A Urokinase-Activated Recombinant Anthrax Toxin Is Selectively Cytotoxic to Many Human Tumor Cell Types

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A urokinase-activated recombinant anthrax toxin is selectively cytotoxic to many human tumor cell types

Ralph J. Abi-Habib,1 Ravibhushan Singh,1 Furthermore, we identify three molecular markers, anthrax Shihui Liu,2 Thomas H. Bugge,3 Stephen H. Leppla,2 toxin receptor, uPA, and uPA receptor, which can be used and Arthur E. Frankel1 as predictors of tumor cell sensitivity to PrAgU2/FP59. [Mol Cancer Ther 2006;5(10):2556–62] 1Cancer Research Institute of Scott & White Memorial Hospital, Temple, Texas; 2Bacterial Toxins and Therapeutics Section, National Institute of Allergy and Infectious Diseases; and Introduction 3Oral and Pharyngeal Cancer Branch, National Institute of A significant number of solid tumors have been shown to Dental and Craniofacial Research, Bethesda, Maryland overexpress extracellular protease systems, which increase their tissue invasiveness and metastatic potential (1, 2). Abstract One of the major proteases overexpressed on such tumors is the urokinase plasminogen activator (uPA; ref. 3). uPA is Urokinase plasminogen activator (uPA) is a tumor-specific a serine protease that is secreted in a single-chain inactive protease highly expressed in several types of solid tumors form (sc-uPA), which is then cleaved by plasmin into a and rarely present on normal cells under physiologic double-chain active uPA. The uPA receptor (uPAR) binds conditions. Due to its high expression on metastatic uPA in both its active and inactive forms and protects it tumors, several different strategies have been used to from being inhibited by the plasminogen activator inhib- target the urokinase system. These have mostly led to itor-1 (PAI-1). The active uPA/uPAR complex cleaves tumor growth inhibition rather than tumor regression. A plasminogen into plasmin, thus leading to extracellular different approach was adopted by replacing the furin matrix degradation and tissue invasiveness and metastasis activation site on a recombinant anthrax toxin with a (4, 5). The uPA system is generally absent on normal cells urokinase activation site. The resulting toxin, PrAgU2/ and is up-regulated only during certain physiologic FP59, was highly potent against tumors both in vitro and processes, such as wound healing and tissue remodeling. in vivo. In this study, we show that PrAgU2/FP59 is toxic Although a number of inhibitors of the uPA/uPAR to a wide range of tumor cell lines, including non–small system have been studied as potential cancer therapies, cell lung cancer, pancreatic cancer, and basal-like breast such inhibitors have proven to be more tumor static than cancer cell lines. Of the few cell lines found to be resistant cytotoxic. An approach that kills rather than inhibits the to PrAgU2/FP59, most became sensitive upon addition of growth of urokinase-expressing cells could provide a more exogenous pro-uPA. PrAgU2/FP59 was much less toxic to effective therapeutic strategy. We, therefore, tookadvan- normal human cells. The potency of PrAgU2/FP59 was tage of this common characteristic of solid tumors by dependent on anthrax toxin receptor, uPA receptor, and specifically targeting a recombinant anthrax toxin (PrAg/ uPA levels but not on total plasminogen activator inhibitor- FP59) to uPA-expressing tumors. 1 levels. In this study, we show that PrAgU2/FP59 is a Anthrax lethal toxin is a binary toxin that consists of wide-range, highly potent, and highly selective toxin that two separate proteins—protective antigen (PrAg), the cell is capable of specifically targeting uPA-expressing tumor binding and translocation moiety, and lethal factor, the cells, independently of the tissue of origin of these cells. catalytic moiety (6). PrAg binds cells through the ubiquitously expressed anthrax toxin receptors (ANTXR), tumor endothelial marker-8, and capillary morphogenesis gene-2 (7). PrAg is then cleaved at the sequence 164RKKR167 by Received 5/26/06; revised 8/8/06; accepted 8/25/06. cell surface furin-like proteases leading to the release of a Grant support: Intramural Research Programs of the National Institute of 20 kDa fragment. The resulting receptor-bound 63 kDa Allergyand Infectious Diseases and the National Institute of Dental and PrAg fragments then heptamerize, bind three molecules of Craniofacial Research, NIH, and a grant from OncoTac, Inc., Copenhagen, Denmark. lethal factor, migrate to lipid rafts, and undergo endocy- The costs of publication of this article were defrayed in part by the tosis. A recently identified coreceptor, low-density lipopro- payment of page charges. This article must therefore be hereby marked tein receptor-related protein 6, has been shown to interact advertisement in accordance with 18 U.S.C. Section 1734 solelyto with tumor endothelial marker-8 and capillary morpho- indicate this fact. genesis gene-2 and mediate toxin-receptor complex inter- Note: R.J. Abi-Habib and R. Singh contributed equallyto this work. nalization (8). In acidic endosomes, PrAg complexes form Requests for reprints: Arthur E. Frankel, Scott & White Memorial Hospital, Cancer Research Institute, 5701 South Airport Road, Temple, TX 76502. pores through which lethal factor reaches the cytosol. Phone: 254-724-9786. E-mail: [email protected] Lethal factor is a zinc metalloprotease that cleaves and Copyright C 2006 American Association for Cancer Research. inhibits mitogen-activated protein kinase kinases, leading doi:10.1158/1535-7163.MCT-06-0315 to cell cycle arrest and cell death (6). We and others have

