cell death and survival - molecular cancer researchlarissa ponomareva 1,2, hongzhu liu , xin duan...

Cell Death and Survival

AIM2, an IFN-Inducible Cytosolic DNA Sensor, in theDevelopment of Benign Prostate Hyperplasia andProstate Cancer

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

AbstractClose 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 molecularmechanisms that contribute to prostatic inflammation remain largely unexplored. Recent studies have indicatedthat the IFN-inducible AIM2 protein is a cytosolic DNA sensor in macrophages and keratinocytes. Upon sensingDNA, AIM2 recruits the adaptor ASC and pro-CASP1 to assemble the AIM2 inflammasome. Activation of theAIM2 inflammasome cleaves pro-interleukin (IL)-1b and pro-IL-18 and promotes the secretion of IL-1b and IL-18proinflammatory cytokines. Given that human prostatic infections are associated with chronic inflammation, thedevelopment of BPH is associated with an accumulation of senescent cells with a proinflammatory phenotype, andthe development of prostate cancer is associated with the loss of IFN signaling, the role of AIM2 in mediating theformation of prostatic diseases was investigated. It was determined that IFNs (a, b, or g) induced AIM2 expressionin human prostate epithelial cells and cytosolic DNA activated the AIM2 inflammasome. Steady-state levels ofthe AIM2 mRNA were higher in BPH than in normal prostate tissue. However, the levels of AIM2 mRNA weresignificantly lower in clinical tumor specimens. Accordingly, constitutive levels of AIM2 mRNA and proteinwere lower in a subset of prostate cancer cells as compared with BPH cells. Further, the cytosolic DNA activated theAIM2 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.MolCancer Res; 11(10); 1193–202. �2013 AACR.

IntroductionThe etiologic agents that contribute to the development of

human prostatic diseases such as benign prostate hyperplasia(BPH) and prostate cancer remain largely unknown (1–4).Studies have suggested close links among certain infectionsof the prostate, chronic inflammation, and the developmentof inflammation-associated prostatic diseases (5–10). It hasbeen proposed that certain infections of the prostate con-tribute to the development of chronic inflammation andregenerative "risk factor" lesions, referred to as proliferativeinflammatory atrophy, contribute to the development ofprostate cancer (3–5). Accordingly, studies have found anincreased relative risk of prostate cancer in men with a prior

history of certain sexually transmitted infections or prosta-titis, which are often associated with intraprostatic inflam-mation (6, 7). Furthermore, studies have indicated possiblelinks between certain prostatic infections and inflammation(6, 7, 11, 12).Prostatic infections, which can induce the production of

type-I IFN-a/b) and the expression of the IFN-induciblegenes (ISG; refs. 13, 14), reduce telomere length (15),resulting in an accumulation of senescent prostate epithelialcells (PrEC) in BPH (16). Accordingly, expression of a set ofISGs is upregulated during the onset of prostate epithelialcellular senescence (17–19). Furthermore, levels of the IFN-b increase in human diploid fibroblasts (HDF) when HDFsapproach cellular senescence (20) and the expression of IFN-inducible proteins is upregulated in senescent PrECs (21)and HDFs (22). Senescent HDFs (and other cell types) areknown to secret proinflammatory cytokines (23). Thisphenotype of senescent cells has been termed senescence-associated secretory phenotype (SASP). The SASP, whichdepends on activation of NF-kB (24), activates the immunesurveillance and exhibits protumorigenic properties (23).Furthermore, the cultures of senescent HDFs contain dead

Authors' Affiliations: 1Cincinnati VA Medical Center; and 2Department ofEnvironmental Health, University of Cincinnati, Cincinnati, Ohio

Corresponding Author: Divaker Choubey, Department of EnvironmentalHealth, University of Cincinnati, 3223 Eden Avenue, PO Box 670056,Cincinnati, OH 45267. Phone: 513-558-1014; Fax: 513-558-0925; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-13-0145

�2013 American Association for Cancer Research.

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cells, exhibit apoptosis, and the senescent HDFs release"exosomes" (microvesicles), which when fused with othercells, deliver dsDNA (a "danger signal") into the cytoplasmof neighboring cells (25, 26).A complex of cytosolic proteins, which is termed "inflam-

masome", is activated in innate immune cells (macrophagesand dendritic cells) in response to sensing "danger signals" byreceptors (27–29). These receptors include members of thenucleotide binding and oligomerization domain (NOD)-likereceptor (NLR) family proteins such as NLRP3 and certainmembers of the IFN-inducible p200-family proteins such asmurine Aim2 and human AIM2 (29–31). The NLRP3receptor can sense cytoplasmic pathogens, self-derived mole-cules, and environmental materials (29). Upon sensing thesemolecules, activated NLRP3 recruits an adaptor protein ASCand pro-caspase-1 to form the NLRP3 inflammasome. Acti-vation of an inflammasome proteolytically cleaves pro-IL-1band pro-IL-18 to the mature forms and potentiates theirsecretion from cells (27, 28). In addition, the activation ofinflammasomes in macrophages induces a rapid highlyinflammatory cell death called pyroptosis (29–31).Activation of inflammasomes has been known in myeloid

