Thrombospondin-1, vascular endothelial growth factor expression and their relationship with p53 status in prostate cancer and benign prostatic hyperplasia

Authors


Abstract

Objective To evaluate the expression of thrombospondin-1 (TSP-1, a potent inhibitor of angiogenesis) and vascular endothelial growth factor (VEGF, an important angiogenic factor in solid tumours) in prostate cancer, and their relationship with p53 status.

Patients and methods Using immunohistochemistry, the expression of VEGF, TSP-1 and p53 was assessed in 82 archival tissue specimens from 23 patients with benign prostatic hyperplasia (BPH), 22 with localized prostate cancer and 37 with metastatic prostate cancer. Seven of the last group had received androgen deprivation therapy. The relationship between the expression of VEGF, TSP-1 and p53 status was also evaluated with tumour grade and stage in patients with prostate cancer.

Results The seven patients receiving hormonal treatment were excluded from the analysis because androgen deprivation significantly increased TSP-1 and decreased VEGF expression (both P < 0.01). Immunohistochemical analysis showed significantly higher VEGF and significantly lower TSP-1 expression (both P < 0.01) in prostate cancer than in BPH tissues. There was also significantly higher VEGF and significantly lower TSP-1 expression (both P < 0.05) in tissues from metastatic than localized prostate cancer. There was no significant correlation between VEGF or TSP-1 expression and Gleason score, but a significant inverse correlation between TSP-1 and VEGF expression. There was a significant association between VEGF expression and p53 status (P < 0.05), but TSP-1 expression was not associated with p53 status.

Conclusions Angiogenic factors, including VEGF and TSP-1, might be important in the development and progression of prostate cancer. These changes seem to be influenced by p53 status. Identifying the angiogenic factors involved in prostate cancer might lead to the development of diagnostic or therapeutic strategies based on anti-angiogenesis.

Introduction

Angiogenesis is the growth of new blood vessels from existing vessels and the mechanism by which tumours induce a blood supply [1]; it is thought to be important in tumour progression and metastasis. Recent work suggests that the tumour angiogenic switch is triggered by a change in the balance of stimulators to inhibitors within a given microenvironment [2].

Prostate adenocarcinoma is a significant cause of morbidity and mortality in older men, and the urological malignancy where angiogenesis may have some role in the development and progression of the tumour. The microvessel density (MVD) is higher and vascular networks more disorganized in malignant than in normal prostate [3,4]. Siegal et al.[5] showed a sequential increase in MVD toward the prostate tumour, with a doubling in malignant compared with benign tissue. However, there is little information about angiogenic factors in the development and progression of prostatic tumour. The most studied angiogenic inducers in prostate cancer are basic fibroblast growth factor and vascular endothelial growth factor (VEGF). However, there are conflicting results for VEGF in prostate cancer, and little evidence supporting the importance of angiogenic inhibitors in regulating angiogenesis in prostate cancer.

Thrombospondin-1 (TSP-1) is a 450 kDa adhesive glycoprotein that was initially discovered in platelets, where it is sequestered within the platelet α granule [6]. In addition to being involved with haemostasis as a component of the platelet α granule, TSP-1 is synthesized and secreted by many normal and transformed cells in culture [7]. There is strong evidence to support an inhibitory role for TSP-1 in cancer cell proliferation and metastasis. Direct evidence for the inhibitory effect of TSP-1 in cancer cell proliferation and metastasis comes from studies in murine melanoma and human lung and breast cancer cell lines, where an inverse correlation was reported between TSP mRNA and protein expression, and malignant progression [8]. Furthermore, transfection studies show that tumour cell production of TSP-1 inhibits tumour progression or metastases [9,10]. These studies strongly suggest that TSP-1 may have a tumour suppressor function. While the mechanism by which TSP-1 has this effect is unknown, it is possible that it may act partly by inhibiting tumour angiogenesis. Campbell et al.[11] suggested that down-regulation of TSP-1 secretion was a key event in the switch from an anti-angiogenic to an angiogenic phenotype, which occurred early in the development of bladder cancer.

p53 has been shown to increase the expression of the major angiogenic inhibitor, TSP-1 [8,12,13], and to decrease the expression of the major angiogenic stimulator VEGF [14,15]. However, the details of this interaction between p53 and TSP-1 and/or VEGF have not yet been determined.

