Malignant Glioma without Affecting Antitumor Efficacy in In vivo Activity Oncolytic Adenovirus Inhibits Matrix Metalloproteinase Tissue Inhibitor of Metalloproteinase-3 Expression from an
چکیده
Oncolytic adenoviruses exhibiting tumor-selective replication are promising anticancer agents. Insertion and expression of a transgene encoding tissue inhibitor of metalloproteinase-3 (TIMP-3), which has been reported to inhibit angiogenesis and tumor cell infiltration and induce apoptosis, may improve the antitumor activity of these agents. To assess the effects of TIMP-3 gene transfer to glioma cells, a replication-defective adenovirus encoding TIMP-3 (Ad.TIMP-3) was employed. Ad.TIMP-3 infection of a panel of glioma cell cultures decreased the proliferative capacity of these cells and induced morphologic changes characteristic for apoptosis. Next, a conditionally replicating adenovirus encoding TIMP-3 was constructed by inserting the TIMP-3 expression cassette into the E3 region of the adenoviral backbone containing a 24-bp deletion in E1A. This novel oncolytic adenovirus, Ad#24TIMP-3, showed enhanced oncolytic activity on a panel of primary cell cultures and two glioma cell lines compared with the control oncolytic virus Ad#24Luc. In vivo inhibition of matrix metalloproteinase (MMP) activity by Ad#24TIMP-3 was shown in s.c. glioma xenografts. The functional activity of TIMP-3 was imaged noninvasively using a near-IR fluorescent MMP-2–activated probe. Tumoral MMP-2 activity was significantly reduced by 58% in the Ad#24TIMP-3–treated tumors 24 hours after infection. A study into the therapeutic effects of combined oncolytic and antiproteolytic therapy was done in both a s.c. and an intracranial model for malignant glioma. Treatment of s.c. (U-87MG) or intracranial (U-87DEGFR) tumors with Ad#24TIMP-3 and Ad#24Luc both significantly inhibited tumor growth and prolonged survival compared with PBS-treated controls. However, expression of TIMP-3 in the context of Ad#24 did not significantly affect the antitumor efficacy of this oncolytic agent. (Cancer Res 2005; 65(20): 9398-405) Introduction Oncolytic viruses are receiving widespread attention as novel therapeutic agents for the treatment of various malignancies (1, 2). Currently, further development of these agents includes combining the oncolytic effects of the virus with the added antitumor activity of a therapeutic transgene. Such ‘‘armed’’ therapeutic viruses carrying a variety of transgenes encoding prodrug-converting enzymes, immune stimulatory molecules, or apoptosis-enhancing proteins have already been shown to exhibit increased antitumor efficacy (3–12). However, contradictory evidence exists regarding the value of additional prodrug-converting enzymes or cytotoxic genes in the context of an oncolytic virus, as they may induce premature killing of the infected tumor cells, thereby limiting the production of progeny virus and counteracting virus-induced oncolysis (13–15). An alternative approach is to insert transgenes that exert their inhibitory effects on the tumor indirectly. This will ensure that sufficient viral replication and enzyme production is maintained. Matrix metalloproteinases (MMP) offer an interesting target for such an approach. MMPs belong to a family of structurally related proteolytic enzymes that mediate degradation of components of the extracellular matrix and basement membrane. Moreover, they mediate the release of sequestered latent growth and angiogenic factors and the activation of latent growth factors (16). Upregulation of MMP activity has been linked to tumor growth and invasion and tumor-associated angiogenesis. Malignant gliomas constitute a relevant target for anti-MMP treatment. Their highly invasive and vascularized nature contributes to the poor prognosis of glioma patients. Immunohistochemical localization studies in surgical specimens have confirmed that MMPs are overexpressed in these tumors (17–19) and correlated tomalignant progression (20, 21). Interference with these proteasesmight therefore inhibit local tumor dissemination and neovascularization. Tissue inhibitors of metalloproteinases (TIMP) are a family of natural inhibitors that control the activity of MMPs and are often concomitantly expressed. Four members of this family have been characterized thus far (i.e., TIMP-1, TIMP-2, TIMP-3, and TIMP-4), which are expressed by a variety of cell types and can be found in most tissues throughout the body (22). In human gliomas, TIMP expression is reported to be down-regulated with increasing malignancy, resulting in an imbalance between the production of MMPs and TIMPs and a shift towards a proproteolytic state with invasive phenotype (21, 23, 24). TIMP-3 is unique within the TIMP Requests for reprints:Martine L.M. Lamfers, Neurosurgery, VU University Medical Center, Boelelaan, Amsterdam, the Netherlands 1007 MB. Phone: 31-20-444-8411; Fax: 31-20-444-8168; E-mail: M.Lamfers@vumc.nl. I2005 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-04-4264 Cancer Res 2005; 65: (20). October 15, 2005 9398 www.aacrjournals.org Research Article American Association for Cancer Research Copyright © 2005 on February 23, 2013 cancerres.aacrjournals.org Downloaded from DOI:10.1158/0008-5472.CAN-04-4264