Expression of the Disintegrin Metalloprotease, ADAM-10, In

314 Vol. 10, 314–323, January 1, 2004 Clinical Cancer Research

Expression of the Disintegrin Metalloprotease, ADAM-10, in Prostate Cancer and Its Regulation by Dihydrotestosterone, Insulin-Like Growth Factor I, and Epidermal Growth Factor in the Prostate Cancer Cell Model LNCaP

Daniel R. McCulloch,1 Pascal Akl,1 Conclusions: This study describes for the first time Hemamali Samaratunga,2 Adrian C. Herington,1 the expression, regulation, and cellular localization of and Dimitri M. Odorico1 ADAM-10 protein in PCa. The regulation and membrane 1 localization of ADAM-10 support our hypothesis that Hormone-Dependent Cancer Program, School of Life Sciences, ADAM-10 has a role in extracellular matrix maintenance Queensland University of Technology, Brisbane, Queensland, Australia, and 2Sullivan Nicolaides Pathology, Brisbane, Queensland, and cell invasion, although the potential role of nuclear Australia ADAM-10 is not yet known.

INTRODUCTION ABSTRACT The disintegrin metalloproteases ADAMs, like the matrix Purpose: The disintegrin metalloprotease ADAM-10 is metalloproteinases (MMPs), are members of the metzincin a multidomain metalloprotease that is potentially significant (zinc-dependent metalloprotease) superfamily. To date, more in tumor progression due to its extracellular matrix-degrad- than 30 ADAMs have been characterized (1), some of which are ing properties. Previously, ADAM-10 mRNA was detected involved in diverse biological functions such as fertilization, in prostate cancer (PCa) cell lines; however, the presence of neurogenesis (2, 3), and the ectodomain shedding of growth ADAM-10 protein and its cellular localization, regulation, factors such as amyloid precursor protein and tumor necrosis and role have yet to be described. We hypothesized that factor ␣ (4, 5). The ADAMs and MMPs share a homologous ADAM-10 mRNA and protein may be regulated by growth metalloprotease catalytic motif (HEXGHNLGXXHG), which is factors such as 5␣-dihydrotestosterone, insulin-like growth responsible for zinc-dependent protease activity (6). In particu- factor I, and epidermal growth factor, known modulators of lar, ADAM-10 has been shown to have substrate specificity PCa cell growth and invasion. overlap with the MMPs, particularly with respect to the degra- Experimental Design: ADAM-10 expression was ana- dation of extracellular matrix (ECM) components. For example, lyzed by in situ hybridization and immunohistochemistry in purified bovine ADAM-10 (synonym, MADM) cleaves native prostate tissues obtained from 23 patients with prostate type IV collagen (7), a major component of the basement disease. ADAM-10 regulation was assessed using quantita- membrane and the ECM. Thus, like the MMPs in aggressive tive reverse transcription-PCR and Western blot analysis in cancer cell types (8), ADAM-10 displays potential ECM remod- the PCa cell line LNCaP. eling capabilities. However, relatively little is known about the Results: ADAM-10 expression was localized to the se- association and roles of ADAM-10 in cancer. To address this, cretory cells of prostate glands, with additional basal cell the present study has examined the expression and hormonal expression in benign glands. ADAM-10 protein was predom- regulation of ADAM-10 in prostate cancer (PCa). inantly membrane bound in benign glands but showed Although many cancers of the prostate are slow-growing marked nuclear localization in cancer glands. By Western and may remain organ-confined for many years, a significant blot, the 100-kDa proform and the 60-kDa active form of number become highly aggressive and generate primary metas- ADAM-10 were synergistically up-regulated in LNCaP cells tases to the bone and lymph nodes. Growth of both the normal treated with insulin-like growth factor I plus 5␣-dihydrotes- prostate and PCa cells is regulated by key growth factors in- tosterone. Epidermal growth factor also up-regulated both cluding androgens, insulin-like growth factor (IGF)-I, and epi- ADAM-10 mRNA and protein. dermal growth factor (EGF) (9). Hence, these three growth factors are key promoters of PCa development. Because members of the MMP family have been shown to be responsive to similar growth factors involved in cancer progression (10, 11), Received 5/30/03; revised 9/24/03; accepted 10/6/03. and given the evidence for functional homology between the Grant support: Grants from the Queensland University of Technology ADAMs and MMPs, we hypothesized that the ADAMs may be and the National Health and Medical Research Council of Australia. The costs of publication of this article were defrayed in part by the regulated in a manner similar to that of the MMPs. payment of page charges. This article must therefore be hereby marked Previously, we described the mRNA expression of five advertisement in accordance with 18 U.S.C. Section 1734 solely to ADAMs in several PCa cell lines (12). In the same study, we indicate this fact. found that the androgen 5␣-dihydrotestosterone (DHT) up- Requests for reprints: Dimitri M. Odorico, Hormone-Dependent Can- cer Program, School of Life Sciences, Queensland University of Tech- regulated the levels of ADAM-10 mRNA in the androgen- nology, GPO Box 2434 Brisbane, Queensland 4001, Australia. Phone: sensitive PCa cell line LNCaP. We now report regulation and 617-3864-5215; Fax: 617-3864-1534; E-mail: [email protected]. cellular localization of expression of this ADAM, at the protein

