2001 Targeting Tumor Angiogenesis with Gene Therapy

MINIREVIEWTargeting Tumor Angiogenesis with Gene Therapy 1Qing-Rong Chen,Lei Zhang,Warren Gasper,and A.James Mixson 2Department of Pathology,University of Maryland at Baltimore,Baltimore,Maryland 21201Received June 22,2001,and revised form July 17,2001;published online September 17,2001A recent target of cancer gene therapy is tumor angiogenesis.An appealing feature of gene therapy targeting the tumor vasculature is that it is readily accessible,particularly when the carrier and its gene are administered systemically.Several gene-based viral and nonviral therapies that target tu-mor angiogenesis have demonstrated the “proof of principle”of antiangiogenic therapy in preclinical models.The utility of antiangiogenic gene therapy in a clinical setting will depend in large part on developing vectors with minimal toxicity and with increased in vivo transfection efficiency.In this re-view,we discuss the current status and future di-rections of antiangiogenic gene therapy.©2001Academic PressKey Words:gene therapy;antiangiogenesis;tu-mor;endothelial cells;VEGF;VEGFR2;angiopoietin 2;nonviral;viral;electroporation.Angiogenesis is a closely regulated physiological process.The human vasculature system of the aver-age person has more than a trillion endothelial cells (1).The lifetime of these normally quiescent cells in vessels surpasses 1000days (2),but once angiogen-esis is induced,endothelial cells can proliferate rap-idly.Except for the corpus luteum,most angiogene-sis in adults occurs in response to pathological insults from wounds or hypoxia (e.g.,stroke and tumors).Tumor growth is dependent on the development of new blood vessels (3).Once the diameter of the tu-mor exceeds 2mm,angiogenesis is required for fur-ther growth (4).Suppression of angiogenic inhibitors together with stimulation of angiogenic growth fac-tors is essential for tumor growth and progression (Fig.1).As a result,therapy targeting tumor vessels attempts either to reduce angiogenic growth factors or to increase angiogenic inhibitors within a tumor (5).Several antiangiogenic proteins/peptides have been shown to be effective in reducing tumor growth.Of particular note,angiostatin and endosta-tin have eliminated tumors in mice completely (6,7).In addition to its antitumor efficacy,an appealing feature of antiangiogenic therapy is that it may avoid the problem of acquired resistance to antican-cer drugs since endothelial cells of tumors have rel-ative genomic stability (8,9).Gene therapy is an attractive means of delivery-ing these antiangiogenic proteins to the tumor site.For most gene therapy approaches,the tumor vas-culature represents an obstacle for therapy to reach tumor cells.The tumor vessels,however,are readily accessible targets for antiangiogenic gene therapy,particularly when it is administered systemically.There are now many studies demonstrating some efficacy of antiangiogenic gene therapy in tumor-bearing animal models.Investigators have success-fully inhibited tumor growth by gene therapy ap-proaches designed to inhibit angiogenic inducers (VEGF,angiopoietin 2)and/or their receptors (10–13).Furthermore,gene therapy with angiogenic in-hibitors has also shown promise with tumor inhibi-tion in animal models (14–16)(Fig.2).Nevertheless,1Supported by the National Institutes of Health (CA70394).2To whom request for reprints should be addressed at Depart-ment of Pathology,University of Maryland at Baltimore,Room 7-59,Building-MSTF,10S.Pine Street,Baltimore,MD 21201.Fax:(410)706-8414.Molecular Genetics and Metabolism 74,120–127(2001)doi:10.1006/mgme.2001.3223,available online at on1201096-7192/01$35.00Copyright ©2001by Academic PressAll rights of reproduction in any form reserved.in vivo gene delivery carriers need improvement before these therapies are clinically useful.Although antiangiogenic gene therapy is an at-tractive gene therapy approach,is this approach more effective than directly injecting the peptide/protein?Several studies with recombinant antian-giogenic proteins have demonstrated marked re-gression of large tumors (6,7).Similar findings,to date,have not been reported with antiangiogenic gene therapy.Nevertheless,there are potential ad-vantages of gene delivery systems for these antian-giogenic proteins.First,gene therapy delivery sys-tems may circumvent the problems of adequate production of certain antiangiogenic proteins (e.g.,angiostatin or endostatin).Adequate production of these proteins by recombinant engineering methods has been problematic