catalytic cleavage of the androgen-regulated tmprss2 ...daniel e. h. afar,1 igor vivanco, rene s....

[CANCER RESEARCH 61, 1686–1692, February 15, 2001]

Catalytic Cleavage of the Androgen-regulated TMPRSS2 Protease Results in ItsSecretion by Prostate and Prostate Cancer Epithelia

Daniel E. H. Afar,1 Igor Vivanco, Rene S. Hubert, James Kuo, Emily Chen, Douglas C. Saffran, Arthur B. Raitano,and Aya Jakobovits2

UroGenesys Inc., Santa Monica, California 90404

ABSTRACT

We identified TMPRSS2as a gene that is down-regulated in androgen-independent prostate cancer xenograft tissue derived from a bone metas-tasis. Using specific monoclonal antibodies, we show that theTMPRSS2-encoded serine protease is expressed as aMr 70,000 full-length form anda cleavedMr 32,000 protease domain. Mutation of Ser-441 in the catalytictriad shows that the proteolytic cleavage is dependent on catalytic activity,suggesting that it occurs as a result of autocleavage. Mutational analysisreveals the cleavage site to be at Arg-255. A consequence of autocatalyticcleavage is the secretion of the protease domain into the media byTMPRSS2-expressing prostate cancer cells and into the sera of prostatetumor-bearing mice. Immunohistochemical analysis of clinical specimensdemonstrates the highest expression of TMPRSS2 at the apical side ofprostate and prostate cancer secretory epithelia and within the lumen ofthe glands. Similar luminal staining was detected in colon cancer samples.Expression was also seen in colon and pancreas, with little to no expressiondetected in seven additional normal tissues. These data demonstrate thatTMPRSS2 is a secreted protease that is highly expressed in prostate andprostate cancer, making it a potential target for cancer therapy anddiagnosis.

INTRODUCTION

Prostate cancer is a hormone-regulated disease that affects men inthe later years of life. Untreated prostate cancer metastasizes to lymphnodes and bone in advanced cases. In such cases, the treatmentconsists of antagonizing the androgenic growth stimulus that feeds thetumor by chemical or surgical hormone ablation therapy (1). Anunfortunate consequence of antiandrogen treatment is the develop-ment of androgen-independent cancer. Androgen-regulated genes,such as the gene encoding PSA,3 are turned off with hormone ablationtherapy but reappear when the tumor becomes androgen independent(2). This suggests that alternative signaling pathways, such as acti-vated tyrosine kinase receptors, can replace the androgen signal (3, 4).

To understand the molecular events surrounding the progression ofprostate cancer to androgen independence, we used LAPC-9, a humanprostate cancer xenograft derived from a bone metastasis, which canmimic the transition from androgen dependence to androgen inde-pendence (5). A subtractive hybridization method (6) was used toidentify androgen-regulated genes by subtracting cDNAs from theLAPC-9 AI subline from cDNAs derived from the original LAPC-9AD xenograft. This strategy resulted in the identification of theTMPRSS2gene, which encodes a putative transmembrane serineprotease (7).

TMPRSS2 was predicted to be a type II transmembrane proteinwith an extracellular COOH terminus containing the protease domainand an intracellular NH2 terminus.TMPRSS2was recently shown tobe regulated by androgens in the LNCaP prostate cancer cell model(8). To characterize the TMPRSS2 protein in prostate and prostatecancer, we generated MAbs toward the protease domain. Using theseMAbs, we demonstrate that TMPRSS2 protein is highly expressed inprostate secretory epithelium and in prostate cancer and that theprotein expression is also dependent on an androgen signal. Theprotease domain is released by an autocatalytic cleavage mechanism,resulting in its secretion into the extracellular space.

MATERIALS AND METHODS

Cell Lines and Human Tissues.All human cancer cell lines used in thisstudy were obtained from the American Type Culture Collection. All cell lineswere maintained in DMEM with 10% FCS. Primary prostate epithelial cells(PrEC) were obtained from Clonetics and grown in prostate epithelial basalmedium (PrEBM) media supplemented with growth factors (Clonetics).

The AD LAPC-4 and LAPC-9 xenografts were routinely passaged as smalltissue chunks in SCID male mice (5, 9). AI sublines of the xenografts werederived as described previously (5, 9) and passaged in castrated males or infemale SCID mice.