level, in prostate tissue sections and in the PCa cell line LNCaP. 37°C). Protease activity was subsequently inhibited by incuba- Our findings suggest IGF-I and DHT together or EGF alone tion in 0.1 M glycine (2 ϫ 5 min at room temperature). Neutral increases ADAM-10 protein expression. Furthermore, in situ buffered formalin (10%, 30 min at room temperature) was added hybridization and immunohistochemistry identified the glandu- to re-fix the tissues. A further dehydration in 70%, 90%, and lar epithelial cells as the predominant ADAM-10-expressing 100% ethanol was performed. The slides were dried for hybrid- cell type in the prostate. No major difference in intensity was ization. Slides were immersed overnight (at 50°C) in hybridiza- observed, but a definitive shift in localization from the cell tion solution [10% dextran sulfate, 1ϫ Denhardt’s solution, membrane to the nucleus occurred as the pathology progressed 50% formamide, 4ϫ SSC (pH 7.0)], 500 ␮g/ml herring sperm from a benign to a cancerous state. DNA, and 200 ␮g/ml tRNA with a 150 ng/ml final concentration of digoxigenin-RNA probe. MATERIALS AND METHODS Washes of increasing stringency were performed [4ϫ SSC, Antibodies 15 min at room temperature; 4ϫ SSC, 2 ϫ 20 min at 50°C, 2ϫ ϫ ϫ The rabbit anti-ADAM-10/KUZBANIAN/MADM poly- SSC, 3 20 min at 50°C; 1 SSC, 10 min room temperature; ϫ ϫ clonal antibody, purchased from Chemicon International (Te- 0.5 SSC, 10 min room temperature; 0.1 SSC, 10 min room mecula, CA), was raised against a COOH-terminal peptide temperature]. Specific binding of the probe to the target RNA corresponding to amino acids 732–748 of human ADAM-10. was detected immunologically by anti-digoxigenin alkaline The anti-␤-tubulin monoclonal antibody was purchased from phosphatase antibody using nitroblue tetrazolium chloride/X- Lab Vision (Fremont, CA). The anti-proliferating cell nuclear phosphate 5-bromo-4-chloro-3-indolyl-phosphate (Roche) as antigen monoclonal antibody was purchased from Zymed (San the chromogenic substrate solution. The sections were then Francisco, CA). counterstained with 1% nuclear fast red and mounted in perma- nent aquamount (Dako, Carpinteria, CA). Chromogenic markers Tissue and Sample Preparation and bright-field microscopy were used for in situ localization of Archival paraffin-embedded prostate tissue blocks from 17 ADAM-10 mRNA. patients with PCa [14 patients with medium-grade disease (Gleason scores of 6–7), 3 patients with high-grade disease Immunohistochemistry (Gleason score of 8–10)], and 6 patients with benign prostatic Procedure. H&E was used for routine histological ex- hyperplasia were obtained from Royal Brisbane Hospital amination. For ADAM-10 staining, antigen retrieval was per- (Queensland, Australia). Ethics approval was obtained from formed by heat pressure-cooking the sections in 0.01 M citrate institutional ethics committees. Tissue blocks were sectioned buffer (pH 6.0) for 3 min. The sections were incubated in 3% (3–5 ␮m) and mounted on 3-aminopropyl triethoxysilane- methanolic peroxide (13 min at room temperature) to block the coated slides. Sections were then dewaxed and rehydrated endogenous peroxidase activity present in the samples. Nonspe- through xylene, graded ethanol (100%, 90%, and 70%), and cific sites were blocked by incubation with 10% Blotto for 1 h water. at room temperature. The optimal antibody dilution (0.7 ␮g/ml) was applied and incubated overnight at 4°C. The immunoreac- In Situ Hybridization tive sites were detected using the Envision system (Dako). The cRNA Probe Preparation. An ADAM-10 cDNA tem- location of the antigen was revealed by addition of the diami- plate was generated by subcloning a 236-bp ADAM-10 reverse nobenzidine substrate-chromogen solution (5–10 min at room transcription-PCR product from LNCaP RNA. PCR was carried temperature), yielding a brown deposit. The nuclei were coun- out using a forward (5Ј-TGGATTGTGGCTTCATTGGTG-3Ј) terstained with hematoxylin. and a reverse (5Ј-TGCAGTTAGCGTCTCATGTGTC-3Ј) As a negative control for the immunostaining, the primary primer under the following conditions: 94°C for 4 min; followed antiserum was omitted from the staining schedule. ADAM-10 by 40 cycles of 94°C for 1 min, 55°C for 30 s (annealing), and primary antibody was also preabsorbed with a purified blocking 72°C for 30 s. The PCR product was ligated into a pGEM-T peptide (Chemicon) to detect nonspecific binding. Easy vector (Promega, Madison, WI), and the insert was con- Scoring. All tissue sections were analyzed in a single run firmed by sequencing. Both antisense and sense (control) cRNA to allow direct comparison of staining intensities. Microscopic transcripts were generated from each strand of the cloned se- analysis was performed in a blind fashion, several times, on quence using T7 or SP6 polymerases, respectively, using tem- different days. Two variables in immunostaining with the plates linearized by SalI and SacII, respectively. cRNA probes ADAM-10 antibody were assessed: (a) location (pattern); and were labeled with digoxigenin using the digoxigenin RNA la- (b) intensity of staining. Intensity on prostate tissue was scored beling kit protocol (Roche, Mannheim, Germany) according to as negative (Ϫ), weak (ϩ), moderate (2ϩ), strong (3ϩ), and the manufacturer’s instructions. very strong (4ϩ). Weak intensity staining was defined as stain- In Situ Hybridization Procedure. Tissue sections were ing that could be detected at ϫ10 objective magnification. Very deparaffinized through several washes in xylene, graded alco- strong intensity was the strongest color obtained, and moderate hols, and diethylpyrocarbonate-treated water. To increase the staining and strong staining were the two intensities detected accessibility of the target RNA, the tissues were treated by between very strong intensity and weak intensity staining. The hydrolysis with 0.2 M HCl (20 min at room temperature) fol- staining pattern was defined as uniform or heterogeneous, de- lowed by 0.3% Triton X-100 in PBS (15 min at room temper- pending on the variability of intensity within or between glands, ature) and proteolysis with proteinase K (10 ␮g/ml; 15 min, for benign or cancerous epithelium.