cells such as monocytes and macrophages (32). Interestingly,activation of an inflammasome has also been reported innonmyeloid cells (33, 34), which are the preferred host cellsformany intracellular pathogens. For example, an infection ofcervical epithelial cells by Chlamydia trachomatis, an intra-cellular pathogen, leads to activation of caspase-1 through theNLRP3 inflammasome (33).The human AIM2 protein from the IFN-inducible p200-

protein family senses cytoplasmic dsDNA (hereafter referredas DNA) in myeloid cells (35–37) and "primed" (treatedwith IFN-g and TNF-a) human keratinocytes (34). Uponsensing DNA, the AIM2 protein recruits an adaptor proteinASC to form the AIM2 inflammasome (31, 35–37). Theactivated AIM2 inflammasome in macrophages throughactivation of caspase-1 promotes the proteolytic cleavageand secretion of proinflammatory cytokines (IL-1b and IL-18). Given that human prostatic infections are associatedwith chronic inflammation (6, 7), the development of BPHis associated with an accumulation of senescent cells with aproinflammatory phenotype (16, 23), and the developmentof prostate cancer is associated with the loss of type I IFNsignaling (18, 21), we investigated the role of AIM2 proteinin human PrECs and prostatic diseases. Here, we report thatcytosolic DNA can activate the AIM2 inflammasome inhuman PrECs. In addition, we found that the expression ofAIM2 mRNA was higher in BPH than normal prostate,whereas levels of AIM2 mRNA were significantly lower inprostate tumors and certain cancer cell lines that were tested.Our observations show a role for the AIM2 inflammasome inthe development of human prostatic diseases.

Materials and MethodsReagentsCompletemini EDTA-free protease inhibitor cocktail was

purchased from Roche Applied Science. Universal recom-

binant IFN-a, recombinant human IFN-b, recombinanthuman IFN-g , and human TNF-a were purchased fromR&D Systems. Caspase inhibitor VI (Z-VAD-FMK) waspurchased fromEMDMillipore. Poly(dA:dT)/LyoVec (syn-thetic double-stranded DNA in complex with LyoVectransfection agent) was purchased from InvivoGen.

Cell lines, culture conditions, and treatmentsHuman normal PrECs prostate cancer cell lines were

cultured at 37�C in a humidified cell culture incubatorcontaining 5% CO2. Human normal PrECs in culture (atpassage 2) were purchased from Lonza and were maintainedin culture as suggested by the supplier. Human prostatecancer cell lines (RWPE-1, RWPE-2, DU-145, PC-3, andLNCaP) were originally purchased from the American TypeCulture Collection (ATCC) and maintained in culture assuggested by the supplier. BPH-1 cell line was generouslyprovided by Dr. Simon Hayward (Vanderbilt UniversityMedical Center, Nashville, TN; ref. 38). Cells were main-tained in Dulbecco's modified Eagle medium (high glucose)culture medium (Invitrogen Life Technologies) supplemen-ted with 10% (v/v) FBS and antibiotics (Invitrogen).When indicated, cells were treated with either IFN-a(1,000 units/ml), IFN-b (1,000 u/ml), or IFN-g (10 ng/mL) for the indicated time (hours). When indicated, PrECsand PC-3 cells in culture were "primed" by incubating withIFN-g (10 ng/mL) and TNF-a (10 ng/mL) for the indicatedtime as described previously (34).

AntibodiesRabbit polyclonal antibodies that were raised against the

keyhole limpet hemocyanin-conjugated hAIM2 C-terminalpeptide (GVHSTIKVIKAKKKT) have been described pre-viously (20). Antibodies specific to detect ASC (sc-22514)and IL-18 (sc-7954) in immunoblotting were purchasedfrom Santa Cruz Biotechnology. Antibodies for b-actin (cat#4967); IkBa (#9247); histone 3 (#9715); caspase 1(#3866); and IL-1b (#2022) were purchased from CellSignaling Technology. Horseradish peroxidase-conjugatedsecondary anti-mouse (NXA-931) and anti-rabbit (NA-934)antibodies were from GE Healthcare Biosciences.

ImmunoblottingCells from subconfluent cultures were lysed with radio-

immunoprecipitation assay (RIPA) buffer (50 mmol/L Tris-Cl (pH 7.4), 150 mmol/L NaCl, 1% Nonidet P-40, 0.5%sodium deoxycholate, 0.1% SDS). The buffer was supple-mented with complete mini EDTA-free protease inhibitorcocktail. Lysates were centrifuged at 12,000 rpm for 5minutes at 4�C. Cell lysates containing equal amounts ofprotein (�50 mg) were separated by SDS-PAGE, transferredto a polyvinylidene difluoride membrane, and immuno-blotted according to the manufacturer's guidelines.

