Activation of the NF-Kb Pathway by the STAT3 Inhibitor JSI-124 in Human Glioblastoma Cells

Published OnlineFirst February 5, 2013; DOI: 10.1158/1541-7786.MCR-12-0528

Molecular Cancer Chromatin, Gene, and RNA Regulation Research

Activation of the NF-kB Pathway by the STAT3 Inhibitor JSI-124 in Human Glioblastoma Cells

Braden C. McFarland, G. Kenneth Gray, Susan E. Nozell, Suk W. Hong, and Etty N. Benveniste

Abstract Glioblastoma tumors are characterized by their invasiveness and resistance to therapies. The transcription factor signal transducer and activator of transcription 3 (STAT3) was recently identified as a master transcriptional regulator in the mesenchymal subtype of glioblastoma (GBM), which has generated an increased interest in targeting STAT3. We have evaluated more closely the mechanism of action of one particular STAT3 inhibitor, JSI-124 (cucurbitacin I). In this study, we confirmed that JSI-124 inhibits both constitutive and stimulus-induced Janus kinase 2 (JAK2) and STAT3 phosphorylation, and decreases cell proliferation while inducing apoptosis in cultured GBM cells. However, we discovered that before the inhibition of STAT3, JSI-124 activates the nuclear factor-kB (NF-kB) pathway, via NF-kB p65 phosphorylation and nuclear translocation. In addition, JSI-124 treatment induces the expression of IL-6, IL-8, and suppressor of cytokine signaling (SOCS3) mRNA, which leads to a corresponding increase in IL-6, IL-8, and SOCS3 protein expression. Moreover, the NF-kB–driven SOCS3 expression acts as a negative regulator of STAT3, abrogating any subsequent STAT3 activation and provides a mechanism of STAT3 inhibition after JSI-124 treatment. Chromatin immunoprecipitation analysis confirms that NF-kB p65 in addition to other activating cofactors are found at the promoters of IL-6, IL-8, and SOCS3 after JSI-124 treatment. Using pharmacological inhibition of NF-kB and inducible knockdown of NF-kB p65, we found that JSI-124–induced expression of IL-6, IL-8, and SOCS3 was significantly inhibited, showing an NF-kB–dependent mechanism. Our data indicate that although JSI-124 may show potential antitumor effects through inhibition of STAT3, other off-target proinflammatory pathways are activated, emphasizing that more careful and thorough preclinical investigations must be implemented to prevent potential harmful effects. Mol Cancer Res; 11(5); 494–505. 2013 AACR.

Introduction GBM tumors that recur are of the mesenchymal subtype (3). k k Glioblastoma (GBM) is the most common malignant Two pathways of interest, the nuclear factor- B (NF- B) tumor of the central nervous system and remains a chal- and Janus Kinase (JAK)/signal transducer and activator of lenging disease to treat because of the high rate of recurrence transcription (STAT), have been linked to this deadly subtype (5–7). and resistance to current treatments (1, 2). Recent efforts k have been made elucidating genetic classifications of GBM The NF- B family consists of 5 members (p65/RelA, RelB, c-Rel, NF-kB1/p50, and NF-kB/p52; ref. 8). The tumors with the hopes of developing more effective therapies k (3, 4). The Cancer Genome Atlas (TCGA) has determined predominant form of NF- B is a dimer composed of p65 that GBM tumors can be genetically divided into 4 subtypes: and p50 proteins and under basal conditions is found in an classical, mesenchymal, proneural, and neural (5). Of these inactive form in the cytosol through interactions with the inhibitor of NF-kB(IkB) proteins, specifically IkBa.In subtypes, mesenchymal tumors have been associated with a k the worst survival. In addition, it is believed that almost all response to stimuli such as TNF- , the upstream kinase, I B Kinase (IKK), phosphorylates IkBa, which leads to ubiqui- tination and degradation of IkBa (8, 9). The NF-kB p65/ p50 dimer then translocates into the nucleus where it Authors' Affiliation: Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama becomes phosphorylated, binds to DNA, and induces the expression of genes involved in inflammation such as IL-8 Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). and IL-6, as well as antiapoptotic genes including cIAP2, Bcl-2, and Bcl-xL (10). Corresponding Author: Braden C. McFarland, Department of Cell, Devel- opmental and Integrative Biology, 1918 University Blvd, MCLM 313, The JAK/STAT pathway is commonly activated in response University of Alabama at Birmingham, Birmingham, AL 35294. Phone: to various immune-mediated and inflammatory stimuli (11). 205-934-7668; Fax: 205-975-6748; E-mail: [email protected] There are 4 JAK kinases (JAK1, 2, 3, and TYK2) that are doi: 10.1158/1541-7786.MCR-12-0528 responsible for phosphorylating 7 STAT proteins (STAT1-5a, 2013 American Association for Cancer Research. -5b, and -6). Cytokines of the interleukin (IL)-6 family