Cell Culture Real-Time PCR Analysis Characterization of ADAM-10 mRNA and Protein Ex- Reverse Transcription-PCR Procedure. Five ␮gof pression. The PCa cell line LNCaP was obtained from Amer- RNA extracted (TRIZOL reagent; Invitrogen) from control and ican Type Culture Collection (Manassas, VA) and cultured in hormone-treated LNCaP cells, from three independent experi- standard T25 and T75 culture flasks (Medos, Brisbane, Austra- ments, were reverse transcribed using Superscript II (Invitrogen) lia) in RPMI 1640 supplemented with 10% heat-inactivated fetal with oligo(dT) priming. All cDNA was found to be free of genomic DNA contamination by screening with ␤-actin primers bovine serum/gentamicin, in an atmosphere of 95% air/5% CO2 at 37°C. Routine tests for Mycoplasma infection were found to that span an intron (12). PCR was carried out on a PE Biosys- be negative. tems 7000 ABI Prism using the following thermocycling protocol (95°C for 10 min, 40 cycles of 95°C for 30 s and 60°C for Cell Proliferation Assay Using the 3-(4,5-Dimethylthiazol- 1 min), and the fluorescence emitted from the SYBR green dye 2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Dye Method. was read by the instrument after the completion of each ther- A 90% confluent cell culture of LNCaP was treated with tryp- mocycle. The following reaction conditions were used: 1ϫ sin/versine and seeded into a 96-well tissue culture plate (Me- SYBR Green master mix (Applied Biosystems, Foster City, dos) at 10,000 cells/well in 200 ␮l of RPMI 1640/10% heat- CA), 50 nM each of ADAM-10 primers (forward, 5Ј-TCCA- inactivated fetal bovine serum/phenol red-free/gentamicin CAGCCCATTCAGCAA-3Ј; reverse, 5Ј-AGGCACTAGGAA- medium. Cells were allowed to attach for 24 h, washed with 200 GAACCAA-3Ј) or 18S rRNA primers (forward, 5Ј-TTCGGAA- ␮ l of warm (37°C) PBS (tissue culture grade; Oxoid, West CTGAGGCCATGAT-3Ј; reverse, 5Ј-CGAACCTCCGACTTTC- Heidelberg, Australia), and serum starved for 24 h in serum-free GTTCC-3Ј, housekeeping gene); and 1 ␮l of cDNA template in RPMI 1640/phenol red-free/0.01% BSA/gentamicin medium. a final reaction volume of 20 ␮l. Each growth factor treatment Test medium containing DHT (Sigma, St. Louis, MO) at con- was run in triplicate. The 18S housekeeping gene (used to centrations of 0 (control) 1.0, and 10 nM Ϯ 50 ng/ml human normalize ADAM-10) was always quantitated in the same PCR, recombinant IGF-I (Gropep, Adelaide, Australia), 50 ng/ml in triplicate, for each treatment in a 96-well PCR plate format. IGF-I alone, or human recombinant EGF (Gropep) alone [0 Quantitation of ADAM-10 mRNA Transcript. Each (control), 5, 10, and 50 ng/ml) was added and replaced every primer set was validated for use in quantitation using the “ct- 24 h for a period of 96 h. A total of 16 wells/concentration were value” direct comparison method as per the manufacturer’s used. Each replacement of medium included a warm wash with (Applied Biosystems) recommendations. Briefly, cDNA from 200 ␮l of PBS. The MTT dye method was carried out as LNCaP cells was serially diluted and subjected to a PCR under described previously (12). Briefly, a standard curve was con- the same conditions described above. The ct value was graphed 2 Ͼ structed by seeding doubling increases in LNCaP cell number against cDNA concentration, and an R value of 0.97 was into a 96-well tissue culture plate (Medos) in phenol red-free/ obtained, showing that these primer sets could effectively detect 10% heat-inactivated fetal bovine serum/RPMI 1640. The cells relative changes of the target message (ADAM-10 or 18S) in the were allowed to plate down for 6 h, followed by treatment with cDNA to be used for subsequent message level quantitation. MTT. Absorbance was plotted against cell number to ensure Western Blot Analysis linearity of the MTT assay across the range of cell numbers used Whole Cell Protein Extraction. LNCaP cell pellets (4,000–40,000 cells). The absorbances obtained at the comple- were resuspended in 500 ␮l of lysis buffer [50 mM Tris (pH 7.5), tion of the proliferation assays were extrapolated to cell number 150 mM NaCl, 1% Triton X-100, 0.1 mM phenylmethylsulfonyl using this standard curve. Statistical analysis was carried out by fluoride, 10 mM EDTA (pH 8.0), and 1 tablet of Roche Com- one-way ANOVA followed by a Tukey post hoc analysis to plete EDTA-free protease inhibitor mixture/25 ml]. The cell determine significance between all treatments and respective lysate was frozen on dry ice, thawed in ice-cold water, and then controls. passed twice through a 26-gauge needle. The cell lysate was ADAM-10 Regulation. The LNCaP cell line was cul- centrifuged at 12,000 ϫ g for 30 min at 4°C, and the resultant tured in T75 culture vessels to 70% confluence under the con- supernatant was collected. Whole cell protein was quantitated in ditions described above. The cells were transferred into serum- triplicate using the BCA protein assay reagent (Pierce, Rock- free RPMI 1640/0.01% BSA/gentamicin for 24 h. Test medium ford, IL) as per the manufacturer’s “microwell” protocol. For was then added to the cells after a warm wash (37°C) with PBS. confirmation of the specificity of the ADAM-10 antibody in To test for androgen regulation, DHT was added at concentra- prostate tissue, fresh frozen prostate tissue obtained from cancer tions of 0 (control), 0.1, 1.0, or 10 nM. For IGF-I regulation, patients and sectioned into 8-␮m samples was homogenized in IGF-I was added at concentrations of 0 (control), 10, or 50 lysis buffer. ng/ml in the presence or absence of 10 nM DHT in serum-free Preparation of LNCaP Nuclear and Cytoplasmic Pro- RPMI 1640/phenol red-free/0.01% BSA/gentamicin. For EGF tein Fractions. Cellular compartmentalization of LNCaP cells regulation, EGF was added at concentrations of 0 (control) and was achieved by differential lysis and centrifugation using the 50 ng/ml. The cells were cultured for an additional 48 h under NePer nuclear/cytoplasmic fractionation kit (Pierce) as per the test conditions, with a change of medium to replenish the manufacturer’s recommendations. Each buffer was adapted to appropriate growth factors after 24 h. Cells were harvested with contain a final concentration of 0.1 mM phenylmethylsulfonyl ice-cold PBS, pelleted by centrifugation at 175 ϫ g (1000 rpm; fluoride and 10 mM EDTA (pH 8.0), and 1ϫ Complete EDTA- CS-6R Centrifuge; Beckman, La Jolla, CA) for 3 min, and free protease inhibitor mixture (Roche) was added to inhibit stored at Ϫ80°C until further manipulation. protease activity, as recommended by the manufacturer.