RNA isolation and PCRTotal RNA was isolated from the cultured cells with

TRIzol reagent (Invitrogen). cDNA synthesis, reversetranscription polymerase chain reaction (RT-PCR), and

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quantitative real-time TaqMan PCR were conducted todetect the expression of the AIM2 gene as described previ-ously (20). The following primers were used for RT-PCR:human AIM2 (forward: 50-ATGTG AAGCCGTCCAGA-30; backward: 50-CATCATTTCTGA TGGCTGCA-30),human ASC (forward: 50-GCCTGCACTTTATAGAC-CAGC-30; backward: 50- GCTTCCGCATCTTGCT-TGG-30), human CASP-1 (forward: 50-TCCAATAATG-GACAAGTC AAGCC-30; backward: 50-GCTGTACCC-CAGATTTTGTAGCA-30), human IL-1b (forward: 50-CTCG CCAGTGAAATGATG GCT-30; backward: 50-GTCGGAGATTCGTAGCTGGAT-30), and actin (for-ward: 50-GCTCGTCGTCGACA ACGGCTC-30; back-ward: 50-CATGATCTGGGTCA TCTTCTC-30). The fol-lowing TaqMan gene-specific assays were purchased fromthe Applied Biosystems and used as suggested by thesupplier: AIM2 (Hs 00175457_m1) and the endogenouscontrol b-actin (assay Id# Hs99999903_ml). TissueScanCancer and Normal Tissue human prostate cDNA Arrayswere purchased from OriGene. The arrays also includedthe information (age, tumor grade, and minimum stage ofthe tumor) about the samples. The PCR plates containingcDNAs from the normal or prostate tumors (Gleasonscore ¼ 6–10) for quantitative polymerase chain reaction(qPCR) were subjected to RT-PCR as suggested by thesupplier. In brief, the quantitative RT-PCR technology(Applied Biosystems) was used to compare the expressionof the AIM2 gene. The PCR cycling program consisted ofdenaturing at 95�C for 10minutes and 40 cycles at 95�C for15 seconds, and annealing and elongation at 60�C for 1minute. The signal in samples was normalized using thehousekeeping genes as suggested by the supplier.

TransfectionsSubconfluent cultures ofPrECs in60mmplateswere either

left untreated (control) or were "primed" as described previ-ously. Control or "primed" cultures of cells were either incu-bated with LyoVec (control) or poly(dA:dT)/LyoVec (5 mg/mL) for the indicated time. At the end of incubations, cellswere harvested to isolate RNA or to prepare total cell lysates.

NucleofectionsPC-3 cells (�2 � 106 cells/nucleofection) were nucleo-

fected as described previously (39). In brief, cells werenucleofected with 2 mg of plasmidDNA (pCMV-GFP) usingthe Nucleofector-II device (Amaxa Biosystems, Nattherman-nalle 1) and the nucleofection kit-V (programU-001). Thesenucleofection conditions resulted in approximately 60% cellsurvival after 24 hours of incubations of cells. After nucleofec-tions, cells were harvested at the indicated times.

Reporter assaysWe have cloned the 50-regulatory region (�1.0 Kb) of the

human AIM2 gene into a basic pGL3 reporter plasmid(plasmid designated as AIM2-luc), which does not containany enhancer and promoter sequences. Therefore, the tran-scription of the reporter gene in the AIM2-luc-reporter isdriven by the AIM2 promoter sequence. Subconfluent

cultures of PC-3 cells in a 6-well plate were transfected withthe AIM2-luc reporter plasmid (1.8 mg) along with the pRL-TK reporter plasmid (0.2 mg) as an internal control, usingFuGene6 Transfection Reagent (Roche). Thirty-six to fortyhours after transfections, the firefly luciferase and Renillaluciferase activities were assayed using dual-luciferase report-er assay kit (Promega). The relative firefly luciferase activity isexpressed as the ratio of the firefly luciferase and Renillaluciferase.

Cell fractionationsCytosolic and nuclear fractions from cultured cells were

prepared as described previously (20). Briefly, cell pelletswere incubated on ice for 10 minutes in cell fractionationbuffer (20 mmol/L Tris-HCl, pH 7.5; 0.65% NP-40, 150mmol/L NaCl) that was supplemented with protease andphosphatase inhibitors. The cytosolic fractions were recov-ered by centrifuging cell lysates at 2,000 rpm for 3minutes ina refrigerated microcentrifuge. The nuclear pellet waswashed with the cell fractionation buffer and the pellet wassuspended into the RIPA buffer followed by brief sonication.The lysates were centrifuged at 12,000 rpm for 10minutes at4�C and supernatants were collected.

Statistical analysesThe measurement values are presented as means� SEM.

The statistical significance of differences in the measuredmean frequencies between the two groups was calculatedusing the Student two-tailed t test.

