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Molecular Oncology, Markers, Clinical Correlates |
The Burnham Institute, La Jolla, California 92037 [M. K., S. K., S. B., X. H., J. C. R.]; Department of Radiation Oncology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 [B. T.]; Institute of Pathology, University of Basel, Switzerland [L. B.]; Laboratory of Cancer Genetics, National Human Genome Research Institute, NIH, Bethesda, Maryland 20892-4470 [O-P. K.]; Department of Pathology, University of California, San Diego, San Diego, California 92103 [A. S.]; The R. W. Johnson Pharmaceutical Research Institute, San Diego, California [A. V.]; Department of Urology, Stanford University, Stanford, California 94305 [D. P.]; and BD Biosciences Pharmingen, San Diego, California 92121 [G-J. G.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: The expression of four members of the IAP family (cellular inhibitor of apoptosis protein 1, cellular inhibitor of apoptosis protein 2, X chromosome-linked IAP, and survivin) was examined by immunohistochemistry and immunoblotting in human prostate cancers and in prostate tissues from transgenic mice expressing SV40 large T antigen under control of a probasin promoter.
Results: Tumor-associated elevations in the levels of all four IAP family members were common in prostate cancers of both humans and mice, suggesting concomitant up-regulation of multiple IAP family proteins. Compared with normal prostatic epithelium, increased IAP expression was often evident even in prostatic intraepithelial neoplasia lesions (carcinoma in situ), suggesting that deregulation of IAP expression occurs early in the pathogenesis of prostate cancer. IAP expression did not correlate with Gleason grade or prostate-specific antigen levels.
Conclusions: The findings demonstrate that tumor- associated elevations in the expression of several IAP family proteins occur as a frequent and early event in the etiology of prostate cancer.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Apoptosis is executed by a family of cysteine proteases known as caspases. Caspases are produced in cells as inactive zymogens and generally must undergo proteolytic processing to become active proteases (reviewed in Ref. 2
). The IAPs3
are the only known endogenous caspase inhibitors (3)
. They contain Baculovirus IAP repeat domains, and some of them bind and potently inhibit activated caspases, including, in mammals, the effector caspases-3 and -7 and the initiator caspase-9 (reviewed in Ref. 4
). In addition to Baculovirus IAP repeat domains, several IAPs also contain a RING domain, which binds ubiquitin-conjugating enzymes that promote degradation of IAP caspase complexes (5)
. Eight human IAPs have been recognized, 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 to the Rel/nuclear factor 
B pathway and mitogen-activated protein kinase signal transduction (7
, 8)
. In particular, cIAP1 and cIAP2 have been shown to activate nuclear factor B (9)
. XIAP, NAIP, and ML-IAP have been reported to modulate apoptosis pathways via the TAK1/c-Jun-NH2-terminal kinase signaling cascade (10)
.
Some IAP family proteins are overproduced in cancers, suggesting that IAP-mediated suppression of apoptosis may contribute to tumor pathogenesis, progression, and resistance to drug treatment. For example, survivin is expressed abundantly in fetal tissues but scarcely present in most adult tissues. High levels of survivin protein have been reported in many types of human cancers, suggesting that reactivation of expression of this gene represents a common event in tumorigenesis (reviewed in Refs. 11 and 12 ). Indeed, genome-wide transcription profiling suggests that survivin is among the most tumor-specific genes thus far identifiable (13) . Similarly, the ML-IAP protein is not expressed at detectable levels in most normal adult tissues but is present in melanomas (14) and perhaps some other types of cancers. Likewise, whereas XIAP is broadly expressed in normal tissues, higher levels of this IAP family member have been demonstrated in patients with acute myelogenous leukemia (15) . Thus, elevations in the levels of certain IAP family proteins may occur in tumors, conferring a selective survival advantage.
