Catalytic Cleavage of the Androgen-Regulated TMPRSS2 Protease Results in Its Secretion by Prostate and Prostate Cancer Epithelia

[CANCER RESEARCH 61, 1686–1692, February 15, 2001] Catalytic Cleavage of the Androgen-regulated TMPRSS2 Protease Results in Its Secretion 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 TMPRSS2 was predicted to be a type II transmembrane protein with an extracellular COOH terminus containing the protease domain We identified TMPRSS2 as a gene that is down-regulated in androgen- and an intracellular NH terminus. TMPRSS2 was recently shown to independent prostate cancer xenograft tissue derived from a bone metas- 2 be regulated by androgens in the LNCaP prostate cancer cell model tasis. Using specific monoclonal antibodies, we show that the TMPRSS2- encoded serine protease is expressed as a M 70,000 full-length form and (8). To characterize the TMPRSS2 protein in prostate and prostate r cancer, we generated MAbs toward the protease domain. Using these a cleaved Mr 32,000 protease domain. Mutation of Ser-441 in the catalytic triad shows that the proteolytic cleavage is dependent on catalytic activity, MAbs, we demonstrate that TMPRSS2 protein is highly expressed in suggesting that it occurs as a result of autocleavage. Mutational analysis prostate secretory epithelium and in prostate cancer and that the reveals the cleavage site to be at Arg-255. A consequence of autocatalytic protein expression is also dependent on an androgen signal. The cleavage is the secretion of the protease domain into the media by protease domain is released by an autocatalytic cleavage mechanism, TMPRSS2-expressing prostate cancer cells and into the sera of prostate resulting in its secretion into the extracellular space. tumor-bearing mice. Immunohistochemical analysis of clinical specimens demonstrates the highest expression of TMPRSS2 at the apical side of prostate and prostate cancer secretory epithelia and within the lumen of MATERIALS AND METHODS the glands. Similar luminal staining was detected in colon cancer samples. Expression was also seen in colon and pancreas, with little to no expression Cell Lines and Human Tissues. All human cancer cell lines used in this detected in seven additional normal tissues. These data demonstrate that study were obtained from the American Type Culture Collection. All cell lines TMPRSS2 is a secreted protease that is highly expressed in prostate and were maintained in DMEM with 10% FCS. Primary prostate epithelial cells prostate cancer, making it a potential target for cancer therapy and (PrEC) were obtained from Clonetics and grown in prostate epithelial basal diagnosis. medium (PrEBM) media supplemented with growth factors (Clonetics). The AD LAPC-4 and LAPC-9 xenografts were routinely passaged as small tissue chunks in SCID male mice (5, 9). AI sublines of the xenografts were INTRODUCTION derived as described previously (5, 9) and passaged in castrated males or in Prostate cancer is a hormone-regulated disease that affects men in female SCID mice. the later years of life. Untreated prostate cancer metastasizes to lymph Human tissues for RNA and protein analyses were obtained from the Human Tissue Resource Center at the University of California Los Angeles nodes and bone in advanced cases. In such cases, the treatment (Los Angeles, CA), from the National Disease Research Interchange (Phila- consists of antagonizing the androgenic growth stimulus that feeds the delphia, PA), and from QualTek, Inc. (Santa Barbara, CA). tumor by chemical or surgical hormone ablation therapy (1). An SSH. Tumor tissue and cell lines were homogenized in Trizol reagent (Life unfortunate consequence of antiandrogen treatment is the develop- Technologies, Inc.) using 10 ml/gram tissue or 10 ml/108 cells to isolate total ment of androgen-independent cancer. Androgen-regulated genes, RNA. Polyadenylated RNA was purified from total RNA using Qiagen’s such as the gene encoding PSA,3 are turned off with hormone ablation Oligotex mRNA Mini and Midi kits. SSH was performed using the PCR-Select therapy but reappear when the tumor becomes