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Loss of neutral endopeptidase and activation of protein kinase B (Akt) is associated with prostate cancer progression
Article first published online: 2 NOV 2006
Copyright © 2006 American Cancer Society
Volume 107, Issue 11, pages 2628–2636, 1 December 2006
How to Cite
Osman, I., Dai, J., Mikhail, M., Navarro, D., Taneja, S. S., Lee, P., Christos, P., Shen, R. and Nanus, D. M. (2006), Loss of neutral endopeptidase and activation of protein kinase B (Akt) is associated with prostate cancer progression. Cancer, 107: 2628–2636. doi: 10.1002/cncr.22312
- Issue published online: 17 NOV 2006
- Article first published online: 2 NOV 2006
- Manuscript Accepted: 14 SEP 2006
- Manuscript Revised: 6 SEP 2006
- Manuscript Received: 8 AUG 2006
- Department of Defense Grant. Grant Number: PC040021
- National Institutes of Health Grant. Grant Number: CA80240
- Robert H. McCooey Memorial Cancer Research Fund
- neutral endopeptidase;
- prostate cancer;
- phosphatase and tensin homolog;
- tumor suppressor protein
Neutral endopeptidase (NEP) is a cell-surface peptidase that can regulate the activation of Akt kinase through catalytic-dependent and independent mechanisms. NEP expression is absent in approximately 50% of prostate cancers. The authors investigated whether NEP loss in vivo would result in Akt phosphorylation and potentially contribute to prostate cancer progression by examining the interaction of NEP, Akt, and phosphatase and tensin homolog (PTEN) in a prostate xenograft model and in clinical specimens from patients with prostate cancer.
Using a tetracycline-repressible expression system to express NEP in a tumor animal xenograft model, the effects of NEP were tested on tumor growth, Akt phosphorylation, and PTEN expression. The clinical relevance of NEP, phosphorylated Akt, and PTEN protein expression also was investigated in 204 patients who had undergone radical prostatectomy.
The results indicated that the induction of NEP expression inhibited established xenograft tumor growth, diminished Akt phosphorylation, and increased PTEN protein levels. In humans, prostate cancers with complete loss of NEP expression were significantly more likely to express phosphorylated Akt (P = .02). Moreover, patients who had prostate cancers with concomitant loss of NEP and expression of phosphorylated Akt had an increased, independent risk of prostate-specific antigen (PSA) recurrence (P = .03). In the study cohort, loss of PTEN protein expression did not correlated significantly with phosphorylated Akt or with patients' clinical outcome.
The findings from this investigation demonstrated that NEP loss leads to Akt activation and contributes to the clinical progression of prostate cancer. Cancer 2006. © 2006 American Cancer Society.
Prostatic epithelial cells normally express neutral endopeptidase (NEP) protein. Lack of NEP expression has been observed in nearly 50% of both primary1-5 and metastatic6 prostate cancers. This loss can result from methylation of the NEP promoter4, 7 or may occur after androgen withdrawal, because NEP transcription is regulated by androgen.6, 8 In our previous investigation of the antitumorigenic effects of NEP in prostate cancer, we demonstrated that the catalytic function of NEP results in the cleavage of the neuropeptides endothelin-1 and bombesin. In the absence of NEP, these neuropeptides activate insulin growth factor-1 receptor (IGF-1R) signaling, leading to downstream protein kinase B (Akt) activation.9 Neuropeptide-induced activation of Akt in these cells is mediated by phosphatidylinositol 3′-kinase (PI3-K) and a Src-dependent signaling pathway.9
The biologic and regulatory effects of NEP were presumed only to result from its enzymatic function. However, we recently reported that NEP also can use a catalytic-independent mechanism that involves the direct binding of NEP with the phosphatase and tensin homolog (PTEN) protein.10 Using an in vitro model, we demonstrated that NEP recruits endogenous PTEN to the membrane, leading to prolonged PTEN protein stability and increased PTEN phosphatase activity. This results in a constitutive down-regulation of Akt activity.10 Thus, NEP regulates Akt activation through several catalytic-dependent and independent mechanisms; and, presumably, NEP loss in vivo would result in Akt phosphorylation and potentially would contribute to prostate cancer progression.11
For the current study, we examined the role of NEP regulation of Akt in tumor growth in vivo by using a cancer xenograft model and then used tumor tissues obtained from a well-characterized cohort of prostate cancer patients to correlate the effects of alterations in NEP, PTEN, and phosphorylated Akt (pAkt) expression with clinical outcome. Here, we report that the induction of NEP expression inhibits established animal tumor growth, diminishes pAkt, and increases PTEN protein expression. In humans, patients with prostate cancer who had tumors that showed complete loss of NEP expression were significantly more likely to express pAkt. Moreover, patients with prostate cancers who had tumors that showed a concomitant loss of NEP with expression of pAkt had an increased, independent risk of prostate-specific antigen (PSA) recurrence after radical prostatectomy. Together, these data demonstrate that NEP loss leads to Akt activation and contributes to the clinical progression of prostate cancer.