In this report, we analyzed the expression of several IAP family proteins, including survivin, XIAP, cIAP1, and cIAP2, in prostate cancers. Our data demonstrate that tumor-associated increases in the expression of several IAP family proteins occur commonly in prostate cancer and probably as an early event. Moreover, overexpression of these IAP family members was also documented in TRAMP, the model further suggesting an important role for deregulation of IAP expression in the pathogenesis of prostate cancer. IAP family proteins thus may be candidate drug discovery targets for restoration of apoptosis sensitivity in prostate cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition, prostate carcinoma specimens were obtained from a well-organized cohort of uniformly treated patients presenting to the Thomas Jefferson University, Department of Radiation Oncology (Philadelphia, PA) for whom clinical follow-up information was available. Needle biopsy specimens included 64 primary tumors derived from these patients, who presented 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 for comparison. Cancer progression during a median follow-up of 66 months was defined as biochemical recurrence [three consecutive rises in PSA concentration]. Of the 16 of 62 (26%) patients who experienced rising PSAs, 15 patients developed metastatic disease as documented by bone scans (94%).
Additional normal prostatic tissues for immunohistochemical analysis were derived from human biopsy and autopsy material (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 in paraffin. Tissue microarrays were constructed as described previously (17)
.
Antibodies.
Polyclonal antisera for survivin (AR-26) and XIAP (AR-27A) were generated in New Zealand white rabbits using recombinant protein immunogens. Survivin (full-length protein) was produced as a glutathione S-transferase-fusion protein and affinity purified essentially as described previously (18)
. Affinity-purified His6-tagged XIAP (BIR2) recombinant protein was produced as described previously (19)
and used as an immunogen for producing XIAP-specific antiserum (AR-27A).4
Polyclonal anti-cIAP1 and anti-cIAP2 antibodies were obtained from R&D Systems Inc.
The monoclonal antibody to human cIAP2, clone F30-2285, was generated according to routine procedures after fusion of hybridoma cell line F0 with spleenocytes from a mouse immunized with full-length recombinant cIAP2 protein. Clonal selection resulted in the isolation of the three specific clones, F30-2285, F30-2295, and F30-591, which reacted with human cIAP2 recombinant protein and were isotyped as IgG1. Only the F30-2285 clone was used in this study. Generation of this antibody was performed at BD BioSciences-PharMingen, Inc. (San Diego, CA), and the antibody will be distributed by the company.
The monospecificity of all antibodies for their intended protein targets was tested by SDS-PAGE/immunoblot analysis, using IAP family proteins in vitro translated from cDNAs or using recombinant proteins produced in bacteria (3 , 15 , 20 , 21) .
Immunohistochemistry.
Dewaxed tissue sections were immunostained by using a DAB-based detection method as described previously, using the Envision-Plus HRP system (DAKO) and an automated immunostainer (Dako Universal Staining System; Ref. 22
). Polyclonal rabbit antisera specific for survivin (AR-26) and XIAP (AR-27A) were applied at 1:10,000 and 1:5,000 (v/v), respectively, whereas rabbit antibodies against cIAP1 (R&D Systems Inc.) and cIAP2 (R&D Systems Inc.) were used at 1:600 (v/v). The cIAP2 monoclonal antibody was used at 0.4 µg/ml. For all polyclonal antisera used, the immunostaining procedure was performed in parallel using preimmune serum to verify specificity of the results. Initial confirmations of antibody specificity also included experiments in which antiserum was preabsorbed with 5–10 µg/ml of either synthetic peptide immunogen or recombinant protein immunogen. The 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 scores of 0–300 (23)
.
Immunoblotting.
Human tissue lysates containing prostate cancer (n = 12) with high ratios of cancer cells relative to stroma (>70%), BPH and PIN (n = 5), or normal prostate (n = 4) were normalized for total protein content (100 µg/lane) and subjected 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 secondary HRP-conjugated goat antirabbit antibody [1:3000 (v/v) dilution; Bio-Rad]. Alternatively, the mouse anti-cIAP2 monoclonal antibody was used at 0.4 µg/ml in conjunction with secondary HRP-conjugated goat antimouse IgG (Bio-Rad). Tissue lysates containing normal, premalignant, or malignant prostatic epithelium derived from TRAMP mice (24)
were subjected to the same procedure. Detection was accomplished using an enhanced chemiluminescence (Amersham-based) multiple antigen detection immunoblotting method that allows for multiple reprobing of blots without antibody stripping, as described previously (25)
.
Animal Model.
