thrombospondin-1 type 1 repeat recombinant proteins inhibit … · tsp-1 protein into mice inhibits...

[CANCER RESEARCH 61, 7830–7839, November 1, 2001]

Thrombospondin-1 Type 1 Repeat Recombinant Proteins Inhibit Tumor Growththrough Transforming Growth Factor-�-dependentand -independent Mechanisms1

Wei-Min Miao, Wen Lin Seng,2 Mark Duquette, Patrick Lawler, Christiane Laus, and Jack Lawler3

The Division of Cancer Biology and Angiogenesis, Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215

ABSTRACT

Thrombospondin-1 (TSP-1) is a potent inhibitor of tumor growth andangiogenesis. The antiangiogenic activity of TSP-1 has been mapped to theprocollagen homology region and the type 1 repeats (TSR) using syntheticpeptides. To elucidate the molecular mechanisms that are involved in theinhibition of tumor growth by the TSRs, we have expressed recombinantversions of these motifs and have assayed their ability to inhibit thegrowth of experimental B16F10 melanomas and Lewis lung carcinomas.Recombinant proteins that contain all three TSRs (3TSR) or the secondTSR with (TSR2�RFK) or without (TSR2) the transforming growthfactor-� (TGF�) activating sequence (RFK) have been expressed inDrosophila S2 cells. In addition, recombinant proteins with mutations ineither the RFK sequence (TSR2�QFK) or the WSHWSPW sequence[TSR2 (W/T)] of the second TSR have been prepared. Similar to plateletTSP-1, these proteins are potent inhibitors of endothelial cell migration,and 3TSR of human TSP-1 (3TSR/hTSP-1) and TSR2�RFK activateTGF�. An 81% inhibition of B16F10 tumor growth is observed at 2.5 mg(135 nmol)/kg/day of the recombinant 3TSR/hTSP-1. A comparable levelof inhibition is observed with 2.5 mg (360 nmol)/kg/day of TSR2�RFK.By contrast, 3TSR of mouse TSP-2 (3TSR/mTSP-2), TSR2�QFK, andTSR2 are significantly less effective. TSR2�RFK and TSR2 reduce tumorvessel density, but TSR2�RFK has a greater effect on B16F10 tumor cellapoptosis and proliferation. Concurrent treatment of B16F10 tumor-bearing mice with TSR2�RFK and either a soluble form of the TGF�receptor or an antibody to active TGF� reduces the inhibition of B16F10tumor growth to levels that are comparable with those of TSR2 andTSR2�QFK. By contrast, the presence of the TGF�-activating sequencedoes not increase the level of inhibition of Lewis lung carcinoma experi-mental tumor growth. These data indicate that the TSRs inhibit tumorgrowth by inhibition of angiogenesis and regulation of tumor cell growthand apoptosis. The regulation of tumor cell growth and apoptosis is TGF�dependent, whereas the inhibition of angiogenesis is not.

INTRODUCTION

Extracellular matrix proteins provide environmental cues that mod-ulate cellular phenotype during development, tissue remodeling, andtumor growth. Neoplasia arise from mutations in oncogenes andtumor suppressor genes and in genes that are involved in the cell cycleand apoptosis (1). Tumor progression is also affected by landscapergenes that make the tumor microenvironment more or less permissivefor growth (2). In vitro and in vivo data indicate that the extracellularmatrix protein TSP4-1 functions as a landscaper gene in that it inhibitstumor growth. The lack of TSP-1 gene expression in p53-deficient

mice results in decreased survival and changes to the spectrum oftumors observed.5 In addition, decreased TSP-1 expression correlateswith the loss of p53 expression in human bladder, skin and coloncancer, and in fibroblasts from patients with Li-Fraumeni syndrome(3–7). In general, decreased TSP-1 expression is observed in trans-formed cells. Transfection of TSP-1 expression vectors into these cellsinhibits the growth of the tumors that form when these cells areimplanted into mice (8–12). Moreover, systemic injection of the intactTSP-1 protein into mice inhibits the growth of B16F10 melanomaexperimental lung metastases (13). Taken together, these data indicatethat TSP-1 gene expression down-regulates tumor growth. The abilityof TSP-1 to inhibit angiogenesis, activate TGF�, induce apoptosis ofendothelial cells, and inhibit tumor cell growth may contribute to thisinhibitory effect.

TSP-1 is a potent inhibitor of angiogenesis (reviewed in Ref. 14). Itinhibits endothelial cell growth, migration, and tube formation in vitro(15). Intact TSP-1, bacterial fusion proteins containing TSP-1 se-quences or synthetic peptides that contain sequences from the type 1repeats (TSRs) of TSP-1 also induce apoptosis of endothelial cells(16). A peptide from the procollagen homology region and severalpeptides from the TSRs reportedly contain antiangiogenic activity(17–19). Recently, it has been reported that the activity of one of thesepeptides is dependent upon an L- to D-amino acid racemization thatoccurs during synthesis (20). The peptide that is synthesized with allL-amino acids is inactive. These data indicate that synthetic peptidesmay not accurately mimic the activities of the native protein.

The antiangiogenic activity of TSP-1 is reportedly mediated byCD36 on the endothelial cell membrane (21, 22). Whereas the inhib-itory peptides from the TSRs are close to the VTCG sequence that isinvolved in CD36 binding, peptides that do not include this sequenceare active as inhibitors of angiogenesis (18–20). These results suggestthat other sequences within TSP-1 may interact with CD36 or thatother membrane proteins are involved.

The boundary between the first and second TSRs of TSP-1 containsa sequence that binds and activates TGF� (23, 24). The sequence RFKhas been shown to be necessary and sufficient for activation of TGF�.Mice that are deficient in TSP-1 display an abnormal phenotype in thelungs that is consistent with decreased levels of TGF� activation (25,26). TGF� has been shown to act to suppress tumor growth. Localinjection of TGF� around experimental A549 human lung carcinomasinhibits tumor growth (27). In mice that lack TGF� and Rag2,spontaneous adenomas and carcinomas occur in the cecum and colon(28). Mice that are heterozygous for a TGF�-null allele exhibitenhanced tumor formation in response to chemical carcinogens (29).In addition, overexpression of a dominant-negative form of the TGF�type II receptor accelerates skin carcinoma (30). By contrast, TGF�has been reported to stimulate the growth of colon, prostate, andmelanoma cells in vitro (31–33). In addition, TGF� reportedly stim-ulates angiogenesis in renal cell carcinoma (34). Thus, the activationof TGF� by TSP-1 may have variable effects on tumor growth.

