PRSS3/Mesotrypsin Is a Therapeutic Target for Metastatic Prostate Cancer

Molecular Cancer Angiogenesis, Metastasis, and the Cellular Microenvironment Research

Alexandra Hockla, Erin Miller, Moh'd A. Salameh, John A. Copland, Derek C. Radisky, and Evette S. Radisky

Abstract PRSS3/mesotrypsin is an atypical isoform of trypsin that has been associated with breast, lung, and pancreatic cancer cell malignancy. In analyses of open source transcriptional microarray data, we find that PRSS3 expression is upregulated in metastatic prostate cancer tissue, and that expression of PRSS3 in primary prostate tumors is prognostic of systemic progression following prostatectomy. Using a mouse orthotopic model with bioluminescent imaging, we show that PRSS3/mesotrypsin is critical for prostate cancer metastasis. Silencing of PRSS3 inhibits anchorage-independent growth of prostate cancer cells in soft agar assays, and suppresses invasiveness in Matrigel transwell assays and three-dimensional (3D) cell culture models. We further show that treatment with recombinant mesotrypsin directly promotes an invasive cellular phenotype in prostate cancer cells and find that these effects are specific and require the proteolytic activity of mesotrypsin, because neither cationic trypsin nor a mesotrypsin mutant lacking activity can drive the invasive phenotype. Finally, we show that a newly developed, potent inhibitor of mesotrypsin activity can suppress prostate cancer cell invasion to a similar extent as PRSS3 gene silencing. This study defines mesotrypsin as an important mediator of prostate cancer progression and metastasis, and suggests that inhibition of mesotrypsin activity may provide a novel modality for prostate cancer treatment. Mol Cancer Res; 10(12); 1555–66. 2012 AACR.

Introduction invasion of host stroma, and escape of tumor cells into the vasculature or lymphatics, and finally dissemination, extrav- Prostate cancer is the most commonly occurring cancer fi – and second leading cause of cancer-related death in Amer- asation, and colonization of speci c metastatic sites (4 6). Many proteases, upregulated in tumor cells or secreted by ican men (1). Mortality results from metastasis of hormone- fi refractory cancer cells to bone, lung, liver, and other vital tumor-associated stromal cells, perform speci c functions to organs (2). Prostate cancer is also a very heterogeneous facilitate the various steps of this cascade. Proteases release angiogenic factors, shed cell-adhesion molecules, degrade disease, in some cases lying latent for decades, whereas in – other cases advancing rapidly to systemic metastasis and basement membranes, stimulate epithelial mesenchymal transition, aid extravasation, and can also be required for death (3). Elucidating the molecular pathways that distin- – guish between indolent cancers and those that progress more colonization of metastatic sites (6 8). Identifying which fi proteases drive the specific steps of tumor progression, and rapidly is critical, both for risk strati cation of patients to fi fi guide clinical management, and for development of better de ning their speci c modes of action, can reveal novel therapies to treat patients at high risk for progression or with druggable targets and unexplored avenues for cancer inter- vention (9). existing metastatic disease. Therapeutic strategies that inhib- PRSS3 it cancer cell invasiveness, or reduce establishment or growth The serine protease mesotrypsin, encoded by the of metastases, could be of considerable benefit. gene, has recently been implicated as an important mediator of progression and metastasis in pancreatic cancer (10). The metastatic cascade of prostate cancer involves a PRSS3 defined sequence of events, first neoangiogenesis or lym- Ectopic expression of transcripts has also been phangiogenesis, followed by loss of tumor cell adhesion, local reported in other epithelial cancers and cancer cell lines including prostate, colon, and lung (11–14). PC-3 cells, derived from a bone metastasis of a grade 4 prostatic adenocarcinoma (15), show very high upregulation of Authors' Affiliation: Department of Cancer Biology, Mayo Clinic Com- PRSS3 prehensive Cancer Center, Jacksonville, Florida (16). Here, we show that PRSS3/mesotrypsin expression is a critical effector of prostate cancer malignancy, and Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). that it plays a key functional role in promoting metastasis. Corresponding Author: Evette S. Radisky, Department of Cancer Biology, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL 32224. Phone: 904- Materials and Methods 953-6372; Fax: 904-953-0277; E-mail: [email protected] Cell culture and reagents doi: 10.1158/1541-7786.MCR-12-0314 RWPE-1 cells and the derivative cell lines NB-14, NB-11, 2012 American Association for Cancer Research. and NB-26 were obtained from American Type Culture

