Human Cancer Biology

Loss of Protein Inhibitors of Activated STAT-3 Expression in Glioblastoma MultiformeTumors: Implications for STAT-3 Activation and Expression Emily C. Brantley,1L. Burton Nabors,2 G. Yancey Gillespie,3 Youn-Hee Choi,5 Cheryl Ann Palmer,2,4 Keith Harrison,4 Kevin Roarty,1and Etty N. Benveniste1

Abstract Purpose: STATs activate transcription in response to numerous , controlling prolifera- tion, , and apoptosis. Aberrant activation of STAT proteins, particularly STAT-3, is implicated in the pathogenesis of many cancers, including GBM, by promoting cell cycle progres- sion, stimulating angiogenesis, and impairing tumor immune surveillance. Little is known about the endogenous STAT inhibitors, the PIAS proteins, in human malignancies.The objective of this study was to examine the expression of STAT-3 and its negative regulator, PIAS3, in human tissue samples from control and GBM brains. Experimental Design: Control and GBM human tissues were analyzed by immunoblotting and immunohistochemistry to determine the activation status of STAT-3 and expression of the PIAS3 protein.The functional consequence of PIAS3 inhibition by small interfering RNA or PIAS3 over- expression in GBM cells was determined by examining cell proliferation, STAT-3 transcriptional activity, and STAT-3 target gene expression.This was accomplished using [3H]TdR incorporation, STAT-3 dominant-negative constructs, reverse transcription-PCR, and immunoblotting. Results and Conclusions: STAT-3 activation, as assessed by tyrosine and serine phosphoryla- tion, was elevated in GBM tissue compared with control tissue. Interestingly, we observed expression of PIAS3 in control tissue, whereas PIAS3 protein expression in GBM tissue was greatly reduced. Inhibition of PIAS3 resulted in enhanced glioblastoma cellular proliferation. Conversely, PIAS3 overexpression inhibited STAT-3 transcriptional activity, expression of STAT- 3^ regulated , and cell proliferation.We propose that the loss of PIAS3 in GBM contributes to enhanced STAT-3 transcriptional activity and subsequent cell proliferation.

Who grade 4 glioblastoma multiforme (GBM) is the most the inability of surgery to cure patients even when surgical aggressive malignant astrocytic glioma because of the high resection is possible. In addition, the majority of GBMs are degree of cellularity, vascular proliferation, and necrosis. resistant to standard radiotherapy and chemotherapy (1). Patients diagnosed with GBMs have a median life expectancy Aberrant signaling through receptor tyrosine kinases, includ- of less than 1 year (1). GBMs are characterized by their ing the epidermal growth factor receptor and platelet-derived propensity to infiltrate throughout the brain, which results in growth factor receptor, is a hallmark of GBM (2). Constitutive activation of epidermal growth factor receptor and platelet- derived growth factor receptor promotes cell growth and evasion of apoptosis, events that lead to maintenance of a Authors’Affiliations: Departments of 1Cell Biology, 2Neurology, and 3Surgery and tumor-promoting environment. Deregulated signaling through 4Division of Neuropathology, Department of Pathology, University of Alabama at the mitogen-activated protein kinase, phosphatidylinositol Birmingham, Birmingham, Alabama and 5Department of Physiology, College of 3-kinase/AKT, protein kinase C, nuclear factor-nB (NF-nB), Medicine, EwhaWomans University, Seoul, Korea and Janus-Activated Kinase (JAK)-Signal Transducers and Received 3/7/08; revised 5/12/08; accepted 5/29/08. Grant support: NIH grants P50 CA-097247 (E.N. Benveniste, L.B. Nabors, C.A. Activators of Transcription (STAT) pathways has also been Palmer,andG.Y.Gillespie),CA-112397(L.B.Nabors),andNS-54158(E.N. implicated in glioma development and progression (2, 3). Benveniste) and University of Alabama at Birmingham Cancer Prevention and The JAK family of receptor-associated tyrosine kinases is ControlTraining Program grant R25 CA-47888 (K. Roarty). activated by phosphorylation after binding and activates The costs of publication of this article were defrayed in part by the payment of page STAT proteins to induce gene expression (4). The STAT family charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. consists of seven members (STAT-1, STAT-2, STAT-3, STAT-4, Note: Supplementary data for this article are available at Clinical Cancer Research STAT-5A, STAT-5B, and STAT-6) and is activated by many stimuli, Online (http://clincancerres.aacrjournals.org/). including the interleukin (IL)-6 family. Members Requests for reprints: EttyN.Benveniste,DepartmentofCellBiology,University include IL-6, oncostatin M (OSM), leukemia inhibitory factor, of Alabama atBirmingham,1918 University Boulevard, MCLM 395A, Birmingham, AL 35294-0005. Phone: 205-934-7667; Fax: 205-975-6748; E-mail: [email protected]. ciliary neurotrophic factor, and IL-11 (5). IL-6 cytokines F 2008 American Association for Cancer Research. preferentially activate STAT-3, leading to dimerization, nuclear doi:10.1158/1078-0432.CCR-08-0618 translocation, and binding to IFN-g–activated site-like DNA