androgen independent cDNA Subtraction Kit (Clontech, Palo Alto, CA; Ref. 6). Tester cDNA was (2). This suggests that alternative signaling pathways, such as acti- derived from the AD LAPC-9 xenograft, whereas driver cDNA was obtained vated tyrosine kinase receptors, can replace the androgen signal (3, 4). from the AI subline of LAPC-9. SSH-derived gene fragments were processed To understand the molecular events surrounding the progression of and analyzed as described previously (10). Expression Analysis. Northern blotting was performed on 10 ␮g of total prostate cancer to androgen independence, we used LAPC-9, a human RNA prepared from cell lines and LAPC xenografts using random hexamer- prostate cancer xenograft derived from a bone metastasis, which can labeled (Boehringer Mannheim) TMPRSS2 cDNA. The human multitissue mimic the transition from androgen dependence to androgen inde- mRNA blots containing 2 ␮g of mRNA per lane were purchased from pendence (5). A subtractive hybridization method (6) was used to Clontech and probed with TMPRSS2 cDNA. identify androgen-regulated genes by subtracting cDNAs from the Constructs. TMPRSS2 cDNA was isolated by screening a human prostate LAPC-9 AI subline from cDNAs derived from the original LAPC-9 cDNA library (Life Technologies, Inc.) using clone 20P1F12, a TMPRSS2 AD xenograft. This strategy resulted in the identification of the SSH-derived fragment, as a probe. For protein expression in 293T cells, TMPRSS2 gene, which encodes a putative transmembrane serine TMPRSS2 was cloned into pcDNA 3.1 Myc-His (Invitrogen, Carlsbad, CA) protease (7). and retroviral vector pSR␣tkneo (11). Cells were transfected with either pSR␣tkneo-expressing TMPRSS2 or a COOH-terminal His-tagged TMPRSS2. Single point mutants of TMPRSS2 were generated using a two-step PCR Received 7/5/00; accepted 12/15/00. method. Arg-240, Arg-252, and Arg-255 were mutated to glutamines, and The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with Ser-441 was mutated to alanine. Using a full-length cDNA clone as template, 18 U.S.C. Section 1734 solely to indicate this fact. a5Ј-TMPRSS2 PCR fragment was generated using one mutagenic primer 1 Present address: EOS Biotechnology, South San Francisco, CA 94080. (5Ј-GGCCCTCCAGCGTCACCC-3Ј for S441A mutant, 5Ј-CCGCAGGC- 2 To whom requests for reprints should be addressed, at UroGenesys Inc., 1701 TATACATTGTAAAGAAACC-3Ј for R240Q, 5Ј-TCCTGCTCTGTTGGCT- Colorado Avenue, Santa Monica, CA 90404. Phone: (310) 820-8029, ext. 216; Fax: Ј Ј Ј (310) 820-8489; E-mail: [email protected]. TGAGTTCA-3 for R252Q, and 5 -CCCACAATCTGGCTCTGGCG-3 for 3 The abbreviations used are: PSA, prostate-specific antigen; MAb, monoclonal anti- R255Q) and one 5Ј-specific TMPRSS2 primer containing the start codon, a body; GST, glutathione S-transferase; AD, androgen-dependent; AI, androgen-indepen- kozak sequence, and an EcoRI site for cloning (5ЈCGAATTCGCAAGATG- dent; SCID, severe combined immunodeficient; SSH, suppression subtractive hybridiza- GCTTTGAAC-3Ј). The 3Ј fragment was generated by PCR using the reverse tion; AR, androgen receptor; SRCR, scavenger receptor cysteine-rich; LDLRA, low- Ј Ј density lipoprotein receptor A; IGFBP-3, insulin-like growth factor-binding protein 3; complement of the mutagenic primer (5 -GGGTGACGCTGGAGGGCC-3 CSS, charcoal-stripped serum. for S441A, 5Ј-GGTTTCTTTACAATGTATAGCCTGCGG-3Ј for R240Q, 5Ј- 1686

RESULTS Identification of TMPRSS2 as a Gene Down-Regulated in AI Prostate Cancer Xenografts. The prostate cancer bone metastasis- derived LAPC-9 xenograft (5) was used to identify genes that are differentially regulated during the transition from androgen dependence to androgen independence. A gene fragment corresponding to the Ј Fig. 1. Schematic representation of TMPRSS2. The TMPRSS2 cDNA was cloned from 3 untranslated region of the TMPRSS2 gene was identified as down- a normal prostate cDNA library. The sequence reveals eight differences in the nucleic acid regulated in LAPC-9 AI tissue. TMPRSS2 was originally discovered sequence that result in six amino acid differences (shown with numbering) compared