EXPRESSION OF ENDOTHELIN RECEPTOR A ASSOCIATED WITH PROSTATE CANCER PROGRESSION

0022-5347/01/1653-1033/0 THE JOURNAL OF UROLOGY® Copyright © 2001 by AMERICAN UROLOGICAL ASSOCIATION, INC.®

Vol. 165, 1033–1036, March 2001 Printed in U.S.A.

EXPRESSION OF ENDOTHELIN RECEPTOR A ASSOCIATED WITH PROSTATE CANCER PROGRESSION KAZUO GOHJI, SOHEI KITAZAWA, HIROSHI TAMADA, YOJI KATSUOKA

AND

MOTOWO NAKAJIMA

From the Department of Urology, Osaka Medical College, Takatsuki, Department of Pathology, Kobe University School of Medicine, Kobe and Biology Research, Tsukuba Research Institute, Novartis Pharma. K. K., Tsukuba, Japan

ABSTRACT

Purpose: We determined the role of endothelin receptors in prostate cancer progression. Materials and Methods: We examined 51 prostate cancer specimens obtained at surgery or biopsy for the relationship of endothelin receptor expression determined by immunohistochemical staining with malignant potential. Results: The positive staining rate of endothelin receptor A in the 51 specimens was significantly higher than of endothelin B (71% versus 24%, p ⬍0.0001). The staining rate of receptor A in Gleason score 5 to 10 disease was significantly higher than in Gleason 2 to 4 disease (91% versus 29%, p ⬍0.0001). The overall staining rate of endothelin receptor A in nonorgan confined disease without bone metastasis but with extraprostatic disease was 87% in 23 cases, including 16 of 19 stage T3 (84%) and all 4 stage T4 (100%) cases. This rate was significantly higher than that of organ confined cancer (29%, p ⫽ 0.0003). All patients with bone metastasis had positive staining for endothelin receptor A. An especially high rate of intensely positive staining was observed for endothelin receptor A in biopsy specimens with bone metastasis or Gleason sum 8 to 10. Moreover, positive staining was stronger in cancer cells penetrating the prostatic capsule than in those at the primary foci. However, the positive staining rate of endothelin receptor B was not significantly different in organ and nonorgan confined cancer without bone metastasis (12% versus 26%, p ⫽ 0.4284), bone metastatic and nonmetastatic cancer (20% versus 36%, p ⫽ 0.2619) or the Gleason sum groups (p ⫽ 0.0874). Conclusions: Our results indicate that endothelin receptor A expression may serve as a marker for and have an important role in prostate cancer progression. KEY WORDS: prostate; prostatic neoplasms; disease progression; receptors, endothelin; tumor marker, biological

Prostate cancer is the most common malignant neoplasm in men in the United States, where each year more than 40,000 die of the disease.1 The growth of prostate cancer cells is stimulated in vivo by several cytokines and growth factors, such as basic fibroblast growth factor, and insulinlike growth factors II and III.2– 4 The 3 subtypes of endothelin include endothelins 1 to 3.5, 6 Endothelin-1 is a potent vasoconstrictor initially isolated from endothelial cells7 and an important factor in the pathophysiology of prostate cancer.8 There are different high affinity endothelin receptors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and competitive displacement studies have revealed that the 2 binding sites, endothelin receptors A and B, are present throughout the prostate at a density exceeding that of cholinergic and adrenergic binding sites.9, 10 Receptor A has a high and equivalent affinity for endothelins 1 and 2 but little cross-reactivity with endothelin-3, whereas receptor B is nonselective with a similar affinity for the 3 subtypes.9 Higher expression of endothelin-1 is demonstrated in more rather than fewer malignant prostate cancer cell lines and the serum level in prostate cancer with bone metastasis is higher than in organ confined prostate cancer.8 However, the relationship of endothelin receptors A and B expression with the malignant potential of surgically resected prostate cancer remains unclear. We evaluated the relationship of endothelin receptor expression with human prostate cancer progression in surgically resected and biopsied prostate cancer tissue. Accepted for publication September 5, 2000.