Transgenic mice (C57BL/6) expressing the SV40 large T antigen controlled by rat probasin promoter regulatory elements were obtained from Baylor University (Houston, TX) and bred in accordance with institutional guidelines. Animals were genotyped for the Tag gene by PCR (24
, 26)
. Additionally, Tag expression in prostate was confirmed by immunohistochemistry using monoclonal antibodies to Tag (Pab100 and Pab101; PharMingen Inc.). TRAMP male mice (n = 44) were examined at 12–28 weeks of age. Prostates were harvested from anesthetized animals after initial perfusion with 2% paraformaldehyde and postfixation in Bouin’s solution. For histological and immunohistochemical investigation, the specimens were embedded in paraffin, and 4-µm sections were cut.
Statistical Analysis.
Data were analyzed using the STATISTICA software package (StatSoft). A log-rank test was used for correlation of immunostaining data with patient survival. Survival distributions were estimated using Kaplan-Meier curves. Multivariate Cox proportional hazards models were fitted to the data to assess which biomarkers were independently associated with disease-free survival. Ninety-five percent CIs for the HR were calculated by the formula exp (ß ± 1.960 SE(ß)), where SE(ß) denotes the SE of the estimated regression coefficient. Gleason score of >7 and pretreatment PSA of >10 ng/ml were defined as "high" for purposes of dichotomizing data. Statistical significance of differences in IAP levels in normal versus malignant prostatic tissue was assessed using the unpaired t test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Rabbit polyclonal antibodies generated in our laboratory against fragments of recombinant IAP family proteins were determined to be specific for XIAP, survivin, and cIAP2, lacking cross-reactivity with other IAP family members and reacting only with proteins of the proper size in cell lysates (Fig. 1)
. Note, for example, in Fig. 1C
that survivin protein was detected in lysates from cancer cell lines but not in lysates from various normal (nontransformed) tissues, consistent with prior reports showing elevations in survivin protein levels in tumors (27)
; reprobing the same blot with an antibody recognizing ß-tubulin confirmed loading of equivalent amounts of total protein from all samples (data not shown). In addition, a mouse monoclonal antibody raised against recombinant cIAP2 as an immunogen was determined to be specific for its intended protein, lacking cross-reactivity with cIAP1, Survivin, or XIAP (Fig. 1D)
. All of these anti-IAP antibodies reacted with the mouse homologues of cIAP1 (MIAP-2), cIAP2 (MIAP-1), XIAP (MIAP-3), and survivin (MIAP-4), reflecting the high percentage of amino acid sequence identity of these proteins in humans and in mice (84–90% of sequence identity for MIAP-2, MIAP-3, and MIAP-4 and 61% of sequence identity for MIAP-1; data not shown).
Using the specific antibodies, immunoblot analysis was performed on another set of tissue lysates derived from normal prostate glands and BPH specimens consisting of histologically confirmed, nontransformed NPE, making comparisons with lysates from resected prostate cancers. This immunoblot analysis provided preliminary evidence of elevated expression of IAP family proteins in prostate cancers (Fig. 1, D–F)
. For example, levels of cIAP2 were higher in four of four cancer specimens tested, compared with NPE specimens (Fig. 1, D and E)
. Likewise, in another group of specimens, at least half of the eight cancer samples examined contained higher levels of cIAP1 and XIAP than normal prostate specimens (Fig. 1F)
. Elevations in survivin protein were also seen in some of these tumor tissue specimens (Fig. 1F)
. Reprobing the blots with antibodies recognizing ß-actin or heat shock protein 60 confirmed loading of approximately equal amounts of total proteins from all lysates tested (Fig. 1, E and F)
.
IAPs Are Commonly Overexpressed in Human Prostate Cancers.
To further characterize the expression of IAPs in human prostate cancers, we used previously constructed tissue microarrays (17)
containing archival prostate tumors reflecting the full range of neoplastic prostate disease, including clinically inapparent (stage T1) tumors and cancers from radical prostatectomies that were either locally confined (stage T2) or locally extensive (stage T3) as well as local recurrences after failed hormonal therapy and metastatic tumor specimens from patients obtained at autopsy. Gleason score data were available for 45% of these tumors, whereas clinical stage information (T1–T4) according to International Union Against Cancer criteria (16)
was known for 40% of tumor specimens. In addition to invasive or metastatic cancer, these microarrays contained 32 separate cases of BPH for comparison with cancers. In addition, roughly 8% of tumor specimens on the arrays contained nonneoplastic prostate epithelium adjacent to tumor tissue, permitting additional comparisons of nontransformed epithelium with invasive cancer.