with by exon trapping for chromosome 21 (7) and subsequently shown to the previously published sequence. The different domains are indicated with different shadings and correspond to the following residues: 255–492 contains the protease; be androgen regulated in the prostate cancer cell line LNCaP (8). We 149–242 contains the SRCR; 113–148 contains the LDLRA; and 84–106 contains the isolated a full-length cDNA for TMPRSS2 from a normal prostate transmembrane (TM) region. The region in the protease domain (residues 362–440) fused to GST and used as antigen is underlined. The nucleotide sequence and protein sequence library. Sequence analysis of the cDNA revealed an open reading for TMPRSS2 have been deposited with GenBank under the accession number AF270487. frame of 492 amino acids, as reported previously (7). However, closer examination of the sequence showed eight differences in the nucleotide sequence that result in six amino acid differences at positions 160, TGAACTCAAGCCAACAGAGCAGGA-3Ј for R252Q mutant, and 5Ј-CGC- 242, 329, 449, 489, and 491 compared with the previously published CAGAGCCAGATTGTGGG-3Ј for R255Q) and a 3Ј-specific TMPRSS2 sequence (Fig. 1). All six differences were confirmed by sequencing primer containing the stop codon and a XbaI site for cloning (5ЈCGTCTA- additional TMPRSS2 cDNA clones and gene fragments derived from GATTAGCCGTCTGCCCTCA-3Ј). For the second round of PCR, the 3Ј and normal prostate and LAPC-4 and LAPC-9 xenograft cDNA libraries. 5Ј fragments were used as templates. The final PCR product was digested with All of these differences occur in the putative extracellular domain; EcoRI and XbaI and cloned into pSR␣MSV-tkNeo. Protein expression was two of them (amino acids 160 and 242) reside in the SRCR domain, analyzed after transfection into 293T cells. whereas the others are all located within the protease domain. To generate PC-3 AR cells, AR cDNA was cloned into pSR␣MSV-tkNeo. TMPRSS2 Is Expressed at High Levels in Prostate Cancer Retrovirus was generated (11) by cotransfection of retroviral expression plas- Tissues and Colon Cancer Cell Lines. To analyze TMPRSS2 ex- Ϫ mid with psi( ) amphotropic packaging plasmid and used to infect PC-3 cells. pression in cancer tissues and cell lines, Northern blotting was per- Stable cell lines were generated by selection in G418-containing media for 1–2 formed on RNA derived from the LAPC xenografts and a panel of weeks. prostate and non-prostate cancer cell lines. The results show high Protein Analysis. In vitro transcription/translation was performed using the TNT Coupled Reticulocyte Lysate System (Promega, Madison, WI). Two expression levels of a 3.8-kb TMPRSS2 transcript in all of the LAPC ␮g of DNA (luciferase or TMPRSS2 cDNA in pCMV-Sport-6) were incubated xenografts and in colon cancer cell lines (Fig. 2). Similar expression with 40 ␮g of TNT lysate and 20 ␮Ci of [35S]methionine Trans-label (ICN, levels were detected in prostate, LAPC-4 AD, LAPC-4 AI, LAPC-9 Costa Mesa, CA). The reaction was terminated with SDS sample buffer and AD, and LNCaP cells. Lower levels of TMPRSS2 were seen in analyzed by SDS-PAGE and autoradiography. LAPC-9 AI and PC-3 cells, with no expression seen in DU145. High Mouse MAbs were generated toward a COOH-terminal region of the levels of TMPRSS2 expression were also detected in three of four protease (residues 362–440) fused to bacterial GST. The GST-fusion protein colon cancer cell lines, including LoVo, T84, and Colo-205 (Fig. 2). was generated by PCR using the following primers to amplify the TMPRSS2 Analysis of other cancer cell lines, including breast, testicular, ovar- Ј Ј Ј sequence: 5 -TTGAATTCCAAACCAGTGTGTCTGCCC-3 and 5 -AAGCT- ian, pancreatic and cervical showed no detectable expression (data not Ј CGAGTCGTCACCCTGGCAAGAAT-3 . The PCR product was inserted into shown). pGEX-4T-3 (Amersham Pharmacia Biotech) using the EcoRI and XhoI cloning sites. GST-fusion protein was purified and used to immunize mice. Hy- bridoma supernatants were screened by Western blotting using cell extracts from 293T cells expressing TMPRSS2-mycHis-tagged protein. 