Human tissues for RNA and protein analyses were obtained from theHuman Tissue Resource Center at the University of California Los Angeles(Los Angeles, CA), from the National Disease Research Interchange (Phila-delphia, PA), and from QualTek, Inc. (Santa Barbara, CA).

SSH. Tumor tissue and cell lines were homogenized in Trizol reagent (LifeTechnologies, Inc.) using 10 ml/gram tissue or 10 ml/108 cells to isolate totalRNA. Polyadenylated RNA was purified from total RNA using Qiagen’sOligotex mRNA Mini and Midi kits. SSH was performed using the PCR-SelectcDNA Subtraction Kit (Clontech, Palo Alto, CA; Ref. 6). Tester cDNA wasderived from the AD LAPC-9 xenograft, whereas driver cDNA was obtainedfrom the AI subline of LAPC-9. SSH-derived gene fragments were processedand analyzed as described previously (10).

Expression Analysis.Northern blotting was performed on 10mg of totalRNA prepared from cell lines and LAPC xenografts using random hexamer-labeled (Boehringer Mannheim)TMPRSS2cDNA. The human multitissuemRNA blots containing 2mg of mRNA per lane were purchased fromClontech and probed withTMPRSS2cDNA.

Constructs. TMPRSS2cDNA was isolated by screening a human prostatecDNA library (Life Technologies, Inc.) using clone 20P1F12, aTMPRSS2SSH-derived fragment, as a probe. For protein expression in 293T cells,TMPRSS2was cloned into pcDNA 3.1 Myc-His (Invitrogen, Carlsbad, CA)and retroviral vector pSRatkneo (11). Cells were transfected with eitherpSRatkneo-expressingTMPRSS2or a COOH-terminal His-taggedTMPRSS2.

Single point mutants ofTMPRSS2were generated using a two-step PCRmethod. Arg-240, Arg-252, and Arg-255 were mutated to glutamines, andSer-441 was mutated to alanine. Using a full-length cDNA clone as template,a 59-TMPRSS2 PCR fragment was generated using one mutagenic primer(59-GGCCCTCCAGCGTCACCC-39for S441A mutant, 59-CCGCAGGC-TATACATTGTAAAGAAACC-39 for R240Q, 59-TCCTGCTCTGTTGGCT-TGAGTTCA-39 for R252Q, and 59-CCCACAATCTGGCTCTGGCG-39forR255Q) and one 59-specific TMPRSS2 primer containing the start codon, akozak sequence, and anEcoRI site for cloning (59CGAATTCGCAAGATG-GCTTTGAAC-39). The 39fragment was generated by PCR using the reversecomplement of the mutagenic primer (59-GGGTGACGCTGGAGGGCC-39for S441A, 59-GGTTTCTTTACAATGTATAGCCTGCGG-39for R240Q, 59-

Received 7/5/00; accepted 12/15/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Present address: EOS Biotechnology, South San Francisco, CA 94080.2 To whom requests for reprints should be addressed, at UroGenesys Inc., 1701

Colorado Avenue, Santa Monica, CA 90404. Phone: (310) 820-8029, ext. 216; Fax:(310) 820-8489; E-mail: [email protected].

3 The abbreviations used are: PSA, prostate-specific antigen; MAb, monoclonal anti-body; GST, glutathioneS-transferase; AD, androgen-dependent; AI, androgen-indepen-dent; SCID, severe combined immunodeficient; SSH, suppression subtractive hybridiza-tion; AR, androgen receptor; SRCR, scavenger receptor cysteine-rich; LDLRA, low-density lipoprotein receptor A; IGFBP-3, insulin-like growth factor-binding protein 3;CSS, charcoal-stripped serum.

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TGAACTCAAGCCAACAGAGCAGGA-39 for R252Q mutant, and 59-CGC-CAGAGCCAGATTGTGGG-39 for R255Q) and a 39-specific TMPRSS2primer containing the stop codon and aXbaI site for cloning (59CGTCTA-GATTAGCCGTCTGCCCTCA-39). For the second round of PCR, the 39 and59 fragments were used as templates. The final PCR product was digested withEcoRI andXbaI and cloned into pSRaMSV-tkNeo. Protein expression wasanalyzed after transfection into 293T cells.

To generate PC-3 AR cells, AR cDNA was cloned into pSRaMSV-tkNeo.Retrovirus was generated (11) by cotransfection of retroviral expression plas-mid with psi(2) amphotropic packaging plasmid and used to infect PC-3 cells.Stable cell lines were generated by selection in G418-containing media for 1–2weeks.