ResultsExpression of AIM2 inflammasome proteins is detectablein human PrECs and the IFN treatment of cells increasesthe expression levelsExpression of AIM2 protein is induced by treatment of

cells with IFNs in a variety of cell types (31, 40). Therefore,

Figure 1. Expression of AIM2 inflammasome proteins is detectable inhuman normal PrECs and the IFN treatment of cells increases theexpression. A, human normal PrECs at an early passage (passage 3)wereeither left untreated (lane 1) or treated with IFN-a (lane 2), b (lane 3), or g(lane 4) for 18 hours. After the treatment, total cell lysates containingequal amounts of proteins were analyzed by immunoblotting usingantibodies specific to the indicated proteins. B, human normal PrECswere treated with the indicated IFN as described in A. After the treatment,total RNA was isolated from cells and reverse transcribed. Steady-statelevels of the mRNAs were analyzed for the indicated genes by RT-PCRusing a pair of the gene-specific primers. Caspase, CASP.

AIM2 Inflammasome in Human Prostatic Diseases

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to investigate the role of the AIM2 inflammasome in humanPrECs, we examined basal and the IFN-induced levels ofAIM2 inflammasome proteins in young PrECs. As shownin Fig. 1A, the basal levels of AIM2protein,which is detectedas two closely migrating protein bands (20), adaptor proteinASC, pro-caspase-1 (p45), and pro-IL-1b (p31) were detect-able in PrECs. Interestingly, the IFN (a, b, or g) treatmentof cells for 24 hours differentially increased the levels of theAIM2, pro-caspase-1, and pro-IL-1b proteins. Notably, thetreatment also increased the levels of the activated caspase-1(p20) to different extents. Consistent with the above obser-vations, treatment of PrECs with either IFN-a, b, or g alsoincreased the steady-state levels of AIM2mRNA to differentextents (Fig. 1B). The maximum increase was noted afterIFN-g treatment. Notably, the treatment of PrECs withIFN-a, b, or g also increased the steady-state levels ofcaspase-1 mRNA. In contrast, treatment of cells withIFN-a or b, but not IFN-g , decreased steady-state levelsof IL-1bmRNA. Together, these observations revealed thathuman normal PrECs in culture express low, but detectable,basal levels of AIM2 inflammasome proteins and the acti-vation of IFN signaling in cells by type I (IFN-a or b) or typeII IFN (IFN-g) differentially regulates the steady-state levelsof the AIM2 and other inflammasome proteins.

Cytosolic DNA activates the AIM2 inflammasome inPrECsCytosolic DNA activates the AIM2 inflammasome in

"primed" (primed with IFN-g and TNF-a treatment)human keratinocytes (34). The "priming" of keratinocytes

increases basal levels of both AIM2 and pro-IL-1b (34).Therefore, to investigate whether AIM2 inflammasome canbe activated by cytosolic DNA in PrECs, we transfectedsynthetic DNA [poly(dA:dT)] into "primed" human PrECs.Surprisingly, transfection of cells with the synthetic DNAdecreased cellular levels of AIM2 protein (Fig. 2A, comparelanes 2 with lane 1). However, pretreatment of cells with z-VAD (a broad-spectrum inhibitor of caspase; 41) decreasedthe extent of decrease in AIM2 protein levels after transfec-tion of synthetic DNA (compare lane 3 with lane 2).Notably, transfection of the synthetic DNA into PrECsdecreased the levels of pro-caspase-1 (p45; ref. Fig. 2A), thus,indicating a proteolytic cleavage of the pro-caspase-1 andactivation of the AIM2 inflammasome (34). Correspond-ingly, treatment of cells with z-VAD increased cellular levelsof procaspase-1 (p45) and cleaved caspase-1 (p20 and p10;compare lane 3 with 2), indicating an inhibition of proteo-lytic cleavage of pro-caspase-1 (p45) and the secretion ofactivated caspase-1 (p20 and p10) into the culture medium.Accordingly, the introduction of synthetic DNA into cellsreduced the cellular levels of IL-18, whereas the secretedlevels of IL-18 increased in themedium (compare lane 2with1). Again, the treatment of cells with z-VAD inhibited adecrease in the cellular levels of IL-18 and an increase inlevels of IL-18 in themedium.Notably, cellular levels of pro-IL-1b did not change after transfection of synthetic DNAinto PrECs. Similarly, transfection of increasing amounts ofplasmid DNA into PrECs increased the cleavage of pro-caspase-1 (indicated by an increase in the levels of the p20form) 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 withIFN-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 withpoly(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,cellswere lysedandcell lysates containing equal amounts of proteinswere analyzedby immunoblotting. B, cells of humanbenignprostate hyperplasia cell lineBPH-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" cellswere 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 andlysates containing equal amounts of proteins were analyzed for the indicated proteins by immunoblotting. Caspase, CASP.

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medium (data not shown). Furthermore, transfection ofsynthetic DNA into untreated (control) or "primed" benignprostate hyperplasia BPH-1 cells also activated the AIM2inflammasome as indicated by a decrease in cellular levels ofthe activated caspase-1 (p20) and corresponding increases incellular levels of IL-18 (Fig. 2B). Together, these observa-tions revealed that the cytosolic DNA can activate the AIM2inflammasome in human normal PrECs and in an immor-talized BPH cell line.

