interleukin-17 receptor-like gene is a novel …...interleukin-17 receptor-like gene is a novel...

Interleukin-17 Receptor-Like Gene Is a Novel Antiapoptotic Gene

Highly Expressed in Androgen-Independent Prostate Cancer

Zongbing You,1Xu-Bao Shi,

2Grayson DuRaine,

1Dominik Haudenschild,

1Clifford G. Tepper,

3

Su Hao Lo,1Regina Gandour-Edwards,

4Ralph W. de Vere White,

2and A. Hari Reddi

1

1Center for Tissue Regeneration and Repair, Department of Orthopaedic Surgery; Departments of 2Urology and Cancer Center,3Biochemistry and Molecular Medicine and Cancer Center, and 4Pathology, School of Medicine,University of California, Davis, Sacramento, California

Abstract

We have recently identified a new gene, interleukin-17receptor-like (IL-17RL), which is expressed in normal prostateand prostate cancer. This investigation is focused on the roleof IL-17RL in prostate cancer. We found that IL-17RL wasexpressed at significantly higher levels in several androgen-independent prostate cancer cell lines (PC3, DU145, cds1,cds2, and cds3) and tumors compared with the androgen-dependent cell lines (LNCaP and MLC-SV40) and tumors. In anin vivo model of human prostate tumor growth in nude mice(CWR22 xenograft model), IL-17RL expression in tumors wasinduced by androgen deprivation. The relapsed androgen-independent tumors expressed higher levels of IL-17RLcompared with the androgen-dependent tumors. Overexpres-sion of IL-17RL in tumor necrosis factor A (TNFA)–sensitiveLNCaP cells inhibited TNFA-induced apoptosis by blockingactivation of caspase-3 downstream to caspase-2 and caspase-8.Reciprocally, knocking down IL-17RL expression by smallinterfering RNA induced apoptosis in all the prostate cancercell lines studied. Taken together, these results show thatIL-17RL is a novel antiapoptotic gene, which may conferpartially the property of androgen-independent growth ofprostate cancer by promoting cell survival. Thus, IL-17RL is apotential therapeutic target in the treatment of prostatecancer. (Cancer Res 2006; 66(1): 175-83)

Introduction

Prostate cancers are initially androgen dependent but progres-sively become androgen independent, and the patients finallysuccumb to widespread metastases especially to bone (1, 2).The molecular mechanisms underlying the progression fromthe androgen-dependent (hormone sensitive) to the androgen-independent (hormone refractory) status of prostate cancerare increasingly being defined. The potential mechanisms involvemutations/amplifications of androgen receptor (AR) or itssignaling pathways, neuroendocrine differentiation, and alterationsof apoptosis-related genes (3–5). Up-regulation of antiapoptoticgenes [e.g., Bcl-2, clusterin , and phosphatidylinositol 3-kinase(PI3K)/Akt] and inactivation of proapoptotic genes (e.g., PTEN

and p53) have been found to play an important role in theandrogen-independent growth of prostate cancer (6–10). Theresults of this investigation show yet another novel mechanismof prostate cancer progression [i.e., inhibition of apoptosis by thegene encoding interleukin-17 receptor-like (IL-17RL) protein].The IL-17 cytokine family has six members (i.e., IL-17A or IL-17,

IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F; refs. 11, 12). IL-17R isthe receptor for IL-17A. Together with the other four homologousreceptor-like molecules [i.e., IL-17Rh1 or IL-17BR, IL-17RL orIL-17RC, IL-17RD or similar expression to fgf genes (Sef ), andIL-17RE], IL-17R has formed a new receptor family that differs fromthe other cytokine receptor families. IL-17Rh1 binds to IL-17B butbinds to IL-17E with higher affinity (13, 14). IL-17RD (or Sef) bindsto fibroblast growth factor (FGF) receptors and inhibits the FGFreceptor–mediated extracellular signal-regulated kinase (ERK)pathway (15, 16). The receptors for IL-17C, IL-17D, and IL-17Fcytokines have not been identified nor the ligands for the receptor-like molecules IL-17RL, IL-17RD, and IL-17RE. In general, the IL-17family members are involved in proinflammatory functions (17).However, depending on the tumor models used, IL-17A has beenfound to either promote (18, 19) or inhibit (20, 21) tumor growth.IL-17A expression is increased in 79% of benign prostatehyperplasia and 58% of prostate cancer specimens (22). Recently,IL-17Rh1 (or IL-17BR) has been found to be overexpressed intamoxifen-treated nonrecurrent breast cancer cases, which may beused as a biomarker together with the HOXB13 gene (23).The IL-17RL protein has 22% identity to IL-17R and is expressed

in prostate cancer (24). The full-length IL-17RL protein is predictedas a type I transmembrane protein, although some of the 11 RNAsplice variants may be secreted proteins due to the lack oftransmembrane and intracellular domains. IL-17RL mRNAs havealso been detected in kidney, cartilage, liver, heart, and skeletalmuscle. During a systematic study to determine the function ofIL-17RL, we unexpectedly found that overexpression of IL-17RL in293 (human embryonic kidney) cells inhibited tumor necrosisfactor a (TNFa)–induced apoptosis. This unexpected discoveryled to a systematic investigation of the antiapoptotic functionof IL-17RL and its role in the androgen-independent growth ofprostate cancer.