and may limit widespread ad-ministration of these proteins.Second,gene therapy can deliver the expressed proteins to the nucleus,cytosol,membrane,or extracellular space.With cur-rent technology,certain antiangiogenic proteins (e.g.,the transmembrane protein,VEGFR2-DN)are only effective if delivered by a gene therapy ap-proach.Thus,gene therapy expands the repertoire of proteins that can be utilized for antiangiogenic therapy.Third,if side effects from the antiangio-genic therapy occur,targeting the therapy to the tumor vessels would be critical.Although significant issues remain to be addressed concerning specificity of ligands for tumor vessels,targeting the tumor vessels with a gene delivery system is much more practical than peptide delivery systems.Because of these problems of protein delivery systems coupled with the improvement of gene delivery systems,we believe that continued investment of time and effort is warranted for the antiangiogenic gene therapy approach.Most studies of antiangiogenic gene therapy have shown their efficacy in preclinical models.Only p53and IL-12,two proteins with multiple tumor inhib-itory mechanisms in addition to antiangiogenesis,have progressed to clinical trial.In subsequent sec-tions,we review the various gene therapy targets and/or the genes encoding antiangiogenic proteins that investigators have used.VEGFVEGF and its cognate receptors (flt-1,or VEGFR1,flk-1or VEGFR2)are essential for tumor angiogen-esis.As a result,it is not surprising that these have frequently been targeted to limit tumor growth.An early study showing the utility ofantiangiogenicFIG.1.The endogenous regulators of tumor angiogenesis:The relative balance of activators and inhibitors of angiogenesis is important to maintain tumors in a quiescent state.Either reducing the inhibitor concentration or increasing the activator level can alter the balance and lead to the tumor angiogenesis and tumor growth.121TUMOR ANGIOGENESIS AND GENE THERAPYgene therapy targeted the VEGF receptor (VEGFR2).In this investigation,injection of a ret-rovirus-truncated VEGFR2construct together with tumor cells resulted in the inhibition of tumor growth (10).It has been suggested that this retrovi-rus-transduced receptor acts through a dominant-negative mechanism to inhibit the wild-type VEGF receptor.This study not only illustrated the poten-tial of this gene therapy approach but also confirmed the essential role of VEGF in tumor development.More recently,antiangiogenic therapy with the truncated VEGF receptor has been shown to sup-press angiogenesis and growth of an intracerebral glioma in a rat model (17).Consequently,rats with these intracerebral tumors survived significantly longer with treatment.In these two studies,the truncated VEGFR2inactivated VEGF-mediated sig-nal transduction by dimerizing with the wild-type receptor of the transfected cell.However,the clinical utility of this method may be limited owing to the low transfection efficiencies of the vector delivery systems injected directly into a tumor.One ap-proach to circumvent the low transfection efficiency of vector delivery systems is to engineer a secreted therapeutic protein.This was demonstrated in a mouse model where tumor cells transduced with a secreted soluble VEGF receptor (sVEGFR1)inhib-ited implantation of a primary tumor (13).Further-more,the mice displayed fewer lung metastases when tumor cells expressing elevated levels of sVEGFR1were injected intravenously.Alternatively,antisense strategies targetingVEGF or its receptor have shown efficacy in vivo .For example,stable transfection of tumor cells with plasmids expressing antisense VEGF did not affect the growth of tumor cells in vitro,but reduced tumor growth in vivo (11).Targeting VEGF with a retrovi-rus-producing antisense RNA also prolonged sur-vival in a glioma-bearing rat model (18).VEGF lev-els may also be decreased indirectly through inhibition of the adenosine 2B receptor with an an-tisense oligonucleotide (19).The major difficulty of targeting VEGF in vivo with antisense approaches either directly or indirectly is that if most of the VEGF is expressed in tumor cells and not in the tumor endothelial cells,it may be difficult to achieve adequate expression of the antisense construct.Re-cently,a ribozyme targeting VEGFR1,upregulated in mitogenic endothelial cells,has been reported to have significant antitumor activity in animal models (20).Angiopoietin 2In addition to VEGF,other ligands with receptors located specifically on endothelial cells have been