractionation lowing TaqMan gene-specific assays were purchased from buffer (20 mmol/L Tris-HCl, pH 7.5; 0.65% NP-40, 150 the Applied Biosystems and used as suggested by the mmol/L NaCl) that was supplemented with protease and supplier: AIM2 (Hs 00175457_m1) and the endogenous phosphatase inhibitors. The cytosolic fractions were recov- control b-actin (assay Id# Hs99999903_ml). TissueScan ered by centrifuging cell lysates at 2,000 rpm for 3 minutes in Cancer and Normal Tissue human prostate cDNA Arrays a refrigerated microcentrifuge. The nuclear pellet was were purchased from OriGene. The arrays also included washed with the cell fractionation buffer and the pellet was the information (age, tumor grade, and minimum stage of suspended into the RIPA buffer followed by brief sonication. the tumor) about the samples. The PCR plates containing The lysates were centrifuged at 12,000 rpm for 10 minutes at cDNAs from the normal or prostate tumors (Gleason 4C and supernatants were collected. score ¼ 6–10) for quantitative polymerase chain reaction (qPCR) were subjected to RT-PCR as suggested by the Statistical analyses supplier. In brief, the quantitative RT-PCR technology The measurement values are presented as means SEM. (Applied Biosystems) was used to compare the expression The statistical significance of differences in the measured of the AIM2 gene. The PCR cycling program consisted of mean frequencies between the two groups was calculated denaturing at 95C for 10 minutes and 40 cycles at 95Cfor using the Student two-tailed t test. 15 seconds, and annealing and elongation at 60Cfor1 minute. The signal in samples was normalized using the Results housekeeping genes as suggested by the supplier. Expression of AIM2 inflammasome proteins is detectable in human PrECs and the IFN treatment of cells increases Transfections the expression levels Subconfluentcultures ofPrECs in60mm plates were either Expression of AIM2 protein is induced by treatment of left untreated (control) or were "primed" as described previ- cells with IFNs in a variety of cell types (31, 40). Therefore, ously. Control or "primed" cultures of cells were either incubated with LyoVec (control) or poly(dA:dT)/LyoVec (5 mg/ mL) for the indicated time. At the end of incubations, cells were harvested to isolate RNA or to prepare total cell lysates.

Nucleofections PC-3 cells (2 106 cells/nucleofection) were nucleofected as described previously (39). In brief, cells were nucleofected with 2 mg of plasmid DNA (pCMV-GFP) using the Nucleofector-II device (Amaxa Biosystems, Nattherman- nalle 1) and the nucleofection kit-V (program U-001). These nucleofection conditions resulted in approximately 60% cell survival after 24 hours of incubations of cells. After nucleofections, cells were harvested at the indicated times. Figure 1. Expression of AIM2 inflammasome proteins is detectable in human normal PrECs and the IFN treatment of cells increases the expression. A, human normal PrECs at an early passage (passage 3) were Reporter assays 0 either left untreated (lane 1) or treated with IFN-a (lane 2), b (lane 3), or g We have cloned the 5 -regulatory region (1.0 Kb) of the (lane 4) for 18 hours. After the treatment, total cell lysates containing human AIM2 gene into a basic pGL3 reporter plasmid equal amounts of proteins were analyzed by immunoblotting using (plasmid designated as AIM2-luc), which does not contain antibodies specific to the indicated proteins. B, human normal PrECs were treated with the indicated IFN as described in A. After the treatment, any enhancer and promoter sequences. Therefore, the tran- total RNA was isolated from cells and reverse transcribed. Steady-state scription of the reporter gene in the AIM2-luc-reporter is levels of the mRNAs were analyzed for the indicated genes by RT-PCR driven by the AIM2 promoter sequence. Subconfluent using a pair of the gene-specific primers. Caspase, CASP.