Microarrays were immunostained using the aforementioned antibodies specific for cIAP1, cIAP2, XIAP, or survivin (15) . The specificity of these immunostaining results was confirmed by control stainings performed using either preimmune serum or immune antisera that had been preabsorbed with the relevant immunogens (data not shown). Immunostaining results were quantified according to the approximate percentage of immunopositive cells (0–100%) and immunointensity on a 0–3 scale, and then an immunoscore was calculated from the product of the percentage immunopositivity and immunointensity (0–300; Ref. 23 ).
Immunohistochemical analysis of tumor tissues on the microarrays revealed cancer-specific elevations in the expression of the four IAP family proteins tested. Fig. 2
shows representative examples of the immunostaining results for tumor specimens, demonstrating higher intensity immunostaining in invasive cancer compared with NPE for all IAP proteins investigated. Calculated immunoscores were also higher for all four IAPs, comparing cancer with NPE (Table 1)
.
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Analysis of IAP Expression in a Cohort of Uniformly Treated Patients with Early-Stage Prostate Cancer.
To more precisely contrast the levels of IAP family protein expression in tumor versus normal prostate tissue, we analyzed skinny-needle biopsies from a small cohort of men (n = 64) with early-stage disease (T2N0M0) who were uniformly treated with external beam irradiation. During needle biopsy before radiotherapy, several cores of tissue were obtained from each patient, providing case-matched tissue samples containing only normal prostatic epithelium for 48 of the 64 tumor biopsies available.
Using the normal and tumor specimens derived from these early-stage patients, we evaluated the expression of the cIAP1, cIAP2, XIAP, and survivin proteins by immunohistochemistry and scored the results as described above (23)
. Whereas immunostaining results varied widely among specimens examined, the overall immunoscores for the cancers displayed clear elevations in immunoreactivity when compared with histologically normal specimens (Fig. 3
; Table 1
). For example, whereas 98% of normal prostate specimens had cIAP1 immunoscores of <100, 50 of 61 (82%) invasive cancer specimens had immunoscores of 
100 (P < 0.0001), thus suggesting that many prostate cancers develop pathological elevations in the levels of this antiapoptotic protein. Similarly, levels of cIAP2 protein in 60 of 61 (99%) of cancers exceeded cIAP2 immunoscores representative of 98% normal prostate specimens (H-score 100; P < 0.0001). Likewise, XIAP immunoscores were 
100 for 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 possessed immunoscores of 40 for survivin, whereas 44 of 62 invasive cancers (71%) had survivin immunoscores of >40 (P < 0.0001).
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In addition to frequent overexpression of IAPs in invasive cancers, we also observed increases in immunostaining for cIAP1, cIAP2, XIAP, and survivin in many precancerous PIN lesions. Based on comparisons with NPE, levels of cIAP1 protein in 12 of 21 (57%) PIN lesions exceeded cIAP1 immunoscores representative of normal prostate epithelium (H-score 100; P < 0.0001), suggesting that elevations of cIAP1 protein occur early in the process of malignant transformation. Similarly, 9 of 22 (41%) PIN specimens had elevated cIAP2 immunoscores compared with NPE (P < 0.0001). Also, whereas XIAP immunoscores were 100 for all NPE specimens, 8 of 26 (31%) PIN lesions showed increased levels of XIAP protein (P < 0.0001). Similarly, compared with NPE, 45% of PIN specimens (10 of 22) had elevated survivin immunoscores (P < 0.0001).
Although representing a relative small cohort, attempts were made to correlate expression of IAP family proteins with clinical outcome using the results obtained for the 64 early-stage patients. To dichotomize data into higher versus lower expression categories, the median immunoscore result for each IAP family protein tested was used to split the data set, thus comparing RFS (PSA relapse) for the patients with immnoscores above versus below the median value. Kaplan-Meier curves and log-rank tests demonstrated a trend of patients with higher cIAP1 and higher cIAP2 to relapse with greater frequency during the follow-up period (median follow-up, 5.5 years), but the results did not reach statistical significance (P = 0.06 for cIAP1 and cIAP2; Fig. 4
; Table 2
). In contrast, higher levels of XIAP were unexpectedly associated with longer RFS (P = 0.0001). Survivin immunostaining data were not correlated with RFS (Fig. 4)
.
