elevated expression of inhibitor of apoptosis proteins in ... · expression occurs early in the...

Elevated Expression of Inhibitor of Apoptosis Proteins inProstate Cancer

Maryla Krajewska,1 Stan Krajewski,1

Steven Banares, Xianshu Huang, Bruce Turner,Lukas Bubendorf, Olli-P. Kallioniemi,Ahmed Shabaik, Antonella Vitiello, Donna Peehl,Guo-Jian Gao, and John C. Reed2

The Burnham Institute, La Jolla, California 92037 [M. K., S. K.,S. B., X. H., J. C. R.]; Department of Radiation Oncology, ThomasJefferson University, Philadelphia, Pennsylvania 19107 [B. T.];Institute of Pathology, University of Basel, Switzerland [L. B.];Laboratory of Cancer Genetics, National Human Genome ResearchInstitute, NIH, Bethesda, Maryland 20892-4470 [O-P. K.];Department of Pathology, University of California, San Diego, SanDiego, California 92103 [A. S.]; The R. W. Johnson PharmaceuticalResearch Institute, San Diego, California [A. V.]; Department ofUrology, Stanford University, Stanford, California 94305 [D. P.]; andBD Biosciences Pharmingen, San Diego, California 92121 [G-J. G.]

ABSTRACTPurpose: Inhibitor of apoptosis (IAP) family proteins

are suppressors of apoptosis that have been implicated inapoptosis resistance in some cancers. Their expression andrelevance to the prognosis of prostate cancer were investi-gated.

Experimental Design: The expression of four membersof the IAP family (cellular inhibitor of apoptosis protein 1,cellular inhibitor of apoptosis protein 2, X chromosome-linked IAP, and survivin) was examined by immunohisto-chemistry and immunoblotting in human prostate cancersand in prostate tissues from transgenic mice expressingSV40 large T antigen under control of a probasin promoter.

Results: Tumor-associated elevations in the levels of allfour IAP family members were common in prostate cancersof both humans and mice, suggesting concomitant up-regu-lation of multiple IAP family proteins. Compared with nor-mal prostatic epithelium, increased IAP expression was of-ten evident even in prostatic intraepithelial neoplasia lesions(carcinoma in situ), suggesting that deregulation of IAPexpression occurs early in the pathogenesis of prostate can-cer. IAP expression did not correlate with Gleason grade orprostate-specific antigen levels.

Conclusions: The findings demonstrate that tumor-associated elevations in the expression of several IAP familyproteins occur as a frequent and early event in the etiologyof prostate cancer.

INTRODUCTIONProstate cancers are generally slow-growing malignancies

that are characterized by an imbalance in the rates of celldivision and cell death. Tissue kinetics studies indicate thatinsufficient programmed cell death represents the chief expla-nation for the gradual accumulation of prostate cancer cells invivo in humans (1). Progression of localized hormone-dependentprostate cancers to metastatic, hormone-refractory disease isalso associated with dysregulation of normal apoptotic mecha-nisms.

Apoptosis is executed by a family of cysteine proteasesknown as caspases. Caspases are produced in cells as inactivezymogens and generally must undergo proteolytic processing tobecome active proteases (reviewed in Ref. 2). The IAPs3 are theonly known endogenous caspase inhibitors (3). They containBaculovirus IAP repeat domains, and some of them bind andpotently inhibit activated caspases, including, in mammals, theeffector caspases-3 and -7 and the initiator caspase-9 (reviewedin Ref. 4). In addition to Baculovirus IAP repeat domains,several IAPs also contain a RING domain, which binds ubiq-uitin-conjugating enzymes that promote degradation of IAPcaspase complexes (5). Eight human IAPs have been recog-nized, including XIAP, cIAP1, cIAP2, survivin, NAIP, apollon(BRUCE), ML-IAP (livin, KIAP), and ILP-2 (reviewed in Ref.6). The antiapoptotic properties of IAPs have also been linked tothe Rel/nuclear factor �B pathway and mitogen-activated pro-tein kinase signal transduction (7, 8). In particular, cIAP1 andcIAP2 have been shown to activate nuclear factor �B (9). XIAP,NAIP, and ML-IAP have been reported to modulate apoptosispathways via the TAK1/c-Jun-NH2-terminal kinase signalingcascade (10).

Some IAP family proteins are overproduced in cancers,suggesting that IAP-mediated suppression of apoptosis maycontribute to tumor pathogenesis, progression, and resistance todrug treatment. For example, survivin is expressed abundantlyin fetal tissues but scarcely present in most adult tissues. Highlevels of survivin protein have been reported in many types ofhuman cancers, suggesting that reactivation of expression of this

Received 7/1/02; revised 5/7/03; accepted 5/7/03.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournal.org).1 Both authors contributed equally to this work.2 To whom requests for reprints should be addressed, at The BurnhamInstitute, 10901 North Torrey Pines Road, La Jolla, CA 92037. Phone:(858) 646-3140; Fax: (858) 646-3194; E-mail: [email protected].

3 The abbreviations used are: IAP, inhibitor of apoptosis; PIN, prostaticintraepithelial neoplasia; PSA, prostate-specific antigen; XIAP, X chro-mosome-linked IAP; cIAP, cellular inhibitor of apoptosis protein;NAIP, neuronal apoptotic inhibitory protein; TRAMP, transgenic mousemodel of prostate cancer; BPH, benign prostatic hyperplasia; DAB,3,3�-diaminobenzidine; HRP, horseradish peroxidase; NPE, nonmalig-nant prostate epithelium; RFS, relapse-free survival; HR, hazard ratio;CI, confidence interval; TAKI, TGFB-activated kinase.