Received 5/12/01; accepted 9/4/01.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 with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grant HL28749 and HL07893 from the National Heart,Lung, and Blood Institute of the NIH.

2 Present address: Phylonix Pharmaceuticals, Inc., 100 Inman Street, Cambridge, MA02139.

3 To whom requests for reprints should be addressed, at Department of Pathology,Beth Israel Deaconess Medical Center, Research North, Room 270C, 99 BrooklineAvenue, Boston, MA 02215. Phone: (617) 667-1694; Fax: (617) 667-3591; E-mail:[email protected].

4 The abbreviations used are: TSP, thrombospondin; TSR, TSP type 1 repeat; TGF,transforming growth factor; HDMEC, human dermal microvessel endothelial cell.

5 J. Lawler, W-M. Miao, M. Duquette, N. Bouck, R. T. Bronson, and R. O. Hynes,Thrombospondin-1 gene expression affects survival and tumor spectrum of p53-deficientmice. Am. J. Pathol., in press, 2001.

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Systemic injection of peptides that contain or lack the RFK sequenceinhibits tumor growth in a mouse xenograft model of breast cancer,showing that activation of TGF� is not essential for the antitumoractivity of TSP-1 (19).

In this study, we have prepared recombinant forms of the TSRs ina eukaryotic expression system. These proteins resemble the nativeprotein in that they bind antibodies to the human TSP-1 TSRs, activateTGF�, and inhibit endothelial cell migration. We have found thatthese proteins are potent inhibitors of tumor growth in vivo. Thisinhibition is attributable to a combination of effects including inhibi-tion of angiogenesis, induction of tumor cell apoptosis, and inhibitionof tumor cell proliferation. A portion of the inhibitory effect ismediated by the activation of TGF� in tumor cells that are responsiveto TGF�.

MATERIALS AND METHODS

Human TSP-1 was purified from the supernatant of thrombin-treated plate-lets as described previously (35). To remove TGF�, the sucrose densitygradient centrifugation step was performed at pH 11 (35, 36). Recombinantproteins that included sequences from the type 1 repeats were prepared by PCRusing the full-length cDNA for human TSP-1 or murine TSP-2 as a template.A recombinant protein containing all three TSRs of TSP-1 (3TSR/hTSP-1,amino acids 361–530) was prepared using the forward primer 475htsp1f (GATGAT CCC GGG GAC GAC TCT GCG GAC GAT GGC) and the reverseprimer 476htsp1r (GAT ACC GGT AAT TGG ACA GTC CTG CTT G). Arecombinant protein containing all three TSRs of mouse TSP-2 (3TSR/mTSP-2, amino acids 381–550) was prepared using the forward primer544mtsp2f (GAT GAT CCC GGG GAT GAG GGC TGG TCT CCG) and thereverse primer 545mtsp2r (GAT ACC GGT AAT AGG GCA GCT TCT CTT).A recombinant protein that contains the second TSR (TSR2, amino acids416–473) was prepared using the forward primer 537htsp1f (GAT GAT CCCGGG CAG GAT GGT GGC TGG AGC) and the reverse primer 515htsp1r(GAT ACC GGT GAT GGG GCA GGC GTC TTT CTT). To evaluate the roleof TGF� activation on the effect of this recombinant protein, a longer versionof the second TSR (TSR2�RFK, amino acids 411–473) that includes the RFKsequence was synthesized using the forward primer 514htsp1f (GAT GATCCC GGG GAC AAG AGA TTT AAA CAG) and the reverse primer515htsp1r. In addition, a mutant protein that contained the sequence QFK wasconstructed using the forward primer 605htsp1f (GAT GAT CCC GGG GACAAG CAA TTT AAA CAG GAT GG) and the reverse primer 515htsp1r.Synthetic peptides that contain glutamine (Q) instead of arginine (R) do notactivated TGF� (26). A mutant protein in which the three conserved trypto-phan residues are mutated to threonine [TSR2 (W/T)] was engineered using theforward primer 592htsp1f (GAT GAT CCC GGG CAG GAT GGT GGC ACGAGC CAC ACG TCC CCG ACG TCA TCT TGT TCT) and the reverseprimer 515htsp1r. All PCR products were cloned between the XmaI and theAgeI sites of the vector pMT/BiP/V5-HisA (Invitrogen, Carlsbad, CA). Therecombinant proteins included the vector-derived amino acid sequenceRSPWG at the NH2 terminal and TGHHHHHH at the COOH terminal. Thefidelity of the PCR products was verified by nucleotide sequencing. Eachexpression vector was cotransfected into Drosophila S2 cells with the selectionvector pCoHYGRO according to the manufacturer’s protocols (Invitrogen).Transfected cells were selected with hygromycin B, and the expression ofrecombinant peptides was monitored by Western blotting using the polyclonalantibody R3 that was raised against a fusion protein that contained all threeTSRs of TSP-1 (37). For large-scale preparation of recombinant protein, S2cells were grown in serum-free medium for 5 days. The culture supernatantwas centrifuged to remove the cells and dialyzed against 20 mM NaPO4 (pH7.8) and 500 mM NaCl. The dialysate was applied to a column of ProBondresin (Invitrogen). The column was eluted with 20 mM NaPO4 (pH 6.0), 500mM NaCl, and 500 mM imidazole. The protein eluted with 500 mM imidazolewas dialyzed against 20 mM NaPO4 (pH 7.0) and 500 mM NaCl, and 1%sucrose was added prior to storage.