Protein Analysis. In vitro transcription/translation was performed usingthe TNT Coupled Reticulocyte Lysate System (Promega, Madison, WI). Twomg of DNA (luciferase orTMPRSS2cDNA in pCMV-Sport-6) were incubatedwith 40 mg of TNT lysate and 20mCi of [35S]methionineTrans-label (ICN,Costa Mesa, CA). The reaction was terminated with SDS sample buffer andanalyzed by SDS-PAGE and autoradiography.

Mouse MAbs were generated toward a COOH-terminal region of theprotease (residues 362–440) fused to bacterial GST. The GST-fusion proteinwas generated by PCR using the following primers to amplify the TMPRSS2sequence: 59-TTGAATTCCAAACCAGTGTGTCTGCCC-39and 59-AAGCT-CGAGTCGTCACCCTGGCAAGAAT-39. The PCR product was inserted intopGEX-4T-3 (Amersham Pharmacia Biotech) using theEcoRI andXhoI clon-ing sites. GST-fusion protein was purified and used to immunize mice. Hy-bridoma supernatants were screened by Western blotting using cell extractsfrom 293T cells expressing TMPRSS2-mycHis-tagged protein. 1F9, a hybri-doma clone that specifically recognizes TMPRSS2 protein, was used for allsubsequent studies. Western blotting of tissue, xenograft, and cancer cell linelysates was performed on 20mg of cell lysate protein. Normalization ofextracts was achieved by probing cell extracts with anti-GRB-2 antibodies(Transduction Laboratories, San Diego, CA).

TMPRSS2 Detection in Medium and Serum Samples.Media was col-lected from TMPRSS2-expressing cells and analyzed directly (20ml) byWestern blotting using 1F9 MAb. For immunoprecipitations, 1F9 MAb wascovalently coupled to protein G beads using the homobifunctional cross-linking reagent dimethyl pimelimidate (Pierce, Rockford, IL). SCID mouseserum (100ml) was diluted to 1 ml with radioimmunoprecipitation assay buffer[25 mM Tris (pH 7.5), 150 mM NaCl, 0.5% sodium deoxycholate, 1% TritonX-100, 1% SDS, and 2 mM EDTA], incubated with 50ml of a 50% (w/v)1F9-coupled protein G bead slurry, and immunoprecipitated at 4°C overnight.Immunoprecipitates were washed with radioimmunoprecipitation assay buffer,eluted in SDS-sample buffer, and analyzed by Western blotting.

Immunohistochemistry. Immunohistochemical analysis of formalin-fixed, paraffin-embedded tissues with 1F9 MAb was performed on 4-mm tissuesections. After steam treatment in sodium citrate (10 mM, pH 6.0), slides wereincubated with 10mg/ml 1F9 MAb followed by an incubation with biotin-ylated rabbit antimouse IgG. Competitive inhibition studies were carried out inthe presence of 50mg/ml GST or GST-TMPRSS2 fusion protein. The reactionswere visualized using avidin-conjugated horseradish peroxidase (Vector Lab-oratories, Burlingame, CA).

RESULTS

Identification of TMPRSS2as a Gene Down-Regulated in AIProstate Cancer Xenografts.The prostate cancer bone metastasis-derived LAPC-9 xenograft (5) was used to identify genes that aredifferentially regulated during the transition from androgen depend-ence to androgen independence. A gene fragment corresponding to the39 untranslated region of theTMPRSS2gene was identified as down-regulated in LAPC-9 AI tissue.TMPRSS2was originally discoveredby exon trapping for chromosome 21 (7) and subsequently shown tobe androgen regulated in the prostate cancer cell line LNCaP (8). Weisolated a full-length cDNA forTMPRSS2from a normal prostatelibrary. Sequence analysis of the cDNA revealed an open readingframe of 492 amino acids, as reported previously (7). However, closerexamination of the sequence showed eight differences in the nucleo-tide sequence that result in six amino acid differences at positions 160,242, 329, 449, 489, and 491 compared with the previously publishedsequence (Fig. 1). All six differences were confirmed by sequencingadditionalTMPRSS2cDNA clones and gene fragments derived fromnormal prostate and LAPC-4 and LAPC-9 xenograft cDNA libraries.All of these differences occur in the putative extracellular domain;two of them (amino acids 160 and 242) reside in the SRCR domain,whereas the others are all located within the protease domain.