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Collection (ATCC) and were maintained according to were housed in a specific pathogen-free barrier facility with ATCC specifications in keratinocyte serum free media 12 hours light/dark cycle. Food (Harlan Irradiated Diet (K-SFM, Invitrogen) supplemented with 0.05 mg/mL T.7912.15), water, and bedding (corncob) were sterilized bovine pituitary extract (BPE) and 5 ng/mL human recom- and cages changed at least once a week. For the orthotopic binant EGF. PC3-M cells were kindly provided by Dr. implantation experiment, PC3-M cells transduced with Raymond Bergan from Northwestern University, Robert H. PRSS3-targeted or nontarget control shRNA lentivirus and Lurie Medical Research Center (Chicago, IL). PC3-M cells superinfected with pSIN-luc, conferring expression of firefly were maintained in RPMI-1640 (Invitrogen) supplemented luciferase, were trypsinized, quenched with RPMI contain- with 10% FBS (Invitrogen). ing 10% FBS, counted, and resuspended in 50% growth factor–reduced Matrigel and 50% serum-free RPMI-1640 RNA extraction, cDNA synthesis, and quantitative real- medium. Mice were anesthetized with Avertin (200 mg/kg) time PCR by intraperitoneal injection and shaved. A small vertical RNA was isolated from cultures using TRIZol reagent incision in the lower abdominal region was made and the (Invitrogen) according to manufacturer's protocols. cDNA prostate exteriorized by exposing the bladder and seminal was synthesized according to kit specifications using the vesicle. PC3-M cells (3 104 cells in 10 mL) were injected High Capacity cDNA Reverse Transcription Kit (Applied into the dorsolateral side of the prostate using a 27.5-gauge Biosystems). Quantitative real-time PCR (qRT-PCR) was needle attached to a 50 mL glass Hamilton Series 1700 conducted using TaqMan gene expression assays (Applied gastight syringe. The peritoneum was closed with a suture Biosystems) on an Applied Biosystems 7900HT Fast Real- and the incision closed with 7 mm wound clips. Saline was Time PCR System according to manufacturer's protocols. administered subcutaneously during recovery on the warm- TaqMan assays used included, glyceraldehyde-3-phosphate ing bed. The study initially included 9 mice in the control dehydrogenase (GAPDH), Hs99999905_m1 and PRSS3, group and 10 mice in the PRSS3 knockdown group. One Hs00605637_m1. mouse in the nontarget control group failed to form a tumor and was excluded from all analyses. Lentiviral shRNA knockdowns A validated lentiviral short hairpin RNA (shRNA) con- Bioluminescent imaging struct NM_002771.2-454s1c1 targeting human PRSS3 was In vivo and ex vivo bioluminescent imaging was conducted obtained from the MISSION TRC-Hs1.0 library (Sigma). A using the IVIS Spectrum three-dimensional (3D) imaging nontarget control lentiviral vector containing a short hairpin system (Xenogen). Twice per week, all mice were injected that does not recognize any human genes (NT) was used as a intraperitoneally with 150 mg/kg potassium D-luciferin negative control in all RNA interference (RNAi) experi- (Gold Biotechnology) and anesthetized with 2.5% isoflur- ments. Conditioned media containing infective lentivirus ane. In the imaging chamber, mice remained sedated with particles were produced using HEK293FT cells and follow- isoflurane administered through nose cones. Up to 5 mice ing supplier protocols. For lentiviral transduction, PC3-M were imaged in vivo at a time with image exposures ranging cells were seeded at 1.5 106 cells per 10-cm dish. After 24 from 1 second to 3 minutes. Regions of interest (ROI) were hours, medium was replaced with a mixture of 3.6 mL RPMI drawn around the primary tumor and quantified as photons/ containing 10% FBS and 10 mg/mL polybrene (Fisher), and second, using Living Image software (Xenogen). For ex vivo 2.4 mL conditioned lentiviral media. Media was changed imaging of tumors and organs harboring metastases, all mice after 24 hours and transduced cells were selected with 2 mg/ were injected with 150 mg/kg potassium D-luciferin 5 mL puromycin. Cells were maintained under puromycin minutes before euthanization by CO2 asphyxiation at 2 selection for up to a week before use in invasion assays or weeks postimplantation. Tissues (lungs, heart, liver, dia- transduction with lentiviral construct pSIN-luc [(gift of Dr. phragm, kidneys, spleen, and primary tumor) were excised Yasuhiro Ikeda, Mayo Clinic (Rochester, MN)] conferring and placed into 15 mm Petri dishes containing 300 mg/mL expression of firefly luciferase for use in the orthotopic tumor potassium D-luciferin in Dulbecco's PBS. The lungs were model. Knockdown was routinely assessed at the transcript imaged first (1–60 seconds exposure), followed by the liver, level by qRT-PCR as described earlier, and in some experi- diaphragm, kidney, spleen (1–2 minutes exposures), and the ments was also assessed at the protein level in cell lysates by primary tumor (0.5–1 seconds exposure). All 8 control Western blotting using a custom rabbit polyclonal antiserum group mice and 10 PRSS3 knockdown group mice were (Cocalico Biologicals) raised against mesotrypsin peptide included in analyses of in vivo and ex vivo bioluminescence acetyl-TQAECKASYPGKITNS-NH2 conjugated to key- and of tumor weight. hole limpet hemocyanin (KLH; EZ Biolab), as described previously (17). Immunohistochemistry Formalin-fixed, paraffin-embedded primary tumors and Animals and orthotopic model of spontaneous metastasis lungs were sectioned and stained with hematoxylin and eosin Six- to 10-weeks old Nod/LtSz-prkds(scid) males from (H&E). Sections were also stained for human cytokeratins Jackson Laboratory (Bar Harbor, ME) were maintained using monoclonal mouse anti-human cytokeratin clones under the guidance of approved Mayo Clinic Institutional AE1/AE3 (Dako) at 1:200 dilution and for human vimentin Animal Care and Use Committee protocol A19709. Mice using antivimentin clone V9 (EMD Millipore) at 1:1,500