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Analysis of IAPs in a Mouse Transgenic Model of Prostate Cancer.
The SV40 T-antigen TRAMP closely resembles the progression of human prostate cancer (28)
.
Histological analysis of prostate tissue from TRAMP transgenic mice revealed age-dependent appearance of PIN and invasive cancer, with 73% of male mice developing multifocal PIN (22 of 30, 73%), and approximately half of mice developing invasive cancer [also often multifocal (12 of 30, 40%) by 28 weeks of age (Table 3)
. In contrast, only 1 of 10 control littermates developed PIN, and no prostate cancers were detected in nontransgenic mice.
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; see supplemental data). Although barely expressed in the normal prostatic epithelium, high levels of MIAP-1 and MIAP-2 immunostaining (assessed using antibodies raised against human cIAP2 and cIAP1), respectively, were found in transformed prostatic epithelium of SV40 large T-antigen transgenic mice (Fig. 5, A, B, and E–G
; summarized in Tables 3
and 4
). In some animals, overexpression of these IAP family proteins was evident in carcinoma in situ (PIN), in addition to invasive adenocarcinomas (Fig. 5, B, D, G, and H
; Table 4
). Similarly, survivin/MIAP-4 immunostaining was not detected in normal prostate epithelium but was found in PIN and in many invasive cancers (Fig. 5, J–L)
. Interestingly, whereas most normal cells were immunonegative for survivin, positive immunostaining was found in occasional nontransformed prostatic epithelial cells of both TRAMP and normal mice, associated with chromosomes or mitotic structures in cells apparently undergoing division. In contrast, intense survivin immunoreactivity was found in interphase nuclei and in the cytosol of most transformed cells within PIN lesion and invasive cancers in TRAMP mice. Heterogeneity in the percentage of transformed cells expressing survivin, however, resulted in some cases scoring as negative, where at least 50% immunopositivity was set as the threshold (see legend of Table 4
for details). The intensity of XIAP/MIAP-3 immunostaining also increased during malignant transformation (Fig. 5, N–Q)
, although more aggressive tumors that had infiltrated seminal vesicles rarely contained high levels of this protein. The specificity of these immunostaining results was confirmed by control stainings performed using either preimmune serum or immune antisera that had been preabsorbed with the relevant immunogens (Fig. 5
, C, F, I, M, and O).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previously, survivin was reported to be aberrantly overexpressed 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 members of the IAP family in cancers. Expression of cIAP1, cIAP2, XIAP, survivin, and NAIP has been examined in the National Cancer Institute collection of 60 human tumor cell lines, revealing widespread expression of cIAP1, XIAP, and survivin in these tumor lines of diverse tissue origins and demonstrating cIAP2 expression primarily in lymphoid malignancies (15) . Importantly, these studies also demonstrated poor correlation of cIAP1, cIAP2, and XIAP protein levels with mRNA levels, consistent with emerging knowledge that expression of many IAPs is regulated predominantly at the level of protein stability (15) . Higher levels of XIAP protein have been correlated with shorter remission duration after chemotherapy and shorter survival in patients with acute myelogenous leukemia (15) . The IAP family member ILP-2 (livin, ML-IAP, KIAP) reportedly is overexpressed in melanoma (14) .
Thus, when combined with these prior reports, our data provide additional evidence that pathological elevations in the expression of antiapoptotic IAP family proteins represent a common event in many types of cancer. Not only were elevations in IAP family proteins seen in archival human prostate cancer specimens, but we also observed increases in the levels of cIAP1/MIAP-2, cIAP2/MIAP-1, XIAP/MIAP-3, and survivin/MIAP-4 during disease progression in a transgenic mouse model of prostate cancer. Up-regulation of IAP expression concomitant with the emergence of PIN implicates a role of these proteins in the early stages of the pathogenesis of prostate cancer and emphasizes the utility of TRAMP for identifying molecular changes in early disease. This finding suggests that overexpression of IAP family proteins is a general concomitant of transformation of prostate epithelium and reinforces the idea that multiple IAP family members become dysregulated in their expression during the pathogenesis of malignancy in the prostate gland. It remains to be seen whether the simultaneous overexpression of several IAP family proteins reflects a commonality in the mechanisms controlling the levels of IAP family proteins in cells versus the possibility that multiple independent signaling pathways that control individual IAP family members become simultaneously deregulated in these neoplasms.
