Endostatin specifically targets both tumor blood vessels and lymphatic vessels

Abstract

Endostatin, a 20 kDa C-terminal fragment of collagen XVIII, was first identified as a potent angiogenic inhibitor. The anti-angiogenic function of endostatin has been well documented during the past decade. Recently, several studies demonstrated that endostatin also inhibits tumor lymphangiogenesis and lymphatic metastasis. However, the exact mechanism that endostatin executes its anti-angiogenic and anti-lymphangiogenic functions remains elusive. In the current mini-review, we briefly summarize recent novel findings, including the functions of endostatin targeting not only angiogenesis but also lymphangiogenesis, and the underlying mechanism by which endostatin internalization regulates its biological functions.

Keywords

endostatin / angiogenesis / lymphangiogenesis / nystatin / internalization / tumor

Introduction

Endostatin, a 183-amino acid proteolytic fragment of the basement membrane collagen type XVIII, is an endogenous tumor angiogenesis inhibitor [1]. It has been proved to inhibit the proliferation, migration, and tube formation of endothelial cells, thus eventually interrupting angiogenesis and tumor growth [1,2]. However, many controversial observations were reported during the past decade, such as its tumor inhibition efficacy and refolding problem. Novel progresses have revealed that the N-terminal integrity, zinc binding, and correct refolding of endostatin are essential to its biologic functions [3-5]. In addition to its well-documented anti-angiogenic function, several studies have demonstrated that endostatin also impedes tumor lymphangiogenesis and subsequently tumor lymphatic metastasis [6-9].

Lymphangiogenesis, the growth of lymphatic vessels, usually occurs in adult tissues during wound healing, inflammation, and tumor metastasis [10,11]. Thus, administration of pro- or anti-lymphangiogenesis factors provides the possibility of targeting lymphatic vasculature in human diseases. Since lymphatic-specific vascular endothelial growth factors (VEGF-C and VEGF-D) and their cell surface receptor VEGFR-3 were identified, lymphatic growth factors have received considerable attentions over the past decade [11,12]. For instance, platelet-derived growth factor BB (PDGF-BB) [13], hepatocyte growth factor (HGF) [14], insulin-like growth factor 1/2 (IGF-1/2) [15], and fibroblast growth factor 2 (FGF-2) [16] have been reported to promote lymphangiogenesis. In comparison, endogenous lymphangiogenesis inhibitors were far less explored. Here we discuss the function of endostatin in tumor lymphangiogenesis inhibition.

Endostatin internalization in endothelial cells has been extensively observed and demonstrated to be important, if not essential, for the biologic activities of endostatin [3,17-20]. Endocytosis is an important cellular process of eukaryotic cells to uptake various compartments and to regulate various signaling pathways. Clathrin-coated pits and caveolae are two of the most recognizable features of the plasma membrane in mammalian cells [21,22]. Accumulating evidence indicates that clathrin-dependant pathway and caveolae/lipid raft pathway are not independent as previously considered. Caveolae/lipid rafts and clathrin-coated pits have been reported to simultaneously participate in the endocytosis process of various proteins, such as integrins [23-25]. Intriguingly, we identified for the first time that the internalization of endostatin requires not only caveolae/lipid raft-dependent endocytosis but also clathrin-mediated internalization [26].