MATERIALS AND METHODS
Cell Culture and Materials
WT-5 cells (TSU-GK27-NEP), which contained full-length NEP combinational DNA (cDNA) subcloned into the tetracycline-repressible, transactivator protein-responsive plasmid pTRE (pTRE/NEP), were derived previously and maintained as described,6, 12 and expressed full-length NEP after tetracycline removal. The following antibodies were used: mouse monoclonal antibody (MoAb) to NEP, NCL-CD10-270 (Novocastra Laboratories Ltd., Newcastle-upon-Tyne, U.K.); MoAb to PTEN, A2B1 (Chemicon, International, Inc., Temecula, CA); rabbit polyclonal antibody (pAb) to PTEN, anti-PTEN (Upstate Biotechnology, Inc., Lake Placid, NY); pAb to Akt, H-136 (Santa-Cruz Biotechnology); pAb to phospho (Ser473)-Akt (Cell Signaling Technology, Inc., Beverly, MA); and pAb to actin (Chemicon).
Swiss Nude mice aged 4 to 6 weeks (Taconic, Germantown, NY) were fed with Dox-Diet (Bio-Serve Inc., Frenchtown, NJ) for 1 week. Semiconfluent WT-5 cells were trypsinized, counted, and resuspended in phosphate-buffered saline on ice; and 5 × 106 cells suspended in 100 μL phosphate-buffered saline were injected subcutaneously into the right flank. After 2 weeks, during which time tumors developed, animals were separated randomly into 2 cages, and Dox-Diet was removed from the diet in 1 of the cages. Tumor measurements were taken 3 times per week, and the tumor volume (mm3) was estimated by using the formula of width2 × length. After 4 weeks, all the animals were killed, and tumor specimens immediately were frozen in liquid nitrogen and stored at −70°C until analysis.