targeted with antiangiogenic gene therapy.In-creased levels of angiopoietin 2,the ligand to the Tie2receptor,in concert with VEGF have been as-sociated with increased tumor angiogenesis.As a result,the ligand angiopoietin 2is a reasonable therapeutic target.An adenovirus expressing a sol-uble Tie 2receptor was recently shown to inhibit tumor growth by binding to angiopoietin 2when injected intravenously (12).With plasmaconcentra-FIG.2.Approaches of antiangiogenic gene therapy:Antiangiogenic gene therapy approaches can be categorized into those that suppress the angiogenic activators and those that enhance the angiogenic inhibitors.122CHEN ET AL.tions of the soluble recombinant Tie2receptor ex-ceeding1mg/ml for an8-day period in this study, established tumors treated by the intravenous in-jectable route were significantly reduced.In addi-tion,coadministration of the adenovirus-Tie2vector with tumor cells greatly reduced the number of lung metastases.A potential disadvantage of this ther-apy is that the adenovirus-Tie2construct may also inactivate angiopoietin1,a protein known primarily for its ability to stabilize blood vessels.CytokinesAlthough earlier in vivo antiangiogenic gene ther-apy experiments had demonstrated antitumor effi-cacy,gene therapy with the angiogenic inhibitor, platelet factor4(PF-4),was thefirst to show efficacy in a clinically relevant model.In this study,an ad-enovirus expressing PF-4(21)was injected into an established tumor and found to inhibit tumor growth of gliomas and prolong animal survival.PF-4 belongs to a family of C-X-C chemokines and may inhibit angiogenesis by altering the affinity of VEGF to its receptors(22).In addition to PF-4,other cytokines including IL-12and IP-10inhibit tumor growth by an antian-giogenic mechanism(23,24).IL-12can inhibit angio-genesis and reduce tumor growth(25),and this an-giostatic response is mediated by interferon-␥(IFN-␥)and IP-10(26,27).Mice receiving IL-12gene therapy were found to survive significantly longer (median survival,43days)than mice treated with control plasmid/lipid complexes(median survival of 35days).These data demonstrated that a nonviral IL-12gene therapy with a cationic liposome carrier could inhibit the development of lung metastases (28).IL-12may mediate its antiangiogenic effect through immune effector cells(29).In addition to its angiostatic mechanism of action,IL-12probably in-hibits tumor growth by several other mechanisms, and it is difficult to distinguish the relative roles of these mechanisms in reducing tumor growth. Angiostatin and EndostatinUnlike the inhibitors that target VEGF and its receptors,the specific molecular targets for the an-giogenic inhibitors,angiostatin and endostatin, have not been clearly identified.Nevertheless,pre-liminary data suggest that tropomyosin binds to endostatin and plays a role in its tumor inhibitory function(30),and it has recently been found that angiostatin binds tightly to an ATP synthase on the endothelial cell surface(31).Both angiostatin and endostatin polypeptides markedly inhibited a vari-ety of tumors in animal models(6,7).Moreover, there has been no evidence of resistance developing to endostatin in these tumor-bearing mice.Identifi-cation of the receptors for endostatin and angiosta-tin may lead to more potent antiangiogenic analogs. Several gene therapy studies with angiostatin and/or endostatin have shown antitumor efficacy in animal models(15,16,32–38).For the most part,pro-teins encoded by genes have been delivered intratu-morally or intravenously by adenoviruses to inhibit tumor growth.However,nonviral delivery via poly-vinylpyrrolidione(PVP),liposomes,or“naked”DNA injection has also demonstrated efficacy.With“na-ked”DNA intratumoral injections of endostatin not only were the tumors decreased as expected but interestingly,VEGF levels increased intratumor-ally.Thus,there appeared to be a compensatory increase in VEGF levels when endostatin was ad-ministered in this manner.Most of these preclinical studies have reported no toxicity with delivery of these peptides by gene therapy.In one study,how-ever,an adenoviral construct expressing endostatin or a fusion IgG/endostatin protein at high doses was associated with severe toxicity in recipient mice, including loss of weight,bleeding,and death(38). Although toxicity was not observed with injection of a control adenovirus that did not contain the en-dostatin,a control adenovirus expressing a protein other than endostatin was not included in these studies.It is possible that nonviral vectors