Cell Culture. HDMECs (kindly provided by Dr. Michael Detmar, Massa-chusetts General Hospital, Boston, MA) were isolated by the procedure ofRichard et al. (38). The cells were cultured in Vitrogen precoated dishes and

maintained in EBM (Clonetics Corp., San Diego, CA) containing 20% fetalbovine serum, 1 �g/ml hydrocortisone acetate, 5 � 10�5 M dibutyryl-cAMP,200 units/ml penicillin, 100 units/ml streptomycin, 250 �g/ml amphotericin,and 2–5 ng/ml vascular endothelial growth factor. Murine B16F10 melanomaand murine Lewis lung carcinoma cells were obtained from the American TypeCulture Collection and were maintained in DMEM supplemented with 10%fetal bovine serum, 50 �g/ml penicillin, 50 units/ml streptomycin, and 2 mM

glutamine (supplemented DMEM). The effect of TGF� and TSR2�RFK onB16F10 melanoma and Lewis lung carcinoma cell proliferation was deter-mined with the CellTiter96 nonradioactive cell proliferation assay according toprotocols supplied by the manufacturer (Promega Biotech, Madison, WI).

Assay for TGF� Activation. B16F10 cells (2.5 � 105) were plated in aT25 flask and grown overnight in supplemented DMEM. The cells were rinsedonce with 1.0 ml of serum-free DMEM, and 2.5 ml of serum-free DMEMcontaining 1.0 �mol of the recombinant protein were added. After an overnightincubation, conditioned medium was collected and centrifuged at 12,000 rpmfor 5 min to remove cellular debris. Undiluted medium was used to determinethe level of active TGF�. Three independent samples of culture medium fromeach peptide treatment group were assayed for the level of active TGF� (seebelow).

To assay the level of active TGF� in tumor tissue, cubes of tissue that were4 mm on a side were homogenized in 1 ml of PBS containing 2 mM phenyl-methylsulfonyl fluoride and 1 �g/ml each of aprotinin, leupeptin, and pepstatinand sonicated for 2 min at 0°C. Homogenates were centrifuged at 12,000 rpmfor 5 min in a microcentrifuge to remove debris. Three independent tumorsamples from each treatment group were evaluated for TGF� level.

The TGF� levels in culture supernatants and tumor extracts were assayedusing an ELISA kit for active TGF� according to the manufacturer’s protocols(R&D Systems, Minneapolis, MN). Briefly, 100 �l of sample or TGF�

standard were mixed with 100 �l of diluent and then added to the individualwells of the assay plate. After incubation for 3 h at 22°C, the plates werewashed four times, 200 �l of TGF� conjugate solution were added to eachwell, and the plates were incubated for 1.5 h at 22°C. The wells were washedfour times, and 200 �l of substrate solution were added to each well. After 20min at 22°C, 50 �l of stop solution were added to each well, and theabsorbency at 450 nm was determined.

In Vitro Migration Assay. HDMECs at passages 7–10 were serum starvedand maintained in EBM with 0.1% BSA (control medium) for 20 h beforetrypsinization to harvest the cells. Cells were washed in EBM twice andresuspended in control medium at the concentration of 1 � 106 cells/ml. Twohundred �l of cells were packed down and kept frozen. They were lysed andserially diluted by CyQuant reagent (Molecular Probe, Eugene, OR) to be usedfor standard curve construction.

Transwell membrane (24-well polycarbonate membrane, 8-�m pore size;Corning Costar Corp., Cambridge, MA) coated with Vitrogen (30 �g/ml;Collagen Corp., Freemont, CA) on both sides was used for chemotacticmigration experiments. Coated transwells were inverted, and 100 �l of cellsuspension were applied to the top of the membrane and covered by the bottomplate carefully so that the cell suspension stayed on top of the membrane. Thecells were allowed to adhere to the coated membrane for 2 h in an incubatorat 37°C with 5% CO2. After the adhesion incubation, the plates were re-inverted, the bottom wells were filled with 0.4 ml of control medium, and thetop wells were filled with 0.1 ml of control medium containing testingreagents. The plate was returned to the incubator for 3.5 h for the cells tomigrate. At the end of the incubation, the transwells were washed in PBS, andthe cells on the bottom side of the membrane (unmigrated cells) were wipedaway with a cotton swab. The membranes were cut out by scalpel and placedinto 96-well plates and frozen at �80°C overnight. Two hundred �l ofCyQuant reagent were added to each well. Fluorescence reading was done 16 hlater with a SpectraFluor plate reader with excitation at 485 nm and emissionat 535 nm. The number of cells migrated was then calculated based on thestandard curve.

Primary Tumor Growth Assay. The proteins for injection were mixedwith Polymyxin B-Agarose (Sigma Chemical Co.) for 30 min at room tem-perature to remove endotoxin. The endotoxin levels were �0.05 EU/�g asdetermined using the QCL-1000 assay kit (BioWhittaker, Walkersville, MD).Proteins were filter sterilized, and the protein concentration was determinedprior to injection.

C57BL/6 mice (Taconic, Germantown, NY), 5–8 weeks of age, were

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INHIBITION OF TUMOR GROWTH BY TSRS



acclimated and caged in groups of four or fewer, and their backs were shaved.Cultured B16F10 melanoma or Lewis lung carcinoma cells (1 � 106) wereinoculated s.c. on the back of each mouse. Each treatment group contained sixmice. Tumors were measured with a dial caliper, and the volumes weredetermined using the formula width2 � length � 0.52. The treatment began 4days after inoculation of the tumor cells. The therapeutic groups receivedTSP-1 or recombinant TSP-1 proteins i.p. daily, and the negative control groupreceived comparable injections of saline alone. The experiments were termi-nated, and mice were sacrificed when the control mice began to die, usually atday 16 after tumor cell injection. In one experiment, the mice were treated witha daily i.v. injection of TSP-1 into the tail vein. To evaluate the role of TGF�activation, mice were treated with a soluble form of the extracellular domainof mouse TGF� type II receptor fused to the Fc region of IgG2a (kindlyprovided by Drs. Phil Gotswal and Victor Koteliansky, Biogen Corp., Cam-bridge, MA; Ref. 39). One hundred �g of this reagent were injected on days1 and 7 of TSR2�RFK [1 mg (144 nmol)/kg/day] or saline treatment. Alter-natively, mice were injected with the anti-TGF� antibody 1D11 that waskindly provided by Dr. Steve Ledbetter (Genzyme Corp., Framingham, MA).This reagent (100 �g/mouse) was injected every other day, beginning on thefirst day of saline or TSR2�RFK treatment. The antibody 13C4 (kindlyprovided by Dr. Steve Ledbetter) was used as a species- and isotype-matchedcontrol.

