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Keywords:

  • prostate cancer;
  • mTOR;
  • Akt;
  • PTEN;
  • 4E-BP1;
  • markers

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

OBJECTIVE

To evaluate the activation level of the mammalian target of rapamycin (mTOR) signalling pathway in Chinese patients with prostate cancer, as this pathway is over-activated in many human cancers and is an attractive target for cancer therapy.

PATIENTS AND METHODS

We used immunohistochemistry to investigate the activation level of five important markers of the mTOR pathway, including PTEN, p-Akt, p-mTOR, p-p70S6K and p-4E-BP1, in tissues from 182 patients with prostate cancer, 20 with benign prostatic hyperplasia (BPH) and 10 with high-grade prostatic intraepithelial neoplasia (HGPIN). The expression levels of these five markers were associated with patient clinical and pathological characteristics.

RESULTS

Expression levels of p-Akt, p-mTOR, p-4E-BP1 and p-p70S6K were significantly higher in prostate cancer tissues than in BPH and HGPIN tissues. In 182 patients with prostate cancer the p-mTOR expression level significantly and positively correlated with its upstream p-Akt and downstream p-4E-BP1 and p-p70S6K expression levels. The cancer Gleason score was significantly correlated with p-Akt and p-mTOR expression level but not with p-4E-BP1 and p-p70S6K expression level. However, the p-4E-BP1and p-p70S6K expression levels in primary cancer lesions were statistically significantly correlated with patient T stage and distant metastases.

CONCLUSIONS

Most patients with prostate cancer have at least one component of the mTOR signalling pathway activated. The activation of the mTOR pathway might be involved in prostate cancer development and progression. The association between activation of mTOR pathway and patient clinicopathological variables suggested that not all patients are equally amenable to treatment strategies targeting the mTOR pathway.


Abbreviations
RP

radical prostatectomy

MAB

maximum androgen blockade

ADT

androgen-deprivation therapy

mTOR

mammalian target of rapamycin

PI3K

phosphatidylinositol 3-kinase

p-

phosphorylated

eIF

eukaryotic translation initiation factor

PTEN

phosphatase and tensin homologue deleted on chromosome 10

(HG)PIN

(high-grade) prostatic intraepithelial neoplasia.

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

Prostate cancer is one of the most common cancers in the world. Due to economic development and changing lifestyles, the incidence of prostate cancer in China is increasing rapidly [1]. Moreover, many newly diagnosed Chinese patients with prostate cancer are symptomatic and have metastatic disease because there is no screening programme in China [1,2]. Metastatic prostate cancer can only be treated palliatively by androgen-deprivation therapy (ADT) [2]. Although ADT can lead to symptomatic improvement and a reduction in serum PSA levels in most patients, almost all these patients ultimately become refractory to ADT [3]. Because there is no consistently effective treatment for androgen-independent metastatic prostate cancer, it continues to be a major cause of death in men, especially in China. Currently, several novel treatment strategies that aim at specific signalling pathways and immunotherapy are being investigated for androgen-independent prostate cancer [4–6].

Recent research has found that the mammalian target of rapamycin (mTOR) pathway is an attractive target for cancer therapy [7–9]. The mTOR is a 289-kDa serine/threonine kinase that controls protein synthesis, angiogenesis and cell-cycle progression [7–9]. As reported in mammalian cells, the upstream molecules phosphatidylinositol 3-kinase (PI3K) and serine/threonine kinase Akt activates mTOR through at least two mechanisms, i.e. direct phosphorylation of mTOR itself and via phosphorylation and inactivation of TSC2 [8,9]. The activated mTOR (p-mTOR) acts through its downstream effectors p70S6K (also termed S6K1) and 4E-BP1 to stimulate protein synthesis and regulate the cell cycle [8,9]. The activated p70S6K (p-p70S6K) promotes protein synthesis by phosphorylating PDCD4 and targeting it for degradation. PDCD4 hinders protein translation by binding and preventing the eukaryotic translation initiation factor (eIF)4A helicase from unwinding secondary structure in the 5′ untranslated region of mRNA [9]. Phosphorylating 4E-BP1 by p-mTOR results in the activation of cap-dependent translation of nuclear mRNA by releasing inhibition of EIF-4E [9]. Therefore, the activation cascade of Akt/mTOR/4E-BP1/p70S6K pathway will result in general protein translation [7]. In experimental studies and clinical trials, the mTOR inhibitors have shown growth inhibitory and anti-angiogenic properties in many types of cancer [7,9–13]. Recently, the mTOR inhibitor temsirolimus improved overall survival among patients with metastatic RCC and a poor prognosis in a phase II clinical trial [14].