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ences in PIAS expression/function depending on the system Translational Relevance under investigation. This study sought to determine the status and functional STAT-3, a cytoplasmic transcription factor that becomes relevance of PIAS3 expression in GBMs. We established a activated in response to a variety of cytokines, chemokines, relation between elevated levels of activated STAT-3 in GBM and growth factors is aberrantly activated in several human and the presence/absence of PIAS3. Additionally, we showed cancers, including GBM. STAT-3 is a promising target for that loss of PIAS3 promoted proliferation of GBM cells. We GBM therapy because it is a convergence point for several propose that PIAS3 loss contributes, in part, to the increased signaling pathways that promote glioma growth and main- STAT-3 activation observed in GBM and proliferation and tenance, and because aberrant STAT-3 activation results survival of glioma cells. from upstream dysregulation, not constitutively active STAT-3 mutations. Pharmacologic inhibitors of STAT-3, including AG490, WP1066, cucurbitacin I, and gefitinib, Materials and Methods have shown promising results in glioblastoma cells in vitro and are in early stages of clinical trials. PIAS3, a negative Cells. U251-MG, U87-MG, SNB-19, M059K-MG, U138-MG, and U118-MG human astroglioma cells were cultured as previously regulator of STAT-3, is a protein whose function is of great described (8). Primary murine astrocytes were >97% positive for GFAP, importance in understanding the regulation of STAT-3 and microglia were >90% positive for Mac1, as previously described signal transduction in vivo. This work provides evidence of (22). Neuronal cultures were prepared by isolating cerebral hemi- PIAS3 dysregulation in GBM and the promotion of STAT-3 spheres from P0 mice and removing the meninges, as described (23). transcriptional activity in human GBM. The presence or GBM cell cultures from four patients were received from the University absence of PIAS3 may determine GBM patients that would of Alabama at Birmingham (UAB) Brain Tumor Bank of the be responsive to STAT-3 inhibitors in future clinical trials. Cooperative Tissue Network, in accordance with the UAB Human Tissue Committee policies, Institutional Review Board Exemption #X050415007. GBM primary cells were obtained after 30 d in culture elements (4). STAT-3 induces expression of genes that regulate and grown in DMEM/F-12 medium supplemented with 2 mmol/L L-glutamine, 100 units/mL penicillin, 100 Ag/mL streptomycin, and antiapoptotic behavior and proliferation, such as survivin, 10% heat-inactivated fetal bovine serum, as previously described (24). vascular endothelial growth factor, c-Myc, and cyclin D1 (6–8). Reagents. Recombinant human OSM, IL-6, and soluble IL-6 Aberrant activation of STAT-3 is observed in primary cancers receptor (sIL-6R) were obtained from R&D Systems. Phorbol 12- (9). In GBM and medulloblastoma tumors, STAT-3 was shown myristate 13-acetate was purchased from Calbiochem. Antibodies to to be constitutively active, as assessed by tyrosine phosphory- STAT-3, PIAS3 (COOH terminus), and p21 were obtained from Santa lation status (10, 11). Activated STAT-3 localized to the vascular Cruz Biotechnology, Inc. Anti-PIAS3 (NH2 terminus) antibody was endothelial growth factor receptor-2 in tumor endothelial cells purchased from Abgent. Antibodies to phosphoserine STAT-3, phos- (11), and immunohistochemical studies of GBM showed that photyrosine STAT-3, and STAT-3 were from Cell Signaling