Histological Examination. The tumor tissues were cut, fixed with neutralbuffered formaldehyde, and embedded in paraffin according to standard his-tological procedures. H&E staining was used for tissue morphology examina-tion. Blood vessels were immunochemically stained by anti-CD31 antibodywith a Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Tumor cellapoptosis was detected by terminal deoxynucleotidyl transferase-mediatednick end labeling assay (35). Tumor cell proliferation was detected by stainingwith a monoclonal anti-proliferating cell nuclear antigen (Santa Cruz Biotech-nology, Santa Cruz, CA). The number of blood vessels was recorded bycounting 10 high-power fields. Tumor cell proliferation and the apoptoticindex were estimated by the percentage of cells scored under a light micro-scope. A minimum of 1000 cells was counted in each tumor sample.

RESULTS

Characterization of Recombinant Proteins. Five recombinantforms of the type 1 repeats of human TSP-1 and one form of the type1 repeats of mouse TSP-2 have been produced in S2 cells. The proteinthat contains all three type 1 repeats of human TSP-1, designated3TSR/hTSP-1, contains amino acids 361–530. This protein has apredicted molecular weight of 20,520 and an apparent molecularweight of 25,000 on SDS-PAGE, suggesting that carbohydrates areadded by posttranslational modification (data not shown). A compa-rable electrophoretic mobility was observed for all three type 1 repeatsof mouse TSP-2 (3TSR/mTSP-2). Each TSR contains six cysteineresidues. Consistent with the presence of intrachain disulfide bonds inthese proteins, 3TSR/hTSP-1 and 3TSR/mTSP-2 migrate more rap-idly in the absence of reducing agent during SDS-PAGE (data notshown; Ref. 40). The constructs, designated TSR2�RFK andTSR2�QFK, contain the second type 1 repeat of human TSP-1(amino acids 411–473) and include the sequence DKRFK orDKQFK, respectively, in the NH2-terminal region. The sequences ofhuman and mouse TSP-1 are identical in this region. The RFKsequence has been shown to mediate the activation of TGF� byTSP-1, and synthetic peptides that contain the sequence QFK areinactive (24). An equivalent region of TSP-1 (amino acids 416–473)that excludes the DKRFK sequence, designated TSR2, and a recom-binant protein in which the three conserved tryptophan residues inTSR2 were mutated to threonine residues [TSR2 (W/T)] have alsobeen prepared. The average level of protein expression is comparablefor all six proteins (�24 �g of purified protein from 1 ml of condi-tioned medium). All six proteins react with a polyclonal antibody,designated R3, that was raised against a bacterial fusion proteincomposed of the type 1 repeats of human TSP-1 fused to �-galacto-

sidase (data not shown; Ref. 37). Thus, the data indicate that therecombinant proteins produced in S2 cells have the expected molec-ular properties based on their content of the type 1 repeats.

Functionally, the recombinant TSR-containing proteins are similarto the native TSRs in that they activate TGF� and inhibit endothelialcell migration. Medium conditioned by B16F10 melanoma cells con-tain 30.3 � 2.3 pg/ml of active TGF� (Fig. 1). Addition of 1 �M

TSR2�RFK to the conditioned medium increases the level of activeTGF� to 180.6 � 23.3 pg/ml. A comparable level of TGF� activationis observed when the conditioned medium is treated with 3TSR/hTSP-1 (Fig. 1). By contrast, addition of recombinant proteins that donot contain the RFK sequence does not result in an increase in thelevel of active TGF� in the conditioned medium (Fig. 1).

As shown in Fig. 2, TSP-1 is a potent inhibitor of endothelial cellmigration in vitro. This inhibition is dose dependent up to �0.5 nM

TSP-1, but concentrations of TSP-1 above this level are less effective.This biphasic response has been reported by others (17). The 3TSR/hTSP-1, TSR2, and TSR2�RFK recombinant proteins also inhibitendothelial cell migration with responses that are similar to TSP-1(Fig. 2).

Inhibition of Experimental Tumor Growth by Intact TSP-1. Inthis study, the B16F10 melanoma model of experimental tumorgrowth has been used to assay the effect of the intact TSP-1 andrecombinant proteins. This model has been used extensively to assaythe activity of the antiangiogenic proteins endostatin, angiostatin, andanti-thrombin III (41–43). Systemic injection of TSP-1 [0.25 mg (0.6nmol)/kg/day] into tumor-bearing mice inhibits tumor growth by 56%on the twelfth treatment day (Fig. 3A). At higher doses, less inhibitionis observed, and a dose of 2.5 mg (6 nmol)/kg/day has no effect ontumor growth. Human platelet TSP-1 reportedly contains low levels ofbound TGF� that can be removed by treatment with pH 11 buffers(36). Removal of TGF� from the TSP-1 preparations did not affect thedose response for inhibition of experimental B16F10 tumor growth(Fig. 3A). To determine whether the higher concentrations of TSP-1are less effective in reaching the circulation, we treated tumor-bearingmice with TSP-1 that lacked TGF� by tail vein injection. Maximuminhibition of tumor growth is observed at the highest concentrations ofTSP-1 [2.5 mg (6 nmol)/kg/day] when the protein is delivered by i.v.injection (Fig. 3B). These data suggest that the biphasic effect ofTSP-1 on tumor growth that is observed with i.p. injection does not

Fig. 1. TGF� activation by recombinant proteins that include the RFK sequence. Salineor the recombinant proteins (1 �M) were incubated with B16F10 cells overnight inserum-free DMEM. The levels of active TGF� were determined using an ELISA. �,P � 0.005 relative to the control saline value; bars, SE.