Materials and Methods

Antibodies and reagents. Rabbit anti-IL-17RL cytoplasmic domain

antibodies were from our lab (24). Anti-V5 antibodies were from Invitrogen(Carlsbad, CA). Anti-GAPDH antibodies were from Chemicon (Temecula,

CA). Antibodies to Myc, TRAF2, caspase-2, and matrix-precoated six-well

plates were from BD Biosciences (San Diego, CA). Antibodies to c-IAP1/2,

pERK (pERK1/2), caspase-10, Bcl-2, and Bcl-XS/L were from Santa CruzBiotechnology (Santa Cruz, CA). Antibodies to caspase-3, caspase-8, and

Fas were from Upstate (Lake Placid, NY). TNFa, IL-17A, IL-17B-Fc, and

Note: The authors have no conflict of interest.Supplementary data for this article are available at Cancer Research Online (http://

cancerres.aacrjournals.org/).Requests for reprints: Zongbing You, Center for Tissue Regeneration and Repair,

Department of Orthopaedic Surgery, School of Medicine, University of California,Davis, Room 2000, 4635 Second Avenue, Sacramento, CA 95817. Phone: 916-734-5750;Fax: 916-734-5750; E-mail: [email protected].

I2006 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-1130

www.aacrjournals.org 175 Cancer Res 2006; 66: (1). January 1, 2006

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antibodies to Bid were from R&D Systems, Inc. (Minneapolis, MN). LY294002and the rest of antibodies used were from Cell Signaling Technology

(Beverly, MA). The Fc-tagged human recombinant IL-17A, IL-17B, IL-17C,

IL-17D, IL-17E, and IgG were from Prite, Inc. (Camarillo, CA). IL-17F was

from Leinco Technologies (St. Louis, MO). The rest of chemicals wereobtained from Sigma-Aldrich (St. Louis, MO).

Cell culture. LNCaP, PC3, DU145, and 293 cells were from the American

Type Culture Collection (Manassas, VA). LNCaP cells and the derivative

LNCaP-C and LNCaP-RL cells were maintained in T-medium (customformula 02-0056) with 5% fetal bovine serum (FBS). The androgen-

independent LNCaP sublines cds1, cds2, and cds3 (25) were maintained in

phenol red–free RPMI 1640 with 5% charcoal/dextran-treated FBS

(HyClone, Logan, UT). PC3 cells were maintained in Ham’s F12K mediumwith 10% FBS. DU145 cells were maintained in Earle’s MEM with 10%

FBS. 293 cells were maintained in DMEM with 10% FBS. MLC-SV40

(an immortalized human prostatic epithelial cell line, androgen dependent)cells (26) were maintained in PrEGM medium (Cambrex, Walkersville, MD).

Medium and supplements were from Invitrogen, unless noted otherwise.

The cells were cultured in a 37jC, 5% CO2 humidified incubator.

Generation of plasmid constructs. Full-length human IL-17RL andIL-17Rh1 cDNAs were subcloned into pcDNA6/V5-His vector (Invitrogen)

by PCR. The V5-His-tagged cDNAs were subcloned into pLNCX2 retro-

virus vector (Clontech, Palo Alto, CA) by PCR and generated pLNCX2-IL-

17RL-V5-His and pLNCX2-IL-17Rh1-V5-His constructs. The insert sequencesof all the constructs were confirmed by DNA sequencing (Davis Sequen-

cing, Inc., Davis, CA). Detailed maps and sequences are available upon

request.Establishment of stable cell lines. LNCaP or 293 cells were transfected

with pLNCX2-IL-17RL-V5-His, pLNCX2-IL-17Rh1-V5-His, or pLNCX2 vectors

by LipofectAMINE (Invitrogen)–mediated transfection. For transient

expression, the cells were harvested 48 hours after transfection. Forestablishing stable cell lines, the transfected cells were selected with 0.6

mg/mL neomycin for 1 week. The resistant clones were pooled and

confirmed by Western blot.

Coimmunoprecipitation, pull-down assay, Western blot, quantita-tive reverse transcription-PCR, and immunohistochemistry. Cell

lysates from 293 cells overexpressing IL-17RL or IL-17Rh1 were prepared

in radioimmunoprecipitation assay (RIPA) lysis buffer. Coimmunoprecipi-

tation of IL-17F with IL-17RL was done essentially as described (27), except

that anti-IL-17F antibodies were used for immunoprecipitation and anti-V5

antibodies were used for Western blot. Pull-down assay was done by

incubating the Fc-tagged IL-17 cytokines with the cell lysates and protein

G-Sepharose beads. The rest of the Western blots were done as described

(28). RNA isolation and quantitative reverse transcription-PCR (RT-PCR)

were done as described (27). Human IL-17RL primer sequences were

5V-CACTGGTCCCACCGCTTT ( forward) and 5V-GCCTTTCAGCAATGG-GAACTC (reverse). Mouse IL-17RL primers and both human and mouse

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were from

Applied Biosystems (Foster City, CA). DC t (cycle threshold) = C t of IL-17RL�� C t of GAPDH. DDC t = DC t of study group � DC t of control group. IL-

17RL mRNA fold change was calculated as 2DDC t. The collection of the

human bone metastatic prostate tumor tissues was approved by University

of California Davis Institutional Review Board (29). Immunohistochemical

staining of IL-17RL was done on paraffin-embedded human tumor tissue

sections as described (27), using rabbit anti-IL17RL antibodies.IL-17RL oligomerization assay. Full-length IL-17RL with V5-His tag

(IL-17RL-V5) or with Myc tag (Myc-IL-17RL) was transfected alone or

cotransfected into 293 cells by LipofectAMINE-mediated transfection.