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previously shown that lethal factor is selectively toxic radiolabel measured using an LKB-Wallac 1205 Betaplate to tumor cells carrying the V599E BRAF mutation and is liquid scintillation counter (Perkin-Elmer, Gaithersburg, only potent against a limited number of tumors, thus the MD) gated for 3H. The percentage maximal [3H]thymidine inherent limited range of this toxin (9, 10). By substituting incorporation was then plotted versus the log of the toxin the zinc metalloprotease domain of lethal factor with the concentration, and nonlinear regression with a variable more potent, protein synthesis inhibitor, Pseudomonas slope sigmoidal dose-response curve was generated along aeruginosa exotoxin A (FP59), a novel, more potent, with IC50 using GraphPad Prism software (GraphPad wider range, anthrax recombinant toxin (PrAg/FP59) was Software, San Diego, CA). All assays were done at least V made (11). twice with an interassay range of 30% for IC50. For the To specifically target uPA-expressing tumors, the furin blocking assay, 10 ng/mL of an anti-uPA monoclonal cleavage sequence of PrAg 164RKKR167 was substituted antibody (American Diagnostica, Stanford, CT) were with a urokinase-specific cleavage sequence 163PGSGRSA169 coincubated with the cells. termed U2 (11, 12). The resulting urokinase-activated Total uPA and PAI-1 Levels recombinant anthrax toxin PrAgU2/FP59 theoretically Total uPA and PAI-1 levels were determined in super- binds to all cells through the ubiquitously expressed natants from all cell lines using ELISA kits (American ANTXRs but becomes activated only on tumor cells Diagnostica). Supernatants were harvested from flat- expressing an active uPA/uPAR system independently of bottomed 96-well plates in which 104 cells were plated in the tumor type. 150 AL medium and incubated for 48 hours at 37jC/ Two recent studies have documented that PrAgU2/FP59 5% CO2. This allowed for the determination of total uPA is potent and selective against certain tumors in vivo and PAI-1 levels in the same conditions as those of the (13, 14). However, our knowledge remains very limited cytotoxicity assay. concerning both the potential usefulness of this toxin uPAR and ANTXR Expression Levels against a broad range of human cancers of different origins The uPAR and ANTXR expression levels were deter- and the molecular markers that can be used to predict mined using a 125I-ligand-receptor binding assay. 125I tumor responsiveness to this urokinase-activated toxin. In labeling of the sc-uPA and full-length PrAg were done as this study, we determine the potency, range, and selectivity described previously (9, 15). The [125I]sc-uPA and [125I]PrAg of PrAgU2/FP59 and we identify molecular markers that binding assays were done as described previously (9). can be used as predictors of tumor sensitivity. Briefly, 106 cells were plated in 12-well plates and incubated with varying amounts of [125I]sc-uPA or [125I]PrAg (2,000–2.5 pmol/L) with or without excess amounts Materials and Methods (4 nmol/L) of cold sc-uPA or PrAg at 37jC for 1 hour; the Tox in s supernatants were then removed, and the cells washed PrAg, PrAgU2, and FP59 were made as described thrice with PBS and harvested using a mammalian protein previously (11). PrAg and PrAgU2 have a molecular extraction buffer (Pierce, Rockford, IL). Experiments were weight of 83 kDa, whereas the molecular weight of FP59 done in duplicate. Receptor number per cell (maximum is 59 kDa. The purity of all three proteins used was >99%. number of binding sites) as well as dissociation constant Cells and Cell Lines (Kd) were calculated using the GraphPad Prism software. All human cancer cell lines were purchased from the Statistical Analysis