such as cationic liposomes administered intravenously can achieve higher selectivity and less toxicity than their viral counterparts.In one preclinical study with cationic liposomes as a carrier,moderate reduction in the tumor size of mice bearing MDA-MB-435cells was observed after the third injection(15).Thurston et al.demonstrated that liposome:DNA complexes se-lectively target the mitogenic tumor endothelial cells rather than quiescent endothelial cells(39). Adding ligands that specifically target the tumor endothelial cells may further increase the specificity and efficacy of cationic liposomes(40,41).An alternative gene therapy strategy for increas-ing angiostatin levels in tumors was recently dem-onstrated by administering the gene encoding pan-creatic elastase I(42).Endothelial cells transduced with elastase showed marked growth inhibition in vitro,but the growth of transduced Lewis lung car-cinoma cells in vitro was not inhibited.However,123TUMOR ANGIOGENESIS AND GENE THERAPYLewis lung cells transduced with elastase and then implanted into mice showed reduced growth com-pared to controls.Increased levels of the antiangio-genic angiostatin,a breakdown product of plasmin-ogen,is the putative mechanism of elastase gene therapy.The Tumor Suppressor,p53In contrast to the preceding angiogenic inhibitors, which are secreted,p53is a nuclear transcriptional protein.Nevertheless,p53may induce secreted an-tiangiogenic proteins or may act directly with the cell cycle of transfected cells(e.g.,endothelial cells). Gene therapy with p53is one of the few gene ther-apy approaches showing efficacy in humans,but initially the antiangiogenic mechanism of p53was not considered(43).In one study,a retroviral vector containing the wild-type p53gene was used to trans-fer p53into human nonsmall cell lung cancers by direct injection(43).Of the nine patients with widely metastatic disease who were treated,tumors regressed in three patients and stabilized in two other patients.Some injected tumors regressed com-pletely despite a transfection efficiency of only20%. Thus,the authors concluded that apoptosis or cell cycle inhibition of the transfected tumor cell could not account for the amount of tumor inhibition ob-served.As a result,it is likely that there may be addi-tional mechanisms to explain tumor inhibition with p53gene therapy.A bystander effect has been sug-gested for the observed difference between the rela-tively low transfection efficiency of the tumors with p53and the reduction of tumor growth,and this effect appears to be due in part to inhibition of angiogenesis(33,44–46).We determined that lipo-somes in complex with a plasmid encoding p53in-hibited tumor growth in a mouse model(47),and subsequently found that blood vessel density was reduced with this therapy(44).Other studies with liposomes carrying p53plasmids have also shown reduction in tumor growth in mouse models (33,48,49).Besides liposomes,adenoviral and retro-viral carriers expressing the p53gene significantly reduced tumor growth and blood vessel density (45,46).Moreover,adenoviral vectors expressing p53significantly reduced VEGF levels within tu-mors(45).P53may also decrease angiogenesis by inhibiting the cell cycle and/or enhancing apoptosis of the endothelial cells(44),inhibiting differentia-tion of endothelial cells(46),or by inducing the antiangiogenic protein,thrombospondin1(33,50).ThrombospondinDameron et al.(51)first identified the binding site on the promoter of thrombospondin by which p53 induces thrombospondin,a secreted protein that po-tently inhibits angiogenesis.Similar to its inducer p53,thrombospondin can be an important prognos-tic indicator.Decreased thrombospondin-1(TSP-1) secreted by a variety of cell lines correlates with a more malignant phenotype.Indeed,the MDA-MB-435cell line,which is a very aggressive and meta-static breast cancer line in nude mice,secretes low levels of thrombospondin1when compared to less malignant breast cell lines tested(52).The antian-giogenic effects of TSP-1are mediated by the CD36 surface receptor.In vitro experiments in which TSP-1was used to reduce endothelial cell growth gave conflicting results.Nonetheless,peptides iso-lated from TSP-1clearly have antiangiogenic effects in vitro(53),but surprisingly,there have been no published reports demonstrating that these natural peptides have antitumor activity in vivo.It is un-clear if the reduced functional activity of these pep-tides in vivo is due to a short half-life.However, synthetic antiangiogenic thrombospondin peptides