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to investigate the role of the AIM2 inflammasome in human increases basal levels of both AIM2 and pro-IL-1b (34). PrECs, we examined basal and the IFN-induced levels of Therefore, to investigate whether AIM2 inflammasome can AIM2 inflammasome proteins in young PrECs. As shown be activated by cytosolic DNA in PrECs, we transfected in Fig. 1A, the basal levels of AIM2 protein, which is detected synthetic DNA [poly(dA:dT)] into "primed" human PrECs. as two closely migrating protein bands (20), adaptor protein Surprisingly, transfection of cells with the synthetic DNA ASC, pro-caspase-1 (p45), and pro-IL-1b (p31) were detect- decreased cellular levels of AIM2 protein (Fig. 2A, compare able in PrECs. Interestingly, the IFN (a, b,org) treatment lanes 2 with lane 1). However, pretreatment of cells with z- of cells for 24 hours differentially increased the levels of the VAD (a broad-spectrum inhibitor of caspase; 41) decreased AIM2, pro-caspase-1, and pro-IL-1b proteins. Notably, the the extent of decrease in AIM2 protein levels after transfec- treatment also increased the levels of the activated caspase-1 tion of synthetic DNA (compare lane 3 with lane 2). (p20) to different extents. Consistent with the above obser- Notably, transfection of the synthetic DNA into PrECs vations, treatment of PrECs with either IFN-a, b,org also decreased the levels of pro-caspase-1 (p45; ref. Fig. 2A), thus, increased the steady-state levels of AIM2 mRNA to different indicating a proteolytic cleavage of the pro-caspase-1 and extents (Fig. 1B). The maximum increase was noted after activation of the AIM2 inflammasome (34). Correspond- IFN-g treatment. Notably, the treatment of PrECs with ingly, treatment of cells with z-VAD increased cellular levels IFN-a, b,org also increased the steady-state levels of of procaspase-1 (p45) and cleaved caspase-1 (p20 and p10; caspase-1 mRNA. In contrast, treatment of cells with compare lane 3 with 2), indicating an inhibition of proteo- IFN-a or b, but not IFN-g, decreased steady-state levels lytic cleavage of pro-caspase-1 (p45) and the secretion of of IL-1b mRNA. Together, these observations revealed that activated caspase-1 (p20 and p10) into the culture medium. human normal PrECs in culture express low, but detectable, Accordingly, the introduction of synthetic DNA into cells basal levels of AIM2 inflammasome proteins and the acti- reduced the cellular levels of IL-18, whereas the secreted vation of IFN signaling in cells by type I (IFN-a or b) or type levels of IL-18 increased in the medium (compare lane 2 with II IFN (IFN-g) differentially regulates the steady-state levels 1). Again, the treatment of cells with z-VAD inhibited a of the AIM2 and other inflammasome proteins. decrease in the cellular levels of IL-18 and an increase in levels of IL-18 in the medium. Notably, cellular levels of pro- Cytosolic DNA activates the AIM2 inflammasome in IL-1b did not change after transfection of synthetic DNA PrECs into PrECs. Similarly, transfection of increasing amounts of Cytosolic DNA activates the AIM2 inflammasome in plasmid DNA into PrECs increased the cleavage of pro- "primed" (primed with IFN-g and TNF-a treatment) caspase-1 (indicated by an increase in the levels of the p20 human keratinocytes (34). The "priming" of keratinocytes form) and increased the levels of secreted IL-18 in the

Figure 2. Cytosolic dsDNA activates the AIM2 inﬂammasome in human PrECs. A, human PrECs at early passage (passage 3) were "primed" by treating with IFN-g (10 ng/mL) for 14 hours and then treated with TNF-a (10 ng/mL) for 3 hours. The "primed" cells were either left untreated (lane 1) or incubated with poly(dA:dT)/LyoVec (5 mg/mL) without any further treatment (lane 2) or with Z-VAD-FMK (10 mmol/L lane 3) for 6 hours. At the end of the incubations, cells were lysed and cell lysates containing equal amounts of proteins were analyzed by immunoblotting. B, cells of human benign prostate hyperplasia cell line BPH-1 were either left without any treatment (lanes 1 and 2) or "primed" with IFN-g and TNF-a treatment (lanes 3 and 4). Control and "primed" cells were either left untreated (lanes 1 and 3) or incubated with poly(dA:dT)/LyoVec (5 mg/mL) for 6 hours. At the end of the incubations, cells were harvested and lysates containing equal amounts of proteins were analyzed for the indicated proteins by immunoblotting. Caspase, CASP.