1F9, a hybri- doma clone that specifically recognizes TMPRSS2 protein, was used for all subsequent studies. Western blotting of tissue, xenograft, and cancer cell line lysates was performed on 20 ␮g of cell lysate protein. Normalization of extracts 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 collected from TMPRSS2-expressing cells and analyzed directly (20 ␮l) by Western blotting using 1F9 MAb. For immunoprecipitations, 1F9 MAb was covalently coupled to protein G beads using the homobifunctional cross- linking reagent dimethyl pimelimidate (Pierce, Rockford, IL). SCID mouse serum (100 ␮l) was diluted to 1 ml with radioimmunoprecipitation assay buffer [25 mM Tris (pH 7.5), 150 mM NaCl, 0.5% sodium deoxycholate, 1% Triton X-100, 1% SDS, and 2 mM EDTA], incubated with 50 ␮l 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-␮m tissue Fig. 2. High expression levels of TMPRSS2 in prostate, prostate cancer, and colon sections. After steam treatment in sodium citrate (10 mM, pH 6.0), slides were cancer cells. Northern blots containing RNA derived from normal tissues, prostate cancer incubated with 10 ␮g/ml 1F9 MAb followed by an incubation with biotin- xenografts and cell lines, and colon cancer cell lines were probed using a TMPRSS2 cDNA ylated rabbit antimouse IgG. Competitive inhibition studies were carried out in fragment. Lane 1, prostate; Lane 2, LAPC-4 AD cells; Lane 3, LAPC-4 AI cells; Lane 4, the presence of 50 ␮g/ml GST or GST-TMPRSS2 fusion protein. The reactions 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; Lane were visualized using avidin-conjugated horseradish peroxidase (Vector Lab- 12, Colo-205 cells. All RNA samples were normalized with a ␤-actin probe. Size oratories, Burlingame, CA). standards (in kb) are indicated on the right. 1687

The first report on TMPRSS2 showed the highest expression of the levels of TMPRSS2 by Northern blotting, showed the same pattern gene in small intestine (7), whereas a second report showed the of protein expression as seen in TMPRSS2-transfected 293T cells. highest expression of the gene in prostate tissue (8). Northern blotting TMPRSS2 expression was detected in protein lysates from LAPC-4 of 16 normal tissues using TMPRSS2 cDNA as a probe showed the AD, LAPC-9 AD, and LNCaP cells, but not in the LAPC-9 AI, AI highest expression of a 3.8-kb transcript in normal prostate (Fig. 2), PC-3, and DU145 AI cells (Fig. 3, B and C). Analysis of three prostate confirming the results obtained by Lin et al. (8). Lower expression cancer specimens and the matched adjacent tissue with apparently levels were also detected in pancreas, kidney, and lung. No significant normal morphology showed significant expression of TMPRSS2 pro- expression was detected in small intestine. tein in all samples (Fig. 3D). High expression was also detected in the TMPRSS2 Protein Is Proteolytically Cleaved in Tissues and colon cancer cell line Colo-205 (Fig. 3B). Cell Lines. In vitro-translated TMPRSS2 appears predominantly as TMPRSS2 Protein Expression Is Dependent on the AR Signal. the predicted Mr 54,000 protein, with a minor breakdown product of Our analysis above shows that expression of TMPRSS2 protein is Mr 32,000 (Fig. 3A). Mouse MAbs were generated toward the prote- undetectable in the AI LAPC-9, PC-3, and DU145 cell lines. To ase domain of TMPRSS2 to study its expression pattern in cells and extend these findings, as well as the observation by Lin et al. (8) that tissues. Western blotting of protein extracts from 293T cells using the TMPRSS2 message is androgen regulated in LNCaP cells, we andro- 1F9 MAb showed equal expression of two protein species with gen-deprived LNCaP cells and an LAPC-9 AD cell line derived from apparent molecular weights of 70,000 and 32,000 only in TMPRSS2- the LAPC-9 AD xenograft, which expresses the wild-type AR and transfected cells and not in control vector-transfected cells (Fig. 3). PSA (data not shown). TMPRSS2 protein expression in the androgen- Similar results were obtained using other MAbs directed against deprived cells was compared with expression in LNCaP cells (Fig.