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dilution, to identify micrometastases of human PC3-M room temperature, and air dried. Stained filters were photo- prostate cancer cells. Lungs of 1 control group mouse and graphed at 2 magnification and cells were counted using 2 PRSS3 knockdown group mice were lost before immu- Image-Pro 6.3 software (Media Cybernetics). Consistent nohistochemical staining; these analyses therefore included results were obtained from 4 experiments using NB26 cells the remaining 7 control group mice and 8 PRSS3 knock- and from more than 10 experiments using PC3-M cells with down group mice. For each mouse, the total number of slightly varying conditions and inhibitor concentrations. metastatic foci in a single 4 mm section through all lung lobes Data shown represent 4 biologic replicates SEM. was counted. Any positive groups of cells were considered to be metastases. 3D culture assays 3D cultures in Matrigel were established using the "on- Anchorage-independent growth assays top" protocol (20). Briefly, in 6-well plates, a base layer of Anchorage-independent growth was assayed by the ability 500 mL 100% Matrigel was polymerized, WPE-1 NB-11 or of cells to form colonies in soft agar following a published NB-26 cells (4 105 cells/well) or PC3-M cells (5 104 procedure (18). PC3-M control cells or PRSS3 knockdown cells/well) were seeded and allowed to attach, excess medium 3 cells (2 10 cells/well) were plated in medium containing was aspirated, and cells were overlaid with medium supple- 10% FBS and 0.75% agarose in 6-well plates. Cells were mented with 10% Matrigel as well as with 100 nmol/L fi incubated at 37 Cin5%CO2 for 14 days, and then xed recombinant mesotrypsin, mesotrypsin-S195A, or cationic with 100% methanol, stained with Giemsa stain, and each trypsin where indicated. Cultures were maintained at 37C well was photographed at 960 960 pixel resolution. in 5% CO2 for up to 5 days, with the media/10% Matrigel Colonies were quantified using Image-Pro Plus 6.3 software overlay (containing fresh protease as appropriate) replaced (Media Cybernetics), using automatic intensity range selec- on day 3. Recombinant proteases were expressed in bacteria, tion for dark objects on bright background. Minimum purified to homogeneity, refolded, activated, and assayed as colony size threshold was set at 7 7 pixels for the graph previously described (21). Photographs shown are represen- shown; identical trends were observed using alternative tative of 6 biologic replicates for PC3-M cells, 2 biologic thresholds down to 3 3 pixels, although the absolute replicates for NB26 cells, and 4 biologic replicates for NB11 number of colonies detected increased with decreasing cells. threshold. Data shown represent the results of 4 biologic replicates of the experiment, each containing 4 technical Statistical analysis replicates per condition. Data for association of PRSS3 expression and prostate cancer progression were obtained from http://www.ncbi. Invasion assays nlm.nih.gov/sites/GDSbrowser?acc¼GDS1439 (probe PC3-M cells transduced with lentiviral shRNA constructs 213421_x_at; ref. 22) and http://www.ncbi.nlm.nih.gov/ were seeded at 1 106 cells per 10-cm dish the day before sites/GDSbrowser?acc¼GDS2545 (probe 40043_at; assay set-up. BD Falcon 24-well cell culture inserts (8.0 mm) ref. 23). Data for PRSS3 expression in patients with prostate were coated with 50 mg Matrigel basement membrane cancer following prostatectomy (24) were obtained from matrix in 100 mL of serum-free RPMI and placed at 37 C http://microarray-pubs.stanford.edu/prostateCA/images/ for 4 hours. Cells were rinsed with cold 1 PBS and lifted fig1data.txt and clinical information was obtained from with 0.05% trypsin, and then suspended in 1 mL of RPMI http://microarray-pubs.stanford.edu/prostateCA/images/ containing 0.1% BSA and counted using a Countess auto- supptab2.pdf. Patients were divided for survival analysis into mated cell counter (Invitrogen). In 24-well invasion cham- PRSS3 n ¼ m groups of high expression (above median; 11) bers, the bottom chambers contained 750 L of NIH/3T3 and low PRSS3 expression (below median; n ¼ 11). All cell conditioned serum-free medium (Dulbecco's modified statistical analyses were conducted using Prism 4 (GraphPad Eagle's medium supplemented with 50 mg/mL ascorbic acid) Software). Pairwise comparisons for gene expression levels, as chemoattractant (19). Suspended cells (2 104 in 400 mL soft agar colony formation, and transwell cellular invasion media) were placed into the upper chambers, and then assays were made using the unpaired t test. Pairwise com- allowed to invade for 18 hours at 37 Cin5%CO.A 2 parisons of bioluminescence or tumor number between similar protocol was followed for invasion assays with NB26 mouse groups were made using the Mann–Whitney test. cells, except that 2 105 cells were added to each chamber Significance in the survival analysis was determined using the and allowed to invade for 24 hours. In some experiments, log-rank test. potent mesotrypsin inhibitor bovine pancreatic trypsin inhibitor (BPTI)–K15R/R17G (0–100 nmol/L), prepared and purified as previously described (17), was added to cell Results suspensions in the upper chamber. Quadruple technical Mesotrypsin gene PRSS3 is upregulated in advanced replicates were set per treatment. Noninvading cells were prostate cancer and is prognostic of recurrence removed from the insert by scrubbing with a cotton swab, While expression of the PRSS3 gene encoding mesotryp- and then cells on the lower surface of the filter were fixed with sin is largely restricted to the pancreas and brain, PRSS3 is 100% methanol for 30 minutes at 20C, stained with transcriptionally upregulated with cancer progression in a 0.1% crystal violet in 20 mmol/L MES, pH 6.0, for 1 hour at number of epithelial cancers, including lung, breast, and

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pancreas (10, 13, 25). To explore a connection between PRSS3 expression and prostate cancer progression, we examined expression profiles in publicly available microarray data from previously reported clinical studies. We identified a correlation between PRSS3 expression and prostate cancer progression in a microarray dataset (22) in which benign prostate (n ¼ 5), clinically localized prostate cancer (n ¼ 5), and metastatic prostate cancer tissue specimens (n ¼ 5) were transcriptionally profiled. We found a trend of increasing PRSS3 expression with progression, and significant upregulation of PRSS3 in metastatic tumors compared with benign and clinically localized tumors (Fig. 1A). We also analyzed a second transcriptional microarray study (23) comparing normal prostate tissue (n ¼ 25), primary prostate tumors (n ¼ 65), and castration-resistant metastatic samples (24 samples obtained from 4 patients). In this study, we found a similar trend of increasing PRSS3 transcription with progression and significant upregulation in metastases relative to normal tissue (Fig. 1B). Another open source microarray study reported clinical follow-up data for patients with prostate cancer following prostatectomy to remove localized primary prostate adenocarcinomas (24). We divided patients (n ¼ 22) into groups representing upper- (n ¼ 11) and lower-half (n ¼ 11) PRSS3 transcript levels, and conducted Kaplan–Meier survival analysis. In this cohort, PRSS3 expression in tumors was strikingly associated with recurrence, defined as systemic progression or rising prostate- specific antigen (Fig. 1C). The association of PRSS3 with prostate cancer metastasis, and the evidence that PRSS3 expression in primary tumors is prognostic of recurrence, suggest that mesotrypsin may play a critical functional role in prostate cancer progression.

