Patterns of Protease Production During Prostate Cancer Progression: Proteomic Evidence for Cascades in a Transgenic Model

Prostate Cancer and Prostatic Diseases (2003) 6, 272–280 & 2003 Nature Publishing Group All rights reserved 1365-7852/03 $25.00 www.nature.com/pcan Patterns of protease production during prostate cancer progression: proteomic evidence for cascades in a transgenic model

RA Bok1*, EJ Hansell2, TP Nguyen1, NM Greenberg3,JH McKerrow2 & MA Shuman1 1Department of Medicine, University of California, San Francisco, California, USA; 2Department of Pathology and Pharmaceutical Chemistry, University of California, San Francisco, California, USA; and 3Department of Cell Biology, Baylor College of Medicine, Houston, Texas, USA

Extracellular proteases are recognized as critical factors in the progression of a number of carcinomas, including prostate cancer. Matrix metalloproteases (MMP) are important in processes of tumor growth, invasion and dissemination, but other classes of proteases, such as serine and cysteine proteases, also contribute. We utilized the TRAMP model for prostate cancer to elucidate proteases involved in prostate cancer progression. General proteomic analysis was performed on normal murine prostate, early TRAMP tumors and advanced TRAMP tumors, as well as normal and involved lymph nodes. Zymography and antigenic analyses revealed increased expression of mainly pro-MMP in early TRAMP tumors but substantial elaboration of activated MMP only in late TRAMP tumors. Progressive increase in cysteine, serine and certain membrane-bound proteases from normal to early to advanced prostate tumors, was also seen. Our results implicate pericellular proteases as initiators of major proteolytic cascades during tumor progression and suggest targets for maximal therapeutic effect. Prostate Cancer and Prostatic Diseases (2003) 6, 272–280. doi:10.1038/sj.pcan.4500676