Whole cell lysate was prepared in RIPA lysis buffer. Equal amounts ofprotein were precipitated with 1 Ag of either anti-V5 or anti-Myc anti-

bodies, together with Protein G-agarose beads for immunoprecipitation.

Western blots were done using anti-Myc or anti-V5 antibodies, respectively.

Whole-cell lysate was probed for the expression of individually taggedIL-17RL.

Cell viability, DNA fragmentation, and cell growth curve. Cells weretreated with 20 ng/mL of TNFa unless noted otherwise for the indicated

time. In some experiments, TNFa was added 30 minutes after addition of

20 Amol/L LY294002, or 16 hours after transfection with small interferingRNA (siRNA) or C-siRNA. Digital images were captured by light

microscopy at �100 magnification. Cell viability was determined by

the trypan blue exclusion assay, in which cell survival was calculated as

(the living cell number of treated group � the living cell number ofuntreated control group) � 100. The DNA fragmentation assay was done

as described (28).

Cell attachment assay. As modified from (30), 2.5 � 105 per well of

LNCaP-C or LNCaP-RL cells in 2-mL serum-free T-medium were plated insix-well plates in triplicate. The regular Costar plate was used as control,

collagen type I–, collagen type IV–, fibronectin-, and laminin-precoated

plates were used to test the cell attachment to these extracellular matrixes.

After 90-minute incubation at 37jC, the cells not attached were washedoff with PBS. The attached cells were fixed with 5% glutaraldehyde for

30 minutes at room temperature. After three washes with PBS, the attached

cells were stained with 0.1% crystal violet dye for 30 minutes. After fivewashes with water, the stain was dissolved in 10% acetic acid and read at

570 nm on a microplate reader. The crystal violet dye stains the cell protein

that is proportional to the number of cells. The number of cells attached to

the matrixes was calculated as percentage of cells attached to the Costarplate.

Tumor xenografts. The CWR22 model of human prostate tumors and

relapsed tumors were generated in nude mice as described (31, 32). For the

time course, tumors were harvested from intact mice (day 0) and from mice3, 7, and 14 days after surgical castration. The animal experiment was

approved by the University of California Davis Institutional Animal Care

and Use Committee, according to NIH guidelines.Synthesis of siRNA and transfection. Both IL-17RL–specific siRNA

and scrambled control C-siRNA were designed by Invitrogen’s Block-iT

RNAi Designer5 and synthesized with Invitrogen’s proprietary Stealth

technology. Their sequences were siRNA 5V-UCGCCUUGGAGAGUACUUA-CUACAA (sense) and C-siRNA 5V-UCGGUUGAGUGAUCACAUAUCCCAA(sense). Fifty percent confluent cells were transfected with the mixtures

of siRNA/LipofectAMINE 2000 (Invitrogen), C-siRNA/LipofectAMINE 2000,

or LipofectAMINE 2000 only as control, according to the manufacturer’sprotocol. The final concentrations used were 100 nmol/L siRNA or

C-siRNA and 5 AL/mL LipofectAMINE 2000.

Statistical analysis. Kruskal-Wallis midrank statistical test was used tocompare the IL-17RL staining on human prostate tumors. Student’s t test

was used to analyze the remaining data.

Results

IL-17RL is a prosurvival molecule in 293 cells. Our initialapproaches to identify a ligand for IL-17RL were not successful(Supplementary Fig. 1A and B). We next overexpressed IL-17RL in293 cells, expecting that overexpression of a ‘‘receptor’’ might causeautoactivation and initiate similar signaling pathways as thoseactivated by IL-17/IL-17R (11, 12). We established a 293 cell linestably overexpressing IL-17RL (named 293RL) and a control cellline transfected with empty vector only (named 293C). Over-expression of IL-17RL in 293 cells did not activate nuclear factor-nB(NF-nB), ERK, c-Jun NH2-terminal kinase, p38, signal transducerand activator of transcription, protein kinase C (PKC), Src, orglycogen synthase kinase 3 h pathways, as shown by the absence ofincrease in the phosphorylated forms of the key proteins(Supplementary Fig. 1B and C).As TNFa is a strong activator of NF-nB pathway, it was used as a

positive control for activation of NF-nB. We were surprised to findthat 293RL cells were more resistant to the cell death inducedby TNFa (Fig. 1A). TNFa killed 293C cells in a dose- and time-dependent manner, whereas overexpression of IL-17RL inhibited

5 http://rnaidesigner.invitrogen.com/sirna.