Protein Extraction, Immunoprecipitation, and Western Blot Analysis
Tumor specimens were extracted in RIPA buffer (10 mM Tris-HCl [pH 7.4] 150 mM NaCl, 1% Triton X-100, 5 mM ethylenediamine tetraacetic acid, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1.2% aprotinin, 5 μM leupeptin, 4 μM antipain, 1 mM phenylmethylsulfonyl fluoride, and 0.1 mM Na3VO4), as described previously.10, 12 Western blot analyses and immunoprecipitations were performed as reported previously reported.10, 12
Patients were identified through a review of the Department of Urology database at the Veterans Administration Medical Center/New York University School of Medicine. This prospective database enrolled patients with prostate cancer from 1990 to the present that documents patient demographics, including race, stage, and grade of the primary tumor. After Institutional Review Board approval and activation of the protocol, a review was performed to gather all relevant clinical and pathologic information, including racial background, age at the time of diagnosis, tumor grade, stage, pretreatment PSA values, PSA recurrence, and survival. Patient selection was based solely on the availability of adequate clinical follow-up and representative pathology specimens for immunohistochemical analysis. Representative hematoxylin and eosin-stained tissue sections were examined to evaluate the histopathologic characteristics of each specimen. Of the 261 patients whose tumors were resected at the Veterans Administration Medical Center, 223 patients had adequate, representative, formalin-fixed, paraffin-embedded primary tumor tissues available for analyses of NEP expression. From the 223 specimens that were analyzed for NEP, 206 specimens were available for analysis of pAkt, and 204 specimens were available for analysis of PTEN. The retrieval rate of the tissues provided confidence in the results, because it minimized the chance of selection bias, which can be a major issue in conducting a retrospective analysis. The clinicopathologic parameters that were investigated included pretreatment PSA, pathologic stage, and Gleason score. Patients were grouped according to whether they had low Gleason scores (<7; n = 122) or high Gleason scores (≥7; n = 101). Patients also were grouped according to pathologic stage into a group with early, organ-confined tumors (pT2; n = 144) or a group with advanced tumors that extended beyond the prostatic capsule (pT >3; n = 79). All patients were hormone-naive at the time of surgery.4, 13, 14
NEP expression was assessed by immunohistochemistry using clone NCL-CD10-270 (1:100 dilution; Novocastra Laboratories Ltd.),4 PTEN (Ab-4; 1:50 dilution; LabVision),15 and pAKT was examined using (Ser 473 rabbit antiserum at 2.2 μg/mL; Cell Signaling Technology, Inc.). An antigen-retrieval protocol for the enhancement of potentially masked epitopes was used. Sections were immersed in boiling 0.01% citric acid, pH 6.0, for 20 minutes under microwave treatment to enhance antigen retrieval, allowed to cool, and incubated with primary antibody or antiserum overnight. The secondary antibody was horse antimouse immunoglobulin G for anti-NEP and anti-PTEN and antirabbit for pAKT used at a dilution of 1:500 followed by avidin-biotin complex (Vector Laboratories) at a dilution of 1:25. The final chromogen was 3,3′-diaminobenzidine. Nuclei were counterstained with hematoxylin before mounting.
NEP membranous immunoreactivity of prostate cancer tissues was classified on a continuous scale with values ranging from undetectable levels (0%) to homogenous membranous staining (100%) of tumor cells. Because the majority of tumors clearly showed that NEP has either homogenous staining of membranous immunoreactivity or undetectable expression, we classified tumors into 3 groups: homogeneous expression (positive), complete lack of expression (negative), and heterogeneous expression, which means NEP expression was observed in focal areas of the tumor. For PTEN (Ab-4; LabVision), endothelial cells and nerves showed strong PTEN expression and were used as internal positive controls.
Expression of PTEN was scored according to signal intensity and proportion of cells with positive nuclear staining. Compared with corresponding normal tissues, specimens that had increased or equal staining intensity compared with the corresponding normal tissue were assigned a score of 2+, and specimens that had decreased intensity were assigned a score of 1+.15–17 A cut-off level of 50% PTEN-immunoreactive cells was established based on data indicating that PTEN is haploinsufficient in tumor suppression and that its dose is a key determinant in cancer progression.18 Therefore, retained PTEN (positive) expression was defined as ≥50% immunoreactive cells and 2+ intensity, whereas altered PTEN (negative) expression was defined as <50% immunoreactive cells or 1+ intensity.
Expression of pAKT was examined using Ser 473 rabbit antiserum (Cell Signaling Technology, Inc.). pAKT also was scored according to signal intensity and the proportion of cells with positive nuclear staining. Compared with corresponding normal tissues, specimens that had increased or equal staining intensity compared with the corresponding normal tissue were assigned a score of 2+, and specimens that had decreased intensity were assigned a score of 1+. pAkt expression was defined as >50% immunoreactivity and 2+ intensity.18, 19 All histopathologic specimens were reviewed and scored by Dr. Peng Lee, an attending pathologist, who was blinded to all patient information at the time of evaluation.
