PI3K-AKT pathway negatively controls EGFR-dependent DNA-binding activity of Stat3 in glioblastoma multiforme cells.

Glioblastoma multiforme (GBM) cells frequently harbor amplification and/or gain-of-function mutation of the EGFR gene leading to the activation of multiple signaling pathways. Blockade of EGFR activation inhibited the activation of both AKT and Stat3 in U87 and D54 GBM cells and induced spontaneous apoptosis, which were associated with reduction in the steady-state level of Mcl-1. Surprisingly, inhibition of PI3 kinase (PI3K) activity, which in turn inhibited AKT activation, significantly increased the DNA-binding activity of Stat3 in U87 and D54 cells. This was not due to an increase in the level of tyrosine-phosphorylated Stat3. Conversely, ectopic expression of constitutively activated AKT significantly decreased the DNA-binding activity of Stat3 in 293T cells. Interestingly, blockade of protein phosphatase 2A activity in GBM or 293T cells by calyculin A, which activated AKT, stabilized the phosphorylation of multiple Ser/Thr residues that were located in the transactivation domain (TAD) of Stat3 and this in turn completely ablated the DNA-binding activity of Stat3. Collectively, these results suggest that both Stat3 and AKT provide survival signals in U87 and D54 cells, and Ser/Thr phosphorylation of Stat3-TAD by the PI3K-AKT pathway negatively controls the DNA-binding function of Stat3.

Inhibition of constitutively active Stat3 suppresses proliferation and induces apoptosis in glioblastoma multiforme cells.

Glioblastoma multiforme (GBM), the most common and malignant central nervous system tumor in humans, is highly proliferative and resistant to apoptosis. Stat3, a latent transcription factor being activated by aberrant cytokine or growth factor signaling, acts as a suppressor of apoptosis in a number of cancer cells. Here we report that GBM tumors and cell lines contain high levels of constitutively activated Stat3 when compared with normal human astrocytes, white matter, and normal tissue adjacent to tumor. The persistent activation of Stat3 is in part, attributable to an autocrine action of interleukin-6 in the GBM cell line U251. Janus kinase inhibitor AG490 inhibits Stat3 activation with a concomitant reduction in steady-state levels of Bcl-X(L), Bcl-2 and Mcl-1 proteins and induces apoptosis in U251 cells as revealed by Poly (ADP-ribose) polymerase cleavage and Annexin-V staining. Expression of a dominant negative mutant Stat3 protein or treatment with AG490 markedly reduces the proliferation of U251 cells by inhibiting the constitutive activation of Stat3. These results provide evidence that constitutive activation of Stat3 contributes to the pathogenesis of glioblastoma by promoting both proliferation and survival of GBM cells. Therefore, targeting Stat3 signaling may provide a potential therapeutic intervention for GBM.

IL-13R(alpha)2, a decoy receptor for IL-13 acts as an inhibitor of IL-4-dependent signal transduction in glioblastoma cells.

Interleukin (IL)-4 and IL-13 share the type II IL-4 receptor for cell signaling. We show that despite expressing the necessary signaling components, glioblastoma cells failed to respond to either IL-4 or IL-13. This was in part because of the expression of a high-affinity IL-13-binding transmembrane protein IL-13R(alpha)2 that inhibited IL-13-mediated Stat6 activation by acting as a decoy receptor. In contrast, normal human astrocytes that did not express the IL-13R(alpha)2 gene efficiently induced Stat6 activation in response to both IL-4 and IL-13. Transient expression of the IL-13R(alpha)2 transgene in nonexpressing heterologous cells inhibited not only IL-13- but also IL-4-mediated signal transduction and Stat6-responsive gene expression. The inhibition was likely mediated through the physical interaction between the short intracellular domain of the IL-13R(alpha)2 protein and the cytoplasmic domain of the IL-4R(alpha) chain that harbors the Stat6 docking sites. Thus, IL-13R(alpha)2 acts as an inhibitor of IL-4-dependent signal transduction pathways via a novel mechanism that is independent of ligand binding.