American Tissue Culture Collection (Manassas, VA) and We defined sensitivity as cell lines with an IC50 to cultured as recommended. Normal human cells were pur- PrAgU2/FP59 lower than 40 pmol/L. The effects of uPAR chased from Cambrex (Baltimore, MD) or American Tissue expression levels and total uPA and PAI-1 levels on cell line Culture Collection and were cultured as recommended. sensitivity to PrAgU2/FP59 were determined using uni- CytotoxicityAssay variate logistic regression. Wilcoxon’s two-sample test was Cytotoxicity was determined using a [3H]thymidine used to compare the two groups of cell lines (sensitive incorporation inhibition assay as described previously versus nonsensitive) with respect to uPAR, uPA, and (15). Briefly, aliquots of 104 cells were incubated with PAI-1. Statistical analyses were done with SAS software À 10 9 mol/L FP59 in 100 AL medium in Costar (Corning, (version 8.02, SAS Institute, Cary, NC). Statistical compar- NY) 96-well flat-bottomed plates in quadruplicates. Fifty isons were made using a 5% level of significance. microliters of wild-type PrAg and urokinase-activated PrAg (PrAgU2) in medium were added to each column À À to yield concentrations ranging from 10 8 to 10 13 mol/L, Results j and the cells were then incubated at 37 C/5% CO2 for Tumor Cell Line Sensitivity to PrAgU2/FP59 48 hours. Then, 1 ACi (0.037 MBq) of [3H]thymidine (NEN A panel of different human tumor cells was tested for A DuPont, Boston, MA) in 50 L medium was added to each sensitivity to PrAgU2/FP59 (Table 1). Cells with an IC50 well and incubation was continued for an additional for PrAgU2/FP59 lower than 40 pmol/L were considered j 18 hours at 37 C/5% CO2. Cells were then harvested sensitive. The most sensitive tumor types were non–small with the Skatron Cell Harvester (Skatron Instruments, Lier, cell lung cancer and pancreatic cancer (four of five and Norway) onto glass fiber mats and cpm of incorporated three of four sensitive cell lines, respectively; Fig. 1). Colon

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Table 1. Cell line sensitivity to PrAg/FP59 and PrAgU2/FP59 as well as uPAR expression, total uPA, and total PAI-1 levels

Cell lines PrAg/FP59 PrAgU2/FP59 uPAR (receptors uPA (ng/mL) PAI-1 (IC50; pmol/L) (IC50; pmol/L) per cell) (ng/mL)

Non–small cell lung cancer H1299 1 2 7,100 20 350 H647 8 3 9,700 23 680 H1915 1 3 1,100 19 390 H358 7 4 1,100 10 41 H650 5 6 2,400 3 750 H1651 2 8 1,200 0.3 0.9 H1734 30 8 4,000 6 37 H23 43 10 2,900 5 8 H1755 1 17 6,100 0.3 490 H2122 1 17 1,400 10 58 H1437 3 21 600 3 1 H1975 1 33 1,140 61 1,700 H1563 6 40 970 1 110 H196 8 40 2,000 0.1 45 H810 70 181 2,300 0.01 0.6 H1048 40 200 100 1 4 H1869* 822 1,245 90 23 8 H2030 3 >10,000 190 0.02 2 H1703 3 >10,000 1,000 0.1 440 H1793* >10,000 >10,000 130 8 16 Pancreatic cancer HPAF-II 28 3 2,200 36 5 AsPC1 1 9 1,100 6 26 Capan-1 23 9 900 20 0.4 BxPC3 15 134 100 23 120 Colon cancer LoVo 7 3 3,000 0.3 0.33 RK0 1 14 6,200 0.4 1,500 CaCo2* >10,000 >10,000 120 0.2 3 Sw480* >10,000 >10,000 270 0.5 130 Prostate cancer PC3 4 3 740 330 370 DU145 43 13 290 170 3 22rv1 20 >10,000 330 0.03 0.4 LNCaP 28 >10,000 70 0.1 0.2 Breast cancer MDA-MB-231 4 5 1,500 0.7 1,500 BT-549 2 8 430 6 350 Hs578T 10 23 540 7 1,100 MDA-MB-435S 4 >10,000 1,300 0.03 78 MCF-7* >10,000 >10,000 490 0.1 8 BT-20* >10,000 >10,000 160 0.02 9 ZR-75-1* >10,000 >10,000 900 0.03 1