in the D-conformation do exhibit in vivo antitumor activity(54).In contrast to peptide therapy,three studies indi-cated that antiangiogenic gene therapy with TSP-1 or peptides derived from TSP-1has antitumor activ-ity(33,55–57).Initially,an ex vivo study demon-strated that a tumor clone expressing high levels of TSP-1peptide reduced tumor growth(55).Recently, an ex vivo study showed that tumor clones express-ing thrombospondin-2(TSP-2)had significantly more antitumor efficacy than clones expressing TSP-1.More impressively,tumor clones expressing TSP-1and-2were synergistic in their antitumor efficacy as demonstrated by the complete inhibition of tumor growth(57).In addition,p53and an anti-angiogenic peptide of TSP-1in complex with lipo-somes were found to be synergistic in their inhibi-tion of tumors(56).These liposome:DNA complexes were delivered intravenously at a site distant from the tumor.Tissue Inhibitory Metalloproteinases(TIMPS) Unlike the previously noted therapies,in vivo gene therapy with TIMPS has not been reported (58,59).In one study,adenovirus-mediated overex-pression of TIMP-1,2,and3inhibited invasion of several different tumor cells through an artificial124CHEN ET AL.basement membrane(58).Nevertheless,only TIMP-3promoted apoptosis,suggesting that this factor may be more effective in reducing tumor growth than either TIMP-1or TIMP-2.Potential Gene Therapy Inhibitors of Angiogenesis There are several angiogenic inhibitors that may be useful with in vivo gene therapy delivery sys-tems.For example,the relaxed form of antithrombin III has demonstrated marked antitumor efficacy but gene therapy with this protein has not yet been attempted.In addition,the kringle5domain of plas-minogen has also been found to be a potent inhibitor of angiogenesis and to be synergistic with angiosta-tin in reducing tumor growth(60–62);similar to antithrombin III,gene therapy with kringle5show-ing antitumor efficacy has not been reported.An-other promising angiogenic inhibitor,PEDF,has not yet demonstrated its antitumor efficacy by either the gene or protein therapy approaches.Pitfalls and Future GoalsSeveral studies have now shown that tumor inhi-bition occurs with antiangiogenic gene therapy. Nevertheless,little is known about the relative an-titumor efficacy of the genes encoding antiangio-genic proteins/peptides.Although comparison stud-ies of antitumor efficacy have been done with some antiangiogenic proteins,the results of protein and gene therapy may diverge because of differences in distribution,half-life,and specificity.Thus,al-though perhaps useful as an initial guide,sole reli-ance on antitumor efficacy of the protein for selec-tion of the appropriate gene may introduce an incorrect bias.Furthermore,it is evident that more studies are required to examine synergistic tumor inhibition between various combinations of genes encoding antiangiogenic proteins.In this context, studies examining the antiangiogenic mechanism of these proteins may facilitate determination of po-tent synergistic combinations.As with other gene therapy approaches,a signifi-cant limitation of antiangiogenic gene therapy lies in the vector delivery systems.Although there has been incremental improvement in these systems, precisely when an adequate vector system will be developed for antiangiogenic genes is difficult to forecast.Since proteins of antiangiogenic gene ther-apy may require secretion,they may be expressed by several tissues other than tumor endothelial cells. Consequently,secretion of antiangiogenic proteins allows the route of gene delivery to be varied,and intravenous,intratumoral,and intramuscular injec-tion have been used.In animal models,all three modes of delivery have demonstrated antitumor ef-ficacy with antiangiogenic genes.Whereas platform deliveries with AAV or electroporation offer the hope of high levels of systemic angiostatic proteins,there is concern that such angiostatic proteins may inter-rupt critical angiogenic processes.In contrast,deliv-ery of the angiostatic gene by intratumoral injec-tions or by tumor-specific carriers may circumvent these concerns.In addition to determining more potent vectors and angiogenic inhibitors,new approaches are on the horizon for antiangiogenic gene therapy.Vac-cines toward tumor angiogenesis are now being de-veloped with partially purified proteins or whole cell endothelial extracts from tumors.Results in preclin-ical models have been encouraging and success of this active immunization 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