PRSS3 silencing suppresses metastasis of prostate cancer cells in an orthotopic model To evaluate a potential functional role for PRSS3 in prostate cancer metastasis, we implemented an orthotopic implantation model of human prostate cancer cells in non- obese diabetic/severe combined immunodeficient (NOD/ SCID) mice to faithfully replicate multiple stages of the metastatic cascade. Our model used PC3-M cells, a subline of PC-3 human prostate adenocarcinoma cells that were selected for enhanced metastatic potential (26). PC3-M cells were transduced with a lentiviral construct conferring Figure 1. PRSS3 expression in human prostate tumors. A and B, PRSS3 fi fl expression correlates with increasing cancer progression. A, metastatic expression of re y luciferase that enabled bioluminescent tumor tissue (n ¼ 5) showed significantly higher levels of PRSS3 detection of as few as 19 cells (Fig. 2A and B), allowing tumor expression than either benign prostate (n ¼ 5; P < 0.001) or clinically progression to be monitored in real time using in vivo localized cancers (n ¼ 5; P < 0.005). The difference in PRSS3 expression bioluminescence imaging (Fig. 2C). Similarly to previous between benign tissue and localized tumors was not significant in this studies in nude mice (27), we found that PC3-M cells dataset. Transcriptional microarray data were from ref. 22. B, normal prostate (n ¼ 25), primary prostate tumors (n ¼ 65), and metastatic tumors implanted orthotopically in NOD/SCID mice formed rap- (n ¼ 24 samples from 4 patients) showed a trend of increasing PRSS3 idly growing tumors characterized by widespread metastasis. expression, with a significant difference between normal prostate and In preliminary studies to characterize the model, we observed metastatic tissue (P ¼ 0.006). The difference in PRSS3 expression metastases first in the lungs; 2 of 5 mice euthanized at 9 days between primary tumors and metastases was not significant in this ex dataset. Transcriptional microarray data were from ref. 23. C, high PRSS3 postimplantation had pulmonary metastases detectable by fi vivo expression in resected prostate tumors was signi cantly associated with bioluminescence imaging at necropsy, and 9 of 9 mice early cancer recurrence following prostatectomy when comparing euthanized at 14 days postimplantation had pulmonary above-median (n ¼ 11) with below-median expressers (n ¼ 11) with metastases. In mice euthanized at 2 weeks or longer post- respect to PRSS3 transcript levels (P ¼ 0.013). Transcriptional implantation, we have detected additional metastases to microarrays and associated survival data were from ref. 24.

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liver, kidney, diaphragm, and spleen (Fig. 2D). We note that lung and liver are common sites of human prostate cancer metastases (2), although like most mouse models of prostate cancer metastasis (28), our model does not recapitulate metastasis to bone. To measure the impact of PRSS3 silencing in this model, PC3-M cells were stably transduced either with a nontarget control virus (NT) or with a specific lentiviral shRNA targeting PRSS3 (KD), which effectively suppressed expression both at the transcript level (Fig. 3A) and at the protein level (Fig. 3B). Cells were superinfected with the firefly luciferase construct and then surgically implanted into the mouse prostate (3 104 cells per animal). In a study of 2- Figure 2. Bioluminescence imaging facilitates detection of tumor growth week duration, with 10 mice in the PRSS3 knockdown and metastasis in mouse orthotopic xenograft model. A, PC3-M cells group and 8 in the control group, it seemed that PRSS3 transduced with lentiviruses for firefly luciferase and GFP expression showed high transduction efficiency by comparing phase contrast (left) knockdown had minimal impact on tumor growth, based on and fluorescence microscopy (right). Scale bar, 50 mm. B, a luciferase total in vivo bioluminescence (Fig. 3C). At the time of assay of cell dilutions detected as few as 19 cells. C, when PC3-M cells euthanasia, assessment of PRSS3 transcript levels by qRT- 4 transduced with luciferase virus were implanted orthotopically (3 10 PCR in a sampling of the primary tumors confirmed that cells), tumor growth could be followed in vivo by bioluminescence PRSS3 imaging. D, ex vivo bioluminescence imaging of organs and tissues was expression remained suppressed in tumors of the used to identify metastases in lung lobes, liver, kidneys, diaphragm, and knockdown group for the duration of the study (Fig. 3D). spleen. Tumor burden from cells in which PRSS3 was silenced, was

Figure 3. PRSS3 silencing slows prostate tumor growth in orthotopic xenografts. A, transduction with shRNA lentivirus targeting human PRSS3 resulted in effective knockdown in PC3-M cells (KD) relative to cells transduced with nontarget control virus (NT), as assessed by qRT-PCR (bars, SEM; n ¼ 3). B, PRSS3 KD cell lysates show reduced expression of mesotrypsinogen (32 kDa) relative to NT cell lysates on a Western blot analysis; the blot was stripped and reprobed for actin (44 kDa) as a loading control. C, in mice implanted with cells with knockdown of PRSS3 (KD) versus nontarget control cells (NT), both superinfected with luciferase virus, PRSS3 knockdown mice (n ¼ 10) showed little change in tumor growth relative to the control mice (n ¼ 8), as assessed by in vivo bioluminescence imaging. D, tumors from 3 PRSS3 knockdown mice harvested after 2 weeks showed persistent suppression of PRSS3 expression relative to a tumor from a control mouse (NT; bars, SEM; n ¼ 3). E, ex vivo imaging of resected prostates with PRSS3 knockdown primary tumors (n ¼ 10) showed reduced luminescence relative to controls (n ¼ 8), although the difference was not statistically signiﬁcant. F, resected tumor-bearing prostates from the PRSS3 knockdown group (n ¼ 10) weighed signiﬁcantly less than resected prostates from the control mice (n ¼ 8). Bars, SEM. , P < 0.01; , P < 0.005.