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Cancer Res 2006; 66: (1). January 1, 2006 176 www.aacrjournals.org



20% to 30% of cell death induced by TNFa (Fig. 1B and C). Similarinhibition of cell death was observed in 293 cells transientlyexpressing IL-17RL (Fig. 1D), which ruled out the possibility thatthe survival advantage was due to clonal selection during theestablishment of the stable cell lines. Taken together, these dataimplied that IL-17RL is a prosurvival molecule in 293 cells.IL-17RL is expressed at higher levels in androgen-indepen-

dent prostate cancer cell lines. The LNCaP cell line is androgendependent and TNFa sensitive. On the other hand, the PC3 cell lineis androgen independent and TNFa resistant (33). Three androgenindependent sublines of LNCaP (i.e., cds1, cds2, and cds3) weregenerated recently (25). These three sublines of LNCaP were alsoresistant to TNFa, similar to PC3 (Fig. 2A). Because we previouslyshowed that IL-17RL was expressed in prostate cancer and IL-17RLis a prosurvival molecule in 293 cells, we hypothesized that theincreased expression of IL-17RL might be responsible for thesurvival advantage of the androgen-independent cell lines. Asexpected, we found that IL-17RL was expressed at significantlyhigher levels in androgen-independent prostate cancer cell linesthan in androgen-dependent MLC-SV40 (26) and LNCaP cell linesat the mRNA level (Fig. 2B and C). As detected by quantitative real-time RT-PCR, the androgen-independent PC3, DU145, cds1, cds2,and cds3 cells expressed 48-, 12-, 10-, 3-, and 2 fold, respectively,more IL-17RL mRNA than LNCaP cells. Consistently, IL-17RLprotein expression was higher in the androgen-independent cellsthan in the androgen-dependent cells, as shown by Western blot(Fig. 2D).IL-17RL is expressed at higher levels in androgen-indepen-

dent prostate tumors. We next examined if IL-17RL expression isincreased in androgen-independent human prostate tumors.Fourteen bone metastatic prostate tumor samples were collectedeither before androgen ablation (samples considered androgendependent, n = 6) or at the time of relapse after androgen ablation(samples considered androgen independent, n = 8), as describedpreviously (29). By immunohistochemical staining with rabbit anti-IL-17RL antibodies, we found that all six androgen-dependent

samples were stained negatively or weakly. In contrast, two out ofeight androgen-independent samples were stained moderately orstrongly, whereas the remaining six samples were stained weakly(Fig. 3A). However, due to the small sample size, there was nostatistical significance by Kruskal-Wallis midrank statistical test(P = 0.0519). As a complementary experimental approach, we usedthe well-established CWR22 androgen-dependent human prostatecancer xenograft mouse model (31, 32, 34, 35). We found thatandrogen-independent CWR22 tumors (relapsed tumors aftercastration) expressed higher levels of IL-17RL mRNA (DC t range,�7.9 to �4.3; average, �6.4; n = 4) compared with androgen-dependent CWR22 tumors from the intact mice (DC t range, �9.0to �8.2; average, �8.6; n =3; Fig. 3B). This indicated that theandrogen-independent tumors on the average expressed 4.6-foldmore IL-17RL than the androgen-dependent tumors, which isstatistically significant (Student’s t test, P < 0.05). In addition, IL-17RL expression was induced in the CWR22 tumors uponcastration, as 3, 7, and 14 days after castration, IL-17RL mRNAincreased 3.3-, 4.7-, and 10.2 fold, respectively, compared with thetumors collected from the intact mice (Fig. 3C). As an internalcontrol, the mouse IL-17RL expression in the stromal cells ofmouse origin that infiltrated into the s.c. xenografted humantumors did not change significantly (Fig. 3C).IL-17RL partially confers TNFA resistance in prostate

cancer cells. To test if the increased IL-17RL in the androgen-independent cells confers TNFa resistance in prostate cancer celllines, we established three independently pooled clones of LNCaPcells stably expressing IL-17RL (named LNCaP-RL) and controlcells transfected with empty vector only (named LNCaP-C; Fig. 4A).Overexpression of IL-17RL in LNCaP cells inhibited about 20% ofcell death induced by 20 ng/mL of TNFa (Fig. 4B). As LNCaP cellsdo not express PTEN and have constitutively active prosurvivalPI3K/Akt signaling pathway (36), we reasoned that the prosurvivalrole of IL-17RL might be partially masked by the active PI3K/Aktsignaling. Indeed, when the PI3K/Akt signaling pathway wasblocked by 20 Amol/L PI3K inhibitor LY294002, IL-17RL expression

Figure 1. Overexpression of IL-17RL in 293 cellsinhibited TNFa-induced apoptosis. A, 293RL cells were293 cells stably overexpressing IL-17RL, whereas293C cells were control cells transfected with emptyvector only. Cells were treated with or without100 ng/mL TNFa for 3 days. The dead cells wereround, floating, and broken. Bar, 60 Am.B, dose-response experiments in stable cell linestreated with or without TNFa. Living cell number wascounted by trypan blue exclusion assay. Cell survivalwas calculated as (the living cell number of treatedgroup � the living cell number of untreated controlgroup) � 100. Column, mean of triplicate (same for all);bars, SD. P < 0.05 between the comparison groups.All in vitro experiments were repeated at least threetimes. Reproducible representative results. C, timecourse experiments in stable cell lines treated with100 ng/mL TNFa. P < 0.05 between the comparisongroups. D, 293 cells transiently overexpressingIL-17RL (IL-17RL group) or control 239 cells (controlgroup) were treated with or without 100 ng/mL of TNFafor 2 days. P < 0.05 between the comparison groups.

