Canstatin Acts on Endothelial and Tumor Cells via Mitochondrial … · Canstatin Acts on Endothelial and Tumor Cells via Mitochondrial Damage Initiated through Interaction with A

Canstatin Acts on Endothelial and Tumor Cells via Mitochondrial

Damage Initiated through Interaction with AvB3 and

AvB5 Integrins

Claire Magnon,1Ariane Galaup,

1Brian Mullan,

1Valerie Rouffiac,

2Jean-Michel Bidart,

3

Frank Griscelli,1Paule Opolon,

1and Michel Perricaudet

1

1UMR 8121 Laboratoire de vectorologie et transfert de genes, 2Laboratoire d’imagerie du petit animal, and 3Departement de biologieclinique, Institut Gustave Roussy, Villejuif cedex, France

Abstract

Canstatin, the noncollagenous domain of collagen type IVA-chains, belongs to a series of collagen-derived angiogenicinhibitors. We have elucidated the functional receptors andintracellular signaling induced by canstatin that explain itsstrong antitumor efficacy in vivo . For this purpose, wegenerated a canstatin-human serum albumin (CanHSA) fusionprotein, employing the HSA moiety as an expression tag. Weshow that CanHSA triggers a crucial mitochondrial apoptoticmechanism through procaspase-9 cleavage in both endothe-lial and tumor cells, which is mediated through cross-talkbetween AvB3- and AvB5-integrin receptors. As a point ofreference, we employed the first three kringle domains ofangiostatin (K1-3), fused with HSA, which, in contrast toCanHSA, act only on endothelial cells through AvB3-integrinreceptor–mediated activation of caspase-8 alone, withoutensuing mitochondrial damage. Taken together, these resultsprovide insights into how canstatin might exert its stronganticancer effect. (Cancer Res 2005; 65(10): 4353-61)

Introduction

Type IV collagen extracellular matrix (ECM) plays a functionalrole in angiogenesis, the process whereby new capillaries sproutfrom existing vessel. Proteolytic remodeling of the ECM not onlydegrades barriers that obstruct vascular cell migration but alsoexposes cryptic sites within collagen IV that promote novelintegrin-ligand interactions required for angiogenesis (1). The typeIV collagen supramolecular structure is composed of a-chains,each containing a noncollagenous (NC1) domain at the COOHterminus which provides the mechanical stability of the ECMstructure (2, 3).ECM is a potential therapeutic target to regulate neovasculari-

zation in cancerous diseases as angiogenesis is required forneoplastic tumor growth (4). Systemic injection of the recombinanta2NC1 domain monomer of type IV collagen, which has beennamed canstatin, inhibits the in vivo growth of xenografted tumorsfollowing daily injection over a period of 15 days as compared withplacebo-treated mice. In vitro , the antiproliferative and antimi-gratory effects of canstatin seem to be due to the induction ofapoptotic events through disruption of the FAK signaling pathwayand induction of Fas ligand expression (5–7).

NC1 globular domain fragments of a-chains of human collagenrepresent a new class of antiangiogenic molecules that differfundamentally in structure to the plasminogen-derived group ofantiangiogenic proteins such as angiostatin kringles 1 to 3 (K1-3;refs. 8–11). Thus, we decided to investigate the relative pharma-cologic properties of one protein from each therapeutic class:canstatin and K1-3.In this study, to improve the pharmacokinetic properties of

canstatin, we investigated in situ the anticancer properties of acanstatin-human serum albumin (CanHSA) fusion protein byadenoviral-mediated gene transfer, which efficiently yields highlevels of transgene expression in many different cell types (12). Tothis end, we constructed a recombinant adenovirus (AdCanHSA)encoding canstatin fused with HSA to decrease in vivo clearance offused protein by increasing its molecular weight (13). The HSAmoiety in the fusion protein carries the added advantage of actingas a tag which allows the expression of canstatin to be analyzedin vitro and in vivo . To evaluate the antitumor potential ofCanHSA, we compared its features both in vitro and in vivo withthat of adenoviral-expressed unfused canstatin (AdCan) and K1-3,either fused (AdK1-3HSA) or unfused (AdK1-3) with HSA (11, 14).Importantly, to characterize the antitumor properties of

CanHSA compared with those of K1-3HSA, we focused our studyon the mechanism of action of each protein. Given that angiostatinand canstatin both induce apoptosis in endothelial cells (7, 11,15, 16), we asked (a) whether tumor cells as well as endothelialcells could be a direct target of canstatin via an integrin-dependentinteraction, and (b) whether the signaling pathways leading toinduction of apoptosis are different following canstatin or K1-3treatment. To this end, we explored cell cycle progression and theactivities of caspases within tumor and endothelial cells.

Materials and Methods

Construction of the recombinant adenoviruses. AdCan and

AdCanHSA express the NC1 domain of the human a2 chain of type IVcollagen, also called canstatin (GenBank accession GI 81011723), either

fused or unfused to HSA moiety. Both canstatin and CanHSA are secreted

through a 24-residue prepro signal sequence from HSA (GenBank accession

E01107), which was inserted into the NH2 terminus of both proteins. AdK1-3 and AdK1-3HSA express respectively the angiostatin kringles 1 to 3 (K1-3)

and K1-3 conjugated with HSA. A recombinant adenovirus not containing a

transgene (AdCO1) was used as a negative control in this study (11, 14).