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Figure 4. PRSS3 silencing inhibits prostate to lung metastasis in orthotopic xenografts. A, mice implanted with PC3-M cells in which PRSS3 was knocked down with viral shRNA (KD; n ¼ 10) showed significantly reduced pulmonary metastasis at 2 weeks postimplantation, as assessed by ex vivo imaging at necropsy, relative to control mice (NT; n ¼ 8; , P ¼ 0.0021). B, lungs from a mouse implanted with nontarget control virus-transduced PC3-M cells (NT) show flux measurement near the group median with extensive metastasis in all lobes (left), in contrast with lungs from a representative mouse implanted with PC3-M cells in which PRSS3 was knocked down with viral shRNA (KD), which show no evidence of metastasis (right). C and D, lung section from a mouse implanted with NT PC3-M cells (left) showed the presence of many small metastases such as that indicated by the black arrow in panels showing H&E staining (C) and immunohistochemical staining for human cytokeratins (D). Lung section from a mouse implanted with PRSS3 KD PC3-M cells (right) illustrates normal lung morphology (C) and background staining for human cytokeratins (D). E, histologic and immunohistochemical analyses found a significantly lower mean number of lung metastases per section in the PRSS3 knockdown group (KD; n ¼ 8) compared with the control group (NT; n ¼ 7; , P ¼ 0.0022). F, lung tissue bearing metastases from mice with PRSS3 KD tumors (n ¼ 5) showed significantly lower expression of human PRSS3 when normalized to human GAPDH, relative to lung tissue from mice bearing NT control tumors (n ¼ 5; bars, SEM; , P ¼ 0.043).

reduced in comparison with NT control; although the described (27). Consistent with the bioluminescent imaging difference did not reach significance as assessed by ex vivo results, tumor counts from stained lung sections showed bioluminescence (Fig. 3E), it was significant when assessed highly significant differences between groups (P ¼ by weight of the resected prostate (P ¼ 0.0044; Fig. 3F). 0.0022; Fig. 4E). We assessed PRSS3 expression in lung We observed a striking effect of PRSS3 silencing on metastases of both groups, and found that PRSS3 remained suppression of metastasis; when metastatic tumor burden suppressed in cells that did metastasize to lung in the KD was assessed at necropsy, 2 weeks postimplantation, by ex group (Fig. 4F), suggesting that metastasis can still occur, vivo bioluminescence imaging of resected organs (Fig. 4A although with reduced incidence, in the absence of and B), all 8 control mice showed extensive pulmonary mesotrypsin. metastases, 2 had metastasis to liver, and 2 had metastasis to the spleen. In contrast, of the 10 mice bearing tumors in PRSS3 silencing suppresses anchorage-independent which PRSS3 expression had been suppressed, only 4 growth of prostate cancer cells showed visible evidence of lung metastasis by biolumines- With clear experimental evidence for the critical role of cent imaging, and none showed evidence of metastasis to PRSS3 expression in prostate cancer metastasis, we next liver or spleen. When we assessed tumor burden in the lungs examined the importance of this gene in individual pheno- as total flux and compared the 2 groups with a Mann– typic characteristics required for metastasis. To assess how Whitney test, the difference in mean tumor burden was PRSS3 expression influences the ability of disseminated highly significant (P ¼ 0.0021; Fig. 4A). Bioluminescence cancer cells to survive and to form metastatic colonies, we results were confirmed by H&E staining of lung specimens compared PC3-M control cells with cells in which PRSS3 (Fig. 4C) and immunohistochemical staining for human expression was suppressed by shRNA knockdown in soft cytokeratins (Fig. 4D) and human vimentin (not shown). agar assays of anchorage-independent growth. We consis- Small PC3-M cell metastases were readily distinguished tently observed approximately 50% reduction in colony from surrounding mouse lung tissue by the presence of formation and growth in cells in which PRSS3 was silenced characteristic, abnormally large nuclei as has been previously (Fig. 5), indicating that PRSS3 expression plays a significant

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PC3-M cell line as a representative of highly aggressive, androgen-independent metastatic prostate cancer (26). Using qRT-PCR to assess transcript levels of PRSS3 in these cell lines, we found that nonmalignant RWPE-1 progenitor cells express very low levels of PRSS3, whereas the MNU- transformed cell lines show increasing levels of PRSS3 expression correlating with their reported invasiveness and malignancy (Fig. 6A). The metastatic PC3-M cells expressed yet higher levels of PRSS3 (Fig. 6A), consistent with the pattern seen in human tumor samples (Fig. 1A and B). We evaluated the invasiveness of cells in this series using Matrigel transwell invasion assays, which confirmed previously reported phenotypes (29). NB14 cells were minimally invasive, NB11 cells were slightly more invasive, and NB26 cells, possessing higher levels of PRSS3 expression, were the most invasive among the MNU cells series, although still far less invasive than PC3-M cells. Knockdown of PRSS3 by stable transduction with a lentiviral shRNA construct led to dramatically reduced invasion in the invasive cell lines PC3- M (Fig. 6B) and NB26 (Fig. 6C). We also assessed the impact of PRSS3 knockdown on migration through uncoat- ed transwell filters, finding significant differences albeit of lesser magnitude than the impact on invasion (Supplemen- tary Fig. S1).