4914 Vol. 9, 4914–4925, October 15, 2003 Clinical Cancer Research

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gene represents a common event in tumorigenesis (reviewed inRefs. 11 and 12). Indeed, genome-wide transcription profilingsuggests that survivin is among the most tumor-specific genesthus far identifiable (13). Similarly, the ML-IAP protein is notexpressed at detectable levels in most normal adult tissues but ispresent in melanomas (14) and perhaps some other types ofcancers. Likewise, whereas XIAP is broadly expressed in nor-mal tissues, higher levels of this IAP family member have beendemonstrated in patients with acute myelogenous leukemia (15).Thus, elevations in the levels of certain IAP family proteins mayoccur in tumors, conferring a selective survival advantage.

In this report, we analyzed the expression of several IAPfamily proteins, including survivin, XIAP, cIAP1, and cIAP2, inprostate cancers. Our data demonstrate that tumor-associatedincreases in the expression of several IAP family proteins occurcommonly in prostate cancer and probably as an early event.Moreover, overexpression of these IAP family members wasalso documented in TRAMP, the model further suggesting animportant role for deregulation of IAP expression in the patho-genesis of prostate cancer. IAP family proteins thus may be

candidate drug discovery targets for restoration of apoptosissensitivity in prostate cancer.

MATERIALS AND METHODSPatient Specimens. Prostate cancer specimens for tissue

microarray preparation were obtained from the archives of theInstitute of Pathology, University of Basel (Basel, Switzerland),the Cantonal Institute of Pathology (Liestal, Switzerland), andthe Tampere University Hospital (Tampere, Finland). The tissuemicroarrays included transurethral resections from 32 patientswith BPH and 725 prostate cancer specimens, containing 646primary tumors derived from 592 patients, and 79 metastases.The primary tumors included 225 specimens representing clin-ically inapparent [stage T1, according to International UnionAgainst Cancer criteria (16)] tumors; 368 cancers from radicalprostatectomies, including both locally confined (stage T2) andlocally advanced disease (stage T3–T4); and an additional 53local recurrences after hormonal therapy failure. Metastatictumor specimens (n � 79) were collected at autopsy from 62

Fig. 1 Characterization of IAP antibodies andanalysis of IAP protein expression in prostate can-cer by immunoblotting. Plasmids encoding humancIAP1, cIAP2, XIAP, survivin, NAIP, mouseBRUCE, or baculovirus Cp-IAP were translated inreticulocyte lysates (IVT) using either T3 or T7RNA polymerase (Promega) according to themanufacturer’s protocol, in the presence ofL-[35S]methionine (�1 mCi/mmol; Amersham).Ten �l were loaded in gels. Autoradiography con-firmed production of all proteins (data not shown).Alternatively, proteins were produced in bacteriaas recombinant proteins (RP) with His6 or gluta-thione S-transferase tags and affinity purified (0.05�g was loaded). Detergent lysates were preparedfrom NPE, prostate adenocarcinomas (CA), or var-ious tumor cell lines (Jurkat and RS11-846) ornormal tissues. The lysates were normalized fortotal protein content (100 �g) and analyzed bySDS-PAGE/immunoblotting. Blots were incu-bated with rabbit polyclonal (Polycl.) antibodiesrecognizing human cIAP1 (A and F), XIAP (B andF), survivin (C and F), or cIAP2 (E). A mousemonoclonal antibody (MAB) recognizing cIAP2was also used (D). Antibody detection was accom-plished by an enhanced chemiluminescencemethod. Blots represent the following: A, recom-binant and in vitro translated proteins; B, in vitrotranslated proteins; C, recombinant proteins(Lanes 1–4), tumor cell lines (Lanes 5 and 6), andnormal tissues (Lanes 7–11); D and E, recombi-nant proteins (Lanes 1–3), NPE specimens (Lanes4 and 5), and prostate cancer specimens (Lanes6–9); and F, two NPE specimens (left) and eightprostate cancer specimens (right). Blots in E and Fwere reprobed with antibodies recognizing �-actinor heat shock protein (HSP60), respectively, asloading controls.

4915Clinical Cancer Research



patients who had undergone androgen deprivation by orchiec-tomy and then subsequently died of end-stage metastatic pros-tate cancer.

In addition, prostate carcinoma specimens were obtainedfrom a well-organized cohort of uniformly treated patients pre-senting to the Thomas Jefferson University, Department ofRadiation Oncology (Philadelphia, PA) for whom clinicalfollow-up information was available. Needle biopsy specimensincluded 64 primary tumors derived from these patients, whopresented with stage T2 peripheral zone carcinomas (T2N0M0)treated by external beam radiation. For 48 of these patients,additional biopsies lacking tumor tissue were also available forcomparison. Cancer progression during a median follow-up of66 months was defined as biochemical recurrence [three con-secutive rises in PSA concentration]. Of the 16 of 62 (26%)patients who experienced rising PSAs, 15 patients developedmetastatic disease as documented by bone scans (94%).

Additional normal prostatic tissues for immunohistochem-ical analysis were derived from human biopsy and autopsymaterial (Department of Pathology, University of California,San Diego).

Tissue Preparation. Tissues were fixed in either neutral-buffered formalin, zinc-buffered formalin (Z-fix; Anatech Inc.),or Bouin’s solution (Sigma, St. Louis, MO) and embedded inparaffin. Tissue microarrays were constructed as described pre-viously (17).