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reflect direct effects on tumor growth but rather reflects the ability ofthe protein to reach the bloodstream.

Inhibition of B16F10 Experimental Tumor Growth by the Type1 Repeats. The recombinant proteins that contain the type 1 repeatswere used to systemically treat B16F10 tumor-bearing mice. The3TSR/hTSP-1 protein is a less effective inhibitor of tumor growth at0.25 mg (13.5 nmol)/kg/day than the intact protein (Fig. 4A). Bycontrast, at dosages of 1.0 mg (54 nmol)/kg/day and greater, the3TSR/hTSP-1 protein is more effective than TSP-1 when the proteinsare injected i.p. Tumor volume is reduced by 81% by the 3TSR/hTSP-1 protein with a dose of 2.5 mg (135 nmol)/kg/day. Increasingthe dose to 10 mg (540 nmol)/kg/day did not result in increasedinhibition of tumor growth (Fig. 4A).

To explore the potential involvement of the RFK sequence in theinhibition of B16F10 tumor growth, TSR2�RFK and TSR2 havebeen assayed. The tumor inhibition effect of the TSR2�RFK iscomparable with that of 3TSR/hTSP-1 on a weight basis at the variousdoses used (Fig. 4, A and B). By contrast, TSR2 is significantly lesseffective (Fig. 4C). At 0.25 mg (40 nmol)/kg/day, the tumor growth inthe mice treated with TSR2 is indistinguishable from the control

group. At 1.0 mg/kg/day, TSR2�RFK inhibits tumor growth by�80%, whereas TSR2 only inhibits growth by �38% on the twelfthday of treatment. Because TSR2�RFK has a higher molecular weightthan TSR2, 1 mg of protein corresponds to 144 nmol of TSR2�RFKand 160 nmol of TSR2. At the higher doses, the difference is lesspronounced; however, TSR2 remains less active (Fig. 4, B and C).Increasing the dose of TSR2 to 5 mg (800 nmol)/kg/day does notproduce a level of inhibition that is achieved with TSR2�RFK at 2.5mg (360 nmol)/kg/day (Fig. 4B and 5). TSR2 (W/T) did not inhibittumor growth, suggesting that the three conserved tryptophan residuesare important for TSR2 activity or stability (Fig. 5).

We prepared a recombinant protein designated TSR2�QFK, inwhich the arginine residue in the RFK sequence is mutated to gluta-mine, to more specifically demonstrate the importance of the RFKsequence. Systemic injection of TSR2�QFK produces levels ofB16F10 tumor growth inhibition that are comparable with TSR2(Fig. 5). In addition, a recombinant protein that contains all three type1 repeats of murine TSP-2 (3TSR/mTSP-2) is less effective as aninhibitor of tumor growth than 3TSR/hTSP-1 (Fig. 5). The aminoacids RIR are found in the murine TSP-2 sequence in the locationwhere the RFK sequence is located in TSP-1.

To further characterize the effect of the recombinant protein treat-ment on tumor growth, we have determined the rate of proliferation,the apoptotic index, and the capillary density in tumors from micetreated with TSR2, TSR2�RFK, or TSR2�QFK at a dose (1.0mg/kg/day), where the largest effect of inclusion of the RFK sequenceis observed. Tumors displayed a 71 and 63% reduction in capillary

Fig. 3. Growth of B16F10 tumors in mice treated with platelet TSP-1 injected i.p. (A)or i.v. (B). Each treatment group contained six mice. Treatment was initiated 4 days afters.c. injection of 1 � 106 tumor cells. The mice received one i.p. or i.v. injection each dayof saline (�) or 0.25 mg (0.6 nmol)/kg/day (E), 1.0 mg (2.4 nmol)/kg/day (Œ), or 2.5 mg(6 nmol)/kg/day (F) of TSP-1. Purification of the TSP-1 was performed at pH 11 toremove TGF�. Tumor volume was determined every other day with a dial caliper. Themeans are plotted; bars, SE.

Fig. 2. The effect of TSP-1 and the TSR-containing recombinant proteins on endo-thelial cell migration. HDMEC migration in the presence of varying concentrations ofTSP-1 (A, �), 3TSR/hTSP-1 (A, �), TSR2 (B, �), or TSR2�RFK (B, �) wasdetermined after 3.5 h. Migration in response to FGF-2 in the absence of inhibitors wasconsidered 100% migration, and migration in the absence of FGF-2 was considered 0%migration.

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density when the mice are treated with TSR2�RFK and TSR2�QFKproteins, respectively (Figs. 6 and 7A). B16F10 tumors treated withTSR2 exhibited a 64% decrease in capillary density. The tumors frommice that are treated with TSR2�RFK protein display a 4-fold in-crease in tumor cell apoptosis (P � 0.005), and the tumors from themice that are treated with TSR2�QFK or TSR2 displayed only a1.9-fold increase in apoptosis (P � 0.025; Figs. 6 and 7B).TSR2�RFK also reduces the percentage of proliferating cell nuclearantigen-positive tumor cells. The number of proliferating cells isdecreased by 35% by treatment with TSR2�RFK (P � 0.005),whereas treatment with TSR2 reduces tumor cell proliferation by7.8% (P � 0.05; Fig. 7C).

The Role of TGF� Activation in the Inhibition of B16F10Tumor Growth by TSR2�RFK. To establish that the additionalinhibitory effect of TSR2�RFK as compared with TSR2 or

TSR2�QFK is attributable to activation of TGF�, we have: (a) addedantagonists of active TGF� concomitant to TSR2�RFK treatment;and (b) assayed the level of active TGF� in tumor tissue. Twoantagonists of active TGF� have been used. Systemic injection of achimeric protein that includes the Fc portion of human IgG2a fused tothe extracellular domain of the type II TGF� receptor along withTSR2�RFK significantly (P � 0.005) reduced the ability ofTSR2�RFK to inhibit tumor growth (Fig. 8). The B16F10 tumor sizeis increased by �15% in the saline-treated control group (Fig. 8).Thus, in the presence of the soluble receptor, TSR2�RFK onlyinhibited tumor growth by 55% as compared with a value of 81% inthe absence of soluble receptor. The apoptotic and proliferative indi-ces in B16F10 tumors from mice that are treated with TSR2�RFKand the soluble TGF� receptor are comparable with those of micetreated with saline or TSR2�QFK (Fig. 7, B and C).

