We demonstrated that a vast majority of patients with high-grade gliomas (HGG) over-express IL13R-α2, an X-linked receptor for Interleukin13 (IL13). We have proposed that epigenetic mechanisms involving activating protein 1 (AP-1) activity regulate the expression of IL13R-α2 in HGG and recent studies demonstrated that the promoter region ofthe receptor’s gene contains both AP-1 and STAT-6 binding sites. We have documented that IL13R-α2 is a very attractive molecular target for anti-HGG targeted therapies. However, little is known whether and how the expression of this receptor is regulated and thus we conducted series of experiments in which either AP-1 or STAT-6 stimulatory cytokines were used, and the levels of receptor monitored. The IL13R-α2 receptor protein levels were examined by Western blot and by immunohistochemistry (IH) in a variety of HGG cells and normal cells. Thus, the cells were treated with epidermal growth factor (EGF) or IL4 or tumor necrosis factor alpha (TNFα), AP-1 and STAT-6 stimulants, respectively. We found that serum-starved HGG cells, such as A-172 MG, U-251 MG, G48a, SNB-19 and U-87 MG, had the levels of immunoreactive IL13R-α2 significantly diminished as detected by both Western blot and IH indicating that this receptor is over-expressed in HGG in a non-constitutive manner. The addition of EGF or TNFα, or IL4 to cells increased prominently the levels of IL13R-α2 protein, usually by three to ten-fold, the extent of which was cytokine-, cell line- and time-dependent. For example, EGF up-regulated the receptor most potently after 24-h treatment in the U-251 MG cells and after 36-h treatment in G48a cells, while IL4 had little effect. We next examined whether an already elevated IL13R-α2 could be further up-regulated by the studied cytokines in the presence of serum in HGG cells. We found that supra-physiologic concentrations of EGF and TNFα, and to a much lesser extent of IL4, could further increase the levels of the receptor in HGG cells. The same experiments were performed on transformed normal glial cells (SVGp12), human endothelial cells (HUVEC) and keratinocytes (HaCat). In general, the background levels of the immunoreactive IL13R-α2 were very low in normal cells when compared with HGG cells. EGF, TNFα or IL4 increased the receptor levels, but they remained still much lower than these found in HGG cells. IL13R-α2 is currently utilized pre-clinically and clinically as a target for targeted recombinant cytotoxins, viruses, cytolytic T cells and vaccines. We demonstrate that a short-term pre-treatment of HGG cells with AP-1 and STAT-6 stimulatory cytokines, some of them with inherent anti-tumor activity, may provide a therapeutic advantage by up-regulating the levels of IL13R-α2.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]