The mTOR inhibitors can not only inhibit the growth of xenografts derived from prostate cancer cell lines, but also enhance the cytotoxic effects of radiation on prostate cancer cell lines [15,16]. Therefore, mTOR might be an important molecular target for therapeutic intervention in prostate cancer, and some clinical trials are currently ongoing. More importantly, we need to identify subsets of patients who will most likely benefit from the mTOR inhibitors. Pantuck et al.[17] found that the mTOR pathway is more significantly altered in clear-cell RCC, high-grade tumours, and tumours with poor prognostic features, and they suggested that these patients with a highly activated mTOR signalling pathway might benefit most from mTOR inhibitors. However, the alteration of the mTOR pathway has not been thoroughly investigated in patients with prostate cancer. Therefore, in the present study we focused on the activation level of several important molecule markers upstream and downstream in the mTOR pathway, including the tumour suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN), Akt, mTOR, p70S6 kinase and 4E-BP1 in Chinese patients with prostate cancer, using immunohistochemical techniques. Because Akt and mTOR are kinases, we examined their phosphorylation status and that of their downstream effectors, using phosphorylation-specific antibodies.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

The study included prostate cancer tissue samples from 182 Chinese patients with prostate cancer who were diagnosed and treated between 2000 and 2006 at Shanghai Cancer Hospital, Fudan University. This study was approved by the Institutional Review Board of our hospital. A written informed consent was obtained from each patient before any study-specific investigations were done. Clinical data, including age, PSA level and TNM staging, were obtained from medical records. The characteristics of these patients are summarized in Table 1. All the patients were hormone-naive before the diagnosis of prostate cancer was established by prostate biopsy in each patient. In this study, 104 (57.1%) patients who had distant metastases at the time of diagnosis were treated by maximum androgen blockade (MAB), with 44 receiving LHRH agonists plus antiandrogen agents, and 60 receiving bilateral orchidectomy plus antiandrogen agents; 51 (28.0%) patients with clinically localized prostate cancer were treated by retropubic radical prostatectomy (RP) and the remaining 27 by MAB and/or radiotherapy. All patients included in this study were routinely followed according to European Association of Urology guidelines. Of the 104 patients with distant metastatic prostate cancer, 48 (46%) died from cancer by the end of the study. Among 51 patients who had RP, 10 (20%) developed biochemical recurrence during the follow-up. The median (range) follow-up was 36  (18–96) months for the censored patients and 18.5 (7–57) months for those who died from prostate cancer.

Table 1.  The characteristics of the patients with prostate cancer
CharacteristicMedian (range) or n (%)
Age, years 70 (43–90)
Gleason score 
 4  3 (1.6)
 5  5 (2.7)
 6 25 (13.7)
 7 67 (36.8)
 8 42 (23.1)
 9 36 (19.8)
 10  4 (2.2)
Serum PSA, ng/mL 52.3 (0.83–6006)
 0–4  6 (3.3)
 4.1–10 10 (5.5)
 10.1–20 34 (18.7)
 20.1–50 38 (20.9)
 50.1–100 29 (15.9)
 >100 65 (35.7)
Clinical stage 
 T1N0M0 10 (5.5)
 T2N0M0 39 (21.4)
 T3N0M0 15 (8.2)
 T4N0M0  6 (3.3)
 T(any)N1M0  8 (4.4)
 T(any)N(any)M1104 (57.1)
Treatment 
 RP 51 (28.0)
 Radical radiotherapy  7 (3.8)
 MAB 113 (62.1)
 MAB + radiotherapy  11 (6.0)

Archival formalin-fixed paraffin-embedded specimens from RPs and prostate biopsies were obtained. First, the haematoxylin and eosin-stained histological sections of each specimen were reviewed by the study pathologists to confirm the diagnosis and re-evaluate the tumour grade according to the Gleason system. One representative tissue block with a high carcinoma content was then chosen for further studies. Samples from 20 BPH tissues and 10 of high-grade prostatic intraepithelial neoplasia (HGPIN) tissues from patients with no prostate cancer were also included in the study for comparison.