The level of active TGF� in extracts of saline-treated B16F10melanomas is 65.4 � 6.5 pg/ml. A comparable level of active TGF�is detected in tumor tissue from mice treated with TSR2 orTSR2�QFK (Fig. 9). By contrast, a 3.2-fold increase in active TGF�is observed in B16F10 melanoma tissue from mice treated withTSR2�RFK. The amount of active TGF� is reduced to control levelswhen the mice are treated concurrently with TSR2�RFK and thesoluble form of the TGF� receptor (Fig. 9).

We have used systemic administration of an antibody to activeTGF� (1D11) as an alternate strategy for inhibiting the activation ofTGF� by TSR2�RFK in vivo. In the control group that received dailysaline injections, treatment with that anti-TGF� antibody increasedtumor size by 17% as compared with the treatment group that receivedno antibody or a species- and isotype-matched control antibody(13C4; Fig. 10). Concomitant treatment with TSR2�RFK and 13C4reduced B16F10 tumor volume by 83% as compared with the tumorsthat grow in mice treated with saline and 13C4. By contrast, concom-itant treatment with TSR2�RFK and 1D11 only reduced tumor vol-ume by 53% as compared with the tumors that grow in mice treatedwith saline and 1D11 (Fig. 10). Histological analysis of the tumorsthat grow in mice treated with TSR2�RFK and 1D11 reveals that the

Fig. 5. Inhibitions of B16F10 tumor growth by recombinant proteins. Each treatmentgroup contained six mice. Treatment was initiated 4 days after s.c. injection of 1 � 106

tumor cells. The mice received one i.p. injection/day of saline, 3TSR/hTSP-1 (2.5mg/kg/day), 3TSR/mTSP-2 (2.5 mg/kg/day), TSR2 (1 or 5 mg/kg/day), TSR2�RFK (1.0mg/kg/day), TSR2�QFK (1.0 mg/kg/day), or TSR2 (W/T) (1.0 mg/kg/day). Tumorvolume was determined on the twelfth treatment day with a dial caliper. The means areplotted; bars, SE.

Fig. 4. Growth of B16F10 tumors in mice treated with 3TSR/hTSP-1 (A) TSR2�RFK(B) or TSR2 (C). Each treatment group contained six mice. Treatment was initiated 4 daysafter s.c. injection of 1 � 106 tumor cells. The mice received one i.p. injection each dayof saline (�) or 0.25 mg/kg/day (E), 1.0 mg/kg/day (Œ), or 2.5 mg/kg/day (F) ofrecombinant protein. One treatment group received 10 mg/kg/day of 3TSR/hTSP-1 (‚; A).Tumor volume was determined every other day with a dial caliper. The means are plotted;bars, SE.

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apoptotic index is not increased, and the proliferative index is notdecreased (Fig. 7, B and C). Taken together, these data indicate thatTSR2�RFK inhibits B16F10 melanoma growth through a combina-tion of TGF�-dependent and -independent mechanisms.

Effect of Type 1 Repeat Recombinant Proteins on Lewis LungCarcinoma Growth. The inhibitory effect of 3TSR/hTSP-1 is alsoobserved in mice bearing experimental tumors that form by s.c.injection of Lewis lung carcinoma cells. At 2.5 mg (135 nmol)/kg/day,3TSR/hTSP-1 inhibits tumor growth by 73% on treatment day 12(data not shown). TSR2�RFK is equally as effective as 3TSR/hTSP-1on a weight basis. By contrast to the B16F10 melanoma tumors, thelevel of inhibition of tumor growth is equivalent for TSR2�RFK,TSR2, and TSR2�QFK (Fig. 11). TSR2 (W/T) is without activity inthe Lewis lung carcinoma model, as it is in the B16F10 model (Fig.11). The capillary density in experimental Lewis lung carcinomas isreduced by 62–64% in mice treated with TSR2, TSR2�RFK, orTSR2�QFK (Fig. 7D). All three proteins produce a comparableincrease (1.8–1.9-fold) in apoptotic index and had no effect on pro-liferative index (Fig. 7, E and F). These data indicate that inclusion ofthe RFK sequence does not increase the ability of the recombinantTSR-containing proteins to inhibit the growth of tumors that formfrom Lewis lung carcinoma cells. To determine whether the lack ofeffect of the RFK sequence is attributable to an inability of the Lewis

lung carcinoma cells to respond to TGF�, we have examined theeffect of TSR2�RFK and TGF� on cell growth in culture. As shownin Fig. 12, TGF� or TSR2�RFK treatment results in a dose-depen-dent suppression of B16F10 melanoma cell growth in vitro. In addi-tion, 3TSR/hTSP-1 inhibits B16F10 melanoma cell growth, whereasTSR2, TSR2�QFK, and 3TSR/mTSR-2 do not (data not shown). Thesuppression of B16F10 melanoma cell growth by TSR2�RFK or3TSR/hTSP-1 is blocked by the soluble TGF� receptor and by theantibody to active TGF� (data not shown). By comparison to theB16F10 melanoma cells, the growth of Lewis lung carcinoma cells isunaffected by the presence of TGF� or TSR2�RFK (Fig. 12).

