NF-κB-induced IL-6 ensures STAT3 activation and tumor aggressiveness in glioblastoma.

Glioblastoma (GBM) is the most aggressive, neurologically destructive and deadly tumor of the central nervous system (CNS). In GBM, the transcription factors NF-κB and STAT3 are aberrantly activated and associated with tumor cell proliferation, survival, invasion and chemoresistance. In addition, common activators of NF-κB and STAT3, including TNF-α and IL-6, respectively, are abundantly expressed in GBM tumors. Herein, we sought to elucidate the signaling crosstalk that occurs between the NF-κB and STAT3 pathways in GBM tumors. Using cultured GBM cell lines as well as primary human GBM xenografts, we elucidated the signaling crosstalk between the NF-κB and STAT3 pathways utilizing approaches that either a) reduce NF-κB p65 expression, b) inhibit NF-κB activation, c) interfere with IL-6 signaling, or d) inhibit STAT3 activation. Using the clinically relevant human GBM xenograft model, we assessed the efficacy of inhibiting NF-κB and/or STAT3 alone or in combination in mice bearing intracranial xenograft tumors in vivo. We demonstrate that TNF-α-induced activation of NF-κB is sufficient to induce IL-6 expression, activate STAT3, and elevate STAT3 target gene expression in GBM cell lines and human GBM xenografts in vitro. Moreover, the combined inhibition of NF-κB and STAT3 signaling significantly increases survival of mice bearing intracranial tumors. We propose that in GBM, the activation of NF-κB ensures subsequent STAT3 activation through the expression of IL-6. These data verify that pharmacological interventions to effectively inhibit the activity of both NF-κB and STAT3 transcription factors must be used in order to reduce glioma size and aggressiveness

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

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 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

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

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