Keywords: proteases; prostate cancer; extracellur transgenic proteomics

Introduction the urogenital tract and this class of proteases is important in prostate biology. The urokinase plasmino- Pericellular proteolysis is thought to be an important gen activator (UPA) was originally isolated from human biochemical event in the progression of a variety of urine and is expressed in PCA specimens.4 Prostate- malignancies, including prostate cancer. Secreted or specific antigen (PSA) is a kallikrein-like serine protease surface-bound proteases from tumor cells or stromal now widely accepted as a useful serum marker for PCA elements help mediate the crucial steps of tumor detection and monitoring.5 Some investigators believe invasion, migration, metastasis and angiogenesis.1 In- that PSA plays a direct role in modulating prostate creased expression of a number of different proteases has cancer cell proliferation.6 A number of more recently been reported in human prostate cancer (PCA). Elevated described serine proteases—pKS2, TMPRSS2, MT-SP1— expression of several matrix metalloproteases (MMPs) have been implicated in PCA evolution, as well.7–9 has been noted in PCA cell lines and primary tumor Clinical investigations of serum levels have also shown specimens.2 Certain preclinical studies have demon- a significant association of the serine protease UPA and strated significant inhibition of xenographic prostate its receptor (UPAR) with PCA progression and prog- tumor growth after treatment with an MMP inhibitor.3 nosis.10 Previous studies in our laboratory have shown Serine protease production has long been associated with pronounced disruption of PCA tumor growth and dissemination by antagonists of this serine protease system.11 Recent studies by other groups also suggest a *Correspondence: R Bok 1270, Department of Medicine, UCSF potential therapeutic role for serine protease inhibitors in Medical Center, 3rd & Parnassus Avenues, San Francisco, CA 94143, 12 USA PCA. E-mail: [email protected] Although these reports on one or two individual Received 4 July 2003; revised 4 July 2003; accepted 4 July 2003 proteases at discordant disease stages are informative, a Patterns of protease production RA Bok et al 273 broader characterization of proteases in PCA at the cially or through collaborators at the University of proteomic level from early to advanced stages has been California San Francisco and Vanderbilt University. lacking. The importance of a more generalized character- Polyclonal antibodies (anti-sera) against MT-SP1 were ization is pointed up by the negative impact of selective raised in rabbits via immunization with synthetic protease inhibitors in recently reported human trials.13 peptides as previously described.15 Paraffin-embedded One hindrance to the elucidation of the role extracellular and frozen sections were cut on microtomes at 7–10 mm proteases play in prostate cancer has been the paucity of thickness. Paraffin sections were deparaffinized accord- human tissue specimens available for such analysis. ing to standard protocols. Frozen sections were generally Although human tissue banks are being established fixed with paraformaldehyde and cold methanol with for prostate cancer, the availability of specimens, from 0.3% H2O2 for quenching of endogenous peroxidases. A various stages for a variety of types of analyses, is still volume of 5% goat serum in phosphate-buffered saline limited. Within the past 5 y, transgenic animal models (PBS) was used as a blocking agent. Primary antibodies have become available, which in many ways, closely were incubated with specimens for 2–3 h; secondary mimic human adenocarcinoma of the prostate. We chose antibody was usually an anti-IgG : biotin conjugate. the TRAMP model developed by Greenberg et al.14 to Washings were carried out for 45–60 min with three carry out a general characterization of protease expres- changes of PBS. Detection was performed with strepta- sion at the protein level across a range of tumor tissue vidin : horseradish peroxidase complex, using