TMPRSS2 Is Expressed at High Levels in Prostate CancerTissues and Colon Cancer Cell Lines.To analyzeTMPRSS2ex-pression in cancer tissues and cell lines, Northern blotting was per-formed on RNA derived from the LAPC xenografts and a panel ofprostate and non-prostate cancer cell lines. The results show highexpression levels of a 3.8-kbTMPRSS2transcript in all of the LAPCxenografts and in colon cancer cell lines (Fig. 2). Similar expressionlevels were detected in prostate, LAPC-4 AD, LAPC-4 AI, LAPC-9AD, and LNCaP cells. Lower levels ofTMPRSS2were seen inLAPC-9 AI and PC-3 cells, with no expression seen in DU145. Highlevels of TMPRSS2expression were also detected in three of fourcolon cancer cell lines, including LoVo, T84, and Colo-205 (Fig. 2).Analysis of other cancer cell lines, including breast, testicular, ovar-ian, pancreatic and cervical showed no detectable expression (data notshown).

Fig. 1. Schematic representation of TMPRSS2. The TMPRSS2 cDNA was cloned froma normal prostate cDNA library. The sequence reveals eight differences in the nucleic acidsequence that result in six amino acid differences (shown with numbering) compared withthe previously published sequence. The different domains are indicated withdifferentshadingsand correspond to the following residues: 255–492 contains the protease;149–242 contains the SRCR; 113–148 contains the LDLRA; and 84–106 contains thetransmembrane (TM) region. The region in the protease domain (residues 362–440) fusedto GST and used as antigen isunderlined. The nucleotide sequence and protein sequencefor TMPRSS2 have been deposited with GenBank under the accession number AF270487.

Fig. 2. High expression levels ofTMPRSS2in prostate, prostate cancer, and coloncancer cells. Northern blots containing RNA derived from normal tissues, prostate cancerxenografts and cell lines, and colon cancer cell lines were probed using aTMPRSS2 cDNAfragment.Lane 1, prostate;Lane 2, LAPC-4 AD cells;Lane 3, LAPC-4 AI cells;Lane 4,LAPC-9 AD cells;Lane 5, LAPC-9 AI cells;Lane 6, LNCaP cells;Lane 7, PC-3 cells;Lane 8, DU145 cells;Lane 9, CaCo-2 cells;Lane 10, LoVo cells;Lane 11, T84 cells;Lane12, Colo-205 cells. All RNA samples were normalized with ab-actin probe. Sizestandards (in kb) are indicated on theright.

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CATALYTIC CLEAVAGE AND SECRETION OF TMPRSS2 PROTEASE



The first report onTMPRSS2showed the highest expression of thegene in small intestine (7), whereas a second report showed thehighest expression of the gene in prostate tissue (8). Northern blottingof 16 normal tissues usingTMPRSS2cDNA as a probe showed thehighest expression of a 3.8-kb transcript in normal prostate (Fig. 2),confirming the results obtained by Linet al. (8). Lower expressionlevels were also detected in pancreas, kidney, and lung. No significantexpression was detected in small intestine.

TMPRSS2 Protein Is Proteolytically Cleaved in Tissues andCell Lines. In vitro-translated TMPRSS2 appears predominantly asthe predictedMr 54,000 protein, with a minor breakdown product ofMr 32,000 (Fig. 3A). Mouse MAbs were generated toward the prote-ase domain of TMPRSS2 to study its expression pattern in cells andtissues. Western blotting of protein extracts from 293T cells using the1F9 MAb showed equal expression of two protein species withapparent molecular weights of 70,000 and 32,000 only inTMPRSS2-transfected cells and not in control vector-transfected cells (Fig. 3).Similar results were obtained using other MAbs directed againstTMPRSS2 (data not shown). TheMr 70,000 cellular isoform ofTMPRSS2 is larger than the predicted andin vitro-translated full-length protein, suggesting that it is modified, possibly by glycosyla-tion. The TMPRSS2 protein sequence contains three possible N-linked glycosylation sites at residues 128, 213, and 249. Preliminarystudies using N-glycosidase F-treated TMPRSS2 protein indicate thatthe protein is indeed glycosylated and that glycosylation accounts forsome of the difference between the predicted and observed molecularweight of cellular TMPRSS2 (data not shown). TheMr 32,000 formmay be a proteolytically cleaved fragment containing the COOH-terminal epitopes recognized by the antibody.