Western Blot Procedure. Five ␮g of protein from each ADAM-10 mRNA expression (Fig. 1B). Staining intensity var- hormone-treated and corresponding control cell pellet or the ied from weak (Fig. 1C), to strong (Fig. 1D) in tumor cells in nuclear/cytoplasmic fractions were suspended in a reducing different samples. High-grade PCa exhibited very strong stain- loading buffer and boiled for 3 min before loading onto a 10% ing intensity in the disrupted epithelial cells as well as in SDS-PAGE gel, followed by standard Western blotting proce- scattered cells in the stroma (Fig. 1E). The negative control dure. The membrane was blocked with 5% Blotto and incubated sense riboprobe gave no positive signal (Fig. 1F). with either 0.2 ␮g/ml rabbit anti-ADAM-10 antibody, 0.2 ␮g/ml Tissue Localization of ADAM-10 Protein. ADAM-10 mouse anti-␤-tubulin monoclonal antibody, or 0.2 ␮g/ml mouse immunostaining was observed across the range of clinical sam- anti-proliferating cell nuclear antigen monoclonal antibody in ples tested, although distinct and differential staining patterns 0.8% Blotto in Tris-buffered saline/0.05% Tween 20 at 4°C were apparent between glands of differing pathology, even overnight. The membrane was incubated for2hatroom tem- within a single section (e.g., Fig. 1K). Only minor differences in perature in either antirabbit horseradish peroxidase (Dako) or staining intensity were observed between benign glands within antimouse peroxidase (Chemicon) in 1% Blotto/Tris-buffered benign prostatic hyperplasia (1ϩ to 3ϩ) or cancer (1ϩ to 4ϩ) saline/0.05%Tween 20. For detection, the membrane was incu- sections. Staining in benign tissue revealed cytoplasmic immu- bated in 2 ml of Femto (Pierce) for 5 min at room temperature noreactivity in nearly all of the basal cells, with weak membra- and exposed to X-ray film. The size of the protein was deter- nous staining in the secretory cells of some glands (Fig. 1G), mined by comparison with Bio-Rad (Hercules, CA) prestained whereas the majority of benign glands showed strong membra- protein markers. nous staining patterns in secretory cells (Fig. 1, H and I). Quantitation of Signal Intensity. For the housekeeping Interestingly, some perinuclear/nuclear staining, along with gene ␤-tubulin, all autoradiographic detections were subjected weaker membrane staining, was evident in some benign glands to over- and underexposures to determine that the blots used for (Fig. 1J, arrow). No reactivity was observed in stroma. In stark subsequent densitometric analysis were in the linear range of contrast, PCa tissues demonstrated heterogeneous nuclear and detection. Densitometric analysis was conducted using a GS- perinuclear intensities, and staining varied from weak to strong 690 image densitometer (Bio-Rad). Signal intensity obtained for (1ϩ to 3ϩ), but across the whole cohort (n ϭ 17) no apparent ADAM-10 expression was normalized for the signal obtained correlation with cancer grade was evident (Fig. 1, K and L). for ␤-tubulin for the same sample on the same Western blot. All Benign glands within neoplastic tissue revealed some perinu- Western blots were performed on protein samples collected clear as well as membrane-bound labeling (Fig. 1K, arrow). The from three separate, treated-cell preparations, giving an exper- localization of ADAM-10 immunostaining to the nucleus was imental value for each set of treatments of n ϭ 3. Western blots strikingly obvious in all cancer glands studied, in all patients. To for each set of treatments were performed at least in duplicate, assess the binding specificity, ADAM-10 primary antibody was giving possible n values of at least 6, and subsequent densito- preabsorbed with purified ADAM-10 peptide. No positive stain- metric analysis was performed for each control and test concen- ing was observed with the preabsorbed antibody (Fig. 1M). tration. Absolute ratios of intensity between control and test Western blotting was performed to assess whether the presence concentrations were calculated, and the entire data set was of ADAM-10 found in the nuclei of prostate tissue via immu- subjected to a one-way ANOVA followed by a Tukey post hoc nohistochemistry could be confirmed in a PCa cell line (Fig. analysis to determine significance between treatments and their 1N). LNCaP cells were subjected to subcellular protein fraction- respective control. ation, and the relative purity of each fraction was evaluated using nuclear (proliferating cell nuclear antigen) and cytoplasmic (␤-tubulin) markers. Fig. 1N shows that the two subcellular RESULTS compartments were successfully isolated with only minor con- In Situ Hybridization and Immunohistochemistry. To tamination of the cytoplasmic fraction with the nuclear marker, determine the cellular localization of ADAM-10 in prostate proliferating cell nuclear antigen. Immunoreactive bands repre- tissue and to assess if benign, non-neoplastic and cancerous senting the unprocessed proform of ADAM-10 (ϳ100 kDa) and prostate tissues differ in their expression of ADAM-10, archived the mature (active) form of ADAM-10 (ϳ60 kDa) were clearly prostate tissue samples from 23 men with differing pathological present in the nuclear as well as cytoplasmic protein fractions. conditions were studied. An additional band (ϳ80 kDa) representing a partially pro- ADAM-10 mRNA Expression in Benign, Nonneoplastic, cessed form was also apparent in this fractionation study. A and Cancerous Prostate Tissue. The hybridization signal similar intermediate band is sometimes seen in whole cell ly- from the ADAM-10 antisense riboprobe was predominantly sates (e.g., Fig. 4). detected in the cytoplasm of glandular epithelial cells and varied LNCaP Cell Proliferation. To test for biological re- in intensity between different patients and different glands. sponsiveness of the LNCaP PCa cell model to each of the Benign glands revealed strong ADAM-10 mRNA expression in hormones/growth factors used, a MTT cell proliferation assay basal cells but mostly weak staining in secretory cells (Fig. 1A, was performed. As expected, and as shown in Fig. 2A, LNCaP inset, arrow). Similarly, glands exhibiting characteristics of cell proliferation was significantly increased after the addition benign prostatic hyperplasia demonstrated strong expression in of DHT (19% increase), with a further increase in proliferation basal cells and heterogeneous staining in secretory cells, which with the addition of IGF-I (an additional 24% increase). For varied from very weak to strong staining (data not shown). Most EGF-treated cells (Fig. 2B), a 4-fold increase in cell prolifera- glands exhibiting characteristics of prostatic intraepithelial ne- tion was observed. These findings parallel the same proliferative oplasia, a putative precursor of PCa (13), showed a high level of response found by Iwamura et al. (14) and Siu et al. (15) and

Fig. 1 In situ hybridization (A–F) and immunohistochemical staining (G–M)ofthe disintegrin metalloprotease ADAM-10 mRNA and protein in prostate tissue sections. In benign epithelium, basal cells stained for ADAM-10 mRNA (A, inset, arrow). A very strong signal was observed in secretory cells of prostatic intraepithelial neoplasia (B, arrow), whereas secretory cells in cancerous epithelium varied in staining intensity from weak to strong (C and D, arrows), to very strong staining in high- grade cancer (E). ADAM-10 sense cRNA probe (negative control) shows no positivity (F). The pattern of immunostaining for ADAM-10 protein in benign epithelium varies from a strong signal in basal cells and weaker staining on the cell membrane of secretory cells (G, arrow) to predominantly strong cell membrane staining of secretory cells (H and I, arrow; I represents a magnification of the boxed area in H) and a mix of uniform basal cell staining, secretory cell membrane staining, and some perinuclear/nuclear (arrow) staining in secretory cells (J). In contrast, a perinuclear/nuclear staining pattern was seen in all cancer epi- thelia (K, arrow) with high-grade cancer cells demonstrating strong nuclear staining (L, arrow). The preabsorbed antibody (negative control) shows no immunoreactivity (M). Scale bar, 30 ␮m. Western blot (N) of ADAM-10, ␤-tubulin (cytoplasmic marker), and proliferating cell nuclear antigen (nuclear marker) on nuclear and cytoplasmic fractions from LNCaP cells, demonstrating both the purity and presence of ADAM-10 in both subcellular fractions.

demonstrate the expected functional activity of DHT, IGF-I, and Regulation of ADAM-10 mRNA Levels. Real-time EGF in the LNCaP PCa cell model. Because it has been shown PCR analysis was performed on mRNA extracted from LNCaP that PSA mRNA is stimulated on DHT treatment (16), the action cells treated with DHT, IGF-I, IGF-I plus DHT, or EGF alone. of DHT in our system was further demonstrated by measuring The changes in ADAM-10 mRNA levels were normalized for the up-regulation of PSA mRNA in LNCaP cells by quantitative levels of mRNA initially reverse transcribed by quantitating 18S 3 PCR. This result also validated our quantitative PCR method. rRNA in the same cDNA in the same PCR. Fig. 3A shows that both DHT (10 nM) and IGF-I alone (50 ng/ml) significantly decreased levels of ADAM-10 mRNA, whereas DHT plus IGF-I significantly elevated (1.8-fold) ADAM-10 mRNA levels. For 3 D. R. McCulloch, A. C. Herington, and D. M. Odorico, unpublished EGF (50 ng/ml), a significant elevation (1.7-fold) of ADAM-10 data. mRNA levels was observed (Fig. 3B).