Cell lines. MDA-MB-231 and human umbilical vascular endothelial cells(HUVEC) have been previously described (17). PC-3 cells (ATCC ref. CRL

1435) were grown in F-12K supplemented with 10% fetal bovine serum

(FBS), 1 mmol/L L-glutamine, and 1.5 g/L sodium bicarbonate (Invitrogen,Carlsbad, CA).

Anticanstatin antibodies. To generate a polyclonal antibody against

canstatin, a peptide spanning the NH2-terminal region of canstatin

Requests for reprints: Claire Magnon, UMR 8121, PR2, Institut Gustave Roussy, 39rue Camille Desmoulins, 94805 Villejuif cedex, France. Phone: 33-1-42115075; Fax: 33-1-42115245; E-mail: [email protected].

I2005 American Association for Cancer Research.

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(VSIGYLLVKHSQTDQEPMC) was synthesized by conventional solid-phasemethodology using an Applied Biosystems Model 431A peptide synthesizer.

The purity of the peptide was ascertained by high-performance liquid

chromatography and its identity verified by mass spectrometry. The

synthetic peptide was conjugated to keyhole limpet hemocyanin, and tworabbits were immunized by two intradermal injections of the synthetic

peptide-carrier conjugate. Antisera were verified by ELISA for their capacity

to react with the canstatin synthetic peptide. Antibodies were purified by

affinity chromatography.Western blot analysis of canstatin, CanHSA, K1-3, and K1-3HSA

expressions in vitro. For canstatin and CanHSA expression analysis, cell

culture supernatants of MDA-MB-231 were collected 96 hours after

adenoviral infection of cell monolayers. Samples (8 Ag) were incubated

with the rabbit anticanstatin antibody and were revealed with a peroxidase-

conjugated donkey antirabbit antibody (Amersham, Buckinghamshire,

United Kingdom).

For CanHSA and K1-3HSA expression analysis, samples were incubated

with a mouse anti-HSA antibody (Biovalley, Marne la Vallee, France; ref.

ab7793). For K1-3 and K1-3HSA expression analysis, membranes were

incubated with an antihuman plasminogen monoclonal antibody (mAb)

A1D12 (11). Both of these antibody complexes were revealed by a goat

antimouse immunoglobulin G1 (IgG1)-horseradish peroxidase secondary

antibody (SouthernBiotech, Birmingham, AL; ref. 1070-05).

For canstatin and CanHSA stability analysis in vivo , MDA-MB-231

xenografted tumors were dissected following intratumoral adenoviral

injection. Tumor protein extracts (20 Ag) were assessed for canstatin and

CanHSA expression as described above.

Quantification of CanHSA and K1-3HSA in vivo. For quantification ofCanHSA and K1-3HSA by ELISA, wells were coated with a mouse anti-HSA

mAb described above. After washing, wells were incubated with mice sera

at the appropriate dilution or a HSA solution standard (Sigma-Aldrich, St.

Louis, MO; ref. A8763). Wells were washed and incubated with a rabbit anti-HSA polyclonal antibody (Sigma-Aldrich; ref. A0433). Antigen-antibody

complexes were revealed with an alkaline phosphatase–conjugated goat

antimouse IgG antibody (Promega, Madison, WI; H + L) and developmentwith an alkaline phosphatase kit (Bio-Rad, Hercules, CA). Absorbance at

405 nm was converted to HSA concentration (ng/mL) via a purified HSA

calibration curve.

Endothelial cell proliferation, migration assays, and contrast-enhanced color Doppler high-frequency ultrasonography of tumorneovascularization. Experiments were done as described (17).

Immunohistology and quantification of the intratumoral micro-vessels.We sacrificed six mice per group at an early stage (day 8) following

intratumoral injection of each adenovirus before the appearance of

necrosis. Paraffin sections of PC-3 tumors were incubated to quantify the

number of intratumoral vessels, as described (17). For each animal (n = 6),

a representative murine CD31-immunostained histologic sample was

analyzed using a Zeiss Axiophot microscope (Zeiss, Oberkochen, Germany)

and a PCO digital camera. Eight fields per animal were digitized (100 �),

excluding fibrotic areas ascertained by HES staining of parallel sections.

Images were then analyzed with custom software run on a Linux-Mandrake

8.2 workstation.In vivo curative and preventive treatment. Human tumors were

induced by s.c. injection of 4 � 106 MDA-MB-231 or 5 � 106 PC-3 cells into

the dorsa of nude mice. When tumors had reached 3 to 4 mm in diameter,

recombinant adenoviruses (n = 6 for each group) were injected into tumors[2 � 109 plaque-forming unit (pfu) per injection, for a total of two injections

separated by a 48-hour interval].

Adenoviruses (5 � 109 pfu) were injected systemically 24 hours before

s.c. implantation of 4 � 106 MDA-MB-231 cells (n = 8 for AdCO1, n = 11 forAdK1-3 and AdK1-3HSA, n = 12 for AdCan, and n = 15 for AdCanHSA).

For each experiment, blood samples were collected at days 8, 15, and 20

to quantify antiangiogenic proteins by ELISA.Immunohistology and apoptotic cell quantification (terminal

deoxyribonucleotidyl transferase–mediated dUTP nick end labeling).An in situ cell death detection kit employing the terminal deoxyribonu-

cleotidyl transferase–mediated dUTP nick end labeling method (TUNEL;

Roche Applied Science, Basel, Switzerland) was used to detect apoptoticcells within paraffin sections. Five fields per animal were digitized (200 �).

The number of stained cells per field was counted and mean values with

their SD were calculated.