*Cell lines that are not sensitive to the furin-activated PrAg/FP59 and therefore not included in the following tables and analyses.

cancer and prostate cancer cell lines were also sensitive to PrAgU2/FP59. As a control for the cell sensitivity to the PrAgU2/FP59 (two of four sensitive cell lines). Only in recombinant anthrax toxin, all cell lines were also tested breast cancer were a minority of cell lines sensitive to for sensitivity to the furin-activated PrAg/FP59 (Table 1). PrAgU2/FP59 (three of seven sensitive cell lines; Table 1). These results show the wide range and high potency of However, closer examination of the breast cancer cell lines PrAgU2/FP59. This toxin is capable of targeting a majority revealed that all the sensitive cell lines were basal-like, of tumor cell lines independently of tumor type and tissue which suggests high potency of PrAgU2/FP59 on basal-like of origin. breast cancer. Non–small cell lung cancer cell lines being uPAR Expression Levels the most sensitive, we expanded that panel to include a Because sensitivity to PrAgU2/FP59 is expected to total of 20 cell lines, 14 of which where sensitive to depend on the uPAR and uPA content of cells, we

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determined the status of the uPA/uPAR system in our panel of cell lines (Table 1). Independently of the tumor type, PrAgU2/FP59–sensitive cell lines had significantly higher expression levels of uPAR compared with nonsensitive cell lines (mean uPAR expression = 2,442 and 678 receptors per cell, respectively; P = 0.0153). These results indicate that high uPAR expression levels are a contributor to the cytotoxicity of PrAgU2/FP59 and a marker for tumor sensitivity to this toxin. uPAR is, therefore, a possible predictor of tumor sensitivity to PrAgU2/FP59. Total uPA and PAI-1 Levels We also determined total uPA and PAI-1 levels in the supernatants of our cell line panel (Table 1). PrAgU2/FP59 Figure 2. The effects of the addition of exogenous pro-uPA on the sensitive cell lines had 10-fold higher mean total uPA levels potencyof PrAgU2/FP59 to MDA-MB-435S cell line as determined by in their supernatants when compared with nonsensitive [3H]thymidine incorporation inhibition. X axis, log of PrAgU2 concentra- Y axis, 3 cell lines (30.71 and 3.02 ng/mL, respectively; P = 0.0058) tion; cell viabilityas expressed bypercentage of [ H]thymidine incorporation in cpm. The addition of 100 ng/mL exogenous pro-uPA (n) independently of the tumor type. Mean total PAI-1 levels, totallyreversed the resistance of MDA-MB-435S cells to PrAgU2/FP59 on the other hand, were not significantly different in (IC50= 54 pmol/L after the addition of pro-uPA compared with an PrAgU2/FP59–sensitive and PrAgU2/FP59–resistant cell IC50 >10,000 pmol/L for PrAgU2/FP59 alone). lines (399.71 and 79.58 ng/mL, respectively; P = 0.08; Table 1). These results indicate that, independently of the tumor type, total PAI-1 levels do not contribute to the explaining why the addition of exogenous sc-uPA in- potency of PrAgU2/FP59 and do not constitute a possible creased their sensitivity to PrAgU2/FP59 by >200-fold marker for tumor