Figure 5. PRSS3 silencing inhibits anchorage independent growth of prostate cancer cells. PC3-M cells transduced with shRNA virus targeting A novel inhibitor of mesotrypsin suppresses invasion of PRSS3 (KD) or with a nontarget control virus (NT) were plated in soft agar prostate cancer cells and grown for 2 weeks. Cells in which PRSS3 expression was Our results showing that PRSS3 silencing inhibits metas- suppressed showed significantly fewer colonies per well. Photographs of tasis in a mouse orthotopic model and anchorage-indepen- representative fields are shown above graphical results. , P < 0.0001; bars, SEM; n ¼ 4 biologic replicates. dent growth and invasiveness in cultured prostate cancer cells suggest that endogenous mesotrypsin activity may help role in the tumor-propagating phenotype of prostate cancer drive invasion and metastasis, and therefore that this protease cells. may offer a potential target for novel antimetastatic therapies. Mesotrypsin is notoriously resistant to inhibition and is unaffected by a number of drugs targeting other serine PRSS3 silencing suppresses invasion in prostate cancer proteases; however, we have recently used structure-guided cells mutagenesis to modify the polypeptide trypsin inhibitor Another essential characteristic of prostate cancer cell BPTI (aprotinin, Trasylol) to generate a novel mesotrypsin metastasis is the capacity to invade through basement mem- inhibitor (17). BPTI binds to the active site and blocks brane and interstitial connective tissue (4). To experimen- activity of most serine proteases of trypsin-like specificity, tally probe the role of mesotrypsin in this phenotype, we but deleterious interactions between BPTI and mesotrypsin identified a series of prostate-derived cell lines spanning a residue Arg-193 greatly diminish binding affinity (21, 30). phenotypic spectrum from benign to aggressively invasive. We have incorporated 2 mutations into the binding loop of The RWPE-1 methylnitrosourea (MNU) cell series shares a BPTI, a Lys-to-Arg mutation at residue 15, which strength- common genetic background and features progressive phe- ens a stabilizing salt bridge in the enzyme primary specificity notypic changes that model prostate cancer progression from pocket (30), and an Arg-to-Gly mutation at residue 17, preneoplastic epithelial cells to prostatic intraepithelial neo- which renders the inhibitor binding loop much more ste- plasia (PIN) to invasive prostatic carcinoma (29). These cell rically complementary to the enzyme active site (Fig. 6D; lines were derived from benign immortalized RWPE-1 ref. 17). In doing so, we have generated a prototype meso- human prostate epithelial cells by treatment with the chem- trypsin inhibitor with 2,400-fold improved mesotrypsin ical carcinogen MNU followed by selection for tumorige- affinity (Ki ¼ 5.9 nmol/L; ref. 17). nicity in nude mice, and have been extensively characterized In Matrigel transwell invasion assays, comparing PC3-M with respect to their karyotype, expression of cancer markers, cells in which PRSS3 has been silenced by shRNA with and growth phenotypes in culture and in mice. They model a control cells and cells treated with varying concentrations of spectrum of phenotypes with regard to loss of cell polarity, BPTI-K15R/R17G, we find that the inhibitor suppresses loss of differentiation, and acquisition of invasive behavior, invasion in a dose-dependent fashion, in which 100 nmol/L from NB14 (least progressed) through NB11 to NB26 (most inhibitor treatment is as equally effective as PRSS3 knock- progressed; ref. 29). We included in this comparison the down (Fig. 6E). These results suggest that cell autonomous

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Figure 6. PRSS3 silencing and pharmacologic inhibition of mesotrypsin inhibit invasion of prostate cancer cells. A, endogenous PRSS3 expression, assessed by qRT-PCR, increases with progression in the RWPE-1– derived cell series, and is highly upregulated in the aggressive metastatic PC3-M cell line (bars, SEM; n ¼ 3). B and C, knockdown of PRSS3 in PC3-M cells (B) or NB26 cells (C) significantly reduced invasion in Matrigel transwell assays (bars, SEM; n ¼ 4). D, structure of mesotrypsin complex with prototype mesotrypsin inhibitor BPTI-K15R/ R17G double mutant, highlighting (in green) inhibitor residues mutated to improve steric complementarity toward mesotrypsin active site residue Arg-193. E, in Matrigel transwell invasion assays, PC3-M NT cells assayed in the presence of 50 or 100 nmol/L BPTI-K15R/R17G showed significantly reduced invasion; 100 nmol/L inhibitor gave results similar to PRSS3 knockdown. Photographs of representative fields from invasion filters are shown above graphical results (bars, SEM; n ¼ 4 biologic replicates). , P < 0.05; , P < 0.01; , P < 0.0005.

endogenous expression of mesotrypsin is a critical mecha- NB11 cells in Fig. 7E. Treatment with 100 nmol/L meso- nism by which prostate cancer cells invade, and that targeting trypsin stimulated a dramatically altered growth phenotype mesotrypsin activity may be a method to suppress prostate characterized by invasive protrusions and a web-like branch- cancer invasion. ing morphology (Fig. 7D and F). Similar effects of mesotrypsin were observed on the 3D growth phenotype of NB14 PRSS3/mesotrypsin promotes an invasive morphology cells (not shown). Critically, we found that stimulation of in prostate cancer cells invasive growth morphology was dependent upon the pro- To further explore the functional impact of PRSS3/meso- teolytic activity of mesotrypsin as the catalytically inactive trypsin expression on prostate cancer cells in a physiologi- mesotrypsin-S195A mutant had no effect (Fig. 7G), and that cally relevant environment, we grew cells in 3D culture in the effect was highly specific to mesotrypsin as it was not Matrigel artificial basement membrane. To examine the recapitulated by the closely homologous human cationic impact of mesotrypsin proteolytic activity on cellular mor- trypsin at equivalent concentration (Fig. 7H). These obser- phology, active purified recombinant mesotrypsin was added vations further suggest a specific and critical function of to the media of some cultures. PC3-M cells, which revealed a mesotrypsin in prostate cancer invasion and metastasis, an native invasive morphology characterized by spiky protru- activity that can potentially be targeted with therapeutic sions when grown in 3D (Fig. 7A), reverted to a less invasive outcome. morphology characterized by small-rounded cell clusters when PRSS3 was silenced using a lentiviral shRNA construct (Fig. 7B). Addition of active recombinant mesotrypsin to 3D Discussion cultures of cells in which PRSS3 was silenced reconstituted Here, we have found that the PRSS3 gene, encoding the the invasive growth morphology (Supplementary Fig. S2). serine protease mesotrypsin, is a gene that is upregulated in The reciprocal phenomenon was observed in cells of the association with prostate cancer metastasis and poor out- MNU series, which grow natively in 3D as a mixture of small come. Our studies using a mouse orthotopic model of amorphous cell clusters and spherical acini as previously metastatic human prostate cancer show that PRSS3 expres- described (31), shown for NB26 cells in Fig. 7C and for sion is a critical factor in prostate to lung metastasis in this