MATERIALS AND METHODS

A total of 51 men 52 to 82 years old (median age 67) with prostate cancer treated from May 1993 to December 1997 at our institution were enrolled in the study. Patients with nodules on the prostate diagnosed by digital rectal examination or with elevated serum prostate specific antigen (PSA) underwent systematic prostate biopsy under transrectal ultrasound guidance.11 Pelvic computerized tomography, prostate magnetic resonance imaging and bone scintigraphy were done to determine disease extent. Of the 51 patients 40 with clinically organ confined disease underwent retropubic radical prostatectomy without previous hormonal therapy, while the remaining 11 with bone metastasis underwent hormonal therapy with luteinizing hormone releasing hormone agonist only. Tumor differentiation and disease extent were determined by the Gleason and 1997 TNM classifications, respectively. In the 40 clinically organ confined cases tumor was stage pT1N0M0 in 3, pT2N0M0 in 14, pT3N0M0 in 11, pT4N0M0 in 2 and pT3, 4N1 to 2M0 in 10. Of the 51 patients overall Gleason sum was 2 to 4 in 17, 5 to 7 in 24 and 8 to 10 in 10. Before collecting prostate tissue we obtained informed patient consent to use the samples in future experimentation. Formalin fixed and paraffin embedded prostate tissue sections were prepared from radical prostatectomy (40 primary lesions) and biopsy (11 bone metastases) specimens. Polyclonal antibodies were raised against endothelin receptors A and B, as described by Sasaki et al.12 Briefly, peptides corresponding to the carboxyl termini 403 to 427 and 405 to 427 of human endothelin receptors A and B, respectively, were used for immunizing the rabbits. Each peptide was coupled to

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EXPRESSION OF ENDOTHELIN RECEPTOR A IN PROSTATE CANCER PROGRESSION

keyhole limpet hemocyanin through cysteine at the amino terminus and antiserum was raised in Japanese White rabbits. In the 25 amino acids of the C-terminal regions of endothelin receptors A and B there was only 28% identity. The C-terminus (23 amino acids) of receptor B was completely conserved in humans, rats, cows and mice. The C-terminus (25 amino acids) of human endothelin receptor A had 92%, 88% and 84% identity against that of bovine, porcine and rat endothelin receptor A, respectively. Therefore, the antibodies produced had subtype specificity and crossreactivity across various species. Serum was affinity purified with gel coupled with the respective peptide. Antibody specificity has previously been demonstrated by Western blot analysis of astrocytes from normal and endothelin receptor B deficient aganglionosis rats.12 Brain tissue and cultured astrocytes were used as controls in immunohistochemical studies, as described by Sasaki et al.12 Sections were stained with rabbit polyclonal antibodies raised against human endothelin receptors A and B by an immunoperoxidase method using a commercially available avidin-biotin complex kit. Briefly, 6 ␮m. paraffin embedded tumor sections were deparaffinized in xylene and rehydrated in a series of graded alcohol (100%, 95%, 70% and 50%). To inhibit endogenous peroxidase sections were immersed in methyl alcohol containing 0.3% hydrogen peroxide for 20 minutes, blocked with 5% normal rabbit serum in phosphate buffered saline (PBS), pH 7.4, at room temperature for 1 hour, washed 3 times in PBS, augmented with rabbit antihuman endothelin receptor A or B antibody (1:200 in 5% blocking serum PBS) and incubated overnight at 4C. Nonimmunized rabbit IgG was applied as a negative control to a matched set of slides. After the incubation period sections were treated with biotinylated goat antirabbit IgG (1:200 in 5% blocking serum) and incubated for 1 hour at room temperature before being washed 3 times in PBS between subsequent steps. Sections were subjected to horseradish peroxidase-streptavidin at a 1:100 dilution in PBS, incubated for 45 minutes and further incubated sequentially with 3-amino-9-ethylcarbazine in 0.1 M. sodium acetate buffer, pH 5, and 0.03% hydrogen peroxide for 20 minutes at room temperature. Sections were then counterstained with hematoxylin for 1 minute, washed in distilled water, dehydrated through a series of graded alcohols and mounted in crystal mounting solution. Under low power microscopy we selected several fields with cancer cells that stained positive more strongly and we evaluated at least 1,000 cells in high power fields. The amount of positive staining was classified semiquantitatively according to the incidence of immunoreactive positive tumor cells as negative—less than 25%, fairly positive—26% to 50%, positive—51% to 75% and intensely

positive—more than 76%. We determined the difference in subjects in the positive staining rate of endothelin receptors A and B using the chi-square test with p ⬍0.05 considered significant. RESULTS