Endostatin inhibits lymphangiogenesis in various manners

The anti-angiogenesis function of endostatin has been extensively studied in the past decade [27]. In addition to its anti-angiogenic properties, recent studies have shown that endostatin also inhibits tumor lymphangiogenesis and lymphatic metastasis [6-9]. Using oral squamous cell carcinoma (SCC) cells transfected with adenovirus expressing recombinant endostatin, Fukumoto et al. [6] first reported that endostatin inhibits lymphangiogenesis and lymph node metastasis by downregulating VEGF-C expression in tumor cells. This discovery provides us a promising prospect that endostatin not only inhibits tumor growth through blocking angiogenesis, but also hinders lymphatic metastasis by abrogating lymphangiogenesis. Subsequently, more attentions were paid to the molecular mechanism for the anti-lymphangiogenesis function of endostatin. Using spontaneous tumor model in transgenic J4 mice overexpressing endostatin, Heljasvaara’s group [7] showed that both the adhesion and migration of VEGF-C-producing mast cells in tumor microenvironment are negatively regulated by endostatin. The reduced level of VEGF-C is responsible for the inhibition of tumor lymphangiogenesis, as VEGF-C has been well documented to stimulate tumor lymphangiogenesis and enhance lymphatic metastasis [6]. These studies demonstrated that endostatin inhibits tumor lymphangiogenesis through downregulating the expression of VEGF-C by targeting tumor cells, as well as mast cells in tumor microenvironment.

Interestingly, Liang’s group [9] suggested a non-classic pathway for endostatin inhibiting tumor lymphangiogenesis. The authors found that the secretion of fibronectin alternative extra domain A (EDA) by SW480 cells and the expression of integrin α₉ in lymphatic endothelial cells (LECs) were decreased by endostatin treatment. Intriguingly, the co-localization between EDA and integrin α₉ on the cell surface of LECs is dramatically reduced by endostatin treatment. It has been reported that the binding of EDA to its receptor integrin α₉ promotes lymphatic valve morphogenesis [28]. The exact signaling pathways responsible for the direct inhibition of endostatin on the expression of integrin α₉ in LEC have yet to be identified. However, we may get a clue from this study that endostatin may directly target LEC and influence the morphology and gene expression. Recently, our group [8] has discovered a novel mechanism of endostatin in lymphangiogenesis and lymphatic metastasis inhibition. We found that endostatin directly inhibits the activities of lymphangiogenic endothelial cells through its cell surface receptor nucleolin. Cell surface nucleolin has been reported to be a potential biomarker of angiogenic endothelial cells and the receptor of endostatin [17,29,30]. Interestingly, cell surface nucleolin is specifically expressed in the lymphangiogenic vessels, but not matured lymphatic vessels. Consequently, endostatin targets tumor lymphangiogenesis without affecting matured lymphatic vessels, which perfectly explains the low-toxicity of endostatin in the clinical application [31,32]. Huber’s group [27] has shown that endostatin regulates up to 12% of the genes in human blood endothelial cells. Thus, it makes sense that the expression and distribution of integrin α₉ on LEC can be regulated by endostatin. The exact variations of gene expression profiles of LECs with or without endostatin treatment remain to be explored. Taken together, in addition to its well-known anti-angiogenic activity, endostatin has been demonstrated to be an endogenous inhibitor of tumor lymphangiogenesis [33,34]. These studies contribute to the novel concept that endostatin controls lymphangiogenesis by a broad spectrum of signaling pathways, via directly targeting lymphangiogenic endothelial cells and regulating tumor cells or mast cells in tumor microenvironment.

Although endostatin has been demonstrated to block tumor lymphangiogenesis, it is not a specific anti-lymphangiogenesis factor. Recently, a secreted alternatively spliced variant of VEGFR-2 (sVEGFR-2) was identified as an endogenous protein that specifically inhibits lymphangiogenesis [35]. Tissue-specific loss of sVEGFR-2 in mice induces lymphatic hyperplasia without affecting blood vessels. This study proposed that sVEGFR-2 is a selective VEGF-C antagonist, which accounts for its specificity on lymphangiogenesis inhibition. The discovery makes a substantial contribution to the knowledge of lymphangiogenesis in various diseases. In tumor tissue, lymphangiogenesis can promote metastasis [11]. It remains mysterious whether sVEGFR-2 can inhibit tumor lymphangiogenesis. We are also curious about whether there would be other specific endogenous inhibitors of lymphangiogenesis existing in tumor microenvironment that directly target lymphatic endothelial cells.