TMPRSS2 (data not shown). The Mr 70,000 cellular isoform of 4A) and LAPC-9 AD cells (Fig. 4B) after treatment with mibolerone, TMPRSS2 is larger than the predicted and in vitro-translated full- a synthetic androgen. The results show that TMPRSS2 expression was length protein, suggesting that it is modified, possibly by glycosyla- significantly reduced during androgen deprivation in both cell lines tion. The TMPRSS2 protein sequence contains three possible N- and reappeared during mibolerone treatment. PSA protein levels were linked glycosylation sites at residues 128, 213, and 249. Preliminary measured in parallel, showing a similar regulation (data not shown). studies using N-glycosidase F-treated TMPRSS2 protein indicate that PC-3 cells, which do not express the AR and grow in an AI manner, the protein is indeed glycosylated and that glycosylation accounts for express only low levels of TMPRSS2 (Fig. 4C). However, PC-3 cells some of the difference between the predicted and observed molecular that have been engineered to express the wild-type AR (PC-3 AR) weight of cellular TMPRSS2 (data not shown). The Mr 32,000 form express significant levels of TMPRSS2 when treated with mibolerone may be a proteolytically cleaved fragment containing the COOH- (Fig. 4C). Interestingly, only the full-length TMPRSS2 protein is terminal epitopes recognized by the antibody. detected in untreated PC-3 AR cells and control PC-3 neo cells. Analysis of human tissue and cell lines, which express varying Treatment with mibolerone not only increases total TMPRSS2 ex-

pression but also induces expression of the Mr 32,000 cleavage product. This indicates that TMPRSS2 expression in prostate cancer cells is dependent on an androgen signal and that cleavage may be a function of expression level. TMPRSS2 Protease Is Released into Cell Culture Medium and Mouse Serum by Prostate Cancer Cells. Structural predictions for TMPRSS2 suggest that it is a type II transmembrane protein with an

extracellular protease domain (Fig. 1). The size of the cleaved Mr 32,000 TMPRSS2 fragment suggests that it contains the entire protease region. Cleavage of this domain is thus predicted to result in the release of the protease into the extracellular space. To test this hypothesis, media were collected from androgen-starved and androgen- stimulated LNCaP cells. The media were then analyzed for the presence of TMPRSS2 protein by Western blotting using anti-TMPRSS2 MAb. The results show a clear detection of cleaved TMPRSS2 protein in the media of androgen-stimulated cells, but not in androgen- deprived cells (Fig. 4D). The amount of protease present in the media is directly correlated to the amount of TMPRSS2 protein present in the cell extracts, which increases with an increased dose of mibolerone (Fig. 4D). Similar release of protease into cell media was also observed for PC-3 AR cells that were treated with mibolerone (Fig. 4D). Secreted TMPRSS2 protease is also detected in the sera of male mice that harbor LNCaP tumors, but not in sera derived from naı¨ve

