10. NPY in Tumor Angiogenesis

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to provide oxygen and nutrients to rapidly growing tumor tissues. Thus, only those tumors, which are able to induce formation of new blood vessels by releasing angiogenic stimulators and downregulating angiogenic inhibitors, can advance to a phase of exponential growth. This phenomenon is often termed the “angiogenic switch.”58−60 The most ubiquitous angiogenic factor involved in tumor vascularization is VEGF, which is upregulated in most of the known solid tumors.58−60 Our recent studies on NPY have revealed that this peptide also promotes tumor angiogenesis and growth.

As a neuronal peptide, NPY is often expressed in neural crestderived tumors. The peptide is particularly abundant in tumors originating in the autonomic nervous system, such as sympathetic neuroblastomas and pheochromocytomas, as well as parasympathetic Ewing’s sarcoma family of tumors (ESFT).13,61−66 High expression of NPY is often associated with its increased release from the tumors leading to elevated plasma levels in patients with neuroblastoma and pheochromocytoma.61,62,64−69 Neuroblastoma cell lines release high amounts of NPY to the culture media13 whereas the peptide is not detectable in conditioned media obtained from rat pheochromocytoma cells, PC12, despite its high mRNA expression,13 unless additional stimulants are present.70 In ESFTs, extracellular release of NPY into the culture media is significantly lower than that of neuroblastomas, in spite of the intracellular NPY being comparable.13 Consequently, no elevated NPY plasma levels in ESFT patients have been reported. Therefore, release of NPY from the tumor cells is an active and tightly regulated process and likely to be an important factor determining functions of the peptide in these tumors.71,72

Since NPY is potently angiogenic, its release from neural crestderived tumors suggests a potential role for the peptide in regulation of their growth. Indeed, endothelial cell proliferation stimulated by neuroblastoma-conditioned media is completely blocked by NPY receptor antagonists, suggesting that the peptide is crucial for vascularization of these tumors.13 In ESFT cells, which release less NPY than neuroblastomas, NPY receptor antagonists only partially reduce angiogenic activity of the conditioned media.13 Thus, in ESFTs, NPY is important, but not the only factor responsible for their vascularization.

The critical role of NPY in tumor angiogenesis is further confirmed by its stimulatory effect on vascularization of neuroblastoma and ESFT xenografts. In both types of tumors, the exogenous peptide significantly increases vessel density within tumor tissues.13 Interestingly, this effect is accompanied by an increased content of pericytes/vascular smooth muscle cells in the vasculature of NPY-treated tumors, indicating its more mature character.13 This effect may be due to the mitogenic activity of the peptide on vascular smooth muscle cells. Thus, as seen in other models, NPY not only stimulates formation of the tumor blood vessels, but also facilitates their maturation, which can improve delivery of oxygen and nutrients to the tumor tissue, and further promote tumor growth.

NPY promotes growth not only of vascular smooth muscle and endothelial cells, but also of a variety of other cells, including neuronal precursors.8,12,21,29 Thus, its angiogenesis-dependent stimulation of tumor growth is additionally modified by direct autocrine effects, which, depending on the type of NPY receptors the tumor cells express, may lead to their proliferation or apoptosis. Neuroblastomas, which are derived from sympathetic neurons, express Y2 receptors and, in some cell lines, also Y5 receptors,13,73,74 and their activation leads to tumor cell proliferation.13 Y2 and Y5 receptor antagonists significantly decrease basal proliferation of neuroblastomas, which further confirms the autocrine growth-promoting effect of endogenous NPY.13 Thus, NPY stimulates growth of neuroblastomas directly, by activation of tumor cell proliferation and indirectly, by its angiogenic effect.

The role of NPY as a critical neuroblastoma growth factor is further supported by clinical data. Elevated plasma levels of NPY have been found mainly in patients with advanced disease, in association with poor clinical outcome.62,67,69 Moreover, expression of Y2 receptors in human neuroblastoma tissues has been detected in both tumor74 and endothelial cells (unpublished data). Since both NPY-induced neuroblastoma cell proliferation and angiogenesis are Y2 receptor-dependent, blocking Y2-NPY pathway may be an effective, bidirectional therapy for these tumors. Such therapies combining angiostatics with low doses of chemotherapeutic agents have been already tested and proven successful in several models, including neuroblastomas.75−80

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Unlike Y2 receptor-bearing neuroblastomas, ESFT cells express Y1 and Y5 receptors, which, interestingly, is associated with an opposite effect of NPY on their growth.13,81 In contrast to its proliferative actions in neuroblastomas, in ESFT cells, endogenous NPY stimulates apoptosis and this requires activation of both Y1 and Y5 receptors.13,82 Therefore, in ESFT the peptide exerts two opposite effects — inhibitory, via Y1/Y5-dependent apoptosis and stimulatory, via Y2 receptor-mediated angiogenesis. The overall effect of NPY on ESFT growth, therefore, depends on balance between these two processes. The activity of the peptide is additionally modified by DPPIV, which is expressed along with NPY and its receptors in ESFT cells and is abundant in tumor vasculature.13 By converting NPY to the non-Y1 receptor agonist, the protease prevents Y1/Y5 receptor-mediated apoptosis of ESFT cells, and shifts activity of the peptide toward Y2 receptor-mediated angiogenesis. Blocking the DPPIV activity with specific inhibitors enhances NPY-induced apoptosis of the ESFT cells.13 Hence, DPPIV acts as a “survival factor” for ESFT by augmenting angiogenesis-dependent, growth-promoting effects of NPY. These activities make the protease a potential target for therapy of these tumors.

Direct effects of NPY on the growth of pheochromocytomas has not been established yet. Clinically, elevated plasma levels of the peptide have been associated with the malignant phenotype of the disease.63 Hence, even if NPY does not exert direct proliferative effects on pheochromocytoma cells, the peptide may facilitate tumor growth and spread via its angiogenic activity. This notion is supported by the fact that nerve growth factor (NGF), which is known to upregulate NPY in PC12 cells, stimulates vascularization of PC12 xenografts in a VEGF-dependent manner.83 Since NPY-induced angiogenesis is also partially driven by VEGF,20 NPY may be a mediator of NGF’s angiogenic actions in pheochromocytomas. However, further studies would be needed to fully elucidate peptide’s functions in these tumors.

The NPY effect on tumor vascularization and growth seems to be most relevant to neural crest-derived tumors, which express their own NPY. However, all tissues of the body have blood vessels, and all of them, except the aorta, are, to a greater or lesser extent, innervated by the sympathetic nerves. Hence, the role of nerve-derived peptide cannot