Based on the data provided here, we cannot conclude whether differences in the expression of one or more IAP family proteins (individually or in combination) are of prognostic significance for men with prostate cancer. In the tissue microarray data set, higher cIAP2 levels were associated with larger tumor size (T stage), hormone-refractory disease, and metastatic disease, but these patients were heterogenous with respect to Gleason grade, pretreatment PSA, and therapy. In the small cohort of early-stage patients treated uniformly with external beam radiation, higher cIAP2 correlated in multivariate analysis with shorter RFS. In contrast, higher XIAP was unexpectedly associated with longer RFS in this small cohort, based on both univariate and multivariate analyses. The association of higher cIAP1 and cIAP2 levels with more aggressive disease is consistent with the documented function of these antiapoptotic proteins as suppressors of caspases (20) . As for XIAP, whereas this protein is a potent caspase inhibitor and suppressor of apoptosis (3) , XIAP has also recently been reported to inhibit cell proliferation by down-regulating levels of cyclins A and D1 and inducing expression of the cyclin-dependent kinase inhibitors p21/Waf1 and p27/Kip1 (30) . Thus, XIAP may provide a selective survival advantage while simultaneously impairing division of cancer cells. Other explanations for the paradoxical association of XIAP with the shorter RFS could be related to expression in the same tumors of endogenous antagonist proteins (which were not measured here), such as SMAC, XAF1, and the serine protease Omi/HtrA2, which can negate apoptosis suppression by XIAP (31, 32, 33, 34, 35) .
The functional importance of IAPs in cancer is beginning to be clarified. Involvement of IAP family proteins in tumor resistance to chemotherapeutic drugs and other apoptotic agents has been demonstrated by use of antisense techniques, helping to validate certain IAPs as potential drug targets for cancer (36, 37, 38, 39, 40, 41, 42) . Also, heptameric peptides from the NH2 terminus of the IAP antagonist protein SMAC have also been applied to reverse caspase inhibition by IAPs in vitro (43, 44, 45, 46, 47, 48, 49) and to overcome IAP-mediated suppression of apoptosis in leukemia cell lines (45) , suggesting a strategy for cancer therapy. Furthermore, a recent observation that cytochrome c microinjection fails to induce apoptosis in prostate cancer LNCaP cells without SMAC also implies a crucial role for proteins of the IAPs in this organ (48) .
Given emerging data suggesting an important role for IAP family proteins in sustaining tumor cell survival and suppressing apoptosis induced by anticancer drugs (38, 39, 40, 41, 42 , 44, 45, 46, 47, 48, 49, 50) , our results demonstrating that overexpression of several IAP family proteins occurs frequently in prostate cancers in humans and transgenic mice provide further validation of IAPs as potential drug discovery targets for the improved treatment of prostate cancer. The observation that more than one IAP family member is often overexpressed simultaneously in prostate cancers, however, raises the possibility that effective strategies will require IAP antagonists that are capable of inhibiting multiple members of this family of apoptosis-suppressing proteins.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournal.org).
1 Both authors contributed equally to this work. 
2 To whom requests for reprints should be addressed, at The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. Phone: (858) 646-3140; Fax: (858) 646-3194; E-mail: jreed{at}burnham.org
3 The abbreviations used are: IAP, inhibitor of apoptosis; PIN, prostatic intraepithelial neoplasia; PSA, prostate-specific antigen; XIAP, X chromosome-linked IAP; cIAP, cellular inhibitor of apoptosis protein; NAIP, neuronal apoptotic inhibitory protein; TRAMP, transgenic mouse model of prostate cancer; BPH, benign prostatic hyperplasia; DAB, 3,3'-diaminobenzidine; HRP, horseradish peroxidase; NPE, nonmalignant prostate epithelium; RFS, relapse-free survival; HR, hazard ratio; CI, confidence interval; TAKI, TGFB-activated kinase.