AIM2 expression in BPH and prostate cancerSenescent cells, which secrete proinflammatory cytokines

(23), accumulate in aging-associated BPH (16, 42). More-over, defects in IFN signaling that result in the loss of theexpression of the IFN-inducible proteins have been associ-ated with the development of human prostate cancer(17, 21). Given that the expression of the AIM2 proteinincreases in senescent human fibroblasts (20), we comparedthe expression levels of AIM2 and other inflammasomeproteins between young proliferating PrECs (passage 3) andsenescent PrECs (passage 8; ref. 21). As shown in Fig. 3A,levels of the AIM2 protein and other inflammasome proteinssuch as ASC, pro-caspase-1 (p45), caspase-1 (p20), and pro-IL-1b (p31) were appreciably higher in the senescent PrECsthan the young proliferating cells. These observationsprompted us to investigate the potential role of AIM2protein in human prostatic diseases such as BPH andprostate cancer. For this purpose, we compared steady-statelevels of the AIM2 mRNA between normal prostate, BPH,

and prostate tumors. As shown in Fig. 3B, steady-state levelsof the AIM2mRNA were measurably higher in BPH tissuesas compared with the normal prostate. However, levels ofAIM2 mRNA were significantly lower in the prostateadenocarcinoma samples (Gleason scores 3–5 þ 3–5 ¼6–9/10; Fig. 3C).The above observations that levels of AIM2 mRNA were

lower in prostate tumors prompted us to investigate thelevels of AIM2 mRNA and protein in prostate epithelial celllines (RWPE-1 and RWPE-2) that differ with respect totheir ability to form tumors in nude mice: the RWPE-1 cellline is an immortalized cell line, which does not grow in softagar and does not form tumors when injected in the nudemice, whereas RWPE-2 cell line is transformed (with acti-vated Ki-Ras oncogene) and forms tumors in nude mice(43). As shown in Fig. 4A, treatment of RWPE-1 andRWPE-2 cells with either IFN-a or IFN-g activated anIFN signaling (as detected by increases in levels of STAT1transcription factor). Levels of STAT1proteinwere higher incells that were treated with IFN-g than IFN-a (compare lane3 with 2 or lane 6 with 5). Notably, treatment of RWPE-1cells with IFN-g , but not IFN-a, measurably increased levelsof AIM2 protein (compare lane 3 with either lane 2 or 1). Incontrast, treatment of RWPE-2 cells with IFN-a or IFN-gdid not appreciably increase levels of AIM2 protein. Accord-ingly, steady-state levels of AIM2 mRNA were detectable inRWPE-1, but not in the RWPE-2, cells (Fig. 4B). Further-more, treatment of RWPE-1 cells with IFN-g increased thelevels of the AIM2mRNA approximately 6-fold, whereas the

Figure 3. AIM2 expression levels in human prostatic diseases. A, total cell lysateswere 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 (fromOrigene) containing normal samples (7 samples) and BPH samples (11 samples) were analyzed by quantitative RT-PCR for the AIM2 mRNA levels assuggested by the supplier. The normalized relative values are indicated. Results are mean values of triplicate experiments and the error bars representthe SD (�,P < 0.05). C, TissueScan Prostate Cancer Tissue qPCRArray II (fromOrigene) containing 48 samples (normal samples: 8; adenocarcinoma samples40; 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 valuesof triplicate experiments and error bars represent the SD (�, P < 0.01). Caspase, CASP.






treatment of RWPE-2 cells only moderately increased themRNA levels (Fig. 4B).We also compared basal and the IFN-induced levels of

AIM2 mRNA and protein in human prostate cancer celllines (DU-145, PC-3, and LNCaP) and a BPH cell line(BPH-1). The basal levels of the AIM2 mRNA and proteinwere very low in DU-145, PC-3, and LNCaP humanprostate cancer cell lines (Fig. 5A–D). However, treatmentof DU-145 and PC-3, but not LNCaP, cells with IFN-a orIFN-g differentially increased levels of the AIM2mRNA andprotein (Fig. 5A–D). Of note, the basal levels of the AIM2protein were measurably higher in the BPH-1, a benignprostate hyperplasia cell line than normal PrECs (Fig. 5E).Together, these observations revealed that the basal levels ofAIM2 mRNA and protein are relatively lower in prostatetumors and prostate cancer cell lines that were tested.However, AIM2 expression is differentially induced inDU-145 and PC-3, but not LNCaP, cells upon treatmentwith IFN-g .

