Oncology/endocrineInhibition of In Vivo Tumor Angiogenesis and Growth Via Systemic Delivery of an Angiopoietin 2-Specific RNA Aptamer
Introduction
Angiogenesis is necessary for successful tumor growth. Although inhibition of vascular endothelial growth factor (VEGF) represents the most validated anti-angiogenic approach described thus far, VEGF works in concert with other components of the angiogenic process [1]. Of these, some of the most important are thought to be the tyrosine kinase receptor Tie2 (Tek), and its ligands, the angiopoietins [1]. Numerous investigations have underscored the significance of Tie2 and the angiopoietins in normal and pathological angiogenesis [1]. The Tie2/angiopoietin (Ang) system comprises a critical signal transduction pathway such that mice deficient in Tie2 exhibit impaired vascular remodeling, disrupted endothelial-mesenchymal interactions, and resultant embryonic lethality [1, 2, 3, 4]. Tie2 is present predominantly, but not exclusively, on endothelial cells during both developmental and adult stages, and its expression is increased during adult vascularization [1, 2]. Ang1 is constitutively expressed by pericytes and smooth muscle cells, and is thought to primarily promote interactions between endothelial and support cells to stabilize the developing vasculature [5, 6, 7]. Effects of Ang1 are antagonized by Ang2, which is expressed at sites of vascular remodeling in adults [8, 9]. Expression of Ang1 in quiescent vasculature and that of Ang2 at sites of active angiogenesis had given rise to the hypothesis that angiopoietins 1 and 2 have opposite functions in vascular development. More recent studies have challenged this concept and emphasized the context in which angiopoietins operate [10, 11, 12, 13, 14]. For example, Ang1 has been shown to act as a negative regulator of VEGF-induced angiogenesis in the heart [13]. Ang2, on the other hand, has been reported to enhance angiogenesis in the presence of VEGF, but to promote vessel regression in its absence [10, 11, 12, 13].
Along with a growing body of knowledge on vascular development, the role of Tie2 and the angiopoietins in angiogenesis and tumor development is becoming better characterized [15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25]. Animal studies have shown Tie2 receptor blockade to inhibit angiogenesis, tumor growth, and metastasis [18, 19, 20, 21, 22]. In a murine model of ischemia-induced retinopathy, systemic administration of an adenovirus encoding the extracellular domain of Tie2 (AdExTek) inhibited retinal neovascularization [17]. Lin et al. demonstrated that administering AdExTek to mice significantly inhibited subcutaneous tumor growth, and intravenous co-injection of tumor cells and AdExTek reduced pulmonary metastatic potential [18]. Human melanoma cells expressing the extracellular domain of Tie2 were shown to inhibit angiogenesis and tumor growth in mouse xenografts [19]. Work specifically directed at the angiopoietins including overexpression studies showed Ang1 to inhibit tumor angiogenesis or growth, and Ang2 to enhance it [22, 23, 25]. Human colon cancer cells overexpressing Ang1 implanted into nude mouse livers inhibited tumor angiogenesis and produced significantly smaller tumors, suggesting potential relevance of the Tie2/angiopoietin pathway in treating metastatic colorectal cancer [25]. Consistent with the notion that the interplay among the angiopoietins and Tie2 dictates vascular stability, Zhang et al. demonstrated VEGF-mediated up-regulation of Ang2 with resultant pericyte loss and instability of host vessels adjacent to tumors, supporting a pro-angiogenic role [23].
Recently, angiogenesis in a non-tumor-bearing rat corneal pocket angiogenesis assay was inhibited by the local delivery of an RNA aptamer specifically designed to bind and inhibit Ang2 [24]. Based on this finding, an Ang2-specific aptamer was similarly synthesized based on the sequence and modifications described by White et al. [24], for use in our study.
By directly inhibiting protein activity, aptamers act similarly to monoclonal antibodies or small molecule drugs, increasing their potential clinical applicability [25, 26, 27, 28, 29, 30, 31]. Advantages of aptamers over monoclonal antibodies include the ability for chemical synthesis in large quantities and low immunogenicity in vivo, while exhibiting high affinity for target proteins [27, 32]. In addition to a therapeutic role, aptamers can be used to escort therapeutic or diagnostic reagents [33]. Aptamers targeting any protein or small molecule can be produced using in vitro selection processes. The Ang2-specific aptamer used in this study had been positively selected for recombinant human Ang2, and negatively selected against recombinant human Ang1 [24]. Optimization of a desired sequence for in vivo use requires manipulating the factors that dictate bioavailability, such as stability in biological fluids and systemic clearance [34]. The former, reliant on nucleic acid backbone, is enhanced by replacing ribonucleotides with 2′-amino, -fluoro, or -O-alkyl nucleotides [27, 35, 36]. Such modifications extend in vitro plasma half-lives of RNA oligonucleotides from seconds to 5 to 15 h. Exonuclease degradation in plasma can be prevented by capping the 3′ end of an oligonucleotide and further reinforced by replacing ribose and deoxyribose with modified nucleotides or non-nucleotide linkers [34]. To allow for practical chemical synthesis, aptamers are currently truncated to fewer than 40 nucleotides [34], promoting rapid systemic clearance. This can be overcome by attaching the aptamer to a polyethylene glycol (PEG) moiety or a liposomal surface [26, 37]. The clinical relevance of aptamers is brought to light by the use of VEGF aptamers in clinical trials for treatment of macular degeneration [38, 39].
It is not known whether inhibiting Tie2 or targeting the angiopoietins is more apt to generate a potent antineoplastic response. We hypothesized that systemic administration of a nuclease-resistant Ang2-specific aptamer, conjugated to 40 kDa PEG to enhance bioavailability, would impair angiogenesis and inhibit growth of primary tumors.
Section snippets
Ang2-Specific Aptamer
The sequence (GAGGACGAUGCGGACUAGCCUCAUCAGCUCAUGUGCCCCUC) had been previously shown by White et al. to bind and inhibit Ang2 function with high affinity and specificity, without affecting Ang1-mediated Tie2 activation [24]. To improve bioavailability, the aptamer was constructed using 41 2′-fluoro pyrimidines and 2′-hydroxy-purines, flanked by 5′ 40 kDa PEG and C6 aminolinker, and 3′ inverted deoxythymidine (Transgenomic, Inc., Omaha, NE). The lyophilized oligonucleotide was suspended in
Effect of Pegylated Aptamer on Ang2-Mediated Tie2 Activity
Ang2 is known to phosphorylate Tie2 that is ectopically expressed in non-endothelial cell lines [9]. The RNA aptamer had previously been shown to inhibit Tie2 phosphorylation by Ang2 in vitro [24]. To verify that following conjugation to 40 kDa PEG the aptamer would inhibit Ang2, immunoprecipitation and WB were performed to detect Tie2 receptor phosphorylation. Two Hundred ninety-three cells stably expressing human Tie2 were treated with Ang2 in the absence and presence of molar excesses of the
Discussion
In an effort to determine if specific targeting of angiopoietin-2 in vivo would affect angiogenesis or tumor growth, we systemically administered a clinically-relevant, nuclease-resistant Ang2-specific RNA aptamer to mice bearing either dorsal window chambers or subcutaneous colorectal tumors. The aptamer had been previously shown to bind Ang2 with high affinity in vitro, and to inhibit angiogenesis in a rat corneal pocket assay when locally delivered [24]. To extend its in vivo applicability,
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