Analysis of human tissue and cell lines, which express varying

levels of TMPRSS2by Northern blotting, showed the same patternof protein expression as seen inTMPRSS2-transfected 293T cells.TMPRSS2 expression was detected in protein lysates from LAPC-4AD, LAPC-9 AD, and LNCaP cells, but not in the LAPC-9 AI, AIPC-3, and DU145 AI cells (Fig. 3,B andC). Analysis of three prostatecancer specimens and the matched adjacent tissue with apparentlynormal morphology showed significant expression of TMPRSS2 pro-tein in all samples (Fig. 3D). High expression was also detected in thecolon cancer cell line Colo-205 (Fig. 3B).

TMPRSS2 Protein Expression Is Dependent on the AR Signal.Our analysis above shows that expression of TMPRSS2 protein isundetectable in the AI LAPC-9, PC-3, and DU145 cell lines. Toextend these findings, as well as the observation by Linet al. (8) thatTMPRSS2message is androgen regulated in LNCaP cells, we andro-gen-deprived LNCaP cells and an LAPC-9 AD cell line derived fromthe LAPC-9 AD xenograft, which expresses the wild-type AR andPSA (data not shown). TMPRSS2 protein expression in the androgen-deprived cells was compared with expression in LNCaP cells (Fig.4A) and LAPC-9 AD cells (Fig. 4B) after treatment with mibolerone,a synthetic androgen. The results show that TMPRSS2 expression wassignificantly reduced during androgen deprivation in both cell linesand reappeared during mibolerone treatment. PSA protein levels weremeasured in parallel, showing a similar regulation (data not shown).

PC-3 cells, which do not express the AR and grow in an AI manner,express only low levels of TMPRSS2 (Fig. 4C). However, PC-3 cellsthat have been engineered to express the wild-type AR (PC-3 AR)express significant levels of TMPRSS2 when treated with mibolerone(Fig. 4C). Interestingly, only the full-length TMPRSS2 protein isdetected in untreated PC-3 AR cells and control PC-3 neo cells.Treatment with mibolerone not only increases total TMPRSS2 ex-pression but also induces expression of theMr 32,000 cleavageproduct. This indicates that TMPRSS2 expression in prostate cancercells is dependent on an androgen signal and that cleavage may be afunction of expression level.

TMPRSS2 Protease Is Released into Cell Culture Medium andMouse Serum by Prostate Cancer Cells.Structural predictions forTMPRSS2 suggest that it is a type II transmembrane protein with anextracellular protease domain (Fig. 1). The size of the cleavedMr

32,000 TMPRSS2 fragment suggests that it contains the entire prote-ase region. Cleavage of this domain is thus predicted to result in therelease of the protease into the extracellular space. To test this hy-pothesis, media were collected from androgen-starved and androgen-stimulated LNCaP cells. The media were then analyzed for the pres-ence of TMPRSS2 protein by Western blotting using anti-TMPRSS2MAb. The results show a clear detection of cleaved TMPRSS2 proteinin the media of androgen-stimulated cells, but not in androgen-deprived cells (Fig. 4D). The amount of protease present in the mediais directly correlated to the amount of TMPRSS2 protein present inthe cell extracts, which increases with an increased dose of mibo-lerone (Fig. 4D). Similar release of protease into cell media was alsoobserved for PC-3 AR cells that were treated with mibolerone (Fig.4D). Secreted TMPRSS2 protease is also detected in the sera of malemice that harbor LNCaP tumors, but not in sera derived from naı¨vemales (Fig. 4D). TheMr 70,000 form of TMPRSS2 is not detected inthe cell media of TMPRSS2-expressing cells or in the sera of LNCaPtumor-bearing mice.

TMPRSS2 Cleavage Is a Consequence of Autocatalytic Ac-tivity. To determine whether the cleavage of TMPRSS2 is dependenton its own catalytic activity, Ser-441 of the catalytic triad in theprotease domain was mutated to alanine (S441A). Mutant TMPRSS2cDNA was cloned into a retroviral vector for expression in 293T cells.Western blot analysis of cell extracts of 293T cells transfected witheither wild-type or S441A mutant TMPRSS2 showed that in contrast

Fig. 3. TMPRSS2 protein is expressed as a full-length and proteolytically cleaved formin tissues and cell lines.A, in vitro-translated proteins (5ml of reaction) were analyzed bySDS-PAGE and autoradiography.B, cell and tissue lysates (20mg of protein) wereprepared in sample buffer and probed on Western blots using the 1F9 MAb. Lysate from293T cells transfected with either control vector orTMPRSS2-expressing vector was usedas a negative and positive control, respectively.C, tissue lysates (20mg) of LAPC-9 AIand LAPC-9 AD xenografts were analyzed by 1F9 MAb Western blotting.D, prostatetumor tissues (T) with matched normal adjacent tissues (N) derived from patients under-going radical prostatectomies, with Gleason scores of 7, 9, and 7, respectively, wereanalyzed by 1F9 MAb Western blotting. The full-length TMPRSS2 protein and thecleavedMr 32,000 protease are indicated witharrows.