IL-17RL Is a Novel Antiapoptotic Gene




alone inhibited 50% of the cell death induced by 20 ng/mL of TNFa(Fig. 4C). Of note, in the absence of LY294002, the LNCaP cells werekilled by TNFa at a slow rate. Significant cell death was observed3 days after the treatment (Fig. 4B). In contrast, dramatic cell deathwas induced 24 hours after TNFa treatment in the presence ofLY294002 (Fig. 4C). However, cell death was inhibited by over-expression of IL-17RL. These results were consistent among all threesets of independently pooled LNCaP-C and LNCaP-RL clones, whichindicated that clonal selection of survival advantage was unlikely.To address the potential bias that might arise from the

overexpression model, we used siRNA (37) to knock down theendogenous and exogenous expression of IL-17RL in the cell lines.As a control, the same nucleotides were scrambled in sequence(C-siRNA). Both siRNA and C-siRNA were designed by Invitrogen’sBlock-iT RNAi Designer and synthesized with Invitrogen’sproprietary Stealth technology to maximally avoid off-target andnonspecific IFN response. IL-17RL–specific siRNA specificallyknocked down the expression of exogenous and endogenous IL-17RL in 293C, 293RL, LNCaP-C, LNCaP-RL, cds1, cds2, cds3, PC3,and DU145 cells (Fig. 5B ; data not shown). Diminution of IL-17RLexpression led to apoptosis of cells, as shown morphologically bymicroscopy (Fig. 5A) and biochemically by cleavage of poly(ADP-ribose) polymerase (PARP; Fig. 5B). Twenty-four hours aftertransfection, IL-17RL–specific siRNA killed about 30% of both theLNCaP-C and LNCaP-RL cells, whereas the scrambled controlsiRNA killed <5% of the cells possibly due to nonspecific stress

response (Fig. 4D). Furthermore, addition of 20 ng/mL TNFa tothe IL-17RL–specific siRNA-transfected cells killed 66% of LNCaP-C cells and 58% of LNCaP-RL cells, whereas TNFa alone killedonly 15% of LNCaP-C cells and 3% of LNCaP-RL cells within24 hours (Fig. 4D). The synergistic cell killing effects of siRNAand TNFa indicated that both the endogenous and overexpressedIL-17RL have antiapoptotic functions. Similar results wereobtained in 293C and 293RL cells, as the combination of siRNAand TNFa induced dramatically more DNA fragmentation (shownas DNA ladder) compared with either treatment alone (Fig. 5C ;similar results were obtained in LNCaP-C and LNCaP-RL cells).Taken together, these data suggested that IL-17RL is a bona fideantiapoptotic gene that provides a cell survival signal to 293 cellsand prostate cancer cells.IL-17RL inhibits apoptosis by blocking activation of

caspase-3 downstream to caspase-2 and caspase-8. Toexamine further the mechanism of how IL-17RL inhibits TNFa-induced apoptosis, we treated LNCaP-C and LNCaP-RL cells with20 ng/mL TNFa, with or without 20 Amol/L LY294002, for up to48 hours. We found that two initiator caspases (caspase-2 andcaspase-8) were activated similarly in LNCaP-C and LNCaP-RLcells, as shown by the cleavage of their proenzymes upon TNFatreatment (Fig. 6A). Addition of LY294002 increased the cleavageof procaspase-2 and procaspase-8, particularly at 24 to 48 hoursafter treatment. The third initiator caspase examined (caspase-10)was not activated in both cells under either treatment (Fig. 6A).

Figure 2. IL-17RL expression was increased inthe androgen-independent and TNFa-resistantprostate cancer cell lines. LNCaP (a humanprostate cancer cell line) and MLC-SV40(an immortalized human prostatic epithelial cellline) cells are androgen dependent. Cds1, cds2,and cds3 are three androgen-independent cell linesderived from LNCaP cells. PC3 and DU145 celllines are androgen-independent human prostatecancer cell lines. A, androgen-independentprostate cancer cells were resistant toTNFa-induced apoptosis. Cells were treatedwith 20 ng/mL TNFa for 3 days. Bar, 60 Am.B and C, IL-17RL mRNA levels detected byquantitative RT-PCR in the cells under normalculture conditions. IL-17RL mRNA foldchange was calculated as 2DDC t, where DC t

(cycle threshold) = C t of IL-17RL � C t ofGAPDH. DDC t = DC t of cancer cell line � DC t

of the MLC-SV40 cell line. D, IL-17RL proteinlevels detected by Western blot in the cells undernormal culture conditions.