volume and density of each signal band allowed us to report ADAM-10 expression as an absolute ratio between the control and test treatments. DHT or IGF-I alone did not significantly alter ADAM-10 protein levels (Fig. 5), despite the reduction in ADAM-10 mRNA levels (Fig. 3). However, IGF-I (10 or 50 ng/ml) in the presence of 10 nM DHT up-regulated both the unprocessed 100-kDa proform (ϳ1.8-fold) and the processed active 60-kDa form (ϳ3–4-fold) of ADAM-10 (Fig. 5), paral- leling the change in ADAM-10 mRNA (Fig. 3). EGF (50 ng/ml) also significantly stimulated the expression of unprocessed 100- kDa proform (ϳ2-fold) and stimulated the processed 60-kDa form (almost 3-fold) of ADAM-10 (Fig. 5), again in complete agreement with observed changes in mRNA (Fig. 3).

DISCUSSION This study describes, for the first time, the expression and cellular localization of ADAM-10 mRNA and protein in prostate tissues and provides novel data regarding the hormonal response of ADAM-10 mRNA and protein in the LNCaP cell model to three key PCa cell growth promoters: DHT; IGF-I; and EGF. The expression of ADAM-10 in the epithelial cells of prostate tumors may provide these cells with the capacity to assist in ECM remodeling and cell mobility, key characteristics of tumor cell invasion. Additionally, our data demonstrating a primarily nuclear localization of ADAM-10 in PCa epithelial cells raises the novel hypothesis that ADAM-10 may have further and distinct actions in this disease. ADAM-10 (protein and/or mRNA) is expressed in all prostate glands, where a variation of staining intensity was observed for ADAM-10 protein in specimens representative of the same histological grade. This may well be a result of the inherent heterogeneity of glands within any given biopsy as being characteristic of normal, benign, prostatic intraepithelial Fig. 2 The effects of 5␣-dihydrotestosterone and growth factors (in- neoplasia, or low- to high-grade cancer. Although no obvious sulin-like growth factor I and epidermal growth factor) on LNCaP cell trend toward an up-regulation of ADAM-10 in high-grade PCa proliferation. Proliferation assays were determined in 96-well plates, seeding 10,000 cells/well. LNCaP cells were serum starved for 24 h and compared with lower grade cancer or benign glands was ob- treated with 5␣-dihydrotestosterone Ϯ insulin-like growth factor I (A)or served, a most striking and unexpected finding was the differ- epidermal growth factor (B) for an additional 96 h. The 3-(4,5-dimeth- ences in the localization of ADAM-10 staining between benign ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye method was used and cancer glands. In benign tissue, epithelial cell staining was to determine change in cell number. Data shown are derived from a largely concentrated on the cell surface with some nuclear/ single experiment with each condition replicated in 16 wells/96-well plate. Similar results were observed in two subsequent cell proliferation perinuclear localization, whereas all tumor cells demonstrated assays. A, b,e, P Ͻ 0.05; a,c,d,f,g ϭ P Ͻ 0.01. B, a,b,c, P Ͻ 0.01. marked nuclear/perinuclear immunoreactivity with membrane staining essentially absent. The absence of membrane staining in high-grade cancer suggests differing roles for ADAM-10 as compared with the initial hypothesis that ADAM-10 may be Western Blot Analysis and Quantification. The ability involved in cell migration and cell invasion. of the ADAM-10 antibody to detect the same protein forms in Benign glands within nonneoplastic and neoplastic tissue LNCaP cells as found in prostate tissue was confirmed by all show consistent membrane localization in secretory epithe- Western blot analysis of LNCaP cells alongside two prostate lial cells. The strong membrane association is consistent with tissue lysates. As depicted in Fig. 4, the banding pattern in the the documented ability of several ADAMs (in particular, prostate tissue sections closely resembles the pattern found in ADAM-9, ADAM-10, and ADAM-17) to act as cell surface LNCaP cells, a 100-kDa proform and a doublet at the expected “sheddases,” cleaving the extracellular (ecto-) domains of mem- position of the active 60-kDa form. Western blots were per- brane-bound proteins from the cell surface (1, 17). This process formed on whole cell protein extracted from LNCaP cells can lead to the activation of growth factors or the solubilization treated with DHT, IGF-I, IGF-I plus DHT, or EGF. Quantitative of extracellularly active molecules. The proteolysis by ADAMs image analysis, using ␤-tubulin as an internal housekeeping can also change the active states of remnant surface molecular marker, was used to correct for protein loading between the complexes and hence affect signaling pathways inside cells (18). control and test lanes in each experiment. Quantitating the Purified ADAM-10 (or its orthologs in species from Drosophila

Fig. 4 Western blot analysis of the disintegrin metalloprotease ADAM-10 in the prostate cancer cell line LNCaP (Lane 1) and in two different prostate cancer tissue section extracts (Lanes 2 and 3). Both LNCaP and tissue extracts depict a similar banding pattern of a 100-kDa ADAM-10 band (proform) and a doublet at ϳ60 kDa (processed active forms).

to human) is highly active as a “sheddase,” being able to proteolytically cleave/degrade, for example, the developmental regulator Notch receptor ligand Delta (19), precursor tumor necrosis factor ␣ (4), amyloid precursor protein (20), and eph- rin-A2 (a well-characterized axon repellent; Ref. 21). Cell membrane-localized ADAM-10 in epithelial cells of benign glands suggests that ADAM-10 may be important in cell-cell and cell-matrix/cell-basement membrane interactions. Although ADAM-10 is not known to interact directly with any integrins (22), it has been shown to indirectly influence integrin- related adhesion activity by cleaving the L1 membrane adhesion molecule, a type I membrane glycoprotein implicated in the cell migration of neural and tumor cells (23). L1 is overexpressed in several carcinomas and is capable of acting as a substrate for integrin-associated cell adhesion (24). L1 has also been shown to trigger cell migration of Chinese hamster ovary cells (24). Human ADAM-10, through the shedding of transmembrane collagen XVII (BP180) from the cell surface of keratino- cytes, leads to the release of a soluble basement membrane collagen and an increased cell motility in vitro (25). Collagen XVII (one component of hemidesmosomes) provides stability for the interactions between the basement membrane and basal cells. Immunohistochemical studies of collagen XVII in human prostate have shown that in normal prostate and prostatic intraepithelial neoplasia, it is expressed along the basal aspect of the basal cells, together with a range of other hemidesmosomal- related proteins. PCa cells, on the other hand, were found to completely lack immunoreactive collagen XVII (26). Whether a direct causal link can be established between expression of ADAM-10 and the loss of collagen XVII in PCa remains to be investigated. The loss of both collagen XVII and ADAM-10 from the membrane of PCa cells may provide a mechanism for loss of cell-cell or cell-basement membrane contact and allow for the regulation of the mobility of such cells. ADAM-10 is known to cleave one of the major constituents of the basement membrane, collagen IV (7), and its activity is regulated by the tissue inhibitors of metalloproteinases (27). Tissue inhibitors of metalloproteinases serve as a key physiological regulatory mechanism for MMP-mediated ECM-degrading activity, pro- viding additional support for functional similarities between the