DISCUSSION

The data presented here show that systemic injection of recombi-nant proteins that contain the TSRs of TSP-1 inhibit the growth ofexperimental tumors. Inhibition has been observed with both B16F10melanoma and Lewis lung carcinoma cell lines. The 3TSR/hTSP-1and TSR2�RFK proteins are relatively potent, showing 70–80%inhibition of tumor volume at 2.5 mg/kg/day. This concentration is100–300 nmol/kg/day. Injection with intact TSP-1 protein at 0.25mg/kg/day (0.6 nmol/kg/day) results in a 56% reduction of tumorgrowth, indicating that the intact protein is considerably more active

Fig. 6. Histology of the tumors. B16F10 tumorsfrom mice treated with saline (A and B) or 1.0mg/kg/day of TSR2 (C and D), TSR2�RFK (E andF), or TSR2�RFK with the anti-TGF� antibody1D11 (G and H) were removed and fixed on thetwelfth treatment day. The antibody 1D11 (100 �g/mouse) was included in the i.p. injection on the firsttreatment day and every other day thereafter. Capil-laries were visualized with anti-CD31 antibody (A,C, E, and G), and apoptotic cells were identified byterminal deoxynucleotidyl transferase-mediated nickend labeling (B, D, F, and H). Only two examples ofapoptotic cells are identified (arrow) in each panel(B, D, F, and H). The scale bar for A, C, E, and G isshown in G, and the scale bar for B, D, F, and H isshown in H. Scale bar, 50 �m.

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than the recombinant proteins. The increased potency of the intactprotein may be attributable to the trimeric structure of TSP-1. Thenine TSRs of TSP-1 may cluster receptors and enhance signal trans-duction. Dimerization of CD36 in response to TSP-1 has been ob-served (44). This receptor reportedly mediates the antiangiogenicactivity of TSP-1 (21, 22). In addition, intact TSP-1 may include otherdomains that inhibit tumor growth. Sequences within the procollagenhomology region and the COOH-terminal domain of TSP-1 report-edly inhibit angiogenesis (17, 45).

When TSP-1 is administered by i.p. injections, higher doses of

TSP-1 are less effective in reducing tumor volume. One other studyhas examined the effect of injection of TSP-1 into tumor-bearing mice(13). In this study, mice with B16F10 melanoma experimental lungmetastases were treated with 5 or 10 mg/kg/day of TSP-1 via i.p.injection. Both doses produced an �90% inhibition in the number ofvisible surface metastases. To reconcile these contradictory effects ofhigher doses of TSP-1, we have used i.v. injection of TSP-1 in micewith s.c. B16F10 melanomas and found that higher doses are indeedactive when the protein is injected directly into the vasculature. Thus,

Fig. 9. Quantitation of active TGF� in tumor extracts from mice treated with recom-binant proteins. The level of active TGF� in tumor tissue from mice from varioustreatment groups was determined by ELISA (see “Materials and Methods”). �, P � 0.01relative to saline control.

Fig. 7. Quantitation of blood vessels (A and D), tumorcell apoptosis (B and E), and tumor cell proliferation (Cand F) in B16F10 melanomas (A–C) and Lewis lungcarcinomas (D–F). Tumors from mice treated with saline,1.0 mg/kg/day of TSR2, TSR2�RFK, TSR2�QFK, orTSR2 (W/T) were fixed, and tissue was prepared forhistology. The recombinant protein that was used to treateach group is indicated below the column. A group ofmice that received TSR2�RFK also received 100 �g/mouse of soluble TGF� receptor (TGFR) on the first andseventh treatment days (TSR2�RFK�TGFR). A group ofmice that received TSR�RFK also received 100 �g/mouse of the anti-TGF� antibody (TGFAB) on the firsttreatment day and every other day thereafter(TSR2�RFK�TGFAB).

Fig. 8. The effect of a soluble form of the TGF� type II receptor on inhibition of tumorgrowth by TSR2�RFK. Each treatment group contained six mice. Treatment was initiated4 days after s.c. injection of 1 � 106 B16F10 melanoma cells. The mice received one i.p.injection each day of saline or recombinant protein (1 mg/kg/day). For one saline and oneTSR2�RFK group, the i.p. injection on treatment days 1 and 7 also contained 100 �g ofsoluble TGF� receptor. Tumor volume was determined on the twelfth treatment day witha dial caliper. The means are plotted; bars, SE.

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the loss of activity at higher doses when the protein is delivered i.p.appears to be attributable to a decreased ability of the TSP-1 to exitthe peritoneal cavity.

Several activities of the TSRs may contribute to the inhibition oftumor growth. These include the ability to: (a) inhibit angiogenesis;(b) reduce tumor cell proliferation; (c) activate TGF�; and (d) regulateextracellular proteases. The results described here are the first to showthat systemic treatment with the TSRs of TSP-1 reduces vessel densityin tumors. They are consistent with the observation that overexpres-sion of intact TSP-1 in tumor cells decreases the capillary density intumors that form from these cells (9, 10). Furthermore, B16F10experimental tumors that are grown in TSP-1-deficient mice displayan increase in capillary density.5 Taken together, these data indicate

that TSP-1 in the tumor or stromal compartments and exogenouslyadded TSRs are potent inhibitors of tumor angiogenesis. TheTSR2�RFK and TSR2 proteins are both effective inhibitors of tumorangiogenesis. This result is consistent with the observation that bothproteins are potent inhibitors of endothelial cell migration. These dataare also consistent with synthetic peptide studies that have beenperformed using the chick chorioallantoic membrane assay (19). Inboth studies, the inhibition of angiogenesis was observed in thepresence or absence of the RFK sequence. Thus, the inhibition ofangiogenesis by TSP-1 is not dependent upon the activation of TGF�.

The rate of tumor cell apoptosis is a key factor in the determination ofthe rate of tumor growth (46, 47). We have found that TSP-1 proteins thatinclude the RFK sequence significantly increase the rate of B16F10tumor cell apoptosis. Whereas TSP-1 has been reported to inhibit tumorcell proliferation and induce endothelial cell apoptosis, this study repre-sents the first demonstration that the TSR-containing proteins inducetumor cell apoptosis (16, 48). This activity is enhanced by the addition ofthe DKRFK sequence at the NH2-terminal because TSR2 did not increasethe level of apoptosis of the tumor cells to the same extent asTSR2�RFK. An increase in tumor cell apoptosis is frequently associatedwith antiangiogenic therapy because of the decrease in nutrients that isassociated with the decrease in blood supply. Our data indicate thatincreased tumor cell apoptosis occurs as a direct result of TSR2�RFKtreatment rather than indirectly through inhibition of angiogenesis.