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to wild-type protein, TMPRSS2 S441A appeared as a single proteinspecies with an apparent molecular weight of 70,000 (Fig. 5). Cellmedia analysis shows the presence of the cleavedMr 32,000 proteasedomain only in media collected from cells transfected with wild-typeTMPRSS2 and not in media from S441A-transfected cells (data notshown). TMPRSS2 with a COOH-terminal myc-His tag also showed

predominant expression of the full-length tagged protein, althoughsome cleavage product is detected (Fig. 5). The myc-His tag at theCOOH terminus of the protease domain may exert some inhibitoryactivity on the proteolytic cleavage. These results suggest that theproteolytic cleavage of TMPRSS2 in cells and tissues is a conse-quence of autocatalytic activity.

The protease domain of TMPRSS2 belongs to the S1 family ofserine proteases with cleavage activity after Arg or Lys residues.Examination of the protein sequence reveals the presence of three Argresidues (amino acids 240, 252, and 255) near the NH2-terminalregion of the TMPRSS2 protease domain. To identify the actualcleavage site of the protease domain, the three Arg residues weremutated to Gln residues. The mutants were expressed in 293T cells bytransient transfection and analyzed by Western blotting. Only theR255Q mutation resulted in a loss of cleavage, identifying the Arg-255-Ile-256 bond as the proteolytic cleavage site.

TMPRSS2 Is Expressed in the Secretory Epithelia of Prostateand Prostate Cancer Cells.The expression of TMPRSS2 in prostatecancer biopsies and surgical samples was examined by immunohis-tochemical analysis. Specific staining of TMPRSS2 protein was val-idated using LNCaP cells that were androgen deprived for 1 week andthen either left untreated or stimulated with mibolerone for 9 h. Thecells were then fixed, embedded in paraffin, and stained with the 1F9MAb. TMPRSS2 staining of androgen-deprived LNCaP showed verylittle staining of the cells. In contrast, the majority of androgen-stimulated cells showed strong intracellular staining (Fig. 6,a andb).LNCaP cells growing in regular media exhibited staining similar tothat seen in androgen-stimulated cells (data not shown). The majorityof staining appeared to be localized to granular structures within thecell.

Analysis of 20 prostate clinical specimens showed moderate tostrong staining in the glandular epithelia of all normal prostate,prostatic intraepithelial neoplasia (PIN), and prostate cancer samplestested (Fig. 6,c andd; Table 1). The signal appeared to be strongestat the apical side of the secretory cells, and in most cases granularstaining was seen, as observed in LNCaP. The prostate tissue stainingwas specific because GST-TMPRSS2 immunogen could competi-tively inhibit staining of prostate cancer tissue, whereas GST alonecould not (Fig. 6,e and f). Similar to PSA, TMPRSS2 protein wasfound to accumulate within the lumen of the epithelial glands (Fig. 6,

Fig. 4. TMPRSS2 protease is androgen regulated andsecreted by prostate cancer cells.A, LNCaP cells were grownin medium containing either 10% fetal bovine serum (NT) orin 2% CSS for 1 week. Cells growing in CSS were leftuntreated (2) or stimulated (1) with mibolerone (10 nM) for9 or 24 h. B, a LAPC-9 AD cell line derived from the LAPC-9AD xenograft was analyzed for AD regulation of TMPRSS2protein as described inA but stimulated with mibolerone for14, 24, and 36 h.C, PC-3 cells and PC-3 cells expressing ARwere stimulated with mibolerone for 9 h. Lysates wereprobed with 1F9 MAb.D, LNCaP cells were androgen de-prived (2) and then treated with either 1 or 10 nM miboleronefor 36 h. Cell extracts and cell media (20ml) were collectedfor anti-1F9 Western blotting. Sera from naı̈ve male SCIDmice or mice bearing LNCaP tumors were harvested, immu-noprecipitated (i.p.) using 1F9 MAb, and then analyzed byWestern blotting using 1F9 MAb. Cell media from androgen-stimulated PC-3 and PC-3 AR cells were immunoprecipitatedusing 1F9 MAb and then analyzed by Western blotting using1F9 MAb. Molecular weight standards are indicated on theright in thousands.