responsiveness to this toxin. High total (IC50= 54, 3.1, and 31 pmol/L, respectively; Fig. 2). On the uPA levels, on the other hand, significantly contributed to other hand, the PrAgU2/FP59 sensitivity of the three the cytotoxicity of PrAgU2/FP59. The presence of uPA is, remaining cell lines (H2030, BxPC3, and LNCaP with 180, therefore, a marker of tumor sensitivity to PrAgU2/FP59 100, and 77 receptors per cell, respectively) was not affected and could be used as a predictor for the toxicity of this by the addition of exogenous sc-uPA, suggesting that other toxin to tumor cells, independently of the tumor type. components of the uPA system were nonfunctional (data Comparison of uPA versus uPAR Dependency not shown). These results show the existence of a threshold Having disregarded the seven cell lines that were of uPAR expression, f200 receptors per cell, below which resistant to both PrAg/FP59 and PrAgU2/FP59, we looked cells are resistant to PrAgU2/FP59 independently of uPA at the remaining six cell lines that were only resistant to levels. uPAR expression is, therefore, an independent PrAgU2/FP59. The addition of exogenous sc-uPA totally predictor of tumor cell sensitivity to PrAgU2/FP59 in reversed the resistance of three of these cell lines to addition to total uPA levels. PrAgU2/FP59. These cell lines (MDA-MB-435, H1703, We also determined the effects of the inhibition of the and 22rv1) had uPAR expression levels similar to those of uPA/uPAR system on the sensitivity of cell lines to sensitive cell lines (1,286, 1,075, and 329 receptors per cell, PrAgU2/FP59. The H650 non–small cell lung cancer cell respectively), but very low levels of uPA expression, line was 200-fold less sensitive to PrAgU2/FP59 when coincubated with an anti-uPA antibody (IC50 = 1.5 and 254 pmol/L, respectively; Fig. 3). These results were also confirmed on pancreatic cancer, colon cancer, prostate cancer, and breast cancer cell lines (data not shown). These results show that PrAgU2/FP59 is specifically activated by the uPA/uPAR system and, therefore, is selectively toxic to uPA/uPAR–expressing cells. ANTXR Expression Levels We controlled for the expression of ANTXR in our panel of cell lines by determining the cell sensitivity to the furin- activated PrAg/FP59. Surprisingly, seven cell lines of different tumor origin (non–small cell lung cancer, colon cancer, and breast cancer) were not sensitive to PrAg/FP59 (Table 1). As expected, these cells were also resistant to Figure 1. PrAg/FP59 and PrAgU2/FP59 cytotoxicity to a human non – PrAgU2/FP59. ANTXR expression levels on all these cell small cell lung cancer cell line H1299 using a [3H]thymidine incorporation lines were <300 receptors per cell and were f20-fold lower X axis, Y axis, inhibition assay. log of the molar PrAgU2 concentration; than ANTXR expression levels on cell lines sensitive to cell viabilityexpressed as percentage control of [ 3H]thymidine incorporation in cpm. H1299 was sensitive to both PrAgU2/FP59 (n) and PrAg/ PrAg/FP59 but not to PrAgU2/FP59 (Table 2). This shows E FP59 ( ;IC50= 2 and 1 pmol/L, respectively). that ANTXR expression is essential for PrAgU2/FP59