Fig. 3 The regulation of the disintegrin metalloprotease ADAM-10 mRNA extracted from LNCaP cells treated with 5␣-dihydrotestosterone Ϯ insulin-like growth factor I or epidermal growth factor alone. ADAM-10 mRNA levels were significantly elevated by a synergistic triplicate in two separate PCR reactions. The changes in ADAM-10 response to treatment with 5␣-dihydrotestosterone ϩ insulin-like mRNA levels were normalized to the levels of the housekeeping gene growth factor I (A) and with epidermal growth factor alone (B). Each 18S rRNA in the same cDNA sample measured in the same PCR. A, a, histogram represents three independent experiments analyzed in P Ͻ 0.05; b,c,d, P Ͻ 0.01. B, a, P Ͻ 0.01.

Fig. 5 Western blot analysis for the disintegrin metalloprotease ADAM-10 expression in whole cell lysates extracted from LNCaP cells treated with 5␣-dihydrotestosterone, insulin-like growth factor I, insulin-like growth factor I in the presence of 5␣-dihydrotestosterone, or epidermal growth factor alone. Quantitative densitometry analysis is shown in the histograms below the Western blots for the 100-kDa ADAM-10 band (proform) and the 60-kDa band (processed, active). These data are representative blots of three separate growth factor-treated LNCaP whole cell lysate preparations, with each test performed at least in duplicate and subsequently analyzed by quantitative image analysis as outlined in “Materials and Methods.” In some cases (where n ϭ 5 rather than 6), the 60-kDa band on the Western blot was too faint for efficient quantitation, whereas the proform (100-kDa) band on the same blot was quantifiable. The signal for ADAM-10 was normalized for protein loading with the signal from ␤-tubulin on the same membrane. For all treatments, a ϭ P Ͻ 0.05.

ADAMs and MMPs (28, 29). The above observations are con- With respect to studies on the regulation of ADAM-10, the sistent with a putative role for membrane-bound ADAM-10 in effects of DHT and IGF-I are particularly relevant in the tran- the early stages of ECM degradation and facilitation of subse- sition from early- to late-stage PCa, when both IGF-I and DHT quent invasion of metastatic cells. may be promoting growth and PCa development synergistically, The primary nuclear localization of ADAM-10 in PCa whereas later stage PCa is known to have elevated levels of EGF tissue sections and the presence of ADAM-10 in the nuclear (9). Previously, we reported the regulation of ADAM-10 mRNA protein fractions of LNCaP cells, however, suggest an additional levels by DHT in the LNCaP cell line (12). The current study but quite different role, which has yet to be defined. We hy- has extended these observations to analyze changes in both pothesize that nuclear ADAM-10 functions by interacting with ADAM-10 mRNA and protein levels in response to DHT and nuclear protein or DNA to directly influence nuclear processes. the growth factors IGF-I and EGF. Alone, IGF-I and DHT Evidence for nuclear localization and function of extracellular/ decreased the levels of ADAM-10 mRNA, but together, they membrane-bound proteins, previously thought to have no role in significantly increased ADAM-10 mRNA. Our previous obser- the nucleus, is becoming more common. Recent examples in- vations showed ADAM-10 mRNA levels significantly elevated clude growth hormone/growth hormone-binding protein (30), at low DHT concentrations (0.1 and 1.0 nM), whereas at 10 nM parathyroid hormone-related peptide (31), IGF-binding pro- DHT, the dose used here, levels of ADAM-10 mRNA declined, tein-3 and -5 (32), EGF receptor (33), and heparin-binding EGF resulting in a bell-shaped dose-response curve (12). Because this (34). Evidence suggests that some of these have direct effects on current study focused on a possible synergistic response to DHT transcriptional activity of target genes (33). The mechanism by ϩ IGF-I, we used the higher 10 nM concentration of DHT, so which ADAM-10 translocates to the nucleus is not known. that any changes detected could be attributed to the addition of However, analysis of the ADAM-10 protein sequence reveals IGF-I. The use of the higher dose of DHT in these experiments several consensus “nuclear localization signals” (35), including two is a likely explanation for the lower response to DHT observed Pattern 4 and two Pattern 7 motifs and a COOH-terminal bipartite in this study, compared with our previous report (12). EGF nuclear localization signal (aa721-RRRPPQPIQQPQRQRPR-). alone also elevated levels of ADAM-10 mRNA significantly, Small proteins [up to 45 kDa (36)] that are not constitutively which is not surprising, given the ability of EGF to regulate gene present in the nucleus can diffuse freely through the nuclear expression of other genes (e.g., syndecan-1, collagens, and pore complex. However, many larger proteins, such as ADAM- MMPs) involved in ECM maintenance (38). 10, require the presence of the largely highly basic nuclear With respect to ADAM-10 protein expression, two distinct localization signal motif and are taken up through one of several bands were observed on Western blots. The larger band (100 regulated, active-transport mechanisms (35, 37). Additional kDa) likely represents the unprocessed proform of ADAM-10, studies are required to determine which nuclear localization whereas the ϳ60-kDa band corresponds to that found in plasma signal-mediated transport system is used by ADAM-10. membrane preparations from bone MG-63 cells (39) and is

