Persistent expression of Aurora-A after neoadjuvant hormonal therapy as a predictor of a poor clinical outcome in patients undergoing radical prostatectomy for prostate cancer

Authors


Hideaki Miyake, Division of Urology, Kobe University, Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650–0017, Japan.
e-mail: hideakimiyake@hotmail.com

Abstract

OBJECTIVES

To characterize the changes in the expression of Aurora-A protein in prostate cancer before and after androgen-withdrawal therapy, and to assess the prognostic significance of the Aurora-A expression in patients undergoing radical prostatectomy (RP) after neoadjuvant hormonal therapy (NHT).

PATIENTS AND METHODS

The study included 97 patients with clinically localized prostate cancer who received NHT followed by RP. Paired needle biopsy and corresponding RP specimens obtained from these patients were analysed for the expression of Aurora-A protein by immunohistochemical staining. These findings were then evaluated in relation to several clinicopathological factors.

RESULTS

There were various levels of Aurora-A protein expression in most prostate cancer tissues before NHT; however, the Aurora-A expression in RP specimens after NHT was significantly down-regulated compared with that in corresponding needle-biopsy specimens. The expression level of Aurora-A in biopsy specimens was significantly associated with the biopsy Gleason score, but not with other factors available before RP. The Aurora-A expression in the RP specimens correlated significantly with the preoperative value of the serum prostate specific antigen and pathological stage, but not with any other clinicopathological factors examined. Furthermore, cell proliferative activity in the RP specimens, measured by Ki-67 immunostaining, was proportional to the expression of Aurora-A. The biochemical recurrence-free survival in patients with a persistent Aurora-A expression in RP specimens was significantly lower than that in those with a weak Aurora-A expression, but the expression level of Aurora-A was not an independent predictor of biochemical recurrence.

CONCLUSIONS

Despite the lack of any independent significance, the expression level of Aurora-A in prostate cancer tissue after NHT, which might inversely reflect the therapeutic effect of NHT, could therefore be a useful variable for predicting biochemical recurrence in patients undergoing RP.

Abbreviations
RP

radical prostatectomy

NHT

neoadjuvant hormonal therapy

HSP

heat-shock protein.

INTRODUCTION

The centrosome ensures an equal segregation of chromosomes to the post-mitotic daughter cells, by organising the bipolar mitotic spindle during normal cell proliferation; however, multipolar mitotic spindles and various types of cetrosomal anomalies are frequent in cancer cells [1]. Such abnormalities might cause the disruption of normal chromosomal segregation and result in the production of aneuploid cells. Although the precise molecular mechanisms regulating segregation of chromosomes have not been well characterized, several genes involved in the function mediating centrosome duplication were recently cloned and analysed, including members of mammalian Aurora homologues [2].

Aurora-A, a serine/threonine protein kinase belonging to the Drosophila aurora and Saccharomyces cerevisiae Ip11 kinase family, has been shown to be crucial in chromosome segregation and centrosome functions [3].

Because of its location on chromosome 20q13, a region frequently amplified in various types of human malignant tumours, Aurora-A has attracted intense interest [4,5]. Indeed, there is marked up-regulation of Aurora-A in several kinds of human cancer specimens [3,6,7]. In addition, recent studies showed that introducing the Aurora-A gene into mouse NIH/3T3 cells and Rat 1 fibroblasts leads to transformation in vitro and tumorigenesis in vivo[8,9]. Collectively, these findings suggest that if overexpressed, Aurora-A could function as an oncogene through the abnormal regulation of centrosome function.

Also in prostate cancer, several studies have shown the important roles of centrosomal defects and chromosomal instability in disease progression [10,11]. Furthermore, recent studies reported that Aurora-A is overexpressed in high-grade prostatic intraepithelial neoplasia and primary prostate cancer lesions, and that expression patterns of Aurora-A correlate with several potential prognostic variables [12,13]. Considering these findings, it would be of interest to investigate whether Aurora-A is involved in adaptive changes in prostate cancer induced by hormonal therapy. However, to our knowledge, there have been no studies characterizing the changes in Aurora-A expression and its prognostic significance in prostate cancer after androgen-withdrawal therapy. Accordingly, we initially compared the expression of Aurora-A protein in paired needle biopsy and radical prostatectomy (RP) specimens from patients who had had neoadjuvant hormonal therapy (NHT) before RP, and further analysed the correlation of Aurora-A expression with several clinicopathological factors.