DAB or specimens, with a variety of techniques. We are also AEC with H2O2 for substrates. Specimens were counter- investigating its use as a preclinical model for evaluating stained with hematoxylin alone or occasionally with novel protease inhibitors. Here we report our findings hematoxylin plus eosin (H&E). in TRAMP from zymography, immunohistochemistry (IHC), Western blotting, and fluorogenic substrate analyses. Our results show a general increase in overall Western analysis. Freshly dissected or frozen tissue proteolytic activity during tumor progression, especially samples from normal organs, primary tumors, tumor in the metalloprotease and cysteine protease classes. metastases, or cell line pellets were homogenized to However, particular species of proteases are modulated 4 mg/ml in ice-cold PBS with 0.1% Triton X-10. The in alternate ways during tumorigenesis. homogenate was centrifuged briefly at 41C to remove insoluble cellular elements. Equal amounts of protein were loaded from different samples into the wells of 7.5–12.5% SDS-polyacrylamide gels. Prestained molecu- Experimental procedures lar weight standards were also included on all gel electrophoreses. Subsequent transfer of protein bands to Transgenic mice and normal murine tissue Immobilon-P membranes (Millipore Corp., Bedford, MA) was usually carried out overnight at 41C. Mem- Generation of early and late TRAMP. Female TRAMP branes were blocked in a casein solution, then incubated mice were provided by NM Greenberg and crossed with with primary antibody, followed by incubation with normal, nontransgenic males to generate affected secondary antibody conjugated directly or indirectly to TRAMP males (C57BL6/FVBN mix). Progeny were peroxidase. Detection was carried out using ECL screened for the PB-Tag transgene by polymerase chain reagents (Amersham, Inc., Piscataway, NJ). reaction (PCR). Certain TRAMP males were allowed to develop only to about 15–16 weeks of age (‘early TRAMP’), when they were euthanized and tissues Protease activity assays harvested for analysis. Other transgenic males were allowed to mature and develop advanced pelvic tumors, Tissue extracts. Freshly dissected tissues or, occassion- with the mice generally killed in the 24–32 weeks of age ally, frozen tissue samples from time-matched prostate range (‘late TRAMP’), when they were in the terminal cancer sets (tumor, adjacent normal tissue, and liver phase of malignancy. Normal murine tissues were metastases stored at À701C), were homogenized to obtained from C57BL6 males that were wild type and 4 mg/ml in ice-cold PBS with 0.1% Triton X-100 using a negative for the PB-Tag transgene. Tenbrock homogenizer on ice. The homogenate was centrifuged in a microfuge at 16 000 g for 10 min at 41C. Protein concentration was determined by Bradford Tissue harvesting. Tissues were dissected immediately analysis using a bovine serum albumin (BSA) standard and processed into tissue extracts, frozen sections with (Bio Rad, Hercules, CA, USA). Pellets and aliquoted OCT, or formalin-fixed for subsequent paraffin embed- supernatants were stored at À701C. ding. Where possible, distinct lobes of the prostate (ventral, lateral, dorsal and anterior), as well as seminal vesicles, were isolated by microdissection. Abdominal and thoracic Protease activity assays. Fluorogenic peptide substrates organs were surveyed grossly for metastatic deposits. were used to assay the activity in the tissue extracts and Lymph nodes (LN) involved by metastases were generally control conditioned media. Release of fluorescent pro- evident due to their enlarged size and easy visibility. duct was followed at room temperature with a 96-well fluorometric plate reader (Fluoroskan II, LabSystems, Franklin, MA, USA). Histology and immunohistochemistry. Polyclonal and General cysteine protease activity was assayed (ex 366, monoclonal antibodies against murine proteases MMP- em 405) in 100 mM Na acetate, pH 6.5, containing 10 mM 2, MMP-3, MMP-9, MT1-MMP were obtained commer- DTT (Buffer A) using either Cbz-Phe-Arg_AMC or