Immunofluorescence. HUVEC and MDA-MB-231 cells were infectedwith adenoviruses 24 hours following seeding of cells. Forty-eight hours

later, cells were washed, fixed in PBS/3% formaldehyde, washed twice with

PBS, and incubated overnight at 4jC in PBS/0.5% FBS. CanHSA and K1-

3HSA proteins were detected using the mouse anti-HSA mAb describedabove. After washing thrice with PBS/0.5% FBS, cells were incubated with a

fluorescein-conjugated secondary antibody (Jackson, West Grove, PA; ref.

111-095-144).

Flow cytometry. For flow cytometric analysis, cells were harvestedfollowing adenoviral infection [coupled, or not, with small inhibitory RNA

(siRNA) transfection] for 48 hours. Cells were stained with fluorescein-

conjugated mAbs (10 Ag/mL) directed against specific integrins: LM609(anti-avh3), P1F6 (anti-avh5), and AMF7 (anti-av). Normal mouse

fluorescein-conjugated IgG1 was used as a negative control antibody.

For cell cycle studies, HUVEC and MDA-MB-231 cells were collected at

96 hours postinfection. Cells were suspended in PBS/0.1% glucose and thenfixed with 70% ethanol. After centrifugation, the supernatants were

discarded and cellular DNA was stained in a solution containing 100 Ag/mL

of RNase A and 50 Ag/mL of propidium iodide.

Determination of caspase-3, -8, and -9 activities. HUVEC, MDA-MB-231, and PC-3 cells were harvested 72 hours after infection with adenovirus,

with or without caspase-3 and -9 inhibitors, during the infection period

(R&D Systems, Minneapolis, MN; ref. FMK004, FMK008). Caspase-3, -8, and -9activities were determined by caspase-3, -8, and -9 colorimetric assays (R&D

Systems). Staurosporine, which induces the mitochondrial caspase-dependent

apoptotic pathway, was used as an apoptotic positive control (50 nmol/L;

Oncogene, Darmstadt, Germany).Av, B3, and B5 integrin gene silencing in HUVEC cells. siRNAs were

purchased as designed, synthesized, purified, and annealed oligos from

Ambion (Huntingdon, United Kingdom). The target sequence names for

human h3, h5, and av integrins are ITGB3_1, ITGB5_1, and ITGAV_1. Cells at50% confluence were treated with 50 nmol/L siRNA in Oligofectamine

reagent (Invitrogen) according to the instructions of the manufacturer. A

nonsilencing siRNA (Ambion), transfected under the same conditions asanti-integrin siRNAs, was used as control. Four hours following siRNA

transfection, cells were infected with AdCO1, AdK1-3HSA, or AdCanHSA.

Expression of integrins was analyzed by flow cytometry 48 hours after

adenoviral transduction as described above. Caspase-8 and -9 activities weredetermined following infection of cells for 72 hours as described above.

Statistical analysis. Each experiment was done twice. Statistical

analysis was done using the Student’s t test (unilateral and unpaired).

Results

In vitro and in vivo characterization of recombinantadenoviruses encoding canstatin, CanHSA, K1-3, and K1-3HSA. Experiments were done to verify expression and secretion ofcanstatin and CanHSA in vitro and in vivo.In vitro , each protein was detected at the expected MW of 24

and 89 kDa, respectively, from AdCan- and AdCanHSA-infected cellsupernatants at 96 hours postinfection (Fig. 1A , 1). No signal wasobserved from AdCO1-infected supernatants. Similar experimentswere carried out for AdK1-3 and AdK1-3HSA (Fig. 1A , 3). Therewas judged to be no quantitative difference in the maximal levelexpression of all four proteins through the hybridization signal tothe shared HSA tag (Fig. 1A , 2).In vivo , the circulating levels of expressed proteins in blood

samples of MDA-MB-231 xenografted mice, following intra-tumoral or systemic injection of AdCanHSA and AdK1-3HSA,were quantified by ELISA (Fig. 1B). CanHSA and K1-3HSAshowed a similar expression pattern in the sera of mice afterboth local and systemic adenoviral injection.

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Cancer Res 2005; 65: (10). May 15, 2005 4354 www.aacrjournals.org



To determine whether fusion with HSA affects the in vivostability of canstatin, Western blot analysis of MDA-MB-231 tumorprotein extracts was done at days 2, 5, and 9 following intra-tumoral injection of AdCan, AdCanHSA, or AdCO1. CanHSA wasthe only protein for which a consistent level of expression wasdetected for 9 days. No signal was observed for unfused canstatinat day 9 (Fig. 1C). Thus, fusion with HSA improves the intra-tumoral bioavailability of canstatin.Canstatin has a more prominent antiangiogenic effect than

angiostatin. The proliferation of HUVEC cells was more stronglyinhibited by AdCan or AdCanHSA (60% on average) than by AdK1-3 or AdK1-3HSA (40% on average; P = 10�6 as compared withAdCO1). Following in vitro infection at different multiplicities ofinfection, canstatin exhibited a dose-dependent inhibition ofendothelial cell proliferation (data not shown). Similarly, canstatinand CanHSA arrested the migration of HUVEC cells (80%; P = 0.02as compared with AdCO1) more strongly than observed with K1-3or K1-3HSA (not significant; Fig. 2A).Having shown in vitro that the inherent activities of canstatin

and K1-3 were not disrupted by fusion with HSA (Fig. 2A), theirantiangiogenic effects were quantified in vivo by anti-CD31 immunohistochemistry following injection of virusesinto PC-3 tumors. As shown in Fig. 2B , the level of intratumoralvascularization was reduced following canstatin treatment. Thethickness of capillary microvessel walls in the K1-3–treated groupwas thinner than those observed in the CO1-treated group.