The positive and intensely positive staining rates of endothelin receptors A and B in prostate cancer tissue obtained at surgery or biopsy were 71% and 24%, respectively (see table). The rate of endothelin receptor A was significantly higher than that of receptor B (p ⬍0.0001, A and B of figure). The A receptor was localized in the cytoplasm of prostate cancer cells (A of figure). The rate of intensely positive staining for receptors A and B in prostate cancer cells was 51% and 6%, respectively, with the former significantly higher than the latter (p ⬍0.0001). The table shows relationships among the degrees of positive staining, pathological stage and Gleason sum. Lymph node metastasis was not pathologically confirmed in 11 men who did not undergo radical prostatectomy due to bone metastasis. The staining rate of endothelin receptor A in Gleason score 5 to 10 disease was significantly higher than in Gleason 2 to 4 disease (91% versus 29%, p ⬍0.0001, A and C of figure). The overall staining rate of endothelin receptor A in 23 cases of nonorgan confined disease without bone metastasis but with extraprostatic disease (capsular penetration and seminal vesical invasion) was 87%, including stage T3 in 16 of 19 (84%) and stage T4 in 4 of 4 (100%). This rate was significantly higher than the 29% in organ confined cancer (p ⫽ 0.0003). The rate of intensely positive staining for endothelin receptor A in nonorgan confined prostate cancer without bone metastasis was 65%, including stage T3 disease in 11 of 19 cases (58%) and stage T4 in 4 of 4 (100%). This incidence was also higher than in organ confined disease (p ⫽ 0.0011). All patients with bone metastasis had positive staining for endothelin receptor A, including about a third with additional positive staining for endothelin receptor B. We observed a high rate of intensely positive staining for endothelin receptor A, especially in biopsy specimens with bone metastasis or Gleason sum 8 to 10 disease. Moreover, positive staining of cancer cells penetrating the prostatic capsule was stronger than that of cancer cells growing at the primary foci (D of figure). However, the positive staining rate of endothelin receptor B in nonorgan confined stage pT3, 4N0 to 2 M0 disease was not significantly higher than that in organ confined stage pT1, 2N0 M0 disease (26% versus 12%, p ⫽ 0.4284). The intensely positive staining rate of endothelin receptor B in nonorgan confined cancer without bone metastasis was also not significantly higher than in organ confined cancer (p ⫽

Positive staining rates of endothelin receptors A and B in patients with prostate cancer Endothelin Receptor A No. Pts.

Stage: Organ confined Nonorgan confined Lymph node metastasis: Neg. Pos. Unknown Bone metastasis: Neg. Pos. Gleason sum: 2–4 5–7 8–10 Totals

No. Pos. (%)

p Value (chi-square test)

No. Intensely Pos. (%)

0.0003

Endothelin Receptor B p Value No. Pos. p Value (chi-square test) (%) (chi-square test) 0.0011

17 23

5 (29) 20 (87)

2 (12) 15 (65)

30 10 11

17 (57) 8 (80) 11 (100)

40 11

25 (63) 11 (100)

17 24 10

5 (29) 21 (80) 10 (100)

⬍0.0001 Vs. 5–10

2 (12) 15 (63) 9 (90)

51

36 (71)

⬍0.0001 Vs. B

26 (51)

0.0201 Vs. pos.

11 (37) 6 (60) 9 (82)

0.0216

0.4284 2 (12) 6 (26)

0.0306 Vs. pos.

5 (17) 3 (30) 4 (36)

0.0385 17 (43) 9 (82)

⬍0.0001 Vs. B

0.4987

0.3633 Vs. pos

1 (3) 1 (20) 1 (9)

0.2619

1 (6) 7 (29) 4 (40) 12 (24)

p Value (chi-square test)

0 2 (9)

8 (20) 4 (36) 0.0001 Vs. 5–10

No. Intensely Pos. (%)

0.6496 Vs. pos. 0.5256

2 (5) 1 (9) 0.0874 Vs. 5–10

0 1 (4) 2 (20) 3 (6)

0.0912 Vs. 5–10

EXPRESSION OF ENDOTHELIN RECEPTOR A IN PROSTATE CANCER PROGRESSION

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Immunohistochemical staining of endothelin receptors in prostate cancer. A, cytoplasm of Gleason sum 4 neoplastic cells stained positive for anti-endothelin receptor A antibody. Reduced from ⫻400. B, neoplastic cells stained negative for anti-endothelin receptor B antibody. Reduced from ⫻400. C, stage D2, Gleason sum 8 prostate cancer stained intensely positive for endothelin receptor A. Reduced from ⫻400. D, endothelin receptor A in moderately differentiated, Gleason sum 4 prostate cancer cells penetrating prostate capsule (arrow). Cancer cells penetrating capsule stained more strongly than those at primary foci (P). Reduced from ⫻100.