Prostate Cancer and Prostatic Diseases Patterns of protease production RA Bok et al 274 Cbz-Arg-Arg-AMC at 20 mM (Bachem (King of Prussia, digestion (clear bands against a dark background), gels PA, USA) from 10 mM stocks in dimethylsulfoxide were stained in 0.5% (weight per volume, w/v) (DMSO)). Controls of extract buffer alone, bovine Coomassie blue R250 (Sigma) in 10% (v/v) acetic acid, cathepsin B (Sigma, St Louis, MO, USA) and recombi- 50% (v/v) methanol for 30 min at room temperature on a nant cruzain (a cathepsin-L-like protease) were included. rocking platform, followed by destaining in 10% (v/v) Specificity of activity was tested by preincubating acetic acid and 40% (v/v) methanol. samples for 30 min at room temperature with the general cysteine protease inhibitor E64 (Sigma; 20 mM stock in DMSO) at 10 mM, or the specific cathepsin B inhibitor CA-074 (a gift of Taisho Pharmaceutical Co., Ltd Results (Santama, Japan); 200 mM stock in DMSO) at 1 mM, or Protease analysis in normal murine prostate DMSO at equivalent % (volume per volume, v/v) alone. General serine protease activity was assayed in 50 mM Zymography of normal murine prostate revealed only Tris-HCl, pH 8.2, 150 mM NaCl, 5 mM CaCl2 (Buffer B) occasional, faint bands of gelatinolytic activity, which using 100 mM Cbz-Arg-Arg-AMC. Specificity of activity contrasts strikingly to the activity seen in TRAMP tumors was determined by preincubating the samples for 30 min (Figure 1a). These faint bands showed up at molecular in 1 mM phenylmethylsulfonyl fluoride (PMSF) (100 mM size/weight positions consistent with the proforms of stock in DMSO, Sigma), 10 mM E64 or DMSO alone. UPA MMP-9 and MMP-2 (pro-MMP-9 and pro-MMP-2). Pro- activity was assayed with 20 mM glutaryl-Gly-Arg-AMC forms of MMPs are readily activated by the SDS (Bachem) in 50 mM Tris, pH 8.8, 50 mM NaCl with 0.01% denaturation process of zymography and so display Tween 80. Controls of human high molecular weight gelatinolytic activity in this assay. Overall, therefore, UPA (American Diagnostics, Greenwich, CT, USA), and minimal levels of protein for these MMPs or their conditioned media from a human prostate cell line (PC3) precursor forms are detectable in normal murine prostate. were included. PSA-like activity was assayed with IHC also shows minimal levels of expression of MMP- 200 mM morpholine carboxyl-His-Ser-Ser-Lys-Leu-Gln- 9 and MMP-2 in normal murine prostate (Figure 3). AMC (20 mM stock in DMSO, Enzyme Systems Products, Stromelysin or MMP-3, on the other hand, is readily Livermore, CA, USA) in Buffer B. Specificity of activity apparent in the stromal compartment of normal prostate, was determined by preincubating samples with PMSF localizing about fibroblast or smooth-muscle cells. MMP- and E64 inhibitors (as described above). 2 staining is also positive in scattered stromal cells in MMP activity for MMP-1 (collagenase I) and MMP-9 normal murine prostate, but otherwise is largely absent (gelatinase B) was assayed with 5 mM DNP-pro-beta- from the prostatic epithelial layer. cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-Lys-(N-Me-Abz)- With regard to the serine protease class, moderate NH2 (ex 365, em 450; 10 mM stock in DMSO; Bachem) in levels of MT-SP1 are seen in normal prostate, but only Buffer B. Cytotrophoblast CM (CTB CM) was used as an minimal levels of UPAR staining are evident in normal activity control. prostate. Surprisingly, Western analysis showed enhanced UPA production in normal dorsal–lateral prostate relative to later stages of TRAMP prostate (Figure 4a). Zymography. Protease activity was detected in 10–15 ml In fact, a gradual decrease in UPA expression with tumor samples from extracts, following separation on SDS- progression was indicated. MT-SP1 and MT1-MMP are polyacrylamide mini-gels copolymerized with 1 mg/ml not readily detectable in normal murine prostate by type I gelatin or 1 mg/ml casein (Novex, Carlsbad, CA). Western, although MT-SP1 did appear to be present by Plasminogen activator acitivity was determined on IHC to some degree and localized to the epithelial layer. zymograms copolymerized with 1 mg/ml casein (Sigma) Fluorogenic substrate assays were also performed on and 13 mg/ml plasminogen (American Diagnostics). normal murine prostatic tissue. These studies showed After electrophoresis, zymogram gels were washed two that low-to-moderate levels of cysteine protease activity times for 15 min in 2.5% Triton X-100 to reactivate (primarily cathepsin B) are present in normal murine enzymes by removal of SDS. Gels were rinsed for prostate (Figure 5). 30 min in developing buffer from Novex, followed by 15–24 h incubation at 371C on a rocking platform in developing buffer. Developing buffer consisted of 50 mM Proteases in early TRAMP tumors Tris-HCl, pH 8.2, 150 mM NaCl, with 5 mM CaCl2.To demonstrate specific protease activities, samples were In comparison to normal prostate, significantly more preincubated for 30 min with class-specific inhibitors. intense bands of activity are seen in gelatin zymography These included 1 mM PMSF (Sigma; 100 mM in DMSO), of early TRAMP prostate/tumor extracts (Figure 1). The 1 mM 1,10-orthophenanthroline (Sigma; 100 mM in major band at B98 kDa is consistent with pro-MMP-9, DMSO), 1 mM E64 (Sigma; 100 mM in DMSO), DMSO but less prominent bands of pro-MMP-2 are also seen (Sigma) at equivalent % (v/v), or 10 mM recombinant (Figure 1b). IHC for MMP-9 also indicates a significant Ecotin (gift of C Craik, University of California, San increase and diffuse distribution of this protease Francisco, CA, USA). Activities were compared with throughout the prostatic gland of early TRAMP. The conditioned media from cell lines with known protease antibody employed for this staining cannot distinguish activities: first-trimester cytotophoblast (CTB) cells med- pro-MMP-9 from its activated-form, but the zymo- ia with MMP-2 and MMP-9 activity (CTB, a gift of Susan graphic analysis indicates that the inactive species is Fisher, University of California, San Francisco, CA, USA) the major form present in early TRAMP tumors. Western and human prostate cancer line (PC3) with UPA and blotting confirmed the identity of this B98 kDa band MMP-2, and MMP-9 activity. To detect areas of protease as pro-MMP-9 (data not shown). A different MMP,