However, microvessels remained numerous and relatively thick incontrast to the vessels observed in the canstatin-treated samples.More precisely, injections of AdCan and AdCanHSA decreased thearea of vascularization (88% on average; P < 10�6 as comparedwith AdCO1). The results are also significant as compared with theangiostatin-treated group (P < 0.01).All tumor vessels are not equal in their ability to provide oxygen

and nutrients to the tumor cells they support (18). Thequantification of intratumoral vessels by contrast-enhanced colorDoppler high-frequency ultrasonography analysis enabled us tofollow the efficacy of treatment over the treatment period byfocusing specifically on the functional vessels in the tumor. At theend of the treatment period (day 42), capillary vessels wereobserved to invade tumors only in the AdCO1-, AdK1-3–, andAdK1-3HSA–treated groups. No intratumoral vessels were detectedfollowing injection of AdCan and AdCanHSA (Fig. 2C). Thethickness of vessel walls (CD31 staining) treated with K1-3 orK1-3HSA at day 8 was thinner by visual inspection than for theCO1 slice (Fig. 2B), which, however, seemed to contain as manyfunctional vessels as the thicker nontreated vessels (Fig. 2C).Interestingly, CanHSA induced a strong and long-lasting inhibitionof the penetration of functional vessels into the tumor mass ascompared with unfused canstatin (90% and 60%, respectively; P <0.01 as compared with AdCO1; Fig. 2C).Thus, unfused canstatin has potent antiangiogenic features

in vitro and in vivo , but we show its fusion with HSA undisputably

Figure 1. Assessment of the expressionlevel in vitro and in vivo of canstatin,CanHSA, K1-3, and K1-3HSA proteins.A, Western blotting of adenoviral infectedMDA-MB-231 cell culture supernatantsconfirms that expression levels ofcanstatin, CanHSA, K1-3, and K1-3HSAare similar. B, quantification of CanHSAand K1-3HSA by ELISA against theHSA moiety of the fusion proteins in thesera of mice at several time pointsfollowing two intratumoral injections of theappropriate adenovirus (1) or systemicinjection (2). C, Western blotting ofprotein extracts of adenoviral-infectedxenografted MDA-MB-231 tumors showthe increased in vivo stability of CanHSA(1 ; for 9 days) as compared withunfused canstatin (2).

Efficacy of Canstatin and Mechanism of Action




improves its efficacy in vivo in inhibiting intratumoral vasculari-zation in the weeks following treatment.CanHSA exhibits a stronger in vivo antitumor effect than

canstatin, K1-3, or K1-3HSA. To evaluate whether the strongantiangiogenic activity of CanHSA was sufficient to broadly affecttumor viability, we followed the growth of xenografted tumor cellsafter curative and prophylactic injection of the recombinantadenoviruses.Adenoviruses were injected directly into MDA-MB-231 xeno-

grafted tumors. Following expression of canstatin and CanHSA,tumor growth was inhibited by 40% (not significant) and 98% (P <0.003), respectively, as compared with AdCO1. Moreover, CanHSAexhibited better efficacy with 20% total tumor regression thanAdCO1- or AdCan-treated group. Similarly, PC-3 tumor growth wasinhibited by CanHSA with better efficacy as compared withcanstatin and CO1 (72%; P = 10�4 at day 19). The antitumor effectof CanHSA is still significant as compared with K1-3 or K1-3HSA(P < 0.04) for both tumor models (Fig. 3A).To evaluate the efficacy of CanHSA in a prophylactic protocol, we

systemically injected each recombinant virus 24 hours before s.c.injection of MDA-MB-231 cells in mice. After 8 weeks of treatment,77% inhibition of tumor growth was observed with CanHSA ascompared with CO1- (P = 0.003) and canstatin-treated groups (P =0.01; Fig. 3A). These results were also significant as compared withK1-3 (P = 0.005) and K1-3HSA (P = 0.006; data not shown).The long-lasting effect of CanHSA in inhibiting tumor penetra-

tion by intratumoral vessels is also correlated with the stronger

antitumor effect of the CanHSA conjugate as compared with thatof free canstatin in vivo .CanHSA induces an apoptotic mechanism in both endothe-

lial and tumoral cells. We showed in situ by TUNEL analysis thatCanHSA induced tumor cell apoptosis in MDA-MB-231 xenograftson nude mice (Fig. 3B). The results showed a marked increaseof apoptotic cells in the AdCanHSA-injected tumors 30 dayspostinfection (11 F 4 cells/field, P < 10�5, for CanHSA as comparedwith 1.7 F 0.9 cells/field observed for the AdCO1-treated group).The results obtained for CanHSA are again significant whencompared with those obtained for the angiostatin-treated group(P < 10�4) and the canstatin-treated group (P < 10�3).To determine whether CanHSA acts directly against tumor cells,

or indirectly by inducing tumor hypoxia, nuclear DNA fragmen-tation, a hallmark of cellular apoptosis, was assessed by propidiumiodide staining of AdCanHSA-infected MDA-MB-231 or HUVECcells. CanHSA induced apoptosis in 31% of tumor cells whereas noapoptotic cells were observed after treatment with AdK1-3HSA orAdCO1 (Fig. 3C). Moreover, CanHSA treatment leads to apoptosisin 42% of the endothelial cells whereas only 17% of K1-3HSA–treated cells were observed in apoptotic (sub-G1) phase. Noapoptotic cells were detected in the CO1-treated population.The strong antiangiogenic and antitumor effects of canstatin