Technology. tumor cells also contained tyrosine-phosphorylated STAT-3 Anti-SOCS-3 antibody was from Zymed and anti-actin antibody was (10, 12, 13). In addition, experimental mouse gliomas express from Sigma. The 3xLy6epZLuc-TK (STAT-3) luciferase construct was obtained from Addgene. The 1,556-bp SOCS-3 promoter has been constitutively activated STAT-3 (13). Due to their ability to previously characterized (25). The matrixmetalloproteinase-9 (MMP- activate STAT-3, IL-6 cytokines have been implicated in 9)-Luc luciferase reporter plasmid containing 670 bp of the human progression of brain tumors (10, 11, 13). Human GBMs MMP-9 promoter was a generous gift from Dr. D. Boyd (M. D. Anderson express elevated levels of OSM and IL-6 (8, 14). Up-regulation CancerCenter,Houston,TX).TheSTAT-3Fdominant-negative of IL-6 and OSM in glioblastoma cells regulates vascular expression construct has been previously described (26). Human endothelial growth factor promoter activity, potentially promot- glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were ing angiogenesis (8, 15). purchased from Clontech. Human PIAS3 cDNA (clone ID: 3528679; Because JAK-STAT signaling is dysregulated in GBM, it is accession number: BC001154) was purchased from Open Biosystems important to characterize negative regulators of this pathway and cloned into the pcDNA3 vector. MG-132 was purchased from to identify potential therapeutic targets. The Protein Inhibitors Calbiochem. Human tissues. Tissues were received from the UAB Brain Tumor of Activated STAT (PIAS) family of proteins (PIAS1, PIAS3, Bank of the Cooperative Tissue Network, in accordance with the UAB PIASx, and PIASy) regulates STAT activity (16). On cytokine Human Tissue Committee policies, Institutional Review Board Exemp- stimulation, PIAS1 and PIAS3 bind activated STAT-1 and tion #X050415007. The nonneoplastic brain tumor biopsy samples STAT-3, respectively, and prevent their ability to bind DNA were obtained from patients diagnosed with epilepsy. The tumor (16). There are limited studies on the expression or function of biopsies were diagnosed as GBM (WHO grade 4 tumors). PIAS proteins in disease states. Increased PIAS3 expression was Small interfering RNA transfection. U87-MG cells were untreated or detected in 100 of 103 human cancers, including lung, breast, transiently transfected using DharmaFECT 1 reagent (Dharmacon) with and brain tumors (17). In contrast, PIAS3 was not detected either 100 nmol PIAS3 siPOOL Reagent or 100 nmol of negative in most samples of anaplastic lymphoma kinase-positive control small interfering RNA (siRNA; Ambion) according to the f T/null-cell lymphoma cells, which correlated with heightened manufacturer’s instructions to achieve 85% transfection efficiency. Twenty-four hours after transfection, cells were collected and analyzed STAT-3 activation (18). Overexpression of PIAS3 induced for target knockdown by reverse transcription-PCR with primers for apoptosis in prostate cancer cells (19) and suppressed growth human PIAS3 and GAPDH. Cells were collected 24, 48, and 72 h after of human lung cancer cells (20). PIAS1 and PIAS3 enhanced transfection and analyzed by immunoblotting for PIAS3 and actin. transcriptional activity of the androgen receptor in prostate Immunoblotting. Nonneoplastic brain biopsy (controls) and GBM cancer cells, whereas PIASy inhibited androgen receptor– biopsy samples were lysed, as described previously (8). Total protein mediated gene expression (21). These results indicate differ- (40 Ag) was resolved on an 8% SDS-PAGE gel, transferred to