A mouse monoclonal antibody against p-p70S6 kinase (Thr389; 1A5) and rabbit monoclonal antibodies against p-Akt (Ser473; 736E11), p-mTOR (Ser2448; 49F9) and p-4E-BP1 (Ser65; 174A9) were purchased from Cell Signalling Technology (Beverly, MA, USA). A rabbit polyclonal antibody against PTEN (clone PN37) was obtained from Zymed (San Fransisco, CA, USA) was also used.

Sections of 4 µm thick were mounted on aminopropyhriethoxy silane-coated slides, the slides heated to 60 °C for 2 h and deparaffinized with xylene and rehydrated through 100% to 85% ethanol. Endogenous peroxidase was quenched in 3% hydrogen peroxide. Antigen was retrieved by soaking the slides in 0.01 mm citrated buffer at pH 6.0 and heating in a 99 °C water bath for 30 min. Nonspecific binding was blocked by treatment with a blocking solution for 1 h at room temperature. The slides were incubated with primary antibodies overnight at 4 °C, at the dilutions indicated: PTEN (1:75), p-Akt (1:20), p-mTOR (1:50), p-4E-BP1 (1:200) and p-p70S6K (1:200). The slides were then incubated with secondary antibody for 30 min, and 3–3′-diaminobenzidine used for colour development, with haematoxylin for counterstaining. Slides of tissues known to express PTEN, p-Akt, p-mTOR, p-4E-BP1 and p-p70S6K were used as positive controls. Omission of the primary antibody and replacement with Tris-buffered saline served as negative controls.

All slides were interpreted by two experienced pathologists in an open discussion, and who were unaware of all clinical data. The p-Akt, p-mTOR, p-4E-BP1 and p-p70S6K expression levels were grouped into three categories based on both staining intensity and staining proportion (percentage of positive cells) according to a previously described scoring method, which has been used widely [10,11,18]. Tumours with <10% cells with weak staining were scored as 0, with >10% cells with weak staining or <20% cells with intermediate to strong staining were scored as 1, and with >20% cells with intermediate to strong staining were scored as 2. For these four markers, a staining score of 1 and 2 was considered positive. Expression for PTEN was also grouped into three categories according to a previously described method, which has been shown to be highly consistent [19,20]. Tumour cells were graded as 0 if staining was undetectable in the tumour cells and present in benign cells, as 1 if the staining intensity was diminished relative to the benign cells, and as 2 if the staining intensity was equal to that of the adjacent benign cells. A staining score of 0, i.e. complete loss of PTEN, was considered negative.

The Pearson chi-square test was used to analyse the differences in PTEN, p-Akt, p-mTOR, p-4E-BP1 and p-p70S6K expression level in BPH, HGPIN and prostate cancer tissues. Spearman’s rank correlation test was used to determine the correlation between PTEN, p-Akt, p-mTOR, p-4E-BP1 and p-p70S6K expression level in 182 patients with prostate cancer; the associations of these five markers with patient clinical and pathological characteristics were evaluated using the Pearson chi-square test (for categorical variables) and nonparametric Kruskal–Wallis H-test (for continuous variables).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

In prostate cancer tissues, the expression of p-Akt was located in both cytoplasm and nucleus, and p-mTOR expression was apparent in both membrane and cytoplasm (Fig. 1D–I). Expression of p-4E-BP1 was mainly in the nucleus and expression of p-p70S6K was apparent in both cytoplasm and nucleus (Fig. 1J–O). On statistical analysis the expression level of p-Akt, p-mTOR, p-4E-BP1 and p-p70S6K all increased significantly as the disease progressed from BPH to HGPIN to prostate cancer (Table 2).