seem to be due to a direct apoptotic mechanism in endothelial andtumoral cells.CanHSA interacts with integrins on the endothelial and

tumor cell surface. To determine whether CanHSA exhibited a

Figure 2. Characterization of the in vitroand in vivo antiangiogenic effects ofcanstatin and CanHSA. A, canstatin- andCanHSA-induced inhibition of HUVECproliferation (1) and migration (2)compared with that of K1-3 andK1-3HSA. Endothelial cell viability wasassessed at 96 hours postinfection by3-(4 c,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Percentage inhibition was calculated withreference to the control adenovirus(AdCO1). Representative of two separateexperiments. B, tumor vessel endothelialcells (arrows ) were immunostained with amurine anti-CD31 antibody followingintratumoral injection of AdCO1- (1 ),AdK1-3– (2), AdK1-3HSA– (3), AdCan-(4 ), or AdCanHSA-injected (5 )PC-3–derived xenografted tumors (100�).6, columns, mean of the area of tumorvascularization for each animal; bars, SD.C, color Doppler high-frequencyultrasonography of MDA-MB-231–derivedtumors (boxed ), following intratumoralinjection of the adenoviruses, listed above,in living mice was used to estimate thenumber of functional intratumoral capillaryvessels (white arrows ) once per weekfor 6 weeks (1 ). Columns, mean number ofvessels for each group of five mice for agiven observation point (week); bars, SD.Representative images of K1-3HSA (2)and CanHSA (3) at day 42 (other data arenot shown). Color Doppler high-frequencyultrasonography was not done for PC-3–derived tumors due to the aggressiveevolution of this tumor (with respect to theslower growing MDA-MB-231–derivedtumors), which prevented pragmaticobservation of functional intratumoralcapillary vessels over the short tumorgrowth period.

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direct antitumor effect, we infected MDA-MB-231 and HUVEC cellsin vitro with adenoviruses. Then, cells were fixed, but notpermeabilized, and the presence of CanHSA and K1-3HSA wasdetected by immunostaining. Endothelial cells infected byAdCanHSA or AdK1-3HSA were clearly labeled compared withAdCO1-infected HUVEC cells (Fig. 4A). On the contrary, only MDA-MB-231 cells infected by AdCanHSA were labeled. Thus, CanHSAseems to localize at the extracellular surface of both HUVEC andMDA-MB-231 cells.Endothelial cell migration and adhesion to the ECM are

mediated by integrins, especially avh3 and avh5 (19, 20), whichalso play a role in tumor progression (21). To identify thereceptor that binds CanHSA, fluorescence-activated cell sorting

(FACS) analysis was done using fluorescein-conjugated anti-avh3

or anti-avh5 mAbs on AdCO1- or AdCanHSA-infected cells.Initially, we characterized the presence of avh3 and avh5 onHUVEC and MDA-MB-231 cells following infection with mockadenovirus. avh3 and avh5 integrins were detected at highlevels on endothelial cells. MDA-MB-231 cells express a lowlevel of avh3 and a high level of avh5, supporting the results ofWong et al. (21). After expression of CanHSA by infected cells,we repeated the FACS analysis to assess the level of integrinsbound to CanHSA, which would be inaccessible to anti-integrinsmAbs. Fifty percent to 66% of the av, h3, or h5 integrins on theHUVEC and MDA-MB-231 cells were not bound to mAbs afterexpression of CanHSA (Fig. 4B). On the contrary, expression of

Figure 3. Tumor growth inhibition and detection of apoptosis following infection with AdCan and AdCanHSA. A, MDA-MB-231– (1 ) or PC-3–derived (2 ) tumorsize on nude mice, following two intratumoral injections of the adenoviruses listed above, was measured once a week. Points, mean of six mice ascompared with the AdCO1-treated group; bars, SE. Asterisks, statistical significance. 3, inhibition of tumor growth by CanHSA in comparison with thecanstatin-, K1-3–, and K1-3HSA–treated groups highlights significant tumor regression mediated by CanHSA as compared with other treatment options versusAdCO1. 4, s.c. injected MDA-MB-231–derived tumor growth over f8 weeks following systemic injection of AdCO1, AdCan, and AdCanHSA. B, TUNELstaining was done on adenoviral-treated MDA-MB-231 tumors (day 30 postinfection) as previously described in A1 . The proportion of apoptotic cells (arrows )per field (five fields per animal) were quantified within sections (200�) from AdCO1-, AdK1-3–, AdK1-3HSA–, AdCan-, or AdCanHSA-injected tumors.Representative fields of AdCO1 (1), AdK1-3HSA (2), and AdCanHSA (3 ). 4, columns, mean of two mice for each treated group; bars, SD. C, cell cycleanalysis. HUVEC and MDA-MB-231 cells were infected with AdCanHSA (1 and 4 ), AdK1-3HSA (2 and 5), or AdCO1 (3 and 6). At 96 hours postinfection,cells were fixed with 70% ethanol overnight and chromatin was stained with propidium iodide. FACS analysis was done twice for each group.