The aim of the present study was to analyse the expression of TSP-1, VEGF and p53 in malignant and benign prostatic tissues, using immunohistochemistry, to investigate the role of TSP-1 in angiogenesis in the prostate and the relationship between TSP-1 and VEGF expression with p53 status, and to assess whether the p53 product regulates the level of TSP-1 or VEGF expression.

Patients and methods

Sections were taken from 82 archival specimens collected from December 1995 to August 1999 from patients with BPH and prostatic adenocarcinoma treated at Seoul National University Hospital, Korea. The patients (mean age 68.6 years, range 47–93) comprised 23 with BPH treated by TURP, 22 with localized prostatic cancer treated by radical prostatectomy and 37 with metastatic prostatic cancer who underwent a TRUS-guided biopsy or TUR-channelling. Seven of the 37 patients with metastatic prostate cancer underwent androgen deprivation therapy (four by orchidectomy and three by LHRH analogue). Two pathologists, independently from the original pathology report, selected the representative formalin-fixed, paraffin-embedded tissue blocks. The clinical data were collected from the patients' records. The primary tumours were staged according to the Whitmore-Jewett system, and graded histologically according to Gleason's scoring system. There were 22 patients with localized (stage B1, B2 or C1) disease and 37 with metastatic (stage D2) disease. The histopathological grade was 3–10; nine were low-grade (Gleason score leqslant R: less-than-or-eq, slant 6) and 50 high-grade (Gleason score of geqslant R: gt-or-equal, slanted 7) tumours.

Immunohistochemical analysis

The unstained sections (4 µm thick) of archival, formalin-fixed, paraffin-embedded tissue were cut and mounted on silane-coated slides (DAKO, Japan). Anti-TSP-1 antibody (Ab-1) and anti-VEGF antibody (Ab-3) were both obtained from Oncogene Research Products (Cambridge, MA), and anti-p53 antibody (DO7) from DAKO (Carpinteria, CA, USA); all were mouse mAbs. In all experiments, the anti-TSP-1 antibody was used at 0.5 µg/mL, the anti-VEGF at 1.0 µg/mL, and the anti-p53 at a dilution of 1 : 1000.

The optimal immunohistochemical staining procedure for TSP-1 involves antigen retrieval using a low-pH buffer with high-temperature microwave heating [16]. All the routinely processed paraffin tissues were deparaffinized with iodine–xylene and rehydrated with graded ethanol solutions. To block endogenous peroxidase activity, the sections were treated with 3% hydrogen peroxide–methanol for TSP-1 staining, or with 0.3% hydrogen peroxide in PBS for VEGF and p53 staining. The slides were then washed with tap water, followed by distilled water and immersed in a 0.1 mol/L Tris–HCl buffer solution (pH 1.0) for TSP-1 staining, or a 0.01 mol/L sodium citrate buffer (pH 6.0) for VEGF and p53 staining. Antigens were then retrieved using microwave heating for 5 min at the highest power setting, after which the slides were cooled for 15 min and 5% normal goat serum (DAKO, USA) used to block nonspecific binding in the tissue sections for 20 min. The primary antibody was incubated overnight at 4 °C for TSP-1 and p53 staining, or incubated for 2 h at 37 °C for VEGF staining. Subsequent immunostaining was carried out by streptavidin peroxidase (DAKO) and 3–3′ diaminobenzidine for TSP-1 and p53 staining, or 3-amino-9-ethylcarbazole for VEGF staining. The primary antibody was replaced with non-immune sera for the negative controls. The positive controls were samples of normal kidney for VEGF and endometrial tissue sections for TSP-1. The sections were counterstained with haematoxylin and eosin in all experiments. The immunohistochemistry for experimental and control specimens was conducted under identical conditions.

Two pathologists unaware of the clinical outcome independently evaluated the degree and pattern of immunostaining, using the classification system developed by Grossfeld et al.[16]. Only extracellular immunoreactivity was considered positive for TSP-1, because cytoplasmic and nuclear reactivities were believed to represent an artefact of the antigen retrieval process [16]. Light microscopy was used to evaluate the intensity and location of the immune reaction; very few cases showed discordant readings.

