Metronomic Low-Dose Chemotherapy Boosts CD95-Dependent Antiangiogenic Effect of theThrombospondin Peptide ABT-510: A Complementation Antiangiogenic Strategy RonaldYap, 1 Dorina Veliceasa, 1 UrbanEmmenegger, 2 RobertS.Kerbel, 2 LauraM.McKay, 3 Jack Henkin, 3 andOlgaV. Volpert 1 Abstract Blocking angiogenesis is a promising approach in cancer therapy. Natural inhibitors of angio- genesis and derivatives induce receptor-mediated signals, which often result in the endothelial cell death. Low-dose chemotherapy, given at short regular intervals with no prolonged breaks (metronomic chemotherapy), also targets angiogenesis by obliterating proliferating endothelial cells and circulating endothelial cell precursors. ABT-510, a peptide derivative of thrombospon- din, kills endothelial cell by increasing CD95L, a ligand for the CD95 death receptor. However, CD95 expression itself is unaffected byABT-510 and limits its efficacy.We found that multiple chemotherapy agents, cyclophosphamide (cytoxan), cisplatin, and docetaxel, induced endo- thelial CD95 in vitro and in vivo at low doses that failed to kill endothelial cells (cytoxan > cisplatin > docetaxel). Thus, we concluded that some of these agents might complement each other and together block angiogenesis with maximal efficacy. As a proof of principle, we designed an antiangiogenic cocktail combining ABT-510 with cytoxan or cisplatin. Cyclophos- phamide and cisplatin synergistically increased in vivo endothelial cell apoptosis and angiosup- pression by ABT-510. This synergy required CD95, as it was reversible with the CD95 decoy receptor. In a mouse model, ABT-510 and cytoxan, applied together at low doses, acted in synergy to delay tumor take, to stabilize the growth of established tumors, and to cause a long-term progression delay of PC-3 prostate carcinoma.These antitumor effects were accom- panied by major decreases in microvascular density and concomitant increases of the vascular CD95, CD95L, and apoptosis. Thus, our study shows a ‘‘complementation’’ design of an optimal cancer treatment with the antiangiogenic peptide and a metronomic chemotherapy. Thrombospondin-1 is a well-known antiangiogenic agent (1). Its mechanism of action and structure-function relationship have been analyzed in considerable depth, resulting in the discovery of a minimal active heptapeptide in which antiangio- genic activity is greatly enhanced by L-isoleucine to D-isoleucine replacement (2). ABT-526 and ABT-510 are modified versions of this minimal peptide with increased potencies (3) and improved clearance; ABT-510 is currently under evaluation in phase II clinical trials (4). Thrombospondin-1 has also been identified as a host-derived mediator of the antiangiogenic action of low-dose metronomic chemotherapy (5, 6). Throm- bospondin-1 and ABT-510 act by inducing endothelial cell apoptosis in some cases via CD36 cell surface receptor (7, 8). Proapoptotic signal elicited by thrombospondin-1 generates CD95L, a ligand for the CD95 death receptor (9). However, CD95 expression on vascular endothelial cell is independent of thrombospondin-1; thus, accessible CD95 limits the rate of apoptosis and antiangiogenesis due to thrombospondin-1 and consequently determines, at least in part, the efficacy of thrombospondin-1-based cancer treatments. Seeking agents to improve the efficacy of ABT-510, we turned to conventional chemotherapy drugs. Metronomic chemother- apy (low-dose chemotherapy given at close regular intervals with no prolonged drug-free breaks) provides a way to inhibit tumor angiogenesis by targeting proliferating endothelial cells in tumor blood vessels and circulating endothelial cell precursors (CEP) that are later integrated in tumor vasculature (10 – 12). Metronomic therapy has lower toxicity compared with pulsatile maximum tolerated dose (MTD) chemotherapy and therefore lessens or removes the need for the growth factors to accelerate recovery from myelosuppression. Moreover, despite the use of lower cumulative doses, the metronomic approach sometimes shows superior results in prolonging survival times compared with the conventional MTD regimens, in preclinical models (13, 14). Concomitant use of metronomic chemotherapy and Cancer Therapy: Preclinical Authors’Affiliations: 1 Department of Urology, Northwestern University Feinberg School of Medicine, Chicago, Illinois; 2 Molecular and Cellular Biology Research, Sunnybrook andWomen’s College Health Sciences Centre,Toronto, Ontario, Canada;and 3 AbbottLaboratories,AbbottPark,Illinois Received3/21/05;revised5/17/05;accepted7/1/05. Grant support: NIHgrantHL068033-04(O.V.Volpert),AmericanCancerSociety grant RSG-01-099-01-CSM (O.V.Volpert), Swiss National Science Foundation (U. Emmenegger), and Swiss Cancer League/Oncosuisse grant BIL SKL1237-02- 2002(U.Emmenegger). Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpage charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely toindicate this fact. Requests for reprints: OlgaV.Volpert, Department of Urology, Northwestern University Feinberg School of Medicine, 303 East ChicagoAvenue,Tarry Research Building Ste16-761, Chicago, IL 60611. Phone: 312-503-5934; Fax: 312-908- 7275; E-mail: olgavolp@northwestern.edu. F 2005AmericanAssociationforCancerResearch. doi:10.1158/1078-0432.CCR-05-0621 www.aacrjournals.org Clin Cancer Res 2005;11(18) September15, 2005 6678 Research. on June 6, 2020. © 2005 American Association for Cancer clincancerres.aacrjournals.org Downloaded from