believed to represent the mature, active form of ADAM-10. An 3. Pan, D., and Rubin, G. M. Kuzbanian controls proteolytic processing additional band (ϳ80 kDa), which may represent a partly pro- of Notch and mediates lateral inhibition during Drosophila and verte- cessed intermediate form of ADAM-10, was inconsistently ob- brate neurogenesis. Cell, 90: 271–280, 1997. served. 4. Rosendahl, M., Ko, S., Long, D., Brewer, M., Rosenzweig, B., Hedl, E., Anderson, L., Pyle, S., Moreland, J., Meyers, M., Kohno, T., Lyons, Neither 10 nM DHT nor 50 ng/ml IGF-I alone resulted in a D., and Lichenstein, H. Identification and characterization of a pro- significant change in either ADAM-10 protein band. However, tumor necrosis factor-␣-processing enzyme from the ADAM family of in response to IGF-I in the presence of DHT, a significant zinc metalloproteases. J. Biol. Chem., 272: 24588–24593, 1997. increase in protein expression was seen for the ADAM-10 5. Skovronsky, D. M., Moore, D. B., Milla, M. E., Doms, R. W., and proform (1.8-fold) and the mature, active form (3–4-fold). The Lee, V. M. Protein kinase C-dependent ␣-secretase competes with addition of EGF also gave rise to a significant up-regulation of ␤-secretase for cleavage of amyloid-␤ precursor protein in the trans- ADAM-10 protein (proform, ϳ2-fold; mature, active form, ϳ3- Golgi network. J. Biol. Chem., 275: 2568–2575, 2000. fold), in the absence of DHT. The apparent differences in 6. Maskos, K., Fernandez-Catalan, C., Huber, R., Bourenkov, G. P., Bartunik, H., Ellestad, G. A., Reddy, P., Wolfson, M. F., Rauch, C. T., regulation of the proform versus the mature form may reflect a Castner, B. J., Davis, R., Clarke, H. R., Petersen, M., Fitzner, J. N., specific effect on transcription (for the proform) and/or an effect Cerretti, D. P., March, C. J., Paxton, R. J., Black, R. A., and Bode, W. on the furin-mediated processing mechanism or stability of the Crystal structure of the catalytic domain of human tumor necrosis mature ADAM proteins (40). factor-␣-converting enzyme. Proc. Natl. Acad. Sci. USA, 95: 3408– The synergistic effect of DHT and IGF-I is consistent with 3412, 1998. known positive interactions between the IGF signaling pathway 7. Millichip, M., Dallas, D., Wu, E., Dale, S., and McKie, N. The and androgen receptor activation (41). The subsequent loss of metallo-disintegrin ADAM10 (MADM) from bovine kidney has type IV collagenase activity in vitro. Biochem. Biophys. Res. Commun., 245: androgen dependence in late-stage PCa might be expected to 594–598, 1998. cause a decrease in ADAM-10 because IGF-I alone was unable 8. Bergers, G., and Coussens, L. Extrinsic regulators of epithelial tumor to increase ADAM-10 in vitro. However, because ADAM-10 is progression: metalloproteinases. Curr. Opin. Genet. Dev., 10: 120–127, maintained at equivalent or greater levels in cancer glands in 2000. vivo (as demonstrated by immunohistochemistry), it would ap- 9. Russell, P. J., Bennett, S., and Stricker, P. Growth factor involvement pear that additional regulatory factors, such as EGF [which has in progression of prostate cancer. Clin. Chem., 44: 705–723, 1998.

also been found to specifically up-regulate the metalloprotease 10. Long, L., Navab, R., and Brodt, P. Regulation of the Mr 72,000 type matrilysin (MMP-7) in LNCaP cells (11)], are involved in IV collagenase by the type I insulin-like growth factor receptor. Cancer regulating ADAM-10 expression. Res., 58: 3243–3247, 1998. The behavior of normal and cancer cells with respect to 11. Sundareshan, P., Nagle, R. B., and Bowden, G. T. EGF induces the expression of matrilysin in the human prostate adenocarcinoma cell line, cellular contacts, cell migration, and invasion of surrounding LNCaP. Prostate, 40: 159–166, 1999. tissues may well be determined, at least in part, by hormone and 12. McCulloch, D. R., Harvey, M., and Herington, A. C. The expres- growth-factor regulation of ADAMs, such as ADAM-10, ex- sion of the ADAMs proteases in prostate cancer cell lines and their pressed and coordinately regulated within those cells. It is clear regulation by dihydrotestosterone. Mol. Cell. Endocrinol., 167: 11–21, from the present study that the particular roles of ADAM-10 in 2000. PCa require further investigation but that ADAM-10 is very 13. Bostwick, D. G., Amin, M. B., Dundore, P., Marsh, W., and specifically expressed in epithelial cells of the prostate and Schultz, D. S. Architectural patterns of high-grade prostatic intraepithe- appears to demonstrate differential regulation and function (nu- lial neoplasia. Hum. Pathol., 24: 298–310, 1993. clear versus membrane) in vivo, as PCa progresses from a 14. Iwamura, M., Sluss, P. M., Casamento, J. B., and Cockett, A. T. benign to a malignant state. Because the ADAMs have a mul- Insulin-like growth factor I: action and receptor characterization in human prostate cancer cell lines. Prostate, 22: 243–252, 1993. tidomain structure and are therefore potentially multifunctional, 15. Siu, S. W., Lau, K. W., Tam, P. C., and Shiu, S. Y. Melatonin and the ADAMs may have multiple roles depending on their cellular prostate cancer cell proliferation: interplay with castration, epidermal localization. growth factor, and androgen sensitivity. Prostate, 52: 106–122, 2002. 16. Montgomery, B. T., Young, C. Y., Bilhartz, D. L., Andrews, P. E., Prescott, J. L., Thompson, N. F., and Tindall, D. J. Hormonal regulation ACKNOWLEDGMENTS of prostate-specific antigen (PSA) glycoprotein in the human prostatic We thank Dr. Lisa Chopin and Loan Bui of the Queensland adenocarcinoma cell line, LNCaP. Prostate, 21: 63–73, 1992. University of Technology for assistance and advice regarding the in situ 17. Moss, M. L., White, J. M., Lambert, M. H., and Andrews, R. C. hybridization and immunohistochemistry methodology and Drs. Erik TACE and other ADAM proteases as targets for drug discovery. Drug. Thompson and Elizabeth Williams from the Bernard O’Brien Institute Discov. Today, 6: 417–426, 2001. of Microsurgery (Victoria, Australia) for the frozen prostate tissue 18. Yan, Y. B., Shirakabe, K., and Werb, Z. The metalloprotease sections. Kuzbanian (ADAM10) mediates the transactivation of EGF receptor by G protein-coupled receptors. J. Cell Biol., 158: 221–226, 2002. 19. Qi, H., Rand, M. D., Wu, X., Sestan, N., Wang, W., Rakic, P., Xu, REFERENCES T., and Artavanis-Tsakonas, S. Processing of the notch ligand Delta by 1. Kheradmand, F., and Werb, Z. Shedding light on sheddases: role in the metalloprotease Kuzbanian. Science (Wash. DC), 283: 91–94, 1999. growth and development. Bioessays, 24: 8–12, 2002. 20. Lammich, S., Kojro, E., Postina, R., Gilbert, S., Preiffer, R., Jas- 2. Inoue, D., Reid, M., Lum, L., Kratzschmar, J., Weskamp, G., Myung, ionowski, M., Haass, C., and Fahrenholz, F. Constitutive and regulated Y. M., Baron, R., and Blobel, C. P. Cloning and initial characterization ␣-secretase cleavage of Alzheimer’s amyloid precursor protein by a of mouse meltrin ␤ and analysis of the expression of four metallopro- disintegrin metalloprotease. Proc. Natl. Acad. Sci. USA, 96: 3922– tease-disintegrins in bone cells. J. Biol. Chem., 273: 4180–4187, 1998. 3927, 1999.