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Table 3. Normal human cell sensitivity to PrAg/FP59 and PrAgU2/FP59 as determined by [3H]thymidine incorporation inhibition

Normal cell type PrAg/FP59 PrAgU2/FP59 (IC50; pmol/L) (IC50; pmol/L)

Human lung fibroblasts 1 68 Human cardiomyocytes 1 305 Human cardiac microvascular 3 636 endothelial cells Renal proximal tubule 33 >10,000 epithelial cells Renal cortical epithelial cells 21 >10,000 Figure 3. The effects of urokinase inhibition on the potencyof PrAgU2/ FP59 on H650 non – small cell lung cancer cell line as determined by [3H]thymidine incorporation inhibition. X axis, log of PrAgU2 concentration; Y axis, cell viabilityas expressed bypercentage of [ 3H]thymidine incorporation in cpm. The addition of anti-uPA antibody( n) decreased the (IC50 = 636 pmol/L) reveals a 300-fold difference in sensitivityof H650 cells to PrAgU2/FP59 by f200-fold (IC50 = 250 and sensitivity and illustrates the tumor selectivity of 1.5 pmol/L, respectively). PrAgU2/FP59 (Fig. 4). These results confirm the selectivity of PrAgU2/FP59 to uPA/uPAR–expressing tumor cells and indicate the existence of a wide in vitro therapeutic potency and could be used as a potential first step marker window for the urokinase-activated recombinant anthrax of cell sensitivity to this toxin. toxin. Normal Cell Sensitivity to PrAgU2/FP59 To determine the selectivity of PrAgU2/FP59 for tumor cells, we tested its toxicity to five normal human cell types. Discussion Cardiomyocytes, cardiac microvascular endothelial cells, The uPA system is overexpressed on a wide variety of renal proximal tubule epithelial cells, and renal cortical tumor types and, along with matrix metalloproteases, is the epithelial cells were, on average, 300-fold less sensitive to most commonly expressed protease system in highly PrAgU2/FP59 than tumor cells (IC50 = 305, 636, and aggressive metastatic tumors. In fact, several studies have >10,000 pmol/L, respectively), while being as sensitive to shown that patients with high circulating levels of uPA and the furin-activated anthrax recombinant toxin (Table 3). soluble uPAR have a very poor prognosis (16–18). Multiple Normal human lung fibroblasts were not as resistant to attempts have been recently made to inhibit the uPA/ PrAgU2/FP59 as the other normal human cells tested uPAR system. These inhibitors, although effective in (IC50 = 68 pmol/L). However, the Hill slope of the sig- limiting tumor growth, did not lead to tumor regression, moidal dose-response curve of all normal cells tested was due to the fact that inhibiting the uPA/uPAR system has significantly different of that of tumor cell lines (data not cytostatic rather than cytotoxic effects on tumor cells shown), indicating a possible difference in binding and (19, 20). We and others favor another approach that consists internalization mechanisms between normal and tumor of taking advantage of the expression of the urokinase cells. The comparison of the potency of PrAgU2/FP59 to system on tumor cells to specifically target these cells H1299 non–small cell lung cancer cell line (IC50 = 2 pmol/L) with fusion toxins (21–23). In this study, we specifically and to human cardiac microvascular endothelial cells target uPAR-expressing tumor cells with the cytotoxic anthrax recombinant toxin (PrAg/FP59) by modifying the furin cleavage site on PrAg to a uPA cleavage site. PrAgU2/FP59 was highly cytotoxic to a majority of the Table 2. Comparison of ANTXR expression levels in cell lines resistant to both PrAg/FP59 and PrAgU2/FP59 and cell lines cell lines present in the large set of tumor cells we tested. resistant only to PrAgU2/FP59 This is highly significant because it shows the ability of PrAgU2/FP59 to selectively target urokinase-expressing Cell line PrAg/FP59 PrAgU2/FP59 ANTXR tumor cells of different tumor types despite binding to both (IC50; pmol/L) (IC50; pmol/L) (receptors per cell) normal and tumor cells through the ubiquitously expressed ANTXRs, tumor endothelial marker-8, and capillary mor- H1703 3 >10,000 1,600 phogenesis gene-2. 22rv1 20 >10,000 1,300 Novel, tumor-selective therapies with well-elucidated LNCaP 28 >10,000 7,600 molecular mechanisms and easily identifiable markers of CaCo2 >10,000 >10,000 110 Sw480 >10,000 >10,000 150 tumor sensitivity have allowed the preselection of patients MCF-7 >10,000 >10,000 140 that are most likely to respond. The most recent examples of BT-20 >10,000 >10,000 280 such therapies are Gleevec, which targets BCR-Abl mutants ZR-75-1 >10,000 >10,000 180 in chronic myeloid leukemia patients and Herceptin, which targets Her2-neu and is therefore used for the treatment of