K1-3HSA impeded binding of anti-avh3 mAb only on the endothelialcell surface. For each cell type, normal fluorescein-conjugatedisotype control antibody IgG1 was used as a negative control,confirming that the observed signals were specific.To confirm that CanHSA binds to avh3 and avh5 integrins at the

cell surface, immunoprecipitations were done from AdCanHSA- or

AdCO1-infected cell lysates withmAbs againstavh3 oravh5.Western

blotting revealed bands characteristic of CanHSA from immuno-precipitated AdCanHSA-infected endothelial and tumor cellular

extracts following hybridization with anti-HSA (data not shown).Taken together, these results show that CanHSA interacts with at

least two kinds of integrins, avh3 and avh5, on the surface of both

human endothelial and tumor cells, whereas K1-3HSA binds avh3

integrins only on the membrane of HUVEC cells. Thus, canstatin

inhibits the broad capacity of endothelial and tumor cells to adhere

and spread by blocking ECM-av-integrin interactions (22).CanHSA-induced apoptosis is sequential to mitochondrial

damage both in endothelial and tumor cells. Antagonists ofavh3 integrins have been shown to participate in inhibition of cell

proliferation and migration as well as apoptosis (23).To determine whether canstatin induces caspase-dependent

apoptotic pathways, caspase-3, -8, and -9 activities in HUVEC and

MDA-MB-231 cellular extracts were quantified. CanHSA treatmentof HUVEC cells led to a 4-fold increase in caspase-9 activity as

compared with CO1-treated cells (P = 0.03). K1-3HSA–treated cells

exhibited no significant activation of caspase-9 (Fig. 5A). Caspase-8 activity was increased less strongly (1.5-fold) by either canstatin

(P = 0.024 as compared with AdCO1) or angiostatin. In addition,

canstatin induced a 3.5-fold increase in caspase-3 activity as

compared with CO1 treatment (P = 0.03), whereas angiostatinincreased this level by only 2.5-fold. Each result was done by

comparison to staurosporine, a known inducer of caspase-

dependent apoptosis. The specificity of caspase cleavage was shownby addition of selective caspase-9 or -3 inhibitors to the cell medium.Our results show that canstatin induced two distinct apoptotic

pathways in endothelial cells through the activation of caspase-8 and caspase-9 leading to cleavage of procaspase-3 (7). K1-3activates only the mitochondrial-independent pathway involvingcleavage of caspase-8 and -3.CanHSA-treated MDA-MB-231 cells showed an activation of the

mitochondrial caspase-9–dependent apoptotic process (increaseof 1.7-fold; P = 0.04 as compared with AdCO1; Fig. 5A).Additionally, PC-3 prostate tumor cells express avh3 and avh5

integrins, and activation of caspase-9 was observed followingCanHSA or staurosporine treatment of PC-3 (P < 0.01 ascompared with AdCO1; Fig. 5A). No CanHSA-induced caspase-8 activation was detected in either tumor cell type (data notshown).Caspase-dependent apoptotic events are mediated by

binding of CanHSA to Av, B3, and B5 integrins. To furthercharacterize the functional receptors mediating the apoptoticsignaling pathways for CanHSA, we used anti-integrin siRNAs todecrease av, h3, or h5 expression in HUVEC cells to inhibit bindingof CanHSA to the cell surface. Incubation of AdCO1-infectedHUVEC cells for 48 hours with anti-h3, anti-h5, or anti-av siRNAsdecreased the level of expression of each integrin population onthe cell surface by 50% on average (Fig. 5B). Identical transfectionwith a random siRNA bearing no homology with any integringenes had no effect on integrin expression.The specific inhibition of av-, h3-, or h5-integrin expression

significantly reduced caspase-9 activity following CanHSA treat-ment (decrease of 1.8- to 3.8-fold; P < 0.01 as compared withtransfection of siRNA control oligo coupled with AdCanHSAinfection; Fig. 5C).As previously shown, K1-3HSA increases caspase-8 activity alone

(by 2-fold; Fig. 5A). Inhibition of av- or h3-integrin expressionimpeded the activation of caspase-8 following K1-3HSA, but notCanHSA, infection (Fig. 5D). Thus, binding of K1-3HSA with avh3

integrins induces a caspase-8–dependent apoptotic pathway,whereas interaction of CanHSA with avh3 and avh5 integrinsselectively triggers the caspase-9–dependent pathway.

Figure 4. Determination of CanHSA binding to HUVEC and MDA-MB-231 cell surface. A, HUVEC and MDA-MB-231 cells were infected with AdCanHSA(1 and 2), AdK1-3HSA (3 and 4), or AdCO1 (5 and 6) at 800 pfu for 48 hours. Cells were fixed but not permeabilized and incubated with a mouse anti-HSAmAb followed by an anti-mouse FITC-labeled antibody. Note the localization of CanHSA at the surface of HUVEC cells (1 ) as well as tumor cells (2). Eachexperiment was done twice. B, to determine which av integrins are associated with CanHSA or K1-3HSA, FACS competition analysis was done on AdCO1- (blue line ),AdCanHSA- (green line ), or AdK1-3HSA–infected (orange line ) HUVEC (1 -4) or MDA-MB-231 (5 and 6) cells using anti-avh3 (1 , 3 , and 5 ) or anti-avh5 (2 , 4 , and 6 )FITC-conjugated mAbs. FACS analysis included a mouse FITC-conjugated IgG1 as negative control (red line ). Y-axis, events (cell number); X-axis,fluorescence intensity.