Antibodies. Polyclonal antisera for survivin (AR-26) andXIAP (AR-27A) were generated in New Zealand white rabbitsusing recombinant protein immunogens. Survivin (full-lengthprotein) was produced as a glutathione S-transferase-fusion pro-tein and affinity purified essentially as described previously(18). Affinity-purified His6-tagged XIAP (BIR2) recombinantprotein was produced as described previously (19) and usedas an immunogen for producing XIAP-specific antiserum

Fig. 2 Examples of immunohistochemical detection of IAP family proteins in normal and malignant human prostate. Prostate cancer biopsies fromstage T2 patients (A–F, I, and J) and tissue microarrays (G, H, K, and L) were immunostained, applying IAP antisera followed by detection using aHRP-based method with DAB colorimetric substrate (brown). Nuclei were counterstained with hematoxylin (blue). Representative data for cIAP1(A–D), cIAP2 (E–H), XIAP (I and J), and survivin (K and L) are shown. Immunostaining results in regions of invasive cancer are shown for cIAP1(C and D; �150–200), cIAP2 (H; �20), and survivin (L; �20). Examples of malignant and adjacent normal prostatic epithelium are presented forcIAP1 (A, �100), cIAP2 (E–G, �100, �400, and �5, respectively), XIAP (I and J, �100–400), and survivin (K, �20). An example of PINimmunostaining is presented for cIAP1 (B, �250).

4916 Overexpression of IAPs in Prostate Cancer



(AR-27A).4 Polyclonal anti-cIAP1 and anti-cIAP2 antibodieswere obtained from R&D Systems Inc.

The monoclonal antibody to human cIAP2, clone F30-2285, was generated according to routine procedures after fu-sion of hybridoma cell line F0 with spleenocytes from a mouseimmunized with full-length recombinant cIAP2 protein. Clonalselection resulted in the isolation of the three specific clones,F30-2285, F30-2295, and F30-591, which reacted with humancIAP2 recombinant protein and were isotyped as IgG1. Only theF30-2285 clone was used in this study. Generation of thisantibody was performed at BD BioSciences-PharMingen, Inc.(San Diego, CA), and the antibody will be distributed by thecompany.

The monospecificity of all antibodies for their intendedprotein targets was tested by SDS-PAGE/immunoblot analysis,using IAP family proteins in vitro translated from cDNAs orusing recombinant proteins produced in bacteria (3, 15, 20, 21).

Immunohistochemistry. Dewaxed tissue sections wereimmunostained by using a DAB-based detection method asdescribed previously, using the Envision-Plus HRP system(DAKO) and an automated immunostainer (Dako UniversalStaining System; Ref. 22). Polyclonal rabbit antisera specific forsurvivin (AR-26) and XIAP (AR-27A) were applied at 1:10,000and 1:5,000 (v/v), respectively, whereas rabbit antibodiesagainst cIAP1 (R&D Systems Inc.) and cIAP2 (R&D SystemsInc.) were used at 1:600 (v/v). The cIAP2 monoclonal antibodywas used at 0.4 �g/ml. For all polyclonal antisera used, theimmunostaining procedure was performed in parallel using pre-immune serum to verify specificity of the results. Initial confir-mations of antibody specificity also included experiments inwhich antiserum was preabsorbed with 5–10 �g/ml of eithersynthetic peptide immunogen or recombinant protein immuno-gen. The scoring of tumor immunostaining was based on thepercentage of immunopositive cells (0–100) multiplied by stain-ing intensity score (0, 1, 2, or 3), yielding scores of 0–300 (23).

Immunoblotting. Human tissue lysates containing pros-tate cancer (n � 12) with high ratios of cancer cells relative tostroma (�70%), BPH and PIN (n � 5), or normal prostate (n �4) were normalized for total protein content (100 �g/lane) andsubjected to SDS-PAGE/immunoblot analysis, using a 1:2000(v/v) dilution for XIAP (AR-27A), a 1:5000 dilution for survivin

(AR-26), and a 1:1000 dilution for cIAP1 (R&D Systems Inc.)and cIAP2 antisera (R&D Systems Inc.) and using secondaryHRP-conjugated goat antirabbit antibody [1:3000 (v/v) dilution;Bio-Rad]. Alternatively, the mouse anti-cIAP2 monoclonal an-tibody was used at 0.4 �g/ml in conjunction with secondaryHRP-conjugated goat antimouse IgG (Bio-Rad). Tissue lysatescontaining normal, premalignant, or malignant prostatic epithe-lium derived from TRAMP mice (24) were subjected to thesame procedure. Detection was accomplished using an enhancedchemiluminescence (Amersham-based) multiple antigen detec-tion immunoblotting method that allows for multiple reprobingof blots without antibody stripping, as described previously(25).

Animal Model. Transgenic mice (C57BL/6) expressingthe SV40 large T antigen controlled by rat probasin promoterregulatory elements were obtained from Baylor University(Houston, TX) and bred in accordance with institutional guide-lines. Animals were genotyped for the Tag gene by PCR (24,26). Additionally, Tag expression in prostate was confirmed byimmunohistochemistry using monoclonal antibodies to Tag(Pab100 and Pab101; PharMingen Inc.). TRAMP male mice(n � 44) were examined at 12–28 weeks of age. Prostates wereharvested from anesthetized animals after initial perfusion with2% paraformaldehyde and postfixation in Bouin’s solution. Forhistological and immunohistochemical investigation, the speci-mens were embedded in paraffin, and 4-�m sections were cut.

Statistical Analysis. Data were analyzed using the STA-TISTICA software package (StatSoft). A log-rank test was usedfor correlation of immunostaining data with patient survival.Survival distributions were estimated using Kaplan-Meiercurves. Multivariate Cox proportional hazards models werefitted to the data to assess which biomarkers were independentlyassociated with disease-free survival. Ninety-five percent CIsfor the HR were calculated by the formula exp (� 1.960SE(�)), where SE(�) denotes the SE of the estimated regressioncoefficient. Gleason score of �7 and pretreatment PSA of �10ng/ml were defined as “high” for purposes of dichotomizingdata. Statistical significance of differences in IAP levels innormal versus malignant prostatic tissue was assessed using theunpaired t test.

