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Smoking increases the risk for lung cancer considerably, and the fear of lung cancer is of great concern to all whom have smoked. In spite of advancement in the understanding and treatment of lung cancer, the long-term prognosis of lung cancer still remains poor, primarily because many lung carcinomas seed metastases before they are diagnosed. Since metastatic microtumors of the lung are particularly resistant to current therapies, novel strategies for their treatment are necessary. One promising novel strategy is to target the blood supply of the tumor.
This project seeks to block critical factors in the growth of new blood vessels (angiogenesis) into the tumor. Both primary and metastatic lung cancer cells depend on an external blood supply for their survival, but while the primary tumor can be resected, this is not true for all the metastases that have spread out from it. Since angiogenesis is so important for the development of both the primary tumor and of all metastases spreading out from it, lung carcinomas produce various angiogenic factors that signal to the endothelial cells that line the blood vessels to migrate towards the tumor cells and sprout new microvessels surrounding the tumor. If this step could be inhibited, metastases could be prevented and the life of many long-time smokers prolonged.
Although it is known that lung cancer cells produce interleukin-8 (IL-8), and that IL-8 is angiogenic in vivo, it has found littleattention in lung cancer research. Interleukin-8 is better known for its role in inflammatory diseases, where it attracts white blood cells into an area of tissue injury. White blood cells express two kinds of interleukin-8 receptors, called CXCR1, which mediates white cell migration, and CXCR2, whose function is poorly understood. Because of experimental difficulties in detecting IL-8 on endothelial cells, it had been assumed that these receptors are lost in culture. Using a method that shows functional changes of the cytoskeleton (the cellular machinery for cell movement), we have recently shown that lung-derived endothelial cells in culture respond to IL-8, that the CXCR1 is activated briefly and the CXCR2 for prolonged periods of time. This opens the possibility to study the molecular mechanisms involved in the endothelial cell response in several in vitro angiogenesis assays, which include cytoskeletal rearrangement, cell migration and sprouting of vessel-like structures in response to IL-8 or to IL-8 containing supernatants of lung cancer cells. Several approaches will then be taken to prevent these responses. These include blocking antibodies to either of the two receptors and inhibitors that interfere with the IL-8 signal transduction cascade, which is not identical for the two receptors. If the angiogenic response of IL-8 on endothelial cells is due to activation of the CXCR2, as suggested by preliminary results, it appears possible to block this effect of IL-8, mediated by the CXCR2 without interfering with the immune protective role of the CXCR1 on white blood cells. While definition of the molecular mechanisms involved in IL-8 mediated angiogenesis is the immediate goal of this project, potent inhibitors of this angiogenic response are likely to be developed into therapeutics, which will hopefully eradicate metastatic lung cancer in the future. |
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Smoking is a major risk factor for lung cancer, and in spite of medical advancements the prognosis of lung cancer patients remains poor, primarily because of metastatic dissemination of the tumor prior to its diagnosis. Because of the poor prognosis of metastatic lung cancer, novel strategies are necessary for the development of therapies that provide better survival and quality of life for those afflicted. A promising new strategy is the development of anti-angiogenic factors, among them the IL-8 family of chemokines. Although it has been known for several years that IL-8 and gro- are angiogenic factors, they have not found proper attention in this respect, in spite of the fact that lung cancer cells produce IL-8.
IL-8 is better known for its role in acute inflammation, where it attracts neutrophils into an area of tissue injury. Human leukocytes express two kinds of IL-8 receptors, the CXCR1, which mediates leukocyte migration and the CXCR2, whose function is poorly understood. Because of experimental difficulties in detecting IL-8 receptors on endothelial cells in vitro, it had been assumed that these receptors are lost in culture. In vitro experiments using human cells are essential, however, because in vivo experiments in mice have limited meaning, since mice express only one IL-8 receptor, which more closely resembles the CXCR2. Thus murine models reflect poorly the conditions present in humans. Here we show that cultured human endothelial cells express both the CXCR1 and CXCR2, but that the CXCR2 mediates the prolonged IL-8 mediated responses in endothelial cells. Blocking antibodies to the CXCR2 inhibited IL-8 or gro- induced cell retraction and gap formation between endothelial cells as well as haptotaxis on collagen in the presence of a gradient of IL-8 or gro-, while antibodies against the CXCR1 failed to show a blocking effect. Several interventions in the downstream signaling cascade of the CXCR2 (dominant negative rac, inhibition of PI-3 kinase) inhibited the IL-8 mediated response. Furthermore, it was observed that inhibition of the epidermal growth factor receptor (EGFR) with a receptor kinase inhibitor (AG1478) or a blocking antibody, which recognizes an extracellular region of the EGFR both prevented IL-8 mediated cell migration and the outgrowth of capillary structures, two in vitro indicators of angiogenesis. Finally an antibody against HB-EGF (heparin-binding epidermal growth factor-like growth factor), but not against EGF blocked the IL-8 mediated response. The inhibition by anti-EGFR and anti-HB-EGF antibodies indicate that inside-out signaling had led to the cleavage of pro-HB-EGF to HB-EGF, which can activate EGFRs in an autocrine or paracrine fashion. By this means IL-8 can activate EGF receptors on endothelial cells or on surrounding cancer cells.
These results suggest that inhibition of the CXCR2 on endothelial cells may block the angiogenic effect of IL-8 without interfering with the IL-8-mediated immune protective role of the CXCR1 on neutrophils. Blocking antibodies against the CXCR2 or small molecular weight compounds that specifically inhibit the CXCR2 or the EGFR are viable options as anti-angiogenic strategies in lung cancer in the future. These options will be explored in an animal model. The unexpected cross-talk scenario between IL-8 receptors and EGF receptors, suggests that IL-8 mediated activation of CXCR2s on endothelial cells, fibroblasts or monocytes, can indirectly activate EGFRs on lung cancer cells and stimulate their growth. This scenario will be further tested in vitro in co-culture experiments between lung cancer cells and ‘accessory’ cells, since it may have effects on cancer cells growth reaching far beyond the initial hypothesis of angiogenesis. |