0.4987). Also, the positive and intensely positive rates of endothelin receptor B in bone metastatic cancer were not significantly different from those in nonbone metastatic disease (36% versus 20%, p ⫽ 0.2619 and 9% versus 5%, p ⫽ 0.5256, respectively). There was no significant difference in the positive staining rate of endothelin receptor B according to Gleason sum (p ⫽ 0.0874). DISCUSSION

The endothelin family has received a great deal of attention since endothelin-1 was first measured in extremely high amounts in the seminal fluid of men who underwent vasectomy.13 Prostate cancer cells need several growth factors to grow, such as basic fibroblast growth factor, insulinlike growth factor-1, hepatocyte growth factor and epidermal growth factor.2– 4 Endothelin-1 is also a significant factor in prostate cancer growth.8 The human prostate epithelium produces a relatively high level of endothelin-1 and metastatic prostate cancer cells secrete ectopic endothelin-1 into the surrounding stroma and blood stream. The amount of endothelin-1 secreted by DU-145 cells after 24 hours is approximately 600-fold that of LNCaP cells, as measured by enzyme-linked immunosorbent assay.8 Endothelin-1 influences cell proliferation directly and has potent synergy with many of the same peptide growth factors implicated in advanced prostate cancer progression.2, 14 Plasma immunoreactive endothelin concentrations are significantly elevated in men with metastatic prostate cancer, while human prostate cancer cells produce endothelin-1 messenger RNA and secrete immunoreactive endothelin in vitro.8 Also, exogenous endothelin-1 is a prostate cancer mitogen in vivo that increases alkaline phosphatase activity in new bone formation at metastatic sites, suggesting that ectopic endothelin-1 may

be a mediator of the osteoblastic response of bone to metastatic prostate cancer.8 The distribution of endothelin receptors A and B in prostatic tissue has been somewhat confusing. Receptors A and B have been identified within prostate tissue with the former predominating in fibromuscular stroma and the latter predominating in the epithelium.8, 15, 16 On the other hand, the zonal distribution of these receptors differs in normal human prostates. In the central zone the main subtypes in the fibromuscular stroma and epithelium are endothelin receptors A and B, respectively.17 In the peripheral zone the opposite is true with receptor A detected on the epithelium, while in the transition zone each receptor subtype is present only in the stroma.17 Moreover, an autoradiography study has shown that in the epithelium of benign prostate hypertrophy (BPH) tissue the binding of endothelin receptor B is significantly higher than that of receptor A, although in the stroma the binding of receptor A is significantly higher than that of receptor B.18 In our study epithelium in BPH expressed endothelin receptor B more intensely than receptor A. However, the stroma expressed each receptor to a similar degree (data not shown). Since we focused on the role of endothelin receptors in prostate cancer progression (invasion and metastasis), which is a late event in cancer growth, we did not determine the expression of these receptors in BPH or precancerous disease (prostatic intraepithelial neoplasia), which may be an initial event in prostate cancer development. In vitro experiments with cell lines have shown that the endothelin receptor A. selective receptor antagonist A-127722 inhibits endothelin-1 stimulated prostate cancer cell growth but the endothelin receptor B selective antagonist BQ-788 does not.18 Receptor B binding predominates in human benign prostatic epithelial tissue but it is not present in

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EXPRESSION OF ENDOTHELIN RECEPTOR A IN PROSTATE CANCER PROGRESSION

metastatic prostate cancer. Furthermore, the expression of endothelin-1 and endothelin receptor A is retained during the progression to metastasis, whereas that of receptor B is decreased.18 The mechanism of endothelin-1 stimulated DNA synthesis through endothelin receptor A or B has been postulated.19 Endothelin-1 activates mitogen activated protein kinase through endothelin receptor A or B and, hence, stimulates DNA synthesis.19 The human lung cell line CCD18Lu has endothelin receptors A and B.20 Endothelin-1 induced DNA synthesis is mediated predominantly through receptor A coupling to pertussis toxin insensitive G protein, postulating that the stimulation of receptor B coupling to pertussis toxin sensitive G protein does not act on the initiation of cell growth directly, but rather modulates receptor A mediated DNA synthesis.20 Our examination of the expression of endothelin receptors A and B in surgically resected and biopsied prostate cancer tissues by immunohistochemical analysis revealed that they localize in the cytoplasm of prostate cancer cells and receptor A expression is higher than that of receptor B. Endothelin receptor A expression was significantly higher in high stage, high grade than in low stage, low grade prostate cancer. In prostate cancer with bone metastasis we noted the highest rate of intensely positive staining for receptor A. Moreover, the positive staining of cancer cells penetrating the prostatic capsule was stronger than that of cancer cells growing at the primary foci. However, endothelin receptor B expression in high grade, high stage prostate cancer was not significantly different from that in low grade, low stage cancer. In addition, receptor B expression in prostate cancer with and without bone metastasis was not significantly different. The results of this study with surgically resected materials suggest that endothelin receptor A may have an important role in prostate cancer invasion and metastasis. CONCLUSIONS

Our results indicate that the expression of endothelin receptor A may be important in prostate cancer cell progression. They suggest that endothelin-1 produced by prostate cancer tissue may stimulate cancer cell growth through the predominantly expressed endothelin receptor A via autocrine pathways. Further investigations of the roles of endothelin-1 and endothelin receptor A in prostate cancer growth, invasion and metastasis are ongoing at our laboratory. REFERENCES

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