Cancer Research




Discussion

The soluble NC1 domain fragment of the a2(IV) chain ofcollagen, called canstatin, is known to potently inhibit tumor-associated angiogenesis (5–7).Initially, we investigated the in vivo antiangiogenic properties

and antitumor efficacy of canstatin using HSA fusion to increasethe bioavailability of canstatin following adenoviral gene transfer.This study sought to evaluate the anticancer efficacy of CanHSAcompared to that of the K1-3HSA as a reference moleculebelonging to the plasminogen-derived class of proteins (10, 11).In vivo , CanHSA strongly inhibited the vascularization of tumorsthrough a stable and long-lasting effect. Our approach provides asolution to maintain an in situ level of canstatin sufficient tostrongly impede the growth of cancer and disrupt implantationof cancer cells and, as such, decrease the number of therapeuticinjections required and thus, potentially, adherence to thetreatment regime. Recently, therapeutic gene transfer becamemore commercially attractive following the debut of adenoviral-mediated p53 gene transfer for head and neck squamouscancers, called Gendicine (Sibiono GeneTech, Shenzhen, China).Next, we attempted to delineate the mechanism of action of

CanHSA to explain its improved efficacy as compared with K1-3HSA. The exact mechanisms by which a(NC1) domains, inparticular canstatin (a2(IV)NC1), inhibit tumor angiogenesis arenot completely understood. We show here that canstatin bindsto the endothelial and tumor cell surface in an integrin-dependent manner. Integrins mediate adhesion to matrixproteins of both endothelial and tumor cells (19–21). Remodelingof the ECM promotes novel integrin-ligand interactions requiredfor angiogenesis (1). As such, CanHSA could compete withcollagen IV ECM for cell surface integrin binding and reverse theproliferative and migratory effects induced by cell-ECM inter-actions. Thus, avh3, and avh5 integrins would seem to mediatethe antiangiogenic and direct antitumor properties of CanHSA.Just two other proteins are known to have a direct effect ontumor cells through interaction with integrins: endostatin (a5h1)and tumstatin (avh3), which are both also collagen-derived NC1domain fragments (24–26). Interestingly, Tarui et al. (27) alsoshowed that K1-3 binds only to avh3 integrins on endothelial cells.Integrin receptors are known to cross-talk with other receptors

and their associated pathways (22). Angiogenesis proceedsthrough two cytokine-dependent pathways: one depending onavh3 that is largely induced by basic fibroblast growth factor andvascular endothelial growth factor (VEGF), and another one whichis potentiated by avh5 and VEGF alone (19, 20). As such, dualantagonists of both avh3 and avh5, such as canstatin, as we haveestablished here, would have a definite therapeutic advantage byblocking the two tumor-induced angiogenesis pathways.Here, we investigated the potential role of integrins as regulators

of apoptosis (23, 28). We have shown that in vitro CanHSA stronglyinhibited the migration and proliferation of HUVEC cells. More-over, our present findings show that these events are mediated byan upstream event involving canstatin binding to avh3 and avh5

integrins, which has not yet been reported. Integrin receptorengagement and subsequent clustering lead to transduction ofpositional cues from the ECM to intracellular signaling phosphor-ylation cascades that promote cell survival, such as the activationof focal adhesion kinase/phosphatidylinositol 3-kinase (FAK/PI3K)pathway (23, 29). Phosphorylated FAK promotes integrin-mediatedcell proliferation and migration events (29). When cell-ECMinteractions are lost, cells undergo a type of apoptosis called

Figure 5. Determination of CanHSA-induced apoptotic pathway and the keyreceptor(s) through siRNA-mediated silencing of av, h3, or h5 expression.A, HUVEC (1 -3), MDA-MB-231 (4), or PC-3 (5) cells were infected with AdCO1,AdK1-3HSA, or AdCanHSA for 48 and 72 hours, respectively. Whole cell lysateswere mixed with reaction buffer and caspase colorimetric substrate asrecommended by R&D Systems. A Bradford assay was done to normalizeabsorbances. Each experiment was done thrice. Columns, mean; bars, SD. Toshow expression of the canstatin-adhesive proteins, avh3 and avh5 integrins,on the PC-3 cell surface, FACS analysis was done on noninfected PC-3 cells(blue line ) using anti-avh3 (6) or anti-avh5 (7) FITC-conjugated mAbs ascompared with IgG1 FITC-conjugated antibody (red line ). B, to assess thesuppression of integrin expression using siRNA, FACS analysis was done onAdCO1-infected HUVEC cells which were transfected with random siRNA(1-3 ; blue line ), or h3 (1 ), h5 (2), or av siRNA (3 ; green line ). A mouseFITC-conjugated IgG1 was included as a negative control (red line ). Y-axis,events (cell number); X-axis, fluorescence intensity. To determine the integrinreceptor(s) that promote caspase-9 (C) or caspase-8 (D) activities followingCanHSA infection, HUVEC cells were pretransfected with each siRNA andthen infected with either AdCO1, AdK1-3HSA, or AdCanHSA for 72 hours.Caspase activities were quantified as described previously in A . Eachexperiment was done twice.





anoıkis (30). The FAK pathway is activated by most integrins.Although both endostatin and tumstatin display differentialintegrin binding characteristics, they both inhibit phosphorylationof FAK, thus leading individually to distinct downstream signalingevents (24). Recently, Panka and Meier (7) have shown thatcanstatin inhibits the phosphorylation of FAK, interfering withdownstream signaling events. Collectively, these data suggest thatthe binding of canstatin to avh3 and avh5 integrins, shown here,could mediate FAK inactivation, leading to suppression of cellspreading and migration and thus apoptosis.To further document endothelial cell-specific apoptosis, our