RESULTSCharacterization of IAP Antibodies and Immunoblot

Analysis of Prostate Tissues. To study expression of the IAPfamily proteins cIAP1, cIAP2, XIAP, and survivin, we used

4 Antisera are available from Biocarta, Inc. (www.biocarta.com) andScience Reagents, Inc. (www.sciencereagents.com).

Table 1 IAP immunoexpression data for tissue microarrayThe scoring of tumor immunostaining was based on the percentage of immunopositive cells (0–100) multiplied by staining intensity score (0,

1, 2, or 3), yielding immunoscores of 0–300. For all biomarkers tested, immunoscores in cancers (CA) were compared with those in NPE, using anunpaired t test.

Marker

NPE CA

Pn Mean SE Median n Mean SE Median

cIAP1 54 31 11 0 653 96 3 80 0.0001cIAP2 40 47 15 25 707 152 3 180 0.0001XIAP 17 18 18 0 80 97 7 80 0.0001Survivin 29 3 13 0 351 73 4 40 0.0001




various antibodies that were either generated in our laboratoryor obtained from commercial sources. Immunoblot analysis wasperformed to characterize the specificity of these antibodies,using IAP family proteins produced by in vitro translation fromcDNAs, recombinant IAP family proteins generated in bacteria,and lysates from cell lines and tissues. These included relatedreference controls comprised of various IAP family proteins(XIAP, survivin, cIAP1, cIAP2, NAIP, BRUCE, and Cp-IAP)and unrelated proteins such as Traf-3 and CD40. Moreover,surgical specimens from normal human spleen, brain, prostate,liver, and kidney were compared by immunoblotting with can-cer cell lines and primary prostate cancer specimens. Represent-ative data are presented in Fig. 1.

A commercially available rabbit polyclonal antibody fromR&D Systems, Inc., was determined to be specific for cIAP1,reacting only with cIAP1 but not with cIAP2, XIAP, NAIP, orsurvivin (Fig. 1A). Rabbit polyclonal antibodies generated in ourlaboratory against fragments of recombinant IAP family pro-teins were determined to be specific for XIAP, survivin, andcIAP2, lacking cross-reactivity with other IAP family membersand reacting only with proteins of the proper size in cell lysates(Fig. 1). Note, for example, in Fig. 1C that survivin protein wasdetected in lysates from cancer cell lines but not in lysates fromvarious normal (nontransformed) tissues, consistent with priorreports showing elevations in survivin protein levels in tumors(27); reprobing the same blot with an antibody recognizing�-tubulin confirmed loading of equivalent amounts of totalprotein from all samples (data not shown). In addition, a mouse

monoclonal antibody raised against recombinant cIAP2 as animmunogen was determined to be specific for its intendedprotein, lacking cross-reactivity with cIAP1, Survivin, or XIAP(Fig. 1D). All of these anti-IAP antibodies reacted with themouse homologues of cIAP1 (MIAP-2), cIAP2 (MIAP-1),XIAP (MIAP-3), and survivin (MIAP-4), reflecting the highpercentage of amino acid sequence identity of these proteins inhumans and in mice (84–90% of sequence identity for MIAP-2,MIAP-3, and MIAP-4 and 61% of sequence identity forMIAP-1; data not shown).

Using the specific antibodies, immunoblot analysis wasperformed on another set of tissue lysates derived from normalprostate glands and BPH specimens consisting of histologicallyconfirmed, nontransformed NPE, making comparisons with ly-sates from resected prostate cancers. This immunoblot analysisprovided preliminary evidence of elevated expression of IAPfamily proteins in prostate cancers (Fig. 1, D–F). For example,levels of cIAP2 were higher in four of four cancer specimenstested, compared with NPE specimens (Fig. 1, D and E). Like-wise, in another group of specimens, at least half of the eightcancer samples examined contained higher levels of cIAP1 andXIAP than normal prostate specimens (Fig. 1F). Elevations insurvivin protein were also seen in some of these tumor tissuespecimens (Fig. 1F). Reprobing the blots with antibodies rec-ognizing �-actin or heat shock protein 60 confirmed loading ofapproximately equal amounts of total proteins from all lysatestested (Fig. 1, E and F).

Fig. 3 Comparison of immunoscores for normal prostate and stage T2 prostate cancers. Immunoscores for normal (N) and malignant prostaticepithelium (T) are shown for normal (n � 34–48) and cancer (n � 61–64) specimens obtained by skinny-needle biopsy from stage T2 patients. Basedon comparisons with normal prostatic epithelium, cutoffs were set so that the immunoscores of �95% of normal specimens fell below this value(horizontal lines), representing immunoscores of 100 for cIAP1, cIAP2, and XIAP and 40 for survivin.




IAPs Are Commonly Overexpressed in Human Pros-tate Cancers. To further characterize the expression of IAPsin human prostate cancers, we used previously constructedtissue microarrays (17) containing archival prostate tumors re-flecting the full range of neoplastic prostate disease, includingclinically inapparent (stage T1) tumors and cancers from radicalprostatectomies that were either locally confined (stage T2) orlocally extensive (stage T3) as well as local recurrences afterfailed hormonal therapy and metastatic tumor specimens frompatients obtained at autopsy. Gleason score data were availablefor 45% of these tumors, whereas clinical stage information(T1–T4) according to International Union Against Cancer crite-ria (16) was known for 40% of tumor specimens. In addition toinvasive or metastatic cancer, these microarrays contained 32separate cases of BPH for comparison with cancers. In addition,roughly 8% of tumor specimens on the arrays contained non-

neoplastic prostate epithelium adjacent to tumor tissue, permit-ting additional comparisons of nontransformed epithelium withinvasive cancer.

