Loss of Global STAT3 Activation but not Tumor-Specific NF-κB Activation Impairs Tumor Growth.

<p><b>A,</b> U251-TR/<i>sh-p65</i> cells were injected subcutaneously into nude mice, and tumor growth was measured on the indicated days. At day 53, mice were randomized into 4 groups (n = 4 per group) to begin treatment (Txt) regimens. Treatment groups consisted of vehicle only (vehicle), food supplemented with doxycycline (Dox, a tetracycline analog to induce p65 shRNA expression), AZD1480 (50 mg/kg) in methylcellulose via oral gavage once a day, or both Dox food and AZD1480 (Dox+AZD1480). Data represent mean ± SEM (*, p<0.05, Students t-test at day 76). <b>B,</b> Frozen tumor samples were homogenized and immunoblotted with the indicated Ab. Representative tumor samples from each group are shown. Densitometric values of total p65 and p-STAT3 were normalized to GAPDH and total STAT3, respectively. <b>C,</b> Frozen tumor samples were homogenized, and RNA isolated, followed by generation of cDNA, and qRT-PCR performed for the indicated genes (n = 2 tumors per condition, replicates of three per tumor). *, p<0.05.</p

Signaling Schematic Illustrating the Cycle of Cooperation Between NF-κB and STAT3.

<p>NF-κB and STAT3 are competent to ensure activation of themselves and each other, either in an autocrine and/or paracrine manner. Upon stimulation with TNF-α, the NF-κB pathway becomes activated, as shown by the phosphorylation and nuclear translocation of NF-κB p65 and transcription of NF-κB genes, including IL-6 and LIF. Newly synthesized IL-6 is secreted by the cells, and binds in an autocrine or paracrine manner to the IL-6 receptor. This leads to activation of the IL-6R/gp130 complex and the intracellular kinases JAK1/2. STAT3 proteins then become phosphorylated by JAK1/2, dimerize, enter the nucleus and begin the transcription of STAT3 driven genes such as SOCS3 and cIAP2.</p

Activation of STAT3 by TNF-α Treatment Induces <i>SOCS3</i> and <i>cIAP2</i> Expression.

<p><b>A</b>, U251-MG cells were stimulated with TNF-α (10 ng/ml) for the indicated times, lysed and immunoblotted with the indicated Ab. <b>B & C</b>, U87-MG cells were stimulated with TNF-α (10 ng/ml) for the indicated times. RNA was isolated, followed by generation of cDNA, and qRT-PCR was performed for the indicated genes. Data are shown as replicates of three and the experiment repeated with similar results observed. *, p<0.05.</p

TNF-α-induced STAT3 Activation is Dependent on NF-κB p65.

<p><b>A & B</b>, U251-TR/<i>sh-p65</i> cells were incubated with tetracycline (Tet) for 48 h prior to stimulation with TNF-α (10 ng/ml) for the indicated times. Cells were lysed and immunoblotted with the indicated Ab (A) or RNA was isolated, followed by generation of cDNA, and qRT-PCR was performed for the indicated genes (B). Densitometric values of p-STAT3, p-p65 and total p65 were normalized to total STAT3, total p65 and GAPDH, respectively. Data are shown as replicates of three. **, p<0.01.</p

TNF-α Recruits p65 to the <i>IL-6</i> Promoter and STAT3 to the <i>IL-6</i> and <i>SOCS3</i> Promoters.

<p>U251-MG cells were grown in the absence or presence of TNF-α (10 ng/ml) for various times, sonicated and soluble chromatin was immunoprecipitated with antibodies specific for p65 or STAT3. Immunoprecipitated DNA was then analyzed by qRT-PCR using primers specific for the <i>IL-6</i> and <i>SOCS3</i> promoters. Each sample was normalized to genomic DNA isolated from cells that were cross-linked and processed, yet did not incur the immunoprecipitation step. The results are shown as percentages of input, replicates of three, and error bars represent standard deviation.</p

TNF-α Induces IL-6 and LIF Expression in Glioma Cells.

<p><b>A&B,</b> U251-MG and X1016 cells were treated with TNF-α (10 ng/ml) for the indicated times. RNA was isolated followed by generation of cDNA, and qRT-PCR was performed for the indicated genes. Data are shown as replicates of three and the experiment repeated with similar results observed. *, p<0.05. <b>C-F,</b> Supernatants were collected from U251-MG and X1016 cells stimulated with TNF-α for the indicated times, and quantitation of secreted IL-6 was measured by ELISA. Data are shown as replicates of three and the experiment repeated with similar results observed. *, p<0.05.</p

TNF-α-induced STAT3 Activation Requires gp130 and JAK Kinases.

<p><b>A & B,</b>U251-MG cells were treated with no IgG, isotype control (1 µg/ml) or anti-gp130 (1 µg/ml) for 2 h followed by treatment with TNF-α (10 ng/ml) for 1 h. RNA was isolated followed by generation of cDNA, and qRT-PCR was performed for the indicated genes. Data are shown as replicates of three. *, p<0.05; **, p<0.01. <b>C & D</b>, U251-MG cells were incubated with AZD1480 (1 µM) for 2 h prior to treatment with TNF-α (10 ng/ml) for 2 h. Cells were lysed and immunoblotted with the indicated Ab (C) or RNA was isolated, followed by generation of cDNA, and qRT-PCR performed for <i>SOCS3</i> (D). Densitometric values of p-STAT3 were normalized to total STAT3. Data are shown as replicates of three. *, p<0.05.</p

Combined Pharmacological Inhibition Disrupts NF-κB and STAT3 Signaling <i>In Vitro</i> and Prolongs Survival <i>In Vivo</i>.

<p><b>A & B,</b> Xenograft X1046 cells were disaggregated into single cells and briefly propagated as neurospheres <i>in vitro</i>. Cells were pre-treated with AZD1480 (1 µM) and/or WA (5 µM) for 2 h prior to TNF-α (10 ng/ml) for 0.25 or 2 h (A) or 2 h (B). Cells were lysed and immunoblotted with the indicated Ab (A), or RNA was isolated followed by generation of cDNA and qRT-PCR was performed for <i>IL-6</i> (B). Densitometric values of p-p65, p-IKKα/β and p-STAT3 were normalized to total p65, total IKKα/β and total STAT3, respectively. Data are shown as replicates of three. *, p<0.05. <b>C,</b> Xenograft X1066 cells were treated with the indicated doses of AZD1480 and/or WA for 48 h, and the WST-1 cell viability assay was performed. Data are shown as replicates of three. *p<0.05. <b>D,</b> Nude mice were injected intracranially with Xenograft X1016 cells. Starting at day 3, mice were treated with vehicle (n = 5), AZD1480 (30 mg/kg, twice a day, n = 5), WA (4 mg/kg, alternate days, n = 5) or both AZD+WA (n = 5) for three weeks. Survival was measured, and mice were euthanized at moribund. *, p<0.05 (LogRank).</p

TNF-α Treatment Activates NF-κB and STAT3 in Glioma Cells.

<p><b>A&B,</b> U87-MG, U251-MG, GL261 and X1016 cells were treated with TNF-α (10 ng/ml) for the indicated times, lysed and immunoblotted with the indicated Ab.</p