Univariate associations between NEP expression categories (negative, 0%; heterogeneous, 5–75%; positive, 100%) and PTEN or pAkt status (positive or negative) were explored using the chi-square test or Fisher exact test. Kaplan-Meier survival analysis was performed to compare PSA recurrence-free survival between the NEP-negative/pAkt-positive and NEP-positive/pAkt-negative patient groups using the log-rank test. The independent effect of several factors, including tumor grade (Gleason scores <7 [referent] vs. ≥7), tumor stage (stage 2 [referent] vs. stage ≥3), pretreatment PSA (<10 ng/mL [referent] vs. ≥10 ng/mL), and concomitant NEP/pAkt expression status (NEP-positive/pAkt-negative [referent] vs. NEP-negative/pAkt-positive), on PSA recurrence-free survival was analyzed by using a multivariate Cox proportional hazards regression model. Adjusted hazard ratios are presented for each parameter of interest, and 95% confidence intervals for the hazard ratios are reported to assess the precision of the obtained estimates. All P values were 2-sided and were considered statistically significant at the .05 α level. All analyses were performed in SAS software (version 9.1; SAS Institute, Inc., Cary, NC).
Expression of NEP Inhibits Xenograft Tumor Growth
We previously demonstrated that NEP inhibits prostate cancer tumorigenicity in an orthotopic model of prostate cancer.20 For the current study, we examined the effect of NEP on the growth of an established orthotopic tumor. For these experiments, we used the WT-5 cell line, which is a derivative of TSU-Pr1 cells that contains tetracycline-repressible, full-length NEP cDNA and expresses NEP in the absence of tetracycline (Tet-off).6, 12 Two weeks after the injection of WT-5 cells into the flanks of athymic mice that were fed with doxycycline to suppress NEP expression (at which time, tumors measured approximately 0.5 cm in greatest dimension), doxycycline was withdrawn from the diet of 50% of the animals. Over the next 30 days, tumors in animals that expressed NEP (doxycycline withdrawn) grew at a significantly slower rate than tumors in animals that had suppressed NEP expression (doxycycline fed; P = .008) (Fig. 1A and 1B). Immunohistochemical analysis with an NEP-specific MoAb for NEP protein expression confirmed NEP expression in animals that were not fed doxycycline (data not shown), and determination of NEP-specific enzymatic activity in tumor lysates showed that catalytically active NEP was expressed strongly in tumors from animals that did not receive doxycycline (median specific activity of tumor lysates from 10 animals, 389 pmol/μg protein per minute) and that NEP was suppressed in tumors from animals that were fed doxycycline (median specific activity of tumor lysates from 7 animals, 89 pmol/μg protein per minute; P < .001) (Fig. 1C).
NEP Expression Modulates Akt Phosphorylation and PTEN Expression
WT-5 cells express wild-type PTEN protein
Protein extracted from representative tumor xenografts was analyzed by Western blot analysis for the expression of NEP, pAkt, and PTEN. Figure 2 shows that tumor lysates from the animals that were fed doxycycline showed that doxycycline suppressed NEP expression (Fig. 2A, top, Lanes 3 and 4) and that doxycycline withdrawal led to high levels of NEP protein expression (Fig. 2A, top, Lanes 1 and 2). PTEN protein expression dramatically increased in tumors in which NEP was expressed (Fig. 2A, middle, Lanes 1 and 2), consistent with our prior studies showing that NEP stabilizes PTEN protein.10 Analysis of Akt showed a marked decrease in Akt phosphorylation in a tumor lysate that expressed NEP (Fig. 2B) compared with tumors in which NEP expression was suppressed. Taken together, these data confirm our in vitro studies and demonstrates that in vivo expression of NEP results in tumor growth inhibition, decreased Akt phosphorylation, and increased PTEN expression.