males (Fig. 4D). The Mr 70,000 form of TMPRSS2 is not detected in the cell media of TMPRSS2-expressing cells or in the sera of LNCaP Fig. 3. TMPRSS2 protein is expressed as a full-length and proteolytically cleaved form tumor-bearing mice. in tissues and cell lines. A, in vitro-translated proteins (5 ␮l of reaction) were analyzed by SDS-PAGE and autoradiography. B, cell and tissue lysates (20 ␮g of protein) were TMPRSS2 Cleavage Is a Consequence of Autocatalytic Ac- prepared in sample buffer and probed on Western blots using the 1F9 MAb. Lysate from tivity. To determine whether the cleavage of TMPRSS2 is dependent 293T cells transfected with either control vector or TMPRSS2-expressing vector was used on its own catalytic activity, Ser-441 of the catalytic triad in the as a negative and positive control, respectively. C, tissue lysates (20 ␮g) of LAPC-9 AI and LAPC-9 AD xenografts were analyzed by 1F9 MAb Western blotting. D, prostate protease domain was mutated to alanine (S441A). Mutant TMPRSS2 tumor tissues (T) with matched normal adjacent tissues (N) derived from patients under- cDNA was cloned into a retroviral vector for expression in 293T cells. going radical prostatectomies, with Gleason scores of 7, 9, and 7, respectively, were analyzed by 1F9 MAb Western blotting. The full-length TMPRSS2 protein and the Western blot analysis of cell extracts of 293T cells transfected with cleaved Mr 32,000 protease are indicated with arrows. either wild-type or S441A mutant TMPRSS2 showed that in contrast 1688

Fig. 4. TMPRSS2 protease is androgen regulated and secreted by prostate cancer cells. A, LNCaP cells were grown in medium containing either 10% fetal bovine serum (NT)or in 2% CSS for 1 week. Cells growing in CSS were left untreated (Ϫ) or stimulated (ϩ) with mibolerone (10 nM) for 9or24h.B, a LAPC-9 AD cell line derived from the LAPC-9 AD xenograft was analyzed for AD regulation of TMPRSS2 protein as described in A but stimulated with mibolerone for 14, 24, and 36 h. C, PC-3 cells and PC-3 cells expressing AR were stimulated with mibolerone for 9 h. Lysates were probed with 1F9 MAb. D, LNCaP cells were androgen deprived (Ϫ) and then treated with either 1 or 10 nM mibolerone for 36 h. Cell extracts and cell media (20 ␮l) were collected for anti-1F9 Western blotting. Sera from naı¨ve male SCID mice or mice bearing LNCaP tumors were harvested, immunoprecipitated (i.p.) using 1F9 MAb, and then analyzed by Western blotting using 1F9 MAb. Cell media from androgen- stimulated PC-3 and PC-3 AR cells were immunoprecipitated using 1F9 MAb and then analyzed by Western blotting using 1F9 MAb. Molecular weight standards are indicated on the right in thousands.

to wild-type protein, TMPRSS2 S441A appeared as a single protein predominant expression of the full-length tagged protein, although species with an apparent molecular weight of 70,000 (Fig. 5). Cell some cleavage product is detected (Fig. 5). The myc-His tag at the media analysis shows the presence of the cleaved Mr 32,000 protease COOH terminus of the protease domain may exert some inhibitory domain only in media collected from cells transfected with wild-type activity on the proteolytic cleavage. These results suggest that the TMPRSS2 and not in media from S441A-transfected cells (data not proteolytic cleavage of TMPRSS2 in cells and tissues is a conse- shown). TMPRSS2 with a COOH-terminal myc-His tag also showed quence of autocatalytic activity. The protease domain of TMPRSS2 belongs to the S1 family of serine proteases with cleavage activity after Arg or Lys residues. Examination of the protein sequence reveals the presence of three Arg