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Caspase-3 is the executioner caspase downstream to caspase-2and caspase-8 (38). In LNCaP-C cells, procaspase-3 was cleaved(thereby activated) by TNFa with or without LY294002 (Fig. 6A).This consequently led to apoptosis, which was shown by cleavageof PARP, with increased PARP cleavage upon addition of LY294002(Fig. 6A). However, in LNCaP-RL cells, the cleavage of procaspase-3 and PARP was blocked when treated with TNFa alone ordramatically diminished with addition of LY294002 (Fig. 6A). Theother executioner caspases, such as caspase-7 and caspase-6, werenot activated under current treatments (Fig. 6A ; data not shown).Bid has been reported to be activated upon caspase-2 or caspase-8 activation, which subsequently activates caspase-9 via inducingcytochrome c release from mitochondria (so-called intrinsic celldeath pathway; refs. 39, 40). In both LNCaP-C and LNCaP-RLcells, Bid and caspase-9 were not activated under currenttreatments (Fig. 6A ; data not shown), which implied that onlythe extrinsic cell death pathway was involved in our cell models.Of note, the cytomegalovirus promoter activity of the pLNCX2vector carrying IL-17RL was inducible by TNFa and other stress

conditions (data not shown), which led to the increase of IL-17RLexpression upon TNFa treatment (Fig. 6A). In addition, we foundthat overexpression of IL-17RL did not change the expression ofseveral proapoptotic or antiapoptotic genes, such as Fas, c-IAP1/2,TRAF2, Bcl-2 , and Bcl-XL , either with or without dihydrotestoster-one treatment (data not shown). These genes regulate apoptosiseither above caspase-2/caspase-8, at mitochondria, or caspase-3levels (39, 41). Furthermore, we found that overexpression of IL-17RL did not stimulate the cellular proliferation of either 293 cellsor LNCaP cells (data not shown), which ruled out the possibilitythat the observed survival advantage of the cells overexpressingIL-17RL was due to enhanced cellular proliferation. Takentogether, our data indicated that IL-17RL inhibits apoptosis byblocking activation of caspase-3 downstream to caspase-2 andcaspase-8.Overexpressed IL-17RL oligomerizes to induce gene

expression that enhances cell attachment to the extracellu-lar matrix. To further delineate the mechanism of how IL-17RLblocks caspase-3 activation, we tested if IL-17RL physically binds

Figure 3. IL-17RL expression was increased in androgen-independent prostate tumors. A, immunohistochemical staining of IL-17RL. The negative staining from anandrogen-dependent (AD ) bone metastatic human prostate tumor and the strong staining from an androgen-independent (AI ) bone metastatic human prostatetumor were presented. The androgen-dependent bone metastatic tumors were collected from the patient before androgen ablation therapy, whereas theandrogen-independent bone metastatic tumors were collected at the time of relapse after androgen ablation therapy. Bar, 60 Am. Kruskal-Wallis midrank statisticaltest showed P = 0.0519 between the androgen-dependent and androgen-independent groups. B, IL-17RL mRNA expression was increased in the xenograftedandrogen-independent human CWR22 tumors. The human androgen-dependent CWR22 tumor cells were xenografted s.c. in male nude mice with a 12.5 mgsustained-release testosterone pellet implanted s.c. When the tumors grew to about 0.75 to 1 cm in diameter (f4 weeks), half of the mice (intact) was euthanizedwhile their tumors were harvested as androgen-dependent tumors. The other half of the mice was castrated by removal of the testosterone pellet and bilateralorchiectomy under anesthesia. The tumors in the castrated mice regressed rapidly but regrew after 3 months. When the regrown tumors reached about 0.75 to 1 cm,they were harvested as androgen-independent tumors. The total RNA was isolated from the tumors, and human IL-17RL expression was detected by quantitativeRT-PCR. DC t (cycle threshold) = C t of human IL-17RL � C t of human GAPDH. The ranges of DC ts of IL-17RL mRNA from the androgen-dependent andandrogen-independent tumors were presented. Of note, the DC ts were in negative values (relative to GAPDH), so the less negative the higher levels of IL-17RL.Androgen dependent versus androgen independent, P < 0.05 by the Student’s t test. C, IL-17RL mRNA expression was induced in the xenografted human CWR22tumors upon castration. The mouse s.c. tumor model was established as described in (B ). The tumors from intact mice (day 0), or 3, 7, and 14 days aftercastration were harvested for detection of IL-17RL mRNA by quantitative RT-PCR. IL-17RL mRNA fold change was calculated as 2DDC t, where DC t (cycle threshold) =C t of IL-17RL � C t of GAPDH. DDC t = DC t of tumors from the castrated mice � DC t of the intact mice. The mouse stromal cells that infiltrated the s.c. xenograftedhuman tumors were used as internal control of the mouse IL-17RL, which was detected by mouse-specific PCR primers for IL-17RL and GAPDH.