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Figure 3. AIM2 expression levels in human prostatic diseases. A, total cell lysates were prepared from human proliferating PrECs (lane 1) and senescent PrECs (lane 2). Cell lysates containing equal amounts of proteins were analyzed by immunoblotting. B, TissueScan Prostate Cancer Tissue qPCR Array I (from Origene) containing normal samples (7 samples) and BPH samples (11 samples) were analyzed by quantitative RT-PCR for the AIM2 mRNA levels as suggested by the supplier. The normalized relative values are indicated. Results are mean values of triplicate experiments and the error bars represent the SD (, P < 0.05). C, TissueScan Prostate Cancer Tissue qPCR Array II (from Origene) containing 48 samples (normal samples: 8; adenocarcinoma samples 40; Gleason score 3–5 þ 3–5 ¼ 6–9/10) were analyzed by RT-PCR for the AIM2 mRNA. The normalized values are indicated. Results are mean values of triplicate experiments and error bars represent the SD (, P < 0.01). Caspase, CASP. medium (data not shown). Furthermore, transfection of and prostate tumors. As shown in Fig. 3B, steady-state levels synthetic DNA into untreated (control) or "primed" benign of the AIM2 mRNA were measurably higher in BPH tissues prostate hyperplasia BPH-1 cells also activated the AIM2 as compared with the normal prostate. However, levels of inflammasome as indicated by a decrease in cellular levels of AIM2 mRNA were significantly lower in the prostate the activated caspase-1 (p20) and corresponding increases in adenocarcinoma samples (Gleason scores 3–5 þ 3–5 ¼ cellular levels of IL-18 (Fig. 2B). Together, these observa- 6–9/10; Fig. 3C). tions revealed that the cytosolic DNA can activate the AIM2 The above observations that levels of AIM2 mRNA were inflammasome in human normal PrECs and in an immor- lower in prostate tumors prompted us to investigate the talized BPH cell line. levels of AIM2 mRNA and protein in prostate epithelial cell lines (RWPE-1 and RWPE-2) that differ with respect to AIM2 expression in BPH and prostate cancer their ability to form tumors in nude mice: the RWPE-1 cell Senescent cells, which secrete proinflammatory cytokines line is an immortalized cell line, which does not grow in soft (23), accumulate in aging-associated BPH (16, 42). More- agar and does not form tumors when injected in the nude over, defects in IFN signaling that result in the loss of the mice, whereas RWPE-2 cell line is transformed (with acti- expression of the IFN-inducible proteins have been associ- vated Ki-Ras oncogene) and forms tumors in nude mice ated with the development of human prostate cancer (43). As shown in Fig. 4A, treatment of RWPE-1 and (17, 21). Given that the expression of the AIM2 protein RWPE-2 cells with either IFN-a or IFN-g activated an increases in senescent human fibroblasts (20), we compared IFN signaling (as detected by increases in levels of STAT1 the expression levels of AIM2 and other inflammasome transcription factor). Levels of STAT1 protein were higher in proteins between young proliferating PrECs (passage 3) and cells that were treated with IFN-g than IFN-a (compare lane senescent PrECs (passage 8; ref. 21). As shown in Fig. 3A, 3 with 2 or lane 6 with 5). Notably, treatment of RWPE-1 levels of the AIM2 protein and other inflammasome proteins cells with IFN-g, but not IFN-a, measurably increased levels such as ASC, pro-caspase-1 (p45), caspase-1 (p20), and pro- of AIM2 protein (compare lane 3 with either lane 2 or 1). In IL-1b (p31) were appreciably higher in the senescent PrECs contrast, treatment of RWPE-2 cells with IFN-a or IFN-g than the young proliferating cells. These observations did not appreciably increase levels of AIM2 protein. Accord- prompted us to investigate the potential role of AIM2 ingly, steady-state levels of AIM2 mRNA were detectable in protein in human prostatic diseases such as BPH and RWPE-1, but not in the RWPE-2, cells (Fig. 4B). Further- prostate cancer. For this purpose, we compared steady-state more, treatment of RWPE-1 cells with IFN-g increased the levels of the AIM2 mRNA between normal prostate, BPH, levels of the AIM2 mRNA approximately 6-fold, whereas the