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(including IL-6, leukemia inhibitory factor [LIF], Oncostatin (S276) were from Cell Signaling Technologies; total IKKa/ M [OSM], and IL-11) preferentially phosphorylate and activ- b, total IkBa, SOCS3, phosphorylated JAK2 (Y1007/ ate JAK2 and STAT3 (12). Cytokine binding to the corre- 1008), total JAK2, and total p65 from Santa Cruz; total sponding extracellular receptor leads to activation of the STAT3, Caspase 3, and PARP from BD Transduction Labs; receptor and in turn causes auto- and trans-phosphorylation and GAPDH from AbCam. For chromatin immunoprecip- of intracellular JAK2 (11). STAT3 then becomes phosphor- itation (ChIP) experiments, Ab to p65 (AbCam), phosphorylated by JAK2, dimerizes, translocates to the nucleus, and ylated p65 (Cell Signaling), phospho-Pol II (S5; Covance), binds to DNA. STAT3 induces the transcription of several and STAT3 (Santa Cruz) were used. JSI-124 and BAY-11- prosurvival genes including IL-6, vascular endothelial growth 7085 were purchased from Calbiochem. OSM was pur- factor, and c-Myc (13). Importantly, STAT3 also induces the chased from R&D Systems. U87-MG and U251-MG cells expression of suppressor of cytokine signaling (SOCS) pro- were maintained as previously described (19). U251-MG teins, specifically SOCS3, which functions as a negative cells were authenticated and are the same as the parent line of regulator preventing prolonged STAT3 activation (14). Dr Darrell Bigner (Duke University, Durham, NC). U87- Numerous studies have showed that the activities of MG cells were purchased from American Type Culture STAT3 and NF-kB are elevated in many cancers, including Collection (ATCC, Manassas, VA) and are authentic and GBM (10, 13, 15, 16). We and others have observed increased consistent with the short tandem repeat profile in the ATCC activity and protein levels of both NF-kB and STAT3 in database. U251-MG cells that stably express the tetracycline GBM (17–20). Moreover, STAT3 has been determined to be repressor (TetR) protein and inducibly express short hairpin a master regulator of the mesenchymal subtype of GBM RNA (shRNA) specific for NF-kB p65 (sh-p65) were gen- (6, 7), and loss of IkBa has also been linked to GBM (21). erated as previously described (19). Briefly, the plasmid Because of the increased interest in these 2 pathways, numer- carrying shRNA specific for p65 was generated by annealing ous pharmacological inhibitors have been developed targeting double-stranded oligonucleotide specific for a 19-bp stretch NF-kB and STAT3 with the hopes of benefiting patients with of the p65 ORF (AA CTG TTC CCC CTC ATC TT) into various cancers including GBM (22, 23). the pBABE-HI-TetO plasmid, which is under dual control JSI-124 (cucurbitacin I), a chemical compound belonging to of the tetracycline operator and the HI polymerase (Pol) III the cucurbitacin family, was first discovered as a potent STAT3 promoter. The pBABE–HI–TetO plasmid was a generous inhibitor in multiple cancer cell lines (24). Inhibition of STAT3 gift of Dr Xinbin Chen (University of California at Davis, activity was attributed to a disruption in STAT3 DNA binding CA). Primary astrocyte cultures from C57BL/6 mice were activity and gene expression, and a reduction in subcutaneous established from neonatal cerebra as described (28). lung and breast xenograft tumors was observed with JSI-124 treatment (24), showing antitumor potential. Furthermore, Immunoblotting JSI-124 has been shown to be a strong activator of apoptosis in Cells were harvested and lysed in radioimmunoprecipita- multiple tumor cell lines including GBM and has a synergistic tion assay buffer lysis buffer with protease inhibitors. Protein effect with other antitumor agents (20, 25–27).However,the concentration was determined using the Pierce BCA Assay. exact mechanism of STAT3 inhibition by JSI-124 remains Equivalent amounts of protein were run on sodium dodecyl unclear. In this study, we set out to examine more closely the sulfate-polyacrylamide gel electrophoresis gels and then effect of JSI-124 treatment on GBM tumor cells in vitro. transferred onto a nitrocellulose membrane. After blocking Consistent with other findings, we found that JSI-124 inhib- with 5% milk in Tris-buffered saline (0.01% Tween) for 1 ited both constitutive and stimulus-induced JAK2 and STAT3 hour, primary Ab were incubated overnight at 4C, followed phosphorylation, decreased cell proliferation, and induced by 1 hour with biotinylated horseradish peroxidase second- apoptosis in human GBM cells. However, before the inhibition ary Ab, and developed with chemiluminescent enhanced of STAT3, time course analysis revealed activation of the NF- chemiluminescence (Pierce), as previously described (29). kB pathway starting at 15 minutes after JSI-124 treatment. In Immunoblots were initially probed with antibodies directed addition, we found that JSI-124–induced mRNA expression of toward the phosphorylated protein followed by stripping IL-8, IL-6,andSOCS3. This increase in mRNA expression and reprobing with antibodies directed against the total translated to protein expression as determined by both IL-8 and protein levels. IL-6 secretion and cellular SOCS3 protein levels. Pharmaco- logical inhibition and inducible knockdown of NF-kBp65 Subcellular fractionation significantly inhibited JSI-124–induced gene expression. These Nuclear and cytosolic fractions were isolated as pre- data reveal that in addition to inhibition of STAT3, additional viously described (30). Briefly, cells were washed twice off-target pathways, including NF-kB, are activated in response with cold PBS followed by the addition of 0.05% NP-40 to JSI-124 treatment. lysis solution to each sample. Cells were collected and centrifuged at 2,700 g for 10 minutes at 4C. Super- natant (cytosol) was removed, centrifuged at 20,000 g Materials and Methods for 15 minutes, and the top 100 mL saved as the pure Reagents and cells cytosolic fraction. The pellet (nuclei) was resuspended in Antibodies (Ab) to phosphorylated STAT3 (Y705), phos- wash buffer and centrifuged at 2,700 g for 5 minutes. phorylated IKKa/b (S176/180), and phosphorylated p65 The supernatant was discarded, pellet resuspended in wash