results show that canstatin induces a profound activation of

caspase-9 as compared with K1-3 treatment of cells. This pathwayis triggered through both avh3 and avh5 integrins. Both canstatinand K1-3 induce the mitochondria-independent pathway with asimilar increase in caspase-8 activity. Our findings show thatcanstatin initiates two apoptotic pathways including activation ofcaspase-8 and -9, both initiators of the downstream apoptoticprocess leading to activation of caspase-3. Canstatin-activatedcaspase-8, by down-regulation of Flip levels and up-regulation ofFas/Fas ligand, triggers not only cell death directly throughcaspase-3 activation but also indirectly through mitochondrialdamage via activation of caspase-9 within the apoptosome (7,31, 32). On the other hand, phosphorylated FAK/PI3K is known toinactivate the mitochondrial apoptotic pathway by inhibition ofcaspase-9 (30). So, canstatin directly activates procaspase-9through inhibition of the FAK/PI3K pathway and amplifies theFas-dependent pathway in mitochondria (ref. 29; Fig. 6).This dual mechanism explains how canstatin strongly enhances

caspase-9 activity and therefore the number of cells enteringapoptosis. Mitochondrial disruption and caspase-9 activation canengage in a circular self-amplification loop that accelerates theapoptotic process. Mitochondrial damage seems to be the criticalpoint of no return of the cell death process (33).Several studies have shown that angiostatin inhibits endothelial

cell function by increasing apoptosis (11, 15, 16). However, themoderate reduction in K1-3–treated endothelial cell number seenin our proliferation assays is due to a single activation of caspase-8 without mitochondrial damage following specific interactionof K1-3HSA with avh3. Recently a study has described thatunligated avh3 integrins (but not h5) or soluble antagonists of thisreceptor initiate endothelial apoptosis, called integrin-mediateddeath, by recruitment of caspase-8 to the plasma membraneindependent of interaction of Fas/Fas ligand with the deathdomain (34). However, CanHSA-induced caspase-8 activity was notdisrupted following av or h3 gene silencing, as we have establishedfollowing treatment with K1-3HSA. In this case, canstatin wouldact only via one of two possible mechanisms: Fas-mediated path-way in caspase-8 activity; the second possible mechanism (i.e.,integrin-mediated death leading to caspase-8 activity) beingabrogated due to siRNA knockdown of avh3 (Fig. 6).Collectively, it would seem that the involvement of distinct

apoptotic pathways is correlated with profound cell death and thusa strong antitumor effect.In our study, we report that canstatin inhibits tumor develop-

ment in vivo and enhances apoptotic induction in tumor cellsthrough generation of active caspase-9. No caspase-dependentapoptotic mechanism was observed in K1-3–treated tumor cells, asdescribed previously by Lucas et al. As confirmed by other authors,fragments derived from NC1 domains, like tumstatin, could act asa specific antitumor antagonist of avh3 integrins in melanomaprogression (26).In conclusion, CanHSA is a powerful anticancer molecule which

triggers two distinct programmed cell death pathways in endothe-lial cells which converge on mitochondrial disruption: the av-integrin-FAK/PI3K/caspase-9 pathway and the Fas/Fas ligand/caspase-8/caspase-9 cascade. These two pathways result inamplification of mitochondrial-linked apoptotic events and activa-tion of caspase-3, the central executioner of the apoptotic process.Our study argues for the delivery in situ of therapeutic gene

with mitochondrial-dependent proapoptotic properties to im-prove antiangiogenic and antitumor features of such selectivedual avh3 and avh5 agonists fused with a carrier protein.

Figure 6. Schematic illustration of distinct signaling pathways induced bycanstatin and angiostatin in endothelial or tumoral cells. A, canstatin bindsto avh3 and avh5, whereas angiostatin (K1-3) binds to avh3 alone on endothelialcell surface. Canstatin initiates two apoptotic pathways, involving activationof caspase-8 and -9, leading to activation of caspase-3. Canstatin activatesprocaspase-9 not only directly through inhibition of the FAK/PI3K/Akt pathway(1) but also by amplifying indirectly the mitochondrial pathway throughFas-dependent caspase-8 activation (2). In contrast, K1-3 binding to avh3 aloneleads to activation of procaspase-8 with no effect on apoptotic mitochondrialevents (3). B, canstatin activates only the mitochondrial pathway in tumoral cells.

Cancer Research




Acknowledgments

Received 9/30/2004; revised 2/10/2005; accepted 3/9/2005.Grant support: Centre National de la Recherche Scientifique, the Institut Gustave

Roussy, the ligue nationale contre le cancer, and the Association pour la Recherche sur

le Cancer. C. Magnon was financed by the Institut National de la Sante et de laRecherche Medicale.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

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In the article on the efficacy of canstatin and mechanism ofaction in the May 15, 2005 issue of Cancer Research (1), CelineBouquet should have appeared in the list of authors. The list ofauthors should have read as the following: Claire Magnon, ArianeGalaup, Brian Mullan, Valerie Rouffiac, Celine Bouquet, Jean-MichelBidart, Frank Griscelli, Paule Opolon, and Michel Perricaudet.Dr. Bouquet’s affiliation is UMR 8121 Laboratoire de vectorologieet transfert de genes, Institut Gustave Roussy, Villejuif cedex,France.

1. Magnon C, Galaup A, Mullan B, Rouffiac V, Bidart JM, Griscelli F, Opolon P,Perricaudet M. Canstatin acts on endothelial and tumor cells via mitochondrialdamage initiated through interaction with avh3 and avh5 integrins. Cancer Res2005;65:4353–61.

www.aacrjournals.org 5989 Cancer Res 2005; 65: (13). July 1, 2005

Correction

2005;65:4353-4361. Cancer Res Claire Magnon, Ariane Galaup, Brian Mullan, et al.

Integrins5βvα and 3βvαMitochondrial Damage Initiated through Interaction with

Canstatin Acts on Endothelial and Tumor Cells via

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