Loss of NEP expression correlates with Akt phosphorylation in localized prostate cancers
To test the clinical validity of the reciprocal correlation of NEP and activated pAkt expression and to examine for an association between NEP and PTEN protein expression in localized prostate cancers, next, we used immunohistochemical analysis to examine the protein expression of NEP, pAkt, and PTEN proteins in 204 prostate cancer tissue specimens from patients who were hormone naive and who uniformly underwent radical prostatectomy for localized prostate cancer. The cohort studied was a well-characterized group of patients who underwent radical prostatectomy at the Veterans Administration Medical Center in New York. In total, 223 patients had adequate tissue for analyses of NEP expression,4, 14 206 patients had the same tissues available for analyses of pAKT expression, and 204 patients for had tissues available for analyses of PTEN expression (Table 1). Examination for an association of NEP, pAkt, and/or PTEN expression (Table 2) revealed a statistically significant, reciprocal correlation between NEP and pAkt (P = .02); 35.6% of tumors demonstrated complete loss of NEP expression and the presence of pAkt (NEP-negative/pAkt-positive), and 23.6% of tumors demonstrated retained NEP expression and undetectable expression of pAkt (NEP-positive/pAkt-negative). This reciprocal correlation between NEP and pAkt, which was detected in 59% of the specimens, is illustrated in Figure 3. Twenty-one percent of tumors showed abundant expression of pAkt concomitant with NEP expression (NEP-positive/pAkt-positive), and the remaining 20% of tumors had decreased levels of pAkt in the absence of NEP expression (NEP-negative/pAkt-negative), with PTEN expression detected in 71% of this subset, suggesting that PTEN can suppress Akt activation in the absence of NEP. In total, PTEN expression was not detected in 79 of 204 tumors (39%), a rate that is in the range of prior immunohistochemical studies, which showed loss of PTEN protein expression in 20% to 30% of localized prostate cancers.21–23 Concomitant expression of NEP and PTEN did not correlate significantly with decreased pAkt expression, and no significant correlation was detected between PTEN and pAkt.
|Variable||NEP (n = 223)||PTEN (n = 204)||pAkt (n = 206)|
|Het (n = 105)||Pos (n = 36)||Neg (n = 82)||Pos (n = 79)||Neg (n = 125)||Pos (n = 86)||Neg (n = 120)|
|NEP expression†||pAkt expression: No. of tumors (%)*|
|0%||34 (19.5)||62 (35.6)||96 (55.2)|
|100%||41 (23.6)||37 (21.3)||78 (44.8)|
|Total||99 (43.1)||75 (56.9)||174 (100)|
NEP loss with pAkt predicts PSA recurrence after prostatectomy
Next, we analyzed the entire cohort of patients for any correlations of NEP, pAkt, and/or PTEN expression with the time to PSA (biochemical) recurrence after prostatectomy. At a mean follow-up of 6.2 years, patients with prostate tumors that had concomitant, complete loss of NEP expression together with overexpression of pAkt had a significantly shorter median time to PSA recurrence compared with patients who had prostate tumors that retained NEP expression but had undetectable pAkt expression (median, 4.91 years vs. 9.62 years; P = .03), whereas no statistically significant difference was observed between any of the other cohorts (Fig. 4). This association remained significant in a multivariate model after controlling simultaneously for grade, stage, and pretreatment PSA (Table 3) (P = .04). Neither PTEN expression nor pAkt expression individually was predictive of PSA recurrence in this patient cohort (P = .72 and P = .14, respectively; log-rank test).
|Variable||Chi-Square test||P||HR||95% CI for HR|
The genetic events that contribute to the development and progression of prostate cancer are complex and likely involve multiple signaling pathways. Alterations of PTEN are common, with loss of protein expression occurring in >50% of advanced prostate tumors. PTEN alterations occur through multiple mechanisms, including gene alterations and mutations, promoter hypermethylation, and transcriptional malfunction.24, 25 The clinical significance of PTEN protein loss, as determined by immunohistochemistry, is unclear. In 1 study, complete loss of PTEN protein expression was correlated with higher Gleason score and advanced tumor stage;22 whereas, in another study, loss of PTEN expression alone did not predict for biochemical recurrence after prostatectomy.23 Studies from prostate cancer animal models suggest that PTEN dose also may contribute to prostate cancer progression,18, 26 suggesting that haplodeficiency of PTEN may contribute to prostate cancer development and progression.