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buffer, layered on top of 1 mL of 1M sucrose and centri- Results fuged at 2,700 g for 10 minutes. The supernatant was JSI-124 inhibits constitutive and stimulus-induced JAK2 discarded, and the pellet was washed in 0.05% NP-40 lysis and STAT3 activation in GBM cells solution and centrifuged at 2,700 g for 5 minutes. The JSI-124 was originally discovered as a STAT3 inhibitor pellet was resuspended in nuclear extraction buffer, incu- (24). First, we validated the inhibition of STAT3 in cultured batedonicefor30minutes,andcentrifugedat20,000 g human GBM cells. U251-MG cells have basal JAK2 and for 15 minutes. The supernatant was saved as pure nuclear STAT3 phosphorylation, and JSI-124 (1 mmol/L) inhibited extract. To verify purity, samples were immunoblotted the constitutive phosphorylation of both JAK2 and STAT3 with caspase 3 and PARP Ab for cytosolic and nuclear (Fig. 1A). JSI-124 pretreatment also prevented OSM- fractions, respectively. induced phosphorylation of JAK2 and STAT3 in a dose- dependent manner (Fig. 1B). Because of the greatly Cell proliferation assay 4 enhanced phosphorylation of STAT3 after OSM stimula- Cells were plated in 96-well plates at a density of 1 10 tion, we have provided an appropriately exposed blot reveal- cells/well, and the WST-1 Cell Proliferation Assay (Roche) ing the constitutive STAT3 phosphorylation (Supplemental was conducted as previously described (31). Fig. 1A). Both constitutive and stimulus-induced JAK2 and STAT3 phosphorylation were also inhibited in U87-MG ELISA cells (Supplementary Figs. S1B and S1C). These results Cells were treated with JSI-124 for the indicated times verify the previously established literature regarding JSI- and supernatants collected. Human IL-6 and IL-8 ELISA 124 inhibition of STAT3 (24). kits were purchased from BioLegend and conducted as previously described (32). JSI-124 decreases cell proliferation and induces cell death Quantitative RT-PCR in GBM cells Cells were washed with PBS, and total RNA was isolated It has been shown that in addition to inhibition of using TRIzol (Invitrogen) as previously described (28). STAT3, JS1-124 decreased cell proliferation and induced Approximately 1 mg of RNA per sample was used to gene- apoptosis (24, 25). It was previously shown that this decrease rate cDNA by reverse transcription for PCR. Predesigned in cell proliferation was due in part to a JSI-124–induced Taqman primers (Applied Biosystems) were used to obtain G2/M arrest and reduction of cyclin D1 (25), but the exact quantitative PCR results using the Applied Biosytems Ste- mechanism remains unclear. We also observed a reduction pOnePlus Real-Time PCR System Thermal Cycling Block in cell proliferation with JSI-124 treatment of both U251- and corresponding software analysis for data quantification MG and U87-MG cells (Fig. 1C and Supplementary Fig. (StepOneSoftware v2.1; Applied Biosystems). The follow- S1D). Dose–response analysis revealed that 0.1 mmol/L ing Taqman primers and the corresponding Gene Ref# JSI-124 was sufficient to significantly inhibit proliferation were used: human IL-6 (Hs00174131_m1), human IL-8 by 24 hours. Furthermore, a cytotoxic drug dose–response (Hs00174103_m1), human NFKBIA (Hs00153283_m1), curve yields an IC50 value of approximately 28 nmol/L and human SOCS3 (Hs02330328_s1). Eukaryotic 18s (Fig. 1D). This reduction in cell proliferation was accom- rRNA (Hs99999901_s1) was used as an endogenous panied by an increase in cell death as measured by the control. presence of cleaved (c) caspase 3 and poly-ADP ribose polymerase (PARP; Fig. 1E). Chromatin immunoprecipitation ChIP assays were conducted as previously described JSI-124 treatment activates the NF-kB pathway (19, 32). Immunoprecipitation was done with 5 mg of the To evaluate other potential effects of JSI-124 treatment, appropriate Ab, and immune complexes absorbed with U251-MG cells were incubated with JSI-124 (1 mmol/L) protein A beads or protein G beads blocked with bovine for various times. Time course analysis revealed phosphor- serum albumin and salmon sperm DNA. Immunoprecipi- ylationofNF-kB p65, starting as early as 15 minutes after tated DNA was subjected to semiquantitative PCR and JSI-124 treatment and decreasing after 1 hour (Fig. 2A). analyzed by gel electrophoresis using the following primers: Moreover, the JSI-124–induced phosphorylation of IL-8 Forward 50-ggg cca tca gtt gca aat c-30; IL-8 Reverse 50- NF-kB was accompanied by an increase in the expression ttc ctt ccg gtg gtt tct tc-30; IL-6 Forward 50-gcg atg gag tca gag of IkBa (Fig. 2B). In addition, both JNK and p38 MAPK, gaa ac-30; IL-6 Reverse 50-tga ggc tag cgc taa gaa gc-30; 2 pathways commonly activated during stress, were SOCS3 Forward 50-ggt ctc ccc tct gga atc tg-30; SOCS3 also found to be activated (data not shown), which has Reverse 50-ccc cca aac ttc tca ttc aca-30. also recently been shown in leukemia cells treated with JSI-124 (33). Activation of the NF-kB pathway involves Statistical analysis nuclear translocation of NF-kB p65, where binding of Student t tests were conducted for comparison of 2 values, DNA and transcriptional regulation occurs. Under basal and analysis of variance (ANOVA) analysis was conducted conditions, NF-kB p65 is found sequestered in the cytosol on appropriate multivariable analyses. P < 0.05 was consid- with minimal to no detection in the nucleus (Fig. 2C, ered statistically significant. Lanes1and5).Asapositivecontrol,weobservedthe