image

Figure 1. Immunostaining to show various levels of PTEN, p-AKT, p-mTOR, p-4E-BP1 and p-p70S6K expression in different human prostate tissues. (A) 2+ cytoplasmic and membranous staining of PTEN in BPH. (B) 1+ cytoplasmic and membranous staining of PTEN in prostate cancer (Gleason grade 3); contrast with 2+ staining in HGPIN peripheral to the cancer. (C) Loss of PTEN expression in prostate cancer (Gleason grade 5). (D) 1+ nuclear staining of p-AKT in HGPIN. (E) 1+ nuclear and cytoplasmic staining of p-AKT in prostate cancer (Gleason grade 3). (F) 2+ nuclear and cytoplasmic staining of p-AKT in prostate cancer (Gleason grade 4). (G) 1+ cytoplasmic and membranous staining of p-mTOR in HGPIN. (H) 1+ cytoplasmic and membranous staining of p-mTOR in prostate cancer (Gleason grade 3). (I) 2+ cytoplasmic and membranous staining of p-mTOR in prostate cancer (Gleason grade 4). (J) Isolated nuclear staining of p-4E-BP1 in HGPIN. (K) 1+ nuclear staining of p-4E-BP1 in prostate cancer (Gleason grade 3). (L) 2+ nuclear staining of p-4E-BP1 in prostate cancer (Gleason grade 3). (M) Weak nuclear staining (1+) of p-p70S6K in HGPIN. (N) Weak nuclear and cytoplasmic staining (1+) of p-p70S6K in prostate cancer (Gleason grade 4). (O) Strong nuclear and weak cytoplasmic staining (2+) of p70S6K in prostate cancer (Gleason grade 4). reduced from ×200.

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Table 2.  Expression pattern of PTEN, p-Akt, p-mTOR, p-4E-BP1 and p-p70S6K in different prostate tissues
ScoreBPHHGPINProstate cancerP*
  • *

    Pearson chi-square test.

No. of patients n (%) or n2010182 
PTEN    
 00040 (22)<0.001
 10180 (44) 
 220 (100)962 (34) 
p-AKT    
 019 (95)7100 (55)0.011
 11 (5)236 (20) 
 20146 (25) 
p-mTOR    
 019 (95)9105 (58)0.006
 11 (5)153 (29) 
 20024 (13) 
p-4E-BP1    
 018 (90)886 (47)0.002
 12 (10)252 (29) 
 20044 (24) 
p-p70S6K    
 020 (100)9101 (56)0.001
 10153 (29) 
 20028 (15) 

PTEN expression was detected in normal prostate epithelial cells and located mainly in membrane and cytoplasm (Fig. 1A–C). In prostate cancer tissues, the PTEN expression was diminished in 80 (44%) cases and completely lost in 40 (22%) cases. The expression level of PTEN decreased significantly as the disease progressed from BPH to HGPIN to prostate cancer (Table 2).

From Spearman’s rank correlation test, PTEN expression level was significantly inversely correlated with p-Akt expression level but not with p-mTOR, p-4E-BP1 and p-p70S6K expression level in the 182 patients with prostate cancer (Table 3). In addition, p-Akt expression level was significantly positively correlated with p-mTOR, p-4E-BP1 and p-p70S6K expression level (Table 3). The expression level of p-mTOR positively correlated with both p-4E-BP1 and p-p70S6K expression level (Table 3).

Table 3.  Correlation between PTEN, p-Akt, p-mTOR, p-4E-BP1 and p-p70S6K expression level in 182 patients with prostate cancer
 Spearman’s correlation coefficientP*
  • *

    Spearman’s rank correlation test.

PTEN
p-AKT−0.1850.012
p-mTOR−0.0920.217
p-4E-BP1−0.0840.258
p-p70S6K−0.0710.341
p-AKT
p-mTOR0.670<0.001
p-4E-BP10.2530.001
p-p70S6K0.1990.007
p-mTOR
p-4E-BP10.240<0.001
p-p70S6K0.323<0.001

From the Kruskal–Wallis H-test, the serum PSA level in patients with prostate cancer increased significantly as their tumour p-4E-BP1 expression level increased from 0 to 1 and 2 (Table 4). However, there was no correlation between PSA level and the other four markers. Patient age did not correlate with any of the five markers (Table 4). From the Pearson chi-square test, tumour Gleason score was significantly correlated with p-Akt and p-mTOR expression level. Gleason score 7–10 tumours tended to have higher p-Akt and p-mTOR expression levels (Table 4). Patients with prostate cancer who had T3-4 stage disease had a significantly higher p-4E-BP1and p-p70S6K expression level in their tumours than those with T1-2 stage disease (Table 4). There was also a significant association of distant metastatic status with p-4E-BP1 and p-p70S6K expression level; patients with distant metastatic prostate cancer tended to have higher p-4E-BP1 and p-p70S6K expression level (Table 4).