Consequently, immunohistochemistry may underestimate the role of PTEN in primary prostate cancer. We also could not detect a correlation between PTEN expression and PSA recurrence, similar to prior reports.22, 23
Although our studies in mice xenografts confirmed previous cell line experiments demonstrating that NEP stabilizes PTEN protein and inhibits Akt activation, we did not detect an association between NEP expression and PTEN expression in primary prostate cancers. This may be explained by the finding that loss of PTEN protein most commonly results from pretranslational alterations24 or by the nonquantitative nature of protein detection by immunohistochemistry. We did detect a significant association between NEP and pAkt expression and a striking difference in the time to biochemical recurrence in patients who had tumors that retained NEP expression and repressed Akt activation compared with patients who had tumors that lost NEP expression and possessed pAkt. We showed previously that NEP can inhibit Akt activation through numerous pathways, including 1) catalytic inactivation of peptide substrates that, in the absence of NEP, would stimulate Akt phosphorylation27; 2) a direct protein-protein interaction between NEP and PTEN, resulting in increased PTEN protein stability and PTEN phosphatase activity10; and 3) an indirect protein-protein interaction in which NEP associates with Lyn kinase, which, is turn, sequesters PI3-K.28 The results reported here confirm the in vitro studies, indicating that NEP inhibits Akt phosphorylation (and that NEP loss results in pAkt) and showing a similar correlation between NEP and activated Akt in patient specimens.
The incidence of activated Akt detected in our patient cohort is in concordance with previously reports, in which pAkt expression was observed in ≈50% of clinical prostate cancer specimens.21, 29 However, our results do not support a strong correlation between pAkt expression and higher Gleason scores29 or recurrence-free survival.30 Our data also demonstrate that the odds that a tumor will be positive for PTEN when it is positive for pAkt is only 0.31, very similar to a recently published report that described an odds ratio of 0.28.21
The WT-5 cell line that we used in the current animal studies originally was believed to be of prostatic origin6, 31; however, subsequent to those experiments, the prostatic origin of the parental TSU-Pr1 cells was disputed. We since have obtained identical results on the effects of NEP on tumor growth and PTEN and pAkt protein expression using 22RV1 prostate cancer cells in which NEP was administered using Lenti virus, and the results suggest that the correlation between NEP, PTEN, and pAkt is valid in prostate cancer (unpublished data).
In the current study, 21% of tumors showed abundant expression of pAkt together with NEP expression (NEP-positive/pAkt-positive). Forty percent of this NEP-positive/pAkt-positive cohort lacked PTEN expression, which may have contributed to Akt phosphorylation in the presence of NEP. Alternatively, other signaling pathways can activate Akt independent of NEP, such as increased IGF-1R signaling. These results highlight the heterogeneity of prostate cancer and that the NEP-Akt pathway is 1 of many molecular events that can result in prostate cancer progression.
In summary, we previously reported that, in a multivariate analysis, complete loss of NEP expression was associated with PSA recurrence after controlling for grade, stage, pretreatment PSA level, and race.4 In the current study, we attempted to decipher the contribution of PTEN and activated Akt to NEP loss and observed that the predictive combination of NEP loss and Akt activation strongly predicted PSA recurrence with a median of 4.91 years compared with 9.62 years in patients who had tumors expressed NEP and lacked pAkt. Although a number of signaling pathways can activate Akt in the presence or absence of NEP, and a myriad of molecular alterations contribute to the development and progression of prostate cancer, our current results suggest that analysis of Akt or PTEN alone may not be adequate to predict clinical outcome. Understanding the biology of prostate cancer (such as the ability of NEP to regulate signaling) can assist in identifying the molecular markers that will allow clinicians to segregate their patients comfortably into prognostic groups that guide therapy.
- 22Loss of PTEN expression in paraffin-embedded primary prostate cancer correlates with high Gleason score and advanced state. Cancer Res. 1999; 59: 4921–4926., , , , , .