to caspase-3. By coimmunoprecipitation assay, we found thatIL-17RL did not bind to caspase-3 (data not shown). Thisindicates that IL-17RL might activate certain intracellularsignaling pathways that are not the common pathways as wealready tested (Supplementary Fig. 1C). By cotransfection ofV5-His-tagged and Myc-tagged IL-17RL in 293 cells, we foundthat IL-17RL protein bound to its self as detected bycoimmunoprecipitation assay (Fig. 6B). When the overexpressedIL-17RL was run on a nonreducing SDS-PAGE gel, a 203-kDacomplex was detected by Western blot, which was not seenunder reducing conditions (either with 25 mmol/L DTT or 7.5%h-mercaptoethanol; Supplementary Fig. 1D). These data indicatethat IL-17RL may form a homotrimer that is bound together bydisulfide bonds among the monomers. To identify the genes thatare regulated by IL-17RL overexpression and mediate IL-17RL’santiapoptotic function in LNCaP cells, we did gene microarrayanalysis on LNCaP-C and LNCaP-RL cells. We found that 74genes were up-regulated, whereas 38 genes were down-regulated(Supplementary Table 1). Of particular interest was that integrinb5 and a-parvin genes were both up-regulated. As these twogenes are involved in cell attachment to the extracellular matrix,we tested if IL-17RL overexpression could enhance cellattachment to the extracellular matrix. We found that therewere 15%, 24%, and 26% more cells attached to the collagen typeI, collagen type IV, and fibronectin matrixes, respectively, inLNCaP-RL cells than in LNCaP-C cells (Fig. 6C). Meanwhile,there was no difference between the attachment of LNCaP-C andLNCaP-RL cells to laminin, to which few of both cells attached(Fig. 6C).

Discussion

The identification of IL-17RL as a novel antiapoptotic gene is anunexpected finding. First, the androgen-independent prostatecancer cell lines expressing high levels of IL-17RL were moreresistant to TNFa-induced apoptosis than the prostate cancer cellline expressing low levels of IL-17RL. Second, overexpression ofIL-17RL inhibited TNFa-induced apoptosis in both 293 cells andTNFa-sensitive LNCaP cells. Third, knocking down the expressionof IL-17RL by IL-17RL–specific siRNA induced apoptosis in 293cells and seven prostate cancer cell lines studied. Finally, TNFakilled more cells when IL-17RL expression was diminished bysiRNA. We must point out that the antiapoptotic IL-17RL may notbe the only cell survival signal in our models, as other antiapoptoticsignals are also present, such as Bcl-2 and PI3K/Akt (6, 36, 42). Thepresence of these additional antiapoptotic signals may mask theeffects of one particular antiapoptotic signal. This explains whyoverexpression of IL-17RL protected only 20% of cell death in thepresence of other antiapoptotic signals, whereas it protected 50%of cell death in the absence of PI3K/Akt signal. These findings alsohighlight the necessity to block several or all survival signals in thedesign of new strategies to treat prostate cancer.The TNFa-induced apoptosis signaling pathway has been well

defined and is one of the paradigms widely used to identify novelantiapoptotic genes or mechanisms (38–40, 43, 44). To determinethe mechanism underlying the inhibition of apoptosis by IL-17RL,we have systematically examined the apoptosis signaling pathways.We identified that IL-17RL did not inhibit the activation of caspase-2and caspase-8 but dramatically blocked the activation of caspase-3.This indicated that IL-17RL suppresses apoptosis at the level

Figure 4. IL-17RL conferred TNFa resistance in LNCaPcells. A, stable cell lines were established by selection withneomycin after transfection with pLNCX2-IL-17RL-V5-His(LNCaP-RL) or pLNCX2 (LNCaP-C), and IL-17RLoverexpression was confirmed by Western blots. B and C,overexpression of IL-17RL in LNCaP cells inhibitedTNFa-induced apoptosis. Cells were treated with 20 ng/mLTNFa alone for 3 days (B ) or plus 20 Amol/L LY294002 for24 hours (C ). Cell survival was calculated as (the livingcell number of treated group � the living cell number ofuntreated control group) � 100. P < 0.05 between thecomparison groups. D, apoptosis was induced by transfectionof the cells with 100 nmol/L siRNA for 24 hours, whichwas synergistically enhanced by adding 20 ng/mL of TNFa.Cell survival was calculated as (the living cell numberof treated group � the living cell number of untreatedcontrol group) � 100. P < 0.05 between the treatment andcontrol groups.

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downstream to caspase-2/caspase8 but at or above the level ofcaspase-3. Currently, we do not know the precise action of IL-17RLin blocking the activation of caspase-3 despite considerable efforts.We have found that IL-17RL does not physically bind to caspase-3;therefore, IL-17RL must act through certain intracellular signalingpathways. We have ruled out the possibility that IL-17RL may actindirectly by modulating the expression of several proapoptotic andantiapoptotic genes, such as Fas, c-IAP1/2, TRAF2, Bcl-2 , and Bcl-XL ,or by activating the common signaling pathways as tested(Supplementary Fig. 1C).However, we propose that the overexpressed IL-17RL forms a

homotrimer complex via intermolecular disulfide bonds. Thisis based on our data that IL-17RL binds to its self, and thenonreduced complex’s molecular weight is about thrice of that ofthe reduced monomer (230 versus 85 kDa), although we can notrule out the possibility that the complex is formed by an IL-17RLhomodimer and an unknown partner. Nevertheless, oligomeriza-tion may activate IL-17RL by autoactivation or by binding to anunknown ligand, which may activate the downstream signalingpathways that lead to regulation of gene expression. Indeed, wefound a total of 112 genes with their expression being up-regulatedor down-regulated by IL-17RL overexpression in LNCaP cells.Among them, integrin b5 and a-parvin are of particular interest, as

both genes are involved in cell attachment and apoptosis (45, 46).These molecular changes are consistent with the biologicalresponse that IL-17RL overexpression enhances cell attachmentto the extracellular matrixes. Cell attachment to the extracellularmatrix is critical for cell survival (47–50). The enhanced cellattachment in LNCaP-RL cells may be partially responsible for theresistance to TNFa-induced apoptosis. In addition, a-parvin (alsonamed as calponin homology domain-containing integrin-linkedkinase–binding protein or actopaxin) has been found as animportant component of the prosurvival signaling pathwayfunctioning primarily by activation of PKB/Akt (46). a-Parvin actsin the cell survival pathway upstream of caspase-3, as depletion ofa-parvin markedly activates caspase-3 and induces apoptosis (46).Therefore, we speculate that the inhibition of caspase-3 activationby IL-17RL is in part due to the induction of a-parvin expression.The androgen-independent prostate cancer cell lines and

tumors express higher levels of IL-17RL compared with theirandrogen-dependent counterparts, hinting at the possible role ofIL-17RL in providing a cellular survival signal. The cancer cellsneed an additional survival signals to compensate for thedeprivation of androgen. On the other hand, once the cancer cellsestablish this additional survival advantage, they are no longerdependent on androgen. Due to difficulties in obtaining large

Figure 5. IL-17RL–specific siRNAknocked down IL-17RL expression andinduced apoptosis. A, apoptosis wasinduced by transfection of the cells with100 nmol/L IL-17RL–specific siRNA for48 hours but not by the control C-siRNA.Bar, 120 Am. B, IL-17RL–specific siRNAknocked down IL-17RL expression andinduced cleavage of PARP in the cell linesin 48 hours as detected by Western blot(cell names of each panel matchedvertically to A). C, DNA fragmentation wasinduced by 100 nmol/L IL-17RL–specificsiRNA transfection in 293 cells in 24 hours,which was synergistically enhanced byadding 20 ng/mL TNFa.





numbers of androgen-independent human prostate tumors, thesample size was not large enough to attain statistical significance.Our in vivo xenograft study complemented this limitation byshowing that IL-17RL expression was significantly increased inandrogen-independent human prostate CWR22 tumors. Further-more, we showed that IL-17RL expression was induced byandrogen deprivation in a time-dependent manner.The present study provides evidence that IL-17RL is a novel

antiapoptotic gene. Because IL-17RL expression is induced byandrogen deprivation and it is highly expressed in androgen-independent prostate cancer cell lines and tumors, it is reasonableto speculate that IL-17RL is potentially associated with androgen-

independent growth of prostate cancer. It is likely that IL-17RLmight be a potential therapeutic target in the treatment ofandrogen-independent prostate cancer.

Acknowledgments

Received 4/4/2005; revised 10/5/2005; accepted 10/25/2005.Grant support: U.S. Army Medical Research and Material Command, Department

of Defense grant DAMD17-03-2-0033 and NIH grant 5R01AR047345-03.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Thomas G. Pretlow (Institute of Pathology, Case Western ReserveUniversity School of Medicine, Cleveland, OH) for generously providing the CWR22xenograft.

Figure 6. IL-17RL inhibited TNFa-inducedapoptosis by blocking caspase-3activation. A, cells were treated with20 ng/mL TNFa, with or without 20 Amol/LLY294002 for up to 48 hours. Western blotswere done using the indicated antibodies.Of note, the decrease or disappearance ofprocaspases indicated their cleavage andactivation. B, IL-17RL protein binds toits self. Full-length IL-17RL withV5-His tag (IL-17RL-V5) or with Myc tag(Myc-IL-17RL) was transfected alone orcotransfected into 293 cells byLipofectAMINE-mediated transfection.Equal amounts of cell lysate wereprecipitated with 1 Ag of either anti-V5 oranti-Myc antibodies, together with ProteinG-agarose beads for immunoprecipitation(IP ). Western blots (WB ) were doneusing anti-Myc or anti-V5 antibodies,respectively. Whole-cell lysate was probedfor the expression of individually taggedIL-17RL. C, IL-17RL enhances cellattachment. Cells (2.5 � 105 per well) ofLNCaP-C or LNCaP-RL cells in 2-mLserum-free T-medium were platedin triplicate in six-well Costar plate, orcollagen type I (Col I ), collagen type IV(Col IV ), fibronectin, and laminin precoatedplates. After a 90-minute incubation at37jC, the attached cells were fixed with 5%glutaraldehyde and stained with 0.1%crystal violet dye. The stain was dissolvedin 10% acetic acid and read at 570 nm on amicroplate reader. The crystal violet dyestains the cell protein that is proportional tothe number of cells. The number of cellsattached to the matrixes was calculatedas percentage of cells attached to theCostar plate. Columns, mean; bars, SD.LNCaP-RL versus LNCaP-C: *, P < 0.05;**, P < 0.01.

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2006;66:175-183. Cancer Res Zongbing You, Xu-Bao Shi, Grayson DuRaine, et al. CancerGene Highly Expressed in Androgen-Independent Prostate

Gene Is a Novel AntiapoptoticInterleukin-17 Receptor-Like

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