residues (amino acids 240, 252, and 255) near the NH2-terminal region of the TMPRSS2 protease domain. To identify the actual cleavage site of the protease domain, the three Arg residues were mutated to Gln residues. The mutants were expressed in 293T cells by transient transfection and analyzed by Western blotting. Only the R255Q 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 Prostate and Prostate Cancer Cells. The expression of TMPRSS2 in prostate cancer biopsies and surgical samples was examined by immunohistochemical analysis. Specific staining of TMPRSS2 protein was val- idated using LNCaP cells that were androgen deprived for 1 week and then either left untreated or stimulated with mibolerone for 9 h. The cells were then fixed, embedded in paraffin, and stained with the 1F9 MAb. TMPRSS2 staining of androgen-deprived LNCaP showed very little staining of the cells. In contrast, the majority of androgen- stimulated cells showed strong intracellular staining (Fig. 6, a and b). LNCaP cells growing in regular media exhibited staining similar to that seen in androgen-stimulated cells (data not shown). The majority of staining appeared to be localized to granular structures within the cell. Analysis of 20 prostate clinical specimens showed moderate to strong staining in the glandular epithelia of all normal prostate, prostatic intraepithelial neoplasia (PIN), and prostate cancer samples tested (Fig. 6, c and d; Table 1). The signal appeared to be strongest Fig. 5. Mutagenic analysis of TMPRSS2 reveals autocatalytic activity and proteolytic at the apical side of the secretory cells, and in most cases granular cleavage site. 293T cells were transfected with the different TMPRSS2 mutants that are staining was seen, as observed in LNCaP. The prostate tissue staining shown schematically. The mutations in Ser-441 of the catalytic triad and Arg-240, Arg-252, and Arg-255 are underlined in bold. The actual cleavage site at Arg-255 is was specific because GST-TMPRSS2 immunogen could competi- indicated by an arrowhead. Western blots of 293T cell lysates (20 ␮g of protein) were tively inhibit staining of prostate cancer tissue, whereas GST alone probed with 1F9 MAb. The full-length Mr 70,000 TMPRSS2 protein and the cleaved Mr 32,000 protease are indicated by arrows. Molecular weight standards are indicated on the could not (Fig. 6, e and f). Similar to PSA, TMPRSS2 protein was right in thousands. found to accumulate within the lumen of the epithelial glands (Fig. 6, 1689

Fig. 6. Immunohistochemical analysis of prostate cancer patient samples with anti-TMPRSS2 MAb. a, LNCaP cells grown in medium containing 2% CSS for 1 week; b, LNCaP cells grown in medium containing 2% CSS for 1 week and stimulated with mibolerone (10 nM)for9h;c, normal prostate tissue; d, grade 3 prostate carcinoma region (Gleason score 7 tissue); e, grade 4 prostate carcinoma stained in the presence of GST-TMPRSS2 protein; f, grade 4 prostate carcinoma stained in the presence of GST protein; g, normal prostate tissue (ϫ1000); h, normal prostate tissue (ϫ1000). Samples a–g were stained with 1F9 MAb; sample h was stained with anti-PSA antibodies. Staining of secreted protein within the prostate gland lumen is indicated by the arrows. All pictures are at ϫ400 magnification, except where indicated otherwise.