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Our orthotopic model, in which extensive metastasis to lung is typically observed within a 2-week window, may be broadly useful for evaluation of genes important for metastasis generally and for metastasis to lung specifically. This model may also prove advantageous for preclinical evaluation of a variety of antimetastatic therapeutics for prostate cancer. An additional feature of our model is the use of tumor cells modified to express firefly luciferase, which enabled tracking of tumor growth in vivo and identification of sites of tumor metastasis ex vivo by bioluminescent imaging. The quantification from ex vivo imaging of primary tumors (Fig. 3E) and of organs containing metastases (Fig. 4B) was highly consistent with traditional methods of assessing tumor burden (Fig. 3F, 4C and D). The key advantage of this technology for our study was the ease of identifying sites of distant small metastases for further study, which otherwise would have required much more labor intensive microscopic histologic examination of all possible metastatic sites. Our cell culture studies indicate that the role of PRSS3 in metastasis is pleiotropic; knockdown of PRSS3 in the aggressively metastatic PC3-M cell line compromised both the capacity for anchorage-independent growth, indicating a potential function in survival at metastatic sites, and the capacity for cellular invasion, suggestive of functions in breaching the integrity of basement membrane barriers, a function often aided by protease activity. The PRSS3 gene encodes the serine protease mesotrypsin, and we find that active recombinant mesotrypsin, but not an inactive mutant, can stimulate prostate cancer cell invasive morphology, suggesting that the effects of PRSS3 expression in metastasis are mediated through the proteolytic activity of mesotrypsin. That the invasive phenotype observed in our 3D culture Figure 7. Endogenous and recombinant mesotrypsin stimulate invasive model cannot be recapitulated by cationic trypsin suggests 3D growth morphology. A and B, PC3-M cells transduced with nontarget that we are not merely seeing an effect of indiscriminate control virus (A) or with PRSS3-targeted shRNA virus (B) were grown in Matrigel in serum-free media for 3 days and then photographed. C and D, matrix degradation, but rather a phenotype induced by NB26 cells were grown in Matrigel and treated with (C) buffer only cleavage of one or more specific substrates of mesotrypsin. or (D) 100 nmol/L active recombinant human mesotrypsin for 5 days and An obvious next question will be, what are those specific then photographed. E–H, NB11 cells were grown in Matrigel and treated mesotrypsin substrates? In prior investigations seeking to with (E) buffer only, (F) 100 nmol/L active recombinant human fi mesotrypsin, (G) 100 nmol/L catalytically inactive mesotrypsin-S195A identify biologic mesotrypsin substrates, we identi ed cleav- mutant, or (H) 100 nmol/L active recombinant human cationic trypsin for 5 age of the cell surface protein CD109 as a mechanism by days and then photographed. Scale bar, 100 mm. which mesotrypsin promotes loss of basal polarity and increased proliferation in malignant breast cells (25), where- model as silencing of PRSS3 led to a dramatic reduction in as in prostate cancer cells we have identified the amyloid formation of lung metastases. While some metastasis genes precursor protein (APP) Kunitz protease inhibitor domain as serve general functions in metastasis initiation and/or infil- a specific mesotrypsin substrate with unclear physiologic tration of the vasculature and distant organs, other genes play consequences (33). As candidate mesotrypsin substrates, we more specialized roles in infiltration and colonization of have individually silenced CD109 and APP in cells of the specific metastatic sites (5). That we also observed metastasis WPE-1 MNU series; knockdown of these genes neither to liver and spleen in mice implanted with control tumor sensitized cells to recombinant mesotrypsin treatment nor cells, but not in mice in which PRSS3 was silenced, suggests blocked responsiveness to mesotrypsin in 3D culture (A. that the effect of PRSS3 expression may extend beyond lung Hockla and E.S. Radisky, unpublished data). This suggests infiltration and colonization to a more general role in that neither of these candidates is likely to be the sole metastasis; studies in additional model systems will be mesotrypsin substrate responsible for mediating the invasive required to evaluate this possibility. Because metastasis to phenotype in prostate cancer cells, and that the immediate bone is particularly common and is a prominent cause of effector through which mesotrypsin drives prostate cancer morbidity and mortality of patients with prostate cancer progression has yet to be discovered. (32), evaluation of a potential role for PRSS3 in bone The protease-activated receptors (PAR) represent another metastasis is an important future direction. group of candidate mesotrypsin substrates. PAR signaling