Fig. 11. The effect of systemic treatment with recombinant proteins on Lewis lungtumor growth. Each treatment group contained six mice. Treatment was initiated 4 daysafter s.c. injection of 1 � 106 Lewis lung carcinoma cells. The mice received one i.p.injection each day with saline or recombinant protein (1 mg/kg/day). The tumor volumewas determined 16 days after injection of the tumor cells with a dial caliper. The meansare plotted; bars, SE.

Fig. 12. The effect of TGF� and TSR2�RFK on the growth of B16F10 melanomacells and Lewis lung carcinoma cell in vitro. Tumor cells (5 � 103) were grow in 96-wellplates in the presence of varying concentrations of TGF� (A) or TSR2�RFK (B). Parallelsamples were also treated with the active TGF� blocking antibody 1D11. The relative cellnumber as measured by absorbance at 570 nm was determined with the Cell Titer 96nonradioactive cell proliferation assay. Bars, SE.

Fig. 10. The effect of an antibody to active TGF� on tumor growth. Each treatmentgroup contained six mice. Treatment was initiated 4 days after s.c. injection of 1 � 106

B16F10 melanoma cells. The mice received one i.p. injection each day of saline,TSR2�RFK, or TSR2�QFK (1.0 mg/kg/day). In some groups, the i.p. injection alsoincluded 100 �g of an anti-active TGF� antibody (1D11) or a species- and isotype-matched control (13C4), starting on the first treatment day and continuing every other daythereafter. The tumor volume was determined on the twelfth treatment day with a dialcaliper. The means are plotted; bars, SE.

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Whereas TSR2�RFK, TSR2�QFK, and TSR2 reduce vessel density tocomparable levels, TSR2�RFK has a more profound effect on B16F10tumor cell apoptosis. In addition, this protein significantly decreasestumor cell proliferation in vivo. By contrast, TSR2, TSR2�RFK, andTSR2�QFK have comparable effects on apoptosis within tumors thatform from Lewis lung carcinoma cells. The increase in apoptosis (1.8–1.9-fold) that is observed in Lewis lung carcinoma tumors with all threeproteins is equivalent to that induced by TSR2 or TSR2�QFK (1.9-fold)in B16F10 melanoma. This level of apoptosis is considerably lower thanthat induced by TSR2�RFK (4-fold) in B16F10 melanoma tumors.

Our data indicate that the ability of RFK-containing type 1 repeatproteins to inhibit B16F10 tumor growth appears to be attributable, inpart, to activation of TGF�. TGF� has pleiotropic effects on tumorgrowth. At early stages of tumorigenesis, TGF� may act as a tumorsuppressor gene (28, 29). TGF� can induce apoptosis of severaldifferent tumor cell lines (Ref. 49 and references therein). At laterstages of tumor growth, TGF� can stimulate angiogenesis through therecruitment of inflammatory and stromal cells (50). We have foundthat deletion or point mutations that affect the RFK sequence result inless active TGF� in vitro and in vivo. These proteins are less effectiveinhibitors of B16F10 tumor growth. In addition, antagonists of activeTGF� reduce the antitumor activity of RFK-containing proteins in theB16F10 model. These data are consistent with the observation that theinjection of TGF� into the tissue surrounding experimental A549 lungadenocarcinomas inhibits tumor growth (27). By contrast, the pres-ence of the RFK sequence does not affect the ability of recombinantproteins to inhibit experimental Lewis lung carcinomas. Furthermore,inclusion of the RFK sequence in a synthetic peptide did not increasethe antitumor activity toward mammary tumors formed by injection ofMDA MB435 cells (19). In vitro, TGF� or TSR2�RFK inhibit thegrowth of B16F10 cells but not Lewis lung carcinoma cells, suggest-ing that the latter have lost the ability to respond to TGF�. Similar toLewis lung carcinoma cells, MDA MB435 cell proliferation is notinhibited by TGF� (18). Many types of tumors have mutations in theTGF� type II receptors or Smads and become resistant to TGF�.Thus, although TSR2�RFK or 3TSR/hTSP-1 are potent inhibitors oftumor growth in general, an enhanced inhibition is obtained in thosetumor cells in which TGF� signaling results in growth suppression.

The data presented here indicate that recombinant proteins thatinclude the TSRs have therapeutic potential as inhibitors of tumorgrowth. They act to inhibit angiogenesis and to induce tumor cellapoptosis. The former activity is TGF� independent, and the latter isTGF� dependent. Thus, a knowledge of the TGF� signaling capacityof the tumor cells is important for the design of therapeutic regimesthat use these proteins. Because these mechanisms of action of theTSRs are probably different from other inhibitors of neoplasia, acombinational approach that includes TSRs with other inhibitors oftumor growth may provide an effective treatment for cancer.

ACKNOWLEDGMENTS

We thank Drs. Sareh Parangi, Zhensheng Wang, Corinne Reimer, andKaren Yee for insightful discussions and Jenny Teece and Mingyan Hu fortechnical support. Dr. Steve Ledbetter kindly provided the anti-TGF� antibody1D11 and the control antibody 13C4. The soluble form of the TGF� type IIreceptor was kindly provided by Drs. Phil Gotwals and Victor Koteliansky.The HDMECs were kindly provided by Dr. Michael Detmar. The Dana-Farber/Harvard Cancer Center, Rodent Histopathology Core provided assist-ance with the histology. The manuscript was prepared by Regina Prout andAlexis Bywater.

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2001;61:7830-7839. Cancer Res Wei-Min Miao, Wen Lin Seng, Mark Duquette, et al. -dependent and -independent Mechanisms

βInhibit Tumor Growth through Transforming Growth Factor-Thrombospondin-1 Type 1 Repeat Recombinant Proteins

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