Selective upregulation of interleukin‐8 by human rhabdomyosarcomas in response to hypoxia: therapeutic implications

Abstract

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of adolescence and childhood. Because RMS tumors are highly vascularized, we sought to determine which factors secreted by RMS cells are crucial in stimulating angiogenesis in response to hypoxia. To address this issue, we evaluated expression of several proangiogenic factors [interleukin (IL)-8, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-2, stromal-derived factor (SDF)-1, hepatocyte growth factor (HGF), and leukemia inhibitory factor (LIF)] in 8 human RMS cell lines in both normal steady-state and hypoxic conditions. We found by real-time quantitative polymerase chain reaction (RQ-PCR) and confirmed by enzyme-linked immunosorbent assay (ELISA) that from all the factors evaluated, IL-8, whose expression is very low in normoxia, had been very highly expressed and secreted by RMS cells lines during hypoxic conditions (~40–170 times). Interestingly, this upregulation was not affected by knocking down hypoxia-inducible factor (HIF)-1α, but was inhibited by mitogen-activated protein kinase (MAPK)p42/44 and phosphatidylinositaol 3-kinase (PI3K)/AKT pathway inhibitors. This suggests that IL-8 expression is regulated in an activating protein (AP)-1- and nuclear factor (NF)-κB-dependent manner. Furthermore, we found that conditioned media (CM) harvested from RMS cells exposed to hypoxia activated and stimulated chemotactic responses in human umbilical vein endothelial cells (HUVECs) and that IL-8 was responsible for hypoxia-related effects. Finally, by employing shRNA, the expression of IL-8 in human RH-30 cells was downregulated. We noticed that such RMS cells, if injected into skeletal muscles of immunodeficient mice, have a reduced ability for tumor formation. We conclude that IL-8 is a pivotal pro-angiogenic factor released by human RMS cells in hypoxic conditions and that the targeting of IL-8 may prove to be a novel and efficient strategy for inhibiting RMS growth.

Macrophage Migration Inhibitory Factor Is Secreted by Rhabdomyosarcoma Cells, Modulates Tumor Metastasis by Binding to CXCR4 and CXCR7 Receptors and Inhibits Recruitment of Cancer-Associated Fibroblasts

“…This supports involvement of MIF signaling in inhibition of the recruitment of fibroblasts (32) by RMS tumors cells. Interestingly, we noticed that MIF inhibits migration of RMS cells to IL-8 gradient (Supplementary Figure 5) that as we reported previously is secreted by RMS cells during hypoxia and may strongly chemoattract CAFs (17). …”

“…The prognosis is poor in particular for the more aggressive and metastatic ARMS type (12, 13). In our previous reports, we focused on the role of chemokines (14, 15, 17), selected growth factors and cytokines (18, 20), as well as the coagulation cascade (16) in RMS metastasis. In particular, we were interested in involvement of the SDF-1–CXCR4/CXCR7 axis in this process (15, 27).…”

Uncovering metabolism in rhabdomyosarcoma

“…74 This molecule is a member of the a (C-X-C) subfamily of chemokines, is secreted by several haematopoietic cells, fibroblasts, hepatocytes and The cartoon depicts the molecular effectors that in cancer cells increase the glucose uptake and glycolytic consumption, nucleotide synthesis, lipogenesis, protein synthesis and glutamine anapleurosis. As observed in different tumors, RMS subsets share many of these molecular hallmarks; moreover, the more aggressive fusion-positive RMS express the Pax3-Foxo1 oncoprotein, which is actively involved in the transcription of different targets, including metabolic genes such as GLUT4 and CPT1A.…”

“…76 Although human RMS cells were found to be negative for CXCR1 and CXCR2 expression, 74 the release of IL-8 by RMS cells was hypothesized to be a paracrine mechanism for stimulating stroma cells to increase de novo vascularization and counteract hypoxia. Consistent with this, shRNA-mediated downregulation of IL-8 expression in the alveolar RH30 cells was sufficient to limit the formation of xenotransplanted tumors in vivo.…”

“…Consistent with this, shRNA-mediated downregulation of IL-8 expression in the alveolar RH30 cells was sufficient to limit the formation of xenotransplanted tumors in vivo. 74 In response to hypoxia, an increased glucose uptake was also observed in the A204 cell line to be protective against cell death 77 ; however, it should be noted that this cell line is no longer retained as truly representative of RMS. 78 Finallly, the cardiac-specific deletion of the von Hippel-Lindau protein (VHL), an E3 ubiquitin ligase responsible of suppression of HIF levels during normoxia, has been described to favor the formation of highly metastatic cardiac RMS in a mouse model.…”