Microarrays were immunostained using the aforemen-tioned antibodies specific for cIAP1, cIAP2, XIAP, or survivin(15). The specificity of these immunostaining results was con-firmed by control stainings performed using either preimmuneserum or immune antisera that had been preabsorbed with therelevant immunogens (data not shown). Immunostaining resultswere quantified according to the approximate percentage ofimmunopositive cells (0–100%) and immunointensity on a 0–3scale, and then an immunoscore was calculated from the productof the percentage immunopositivity and immunointensity (0–300; Ref. 23).

Fig. 4 Correlations of biomarker immunostaining data with RFS for stage T2 prostate cancer patients. The log-rank test was used for correlation ofimmunoscore data with patient survival. Kaplan-Meier curves illustrate correlations of the investigated biomarkers with RFS for this cohort of patients.RFS curves for patients expressing high levels of markers are denoted in black symbols. White symbols indicate patients whose tumors contain lowerprotein levels, using the median immunoscore for dichotomizing data. Median immunoscores were 160 for cIAP1, 225 for cIAP2, 60 for XIAP, and100 for survivin.

Table 2 Comparison of IAP expression data with RFS in stage T2

prostate cancer patientsIAP immunostaining data were dichotomized using the median

immunoscore for segregating patients into high versus low expressioncategories (cutpoint indicated). Data represent the proportion of patients(percentage in parentheses) with low versus high expression remainingrelapse free after median follow-up of 66 months. Significance (P) wascalculated based on a log-rank test.

BiomarkerMedianscore

Proportion (%)

PLow High

cIAP1 160 19/24 (80%) 25/37 (68%) 0.06cIAP2 225 24/29 (83%) 18/30 (60%) 0.06XIAP 60 23/38 (61%) 22/24 (92%) 0.0001Survivin 100 25/32 (78%) 21/30 (70%) 0.9

Table 3 Summary of histopathology for prostate tissue fromTRAMP mice

Summary of H&E histology. The prostate glands of a total of 44TRAMP male mice and 10 age-matched control mice were investigatedby H&E histology. The numbers of animals developing PIN or invasivecancer are indicated at various ages. PIN and cancer lesions weremultifocal in nearly all animals in which transformation of prostateepithelium was found.

Age(weeks)

TRAMP Controls

Animals/group PIN Cancer

Animals/group

PIN(cases/foci)

Cancer(cases/foci)

12–16 6 2 0 4 0 018–24 8 4 1 3 0 028 30 22 12 3 1 0

Total 44 28 13 10 1 0




Fig. 5 Examples of immunohistochemistry data for IAPs in the TRAMP model. To characterize the expression of cIAP1, cIAP2, XIAP, and survivinin TRAMP mice, paraffin sections from male urogenital system were immunostained. Immunodetection of specific reactions was accomplished bya DAB-based colorimetric method (brown), and the sections were either not counterstained or counterstained with methyl green or hematoxylin (blue).Expression of cIAP1/MIAP-2 protein (A, B, and D) is demonstrated in the transformed prostatic epithelium. PIN lesions are indicated by arrows inA and B (�10 and �80, respectively). Note elevated immunostaining in invasive adenocarcinoma (D, �200). Elevated cIAP2/MIAP-1 levels areshown during progression of malignant transformation of the prostate gland in E–I [E (�200), normal prostatic epithelium (arrow) and PIN (brown);G (�400), PIN; H (�150), invasive cancer]. Survivin/MIAP-4 expression data are shown in J–L. Survivin immunostaining of prostate tissue ofTRAMP mice reveals marked increase in immunointensity in PIN compared with nontransformed prostatic epithelium (J and K; arrow). Immuno-




Immunohistochemical analysis of tumor tissues on the mi-croarrays revealed cancer-specific elevations in the expressionof the four IAP family proteins tested. Fig. 2 shows represent-ative examples of the immunostaining results for tumor speci-mens, demonstrating higher intensity immunostaining in inva-sive cancer compared with NPE for all IAP proteinsinvestigated. Calculated immunoscores were also higher for allfour IAPs, comparing cancer with NPE (Table 1).

Using the tissue microarray data, we compared IAP immu-noscores with a variety of variables, including Gleason grade,clinical stage (T1–T4), and hormone-refractory disease. IAPfamily immunoscore data did not correlate with Gleason grade.However, higher cIAP2 immunoscores correlated with higher Tstage, suggesting that higher levels of this IAP family proteintend to occur in larger or more invasive tumors. For example,cIAP2 immunoscores were 83 5 (mean SE) for T1–T2 (n �221) tumors compared with 168 18 for T3–T4 tumors (n �18; P 0.0001). Survivin immunoscores also tended to behigher in more invasive tumors, with immunoscores of 65 6for T1–T2 tumors (n � 152) versus 97 16 for T3–T4 tumors(n � 19), but this difference did not reach statistical significance(P � 0.06). Comparisons of untreated tumors with hormone-refractory specimens on the microarray revealed significantcorrelations of refractory disease with higher cIAP2 [139 4(n � 563) versus 202 8 (n � 144)] and with lower cIAP1[104 4 (n � 516) versus 67 7 (n � 137)] and lowersurvivin [77 4 (n � 319) versus 34 13 (n � 32)]. Furtherevidence of a correlation of higher cIAP2 expression withaggressive disease was found by comparing the immunoscoresof 79 metastases collected at the autopsies from 62 patients, whohad undergone androgen deprivation by orchiectomy and hadsubsequently died of end-stage metastatic prostate cancer, re-vealing higher levels of cIAP2 protein (mean immunoscore,215 10) compared with primary tumors (mean immunoscore,142 4; n � 591; P 0.0001).