PATIENTS AND METHODS

This study included 97 patients who were diagnosed by transrectal needle biopsy as having prostate cancer, and subsequently received NHT using a LHRH agonist (3.6 mg goserelin acetate or 3.75 mg leuprorelin acetate every 4 weeks) plus antiandrogen (80 mg bicalutamide or 375 mg flutamide daily) followed by RP, at our institution between April 1993 and December 2004. The median (range) duration of the follow- up after RP was 47 (10–152) months. Informed consent to the study was obtained from each patient, and the study design was approved by the Research Ethics Committee of our institution. Specimens were examined pathologically by a one pathologist, according to the 2002 TNM system. After RP, patients were usually followed by measurements of serum PSA level every ≤3 months for the first 2 years and every 6 months thereafter. Biochemical recurrence was defined as a PSA level persistently >0.2 ng/mL. Irrespective of pathological findings suggesting a poor prognosis, none of the patients received any adjuvant therapy until biochemical recurrence was detected.

The prostate cancer specimens were stained immunohistochemically as previously described [14]; briefly, sections from formaldehyde-fixed, paraffin-embedded tissue from 97 specimens were deparaffinized by xylene and rehydrated in decreasing concentrations of ethanol. After blocking endogenous peroxidase with 3% hydrogen peroxidase in methanol, sections were boiled in 0.01 m citrate buffer for 10 min and incubated with 5% normal blocking serum for 20 min The sections were then incubated with antihuman Aurora-A rabbit polyclonal antibody (Abcam, Cambridge, UK) and antihuman Ki-67 mouse monoclonal antibody (Dako, Carpinteria, CA, USA) for 2 h at room temperature, followed by incubation with biotinylated goat anti-rabbit or mouse IgG (Vector Laboratories, Burlingame, CA, USA) for 30 min. After incubation in an avidin-biotin peroxidase complex for 30 min, the samples were exposed to diaminobenzidine tetrahydrochloride solution and counterstained with haematoxylin. Negative controls, in which PBS was used instead of the primary antibody, were run with each batch of staining samples, while s.c. inoculated human androgen-independent prostate PC3 tumours were used as a positive control, as previously described [13]. Staining results were interpreted by two independent observers (J.F. and A.T.) who were unaware of the clinicopathological data of the patients. For Aurora-A analysis, staining intensity was scored from +1 (no staining), +2 (weak), +3 (medium) to +4 (strong), and staining extent was also scored from +1 (0–10%), +2 (11–25%), +3 (26–50%) to +4 (51–100%). The product of staining intensity and staining extent was defined as the staining score. In addition, only nuclear staining was considered when evaluating Ki-67 staining, and strong expression of Ki-67 was defined as the proportion of positively stained tumour cells >5%.

Several clinicopathological factors of the patients were analysed using the chi-square test. The biochemical recurrence-free survival rates were calculated by the Kaplan-Meier method, and differences assessed using the log-rank test. The prognostic significance of some factors was assessed by the Cox proportional hazards regression model; in all tests P < 0.05 was considered to indicate significance.

RESULTS

The findings of Aurora-A immunostaining in paired needle biopsy and corresponding RP specimens from 97 patients are shown in Table 1. Although Aurora-A protein was present in the cytoplasm of prostate cancer cells in most biopsy specimens, there was no detectable Aurora-A expression in 24 RP specimens (25%). Furthermore, both staining intensity and the extent of Aurora-A expression in RP specimens after NHT were significantly lower than those in biopsy specimens. When we defined any staining score of >8 as indicating strong expression, 56 (58%) and 37 (38%) patients were regarded as having prostate cancer with strong Aurora-A expression on biopsy and RP specimens, respectively. Representative findings of immunohistochemical studies based on this system are shown in Fig. 1.

Table 1. 
Results of Aurora-A immunostaining on paired needle biopsy and RP specimens from 97 patients who had NHT
ImmunostainingN(%)P
Needle biopsyRP
Intensity  <0.001
 +1 1 (1)24 (25) 
 +235 (36)31 (32) 
 +341 (42)37 (38) 
 +420 (21) 5 (5) 
Extent  <0.001
 +1 1 (1)12 (12) 
 +2 5 (5)16 (17) 
 +3 11 (11)20 (21) 
 +480 (83)49 (51) 
Score  0.006
 ≤841 (42)60 (62) 
 ≥956 (58)37 (38) 
Figure 1.

Representative findings of immunohistochemical staining of Aurora-A expression in prostate cancer. Biopsy specimens showing (A) weak, (B) strong expression; and RP specimens showing (C) weak and (D) strong expression of Aurora-A.

We subsequently assessed the correlation of Aurora-A staining on needle biopsy and RP specimens with several clinicopathological variables. In biopsy specimens, the expression level of Aurora-A protein was significantly associated with biopsy Gleason score, but there was no significant relation of the Aurora-A expression level to other factors available before RP, including age, serum PSA level and percentage of positive biopsy cores (Table 2). On RP specimens after NHT, Aurora-A protein expression was significantly associated with preoperative serum PSA level and pathological stage, but not other pathological factors, including Gleason score, perineural invasion and lymph node metastasis. Furthermore, cell proliferative activity in the RP specimens, measured by Ki-67 immunostaining, was in proportion to the expression level of Aurora-A (Table 2).

Table 2.  Correlation of Aurora-A expression with several factors in needle-biopsy specimens before NHT and RP specimens after NHT
VariablesNo. of patientsStaining intensityStaining extentStaining score
+1–2+3–4P+1–2+3–4P≤8≥9P
Needle biopsy
Age, years   0.770  0.850  0.400
 ≤69522032  349 2428 
 ≥70451629  342 1728 
Serum PSA (ng/mL)   0.620  0.390  0.960
 ≤9.912 5 7  1 11  5 7 
 ≥10–19.9411724  338 1823 
 ≥20.0441430  242 1826 
Biopsy Gleason score   0.030  0.001  0.029
 ≤6221210  517 14 8 
 7481929  147 2028 
 ≥827 522  027  720 
% of positive biopsy core   0.370  0.570  0.410
 ≤49592435  356 2336 
 ≥50381226  335 1820 
RP specimens
Age, years   0.830  0.990  0.730
 ≤69523022 1537 3319 
 ≥70452520 1332 2718 
Preop. serum PSA (ng/mL)   0.010  0.028  0.023
 ≤0.09563818 2135 4016 
 ≥0.1411724  734 2021 
Pathological stage   0.021  0.013  0.011
 ≤pT2503416 2030 3713 
 ≥pT3472126  839 2324 
Gleason score   0.480  0.980  0.960
 ≤6251510  718 1510 
 7492920 1435 3118 
 ≥823 1112  716 14 9 
Perineural invasion   0.620  0.690  0.390
 Negative422517 1329 2814 
 Positive553025 1540 3223 
Lymph node metastasis   0.620  0.800  0.140
 pN0895138 2663 5732 
 pN1 8 4 4  2 6  3 5 
Ki-67 expression   0.021  0.010  0.030
 weak835132 2855 5528 
 strong14 410  014  5 9 

There was no significant difference in overall survival between patients with strong and weak Aurora-A expression (data not shown). During the present observation period biochemical recurrence developed in 21 of 97 patients (22%); there was biochemical recurrence occurred in 13 of the 37 (35%) with strong and in eight of 60 (13%) with weak Aurora-A expression in RP specimens. Biochemical recurrence-free survival in patients with strong Aurora-A expression was significantly lower than that in those with weak expression (Fig. 2). Furthermore, to evaluate the predictive value of several clinicopathological factors for biochemical recurrence, we used a multivariate analysis using the Cox proportional hazard regression model. As shown in Table 3, only lymph-node metastasis was independently associated with biochemical recurrence, irrespective of other factors examined.

Figure 2.

Biochemical recurrence-free survival of patients with prostate cancer according to Aurora-A expression status in RP specimens after NHT, by Kaplan-Meier analysis.

Table 3.  Association of variables with biochemical recurrence-free survival in patients with prostate cancer who had RP after NHT
VariableHazard ratio (95% CI)P
Age, years (≤69 vs ≥70)1.12 (0.71–4.10)0.520
Preop. serum PSA level, ng/mL (≤0.09 vs ≥0.1)1.33 (0.60–3.77)0.290
Pathological stage (≤pT2 vs ≥pT3)1.71 (0.57–2.44)0.110
Gleason score (≤6 vs ≥7)1.37 (0.20–4.92)0.610
Perineural invasion (−ve vs +ve)1.70 (0.44–6.89)0.420
Lymph node metastasis (pN0 vs pN1)3.45 (1.08–8.23)0.037
Ki-67 expression in RP (weak vs strong)1.50 (0.37–3.44)0.230
Aurora-A score in RP (weak vs strong)1.55 (0.42–3.15)0.200

DISCUSSION

Although >80% of patients with prostate cancer initially respond to androgen-withdrawal therapy, progression to androgen-independence ultimately occurs within a few years in most of these patients. To date, various molecular mechanisms involved in androgen-independent progression have been reported, amongst which adaptive up-regulation of anti-apoptotic genes and/or persistent overexpression of cell survival genes, e.g. bcl-2, bcl-xL, clusterin, IGF binding protein-2 and -5 and heat-shock protein 27 (HSP27), is currently regarded as one of the most important events contributing to the acceleration of the acquisition of an androgen-independent phenotype [15]. Furthermore, our previous studies identified changes in the expression levels of these genes after NHT as useful predictors of biochemical outcome in patients undergoing RP [16,17].