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Figure 1 Gelatin zymography of tissues from normal C57BL/6 male mice (a) and from various tissues of advanced TRAMP males involved with tumor (b), including ventral prostate (VP), dorsal–lateral prostate (DLP), anterior prostate (AP), seminal vesicles (SV), lymph nodes (LN) of mesenteric (Mes), pelvic (Pel), and aortic (Aor) location, as well as gross primary tumor (Tum). Gelatin zymography of tissues from early and late TRAMP tissues: (c) isolated individual prostatic lobes from an early stage and late stage TRAMP male—AP, lateral prostate (LP), VP, dorsal prostate (DP), as well as SV; (d) side-by-side comparison of early and late TRAMP (TR) extracts from LP and SV. Additional samples from involved LNs are also shown.

Prostate Cancer and Prostatic Diseases Patterns of protease production RA Bok et al 276 matrilysin (MMP-7), shows focal areas of increased Proteases in late TRAMP tumors expression by IHC (Figure 3), as do certain serine protease members, MT-SP1 and UPAR. The areas of increased In advanced or late TRAMP tumor tissues, a most staining for these proteases appear to correspond to striking finding was that the activated forms of the major niduses of epithelial transformation/hyperproliferation, MMPs (MMP-9 and MMP-2) predominate in zymo- supporting the supposition that upregulation of certain graphic analysis (Figure 1b). There is therefore a clear proteases is concomitant with tumor development. shift from the latent, proform in early TRAMP to the Western analysis of early TRAMP tumor extracts active protease in late TRAMP, especially with respect to indicates that expression of a membrane-type MMP, MMP-9. Figure 2 shows zymography of LN specimens MT1-MMP, is increased relative to its level in normal from various advanced TRAMP mice. These dissected prostate (Figure 4e). The serine protease UPA (another LNs were all abnormally enlarged and easily identifiable membrane-localized protease), in contrast, shows de- at necropsy, indicating pathological involvement. As creased expression in early TRAMP tumors relative to with primary prostate tumor extracts, a general upregu- normal prostate. The respective trends in expression lation of protease activity is seen. Pro-MMP-9 and level continue into late TRAMP for these two proteases activated-MMP-2 bands appear prominently in most of (see below). the metastatic LN specimens, but the most notable band Fluorogenic substrate analysis for cysteine protease of increased intensity corresponds to a o50 kDa species, activity in early TRAMP revealed a level of activity for which is a molecular size suggestive of UPA. Analysis of this class of protease that is very similar to that seen in these same specimens in casein–plasminogen zymo- normal murine prostate (Figure 5). By use of the selective grams, along with Western analysis of LN metastases inhibitor CA074, the most prominent component of this from other advanced TRAMP animals (see Figure 4b), cysteine protease activity was shown to be cathepsin B substantiated this identification. A fair amount of (B80%). Therefore, cathepsin B activity in particular or heterogeneity is seen between the different TRAMP LN cysteine protease activity in general does not appreciably specimens. This may reflect, in part, the contribution increase in the early stages of TRAMP tumor develop- of inflammatory reactions in the involved LNs. It should ment. be mentioned that gelatin zymography reveals only a

Figure 2 Comparison of various LN samples from normal mice and advanced TRAMP (TR) that had metastases: (a) single zymogram of LN samples from pelvic, mesenteric, and periaortic locations; (b) second zymogram, allowed to develop for shorter period, of LN samples from two advanced TR—renal, gastric, pelvic, and aortic areas. (c) Western blotting for UPA (urokinase) in involved LN from late TRAMP mice. Broad range of bands in the aortic LN may be indicative of substantial amounts of unactivationed, pro-UPA and various intermediates. The lower molecular weight, heavy band (arrow head; o50 kDa) probably corresponds to the prominent band of activity seen in zymograms. Normal LN extract is also included for comparison.

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Figure 3 Detection and localization of various proteases in normal, early and late TRAMP prostate tissue sections by IHC with DAB staining. Progressive loss of normal glandular architecture from normal to early to late stage is evident. See text for further discussion. Counterstain was performed with hematoxylin plus eosin in late TRAMP sections of MMP-9 and UPAR and with hematoxylin alone in other sections. Arrows indicate areas of specific, punctate DAB staining.