Table 4.  The association of PTEN, p-Akt, p-mTOR, p-4E-BP1 and p-p70S6K with clinical and pathological characteristics in 182 patients with prostate cancer
MarkerNo. of patientsMedian (range)Gleason score, n (%)T stage (any NM)Distant metastasis (any TN)
Age, years, P*PSA, ng/mL4–67–10T1-2T3-4M0M1
  • *

    Kruskal–Wallis H-test;

  • †Pearson chi-square test.

PTEN
 04070 (48–80)67.8 (6.6–5577)7 (21.2)33 (22.1)20 (23.8)20 (20.4)15 (19.2)25 (24.0)
 18068 (48–90)55.2 (2.4–3808)15 (45.5)65 (43.6)35 (41.7)45 (45.9)36 (46.2)44 (42.3)
 26271 (43–82)45.1 (0.8–6006)11 (33.3)51 (34.2)29 (34.5)33 (33.7)27 (34.6)35 (33.7)
P 0.597*0.860*0.981 0.805 0.729 
p-Akt
 010070 (43–82)42.0 (0.8–6006)25 (75.8)75 (50.3)53 (63.1)47 (48.0)50 (64.1)50 (48.1)
 13670 (48–90)84.5 (3.0–1213)2 (6.1)34 (22.8)16 (19.0)20 (20.4)10 (12.8)26 (25.0)
 24669 (48–88)58.2 (5.7–5577)6 (18.2)40 (26.8)15 (17.9)31 (31.6)18 (23.1)28 (26.9)
P 0.868*0.228*0.020 0.070 0.058 
p-mTOR
 010571 (48–90)50.0 (0.8–6006)25 (75.8)80 (53.7)56 (66.7)49 (50.0)52 (66.7)53 (51.0)
 15369 (43–89)99.8 (2.8–2809)7 (21.2)46 (30.9)21 (25.0)32 (32.7)17 (21.8)36 (34.6)
 22470 (48–80)35.9 (5.7–5577)1 (3.0)23 (15.4)7 (8.3)17 (17.3)9 (11.5)15 (14.4)
P  0.520*0.061*0.043 0.053 0.095 
p-4E-BP1
 08670 (48–82)30.8 (0.8–6006)17 (51.5)69 (46.3)49 (58.3)37 (37.8)50 (64.1)36 (34.6)
 15269 (48–90)58.7 (5.7–3808)10 (30.3)42 (28.2)18 (21.4)34 (34.7)18 (23.1)34 (32.7)
 24472 (43–88)95.5 (4.9–5577)6 (18.2)68 (25.5)17 (20.2)27 (27.6)10 (12.8)34 (32.7)
P 0.401*0.002*0.672 0.020 <0.001 
p-p70S6K
 010170 (48–90)40.9 (0.8–4001)20 (60.6)81 (54.4)57 (67.9)44 (44.9)52 (66.7)49 (47.1)
 15369 (56–89)90.0 (6.6–6006)9 (27.3)44 (29.5)18 (21.4)35 (35.7)17 (21.8)36 (34.6)
 22868 (43–88)62.5 (2.8–3739)4 (12.1)24 (16.1)9 (10.7)19 (19.4)9 (11.5)19 (18.3)
P 0.412*0.077*0.773 0.008 0.032 

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

The mTOR signalling pathway, which serves as a central regulator of cell growth, is being actively pursued as a therapeutic target for cancer therapy [7,8]. In previous studies, the activation of mTOR pathway in both androgen- dependent and -independent prostate cancer cell lines was confirmed [15,21]. Moreover, the mTOR inhibitor CCI-779 inhibited the growth of xenografts derived from both PTEN mutant PC-3 cells and PTEN wild-type DU145 cells, with greater effects against PC-3 than DU145 tumours [15]. However, compared with cancer cell lines, the clinical situation is more complicated. Clinical samples of human prostate cancers are not homogeneous. Determining the activation level of the mTOR signalling pathway in prostate cancer tissues might help to identify subsets of patients who will most likely benefit from mTOR inhibitors. Previously, Kremer et al.[22] studied the expression level of mTOR signalling pathway markers in prostate cancer tissues, but their study only included tissue samples from 33 patients, and their results had limited value in guiding clinical practice.