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AIM2 protein (Fig. 5), to investigate the role of AIM2 protein in prostate cancer cells, we first investigated how treatment of prostate cancer cells with IFN-g increases levels of AIM2 protein and its subcellular localization. For this purpose, we chose PC-3 cancer cell line because we had used this cell line to investigate the localization of the IFI16 protein (a member of the p200 protein family; ref. 21). As shown in Fig. 6A, treatment of PC-3 cells with IFN-g for the indicated time increased the levels of STAT1, an IFN- inducible transcription factor (13). In addition, the treatment also increased an activating phosphorylation of STAT1 on Tyr-701 (PY-STAT1), indicating activation of the IFN signaling in PC-3 cells. Interestingly, the activation of an IFN signaling in PC-3 cells was accompanied by increases in AIM2 protein levels. Furthermore, the activity of the AIM2- luc-reporter plasmid (in which the transcription of the reporter gene was driven by the 50-regulatory region of the AIM2 gene) was induced approximately 3-fold upon treatment of PC-3 cells with IFN-g (Fig. 6B). Together, these observations indicated that treatment of PC-3 prostate cancer cells with IFN-g activated an IFN signaling in cells, Figure 4. AIM2 expression decreases in human prostatic epithelial cell line which increased the steady-state levels of the AIM2 protein RWPE-1 upon transformation with activated Ki-Ras oncogene. A, through transcriptional activation of the AIM2 gene. subconfluent cultures of RWPE-1 and RWPE-2 cells were either left In transfected cells that overexpress the human AIM2 without any treatment (control) or treated with IFN-a (1,000 m/mL) or IFN-g (10 ng/mL) for 18 hours. Total cell lysates from control and IFN-treated protein, the protein is primarily detected in the cytoplasm cells were analyzed by immunoblotting for the levels of the indicated proteins. B, subconfluent cultures of RWPE-1 and RWPE-2 cells were either left without any treatment (control) or treated with IFN-g for 18 hours. Total RNA isolated from control and IFN-treated cells were analyzed by quantitative RT-PCR. The ratio of the test gene to actin mRNA was calculated in units (one unit being the ratio of the AIM2 mRNA to actin mRNA). The relative steady-state levels of AIM2 mRNA in RWPE-1 cells are indicated as 1.

treatment of RWPE-2 cells only moderately increased the mRNA levels (Fig. 4B). We also compared basal and the IFN-induced levels of AIM2 mRNA and protein in human prostate cancer cell lines (DU-145, PC-3, and LNCaP) and a BPH cell line (BPH-1). The basal levels of the AIM2 mRNA and protein were very low in DU-145, PC-3, and LNCaP human prostate cancer cell lines (Fig. 5A–D). However, treatment of DU-145 and PC-3, but not LNCaP, cells with IFN-a or IFN-g differentially increased levels of the AIM2 mRNA and protein (Fig. 5A–D). Of note, the basal levels of the AIM2 protein were measurably higher in the BPH-1, a benign prostate hyperplasia cell line than normal PrECs (Fig. 5E). Together, these observations revealed that the basal levels of Figure 5. Constitutive AIM2 expression decreases in human prostate AIM2 mRNA and protein are relatively lower in prostate cancer cell lines. A, subconﬂuent cultures of DU-145, PC-3, or LNCaP tumors and prostate cancer cell lines that were tested. cells were either left without any treatment (control) or treated with IFN-a However, AIM2 expression is differentially induced in or IFN-g for 18 hours. Total cell lysates from control or IFN-treated cells DU-145 and PC-3, but not LNCaP, cells upon treatment were analyzed by immunoblotting for the levels of the indicated proteins. B–D, subconﬂuent cultures of DU-145, PC-3, or LNCaP cells with IFN-g. were either left without any treatment (control) or treated with IFN-a or IFN-g for 18 hours. Total RNA isolated from control or IFN-treated IFN-g treatment of PC-3 cells induces the expression of cells were analyzed by RT-PCR for steady-state levels of AIM2 mRNA. AIM2 and the induced levels of the AIM2 are detected Total RNA from the human THP-1 cell line was used as a positive control for RT-PCR in the D (lane 4; Cont). E, total cell lysates prepared from primarily in the cytoplasm human proliferating PrECs (passage 3), BPH-1, and LNCaP cells. Cell Because IFN-g treatment of human prostate cancer cell lysates containing equal amounts of proteins were analyzed by lines (such as DU-145 and PC-3) increased the levels of the immunoblotting.