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breast cancer patients with Her2-neu–overexpressing different mechanism of toxin activation and internalization tumors (24–27). in normal cells, which might provide the opportunity for We looked at four molecular markers that would predict novel ways of specific tumor targeting using this family of tumor sensitivity to PrAgU2/FP59 and therefore may toxins. Furthermore, the inhibition of the uPA protease eventually allow preselection of patients with sensitive system significantly decreased the potency of PrAgU2/ tumors. All four of these markers, ANTXR, uPA, uPAR, FP59 independently of tumor type. This shows the absolute and PAI-1, are involved in the primary binding, activation, requirement of this toxin for the presence of an active and internalization of PrAgU2/FP59, which constitute the urokinase system at the cell surface for its activation, thus control points for the potency and selectivity of this toxin. confirming uPA and uPAR as molecular markers and The subsequent translocation of FP59 into the cytosol, potent predictors of tumor cell sensitivity to PrAgU2/FP59. ADP-ribosylation of EF2, protein synthesis inhibition, and The remarkable potency, wide range, and selectivity cell death occur independently of tumor cell characteristics. of PrAgU2/FP59 in vitro indicate the high potential of Interestingly, three of four markers proved to be excellent this approach in the targeting of urokinase-expressing predictors of tumor sensitivity to PrAgU2/FP59. The solid tumors. Recent studies have shown the relative safety expression of ANTXR and uPAR as well as the presence and selective potency of the systemic administration of of uPA predict tumor cell sensitivity to PrAgU2/FP59, PrAgU2/FP59 in a mouse xenograft model (14). However, whereas the absence of any one of these markers, especially more extensive investigation is needed to further our ANTXR and uPAR, greatly diminishes that sensitivity. understanding of the in vivo safety, potency, range, and Moreover, these markers are very strong predictors selectivity of this toxin. because relatively low levels of ANTXR and uPAR A growing body of workis increasing our understanding expression (>300 and 200 receptors per cell, respectively) of the selective targeting of tumors with urokinase and as well as total uPA levels (>1 ng/mL) are needed matrix metalloprotease–activated fusion toxins. In a recent to predict sensitivity of tumor cells to PrAgU2/FP59. paper, Liu et al. (12) showed the potency and high tumor Although we did not perform an in-depth analyses of the specificity of a dual protease complementation system complex interactions between the four previously men- using anthrax toxin activated by both uPA and matrix tioned markers, we can conclude the following. ANTXR metalloproteases, which further confirms the potential being the primary binding site of PrAg its presence is usefulness of tumor protease-activated toxins and validates essential to the potency of PrAgU2/FP59 independently of this approach for the specific targeting of tumors. uPA and uPAR levels. Also, our data shows that in the PrAgU2/FP59 is a wide-range, highly selective, and absence of uPAR, cell lines are resistant to PrAgU2/FP59 highly potent toxin that specifically targets uPA-expressing even in the presence of excess amounts of pro-uPA, which tumors, regardless of their tissue origin. This toxin is a very indicates that uPAR expression levels are a stronger promising potential treatment for all types of urokinase- predictor of the potency of PrAgU2/FP59 than uPA levels. expressing solid tumors and it deserves further investiga- PrAgU2/FP59 proved to be a highly selective compound tion and development. because, unlike tumor cell lines, most normal human cells tested were relatively resistant to this toxin. Moreover, the References significant difference in the Hill slope of the sigmoidal 1. Johansson N, Ahonen M, Kahari VM. Matrix metalloproteinases in dose-response curves between normal and tumor cells tumor invasion. Cell Mol Life Sci 2000;57:5 – 15. indicates a possible difference in the kinetics of toxin 2. Andreasen PA, Egelund R, Petersen HH. The plasminogen activation activation and cell entry (27). This suggests a potentially system in tumor growth, invasion, and metastasis. Cell Mol Life Sci 2000; 57:25 – 40. 3. Dano K, Romer J, Nielsen BS, et al. Cancer invasion and tissue remodeling-cooperation of protease systems and cell types. APMIS 1999; 107:120 – 7. 4. Ploug M, Ostergaard S, Hansen LB, Holm A, Dano K. Photoaffinity labeling of the human receptor for urokinase-type plasminogen activator using a decapeptide antagonist. Evidence for a composite ligand binding site and a short interdomain separation. Biochemistry1998;37:3612 – 22. 5. Garsdvall H, Dano K, Ploug M. Mapping part of the functional epitope for ligand binding on the receptor for urokinase type plasminogen activator bysite directed mutagenesis. J Biol Chem 1999;274:37995 – 8003. 6. DuesberyNS, Webb CP, Leppla SH, et al. Proteolyticinactivation of MAP kinase kinase byanthrax lethal factor. Science 1998;280:734 – 7. 7. BradleyKA, Mogridge J, Mourez M, Collier RJ, Young JA. Identification of the cellular receptor for anthrax toxin. Nature 2001;414:225 – 9. 8. Wei W, Lu Q, ChaudryGJ, Leppla SH, Cohen SN. The LDL receptor related protein LRP6 mediates internalization and lethalityof anthrax toxin. Figure 4. PrAgU2/FP59 cytotoxicity on normal cells versus tumor cells Cell 2006;124:1141 – 54. 3 as determined by[ H]thymidine incorporation inhibition. PrAgU2/FP59 9. Abi Habib RJ, Urieto JO, Liu S, Leppla SH, DuesberyNS, Frankel AE. was f300-fold more toxic to H1299 non – small cell lung cancer cell line BRAF status and mitogen-activated protein/extracellular signal-regulated (n) than it was to normal human cardiac microvascular endothelial cells kinase kinase 1/2 activityindicate sensitivityof melanoma cells to anthrax (E;IC50= 2 and 636 pmol/L). lethal toxin. Mol Cancer Ther 2005;4:1303 – 10.

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Ralph J. Abi-Habib, Ravibhushan Singh, Shihui Liu, et al.

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