g and h), indicating that TMPRSS2 protein is secreted by the secretory DISCUSSION epithelia. This staining pattern is in contrast to the RNA in situ hybridization analysis, which showed expression of TMPRSS2 RNA TMPRSS2 was identified during an effort to discover genes that primarily in the basal cell layer of normal prostate glands and not in are differentially expressed between hormone-dependent and hor- the secretory epithelium (8). It is possible that TMPRSS2 RNA is mone-independent prostate cancer tissue. We found TMPRSS2 to present in both compartments, but that basal cell RNA is more stable. be down-regulated in LAPC-9 AI, a prostate cancer xenograft Analysis of 10 non-prostate tissues showed no staining in most originally derived from a bone metastasis. Our sequence, which tissues (Table 1), including kidney and lung, which express some was derived from a normal prostate library, contains several dif- TMPRSS2 message. Protein expression was detected in normal pan- ferences compared with the previously published sequence, which creas samples and normal colon and colon cancer tissues (Table 1). was cloned from a heart library (7). These differences occur The pancreatic staining was restricted to the pyramidal exocrine primarily in the protease domain and could have functional im- acinar cells, with no staining detected in the islets of Langerhans (data plications for protease activity or substrate specificity of the not shown). The staining in normal colon appeared primarily within TMPRSS2 protein. Extensive expression profiling by us and others crypts of the mucosal lining but was significantly less intense than (8) has determined that TMPRSS2 is most highly expressed in normal prostate staining (data not shown). The staining in colon prostate and prostate cancer cell lines and tissues. However, our cancer seemed to concentrate in luminal areas, indicating possible studies show that TMPRSS2 protein is also expressed in pancre- secretion of the antigen by colon cancer cells. atic, colon, and colon cancer tissue. High expression levels were 1690

Table 1 Immunohistochemical staining of human tissues with anti-TMPRSS2 MAb of normal and cancerous prostate tissues is the cleaved protease Tissue sections were stained with 1F9 MAb as described in “Materials and Methods.” fragment. Proliferation and metastasis of cancer cells, associated with The number of samples that stained positive per tissue are indicated in parentheses. destruction of the normal tissue architecture, may cause a rise in Staining intensity Tissuea serum TMPRSS2 levels that would signify the presence of cancer None Kidney cells. These data suggest that released TMPRSS2 protease may be Skin Liver useful as a potential serum diagnostic or prognostic marker for pros- Lung tate and possibly colon cancer. Testis Secreted TMPRSS2 protease may be involved in processing and Spleen Light Fallopian tubes activating growth factors present in the extracellular space. Recent Colon cancer (1/4) work with PSA and human glandular kallikrein have shown that they Moderate Colon (4/4) may play a role in activating growth factors important in the osteo- Strong Prostate (7/7) BPH (7/7) blastic response of bone-metastasized prostate cancer (21). One of PIN (1/1) these factors, parathyroid hormone-related protein, has been shown to Prostate cancer (5/5) Colon cancer (3/4) increase the rate of prostate tumor growth in vivo and protect LNCaP Pancreas (4/4) cells from apoptosis (22). IGFBP-3, an inhibitor of insulin-like growth a PIN, prostatic intraepithelial neoplasia. factor 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-like growth factor I, a growth factor implicated in prostate cancer cell detected particularly in colon cancer cell lines, indicating a possi- growth (23, 25). It remains to be seen whether TMPRSS2 is capable ble up-regulation of TMPRSS2. of acting on parathyroid hormone-related protein, IGFBP-3, and/or In prostate cancer cells and tissues, TMPRSS2 is regulated by an other factors that could influence prostate cancer growth. The expres- androgen signal. Androgen deprivation and stimulation experiments sion pattern and localization of TMPRSS2 make it a potential target in LAPC-9 xenograft and LAPC-9 cell line, which express the wild- for therapy in cancers of the prostate. type AR (5), and in LNCaP cells show that TMPRSS2 protein expression is increased with androgen stimulation. PC-3 cells, which do not normally express the AR (12) and grow in an AI manner, ACKNOWLEDGMENTS express little TMPRSS2. However, constitutive expression of the wild-type AR in PC-3 cells and simultaneous stimulation with andro- We thank Drs. Robert Eisenman, Owen Witte, and William Isaacs for helpful suggestions and critical reading of the manuscript; Frank Lynch and gen induce the expression of TMPRSS2 protein. PSA expression in Page Erickson at QualTek Molecular Laboratories for their expertise in im- PC-3 cells can also be induced by heterologous expression of the AR munohistochemistry; and Celeste Jones for help in preparation of the manu- and concomitant androgen stimulation (13). These studies demon- script. strate that, similar to PSA, TMPRSS2 protein expression is critically dependent on the AR pathway. Prostate tissue expresses a number of androgen-regulated proteases, REFERENCES including PSA, human glandular kallikrein and prostase/KLK-L1 1. Galbraith, S. M., and Duchesne, G. M. Androgens and prostate cancer: biology, (14–16). TMPRSS2 is unique among these proteases due to its pathology and hormonal therapy. Eur. J. Cancer, 33: 545–554, 1997. structural features. 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Daniel E. H. Afar, Igor Vivanco, Rene S. Hubert, et al.

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