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Fig. 1. STAT-3 protein activation is elevated and PIAS3 protein expression is reduced in GBM tissues compared with control brain tissue. A, immunoblot of phospho-Tyr705 STAT-3, phospho-Ser727 STAT-3, total STAT-3, and actin levels in control tissues (n = 8) and GBM tissues (n =14). Representative of three experiments. B, quantitative real-time PCR was done for PIAS3 mRNAin control samples and GBM samples. PIAS3 mRNAexpression is represented as a ratio to the housekeeping gene S9.Columns,meanof three experiments on control (n =11)andGBM(n =11) tissue samples; bars, SD. C, immunoblot of PIAS3 protein in control and GBM tissues. Blots were reprobed for actin to verify protein loading between samples. Representative of four experiments.

nitrocellulose membrane, and blocked for 1 h in 5% milk. The follows: PIAS3, 5¶-CACTCGCGGGCACTGATCAAGGAGA-3¶; SOCS-3, membranes were then probed with antibodies against phosphotyrosine 5¶-ACCAGCTGGTGGTGAACGCAGTGCG-3¶; GAPDH, 5¶ TGGAGGG- STAT-3, phosphoserine STAT-3, total STAT-3 (1:1,000), PIAS3 (1:100 CAAGTCTGGTGCCAGCAG-3¶; and S9, 5¶-AGCAGGTGGTGGTGAACA- dilution; Abgent), SOCS-3 (1:500), and actin (1:1,000 dilution) to TCCCGTCCTT-3¶. For reverse transcription-PCR, 2 Ag RNA was reverse control for loading. The enhanced chemiluminescence method was transcribed to cDNA, which was amplified using primers specific used for protein detection, as previously described (25). For SOCS-3, for human PIAS3 (forward, 5¶-ATTGACTGCTGACCCTGACA-3¶; 100 Ag protein was used. reverse, 5¶-GGGACAGCGAAGTTTCCATA-3¶), human Bcl-xL (forward, U251-MG cells were treated with either 50 ng/mL OSM or 10 ng/mL 5¶-GGAGCTGGTGGTTGACTTTC-3¶; reverse, 5¶-ACAATGCGACCC- IL-6 plus 50 ng/mL sIL-6R for various times. Cells were lysed as CAGTTTAC-3¶), human survivin (forward, 5¶-TTTCTGCCACATCT- previously described (25). Total cell lysate (40 Ag) was resolved on an GAGTCG-3¶; reverse, 5¶-TGTCGAGGAAGCTTTCAGGT-3¶), and 8% SDS-PAGE gel, transferred to nitrocellulose membrane, and then GAPDH (Clontech). Densitometry was used to quantify the PCR blocked for 1 h in 5% milk. Membranes were probed as described above. results. GAPDH mRNA served as an internal control for sample For proteasome inhibition, U251-MG, SNB-19, and GBM primary loading and mRNA integrity, and all results were normalized by the cells were incubated with MG-132 (10 Amol/L) for various times and respective GAPDH values. then collected as described above. Total cell lysate (40 Ag) was resolved Immunohistochemistry. Formalin-fixed, paraffin-embedded tissues on an 8% SDS-PAGE gel and analyzed as described above. Densitom- were obtained from the UAB Brain Tumor Bank, in accordance with etry was used to quantify the immunoblotting results, and actin served the UAB Human Tissue Committee policies, Institutional Review as an internal control for sample loading. All results were normalized Board Exemption #X050415007. Briefly, tissue sections were deparaffi- by the respective actin values. nized, and endogenous peroxidase activity was blocked with 3% RNA isolation, quantitative real-time PCR, and reverse transcription- hydrogen peroxide in PBS. Sections were then incubated in PBS PCR. Total RNA from human tissues was extracted using a PowerGen blocking buffer for 30 min at room temperature. PIAS3 antibody 125 with Trizol. The GeneAmp 7700 Sequence Detection System (1:300) was applied to sections overnight at 4jC. After washes with (Applied Biosystems) was used for detection of real-time PCR products PBS, sections were incubated with a donkey anti-rabbit horseradish amplified from reverse-transcribed total RNA (25 ng). Total RNA from peroxidase–conjugated secondary antibody (1:1,500; Jackson Immu- U251-MG cells was extracted using Trizol. PCRs were done in triplicate noResearch) for 1 h at room temperature. Tyramide signal amplifica- for each sample on a 96-well plate that included separate wells to tion detection followed and biotin-tyramide was applied (1:500) to the quantify an internal RNA control (S9) and a standard curve. The slides for 10 min. Tissues were then subjected to incubation for 30 min cycling conditions were 48jC for 30 min and 95jC for 10 min with the Vectastain ABC kit (Vector Laboratories). Immunostaining was followed by 40 cycles of 95jC for 15 s and 60jC for 1 min. FAM- detected using a metal-enhanced 3,3¶-diaminobenzidine substrate kit conjugated Taqman probes (Applied Biosystems) were synthesized as (Pierce) according to the manufacturer’s instructions.

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Fig. 1 Continued. D, human control (a-f ; n =2)andGBM(g-o ; n = 3) tissues were analyzed by histochemistry. a, d, g, j, and m, tissue integrity was verified by H&E staining. b, e, h, k, and n, immunohistochemistry using normal rabbit serum (NRS) served as a negative control for staining in control and GBM tissues. c, f, i, l, and o, control and GBM tissues were stained with anti-PIAS3 antibody. Nuclei were counterstained with hematoxylin. Magnifications, Â100 and Â400 (insets).