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has been implicated in the proliferation, migration, invasion, these drugs inhibited too wide a swath of this large protease and metastasis of a number of tumor types (34, 35). In family, and in doing so, caused troubling side effects and prostate cancer, PAR-1, -2, and -4 are upregulated (36, 37) failed to improve clinical outcome (49, 52, 53). Serine and have been shown to enhance proliferation (38) and to proteases also represent a large family, with more than stimulate cell migration (39) and RhoA-induced cytoskeletal 100 members; as with MMPs, identification and selective reorganization (40), processes important for invasion and targeting of the right cancer promoting serine proteases will metastasis. There have been conflicting reports of the ability likely be essential, because these enzymes also have essential of mesotrypsin to cleave and activate PAR-1, -2, and -4 on functions in the coagulation, fibrinolysis, and complement the surface of epithelial cells; 1 group has reported significant pathways. In addition, similar to the case with the MMP cleavage and downstream signaling, albeit with greatly family, some serine proteases have been found to have tumor reduced potency compared with other trypsins (11, 41), suppressing activities (54–56). whereas another group has reported that mesotrypsin cannot Toward development of potent and selective mesotrypsin activate epithelial PARs (42). Further study will be required inhibitors, we have undertaken structural and biochemical to determine whether PAR signaling may be involved in studies to define the binding specificity of mesotrypsin. The promotion of prostate cancer cell invasion and metastasis by mesotrypsin active site possesses steric and electrostatic mesotrypsin. features unique among human serine proteases that mod- Another mechanistic question brought up by our data ulate interactions with the "primed-side" residues (those concerns the regulation of PRSS3 itself; what drives increased residues located proximal to the cleavage site in the direction expression of this gene in prostate cancers of increasing of the protein C-terminus) of a polypeptide substrate or malignancy? The PRSS3 gene encodes several precursor inhibitor (21, 30, 57). These distinctive active site features splice isoforms of the serine protease mesotrypsin, initiated are responsible for the resistance of mesotrypsin to many from 2 different promoters resulting in different first exons polypeptide trypsin inhibitors (58, 59), and are also likely (43–45). The transcript encoding mesotrypsinogen, most responsible for the unique substrate specificity of meso- abundant in normal pancreas, is transcribed from a promoter trypsin that we infer from the inability of cationic trypsin regulated by pancreatic transcription factor PTF1, whereas to substitute for mesotrypsin (Fig. 7). Significantly, these the transcripts encoding trypsinogens 4a, 4b, and 5, more distinctive features may also provide an opportunity to commonly found outside the pancreas, are transcribed from develop new inhibitors, complementary in shape and an alternative promoter that is likely responsible for expres- charge to the mesotrypsin active site, which will selectively sion of PRSS3/mesotypsin in tumors (43, 46). Little is target mesotrypsin for therapeutic purposes. Our proto- known about the mechanisms of transcriptional regulation type inhibitor, while capable of potently inhibiting meso- of the extrapancreatic PRSS3 isoforms, but in colon cancer trypsin catalytic function and biologic activity, does not cells, PRSS3 transcription seems to be upregulated in possess the selectivity that would ultimately be desirable in response to the oncogenic zinc finger transcription factor a therapeutic inhibitor (17); additional efforts to optimize ZKSCAN3 (14). Another clue may be found in cell surface selectivity are required. Finally, mesotrypsin inhibition as a protein cytokeratin-associated protein in cancer (CAPC), therapeutic strategy would also require evaluation of the also known as LRRC26, a protein with tumor suppressor prevalence of mesotrypsin expression as a mechanism driving activities that is highly expressed in normal prostate cells progression and metastasis in patients with prostate cancer, (47). Overexpression of CAPC in MDA-MB-231 breast and identification of the subset of patients most likely to cancer cells suppressed tumor development and lung metas- benefit from such molecularly targeted therapy. tasis in mice; transcriptional profiling showed 10- to 20-fold reduced expression of PRSS3 along with several other poten- Disclosure of Potential Conflicts of Interest tially important genes in the CAPC-expressing cultured cells No potential conflicts of interest were disclosed. and tumors (47). The suppression of prostate cancer metastasis through Authors' Contributions silencing of PRSS3, and the finding that pharmacologic Conception and design: M.A. Salameh, D.C. Radisky, E.S. Radisky Development of methodology: A. Hockla, E. Miller, M.A. Salameh, J.A. Copland, inhibition of mesotrypsin can recapitulate the effect of D.C. Radisky, E.S. Radisky PRSS3 knockdown on cellular invasion, suggests mesotryp- Acquisition of data (provided animals, acquired and managed patients, provided sin as a potential target for antimetastatic therapies. The facilities, etc.): A. Hockla, E. Miller, M.A. Salameh, D.C. Radisky, E.S. Radisky Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- human proteome contains over 500 distinct proteolytic tational analysis): A. Hockla, E. Miller, M.A. Salameh, D.C. Radisky, E.S. Radisky enzymes, and in fact proteases represent one of the largest Writing, review, and/or revision of the manuscript: A. Hockla, J.A. Copland, D.C. classes of druggable targets (48–50). A caveat is that the Radisky, E.S. Radisky fi Administrative, technical, or material support (i.e., reporting or organizing data, protease inhibitors will probably require ne selectivity constructing databases): M.A. Salameh toward the right cancer-promoting proteases, and the ability Study supervision: E.S. Radisky to spare "antitarget" proteases involved in critical biologic processes or tumor suppressive functions, for efficacy as Acknowledgments cancer therapeutics (9, 51). Examples of the dangers of The authors thank Drs. Mukta Webber, Professor Emeritus of Michigan State University (Lansing, MI), for sharing information about the RWPE-1 MNU pro- poorly selective protease inhibitors are found in the cancer gression series; Raymond Bergan, Northwestern University Robert H. Lurie Medical clinical trials of matrix metalloproteinase (MMP) inhibitors; Research Center, for providing the PC3-M cells; and Yasuhiro Ikeda, Mayo Clinic

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(Rochester, MN), for the pSIN-luc lentiviral construct. The authors also thank Brandy Radisky, and from the US National Cancer Institute (R01CA104505, Edenﬁeld for immunohistochemical analyses. R01CA136665, 3R01CA104505-05S1) to J.A. Copland. The costs of publication of this article were defrayed in part by the payment of page Grant Support charges. This article must therefore be hereby marked advertisement in accordance with This work was supported by grants from the Bankhead-Coley Florida Biomedical 18 U.S.C. Section 1734 solely to indicate this fact. Research Program (07BN-07), US Department of Defense (PC094054), and US National Cancer Institute (CA091956; Principal Investigator Don Tindall) to E.S. Radisky, from the US National Cancer Institute (CA122086, CA116201; Principal Received May 16, 2012; revised August 17, 2012; accepted September 8, 2012; Investigator James Ingle) and the Susan B. Komen Foundation (KG110542) to D.C. published online December 18, 2012.

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