4 Antisera are available from Biocarta, Inc. (www.biocarta.com) and Science Reagents, Inc. (www.sciencereagents.com).
Received 7/ 1/02; revised 5/ 7/03; accepted 5/ 7/03.
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L. A. Gomez, A. de las Pozas, and C. Perez-Stable Sequential combination of flavopiridol and docetaxel reduces the levels of X-linked inhibitor of apoptosis and AKT proteins and stimulates apoptosis in human LNCaP prostate cancer cells Mol. Cancer Ther., May 1, 2006; 5(5): 1216 - 1226. [Abstract] [Full Text] [PDF]
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M. Stanbrough, G. J. Bubley, K. Ross, T. R. Golub, M. A. Rubin, T. M. Penning, P. G. Febbo, and S. P. Balk Increased expression of genes converting adrenal androgens to testosterone in androgen-independent prostate cancer. Cancer Res., March 1, 2006; 66(5): 2815 - 2825. [Abstract] [Full Text] [PDF]
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Z. Liu, H. Li, M. Derouet, J. Filmus, E. C. LaCasse, R. G. Korneluk, R. S. Kerbel, and K. V. Rosen ras Oncogene Triggers Up-regulation of cIAP2 and XIAP in Intestinal Epithelial Cells: EPIDERMAL GROWTH FACTOR RECEPTOR-DEPENDENT AND -INDEPENDENT MECHANISMS OF ras-INDUCED TRANSFORMATION J. Biol. Chem., November 11, 2005; 280(45): 37383 - 37392. [Abstract] [Full Text] [PDF]
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K. Yamanaka, P. Rocchi, H. Miyake, L. Fazli, B. Vessella, U. Zangemeister-Wittke, and M. E. Gleave A novel antisense oligonucleotide inhibiting several antiapoptotic Bcl-2 family members induces apoptosis and enhances chemosensitivity in androgen-independent human prostate cancer PC3 cells Mol. Cancer Ther., November 1, 2005; 4(11): 1689 - 1698. [Abstract] [Full Text] [PDF]
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O. Berezovskaya, A. D. Schimmer, A. B. Glinskii, C. Pinilla, R. M. Hoffman, J. C. Reed, and G. V. Glinsky Increased Expression of Apoptosis Inhibitor Protein XIAP Contributes to Anoikis Resistance of Circulating Human Prostate Cancer Metastasis Precursor Cells Cancer Res., March 15, 2005; 65(6): 2378 - 2386. [Abstract] [Full Text] [PDF]
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Z. Wang, M. Cuddy, T. Samuel, K. Welsh, A. Schimmer, F. Hanaii, R. Houghten, C. Pinilla, and J. C. Reed Cellular, Biochemical, and Genetic Analysis of Mechanism of Small Molecule IAP Inhibitors J. Biol. Chem., November 12, 2004; 279(46): 48168 - 48176. [Abstract] [Full Text] [PDF]
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1 mCi/mmol; Amersham). Ten µl were loaded in gels. Autoradiography confirmed production of all proteins (data not shown). Alternatively, proteins were produced in bacteria as recombinant proteins (RP) with His6 or glutathione S-transferase tags and affinity purified (0.05 µg was loaded). Detergent lysates were prepared from NPE, prostate adenocarcinomas (CA), or various tumor cell lines (Jurkat and RS11-846) or normal tissues. The lysates were normalized for total protein content (100 µg) and analyzed by SDS-PAGE/immunoblotting. Blots were incubated with rabbit polyclonal (Polycl.) antibodies recognizing human cIAP1 (A and F), XIAP (B and F), survivin (C and F), or cIAP2 (E). A mouse monoclonal antibody (MAB) recognizing cIAP2 was also used (D). Antibody detection was accomplished by an enhanced chemiluminescence method. Blots represent the following: A, recombinant and in vitro translated proteins; B, in vitro translated proteins; C, recombinant proteins (Lanes 1–4), tumor cell lines (Lanes 5 and 6), and normal tissues (Lanes 7–11); D and E, recombinant proteins (Lanes 1–3), NPE specimens (Lanes 4 and 5), and prostate cancer specimens (Lanes 6–9); and F, two NPE specimens (left) and eight prostate cancer specimens (right). Blots in E and F were reprobed with antibodies recognizing ß-actin or heat shock protein (HSP60), respectively, as loading controls.
