An In Vitro and In Vivo Evidence That Interleukin-8 Is a Pivotal Hypoxia-Regulated Pro-Angiopoietic Factor Secreted by Human Rhabdomyosarcomas (RMS) - Therapeutic Implications.

Abstract

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of adolescence and childhood that frequently infiltrates bone marrow (BM). We have demonstrated that the stromal-derived factor (SDF)-1 (Blood 2002; 100:2597), hepatocyte growth factor (HGF) (Cancer Res . 2003; 63:7926) and leukemia inhibitory factor (LIF) (Cancer Res . 2007; 67:2131) play an important role in RMS metastasis. Since RMS tumors are very well vascularized we asked if these factors are secreted by RMS cells and if they stimulate angiogenesis. To address this issue, first we evaluated expression of SDF-1, HGF and LIF as well as several other proangiopoietic factors (IL-8, VEGF, FGF-2) in 9 human RMS cell lines. Expression of these factors was evaluated in both normal steady state conditions and in conditions that lead to overexpression of hypoxia inducible factor-1 (HIF-1a) due to hypoxia or exposure to CoCl2. We found by RQ-PCR and confirmed by ELISA assays that from all the factors evaluated, IL-8, which expression was very low in steady state conditions was very highly upregulated in all RMS cells lines during hypoxic conditions (∼30–135 times). This selective upregulation of IL-8 was somehow unique for RMS cells, and we did not observe similar phenomenon in several other solid tumor cell lines, where IL-8 was usually already very highly expressed in steady state conditions. To evaluate a biological significance of IL-8 in RMS angiogenesis, we collected conditioned media (CM) from RMS cells and found that they stimulate in IL-8 dependent manner in HUVEC i) phosphorylation of MAPKp42/44 and AKT, and ii) chemotactic responses. Pre-clearing of RMS-derived CM with antibodies against IL-8 significantly inhibited chemotaxis of HUVEC. Finally, by employing shRNA we inhibited expression of IL-8 in human RH-30 cells and noticed that such cells in vivo, if injected into skeletal muscles of immunodeficient mice, have the reduced ability to grow tumors and chemoattract co-transplanted human endothelial cells. We conclude that IL-8 is a pivotal pro-angiopoietic factor that is released by human RMS cells in hypoxic conditions, and that the targeting of IL-8 may prove to become a novel efficient strategy to inhibit RMS growth.