IFN-g treatment of PC-3 cells induces the expression ofAIM2 and the induced levels of the AIM2 are detectedprimarily in the cytoplasmBecause IFN-g treatment of human prostate cancer cell

lines (such as DU-145 and PC-3) increased the levels of the

AIM2 protein (Fig. 5), to investigate the role of AIM2protein in prostate cancer cells, we first investigated howtreatment of prostate cancer cells with IFN-g increases levelsof AIM2 protein and its subcellular localization. For thispurpose, we chose PC-3 cancer cell line because we had usedthis cell line to investigate the localization of the IFI16protein (a member of the p200 protein family; ref. 21). Asshown in Fig. 6A, treatment of PC-3 cells with IFN-g for theindicated time increased the levels of STAT1, an IFN-inducible transcription factor (13). In addition, the treat-ment also increased an activating phosphorylation of STAT1on Tyr-701 (PY-STAT1), indicating activation of the IFNsignaling in PC-3 cells. Interestingly, the activation of anIFN signaling in PC-3 cells was accompanied by increases inAIM2 protein levels. Furthermore, the activity of the AIM2-luc-reporter plasmid (in which the transcription of thereporter gene was driven by the 50-regulatory region of theAIM2 gene) was induced approximately 3-fold upon treat-ment of PC-3 cells with IFN-g (Fig. 6B). Together, theseobservations indicated that treatment of PC-3 prostatecancer cells with IFN-g activated an IFN signaling in cells,which increased the steady-state levels of the AIM2 proteinthrough transcriptional activation of the AIM2 gene.In transfected cells that overexpress the human AIM2

protein, the protein is primarily detected in the cytoplasm

Figure 4. AIM2 expression decreases in humanprostatic epithelial cell lineRWPE-1 upon transformation with activated Ki-Ras oncogene. A,subconfluent cultures of RWPE-1 and RWPE-2 cells were either leftwithout any treatment (control) or treatedwith IFN-a (1,000m/mL) or IFN-g(10 ng/mL) for 18 hours. Total cell lysates from control and IFN-treatedcells were analyzed by immunoblotting for the levels of the indicatedproteins. B, subconfluent cultures of RWPE-1 and RWPE-2 cells wereeither left without any treatment (control) or treated with IFN-g for 18hours. Total RNA isolated from control and IFN-treated cells wereanalyzed by quantitative RT-PCR. The ratio of the test gene to actinmRNA was calculated in units (one unit being the ratio of the AIM2mRNA to actinmRNA). The relative steady-state levels of AIM2mRNA inRWPE-1 cells are indicated as 1.

Figure 5. Constitutive AIM2 expression decreases in human prostatecancer cell lines. A, subconfluent cultures of DU-145, PC-3, or LNCaPcells were either left without any treatment (control) or treated with IFN-aor IFN-g for 18 hours. Total cell lysates from control or IFN-treated cellswere analyzed by immunoblotting for the levels of the indicatedproteins. B–D, subconfluent cultures of DU-145, PC-3, or LNCaP cellswere either left without any treatment (control) or treated with IFN-a orIFN-g for 18 hours. Total RNA isolated from control or IFN-treatedcells were analyzed by RT-PCR for steady-state levels of AIM2 mRNA.Total RNA from the human THP-1 cell line was used as a positive controlfor RT-PCR in the D (lane 4; Cont). E, total cell lysates prepared fromhuman proliferating PrECs (passage 3), BPH-1, and LNCaP cells. Celllysates containing equal amounts of proteins were analyzed byimmunoblotting.

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(35–37). Given that the IFI16 protein, which can bind tothe AIM2 protein (44), is detected both in the cytoplasmand nucleus (45), we investigated the subcellular locali-zation of the endogenous AIM2 protein in IFN-g-treatedPC-3 cells. As shown in Fig. 6C, the induced levels of theAIM2 protein were primarily detected in the cytoplasm,whereas the IFI16 protein was detected both in thecytoplasm and nucleus (albeit more in the cytoplasm).However, basal and IFN-g–induced AIM2 protein wasdetected in the cytoplasmic as well as in the nuclearfraction of BPH-1 cells (data not shown). Together, theseobservations revealed that IFN-g treatment of PC-3 cells

induces the expression of AIM2 protein and the inducedlevels of the AIM2 protein are primarily detected in thecytoplasm of PC-3 cells.

Cytosolic DNA activates the AIM2 inflammasome inPC-3 cellsIn our preliminary experiments, PC-3 cells expressed

detectable levels of ASC adaptor protein (which is necessaryto assemble an inflammasome) and pro-caspase-1. There-fore, our observations (Figs. 5 and 6) that the expression ofAIM2 protein can be induced by activation of the IFN-gsignaling in PC-3 cells and AIM2 protein is primarilydetected in the cytoplasmic fraction prompted us to inves-tigate whether cytosolic DNA can activate the AIM2 inflam-masome in PC-3 prostate cancer cells. As expected (34),"priming" of PC-3 cells increased levels of both AIM2 andpro-IL-1b (p31; Fig. 7). However, the "priming" decreasedthe levels of pro-caspase-1 and ASC moderately (comparelane 3 with 1). Furthermore, nucleofection of plasmid DNA(pCMV-GFP) into control and "primed" PC-3 cells measur-ably decreased the levels of cellular IL-1b (p17; compare lane2 with 1 or lane 4 with 3). Notably, the decrease in thecellular levels of mature IL-1b (p17) was appreciably higherin "primed" cells as compared with control cells. Thisobservation is consistent with higher steady-state levels oftheAIM2protein in "primed" cells as comparedwith controlcells (compare lane 3 with lane 1). Although a decrease incellular levels of the mature IL-1b (p17) is consistent with itssecretion into the culture medium (44), we were unable todetect the secreted lower levels of IL-1b (p17) in the culturemedium by immunoblotting (data not shown). Together,these observations revealed that nucleofected DNA can