certain number of metalloproteases, that is, those with reported alternative transcript for murine UPAR, termed gelatinolytic activity, so other MMPs (such as MT1- muPAR2,17 and is probably a secreted variant. MMP) could be present and just not detected under these Fluorogenic substrate analysis of late and early assay conditions. TRAMP primary prostate tumors and involved SV By IHC, the pattern of staining for MMP-9 becomes specimens showed that cysteine protease activity is more diffuse in advanced tumor lesions and appears about 10-fold higher than serine protease activity (data to be present on or around both stromal and tumor not shown). A comparison of normal, early TRAMP and elements in the anaplastic tissue (Figure 3, bottom panel). late TRAMP prostate specimens (see Figure 5) indicates MMP-2 staining, in contrast, appears to be localized that, while overall cysteine protease activity is not to the stroma in advanced TRAMP tumor. This is enhanced in early TRAMP tumors relative to normal, consistent with previous reports on the localization cysteine protease activity is significantly elevated in late of this MMP in other tumor types.16 Similar to MMP-9, TRAMP tumors (B2.5-fold). In late TRAMP tissue, as in the serine protease MT-SP1 displays diffuse staining normal and early TRAMP, 80% of this activity is over both epithelial and stromal components in ad- attributable to cathepsin B, with 20% attributable to vanced TRAMP. For UPAR, small but distinct foci of cathepsin-L-like proteases. staining are seen in advanced TRAMP tumor—similar to the pattern seen in early TRAMP, but more limited in size. These could represent foci of inflammatory cell infiltrates, but morphology of the cells in these foci is not Discussion obviously different from adjacent tumor cells. No expression of MMP-3 could be detected by IHC in The TRAMP transgenic mouse is an autochthonous advanced TRAMP tumor, even in stromal elements, model for adenocarcinoma of the prostate, which reason- indicating downregulation of this particular MMP in ably recapitulates major features of human prostate late-stage disease. cancer. Histological hallmarks, similar to those seen in By Western analysis, UPA appears to decrease further the human gland, ranging from early prostatic intrae- in expression and is largely absent from advanced pithelial neoplasia (PIN) like lesions to well-differen- tumors in late TRAMP mice (Figure 4a). MT-SP1 was tiated carcinoma to poorly differentiated carcinoma, readily detectable in late TRAMP tumor extracts by have been identified in this animal model. For these Western blotting (Figure 4c), corroborating the IHC initial studies, we focused on a few easily distinguishable findings for this membrane-tethered protease. MT1- stages of disease to evaluate broadly the changes in MMP, which was minimally expressed in normal murine several different classes of protease. The data, obtained prostate and moderately expressed in early TRAMP, by various enzymatic and antigen detection techniques shows continued increased expression in late TRAMP from the different stages and tissues, are organized and tumor by Western analysis (Figure 4e). Interestingly, summarized in Table 1. Our investigations reveal a UPAR, the GPI-membrane anchored receptor for UPA, significant increase of functionally active proteases, in a shows a significant shift in molecular weight form in late sequential, nonlinear pattern across time, for several TRAMP (Figure 4b). The size of the major UPAR antigen categories of protease. However, a progressive decline in detected by Western corresponds well to the previously certain proteases is also evident.

Prostate Cancer and Prostatic Diseases Patterns of protease production RA Bok et al 278

Figure 4 Western analysis for various cell-surface-associated proteases across tumor stage. (a) UPA in normal murine prostate (Nl DLP), early and late TRAMP (TR) tumors, and normal ovary (positive control). After initial blotting for UPA, membrane was reprobed for actin to check the level of total protein loading per lane. (b) Western analysis for UPAR in extracts from a human PCA (PC3) and a rat PCA (MatLyLu) cell line compared to late TRAMP tumor extract. The heavily glycosylated MW 55–60 kDa human receptor is evident in the PC3 lane. Rat UPAR shows a size very similar to that of the human receptor. TRAMP tumor seems to express primarily the alternative splice variant muPAR2 (see text and refer Kristensen et al17), with an MW of 26–28 kDa, although fainter bands of higher MW forms are evident. (c) Western analysis for MT-SP1 in normal murine prostate and late TRAMP tumor samples. Expected activation fragment of MT-SP1 is evident with higher percentage gels. (d) Initial Western analysis for MT1-MMP in normal prostate, early and late TRAMP tumor, as well as normal ovary (positive control) and aorta. (e) Additional Western analysis of four late TRAMP tumors for MT1– MMP, under nonreduced and reduced conditions, showing moderately high levels of protease in three out of four tumors.

Prostate Cancer and Prostatic Diseases Patterns of protease production RA Bok et al 279 Table 1 Semiquantitative summary of protease expression at protein level in normal, early and late TRAMP prostate tumors

Normal Early TRAMP Late TRAMP LN Met

pMMP-9/2 — +++ ++ + aMMP-9/2 — 7 +++ + MT-MMP1 7 +++NA

UPA + 7 — ++++ MT-SP1 7 +++NA

Cat-B/L ++ ++ +++ NA

An assessment of enzymatic and/or antigenic levels was made for each of the indicated proteases, based on the overall results from zymography, IHC, Western and/or fluorometric assay. Scores from negative (undetectable) to 4+ (high expression) were assigned (NA=not available).