TSP-1 immunostaining was graded as negative or positive, based on the extent of extracellular immunoreactivity. Tissue sections were classified as having negative TSP-1 expression when they showed no or negligible/equivocal immunoreactivity in the intratumoral or immediate peritumoral areas. Tissue sections with detectable TSP-1 immunoreactivity were classified as having positive TSP-1 expression for the statistical analysis when they showed moderate or high immunoreactivity in the intratumoral or immediate peritumoral areas. Cytoplasmic immunoreactivity was considered positive for VEGF reactivity. Tissue sections were classified as having negative VEGF expression when < 10% of the cells showed positive immunoreactivity, and as positive when > 10% of the cells showed VEGF immunoreactivity. Immunohistochemical staining for p53 was considered negative when there was < 20% and as positive with geqslant R: gt-or-equal, slanted 20% reactivity, and only where there was nuclear staining.

For the statistical analysis the patients were divided into two groups, i.e. positive and negative, based on the intensity of TSP-1 immunoreactivity and the percentage of positive VEGF and p53 immunoreactivity. The chi-square test, Fisher's exact test and Student's t-test were used to evaluate the relationships among the variables, as appropriate, with P values based on two-sided testing and P< 0.05 regarded as statistically significant.

Results

In the seven patients with metastatic prostate cancer treated by androgen deprivation, VEGF immunoreactivity was significantly lower and TSP-1 immunoreactivity significantly higher (both P < 0.01), but p53 immunoreactivity was not significantly different. Because such hormonal therapy could potentially influence the immunoreactivities of VEGF and TSP-1 these seven patients were excluded from further analysis.

Of the 75 remaining patients (mean age 68.6 years, range 47–93), 23 had BPH, 22 localized (stage B1, B2, or C1) prostate cancer and 30 metastatic (stage D2) disease. The histopathological grade was 3–10, with nine low-grade (Gleason score leqslant R: less-than-or-eq, slant 6) and 43 high-grade (Gleason score geqslant R: gt-or-equal, slanted 7) tumours.

TSP-1 immunoreactivity was detected in 18 (78%) of 23 BPH and in 17 (33%) of 52 prostate cancer specimens (Table 1). TSP-1 immunoreactivity was significantly lower in malignant than in benign prostate, as determined by the chi-square test for linear trends (P < 0.01). The presence of TSP-1 was confirmed by its characteristic extracellular staining pattern (Fig. 1). The BPH tissues showed strong immunohistochemical staining for TSP-1 confined to the stroma (Fig. 1a). The peritumoral stromal tissue of metastatic prostate cancer showed moderately positive TSP-1 immunoreactivity (Fig. 1b). No staining was detected in negative control samples (data not shown).

Table 1.  TSP-1, VEGF and p53 expression in BPH and prostate cancer
GroupNo. of patients (%) by expression level ofTotal
TSP-1VEGFp53
negativepositivenegativepositivenegativepositive
BPH5 (22)18 (78)20 (87)3 (13)22 (96)1 (4)23
Cancer35 (67)17 (33)18 (35)34 (65)45 (87)7 (13)52
Total40 (53)35 (47)38 (51)37 (49)67 (89)8 (11)75
Localized11 (50)11 (50)12 (55)10 (45)22 (100)022
Metastatic24 (80)6 (20)6 (20)24 (80)23 (77)7 (23)30
Gleason score
 Low (leqslant R: less-than-or-eq, slant 6)5 (56)4 (44)3 (33)6 (67)9 (100)09
 High (geqslant R: gt-or-equal, slanted 7)30 (70)13 (30)15 (35)28 (65)36 (84)7 (16)43
 Total35 (67)17 (33)18 (35)34 (65)45 (87)7 (13)52
Figure 1.

TSP-1 immunostaining in BPH and prostate cancer tissues. a, highly positive TSP-1 immunoreactivity in the stromal tissue of BPH (× 400); b, weakly positive TSP-1 immunoreactivity in the peritumoral tissue of metastatic prostate cancer (× 200).