Analysis of IAP Expression in a Cohort of UniformlyTreated Patients with Early-Stage Prostate Cancer. Tomore precisely contrast the levels of IAP family protein expres-sion in tumor versus normal prostate tissue, we analyzed skinny-needle biopsies from a small cohort of men (n � 64) withearly-stage disease (T2N0M0) who were uniformly treated withexternal beam irradiation. During needle biopsy before radio-therapy, several cores of tissue were obtained from each patient,providing case-matched tissue samples containing only normalprostatic epithelium for 48 of the 64 tumor biopsies available.

Using the normal and tumor specimens derived from theseearly-stage patients, we evaluated the expression of the cIAP1,cIAP2, XIAP, and survivin proteins by immunohistochemistryand scored the results as described above (23). Whereas immu-

nostaining results varied widely among specimens examined,the overall immunoscores for the cancers displayed clear eleva-tions in immunoreactivity when compared with histologicallynormal specimens (Fig. 3; Table 1). For example, whereas 98%of normal prostate specimens had cIAP1 immunoscores of100, 50 of 61 (82%) invasive cancer specimens had immu-noscores of �100 (P 0.0001), thus suggesting that manyprostate cancers develop pathological elevations in the levels ofthis antiapoptotic protein. Similarly, levels of cIAP2 protein in60 of 61 (99%) of cancers exceeded cIAP2 immunoscoresrepresentative of 98% normal prostate specimens (H-score �100; P 0.0001). Likewise, XIAP immunoscores were �100for nonmalignant epithelium, in contrast to invasive cancers,where 15 of 64 (23%) had immunoscores of �100 (P 0.0001). Finally, all normal prostatic epithelium samples pos-sessed immunoscores of �40 for survivin, whereas 44 of 62invasive cancers (71%) had survivin immunoscores of �40(P 0.0001).

We also looked at the data an alternative way, where onlycase-matched samples of normal and tumor tissue from the samepatient were evaluated (n � 48), thus excluding the 14 tumorspecimens for which normal tissue was unavailable. In pairwiseanalyses, expression of IAP family proteins as defined by im-munoscore was greater in tumor compared with matching nor-mal prostate tissue for cIAP1 in 35 of 36 (97%) cases, cIAP2 in32 of 32 (100%) cases, XIAP in 26 of 40 (65%) cases, andsurvivin in 36 of 44 (82%) cases (all Ps 0.001). Thus, for allIAP family members examined, evidence of tumor-associatedup-regulation of expression was observed by comparisons ofnormal versus tumor tissues from these early-stage prostatecancer patients. These patient materials thus provide indepen-dent confirmation of the impressions derived from archivaltissue microarrays that multiple IAP family proteins are oftensimultaneously overexpressed in prostate cancers.

In addition to frequent overexpression of IAPs in invasivecancers, we also observed increases in immunostaining forcIAP1, cIAP2, XIAP, and survivin in many precancerous PINlesions. Based on comparisons with NPE, levels of cIAP1protein in 12 of 21 (57%) PIN lesions exceeded cIAP1 immu-noscores representative of normal prostate epithelium (H-score � 100; P 0.0001), suggesting that elevations of cIAP1protein occur early in the process of malignant transformation.Similarly, 9 of 22 (41%) PIN specimens had elevated cIAP2immunoscores compared with NPE (P 0.0001). Also,whereas XIAP immunoscores were �100 for all NPE speci-mens, 8 of 26 (31%) PIN lesions showed increased levels ofXIAP protein (P 0.0001). Similarly, compared with NPE,45% of PIN specimens (10 of 22) had elevated survivin immu-noscores (P 0.0001).

staining of invasive cancer with anti-survivin antibody (L) or with preimmune serum (M) is also shown, revealing intense survivin immunoreactivityin nuclei and cytosol of tumor cells and confirming specificity of the immunostaining, respectively. N–Q show XIAP/MIAP-3 staining results.Elevated XIAP levels were observed in PIN (N, �80; P, �200) and invasive cancer (Q, �250), whereas normal prostatic epithelium contained barelydetectable XIAP/MIAP-3 immunostaining (N, �80; P, �200; arrows). The specificity of the immunostaining results was confirmed by controlstainings performed using either preimmune serum or immune antisera that had been preabsorbed with the relevant immunogens [C (�80),preabsorbed MIAP-2; F (�200), preabsorbed MIAP-1; I (�150), preimmune serum; M (�250), preabsorbed survivin/MIAP-4; O (�80), preabsorbedXIAP/MIAP-3].




Although representing a relative small cohort, attemptswere made to correlate expression of IAP family proteins withclinical outcome using the results obtained for the 64 early-stagepatients. To dichotomize data into higher versus lower expres-sion categories, the median immunoscore result for each IAPfamily protein tested was used to split the data set, thus com-paring RFS (PSA relapse) for the patients with immnoscoresabove versus below the median value. Kaplan-Meier curves andlog-rank tests demonstrated a trend of patients with highercIAP1 and higher cIAP2 to relapse with greater frequencyduring the follow-up period (median follow-up, 5.5 years), butthe results did not reach statistical significance (P � 0.06 forcIAP1 and cIAP2; Fig. 4; Table 2). In contrast, higher levels ofXIAP were unexpectedly associated with longer RFS (P �0.0001). Survivin immunostaining data were not correlated withRFS (Fig. 4).

Immunostaining data did not correlate with Gleason scoresor PSA nadir for all IAPs tested (data not shown). However, lowlevels of XIAP protein were associated with higher pretreatmentPSA (mean, 26.4 ng/ml; Hybritech assay) compared with tu-mors containing higher XIAP (mean PSA, 11.7 ng/ml; P �0.009), which may be related to the unexpected association ofhigher XIAP with longer RFS. Gleason grade also was signif-icantly correlated with RFS (P � 0.01) in this patient cohort.When stepwise multivariate analyses were conducted using aCox proportional hazards regression model (variables includedcIAP1, cIAP2, XIAP, survivin, Gleason score, and pretreatmentPSA), the factors that remained predictive of relapse in thiscohort were high cIAP1 immunoscore (HR, 3.8; 95% CI, 1.16–12.53; P � 0.03), lower XIAP (HR, 0.06; 95% CI, 0.01–0.31;P � 0.0007), and high Gleason score [HR, 2.8; 95% CI, 1.1–7.25; P � 0.03 (see supplemental data)].

Analysis of IAPs in a Mouse Transgenic Model of Pros-tate Cancer. The SV40 T-antigen TRAMP closely resemblesthe progression of human prostate cancer (28).

Histological analysis of prostate tissue from TRAMP trans-genic mice revealed age-dependent appearance of PIN and in-vasive cancer, with 73% of male mice developing multifocalPIN (22 of 30, 73%), and approximately half of mice developinginvasive cancer [also often multifocal (12 of 30, 40%) by 28weeks of age (Table 3). In contrast, only 1 of 10 controllittermates developed PIN, and no prostate cancers were de-tected in nontransgenic mice.

We then explored the expression of IAP family proteins in

the prostates of male transgenic and aged-matched littermatecontrol mice, using immunohistochemistry and immunoblottingmethods (Fig. 5, A–Q; see supplemental data). Although barelyexpressed in the normal prostatic epithelium, high levels ofMIAP-1 and MIAP-2 immunostaining (assessed using antibod-ies raised against human cIAP2 and cIAP1), respectively, werefound in transformed prostatic epithelium of SV40 large T-antigen transgenic mice (Fig. 5, A, B, and E–G; summarized inTables 3 and 4). In some animals, overexpression of these IAPfamily proteins was evident in carcinoma in situ (PIN), inaddition to invasive adenocarcinomas (Fig. 5, B, D, G, and H;Table 4). Similarly, survivin/MIAP-4 immunostaining was notdetected in normal prostate epithelium but was found in PIN andin many invasive cancers (Fig. 5, J–L). Interestingly, whereasmost normal cells were immunonegative for survivin, positiveimmunostaining was found in occasional nontransformed pros-tatic epithelial cells of both TRAMP and normal mice, associ-ated with chromosomes or mitotic structures in cells apparentlyundergoing division. In contrast, intense survivin immunoreac-tivity was found in interphase nuclei and in the cytosol of mosttransformed cells within PIN lesion and invasive cancers inTRAMP mice. Heterogeneity in the percentage of transformedcells expressing survivin, however, resulted in some cases scor-ing as negative, where at least 50% immunopositivity was set asthe threshold (see legend of Table 4 for details). The intensity ofXIAP/MIAP-3 immunostaining also increased during malignanttransformation (Fig. 5, N–Q), although more aggressive tumorsthat had infiltrated seminal vesicles rarely contained high levelsof this protein. The specificity of these immunostaining resultswas confirmed by control stainings performed using either pre-immune serum or immune antisera that had been preabsorbedwith the relevant immunogens (Fig. 5, C, F, I, M, and O).

Immunoblot analysis at least partially corroborated thesefindings. Expression of M IAP-1 (orthologue of human cIAP2),MIAP-2 (orthologue of human cIAP1), and XIAP/MIAP-3 wasdetected in every tumor specimen evaluated (n � 8; data notshown). Elevated levels of MIAP-2 protein (compared withnormal prostate tissue) were readily documented by immuno-blotting for all tumor specimens evaluated. Cancer-associatedincreases in MIAP-1, XIAP/MIAP-3, and survivin/MIAP-4were more heterogeneous, as measured by comparisons of nor-mal and tumor tissue by immunoblotting, possibly because ofadmixture of normal and malignant cells. Nevertheless, at leasthalf the tumor specimens analyzed contained elevated levels of

Table 4 Summary of IAP expression data for prostate tissue from TRAMP miceSummary of immunohistochemical results of IAP family protein expression in prostate tissue of TRAMP mice. Prostate tissue from male

TRAMP mice and age-matched littermate control animals was analyzed by immunohistochemistry using antibodies specific for cIAP1/MIAP-2,cIAP2/MIAP-1, XIAP/MIAP-3, or survivin/MIAP-4. Positive immunostaining was defined as intensity equal or stronger than that observed in NPE,which was present in more than 50% of cells. A total of 164 transformed foci were found in TRAMP mice compared with only 3 in age-matchedcontrol mice. The proportion of foci of transformed tissue exhibiting immunopositivity is indicated in the table for both PIN and invasive cancer (CA).

Marker

TRAMP transgenics Controls

PIN POS/total foci CA POS/total foci PIN POS/total foci CA POS/total foci

cIAP1/MIAP-2 148/164 (90.3%) 27/30 (90%) 1/3 (33%) 0cIAP2/MIAP-1 164/164 (100%) 25/30 (83%) 2/3 (67%) 0XIAP/MIAP-3 135/164 (82.3%) 22/30 (73%) 3/3 (100%) 0Survivin/MIAP-4 158/164 (96.3%) 14/30 (47%) 1/3 (33%) 0




MIAP-1, XIAP/MIAP-3, and survivin/MIAP-4, as determinedby immunoblotting. Thus, we conclude that tumor-associatedoverexpression of several IAP family proteins occurs duringprostate cancer development in TRAMP mice.

DISCUSSIONIn the normal prostate, tissue homeostasis is achieved by

matching the 1–2% daily rate of cell proliferation with an equalrate of cell death (29). Deregulation of apoptosis contributes totumor initiation, progression to the androgen-insensitive state,and metastasis. IAP family proteins represent critical regulatorsof apoptosis that serve as endogenous inhibitors of caspasefamily cell death proteases (3). Our immunohistochemical anal-ysis demonstrates for the first time that alterations in the ex-pression of several IAP family proteins (cIAP1, cIAP2, XIAP,and survivin) occur commonly and often simultaneously inprostate cancers.