Fig. 5. Mutagenic analysis of TMPRSS2 reveals autocatalytic activity and proteolyticcleavage site. 293T cells were transfected with the differentTMPRSS2mutants that areshown schematically. The mutations in Ser-441 of the catalytic triad and Arg-240,Arg-252, and Arg-255 areunderlined in bold. The actual cleavage site at Arg-255 isindicated by anarrowhead. Western blots of 293T cell lysates (20mg of protein) wereprobed with 1F9 MAb. The full-lengthMr 70,000 TMPRSS2 protein and the cleavedMr

32,000 protease are indicated byarrows. Molecular weight standards are indicated on theright in thousands.

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g andh), indicating that TMPRSS2 protein is secreted by the secretoryepithelia. This staining pattern is in contrast to the RNAin situhybridization analysis, which showed expression ofTMPRSS2RNAprimarily in the basal cell layer of normal prostate glands and not inthe secretory epithelium (8). It is possible thatTMPRSS2RNA ispresent in both compartments, but that basal cell RNA is more stable.

Analysis of 10 non-prostate tissues showed no staining in mosttissues (Table 1), including kidney and lung, which express someTMPRSS2message. Protein expression was detected in normal pan-creas samples and normal colon and colon cancer tissues (Table 1).The pancreatic staining was restricted to the pyramidal exocrineacinar cells, with no staining detected in the islets of Langerhans (datanot shown). The staining in normal colon appeared primarily withincrypts of the mucosal lining but was significantly less intense thannormal prostate staining (data not shown). The staining in coloncancer seemed to concentrate in luminal areas, indicating possiblesecretion of the antigen by colon cancer cells.

DISCUSSION

TMPRSS2was identified during an effort to discover genes thatare differentially expressed between hormone-dependent and hor-mone-independent prostate cancer tissue. We foundTMPRSS2tobe down-regulated in LAPC-9 AI, a prostate cancer xenograftoriginally derived from a bone metastasis. Our sequence, whichwas derived from a normal prostate library, contains several dif-ferences compared with the previously published sequence, whichwas cloned from a heart library (7). These differences occurprimarily in the protease domain and could have functional im-plications for protease activity or substrate specificity of theTMPRSS2 protein. Extensive expression profiling by us and others(8) has determined thatTMPRSS2is most highly expressed inprostate and prostate cancer cell lines and tissues. However, ourstudies show that TMPRSS2 protein is also expressed in pancre-atic, colon, and colon cancer tissue. High expression levels were

Fig. 6. Immunohistochemical analysis of prostate cancerpatient samples with anti-TMPRSS2 MAb.a, LNCaP cellsgrown in medium containing 2% CSS for 1 week;b, LNCaPcells grown in medium containing 2% CSS for 1 week andstimulated with mibolerone (10 nM) for 9 h; c, normal prostatetissue;d, grade 3 prostate carcinoma region (Gleason score 7tissue);e, grade 4 prostate carcinoma stained in the presence ofGST-TMPRSS2 protein;f, grade 4 prostate carcinoma stained inthe presence of GST protein;g, normal prostate tissue (31000);h, normal prostate tissue (31000). Samplesa–g were stainedwith 1F9 MAb; sampleh was stained with anti-PSA antibodies.Staining of secreted protein within the prostate gland lumen isindicated by thearrows. All pictures are at3400 magnification,except where indicated otherwise.

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detected particularly in colon cancer cell lines, indicating a possi-ble up-regulation of TMPRSS2.

In prostate cancer cells and tissues, TMPRSS2 is regulated by anandrogen signal. Androgen deprivation and stimulation experimentsin LAPC-9 xenograft and LAPC-9 cell line, which express the wild-type AR (5), and in LNCaP cells show that TMPRSS2 proteinexpression is increased with androgen stimulation. PC-3 cells, whichdo not normally express the AR (12) and grow in an AI manner,express little TMPRSS2. However, constitutive expression of thewild-type AR in PC-3 cells and simultaneous stimulation with andro-gen induce the expression of TMPRSS2 protein. PSA expression inPC-3 cells can also be induced by heterologous expression of the ARand concomitant androgen stimulation (13). These studies demon-strate that, similar to PSA, TMPRSS2 protein expression is criticallydependent on the AR pathway.