VEGF immunoreactivity was detected in 34 (65%) of 52 prostate cancer specimens and in three (13%) of 23 BPH specimens (Table 1). VEGF immunoreactivity was statistically significantly higher in malignant than in benign prostate (P < 0.01). Positive staining for VEGF was cytoplasmic and the immunoreactivity ranged from negative to strongly positive (Fig. 2). The cytoplasm of renal tubular cells was stained in normal kidney, as a positive control, and no staining was detected in negative control samples (data not shown). Similar to the malignant prostate tissues, some BPH specimens contained foci of positively stained glands and stromal cells (data not shown). VEGF immunoreactivity was weak in the cytoplasm of tumour cells in localized prostate cancer (Fig. 2a). There was diffuse and highly positive VEGF staining in metastatic prostate cancer (Fig. 2b).

Figure 2.

VEGF immunostaining in BPH and prostate cancer tissues. a, weakly positive staining for VEGF in localized prostate cancer (× 200); b, diffuse and highly positive staining for VEGF in metastatic prostate cancer (× 100).

p53 immunoreactivity was detected in seven (13%) of 52 prostate cancer and in only one (4%) of 23 BPH specimens (Table 1). p53 immunoreactivity was not significantly higher in malignant than in benign prostate (P > 0.05). p53 expression was exclusively nuclear (Fig. 3) and extensively distributed in the tumour.

Figure 3.

Strong p53 immunoreactivity in a sample of metastatic prostate cancer tissue (× 200).

TSP-1 immunoreactivity was significantly higher in patients with localized (stage B1, B2, and C1) disease than in those with metastatic (stage D2) disease (P < 0.05; Table 1). VEGF and p53 immunoreactivity was significantly higher in metastatic than in localized prostate cancer tissue (both P < 0.05; Table 1). Analysis of the 52 patients with prostate cancer showed that TSP-1 and p53 were associated with the histological grade of cancer, but not significantly, whereas VEGF was not associated with histological grade (Table 1).

The mean (sd) preoperative serum PSA level in the TSP-1-negative group was 65.9 (103.0) ng/mL and higher than that of the TSP-1-positive group, at 33.4 (66.2) ng/mL, although the difference was not significant. The mean serum PSA serum level of the VEGF-positive group, at 80.9 (98.6) ng/mL, was significantly higher than that of the VEGF-negative group, at 22.6 (67.5) ng/mL (P < 0.01), and that of the p53-positive groups, at 118.5 (115.9) ng/mL, was higher than in the p53-negative group, at 43.7 (83.3) ng/mL, but again the difference was not statistically significant.

There was no significant difference between the mean preoperative haemoglobin or alkaline phosphatase levels in the TSP-1-, VEGF- or p53-positive groups and those in the corresponding negative groups (data not shown).

There was a significant inverse correlation between TSP-1 and VEGF protein expression (P < 0.05) in all specimens (Table 2). TSP-1 expression was not associated with p53 status, but VEGF expression was (Table 2).

Table 2.  The association between TSP-1, VEGF and p53 expression in whole specimens
GroupNo. of
patients
No. of patients (%) by expression level of
TSP-1VEGF
negativepositivePnegativepositiveP
VEGF expression
 Negative3815 (40)23 (60)0.015   
 Positive3725 (68)12 (32)    
p53 expression
 Negative6735 (52)32 (48)0.71637 (55)30 (45)0.028
 Positive85 (62)3 (38) 1 (12)7 (88) 

Discussion

Prostate cancer is the urological malignancy in which angiogenesis may have the greatest potential as a prognostic indicator and a therapeutic alternative, because any additional prognostic indicator to identify which patients to treat aggressively and which to treat conservatively will be beneficial. Anti-angiogenic strategies should offer new ways to treat hormone-resistant or chemo-insensitive prostate cancer. However, the exact role of angiogenesis in prostate cancer has not yet been determined.

Currently, angiogenesis is thought to be controlled by a balance between angiogenic stimulators and inhibitors rather than by the activity of a single regulator [2]. Solid tumours can express one or more of these angiogenic factors, which can work synergistically in promoting tumour growth. Angiogenesis can be studied by examining new vessel growth within tumour specimens, i.e. the MVD. The role of angiogenesis in the development and progression of prostatic malignancy is supported, in that the MVD is higher and the vascular networks more disorganized in malignant than in normal prostate [3,4]. In addition, angiogenesis can be assessed by analysing the levels of various angiogenic stimulators or inhibitors, because there is a dynamic balance between them.