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induces the expression of AIM2 protein and the induced levels of the AIM2 protein are primarily detected in the cytoplasm of PC-3 cells.

Cytosolic DNA activates the AIM2 inflammasome in PC-3 cells In our preliminary experiments, PC-3 cells expressed detectable levels of ASC adaptor protein (which is necessary to assemble an inflammasome) and pro-caspase-1. There- fore, our observations (Figs. 5 and 6) that the expression of AIM2 protein can be induced by activation of the IFN-g signaling in PC-3 cells and AIM2 protein is primarily detected in the cytoplasmic fraction prompted us to investigate whether cytosolic DNA can activate the AIM2 inflammasome in PC-3 prostate cancer cells. As expected (34), "priming" of PC-3 cells increased levels of both AIM2 and pro-IL-1b (p31; Fig. 7). However, the "priming" decreased the levels of pro-caspase-1 and ASC moderately (compare lane 3 with 1). Furthermore, nucleofection of plasmid DNA (pCMV-GFP) into control and "primed" PC-3 cells measurably decreased the levels of cellular IL-1b (p17; compare lane 2 with 1 or lane 4 with 3). Notably, the decrease in the cellular levels of mature IL-1b (p17) was appreciably higher in "primed" cells as compared with control cells. This observation is consistent with higher steady-state levels of the AIM2 protein in "primed" cells as compared with control cells (compare lane 3 with lane 1). Although a decrease in cellular levels of the mature IL-1b (p17) is consistent with its secretion into the culture medium (44), we were unable to Figure 6. IFN-g treatment of PC-3 cells induces the expression of AIM2 and the induced levels of the AIM2 are detected primarily in the detect the secreted lower levels of IL-1b (p17) in the culture cytoplasm. A, subconfluent cultures of PC-3 cells were either left medium by immunoblotting (data not shown). Together, untreated (lane 1) or treated with IFN-g for the indicated times (lanes 2–4). these observations revealed that nucleofected DNA can Total cell lysates containing equal amounts of proteins were analyzed by immunoblotting using antibodies specific to the indicated proteins. B, PC-3 cells were transfected with the AIM2-luc reporter plasmid (1.8 mg) along with pRL-TK (0.2 mg) plasmid using FuGene 6 transfection agent. After 24 hours of transfections, cells were either left untreated or treated with IFN-g for 14 hours. Thirty-six to forty hours of transfections, the firefly and Renilla luciferase activities as were determined as described in Materials and Methods. Normalized relative firefly luciferase activity is shown. C, subconfluent cultures of PC-3 cells were either left without any treatment or treated with IFN-g for 18 hours. Control and IFN-treated cells were fractionated into cytoplasmic (C) and nuclear (N) fractions. Fractions containing equal amounts of proteins were analyzed by immunoblotting using antibodies specific to the indicated proteins. Detection of IkBa protein primarily in the cytoplasm, whereas histone H3 in the nuclear fraction served as the quality control for the cell fractionations.

(35–37). Given that the IFI16 protein, which can bind to the AIM2 protein (44), is detected both in the cytoplasm and nucleus (45), we investigated the subcellular localization of the endogenous AIM2 protein in IFN-g-treated PC-3 cells. As shown in Fig. 6C, the induced levels of the Figure 7. Cytosolic DNA in PC-3 cells activates the AIM2 inflammasome. AIM2 protein were primarily detected in the cytoplasm, Subconfluent cultures of PC-3 cells were either left untreated or cells whereas the IFI16 protein was detected both in the were "primed" by treating them with IFN-g and TNF-a. Control and cytoplasm and nucleus (albeit more in the cytoplasm). "primed" cells were nucleofected without DNA (lanes 1 and 3) or with 2 mg However, basal and IFN-g–induced AIM2 protein was of plasmid DNA (pCMV-GFP) provided with the Amaxa Nucleofection Kit. detected in the cytoplasmic as well as in the nuclear Twenty-four hours after nucleofections, cells were lysed and cell lysates containing equal amounts of proteins were analyzed by immunoblotting fraction of BPH-1 cells (data not shown). Together, these using antibodies specific to the indicated proteins (C indicates cell- observations revealed that IFN-g treatment of PC-3 cells associated levels of IL-1b). Caspase, CASP.

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Ponomareva et al.

Figure 8. Proposed model for the role of the AIM2 protein in prostatic inﬂammation and the development of prostatic diseases.

activate the AIM2 inflammasome in control as well as the cancer cells. Accordingly, overexpression of the AIM2 "primed" PC-3 cells. In addition, our observations indicated protein in certain human cancer cell lines (including the that PC-3 cancer cells retain the ability to assemble the breast and colon cancer cell lines) suppressed cell prolif- cytosolic DNA-responsive AIM2 inflammasome. eration (49–51). Consistent with the above observations, we found that basal levels of the AIM2 mRNA were significantlylowerinprostatetumorsascomparedwith Discussion normal prostate (Fig. 3). Similarly, the basal levels of We show that the cytosolic DNA, a "danger signal", can AIM2 mRNA and protein were either very low or unde- activate the AIM2 inflammasome in human normal PrECs tectable in certain prostate cancer cell lines that were tested and PC-3 prostate cancer cells. These observations are con- (Figs. 4 and 5). Further work is in progress to investigate sistent with the idea that activation of the AIM2 inflamma- the potential role of AIM2 protein in the regulation of some in human prostate by certain pathogens or self-derived prostate epithelial cell growth. DNA contributes to the prostatic inflammation (Fig. 8). In AIM2 gene is constitutively expressed in the spleen, small addition, our observations revealed that steady-state levels of intestine, and peripheral leukocytes (49). Furthermore, AIM2 protein were appreciably higher in senescent PrECs IFN-g treatment of human HL-60 cell line (52) or IFN- (Fig. 3) and that AIM2 mRNA levels were higher in BPH b treatment of human THP-1 monocytic cell line (44) tissues as compared with normal prostate tissues. Further- increased steady-state levels of the AIM2 mRNA. Consistent more, our observations indicated that the steady-state levels of with these observations, our observations indicated that the the AIM2 mRNA were significantly lower in prostate tumors AIM2 expression is induced by IFN-a or IFN-g treatment of (as compared with normal prostate) and certain prostate human normal PrECs (Fig. 1) and certain prostate cancer cancer cell lines that were tested. Together, these observations cell lines that were tested (Figs. 4 and 5). Furthermore, the are consistent with the idea that accumulation of senescent IFN-g treatment of PC-3 cells, which activated the IFN- PrECs in BPH contributes to an increased levels of the AIM2 activatable transcription factor STAT1 (Fig. 6), also inc- protein in BPH, whereas reduced levels of the AIM2 protein reased the levels of the AIM2 protein and stimulated the in PrECs are associated with the development of prostate activity of the AIM2-luc reporter. However, it remains cancer. unclear how activation of IFN-g signaling in PrECs and AIM2 gene contains a microsatellite instability site that PC-3 cells increases the steady-state levels of the AIM2 results in inactivation of the gene in approximately 47% of mRNA and protein. Given that treatment of prostate cancer colorectal tumors with high microsatellite instability (46). cell lines such as PC-3 with either IFN-a or IFN-g increased In addition, mutations in the AIM2 gene have also been steady-state levels of AIM2 mRNA (Fig. 5C) and only IFN-g associated with the development of gastric and endome- treatment of cells increased the AIM2 protein levels (Fig. trial cancers (47). Moreover, the AIM2 gene is frequently 5A), suggest that additional mechanisms involving a post- silenced by DNA methylation in human cancers (46, 48). transcriptional mechanisms may contribute to the IFN- These observations suggest that the loss of AIM2 protein induced levels of the AIM2 protein in PC-3 prostate cancer function in cancer cells may provide a growth advantage to cells (and possibly other cancer cells). Further work is in