Figure 6. IFN-g treatment of PC-3 cells induces the expression of AIM2and the induced levels of the AIM2 are detected primarily in thecytoplasm. A, subconfluent cultures of PC-3 cells were either leftuntreated (lane 1) or treated with IFN-g for the indicated times (lanes 2–4).Total cell lysates containing equal amounts of proteins were analyzed byimmunoblotting 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 treatedwith IFN-g for 14 hours. Thirty-six to forty hours of transfections, the fireflyand Renilla luciferase activities as were determined as described inMaterials and Methods. Normalized relative firefly luciferase activity isshown. C, subconfluent cultures of PC-3 cells were either left without anytreatment or treated with IFN-g for 18 hours. Control and IFN-treatedcells were fractionated into cytoplasmic (C) and nuclear (N) fractions.Fractions containing equal amounts of proteins were analyzed byimmunoblotting using antibodies specific to the indicated proteins.Detection of IkBa protein primarily in the cytoplasm, whereas histone H3in the nuclear fraction served as the quality control for the cellfractionations.

Figure 7. Cytosolic DNA in PC-3 cells activates the AIM2 inflammasome.Subconfluent cultures of PC-3 cells were either left untreated or cellswere "primed" by treating them with IFN-g and TNF-a. Control and"primed" cells were nucleofectedwithout DNA (lanes 1 and 3) or with 2mgof plasmid DNA (pCMV-GFP) providedwith the AmaxaNucleofection Kit.Twenty-four hours after nucleofections, cells were lysed and cell lysatescontaining equal amounts of proteins were analyzed by immunoblottingusing antibodies specific to the indicated proteins (C indicates cell-associated levels of IL-1b). Caspase, CASP.






activate the AIM2 inflammasome in control as well as"primed" PC-3 cells. In addition, our observations indicatedthat PC-3 cancer cells retain the ability to assemble thecytosolic DNA-responsive AIM2 inflammasome.

DiscussionWe show that the cytosolic DNA, a "danger signal", can

activate the AIM2 inflammasome in human normal PrECsand PC-3 prostate cancer cells. These observations are con-sistent with the idea that activation of the AIM2 inflamma-some in human prostate by certain pathogens or self-derivedDNA contributes to the prostatic inflammation (Fig. 8). Inaddition, our observations revealed that steady-state levels ofAIM2 protein were appreciably higher in senescent PrECs(Fig. 3) and that AIM2 mRNA levels were higher in BPHtissues as compared with normal prostate tissues. Further-more, our observations indicated that the steady-state levels ofthe AIM2mRNAwere significantly lower in prostate tumors(as compared with normal prostate) and certain prostatecancer cell lines that were tested. Together, these observationsare consistent with the idea that accumulation of senescentPrECs in BPH contributes to an increased levels of the AIM2protein in BPH, whereas reduced levels of the AIM2 proteinin PrECs are associated with the development of prostatecancer.AIM2 gene contains a microsatellite instability site that

results in inactivation of the gene in approximately 47% ofcolorectal tumors with high microsatellite instability (46).In addition, mutations in the AIM2 gene have also beenassociated with the development of gastric and endome-trial cancers (47). Moreover, the AIM2 gene is frequentlysilenced by DNA methylation in human cancers (46, 48).These observations suggest that the loss of AIM2 proteinfunction in cancer cells may provide a growth advantage to

the cancer cells. Accordingly, overexpression of the AIM2protein in certain human cancer cell lines (including thebreast and colon cancer cell lines) suppressed cell prolif-eration (49–51). Consistent with the above observations,we found that basal levels of the AIM2 mRNA weresignificantly lower in prostate tumors as compared withnormal prostate (Fig. 3). Similarly, the basal levels ofAIM2 mRNA and protein were either very low or unde-tectable in certain prostate cancer cell lines that were tested(Figs. 4 and 5). Further work is in progress to investigatethe potential role of AIM2 protein in the regulation ofprostate epithelial cell growth.AIM2 gene is constitutively expressed in the spleen, small

intestine, and peripheral leukocytes (49). Furthermore,IFN-g treatment of human HL-60 cell line (52) or IFN-b treatment of human THP-1 monocytic cell line (44)increased steady-state levels of the AIM2mRNA. Consistentwith these observations, our observations indicated that theAIM2 expression is induced by IFN-a or IFN-g treatment ofhuman normal PrECs (Fig. 1) and certain prostate cancercell lines that were tested (Figs. 4 and 5). Furthermore, theIFN-g treatment of PC-3 cells, which activated the IFN-activatable transcription factor STAT1 (Fig. 6), also inc-reased the levels of the AIM2 protein and stimulated theactivity of the AIM2-luc reporter. However, it remainsunclear how activation of IFN-g signaling in PrECs andPC-3 cells increases the steady-state levels of the AIM2mRNA and protein. Given that treatment of prostate cancercell lines such as PC-3 with either IFN-a or IFN-g increasedsteady-state levels of AIM2mRNA (Fig. 5C) and only IFN-gtreatment of cells increased the AIM2 protein levels (Fig.5A), suggest that additional mechanisms involving a post-transcriptional mechanisms may contribute to the IFN-induced levels of the AIM2 protein in PC-3 prostate cancercells (and possibly other cancer cells). Further work is in