In the present study, using immunohistochemistry with phosphorylation-specific antibodies to detect Akt, mTOR, 4E-BP1 and p70S6K phosphorylation status in BPH, HGPIN and prostate cancer tissues, only a small part of the BPH tissues had p-Akt, p-mTOR, p-4E-BP1 and p-p70S6K positive staining, indicating low activity of this pathway in the benign proliferating cells in BPH. There was increased expression of p-Akt, p-mTOR, p-4E-BP1 and p-p70S6K in HGPIN, which has a high possibility of developing into prostate cancer. Moreover, the expression level of these four markers was even higher in prostate cancer tissues (Table 2); of the 182 samples, 77 (42.3%), 96 (52.7%) and 81 (44.5%) patients had positive p-mTOR, p-4E-BP1 and p-p70S6K expression, respectively, which means that activation of mTOR protein and its downstream targets is quite common in these Chinese patients with prostate cancer. The p-mTOR expression level significantly and positively correlated with its upstream p-Akt and downstream p-4E-BP1 and p-p70S6K expression level in the 182 patients (Table 3). These results confirmed the activation cascade of the Akt/mTOR/4E-BP1/p70S6K pathway in prostate cancer. In the present study, the increased expression level of these four markers was apparent in the preneoplastic lesion HGPIN, which suggested that activation of this pathway might be an early event in prostate carcinogenesis. Majumder et al.[23] reported that overexpression of Akt leads to the formation of PIN lesions in a transgenic mouse model, which can be reversed through mTOR inhibition. Furthermore, several previous studies found that activation of the mTOR pathway can occur rapidly in normal cells in response to carcinogen exposure, and can be detected in preneoplastic lesions of other malignancies [7]. Therefore, we believe that activation of the mTOR pathway might be an early event in the oncogenic transformation of prostate epithelium, and it can cause abnormal initiation of protein synthesis and increased cell proliferation and survival.

The tumour suppressor PTEN is a dual lipid/protein phosphatase that antagonises the action of PI3K and suppresses the phosphorylation of its downstream targets, including Akt [7]. Mutation, deletion or epigenetic silencing of PTEN, which leads to reduced or absent of PTEN expression at the mRNA or protein level, has been reported in numerous primary cancers, including prostate cancer [7,20,22]. Our data also suggest that loss of expression of the tumour suppressor PTEN is a frequent event in Chinese patients with prostate cancer, because the PTEN protein was lost or decreased in 120 (65.9%) of the 182 patients (Table 2). In addition, PTEN expression decreased occasionally in HGPIN (10%), compared with no change in BPH. Previously, Yoshimoto et al.[24] reported that PTEN gene deletions were found in 23% of HGPIN and 68% of prostate cancer samples. They considered that the high frequency of PTEN deletion observed in prostate cancer vs precursor lesions implies a pivotal role for PTEN haplo-insufficiency in the transition from preneoplastic HGPIN to prostate cancer [24]. In the present patients with prostate cancer the PTEN expression level was significantly inversely correlated with p-Akt level (Table 3). This phenomenon was reported previously and suggests that loss of PTEN protein is involved in regulation of the Akt activation in prostate cancer [19]. Although the p-Akt expression level was significantly positively correlated with its three downstream effectors (p-mTOR, p-4E-BP1 and p-p70S6K) in the 182 patients, PTEN expression did not correlate with them. This might be because mTOR and its downstream 4E-BP1 and p70S6K can also be activated by other pathways, such as the Raf/mitogen-activated protein kinase signalling pathway, which is not regulated by PTEN [8]. Furthermore, the similar p-mTOR level in PTEN mutant PC-3 cells and PTEN wild-type DU145 cells gives another example of no correlation between PTEN and p-mTOR expression in prostate cancer [16]. Although there is merit to considering whether reconstitution of PTEN might have therapeutic value in prostate cancer, the safety concerns over gene therapy will limit clinical application of this approach in the near future [7].