Recently, Aurora-A, a key regulator of centrosome function, has attracted great interest, as several studies recently showed that overexpression of Aurora-A results in disruption of normal cell-cycle progression, and subsequently promotes oncogenic transformation [2,3,8,9]; however, the effects of NHT on changes in Aurora-A expression in prostate cancer, and its clinical significance, remain largely unknown. Hence, we evaluated the expression of Aurora-A protein by immunohistochemical staining in paired needle biopsy and corresponding RP specimens obtained from patients who had had NHT before RP.

In this series, despite the expression of Aurora-A protein in most prostate cancer tissues in needle-biopsy specimens before NHT, the markedly lower Aurora-A expression in the RP specimens than in the corresponding biopsy specimens was confirmed. However, there was persistent strong expression in ≈ 40% of RP specimens. The patterns of change in Aurora-A expression before and after androgen withdrawal do not parallel the patterns of changes in proteins with anti-apoptotic activity, e.g. clusterin and HSP27, suggesting the involvement of Aurora-A in the process of progression to androgen-independence in a manner differing from those of previously characterized anti-apoptotic genes [15–17].

There was a significant correlation of Aurora-A expression in biopsy specimens with the Gleason score, but no correlation with other variables before RP. This finding is supported by the results of Lee et al.[13], who identified a strong association between Aurora-A expression in human prostate cancer cell lines and its corresponding biological malignant potential. We also found that persistent overexpression of Aurora-A in RP specimens after NHT was marked in patients with a higher preoperative serum PSA and pathological stage. Cell proliferative activity in the RP specimens, measured by Ki-67 immunostaining, was also in proportion to the expression level of Aurora-A, suggesting that down-regulation of Aurora-A after NHT would reflect an indirect effect of reduced cell proliferation rather than that of androgen regulation of this gene. Considering these findings, it would be interesting to investigate the value of a novel therapeutic strategy targeting the Aurora-A gene, to further enhance the pro-apoptotic effect of NHT on prostate cancer cells.

Despite the lack of independent significance, biochemical recurrence-free survival in patients with strong Aurora-A expression in the RP specimens was significantly lower than that in those with weak expression. Although the expression level of Aurora-A protein in prostate cancer specimens from patients who were not treated by NHT has already been shown to be associated with several prognostic factors, to our knowledge, this is the first study to identify persistent expression of Aurora-A after NHT as a significant prognostic predictor. These findings indicate that persistent overexpression of Aurora-A after NHT might inversely reflect its therapeutic effect, and that persistent expression of Aurora-A after NHT could be a potential adjunct to conventional predictors of biochemical recurrence in patients undergoing RP.

The present study has some limitations; at our institution, RP is usually used with no previous NHT, resulting in a recruitment time for the 97 patients of >10 years. Such a long recruitment time could affect clinical outcomes, due to recent changes associated with the treatment of prostate cancer, e.g. progress in surgical techniques and the introduction of novel therapeutic methods for recurrent disease. In addition, there are several factors that cause biases in the present results; e.g. the evaluation of Aurora-A expression in biopsy specimens might not completely reflect that of the RP specimens in each case. It would also bias the biochemical recurrence-free survival to combine RP with NHT. Finally, the expression level of Aurora-A in RP specimens was shown to lack independent significance as a predictor of biochemical recurrence, which could be explained by the significant correlation of Aurora-A expression with potential prognostic indicators. Collectively, these findings suggest that it would be necessary to include more patients with a longer follow-up to draw definitive conclusions about the significance of Aurora-A expression level in RP specimens after NHT.

In conclusion, the expression of Aurora-A was significantly lower in RP specimens than in biopsy specimens before NHT, but persistent overexpression of Aurora-A was detected in ≈ 40% of RP specimens after NHT. Moreover, despite the lack of independent significance, the expression level of Aurora-A in prostate cancer tissue after NHT, which might inversely reflect the therapeutic effect of NHT, could be a useful variable predicting biochemical recurrence in patients undergoing RP.

CONFLICT OF INTEREST

None declared.

Ancillary