Figure 5 Cysteine protease activity in normal, early TRAMP and late TRAMP prostate/tumor tissues, as determined by fluorogenic (Z-Phe-Arg- for prostate cancer has also been suggested by other AMC) substrate. Group mean values indicate similar levels of cysteine rodent model studies.11 In other instances, alternative protease activity in normal and early TRAMP tissues, but a clear increase transcription products of a protease factor may come into in late TRAMP tumor. Four to six mice were assayed for each stage; play, for example, the lower molecular weight form of individual mouse tumors were assayed in duplicate. Group means are depicted with standard errors. Additional assays with CA-074, a cathepsin UPAR detected on Western. Perhaps such a shift in B inhibitor (data not shown), indicate that 80% of activity is attributable expression to a smaller, soluble form of this protease to cathepsin B with the other 20% attributable to cathepsin-L-like receptor explains the shift to more limited, punctate proteases. staining seen by IHC in advanced TRAMP tumor. The major, low molecular weight species of UPAR seen in advanced TRAMP tumor extracts by Western blotting A general upregulation of proteolytic activity in suggests that the alternative spliced transcript for UPAR tumors has been appreciated for decades.18 However, is more abundantly expressed in later stages of prostate in vivo investigations of changes in protease expression tumor development. This alternative spliced variant during prostate tumor progression have been lacking. probably corresponds to a secreted, non-cell-surface- Our analysis in TRAMP also reveals a general upregula- associated form of UPAR.17 This observation in the tion of proteolytic activity during prostate tumor transgenic mouse model is consistent with the recent progression. Members within the MMP, serine protease, reports of Miyake et al,10 who found significant correla- and cysteine protease classes are all increased in prostate tions between serum levels of UPAR/UPA and metas- tumors from this murine model. However, some distinct tases and survival in PCA patients. In prostate and phases in the upregulation of certain proteases, as lesions genitourinary physiology more broadly, the serine progress from early, well-differentiated to late, undiffer- proteases appear to be a very important class of protease. entiated masses, are seen. Inactive or proforms of the This probably reflects the secretory function of the gland. major MMPs (MMP-9 and MMP-2) are substantially Urokinase, one of the best studied plasminogen activa- increased in early TRAMP tumor specimens, but their tors, was first isolated from urine. PSA, a kallikrein-like activated forms predominate mainly in late TRAMP serine protease, is a remarkably specific prostate secre- tumors. Similarly, cysteine proteases, as detected by tory product and has been validated as a diagnostic and functional fluorogenic substrate assays, display sharp prognostic marker for PCA. increases in levels of activity in the more advanced Of particular interest, especially with regard to drug TRAMP tumors. These findings indicate an initial build- targeting, is our observation that certain membrane- up of inactive precursors in the early tumor stage, with a bound proteases appear prominently and early in subsequent cascade of activation in late tumors, for these prostatic carcinoma relative to normal prostate. MT-SP1 major classes of proteases. and MT1-MMP are both proteases with classical, hydro- At the same time, distinct instances of downregulation phobic transmembrane domains. Their surface localiza- are also evident, for example, MMP-3 or stromelysin. The tion has been confirmed by immunofluorescent latter finding could be reflective of a tumor-suppressive staining15 and indicates a function in upstream, imme- role for this particular MMP in prostatic carcinoma. diately perimembrane proteolytic events (see Figure 6). Indirect evidence for a tumor-suppressive function of Several members of the membrane-associated pro- stromelysin-related proteases has been reported.19 Alter- teases—MT-SP1, MT1-MMP, and UPAR/UPA—are natively, MMP-3 may be important only in very early either already present or upregulated early in the tumor phases of tumor initiation, as was suggested in studies of development process in TRAMP. As these are all a murine mammary carcinoma model by Sternlicht et al.20 activators of other proteases in the pericellular/extra- UPA activity also appears to decline in advanced, cellular environment, their elaboration early in the anaplastic primary tumors, although it appears substan- transformation process may be important temporally tially increased in metastatic LN deposits. These results for the subsequent wave of metalloprotease and cysteine suggest distinct roles for this plasminogen activator in protease activity seen in advanced tumors. MT1-MMP is the metastatic vs primary tumor growth process. The a known activator of pro-MMP-2, and a coordinated importance of increased UPA in the metastatic process expression of these two proteins has been demonstrated

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