Previously, survivin was reported to be aberrantly overex-pressed in most cancers, including prostate, lung, colon, breast,pancreatic, and gastric cancer and others (reviewed in Ref. 11).However, little is known about the expression of other membersof the IAP family in cancers. Expression of cIAP1, cIAP2,XIAP, survivin, and NAIP has been examined in the NationalCancer Institute collection of 60 human tumor cell lines, reveal-ing widespread expression of cIAP1, XIAP, and survivin inthese tumor lines of diverse tissue origins and demonstratingcIAP2 expression primarily in lymphoid malignancies (15).Importantly, these studies also demonstrated poor correlation ofcIAP1, cIAP2, and XIAP protein levels with mRNA levels,consistent with emerging knowledge that expression of manyIAPs is regulated predominantly at the level of protein stability(15). Higher levels of XIAP protein have been correlated withshorter remission duration after chemotherapy and shorter sur-vival in patients with acute myelogenous leukemia (15). TheIAP family member ILP-2 (livin, ML-IAP, KIAP) reportedly isoverexpressed in melanoma (14).

Thus, when combined with these prior reports, our dataprovide additional evidence that pathological elevations in theexpression of antiapoptotic IAP family proteins represent acommon event in many types of cancer. Not only were eleva-tions in IAP family proteins seen in archival human prostatecancer specimens, but we also observed increases in the levelsof cIAP1/MIAP-2, cIAP2/MIAP-1, XIAP/MIAP-3, and sur-vivin/MIAP-4 during disease progression in a transgenic mousemodel of prostate cancer. Up-regulation of IAP expression con-comitant with the emergence of PIN implicates a role of theseproteins in the early stages of the pathogenesis of prostatecancer and emphasizes the utility of TRAMP for identifyingmolecular changes in early disease. This finding suggests thatoverexpression of IAP family proteins is a general concomitantof transformation of prostate epithelium and reinforces the ideathat multiple IAP family members become dysregulated in theirexpression during the pathogenesis of malignancy in the pros-tate gland. It remains to be seen whether the simultaneousoverexpression of several IAP family proteins reflects a com-monality in the mechanisms controlling the levels of IAP familyproteins in cells versus the possibility that multiple independent

signaling pathways that control individual IAP family membersbecome simultaneously deregulated in these neoplasms.

Based on the data provided here, we cannot concludewhether differences in the expression of one or more IAP familyproteins (individually or in combination) are of prognostic sig-nificance for men with prostate cancer. In the tissue microarraydata set, higher cIAP2 levels were associated with larger tumorsize (T stage), hormone-refractory disease, and metastatic dis-ease, but these patients were heterogenous with respect to Glea-son grade, pretreatment PSA, and therapy. In the small cohort ofearly-stage patients treated uniformly with external beam radi-ation, higher cIAP2 correlated in multivariate analysis withshorter RFS. In contrast, higher XIAP was unexpectedly asso-ciated with longer RFS in this small cohort, based on bothunivariate and multivariate analyses. The association of highercIAP1 and cIAP2 levels with more aggressive disease is con-sistent with the documented function of these antiapoptoticproteins as suppressors of caspases (20). As for XIAP, whereasthis protein is a potent caspase inhibitor and suppressor ofapoptosis (3), XIAP has also recently been reported to inhibitcell proliferation by down-regulating levels of cyclins A and D1and inducing expression of the cyclin-dependent kinase inhibi-tors p21/Waf1 and p27/Kip1 (30). Thus, XIAP may provide aselective survival advantage while simultaneously impairingdivision of cancer cells. Other explanations for the paradoxicalassociation of XIAP with the shorter RFS could be related toexpression in the same tumors of endogenous antagonist pro-teins (which were not measured here), such as SMAC, XAF1,and the serine protease Omi/HtrA2, which can negate apoptosissuppression by XIAP (31–35).

The functional importance of IAPs in cancer is beginningto be clarified. Involvement of IAP family proteins in tumorresistance to chemotherapeutic drugs and other apoptotic agentshas been demonstrated by use of antisense techniques, helpingto validate certain IAPs as potential drug targets for cancer(36–42). Also, heptameric peptides from the NH2 terminus ofthe IAP antagonist protein SMAC have also been applied toreverse caspase inhibition by IAPs in vitro (43–49) and toovercome IAP-mediated suppression of apoptosis in leukemiacell lines (45), suggesting a strategy for cancer therapy. Further-more, a recent observation that cytochrome c microinjectionfails to induce apoptosis in prostate cancer LNCaP cells withoutSMAC also implies a crucial role for proteins of the IAPs in thisorgan (48).

Given emerging data suggesting an important role forIAP family proteins in sustaining tumor cell survival andsuppressing apoptosis induced by anticancer drugs (38 – 42,44 –50), our results demonstrating that overexpression ofseveral IAP family proteins occurs frequently in prostatecancers in humans and transgenic mice provide further vali-dation of IAPs as potential drug discovery targets for theimproved treatment of prostate cancer. The observation thatmore than one IAP family member is often overexpressedsimultaneously in prostate cancers, however, raises the pos-sibility that effective strategies will require IAP antagoniststhat are capable of inhibiting multiple members of this familyof apoptosis-suppressing proteins.




ACKNOWLEDGMENTSWe thank Judie Valois for manuscript preparation.

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2003;9:4914-4925. Clin Cancer Res Maryla Krajewska, Stan Krajewski, Steven Banares, et al. Prostate CancerElevated Expression of Inhibitor of Apoptosis Proteins in

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