21. Hattori, M., Osterfield, M., and Flanagan, J. G. Regulated cleavage cytes under conditions that promote apoptotic cell death. Mol. Cell. of a contact-mediated axon repellent. Science (Wash. DC), 289: 1360– Biol., 15: 4064–4075, 1995. 1365, 2000. 32. Schedlich, L. J., Le Page, S. L., Firth, S. M., Briggs, L. J., Jans, 22. White, J. M., Bigler, D., Chen, M., Takahashi, Y., and Wolfsberg, D. A., and Baxter, R. C. Nuclear import of insulin-like growth factor- T. G. ADAMs. In: M. C. Beckerle (ed.) Cell Adhesion, pp. 189–216. binding protein-3 and -5 is mediated by the importin ␤ subunit. J. Biol. New York: Oxford University Press, 2001. Chem., 275: 23462–23470, 2000. 23. Mechtersheimer, S., Gutwein, P., Agmon-Levin, N., Stoeck, A., 33. Lin, S. Y., Makino, K., Xia, W., Matin, A., Wen, Y., Kwong, K. Y., Oleszewski, M., Riedle, S., Fogel, M., Lemmon, F., and Altevogt, P. Bourguignon, L., and Hung, M. C. Nuclear localization of EGF receptor Ectodomain shedding of L1 adhesion molecule promotes cell migration and its potential new role as a transcription factor. Nat. Cell. Biol., 3: by autocrine binding to integrins. J. Cell Biol., 155: 661–673, 2001. 802–808, 2001. 24. Gutwein, P., Mechtersheimer, S., Riedle, S., Stoeck, A., Gast, D., 34. Adam, R. M., Danciu, T., McLellan, D. L., Borer, J. G., Lin, J., Joumaa, S., Zentgraf, H., Fogel, M., and Altevogt, P. ADAM10-medi- Zurakowski, D., Weinstein, M. H., Rajjayabun, P. H., Mellon, J. K., and ated cleavage of L1 adhesion molecule at the cell surface and in released Freeman, M. R. A nuclear form of the heparin-binding epidermal membrane vesicles. FASEB J., 17: 292–294, 2003. growth factor-like growth factor precursor is a feature of aggressive 25. Franzke, C. W., Tasanen, K., Schacke, H., Zhou, Z., Tryggvason, transitional cell carcinoma. Cancer Res., 63: 484–490, 2003. K., Mauch, C., Zigrino, P., Sunnarborg, S., Lee, D. C., Fahrenholz, F., 35. Jans, D. A., Xiao, C. Y., and Lam, M. H. Nuclear targeting signal and Bruckner-Tuderman, L. Transmembrane collagen XVII, an epithe- recognition: a key control point in nuclear transport? Bioessays, 22: lial adhesion protein, is shed from the cell surface by ADAMs. EMBO 532–544, 2000. J., 21: 5026–5035, 2002. 36. Jans, D. A., and Hubner, S. Regulation of protein transport to the 26. Nagle, R. B., Hao, J., Knox, J. D., Dalkin, B. L., Clark, V., and nucleus: central role of phosphorylation. Physiol. Rev., 76: 651–685, Cress, A. E. Expression of hemidesmosomal and extracellular matrix 1996. proteins by normal and malignant human prostate tissue. Am. J. Pathol., 37. Quimby, B. B., and Corbett, A. H. Nuclear transport mechanisms. 146: 1498–1507, 1995. Cell. Mol. Life Sci., 58: 1766–1773, 2001. 27. Amour, A., Knight, C. G., Webster, A., Slocombe, P. M., Stephens, 38. Yarwood, S. J., and Woodgett, J. R. Extracellular matrix composi- P. E., Knauper, V., Docherty, A. J., and Murphy, G. The in vitro activity tion determines the transcriptional response to epidermal growth factor of ADAM-10 is inhibited by TIMP-1 and TIMP-3. FEBS Lett., 473: receptor activation. Proc. Natl. Acad. Sci. USA, 98: 4472–4477, 2001. 275–279, 2000. 39. Dallas, D. J., Genever, P. G., Patton, A. J., Millichip, M. I., McKie, 28. Bauvois, B. Transmembrane proteases in focus: diversity and re- N., and Skerry, T. M. Localization of ADAM10 and notch receptors in dundancy? J. Leukocyte Biol., 70: 11–17, 2001. bone. Bone, 25: 9–15, 1999. 29. Chang, C., and Werb, Z. The many faces of metalloproteases: cell 40. Endres, K., Anders, A., Kojro, E., Gilbert, S., Fahrenholz, F., and growth, invasion, angiogenesis and metastasis. Trends Cell Biol., 11: Postina, R. Tumor necrosis factor-␣ converting enzyme is processed by S37–S43, 2001. proprotein-convertases to its mature form which is degraded upon 30. Lobie, P. E., Garcia-Aragon, J., Wang, B. S., Baumbach, W. R., and phorbol ester stimulation. Eur. J. Biochem., 270: 2386–2393, 2003. Waters, M. J. Cellular localization of the growth hormone binding 41. Culig, Z., Hobisch, A., Cronauer, M. V., Radmayr, C., Trapman, J., protein in the rat. Endocrinology, 130: 3057–3065, 1992. Hittmair, A., Bartsch, G., and Klocker, H. Androgen receptor activation 31. Henderson, J. E., Amizuka, N., Warshawsky, H., Biasotto, D., in prostatic tumor cell lines by insulin-like growth factor-I, keratinocyte Lanske, B. M., Goltzman, D., and Karaplis, A. C. Nucleolar localization growth factor, and epidermal growth factor. Cancer Res., 54: 5474– of parathyroid hormone-related peptide enhances survival of chondro- 5478, 1994.

Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2004 American Association for Cancer Research. Expression of the Disintegrin Metalloprotease, ADAM-10, in Prostate Cancer and Its Regulation by Dihydrotestosterone, Insulin-Like Growth Factor I, and Epidermal Growth Factor in the Prostate Cancer Cell Model LNCaP

Daniel R. McCulloch, Pascal Akl, Hemamali Samaratunga, et al.

Clin Cancer Res 2004;10:314-323.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/10/1/314

Cited articles This article cites 40 articles, 17 of which you can access for free at: http://clincancerres.aacrjournals.org/content/10/1/314.full#ref-list-1

Citing articles This article has been cited by 12 HighWire-hosted articles. Access the articles at: http://clincancerres.aacrjournals.org/content/10/1/314.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/10/1/314. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.