1200 Mol Cancer Res; 11(10) October 2013 Molecular Cancer Research

AIM2 Inﬂammasome in Human Prostatic Diseases

progress to investigate how IFN-a and IFN-g transcription- experimental BPH (56). Therefore, our observations that ally activate the expression of the AIM2 gene in PrECs. steady-state levels of AIM2 mRNA were relatively higher in Caspase-1 activation is required for human prostate cancer BPH than normal prostate are consistent with the idea that cells to undergo apoptosis in response to TGF-b treatment increased levels of the AIM2 protein in senescent PrECs (53). Notably, the majority of primary prostate cancer contribute to senescence-associated secretory phenotype that specimens do not express detectable levels of caspase-1 is associated with the development of BPH. (54). Moreover, certain prostate cancer cell lines express Inflammasomes contribute to tissue homeostasis, inflam- reduced levels of caspase-1 (54). Accordingly, we found that mation, and immune responses (both innate and adaptive; levels of AIM2 mRNA were significantly lower in prostate ref. 29). Therefore, it is likely that inflammasomes also tumors as compared with normal prostate (Fig. 3) and influence the formation, progression, and therapeutic certain prostate cancer cell lines did not express detectable responses of the human prostate cancer. Consequently, our basal levels of the AIM2 protein (Figs 4 and 5). Together, observations (Fig. 8) that the presence of cytosolic DNA these observations suggest that the pattern of caspase-1 and (derived from pathogens, necrotic cells, or senescent cells- AIM2 expression in prostate tumors may have prognostic derived "exosomes") in normal PrECs and PC-3 prostate significance in the disease progression. cancer cell line can activate the AIM2 inflammasome will Methylation-mediated silencing of the gene encoding for serve as a basis to identify the molecular mechanisms through the ASC, an adaptor protein that is required to assemble which certain prostatic infections and/or sterile inflamma- certain inflammasome (including the AIM2 inflammasome; tion contribute to the development of human prostatic refs. 35–37), has been associated with the initial develop- diseases. ment of the human prostate cancers (55). Furthermore, a complete or partial methylation of the ASC gene has been Disclosure of Potential Conflicts of Interest noted in certain human prostate cancer cell lines (including No potential conflicts of interest were disclosed. LNCaP, DU145, PC-3, MDAPCa2b, and LAPC4). Nota- bly, the PC-3 cells were reported to express detectable levels Authors' Contributions Conception and design: X. Duan, D. Choubey of ASC mRNA (55). Consistent with these observations, we Development of methodology: L. Ponomareva, H. Liu, H. Shen, R. Panchanathan were able to detect the expression of ASC protein in PC-3 Acquisition of data (provided animals, acquired and managed patients, provided cells (Fig. 7) and treatment of cells with 5-aza-2-deoxyciti- facilities, etc.): L. Ponomareva, H. Liu, X. Duan, E. Dickerson, R. Panchanathan Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- dine, an inhibitor of methylation, increased the protein levels tational analysis): L. Ponomareva, R. Panchanathan, D. Choubey further (data not shown). Moreover, nucleofection of plas- Writing, review, and/or revision of the manuscript: L. Ponomareva, H. Shen, D. mid DNA into "primed" PC-3 cells activated the AIM2 Choubey Administrative, technical, or material support (i.e., reporting or organizing data, inflammasome (Fig. 7). Therefore, these observations indi- constructing databases): X. Duan, E. Dickerson, H. Shen cated that PC-3 cells can assemble the AIM2 inflammasome Study supervision: D. Choubey upon sensing the cytosolic DNA. We have reported previously that increased levels of AIM2 Grant Support fi This research was supported by the Merit Award from the Department of Veteran protein in senescent human broblasts are associated with an Affairs, Medical Research and Development Service (to D. Choubey). increased production of IL-1b and an inflammatory phe- The costs of publication of this article were defrayed in part by the payment of page notype (20). Notably, the accumulation of senescent pros- charges. This article must therefore be hereby marked advertisement in accordance with tate epithelial cells in elderly patients plays an important role 18 U.S.C. Section 1734 solely to indicate this fact. in the development of BPH (16, 42) and increased expres- Received March 25, 2013; revised June 17, 2013; accepted July 9, 2013; sion levels of IL-1b are associated with the development of published OnlineFirst July 17, 2013.

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1202 Mol Cancer Res; 11(10) October 2013 Molecular Cancer Research

AIM2, an IFN-Inducible Cytosolic DNA Sensor, in the Development of Benign Prostate Hyperplasia and Prostate Cancer

Larissa Ponomareva, Hongzhu Liu, Xin Duan, et al.

Mol Cancer Res 2013;11:1193-1202. Published OnlineFirst July 17, 2013.

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