TSP-1 is a large protein secreted by many cell types and is a potent inhibitor of neovascularization [17]. The inhibitory effect of TSP-1 in solid tumours has been confirmed by many [8–10,18], but the expression of TSP-1 in a benign or malignant prostate, or the effect of TSP-1 in prostate cancer, has not previously been reported. TSP-1 has been localized to the peritubular connective tissue of kidney, the basement membrane regions beneath glandular epithelium in skin and lung, the dermal-epidermal junction in skin, and the intestinal areas in skeletal muscle, confirming the extracellular location of TSP in tissues [19]. This is consistent with the present results, in which TSP-1 was primarily localized to the extracellular matrix. This is the first report showing that the expression of TSP-1 was significantly lower in prostate cancer than in BPH tissue (P < 0.01). Also, TSP-1 immunoreactivity was significantly lower in metastatic than in localized prostate cancer (P < 0.05). The present results suggest that TSP-1 could inhibit the development and progression of prostate cancer.

One of the most important angiogenic factors in a solid tumour is VEGF. Further evidence is required to confirm its precise role in prostate cancer because there are conflicting results for VEGF. One immunohistochemical study reported significantly higher levels of VEGF staining in prostate cancer cells [20], whereas others confirmed high levels of VEGF mRNA and protein in normal glands [21], and in BPH and malignant glands [22]. The present results showed that VEGF immunoreactivity was higher in prostate cancer (65%) than in BPH (13%); BPH tissues showed significantly less staining than malignant tissues (P < 0.01). VEGF immunoreactivity was significantly higher in metastatic (80%) than in localized prostate cancer (45%), suggesting that the expression of VEGF increases during the progression of prostatic malignancy.

The mean serum PSA level of the VEGF-positive group was significantly higher than that of the corresponding negative group, although the serum PSA level was not associated with TSP-1 expression. The Gleason score was ≤ 6 in nine patients (17%) and ≥ 7 in 43 (83%); TSP-1 expression tended to be lower and p53 expression higher in prostate cancer of high Gleason score, but these associations were not significant. However, there was no association between the expressions of VEGF and the histological grade of tumour. With only a few patients having a low Gleason score it was not possible to clarify the association between the expression of angiogenic factors and the histological grade of the tumour.

There are several studies in which untreated primary prostate cancer samples have been assessed for p53 accumulation or screened for specific mutations in p53. Most of these, including the two largest, concluded that the frequency of p53 accumulation is 10–20% in primary untreated prostatic carcinoma [23,24]. In the present study, p53 was over-expressed in 23% of metastatic but in no localized prostate cancer samples, which suggests that mutations of p53 could coincide with an aggressive form of prostate cancer. Dameron et al.[12] reported that wild-type p53 inhibited angiogenesis through the up-regulation of TSP-1 synthesis. In the present study, TSP-1 expression decreased in cases of p53 mutation, but this decrease was insignificant. In addition, Mukhopadhyay et al.[15] showed that VEGF gene expression was suppressed in an adenovirus-transformed human fetal kidney cell line by wild-type p53. The present results showed that VEGF expression was associated with p53 status. The tumour suppressor gene p53 could be important in controlling neoangiogenesis via TSP-1 or VEGF in the prostate.

There was an inverse association between TSP-1 and VEGF expression, suggesting that VEGF and TSP-1 could interact in the control of tumour angiogenesis, although the exact mechanism is unknown. Further investigations are needed to determine if the inverse association in the present study between TSP-1 and VEGF is the result of an interaction between them or the effect of an upstream inducer, e.g. p53, that could control the expression of TSP-1 and VEGF. That the expression of TSP-1 was lower and that of VEGF higher in prostate cancer than in BPH, and in metastatic than localized prostate cancer, may offer a basis for developing anti-angiogenic strategies useful for therapeutic intervention in prostate cancer, e.g. anti-VEGF antibody and TSP-1 transfection using a delivery system. Identifying the angiogenesis factors involved in prostate cancer and understanding their regulation could potentially lead to the development of anti-angiogenic strategies useful for diagnostic studies and therapeutic interventions in prostate cancer.

Acknowledgements

This study was supported in part by year 2000 BK21 project for Medicine, Dentistry and Pharmacy and in part by Korean Prostate Cancer Society.

S.E. Lee, 28 Yongon Dong Jongno Ku, Department of Urology, Seoul National University Hospital, Seoul, Korea 110–744.
e-mail: urology@snu.ac.kr

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