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ABJSI-124 (1 μmol/L) OSM

0 1 2 4 6 8 12 24 Time (h) – – 0.1 0.5 1 10 JSI-124 (μmol/L)

p-STAT3 (Y705) p-STAT3 (Y705)

STAT3 STAT3

p-JAK2 (Y1007/1008) p-JAK2 (Y1007/1008)

JAK2 JAK2

GAPDH GAPDH

C D 500 1.0 Untreated 1 nmol/L JSI-124 400 10 nmol/L JSI-124 0.8 0.1 μmol/L JSI-124 1 μmol/L JSI-124 μ 300 10 mol/L JSI-124 0.6

200 0.4 (% of control) Cell proliferation 100 Cell viability

(raw absorbance) (raw 0.2 0 0.0 0244872 0 1 10 100 1,000 10,000 Time (h) JSI-124 (nmol/L)

– 1 nmol/L10 nmol/L0.1 μmol/L1 μmol/L10 μmol/LJSI-124 (24 h)

Caspase 3 (f)

(c)

PARP (f) (c) GAPDH

Figure 1. JSI-124 inhibits constitutive and stimulus-induced JAK2 and STAT3 phosphorylation, decreases cell proliferation, and induces apoptosis in glioblastoma cells. A, U251-MG cells were treated with 1 mmol/L JSI-124 for the indicated times, lysed and immunoblotted with the indicated Ab. B, U251-MG cells were pretreated with various concentrations of JSI-124 for 2 hours, followed by treatment with 1 ng/mL OSM for 15 minutes. Cells were lysed and immunoblotted with the indicated Ab. C, U251-MG cells were plated in 96-well plates and incubated with various concentrations of JSI-124 or vehicle control (dimethyl sulfoxide; DMSO) for the indicated times, and the WST-1 cell proliferation assay was conducted. Data represent mean standard deviation (SD), replicates of 3. Beginning at 24 hours, the 0.1, 1, and 10 mmol/L concentrations of JSI-124 reached statistical signiﬁcance (P < 0.005; ANOVA). D, cytotoxic drug dose–response curve of U251-MG cells with a 24 hours treatment with JSI-124. E, U251-MG cells were treated with various concentrations of JSI-124 for 24 hours, lysed and immunoblotted with the indicated Ab. presence of nuclear NF-kBp65afterTNF-a treatment the phosphorylation of NF-kB p65 as well as nuclear in U251-MG cells (Fig. 2C, Lane 6). Moreover, we translocation. found that JSI-124 treatment also induced nuclear trans- The NF-kB pathway is activated in response to stimuli location of NF-kB p65 within 30 minutes (Fig. 2C, Lane such as TNF-a, which leads to phosphorylation of IKK and 8). These results indicate that JSI-124 treatment results in the degradation of IkBa by the proteasome (8, 9). Using

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AB JSI-124 (1 μmol/L) 12 * 0 0.08 0.25 0.5 1 2 4 16 Time (h) 10 8 p-p65 6

p65 4 * * (fold change) (fold I κ B α expression 2 GAPDH 0 0 0.5 1 2 416 CDTime (h) Cytosolic Nuclear

––15 30 – – 15 30 JSI-124 (min) TNF-α JSI-124 α –––+–––+ TNF- 0 5 10 15 0 5 10 15 Time (min) p-IKK α/β p65

IKK α/β Caspase 3 IκBα

PARP GAPDH 12345678Lane 123 45 6 7 8Lane

Figure 2. JSI-124 treatment induces NF-kB p65 phosphorylation and nuclear translocation independent of IKK phosphorylation. A, U251-MG cells were treated with JSI-124 (1 mmol/L) for the indicated times. Cells were lysed and immunoblotted with the indicated Ab. B, U251-MG cells were treated with JSI-124 (1 mmol/L) for the indicated times, followed by isolation of RNA, generation of cDNA, and quantitative RT-PCR using Taqman primers. Data represent mean SD, replicates of 3 (P < 0.001; ANOVA). C, U251-MG cells were treated with TNF-a (10 ng/mL) for 15 minutes or JSI-124 (1 mmol/L) for 15 or 30 minutes. Cells were lysed and nuclear and cytosolic fractions were isolated, followed by immunoblotting with the indicated Ab. Caspase 3 and PARP Ab were used to verify purity of cytosolic and nuclear fractions, respectively. D, U251-MG cells were treated with TNF-a (10 ng/mL) or JSI-124 (1 mmol/L) for the indicated times. Cells were lysed and immunoblotted with the indicated Ab.