Figure 8. Proposed model for therole of the AIM2 protein in prostaticinflammation and the developmentof prostatic diseases.

Ponomareva et al.





progress to investigate how IFN-a and IFN-g transcription-ally activate the expression of the AIM2 gene in PrECs.Caspase-1 activation is required for human prostate cancer

cells to undergo apoptosis in response to TGF-b treatment(53). Notably, the majority of primary prostate cancerspecimens do not express detectable levels of caspase-1(54). Moreover, certain prostate cancer cell lines expressreduced levels of caspase-1 (54). Accordingly, we found thatlevels of AIM2 mRNA were significantly lower in prostatetumors as compared with normal prostate (Fig. 3) andcertain prostate cancer cell lines did not express detectablebasal levels of the AIM2 protein (Figs 4 and 5). Together,these observations suggest that the pattern of caspase-1 andAIM2 expression in prostate tumors may have prognosticsignificance in the disease progression.Methylation-mediated silencing of the gene encoding for

the ASC, an adaptor protein that is required to assemblecertain inflammasome (including the AIM2 inflammasome;refs. 35–37), has been associated with the initial develop-ment of the human prostate cancers (55). Furthermore, acomplete or partial methylation of the ASC gene has beennoted in certain human prostate cancer cell lines (includingLNCaP, DU145, PC-3, MDAPCa2b, and LAPC4). Nota-bly, the PC-3 cells were reported to express detectable levelsof ASCmRNA (55). Consistent with these observations, wewere able to detect the expression of ASC protein in PC-3cells (Fig. 7) and treatment of cells with 5-aza-2-deoxyciti-dine, an inhibitor ofmethylation, increased the protein levelsfurther (data not shown). Moreover, nucleofection of plas-mid DNA into "primed" PC-3 cells activated the AIM2inflammasome (Fig. 7). Therefore, these observations indi-cated that PC-3 cells can assemble the AIM2 inflammasomeupon sensing the cytosolic DNA.We have reported previously that increased levels of AIM2

protein in senescent human fibroblasts are associated with anincreased production of IL-1b and an inflammatory phe-notype (20). Notably, the accumulation of senescent pros-tate epithelial cells in elderly patients plays an important rolein the development of BPH (16, 42) and increased expres-sion levels of IL-1b are associated with the development of

experimental BPH (56). Therefore, our observations thatsteady-state levels of AIM2 mRNA were relatively higher inBPH than normal prostate are consistent with the idea thatincreased levels of the AIM2 protein in senescent PrECscontribute to senescence-associated secretory phenotype thatis associated with the development of BPH.Inflammasomes contribute to tissue homeostasis, inflam-

mation, and immune responses (both innate and adaptive;ref. 29). Therefore, it is likely that inflammasomes alsoinfluence the formation, progression, and therapeuticresponses of the human prostate cancer. Consequently, ourobservations (Fig. 8) that the presence of cytosolic DNA(derived from pathogens, necrotic cells, or senescent cells-derived "exosomes") in normal PrECs and PC-3 prostatecancer cell line can activate the AIM2 inflammasome willserve as a basis to identify themolecularmechanisms throughwhich certain prostatic infections and/or sterile inflamma-tion contribute to the development of human prostaticdiseases.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: X. Duan, D. ChoubeyDevelopment of methodology: L. Ponomareva, H. Liu, H. Shen, R. PanchanathanAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): L. Ponomareva, H. Liu, X. Duan, E. Dickerson, R. PanchanathanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): L. Ponomareva, R. Panchanathan, D. ChoubeyWriting, review, and/or revision of the manuscript: L. Ponomareva, H. Shen, D.ChoubeyAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): X. Duan, E. Dickerson, H. ShenStudy supervision: D. Choubey

Grant SupportThis research was supported by the Merit Award from the Department of Veteran

Affairs, Medical Research and Development Service (to D. Choubey).The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received March 25, 2013; revised June 17, 2013; accepted July 9, 2013;published OnlineFirst July 17, 2013.

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2013;11:1193-1202. Published OnlineFirst July 17, 2013.Mol Cancer Res Larissa Ponomareva, Hongzhu Liu, Xin Duan, et al. of Benign Prostate Hyperplasia and Prostate CancerAIM2, an IFN-Inducible Cytosolic DNA Sensor, in the Development

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