Prostate tissue expresses a number of androgen-regulated proteases,including PSA, human glandular kallikrein and prostase/KLK-L1(14–16). TMPRSS2 is unique among these proteases due to itsstructural features. It is a putative type II transmembrane protease witha SRCR and a LDLRA domain (7). The protease domain is located atthe COOH terminus, which is postulated to be extracellular. Thesefeatures suggested that TMPRSS2 protein is expressed at the cellmembrane and could function as a receptor for other proteins or smallmolecules. The TMPRSS2 protease domain is most homologous tohepsin (also a type II transmembrane protease), which lacks the SRCRand LDLRA domains (17) and is up-regulated in ovarian cancer (18).Subcellular fractionation studies localized hepsin to the membraneousfraction of cultured HepG2 cells (19). Our studies show that a signif-icant fraction of TMPRSS2 protein is proteolytically cleaved andsecreted. The remaining regions, including the LDLRA and the SRCRdomains, are possibly still membrane associated and could function asreceptors independent of the protease domain.

Mutational inactivation of the TMPRSS2 protease shows that thecleavage of the protease is a consequence of its own catalytic activity,suggesting that TMPRSS2 may be its own substrate. Alterna-tively, TMPRSS2 activates a secondary protease that then cleavesthe TMPRSS2 protease. Both interpretations require an activeTMPRSS2 protease. Similar autocatalytic cleavage has also beenobserved for hepsin (20). Site-directed mutagenesis shows that thecleavage of TMPRSS2 occurs at Arg-255 and results in the release ofthe protease domain. Western blot analysis using anti-TMPRSS2MAb, directed to the protease domain, demonstrated that only theMr

32,000 protease fragment is secreted by prostate cancer cells andxenografts into the media or sera, respectively (Fig. 4D). Thesefindings suggest that the protein accumulated in the glandular lumen

of normal and cancerous prostate tissues is the cleaved proteasefragment. Proliferation and metastasis of cancer cells, associated withdestruction of the normal tissue architecture, may cause a rise inserum TMPRSS2 levels that would signify the presence of cancercells. These data suggest that released TMPRSS2 protease may beuseful as a potential serum diagnostic or prognostic marker for pros-tate and possibly colon cancer.

Secreted TMPRSS2 protease may be involved in processing andactivating growth factors present in the extracellular space. Recentwork with PSA and human glandular kallikrein have shown that theymay play a role in activating growth factors important in the osteo-blastic response of bone-metastasized prostate cancer (21). One ofthese factors, parathyroid hormone-related protein, has been shown toincrease the rate of prostate tumor growthin vivo and protect LNCaPcells from apoptosis (22). IGFBP-3, an inhibitor of insulin-like growthfactor I, has also recently been shown to be a substrate for PSA (23,24). Cleavage of IGFBP-3 results in an increase in active insulin-likegrowth factor I, a growth factor implicated in prostate cancer cellgrowth (23, 25). It remains to be seen whether TMPRSS2 is capableof acting on parathyroid hormone-related protein, IGFBP-3, and/orother factors that could influence prostate cancer growth. The expres-sion pattern and localization of TMPRSS2 make it a potential targetfor therapy in cancers of the prostate.

ACKNOWLEDGMENTS

We thank Drs. Robert Eisenman, Owen Witte, and William Isaacs forhelpful suggestions and critical reading of the manuscript; Frank Lynch andPage Erickson at QualTek Molecular Laboratories for their expertise in im-munohistochemistry; and Celeste Jones for help in preparation of the manu-script.

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Table 1 Immunohistochemical staining of human tissues with anti-TMPRSS2 MAb

Tissue sections were stained with 1F9 MAb as described in “Materials and Methods.”The number of samples that stained positive per tissue are indicated in parentheses.

Staining intensity Tissuea

None KidneySkinLiverLungTestisSpleen

Light Fallopian tubesColon cancer (1/4)

Moderate Colon (4/4)Strong Prostate (7/7)

BPH (7/7)PIN (1/1)Prostate cancer (5/5)Colon cancer (3/4)Pancreas (4/4)

a PIN, prostatic intraepithelial neoplasia.

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2001;61:1686-1692. Cancer Res Daniel E. H. Afar, Igor Vivanco, Rene S. Hubert, et al. Cancer EpitheliaProtease Results in Its Secretion by Prostate and Prostate Catalytic Cleavage of the Androgen-regulated TMPRSS2

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