TNF-a as a positive control, we observed IKK phosphor- endogenous negative regulator of the JAK/STAT3 pathway, ylation and IkBa degradation within 5 minutes of TNF-a which is most often induced by JAK/STAT3 activation treatment (Fig. 2D). However, we did not observe phos- (ref. 14; Fig. 3C; Supplementary Fig. S2). The JSI-124– phorylation of IKK in response to JSI-124 treatment. This induced gene expression was also validated in human GBM indicates that activation of the NF-kB pathway in response neurospheres (X1066 cells) as well as murine primary to JSI-124 is not mediated through IKK phosphorylation, astrocytes (Supplementary Fig. S3). which will be further explained in the discussion. Modest To verify the induction of IL-6 and IL-8 translation to degradation of IkBa by JSI-124 was observed by 15 minutes protein, we treated U251-MG cells with JSI-124 for various (Fig. 2D, Lane 8), which is necessary to allow NF-kB p65 times, collected supernatants, and measured secreted IL-6 translocation into the nucleus. Overall, these results conﬁrm and IL-8 by ELISA. We found a signiﬁcant increase in the that JSI-124 treatment activates the NF-kB pathway. amount of secreted IL-6 and IL-8 after JSI-124 treatment (Fig. 3D and E). In addition, beginning at 2 hours after JSI- JSI-124 treatment induces IL-6, IL-8, and SOCS3 124 treatment, SOCS3 protein expression was increased expression (Fig. 3F). As JSI-124 activates intracellular signaling cascades including NF-kB, we evaluated the induction of several Phosphorylated p65 and RNA polymerase (Pol) II are potential downstream genes. We found that JSI-124 treat- found at the promoters of IL-6, IL-8, and SOCS3 in cells ment induced mRNA expression of IL-6 and IL-8 in both treated with JSI-124 U251-MG (Fig. 3A and B) and U87-MG cells (Supple- To further characterize the role of NF-kB p65 in JSI- mentary Fig. S2) as measured by quantitative RT-PCR. Both 124–induced gene expression, the presence of several IL-6 and IL-8 are known targets of NF-kB p65 (13). We also transcription factors at the promoters of IL-6, IL-8, and observed an increase in the mRNA expression of SOCS3, an SOCS3 was evaluated. Analysis reveals the presence of total

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A D 7 50 ** 6 ** 40 5 4 ** 30 3 20 (fold change) (fold

IL-6 expression 2 10 1 Secreted IL-6 (pg/mL) 0 0 0 12 416 0 12424 Time (h) Time (h) BE 100 ** 1,750 90 ** 1,500 80 70 1,250 60 1,000 50 40 750 (fold change) (fold IL-8 expression 30 500 20 ** **

Secreted IL-8 (pg/mL) 250 10 0 0 0 12 416 0 12424 Time (h) Time (h) CF 7 ** 6 JSI-124 (1 μmol/L) 5 0 12424 Time (h) 4 ** SOCS3 3

(fold change) (fold 2 ** GAPDH SOCS3 expression 1 0 0 12 416 Time (h)

Figure 3. JSI-124 induces the expression of IL-6, IL-8, and SOCS3.A–C, U251-MG cells were treated with JSI-124 (1 mmol/L) for the indicated times, followed by isolation of RNA, generation of cDNA, and quantitative RT-PCR using Taqman primers. Data represent mean SD, replicates of 3 (P < 0.001; ANOVA). D and E, supernatants were collected from U251-MG cells treated with JSI-124 (1 mmol/L) for the indicated times, and ELISA done for detection of IL-6 and IL-8 secreted protein. Data represent mean SD, replicates of 3 (P < 0.001; ANOVA). F, U251-MG cells were treated with JSI-124 (1 mmol/L) for the indicated times. Cells were lysed and immunoblotted with the indicated Ab. and phosphorylated p65 as well as phosphorylated Blockade of the NF-kB pathway inhibits JSI-124– RNA Pol II at the promoters of IL-6, IL-8, and SOCS3 induced gene expression after JSI-124 treatment (Fig. 4A–C). Phosphorylation of To verify the role of the NF-kB pathway in JSI-124– RNA Pol II at Serine 5 (S5) is necessary and indicative induced gene expression, we used a pharmacological inhib- of transcriptional initiation and activation (34). STAT3 itor of the NF-kB pathway, BAY-11-7085 (BAY-11; was not at the promoters of the genes analyzed upon JSI- Supplementary Fig. S4A; ref. 35). U251-MG cells were 124 treatment, indicating that STAT3 is not activated pretreated with BAY-11 for 2 hours followed by treatment or responsible for the increase in expression of IL-6, IL-8, with JSI-124 for 1 hour, and quantitative RT-PCR was or SOCS3 observed after JSI-124 treatment (Fig. 4D). This conducted. JSI-124 treatment induced mRNA expression conﬁrms that in response to JSI-124 treatment, activated of IL-6, IL-8, and SOCS3, which was inhibited by pre- NF-kB p65 is recruited to the promoters of IL-6, IL-8, treatment with BAY-11 (Fig. 5A and B). This suggests that and SOCS3, and is responsible for the increase in gene the NF-kB pathway is necessary for JSI-124 induction of expression. IL-6, IL-8, and SOCS3. To examine the necessity of NF-kB

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Figure 4. ChIP reveals the presence of phosphorylated p65 and RNA Pol II at the promoters of IL-6, IL-8, and SOCS3 of JSI-124–treated cells. U251-MG cells were left untreated (UT) or treated with JSI- 124 (1 mmol/L) or DMSO (vehicle) for 1 hour. Immunoprecipitation was done with 5 mgofAbto phospho-p65 (A), total p65 (B), phospho-RNA Pol II (C), and STAT3 (D). The immune complexes were absorbed with protein A beads or protein G beads blocked with bovine serum albumin and salmon sperm DNA. Immunoprecipitated DNA was then subjected to semiquantitative PCR and analyzed by gel electrophoresis for IL-6, IL-8,andSOCS3. The experiment was repeated, and similar results were observed.

p65 in JSI-124–induced gene expression, we used an induc- decrease in proliferation with knockdown of NF-kB p65 ible p65 knockdown U251-MG–derived cell line, U251- (Fig. 6C). Using this information, we combined knockdown TR/sh-p65 (19). U251-TR/sh-p65 cells were grown in the (þTet) with 2 doses of JSI-124 (low and high). Similar to the absence or presence of Tetracycline (Tet) for 48 hours to pharmacological inhibition results, we observed that com- effectively decrease p65 levels before treatment with JSI-124 bination treatment does not enhance or prevent the decrease (Supplementary Fig. S4B). U251-TR/sh-p65 cells treated in proliferation with JSI-124 treatment (Fig. 6D). Further- with Tet exhibit significantly decreased levels of p65 mRNA more, pharmacological inhibition or shRNA knockdown of (Fig. 5C). Furthermore, loss of p65 significantly inhibited NF-kB p65 does not prevent JSI-124–induced cell death JSI-124–induced IL-8, IL-6, and SOCS3 expression (Fig. (Supplementary Fig. 5A and B). Therefore, JSI-124 effi- 5D–F). Similar to the inhibitor results in Fig. 5A and B, JSI- ciently inhibits proliferation and induces cell death in the 124 gene expression requires the presence of NF-kB p65. absence of NF-kB. In this report, the induction of SOCS3, a negative regulator Blockade of the NF-kB pathway does not inhibit JSI- of STAT3, via JSI-124–induced NF-kB activation could 124–induced phenotypic changes or STAT3 inhibition provide a potential mechanism of the observed STAT3 Next, we used functional analyses to better understand the inhibition. To test this, we evaluated whether blockade of relationship between NF-kB signaling and the effects of JSI- NF-kB would prevent JSI-124–induced STAT3 inhibition. 124 treatment. First, we evaluated a dose–response of BAY- U251-MG as well as U251-TR/sh-p65 cells were treated with 11 in GBM cells and observed a dose-dependent decrease in JSI-124 and a noticeable inhibition of STAT3 was achieved proliferation at 24 hours (Fig. 6A). Using 5 mmol/L BAY-11, (Fig. 6E and F, Lane 2). However, we observed that blockade we tested the combination using 2 doses of JSI-124 (low and (BAY-11) or downregulation of NF-kB p65 (U251-TR/ high) to evaluate any possible additive or synergistic effects sh-p65 cells) does not prevent JSI-124–induced inhibition of (Fig. 6B). We observed no enhanced decrease in prolifera- STAT3 (Fig. 6E and F, Lanes 4 and 3, respectively). tion with the combination of JSI-124 and BAY-11. In addition, pretreatment with BAY-11 does not prevent the dramatic decrease in cell proliferation with the higher dose of Discussion JSI-124 (0.1 mmol/L; Fig. 6B). In the NF-kB p65 knock- In this report, we have determined that the STAT3 down cells (U251-TR/sh-p65), there is no significant inhibitor JSI-124 has the ability to activate signaling

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12 IL-6 2.5 10 IL-8 2 8 1.5 6 1 4 Average RQ Average (fold change) (fold (fold change) (fold 0.5 2 SOCS3 expression 0 0 UT JSI-124 BAY-11 JSI-124 + UT JSI-124 BAY-11 JSI-124 + BAY-11 BAY-11 C D 1.2 5

1 4 0.8 3 0.6 2

0.4 change) (fold (fold change) (fold IL-6 expression p65 expression 0.2 1

0 0 –Tet +Tet –Tet +Tet –Tet +Tet –Tet +Tet UT JSI-124 UT JSI-124 E F 4 4.5 4 3 3.5 3 2.5 2 2

(fold change) (fold 1.5 IL-8 expression 1 change) (fold 1 SOCS3 expression 0.5 0 0 –Tet +Tet –Tet +Tet –Tet +Tet –Tet +Tet UT JSI-124 UT JSI-124

Figure 5. Blockade of the NF-kB pathway inhibits JSI-124–induced expression of IL-6, IL-8,andSOCS3. A and B, U251-MG cells were treated with BAY-11-7085 (BAY-11; 10 mmol/L) for 2 hours before JSI-124 (1 mmol/L) treatment for 1 hour. Isolation of RNA, generation of cDNA, and quantitative RT-PCR was conducted for IL-6 (A), IL-8 (A), and SOCS3 (B) expression. Data represent mean SD, replicates of 3 (P < 0.01; P < 0.001; Students t test). The experiment was repeated, and similar results were observed. C–F, U251-TR/sh-p65 cells were treated with tetracycline (Tet) for 48 hours to decrease p65 mRNA expression (C). After 48 hours of Tet, cells were then treated with JSI-124 (1 mmol/L) for 1 hour. RNA was isolated, followed by generation of cDNA and quantitative RT-PCR was conducted for IL-6 (D), IL-8 (E), and SOCS3 (F). Data represent mean SD, replicates of 3 (P < 0.05; P < 0.005; Students t test). pathways in addition to the inhibition of STAT3 in human also observed after JSI-124 treatment. These data confirm GBM cells. A signaling schematic illustrating the observed the literature regarding inhibition of STAT3 signaling, effects of JSI-124 treatment is provided in Fig. 7. JSI-124 decreased proliferation, and induction of apoptosis in cells was originally discovered as a STAT3 inhibitor in multiple in vitro (24–26). cancer cells (24). We first verified the previously established However, we observed that the time needed to inhibit literature regarding the effects of JSI-124, and observed an STAT3 phosphorylation was much longer than other known inhibition of constitutive and stimulus-induced JAK2 and JAK/STAT inhibitors. For example, we and others have STAT3 phosphorylation in cultured human GBM cells. shown that AZD1480, a specific JAK1/2 inhibitor, decreases Inhibition of proliferation and induction of apoptosis was constitutive STAT3 phosphorylation as early as 30 minutes

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AB 120 120 100 100 Figure 6. Inhibition of NF-kB 80 80 signaling does not prevent JSI- 124–induced phenotypic changes 60 * 60 * or inhibition of STAT3. A and B, 40 40 * U251-MG cells were plated in 96- (% of control) (% of control) Cell proliferation Cell proliferation well plates and incubated with 20 * 20 * * various concentrations of JSI-124 0 0 and/or BAY-11 for 24 hours, and BAY-11 (μmol/L) – 0.1 1510 BAY-11 (μmol/L) –5–5–5 the WST-1 cell proliferation assay JSI-124 (μmol/L) – – 0.01 0.01 0.1 0.1 was conducted. Data represent mean SD, replicates of 3 CD (P < 0.001; ANOVA). C and D, 800 600 sh-p65 +Tet U251-TR/ cells were plated – Tet μ 700 500 +Tet + JSI-124 (0.01 mol/L) in 96-well plates and incubated + Tet +Tet + JSI-124 (0.1 μmol/L) 600 with Tet for the indicated times (C) 400 or pretreated with Tet for 48 hours 500 before plating and treatment with 400 300 various concentrations of JSI-214 300 200 for the indicated times (D), and the (% of control) (% of control)

Cell proliferation 200 Cell proliferation WST-1 cell proliferation assay was 100 conducted. Data represent mean 100 * * SD, replicates of 3 ( P < 0.001; 0 0 ANOVA). E and F, U251-MG cells 0244872 02448 were incubated with BAY-11 Time (h) Time (h) (5 mmol/L) for 2 hours before EF treatment with JSI-124 (1 mmol/L) U251-MG U251-TR/sh-p65 for 2 hours (E), or U251-TR/sh-p65 cells were pretreated with or ––++BAY-11 ––+Tet without Tet for 48 hours before –+–+JSI-124 –++JSI-124 treatment with JSI-124 (1 mmol/L) p-STAT3 (Y705) p-STAT3 (Y705) for 8 hours (F). Cells were lysed and immunoblotted with the STAT3 STAT3 indicated Ab. GAPDH GAPDH

1 2 3 4 Lane 1 2 3 Lane

posttreatment (31, 36). Because the exact mechanism of JSI- was through an IKK-independent mechanism, also known 124 remains unknown, this suggested that the JSI-124 as the atypical pathway (9). In the atypical pathway, reports mechanism of STAT3 inhibition was less speciﬁc and could have suggested that other kinases can also phosphorylate be exerting effects on other signaling pathways. Moreover, IkBa, leading to dissociation and proteasomal degradation we observed that before the inhibition of STAT3, the NF- independent of IKK activation (9). For example, in response kB pathway becomes activated, as observed via phosphor- to UV light or expression of the Her2/Neu oncogene, ylation of NF-kB p65 as well as nuclear translocation and casein kinase II (CK2) phosphorylates IkBa leading to its IkBa degradation (Fig. 7). Similarly, a recent study by degradation and subsequent activation of NF-kB that is Nefedova and colleagues (37) found that JSI-124 treatment dependent on p38 MAPK pathway activation (38, 39). of dendritic cells led to the activation of NF-kB. However, in Interestingly, we did observe modest activation of the p38 this study, the activation of NF-kB was observed after the MAPK pathway in response to JSI-124 treatment (data not inhibition of STAT3 signaling, was IkBa degradation shown). Other kinases, including Syk, c-Src, and Ribosomal independent, and was attributed to the loss of a dominant S6 Kinase 1 (RSK1) have also been linked to activation of negative effect of STAT3 binding to NF-kB family members NF-kB, independent of IKK activation (40–42). Therefore, in these cells. we suspect other kinases may also become activated after There are several mechanisms that ultimately lead to the JSI-124 treatment that might lead to the activation of activation of NF-kB (9, 22). Activation of NF-kB through NF-kB p65, independent of classical IKK activation. the canonical pathway, via stimuli such as TNF-a or LPS, After JSI-124 treatment and NF-kB activation, we mea- involves the phosphorylation and activation of the IKK sured the expression of inﬂammatory NF-kB–regulated complex. IkBa is then phosphorylated by IKK, which genes including IL-6 and IL-8. This expression was trans- targets IkBa for degradation via the proteasome, and allows lated into protein as measured by secreted IL-6 and IL-8 in NF-kB p65 to translocate to the nucleus. Interestingly, we the conditioned medium of cells treated with JSI-124. Both found that the JSI-124–induced activation of NF-kB p65 IL-6 and IL-8 expression have been shown to be upregulated

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JSI-124–Induced NF-kB Activation

Figure 7. Signaling schematic illustrating the effects of JSI-124 treatment in GBM cells. JSI-124 treatment activates the NF-kB pathway by phosphorylation (P) of NF-kB p65 protein, subsequent nuclear localization, and IkBa degradation (Ub, ubiquitin). Once in the nucleus, phosphorylated NF-kB dimers bind to the promoters of IL-6, IL-8, and SOCS3 and begin gene transcription. IL-6 and IL-8 mRNA are translated into protein and secreted into the medium. SOCS3 mRNA is also translated into protein and acts as a negative regulator of JAK/ STAT3, preventing STAT3 activation.

in GBM and the corresponding tumor microenvironment induced inhibition of STAT3 (Fig. 6E and F). This could both in vitro and in vivo (43–45), and IL-6 gene amplifi- be due, in part, to the fact that NF-kB inhibition before JSI- cation has been observed in 40% to 50% of GBM patients 124 treatment significantly reduces, but does not completely and is associated with decreased patient survival (46). abrogate SOCS3 expression (Fig. 5B and F). Therefore, after Although the role of IL-8 is less characterized in GBM JSI-124 treatment, the NF-kB–driven SOCS3 expression biology, studies have shown that IL-8 acts predominantly as acts as a negative regulator of STAT3, abrogating any an inflammatory chemoattractant and proangiogenic factor subsequent STAT3 activation and provides a mechanism (45). This indicates that JSI-124 induction of IL-6 and IL-8 of STAT3 inhibition (Fig. 7). could have potentially counter-productive effects with In conclusion, there have been several papers evaluating regards to inhibiting tumor growth and progression in combination therapies with other anticancer agents and JSI- GBM. 124. Premkumar and colleagues (27) illustrated that Dasa- We also observed the induction and expression of SOCS3 tinib, a small molecule tyrosine kinase inhibitor, synergizes after JSI-124 treatment. Upon typical JAK/STAT activa- with JSI-124 to increase apoptosis and inhibit growth and tion, SOCS3 is expressed to provide a mechanism of negative migration of GBM cells. However, several chemotherapeutic feedback. Once expressed, SOCS3 interacts with various agents including doxorubicin and cisplatin have also been cytokine receptors and/or JAKs to prevent subsequent shown to activate NF-kB, similar to JSI-124 (50). Overall, STAT3 phosphorylation. In this study, we found the this report reveals additional off-target effects of JSI-124 SOCS3 induction was driven by NF-kB, not STAT3. treatment, in addition to the inhibition of STAT3. Given the Although NF-kB–induced SOCS3 is uncommon, we and link between NF-kB, inflammation, and cancer progression, others have shown that under certain conditions, SOCS3 continued research of these preclinical therapeutic agents expression can be induced by NF-kB activation (47–49). must be conducted to fully understand and anticipate We found that the expression of SOCS3 was induced by NF- potential harmful effects. kB as observed by the presence of phosphorylated NF-kB p65 at the promoter of SOCS3 (Fig. 4A). The requirement Disclosure of Potential Conflicts of Interest for NF-kB in SOCS3 expression was validated using phar- No potential conflicts of interest were disclosed. macological inhibition and shRNA knockdown of NF-kB. The induction of SOCS3, a negative regulator of STAT3, via Authors' Contributions JSI-124–induced NF-kB activation could provide a poten- Conception and design: B. C. McFarland Development of methodology: B. C. McFarland, S. E. Nozell tial mechanism of the observed STAT3 inhibition. There- Acquisition of data (provided animals, acquired and managed patients, provided fore, we evaluated whether inhibition of NF-kB would facilities, etc.): B. C. McFarland, S. E. Nozell, S. W. Hong prevent JSI-124–induced STAT3 inhibition, and observed Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- k tational analysis): B. C. McFarland, S. E. Nozell that the blockade (BAY-11) or downregulation of NF- B Writing, review, and/or revision of the manuscript: B. C. McFarland, G. K. Gray, p65 (U251-TR/sh-p65 cells) does not prevent JSI-124– E. N. Benveniste

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Administrative, technical, or material support (i.e., reporting or organizing data, of Paul Fabbri (B. C. McFarland), and funding from the Southeastern Brain Tumor constructing databases): G. K. Gray, S. W. Hong Foundation (E. N. Benveniste). Study supervision: E. N. Benveniste The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement Grant Support in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This work was supported by T32NS048039 (B. C. McFarland), R01CA158534 (E. N. Benveniste), and R01CA138517 (S. E. Nozell) from the National Institutes Received September 6, 2012; revised January 8, 2013; accepted January 24, 2013; of Health, American Brain Tumor Association Basic Research Fellowship in Honor published OnlineFirst February 5, 2013.

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Activation of the NF-κB Pathway by the STAT3 Inhibitor JSI-124 in Human Glioblastoma Cells

Braden C. McFarland, G. Kenneth Gray, Susan E. Nozell, et al.

Mol Cancer Res 2013;11:494-505. Published OnlineFirst February 5, 2013.

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