The anti-endothelial function of endostatin is regulated by its internalization

Although a variety of endostatin binding proteins on endothelial cell surface, including integrins, cell surface nucleolin, glypicans, tropomyosin and laminin, have been identified as potential endostatin receptors [17,19,36-40], the mechanism of how these proteins mediate endostatin internalization still remains largely unknown. Alitalo and colleagues have shown that endostatin can associate with lipid rafts through its interaction with integrin α₅β₁ and caveolin-1 [41,42]. Caveolin-1 is the main coat component of caveolae [43], which is especially abundant in endothelial cells, smooth muscle cells, adipocytes, and fibroblasts [22]. In addition, endostatin-induced lipid raft clustering in endothelial cells has been reported by Li and colleagues and such clustering can be disrupted by cholesterol depleting reagent such as methyl-β-cyclodextrin [44,45]. More recently, we report for the first time that both caveolae/lipid rafts and clathrin-coated pits are involved in endostatin internalization by endothelial cells (Fig. 1). Interestingly, cholesterol sequestration with nystatin or amphotericin B facilitates the translocation of endostatin out of caveolae/lipid rafts and dramatically increases endostatin overall uptake through switching endostatin internalization predominantly to the high-efficient clathrin-dependent pathway. Furthermore, nystatin-enhanced internalization of endostatin promotes its anti-angiogenic effects on endothelial cell activities such as tube formation and migration. More importantly, combined treatment with nystatin and endostatin selectively enhances the bio-distribution and uptake of endostatin in tumor blood vessels and tumor tissues, but not in normal tissues of tumor-bearing mice, eventually resulting in elevated anti-angiogenic and anti-tumor efficacies of endostatin in vivo [26]. In addition, we also examined the effect of nystatin on endostatin internalization in LECs, and similar result was obtained (data not published).

Taken together, these studies demonstrate that the internalization of endostatin is important for its anti-angiogenic, possibly also anti-lymphangiogenic functions and involves not only caveolae/lipid rafts but also clathrin-coated pits. Existing evidences also support the notion that those previously identified receptors and interacting proteins of endostatin may act in a correlative and dynamic pattern. Different receptors of endostatin may form various complexes and combinations, regulating endostatin internalization via clathrin pathway and/or caveolae. For example, integrin α₅β₁ has been shown to be internalized through not only caveolar but also clathrin-mediated endocytosis [23-25]. Interestingly, cell surface nucleolin has also been found to associate with both lipid rafts and clathrin-dependent internalization [46,47]. There is a large reservoir of non-raft-localized nucleolin and integrin α₅β₁ which can undergo clathrin-mediated endocytosis [25,47]. These non-raft-localized cell surface nucleolin and integrin α₅β₁ may conduct the clathrin-dependent internalization of endostatin. Meanwhile, raft-localized endostatin receptors such as integrin, nucleolin, caveolin-1, and heparan sulfate proteoglycan are presumably responsible for endostatin association with caveolae/lipid rafts and subsequent internalization of endostatin through raft-dependent pathway. Further understanding of endostatin internalization may help us to better explain the anti-angiogenic and anti-lymphangiogenic functions of endostatin.

Summary

Endostatin has been applied in clinical cancer therapy and shows a broad anti-cancer spectrum and low toxicity. In addition to our traditional understanding, endostatin inhibits both angiogenesis and lymphangiogenesis. Besides the primary tumor shrinkage, we should pay more attention to the tumor lymphatic metastasis related to the efficiency of endostatin. It is intriguing that tumor recurrence and/or metastasis may be dramatically reduced after probable application of endostatin in clinical. Furthermore, enhancing the internalization and uptake of endostatin by regulating related endocytic pathways seems to provide a novel route to promote the anti-tumor activities of endostatin. As a regulator of caveola/lipid raft endocytic pathway in cellular biology, nystatin (approved by the Food and Drug Administration of the USA) is widely utilized as a polyene antifungal drug in human patients through oral or topical administration. Since recombinant human endostatin (approved by the State Food and Drug Administration of China) is being used for the treatment of non-small cell lung cancer, it would be intriguing to further examine the efficacy of combined treatment with endostatin and nystatin in clinical studies.

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