Elsevier

Human Pathology

Volume 37, Issue 11, November 2006, Pages 1442-1451
Human Pathology

Original contribution
Evaluation of urokinase plasminogen activator and its receptor in different grades of human prostate cancer

https://doi.org/10.1016/j.humpath.2006.05.002Get rights and content

Summary

Increased expression of urokinase plasminogen activator (uPA) and its receptor (uPAR) has been reported in various malignancies including prostate cancer (CaP). However, their expression in the different grades of CaP remains poorly understood. Here, we use tissue microarrays to examine the expression of uPA and uPAR in different grades of human CaP and to establish the potential of these tumor-associated antigens as candidates for targeted therapy. One hundred twenty paraffin-embedded specimens were selected from patients who underwent radical retropubic prostatectomy or transurethral resection of the prostate for primary untreated CaP and 10 matched lymph node metastases. Monoclonal antibodies #394 and #3936 were used on tissue microarrays with standard immunohistochemistry to examine uPA and uPAR expression, respectively. Overexpression of uPA and uPAR was detected in 53% and 64% of primary CaP tissues, respectively, and in more than 90% of lymph node metastases, but not in normal prostates or benign tissues. Of the uPA and uPAR positive tumors, 76% and 68% were Gleason score 7 or higher, respectively, and most of these tumors also showed stromal staining. The overexpression of uPA and uPAR was highly related to tumor differentiation in patients with CaP. Both uPA and uPAR proteins are candidate therapeutic targets for cancer therapy to control micrometastases and hormone refractory disease in CaP.

Introduction

Prostate cancer (CaP) is the most commonly diagnosed malignancy and the second leading cause of cancer mortality in males in the United States [1]. The high rate of mortality is associated with widespread metastatic disease. The exact mechanisms involved in CaP progression remain unclear. Several proteolytic enzyme systems are reported to be involved in the degradation of the extracellular matrix (ECM) and basement membrane. Among them, the urokinase plasminogen activator (uPA) system is believed to play a key role in tissue degradation, cell migration, angiogenesis, cancer invasion, and metastasis [2].

uPA is a member of the serine protease family and is strongly implicated as a promoter of tumor progression in various human malignancies [3], including CaP [4], [5]. The uPA is synthesized and secreted as a proenzyme, the activation of which is markedly accelerated upon binding with high affinity (∼0.1-1 nmol/L) to specific cell surface uPA receptors (uPAR). The density of receptors varies depending on cell type (∼103-106 sites per cell). When bound to uPAR, uPA efficiently converts the inactive zymogen, plasminogen, into the active serine protease, plasmin, which then directly or indirectly cleaves ECM components including laminin, fibronectin, fibrin, vitronectin, and collagen [6]. In addition, plasmin can activate latent elastase and matrix metalloproteinases (MMPs), potent enzymes that can also digest a variety of ECM components. Overwhelming evidence demonstrates that the cell surface–associated uPA/uPAR complex is causatively involved in tumor invasion and metastasis of many types of cancers by exerting multifaceted functions via either direct or indirect interactions with integrins, endocytosis receptors, and growth factors [3].

The activity of uPA is physically regulated by plasminogen activator inhibitors type 1 (PAI1) and 2 (PAI2) and by uPAR [3]. Both PAI1 and PAI2 belong to the serine protease inhibitor superfamily and form sodium dodecyl sulfate–stable 1:1 complexes with uPA [7], [8]. Plasminogen activator inhibitor 1 is a single-chain glycoprotein with an Mr of 43 kd and is thought to be the primary inhibitor of uPA, whereas PAI2 exists in 2 forms, that is, a 47-kd intracellular nonglycosylated form and a 60-kd extracellular glycosylated form [8]. Interaction of PAI1 with the uPA/uPAR complexes leads to internalization of the ternary complex, which then stimulates cell proliferation. The process of internalization degrades both uPA and PAI1, but uPAR is recycled to the cell surface [9]. In contrast, previous studies have indicated that the trimeric PAI2/uPA/uPAR complex is not internalized but is processed on the cell surface [9], [10]. However, a recent study found that the efficient and rapid formation of uPA-PAI2 complexes was associated with specific and rapid internalization of PAI2, which could be localized within endosomes and lysosomes, and that PAI2 binding capacity and internalization were uPA-dependent [11].

The serum levels of uPA and uPAR are moderately elevated in individuals with benign prostate hyperplasia (BPH) and significantly elevated in patients with CaP [4], [5]. The elevated serum levels of uPA and uPAR correlate directly with the serum level of prostate-specific antigen (PSA) and the development of CaP metastasis and inversely correlate with the overall survival rate among patients with CaP [12]. Consistently, the density of uPA and uPAR in prostate tumor tissues is significantly higher than that in prostate tissues from healthy individuals [13].

The increased expression of uPA and uPAR in patients with cancer suggests these proteins as suitable candidates for targeting novel therapeutic agents to prevent cancer invasion and metastasis. Although overexpression of uPA and uPAR has been reported in a limited number of CaP specimens [14], [15], the distribution of uPA and uPAR produced by normal prostatic tissues, prostatic carcinomas, and metastatic lesions, especially in different grades of CaP, has not been reported. In this study, we selected a uniform group of patients with primary untreated CaP of different Gleason scores and some matched lymph node metastases. We investigated tissue localization, CaP grade, and expression levels of uPA and uPAR to establish the potential of these proteins as targets for cancer-targeted therapy.

Section snippets

Tissues and clinical data

With informed consent, a total of 120 CaP tissues were obtained from patients treated for clinically localized CaP by radical retropubic prostatectomy (RRP) or transurethral resection of the prostate at Urology Sydney, St George Private Hospital, from 2000 to 2005. Normal controls (n = 40) were obtained from patients with normal biopsies or from the morphologically normal parts of CaP tissue. Ethical approval was obtained from the South East Health Human Research Ethics Committee, South

Expression of uPA and uPAR in normal and benign prostate tissues

The immunostaining results are summarized in Table 1. Normal prostate tissues from the control group were negative for uPA expression (Fig. 2A), whereas 15% (6/40) were weakly positive for uPAR expression (Fig. 2B). The positive staining for uPAR in normal prostates was located in epithelial cells, but not in stromal cells, and was homogeneous.

In the BPH group, 27% (4/15) and 47% (7/15) of benign tissues were weakly positive for uPA (1+) (Fig. 2C) and uPAR (Fig. 2D), respectively. The positive

Discussion

In the present study, we examined the expression of uPA and uPAR proteins in CaP, PIN, benign tissues, and normal prostates using TMAs. Overexpression of uPA and uPAR was observed in advanced CaP specimens but not in normal and benign prostate tissues. Most of the lymph node metastases and the matched primary cancer tissues expressed uPA and uPAR. To our knowledge, this is the first study to report uPA and uPAR expression in different grades of CaP, especially in lymph node metastasis, in a

Acknowledgments

The authors thank Sarah Eggleton (Garvan Institute of Medical Research, Sydney, Australia) for her technical assistance in preparing the TMAs and Lisa Marie (Urology Sydney) for her help in data collection. Specific thanks to the staff in Urology Sydney, St George Private Hospital, NSW, Australia, for tumor specimen collection and to Professor J. Kearsley and Professor B. J. Allen for their ongoing support.

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    This study was sponsored by a grant from the U.S. Department of Defense Prostate Cancer Research Program (Y. L.) (New Investigator Award: W81XWH-04-1-0048).

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