In the present 182 patients, p-4E-BP1 and p-p70S6K expression levels in primary cancer lesions were statistically significantly correlated with patient T stage and distant metastases (Table 4). Patients with higher T stage (T3-4) and distant metastasis tended to have higher p-4E-BP1 and p-p70S6K expression levels. To the best of our knowledge, these correlations have not been reported previously in prostate cancer. Previously, Rojo et al.[25] reported correlations of p-4E-BP1 expression with tumour size, lymph node metastasis and locoregional recurrence in breast cancer. They considered that phosphorylation of 4E-BP1 was the consequence of several oncogenic events in breast cancer, including amplification or mutation of growth factor receptors, PI3K mutations, Ras mutations, etc. [25]. They also proposed that the phosphorylated form of 4E-BP1 might act as a bottleneck factor through which many upstream proliferative oncogenic signals converge [25]. Pantuck et al.[17] found that in RCC the expression level of ribosomal protein S6, which is activated by p-p70S6K and reflects p70S6K activity, was significantly higher in metastatic patients and correlated with T stage. Recently, Zhou et al.[26] discovered that activation of p70S6K stimulated expression and proteolytic activity of matrix metalloproteinase-9 and cellular invasion in human ovarian cancer cells. In addition, Wan et al.[27] found that the mTOR/4E-BP1/p70S6K pathway was important in ezrin protein-mediated cancer metastatic behaviour, and suppression of 4E-BP1 and p70S6K by rapamycin led to decreased osteosarcoma cell migration and invasion. All these findings suggest that phosphorylation of 4E-BP1 and p70S6K by Akt/mTOR pathway might lead to protein synthesis, cell proliferation, cell-cycle progression, and increased invasive and metastatic ability of various cancer cells, including prostate cancer. It will be interesting to investigate in the future whether mTOR inhibitors which down-regulate both p-4E-BP1and p-p70S6K expression levels can inhibit prostate cancer cell invasion and metastasis in animal models.

In the present study high Gleason score tumours tended to have higher p-Akt and p-mTOR expression levels (Table 4). The correlation between tumour grade and p-Akt expression level was previously reported in prostate cancer [28–30]. The first study of the immunohistochemical detection of p-Akt using a phospho-specific antibody in paraffin-embedded human prostate cancer tissues was reported by Malik et al.[28]. They found that the staining intensity for p-Akt was significantly greater in Gleason score 8–10 tumours than in PIN and all other grades of prostate cancer [28]. After several years of follow-up in the same group of patients, Kreisberg et al.[29] reported that increased expression of p-Akt in prostate cancer was a very good predictor of biochemical recurrence after RP. Recently, the first study from an Asian country in this field was reported by Shimizu et al.[30], who found that increased expression of both Akt and p-Akt were associated with higher tumour grade as well as a higher androgen receptor staining score and Ki67 labelling index (the percentage of the nuclear area stained for Ki67) in Japanese patients with prostate cancer. All these findings and the present results suggest that p-Akt might be a molecular marker for malignant biological features of prostate cancer. Previously, the correlation between p-mTOR expression level and tumour grade in breast cancer was reported by Bose et al.[31]; they also discovered that patients with invasive breast carcinomas overexpressing p-mTOR had three times the risk of recurrence after operation [31]. After searching PubMed, we consider that the present study is the first to show a significant association between the p-mTOR expression and prostate cancer Gleason score. The clinical value of this association merit further investigation.

In conclusion, using simple and reproducible immunohistochemical staining, most patients with prostate cancer had at least one component of the mTOR signalling pathway activated. The frequent deregulation of this pathway in HGPIN and prostate cancer suggested that activation of mTOR pathway might be involved in prostate cancer development and progression. The association between activation of the mTOR pathway and patient clinicopathological variables suggested that not all patients with prostate cancer are equally amenable to treatment strategies targeting the mTOR pathway. The notion of using mTOR inhibitors as an additional treatment for patients with prostate cancer and a highly activated mTOR pathway is reasonable, and should be evaluated in future clinical trials.

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

This study was supported by grants from National Natural Science Foundation of China (30772162).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES