activation of the nf-kb pathway by the stat3 inhibitor jsi...

Chromatin, Gene, and RNA Regulation

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

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

AbstractGlioblastoma 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 mastertranscriptional regulator in the mesenchymal subtype of glioblastoma (GBM), which has generated an increasedinterest in targeting STAT3. We have evaluated more closely the mechanism of action of one particular STAT3inhibitor, JSI-124 (cucurbitacin I). In this study, we confirmed that JSI-124 inhibits both constitutive andstimulus-induced Janus kinase 2 (JAK2) and STAT3 phosphorylation, and decreases cell proliferation whileinducing apoptosis in cultured GBM cells. However, we discovered that before the inhibition of STAT3, JSI-124activates the nuclear factor-kB (NF-kB) pathway, via NF-kB p65 phosphorylation and nuclear translocation. Inaddition, 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, theNF-kB–driven SOCS3 expression acts as a negative regulator of STAT3, abrogating any subsequent STAT3activation and provides a mechanism of STAT3 inhibition after JSI-124 treatment. Chromatin immunopre-cipitation analysis confirms that NF-kB p65 in addition to other activating cofactors are found at the promotersof IL-6, IL-8, and SOCS3 after JSI-124 treatment. Using pharmacological inhibition of NF-kB and inducibleknockdown of NF-kB p65, we found that JSI-124–induced expression of IL-6, IL-8, and SOCS3 wassignificantly inhibited, showing an NF-kB–dependent mechanism. Our data indicate that although JSI-124may show potential antitumor effects through inhibition of STAT3, other off-target proinflammatory pathwaysare activated, emphasizing that more careful and thorough preclinical investigations must be implemented toprevent potential harmful effects. Mol Cancer Res; 11(5); 494–505. �2013 AACR.

IntroductionGlioblastoma (GBM) is the most common malignant

tumor of the central nervous system and remains a chal-lenging disease to treat because of the high rate of recurrenceand resistance to current treatments (1, 2). Recent effortshave been made elucidating genetic classifications of GBMtumors with the hopes of developingmore effective therapies(3, 4). The Cancer Genome Atlas (TCGA) has determinedthat GBM tumors can be genetically divided into 4 subtypes:classical, mesenchymal, proneural, and neural (5). Of thesesubtypes, mesenchymal tumors have been associated withthe worst survival. In addition, it is believed that almost all

GBM tumors that recur are of themesenchymal subtype (3).Two pathways of interest, the nuclear factor-kB (NF-kB)and Janus Kinase (JAK)/signal transducer and activator oftranscription (STAT), have been linked to this deadlysubtype (5–7).The NF-kB family consists of 5 members (p65/RelA,

RelB, c-Rel, NF-kB1/p50, and NF-kB/p52; ref. 8). Thepredominant form of NF-kB is a dimer composed of p65and p50 proteins and under basal conditions is found in aninactive form in the cytosol through interactions with theinhibitor of NF-kB (IkB) proteins, specifically IkBa. Inresponse to stimuli such asTNF-a, the upstreamkinase, IkBKinase (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 itbecomes phosphorylated, binds to DNA, and induces theexpression of genes involved in inflammation such as IL-8and IL-6, as well as antiapoptotic genes including cIAP2,Bcl-2, and Bcl-xL (10).The JAK/STATpathway is commonly activated in response

to various immune-mediated and inflammatory stimuli (11).There are 4 JAK kinases (JAK1, 2, 3, and TYK2) that areresponsible for phosphorylating 7 STAT proteins (STAT1-5a,-5b, and -6). Cytokines of the interleukin (IL)-6 family

Authors' Affiliation: Department of Cell, Developmental and IntegrativeBiology, University of Alabama at Birmingham, Birmingham, Alabama

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Braden C. McFarland, Department of Cell, Devel-opmental and Integrative Biology, 1918 University Blvd, MCLM 313,University of Alabama at Birmingham, Birmingham, AL 35294. Phone:205-934-7668; Fax: 205-975-6748; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-12-0528

�2013 American Association for Cancer Research.

MolecularCancer

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(including IL-6, leukemia inhibitory factor [LIF], OncostatinM [OSM], and IL-11) preferentially phosphorylate and activ-ate JAK2 and STAT3 (12). Cytokine binding to the corre-sponding extracellular receptor leads to activation of thereceptor and in turn causes auto- and trans-phosphorylationof intracellular JAK2 (11). STAT3 then becomes phosphor-ylated by JAK2, dimerizes, translocates to the nucleus, andbinds to DNA. STAT3 induces the transcription of severalprosurvival genes including IL-6, vascular endothelial growthfactor, and c-Myc (13). Importantly, STAT3 also induces theexpression of suppressor of cytokine signaling (SOCS) pro-teins, specifically SOCS3, which functions as a negativeregulator preventing prolonged STAT3 activation (14).Numerous studies have showed that the activities of

STAT3 and NF-kB are elevated in many cancers, includingGBM(10, 13, 15, 16).We andothers have observed increasedactivity and protein levels of both NF-kB and STAT3 inGBM (17–20).Moreover, STAT3 has been determined to bea master regulator of the mesenchymal subtype of GBM(6, 7), and loss of IkBa has also been linked to GBM (21).Because of the increased interest in these 2 pathways, numer-ous pharmacological inhibitors have been developed targetingNF-kB and STAT3with the hopes of benefiting patients withvarious cancers including GBM (22, 23).JSI-124 (cucurbitacin I), a chemical compound belonging to

the cucurbitacin family, was first discovered as a potent STAT3inhibitor inmultiple cancer cell lines (24). Inhibitionof STAT3activity was attributed to a disruption in STAT3DNAbindingactivity and gene expression, and a reduction in subcutaneouslung and breast xenograft tumors was observed with JSI-124treatment (24), showing antitumor potential. Furthermore,JSI-124 has been shown to be a strong activator of apoptosis inmultiple tumor cell lines including GBM and has a synergisticeffect with other antitumor agents (20, 25–27). However, theexact mechanism of STAT3 inhibition by JSI-124 remainsunclear. In this study, we set out to examine more closely theeffect of JSI-124 treatment on GBM tumor cells in vitro.Consistent with other findings, we found that JSI-124 inhib-ited both constitutive and stimulus-induced JAK2 and STAT3phosphorylation, decreased cell proliferation, and inducedapoptosis in humanGBMcells.However, before the inhibitionof STAT3, time course analysis revealed activation of the NF-kB pathway starting at 15 minutes after JSI-124 treatment. Inaddition,we found that JSI-124–inducedmRNAexpression ofIL-8, IL-6, and SOCS3. This increase in mRNA expressiontranslated to protein expression as determinedbyboth IL-8 andIL-6 secretion and cellular SOCS3 protein levels. Pharmaco-logical inhibition and inducible knockdown of NF-kB p65significantly inhibited JSI-124–induced gene expression.Thesedata reveal that in addition to inhibition of STAT3, additionaloff-target pathways, includingNF-kB, are activated in responseto JSI-124 treatment.

Materials and MethodsReagents and cellsAntibodies (Ab) to phosphorylated STAT3 (Y705), phos-

phorylated IKKa/b (S176/180), and phosphorylated p65

(S276) were from Cell Signaling Technologies; total IKKa/b, total IkBa, SOCS3, phosphorylated JAK2 (Y1007/1008), total JAK2, and total p65 from Santa Cruz; totalSTAT3, Caspase 3, and PARP from BDTransduction Labs;and GAPDH from AbCam. For chromatin immunoprecip-itation (ChIP) experiments, Ab to p65 (AbCam), phosphor-ylated p65 (Cell Signaling), phospho-Pol II (S5; Covance),and STAT3 (Santa Cruz) were used. JSI-124 and BAY-11-7085 were purchased from Calbiochem. OSM was pur-chased from R&D Systems. U87-MG and U251-MG cellswere maintained as previously described (19). U251-MGcells were authenticated and are the same as the parent line ofDr Darrell Bigner (Duke University, Durham, NC). U87-MG cells were purchased from American Type CultureCollection (ATCC, Manassas, VA) and are authentic andconsistent with the short tandem repeat profile in the ATCCdatabase. U251-MG cells that stably express the tetracyclinerepressor (TetR) protein and inducibly express short hairpinRNA (shRNA) specific for NF-kB p65 (sh-p65) were gen-erated as previously described (19). Briefly, the plasmidcarrying shRNA specific for p65 was generated by annealingdouble-stranded oligonucleotide specific for a 19-bp stretchof the p65 ORF (AA CTG TTC CCC CTC ATC TT) intothe pBABE-HI-TetO plasmid, which is under dual controlof the tetracycline operator and the HI polymerase (Pol) IIIpromoter. The pBABE–HI–TetO plasmid was a generousgift of Dr Xinbin Chen (University of California at Davis,CA). Primary astrocyte cultures from C57BL/6 mice wereestablished from neonatal cerebra as described (28).

ImmunoblottingCells were harvested and lysed in radioimmunoprecipita-

tion assay buffer lysis buffer with protease inhibitors. Proteinconcentration was determined using the Pierce BCA Assay.Equivalent amounts of protein were run on sodium dodecylsulfate-polyacrylamide gel electrophoresis gels and thentransferred onto a nitrocellulose membrane. After blockingwith 5% milk in Tris-buffered saline (0.01% Tween) for 1hour, primary Ab were incubated overnight at 4�C, followedby 1 hour with biotinylated horseradish peroxidase second-ary Ab, and developed with chemiluminescent enhancedchemiluminescence (Pierce), as previously described (29).Immunoblots were initially probed with antibodies directedtoward the phosphorylated protein followed by strippingand reprobing with antibodies directed against the totalprotein levels.

Subcellular fractionationNuclear and cytosolic fractions were isolated as pre-

viously described (30). Briefly, cells were washed twicewith cold PBS followed by the addition of 0.05% NP-40lysis solution to each sample. Cells were collected andcentrifuged at 2,700 � g for 10 minutes at 4�C. Super-natant (cytosol) was removed, centrifuged at 20,000 � gfor 15 minutes, and the top 100 mL saved as the purecytosolic fraction. The pellet (nuclei) was resuspended inwash buffer and centrifuged at 2,700 � g for 5 minutes.The supernatant was discarded, pellet resuspended in wash

JSI-124–Induced NF-kB Activation

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buffer, layered on top of 1 mL of 1M sucrose and centri-fuged at 2,700 � g for 10 minutes. The supernatant wasdiscarded, and the pellet was washed in 0.05%NP-40 lysissolution and centrifuged at 2,700 � g for 5 minutes. Thepellet was resuspended in nuclear extraction buffer, incu-bated on ice for 30 minutes, and centrifuged at 20,000� gfor 15 minutes. The supernatant was saved as pure nuclearextract. To verify purity, samples were immunoblottedwith caspase 3 and PARP Ab for cytosolic and nuclearfractions, respectively.

Cell proliferation assayCells were plated in 96-well plates at a density of 1� 104

cells/well, and the WST-1 Cell Proliferation Assay (Roche)was conducted as previously described (31).

ELISACells were treated with JSI-124 for the indicated times

and supernatants collected. Human IL-6 and IL-8 ELISAkits were purchased from BioLegend and conducted aspreviously described (32).

Quantitative RT-PCRCells were washed with PBS, and total RNA was isolated

using TRIzol (Invitrogen) as previously described (28).Approximately 1 mg of RNA per sample was used to gene-rate cDNA by reverse transcription for PCR. PredesignedTaqman primers (Applied Biosystems) were used to obtainquantitative PCR results using the Applied Biosytems Ste-pOnePlus Real-Time PCR System Thermal Cycling Blockand corresponding software analysis for data quantification(StepOneSoftware v2.1; Applied Biosystems). The follow-ing Taqman primers and the corresponding Gene Ref#were used: human IL-6 (Hs00174131_m1), human IL-8(Hs00174103_m1), human NFKBIA (Hs00153283_m1),and human SOCS3 (Hs02330328_s1). Eukaryotic 18srRNA (Hs99999901_s1) was used as an endogenouscontrol.

Chromatin immunoprecipitationChIP assays were conducted as previously described

(19, 32). Immunoprecipitation was done with 5 mg of theappropriate Ab, and immune complexes absorbed withprotein A beads or protein G beads blocked with bovineserum albumin and salmon sperm DNA. Immunoprecipi-tated DNA was subjected to semiquantitative PCR andanalyzed by gel electrophoresis using the following primers:IL-8 Forward 50-ggg cca tca gtt gca aat c-30; IL-8 Reverse 50-ttc ctt ccg gtg gtt tct tc-30; IL-6 Forward 50-gcg atg gag tca gaggaa ac-30; IL-6 Reverse 50-tga ggc tag cgc taa gaa gc-30;SOCS3 Forward 50-ggt ctc ccc tct gga atc tg-30; SOCS3Reverse 50-ccc cca aac ttc tca ttc aca-30.

Statistical analysisStudent t tests were conducted for comparison of 2 values,

and analysis of variance (ANOVA) analysis was conductedon appropriate multivariable analyses. P < 0.05 was consid-ered statistically significant.

ResultsJSI-124 inhibits constitutive and stimulus-induced JAK2and STAT3 activation in GBM cellsJSI-124 was originally discovered as a STAT3 inhibitor

(24). First, we validated the inhibition of STAT3 in culturedhuman GBM cells. U251-MG cells have basal JAK2 andSTAT3 phosphorylation, and JSI-124 (1 mmol/L) inhibitedthe constitutive phosphorylation of both JAK2 and STAT3(Fig. 1A). JSI-124 pretreatment also prevented OSM-induced phosphorylation of JAK2 and STAT3 in a dose-dependent manner (Fig. 1B). Because of the greatlyenhanced phosphorylation of STAT3 after OSM stimula-tion, we have provided an appropriately exposed blot reveal-ing the constitutive STAT3 phosphorylation (SupplementalFig. 1A). Both constitutive and stimulus-induced JAK2 andSTAT3 phosphorylation were also inhibited in U87-MGcells (Supplementary Figs. S1B and S1C). These resultsverify the previously established literature regarding JSI-124 inhibition of STAT3 (24).

JSI-124 decreases cell proliferation and induces cell deathin GBM cellsIt has been shown that in addition to inhibition of

STAT3, JS1-124 decreased cell proliferation and inducedapoptosis (24, 25). It was previously shown that this decreasein cell proliferation was due in part to a JSI-124–inducedG2/M arrest and reduction of cyclin D1 (25), but the exactmechanism remains unclear. We also observed a reductionin cell proliferation with JSI-124 treatment of both U251-MG and U87-MG cells (Fig. 1C and Supplementary Fig.S1D). Dose–response analysis revealed that 0.1 mmol/LJSI-124 was sufficient to significantly inhibit proliferationby 24 hours. Furthermore, a cytotoxic drug dose–responsecurve yields an IC50 value of approximately 28 nmol/L(Fig. 1D). This reduction in cell proliferation was accom-panied by an increase in cell death as measured by thepresence of cleaved (c) caspase 3 and poly-ADP ribosepolymerase (PARP; Fig. 1E).

JSI-124 treatment activates the NF-kB pathwayTo evaluate other potential effects of JSI-124 treatment,

U251-MG cells were incubated with JSI-124 (1 mmol/L)for various times. Time course analysis revealed phosphor-ylation of NF-kB p65, starting as early as 15 minutes afterJSI-124 treatment and decreasing after 1 hour (Fig. 2A).Moreover, the JSI-124–induced phosphorylation ofNF-kB was accompanied by an increase in the expressionof IkBa (Fig. 2B). In addition, both JNK and p38 MAPK,2 pathways commonly activated during stress, werealso found to be activated (data not shown), which hasalso recently been shown in leukemia cells treated withJSI-124 (33). Activation of the NF-kB pathway involvesnuclear translocation of NF-kB p65, where binding ofDNA and transcriptional regulation occurs. Under basalconditions, NF-kB p65 is found sequestered in the cytosolwith minimal to no detection in the nucleus (Fig. 2C,Lanes 1 and 5). As a positive control, we observed the

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presence of nuclear NF-kB p65 after TNF-a treatmentin U251-MG cells (Fig. 2C, Lane 6). Moreover, wefound that JSI-124 treatment also induced nuclear trans-location of NF-kB p65 within 30 minutes (Fig. 2C, Lane8). These results indicate that JSI-124 treatment results in

the phosphorylation of NF-kB p65 as well as nucleartranslocation.The NF-kB pathway is activated in response to stimuli

such as TNF-a, which leads to phosphorylation of IKK andthe degradation of IkBa by the proteasome (8, 9). Using

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Figure 1. JSI-124 inhibits constitutive and stimulus-induced JAK2 and STAT3 phosphorylation, decreases cell proliferation, and induces apoptosis inglioblastoma cells. A, U251-MGcellswere treatedwith 1mmol/L JSI-124 for the indicated times, lysed and immunoblottedwith the indicated Ab. B, U251-MGcells were pretreated with various concentrations of JSI-124 for 2 hours, followed by treatment with 1 ng/mL OSM for 15 minutes. Cells were lysedand immunoblotted with the indicated Ab. C, U251-MG cells were plated in 96-well plates and incubated with various concentrations of JSI-124 or vehiclecontrol (dimethyl sulfoxide; DMSO) for the indicated times, and the WST-1 cell proliferation assay was conducted. Data represent mean � standarddeviation (SD), replicates of 3. Beginning at 24 hours, the 0.1, 1, and 10mmol/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 concentrationsof JSI-124 for 24 hours, lysed and immunoblotted with the indicated Ab.






TNF-a as a positive control, we observed IKK phosphor-ylation and IkBa degradation within 5 minutes of TNF-atreatment (Fig. 2D). However, we did not observe phos-phorylation of IKK in response to JSI-124 treatment. Thisindicates that activation of the NF-kB pathway in responseto JSI-124 is not mediated through IKK phosphorylation,which will be further explained in the discussion. Modestdegradation of IkBa by JSI-124 was observed by 15minutes(Fig. 2D, Lane 8), which is necessary to allow NF-kB p65translocation into the nucleus. Overall, these results confirmthat JSI-124 treatment activates the NF-kB pathway.

JSI-124 treatment induces IL-6, IL-8, and SOCS3expressionAs JSI-124 activates intracellular signaling cascades

including NF-kB, we evaluated the induction of severalpotential downstream genes. We found that JSI-124 treat-ment induced mRNA expression of IL-6 and IL-8 in bothU251-MG (Fig. 3A and B) and U87-MG cells (Supple-mentary Fig. S2) asmeasured by quantitative RT-PCR. BothIL-6 and IL-8 are known targets ofNF-kBp65 (13).We alsoobserved an increase in the mRNA expression of SOCS3, an

endogenous negative regulator of the JAK/STAT3 pathway,which is most often induced by JAK/STAT3 activation(ref. 14; Fig. 3C; Supplementary Fig. S2). The JSI-124–induced gene expression was also validated in human GBMneurospheres (X1066 cells) as well as murine primaryastrocytes (Supplementary Fig. S3).To verify the induction of IL-6 and IL-8 translation to

protein, we treated U251-MG cells with JSI-124 for varioustimes, collected supernatants, and measured secreted IL-6and IL-8 by ELISA. We found a significant increase in theamount of secreted IL-6 and IL-8 after JSI-124 treatment(Fig. 3D and E). In addition, beginning at 2 hours after JSI-124 treatment, SOCS3 protein expression was increased(Fig. 3F).

Phosphorylated p65 and RNA polymerase (Pol) II arefound at the promoters of IL-6, IL-8, and SOCS3 in cellstreated with JSI-124To further characterize the role of NF-kB p65 in JSI-

124–induced gene expression, the presence of severaltranscription factors at the promoters of IL-6, IL-8, andSOCS3 was evaluated. Analysis reveals the presence of total

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Figure 2. JSI-124 treatment induces NF-kB p65 phosphorylation and nuclear translocation independent of IKK phosphorylation. A, U251-MG cellswere 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 withJSI-124 (1 mmol/L) for the indicated times, followed by isolation of RNA, generation of cDNA, and quantitative RT-PCR using Taqman primers. Datarepresentmean�SD, replicates of 3 (�P < 0.001; ANOVA). C, U251-MGcellswere treatedwith TNF-a (10 ng/mL) for 15minutes or JSI-124 (1 mmol/L) for 15 or30 minutes. Cells were lysed and nuclear and cytosolic fractions were isolated, followed by immunoblotting with the indicated Ab. Caspase 3 andPARPAbwere used to verify purity of cytosolic and nuclear fractions, respectively. D,U251-MGcellswere treatedwith TNF-a (10 ng/mL) or JSI-124 (1mmol/L)for the indicated times. Cells were lysed and immunoblotted with the indicated Ab.

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and phosphorylated p65 as well as phosphorylatedRNA Pol II at the promoters of IL-6, IL-8, and SOCS3after JSI-124 treatment (Fig. 4A–C). Phosphorylation ofRNA Pol II at Serine 5 (S5) is necessary and indicativeof transcriptional initiation and activation (34). STAT3was not at the promoters of the genes analyzed upon JSI-124 treatment, indicating that STAT3 is not activatedor responsible for the increase in expression of IL-6, IL-8,or SOCS3 observed after JSI-124 treatment (Fig. 4D). Thisconfirms that in response to JSI-124 treatment, activatedNF-kB p65 is recruited to the promoters of IL-6, IL-8,and SOCS3, and is responsible for the increase in geneexpression.

Blockade of the NF-kB pathway inhibits JSI-124–induced gene expressionTo verify the role of the NF-kB pathway in JSI-124–

induced gene expression, we used a pharmacological inhib-itor of the NF-kB pathway, BAY-11-7085 (BAY-11;Supplementary Fig. S4A; ref. 35). U251-MG cells werepretreated with BAY-11 for 2 hours followed by treatmentwith JSI-124 for 1 hour, and quantitative RT-PCR wasconducted. JSI-124 treatment induced mRNA expressionof IL-6, IL-8, and SOCS3, which was inhibited by pre-treatment with BAY-11 (Fig. 5A and B). This suggests thatthe NF-kB pathway is necessary for JSI-124 induction ofIL-6, IL-8, and SOCS3. To examine the necessity of NF-kB

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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 donefor 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 withJSI-124 (1 mmol/L) for the indicated times. Cells were lysed and immunoblotted with the indicated Ab.






p65 in JSI-124–induced gene expression, we used an induc-ible p65 knockdown U251-MG–derived cell line, U251-TR/sh-p65 (19). U251-TR/sh-p65 cells were grown in theabsence or presence of Tetracycline (Tet) for 48 hours toeffectively decrease p65 levels before treatment with JSI-124(Supplementary Fig. S4B). U251-TR/sh-p65 cells treatedwith Tet exhibit significantly decreased levels of p65 mRNA(Fig. 5C). Furthermore, loss of p65 significantly inhibitedJSI-124–induced IL-8, IL-6, and SOCS3 expression (Fig.5D–F). Similar to the inhibitor results in Fig. 5A and B, JSI-124 gene expression requires the presence of NF-kB p65.

Blockade of the NF-kB pathway does not inhibit JSI-124–induced phenotypic changes or STAT3 inhibitionNext, we used functional analyses to better understand the

relationship between NF-kB signaling and the effects of JSI-124 treatment. First, we evaluated a dose–response of BAY-11 in GBM cells and observed a dose-dependent decrease inproliferation at 24 hours (Fig. 6A). Using 5mmol/L BAY-11,we tested the combination using 2 doses of JSI-124 (low andhigh) to evaluate any possible additive or synergistic effects(Fig. 6B). We observed no enhanced decrease in prolifera-tion with the combination of JSI-124 and BAY-11. Inaddition, pretreatment with BAY-11 does not prevent thedramatic decrease in cell proliferation with the higher dose ofJSI-124 (0.1 mmol/L; Fig. 6B). In the NF-kB p65 knock-down cells (U251-TR/sh-p65), there is no significant

decrease in proliferation with knockdown of NF-kB p65(Fig. 6C). Using this information, we combined knockdown(þTet) with 2 doses of JSI-124 (low and high). Similar to thepharmacological inhibition results, we observed that com-bination treatment does not enhance or prevent the decreasein proliferation with JSI-124 treatment (Fig. 6D). Further-more, pharmacological inhibition or shRNA knockdown ofNF-kB p65 does not prevent JSI-124–induced cell death(Supplementary Fig. 5A and B). Therefore, JSI-124 effi-ciently inhibits proliferation and induces cell death in theabsence of NF-kB.In this report, the induction of SOCS3, a negative regulator

of STAT3, via JSI-124–induced NF-kB activation couldprovide a potential mechanism of the observed STAT3inhibition. To test this, we evaluated whether blockade ofNF-kB would prevent JSI-124–induced STAT3 inhibition.U251-MG as well as U251-TR/sh-p65 cells were treated withJSI-124 and a noticeable inhibition of STAT3 was achieved(Fig. 6E and F, Lane 2). However, we observed that blockade(BAY-11) or downregulation of NF-kB p65 (U251-TR/sh-p65 cells) does not prevent JSI-124–induced inhibition ofSTAT3 (Fig. 6E and F, Lanes 4 and 3, respectively).

DiscussionIn this report, we have determined that the STAT3

inhibitor JSI-124 has the ability to activate signaling

Figure 4. ChIP reveals the presenceof phosphorylated p65 and RNAPol II at the promoters of IL-6, IL-8,and SOCS3 of JSI-124–treatedcells. U251-MG cells were leftuntreated (UT) or treated with JSI-124 (1 mmol/L) or DMSO (vehicle)for 1 hour. Immunoprecipitationwas done with 5 mg of Ab tophospho-p65 (A), total p65 (B),phospho-RNAPol II (C), andSTAT3(D). The immune complexes wereabsorbed with protein A beads orprotein G beads blocked withbovine serum albumin and salmonsperm DNA. ImmunoprecipitatedDNA was then subjected tosemiquantitative PCR andanalyzed by gel electrophoresis forIL-6, IL-8, and SOCS3. Theexperiment was repeated, andsimilar results were observed.

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pathways in addition to the inhibition of STAT3 in humanGBM cells. A signaling schematic illustrating the observedeffects of JSI-124 treatment is provided in Fig. 7. JSI-124was originally discovered as a STAT3 inhibitor in multiplecancer cells (24). We first verified the previously establishedliterature regarding the effects of JSI-124, and observed aninhibition of constitutive and stimulus-induced JAK2 andSTAT3 phosphorylation in cultured human GBM cells.Inhibition of proliferation and induction of apoptosis was

also observed after JSI-124 treatment. These data confirmthe literature regarding inhibition of STAT3 signaling,decreased proliferation, and induction of apoptosis in cellsin vitro (24–26).However, we observed that the time needed to inhibit

STAT3phosphorylationwasmuch longer than other knownJAK/STAT inhibitors. For example, we and others haveshown that AZD1480, a specific JAK1/2 inhibitor, decreasesconstitutive STAT3 phosphorylation as early as 30 minutes

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Figure 5. Blockade of the NF-kB pathway inhibits JSI-124–induced expression of IL-6, IL-8, and SOCS3. A and B, U251-MG cells were treatedwith 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, andquantitative 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 withtetracycline (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 representmean � SD, replicates of 3 (�P < 0.05; ��P < 0.005; Students t test).






posttreatment (31, 36). Because the exact mechanism of JSI-124 remains unknown, this suggested that the JSI-124mechanism of STAT3 inhibition was less specific and couldbe exerting effects on other signaling pathways. Moreover,we observed that before the inhibition of STAT3, the NF-kB pathway becomes activated, as observed via phosphor-ylation of NF-kB p65 as well as nuclear translocation andIkBa degradation (Fig. 7). Similarly, a recent study byNefedova and colleagues (37) found that JSI-124 treatmentof dendritic cells led to the activation ofNF-kB.However, inthis study, the activation of NF-kB was observed after theinhibition of STAT3 signaling, was IkBa degradationindependent, and was attributed to the loss of a dominantnegative effect of STAT3 binding toNF-kB familymembersin these cells.There are several mechanisms that ultimately lead to the

activation of NF-kB (9, 22). Activation of NF-kB throughthe canonical pathway, via stimuli such as TNF-a or LPS,involves the phosphorylation and activation of the IKKcomplex. IkBa is then phosphorylated by IKK, whichtargets IkBa for degradation via the proteasome, and allowsNF-kB p65 to translocate to the nucleus. Interestingly, wefound that the JSI-124–induced activation of NF-kB p65

was through an IKK-independent mechanism, also knownas the atypical pathway (9). In the atypical pathway, reportshave suggested that other kinases can also phosphorylateIkBa, leading to dissociation and proteasomal degradationindependent of IKK activation (9). For example, in responseto UV light or expression of the Her2/Neu oncogene,casein kinase II (CK2) phosphorylates IkBa leading to itsdegradation and subsequent activation of NF-kB that isdependent on p38 MAPK pathway activation (38, 39).Interestingly, we did observe modest activation of the p38MAPK pathway in response to JSI-124 treatment (data notshown). Other kinases, including Syk, c-Src, and RibosomalS6 Kinase 1 (RSK1) have also been linked to activation ofNF-kB, independent of IKK activation (40–42). Therefore,we suspect other kinases may also become activated afterJSI-124 treatment that might lead to the activation ofNF-kB p65, independent of classical IKK activation.After JSI-124 treatment and NF-kB activation, we mea-

sured the expression of inflammatory NF-kB–regulatedgenes including IL-6 and IL-8. This expression was trans-lated into protein as measured by secreted IL-6 and IL-8 inthe conditioned medium of cells treated with JSI-124. BothIL-6 and IL-8 expression have been shown to be upregulated

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signaling does not prevent JSI-124–induced phenotypic changesor inhibition of STAT3. A and B,U251-MG cells were plated in 96-well plates and incubated withvarious concentrations of JSI-124and/or BAY-11 for 24 hours, andthe WST-1 cell proliferation assaywas conducted. Data representmean � SD, replicates of 3(�P < 0.001; ANOVA). C and D,U251-TR/sh-p65 cells were platedin 96-well plates and incubatedwith Tet for the indicated times (C)or pretreated with Tet for 48 hoursbefore plating and treatment withvarious concentrations of JSI-214for the indicated times (D), and theWST-1 cell proliferation assay wasconducted. Data represent mean� SD, replicates of 3 (�P < 0.001;ANOVA). E and F, U251-MG cellswere incubated with BAY-11(5 mmol/L) for 2 hours beforetreatment with JSI-124 (1 mmol/L)for 2 hours (E), or U251-TR/sh-p65cells were pretreated with orwithout Tet for 48 hours beforetreatment with JSI-124 (1 mmol/L)for 8 hours (F). Cells were lysedand immunoblotted with theindicated Ab.

McFarland et al.





in GBM and the corresponding tumor microenvironmentboth in vitro and in vivo (43–45), and IL-6 gene amplifi-cation has been observed in 40% to 50% of GBM patientsand is associated with decreased patient survival (46).Although the role of IL-8 is less characterized in GBMbiology, studies have shown that IL-8 acts predominantly asan inflammatory chemoattractant and proangiogenic factor(45). This indicates that JSI-124 induction of IL-6 and IL-8could have potentially counter-productive effects withregards to inhibiting tumor growth and progression inGBM.We also observed the induction and expression of SOCS3

after JSI-124 treatment. Upon typical JAK/STAT activa-tion, SOCS3 is expressed to provide amechanism of negativefeedback. Once expressed, SOCS3 interacts with variouscytokine receptors and/or JAKs to prevent subsequentSTAT3 phosphorylation. In this study, we found theSOCS3 induction was driven by NF-kB, not STAT3.Although NF-kB–induced SOCS3 is uncommon, we andothers have shown that under certain conditions, SOCS3expression can be induced by NF-kB activation (47–49).We found that the expression of SOCS3was induced byNF-kB as observed by the presence of phosphorylated NF-kBp65 at the promoter of SOCS3 (Fig. 4A). The requirementfor NF-kB in SOCS3 expression was validated using phar-macological inhibition and shRNA knockdown of NF-kB.The induction of SOCS3, a negative regulator of STAT3, viaJSI-124–induced NF-kB activation could provide a poten-tial mechanism of the observed STAT3 inhibition. There-fore, we evaluated whether inhibition of NF-kB wouldprevent JSI-124–induced STAT3 inhibition, and observedthat the blockade (BAY-11) or downregulation of NF-kBp65 (U251-TR/sh-p65 cells) does not prevent JSI-124–

induced inhibition of STAT3 (Fig. 6E and F). This couldbe due, in part, to the fact that NF-kB inhibition before JSI-124 treatment significantly reduces, but does not completelyabrogate SOCS3 expression (Fig. 5B and F). Therefore, afterJSI-124 treatment, the NF-kB–driven SOCS3 expressionacts as a negative regulator of STAT3, abrogating anysubsequent STAT3 activation and provides a mechanismof STAT3 inhibition (Fig. 7).In conclusion, there have been several papers evaluating

combination therapies with other anticancer agents and JSI-124. Premkumar and colleagues (27) illustrated that Dasa-tinib, a small molecule tyrosine kinase inhibitor, synergizeswith JSI-124 to increase apoptosis and inhibit growth andmigration ofGBMcells.However, several chemotherapeuticagents including doxorubicin and cisplatin have also beenshown to activate NF-kB, similar to JSI-124 (50). Overall,this report reveals additional off-target effects of JSI-124treatment, in addition to the inhibition of STAT3.Given thelink betweenNF-kB, inflammation, and cancer progression,continued research of these preclinical therapeutic agentsmust be conducted to fully understand and anticipatepotential harmful effects.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: B. C. McFarlandDevelopment of methodology: B. C. McFarland, S. E. NozellAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): B. C. McFarland, S. E. Nozell, S. W. HongAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): B. C. McFarland, S. E. NozellWriting, review, and/or revision of the manuscript: B. C. McFarland, G. K. Gray,E. N. Benveniste

Figure 7. Signaling schematicillustrating the effects of JSI-124treatment in GBM cells. JSI-124treatment activates the NF-kBpathway by phosphorylation (P) ofNF-kB p65 protein, subsequentnuclear localization, and IkBadegradation (Ub, ubiquitin). Once inthe nucleus, phosphorylated NF-kBdimers bind to the promoters of IL-6,IL-8, and SOCS3 and begin genetranscription. IL-6 and IL-8mRNAaretranslated into protein and secretedinto the medium. SOCS3 mRNA isalso translated into protein and actsas a negative regulator of JAK/STAT3, preventing STAT3 activation.






Administrative, technical, or material support (i.e., reporting or organizing data,constructing databases): G. K. Gray, S. W. HongStudy supervision: E. N. Benveniste

Grant SupportThis work was supported by T32NS048039 (B. C. McFarland), R01CA158534

(E. N. Benveniste), and R01CA138517 (S. E. Nozell) from the National Institutesof Health, American Brain Tumor Association Basic Research Fellowship in Honor

of Paul Fabbri (B. C. McFarland), and funding from the Southeastern Brain TumorFoundation (E. N. Benveniste).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 6, 2012; revised January 8, 2013; accepted January 24, 2013;published OnlineFirst February 5, 2013.

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2013;11:494-505. Published OnlineFirst February 5, 2013.Mol Cancer Res Braden C. McFarland, G. Kenneth Gray, Susan E. Nozell, et al. Human Glioblastoma Cells

B Pathway by the STAT3 Inhibitor JSI-124 inκActivation of the NF-

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