Transfection and luciferase assay. U251-MG cells were transiently activity (28) and normalized to total protein. The luciferase activity transfected using the Fugene 6 reagent (Roche Diagnostics) with either from the untreated condition was arbitrarily set at 1 for calculation of 500 ng of 3xLy6epZLuc-TK, which contains three STAT-3 sites (27), fold induction. 500 ng of the 1,556-bp SOCS-3 promoter (25), or 200 ng of the [3H]TdR incorporation. [3H]TdR incorporation was done as previ- MMP-9 promoter. Cells were also transfected with either the STAT-3F ously described (24). Briefly, U87-MG and U251-MG cells were labeled plasmid or the PIAS3-pcDNA (100-500 ng). Cells were incubated for with 1 ACi of [3H]TdR for 16 h before terminating the culture. 16 h in the presence or absence of either OSM or phorbol 12-myristate Radioactivity was counted in duplicate samples using a scintillation 13-acetate, and then cell lysates were assayed in triplicate for luciferase counter (Beckman).

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Statistical analysis. Levels of significance for comparison between protein levels were examined by real-time reverse transcription- samples were determined by the Student’s t test distribution. P values of PCR and immunoblotting, respectively. Similar levels of PIAS3 V 0.05 were considered to be statistically significant. mRNA expression were observed in control and GBM tissues (Fig. 1B). Due to the instability in housekeeping gene expres- Results sion in human tumor tissues, the levels of PIAS3 were com- pared with both S9 (Fig. 1B) and GAPDH (data not shown), The STAT-3 protein is constitutivelyactivated and PIAS3 and comparable results were obtained. The 68-kDa PIAS3 protein expression is diminished in human GBM tissue. There protein was expressed in all control tissues tested; however, are conflicting reports about the activation status of STAT-3 in little to no expression of the PIAS3 protein was detected in GBM. Although tyrosine-phosphorylated STAT-3 was detected GBM tissues (Fig. 1C). In 35 GBM samples tested, PIAS3 in GBM samples by several investigators (10–13), a tissue protein expression was detected in only 4 samples, translating microarray study failed to confirm these findings (29). STAT-3 to 89% of GBM samples with loss of PIAS3 protein expression, activation is reflected by phosphorylation of both tyrosine and whereas 100% of control samples (n = 33) expressed PIAS3 serine residues; thus, we examined both variables in this study. (data not shown). Because of the striking difference in PIAS3 Tissue samples were analyzed by immunoblotting, and higher protein levels between control and GBM groups, a second levels of tyrosine- and serine-phosphorylated STAT-3 protein antibody recognizing a different epitope on the PIAS3 protein were detected in GBM tissue compared with controls (Fig. 1A). was used to confirm the results (data not shown). To determine This is the first demonstration of serine-phosphorylated STAT-3 if any correlates could be established between the absence/ in GBM tissues. These results indicate that GBM samples presence of PIAS3 in GBM samples and STAT-3 activation, contain elevated levels of activated STAT-3. we compared these two variables. Of the 14 GBM samples PIAS3 functions as a negative regulator of STAT-3 signaling analyzed in Fig. 1A, we were able to obtain PIAS3 status on by interfering with its ability to bind DNA (16). Using tissue 11 samples. Two of the 11 GBM samples expressed PIAS3 samples from both GBM and control brains, PIAS3 mRNA and (samples 14 and 18), and those samples also had low levels of

Fig. 2. PIAS3 is constitutively expressed in glioblastoma cell lines and primary central nervous system cells. A, U251-MG cells were incubated in the presence or absence of IL-6 (10 ng/mL) plus sIL-6R (50 ng/mL) for up to 24 h, and then cell lysates were assessed by immunoblotting for phospho-Tyr705 STAT-3, STAT-3, and PIAS3 protein expression. Immunoblots were reprobed for actin. Representative of three experiments. B, constitutive PIAS3 protein expression in four glioblastoma cell lines, SNB-19, M059K-MG, U138-MG, and U118-MG, is shown by immunoblotting. Cells were maintained in 10% fetal bovine serum (FBS) or serum starved (no fetal bovine serum) before collection. Blots were stripped and reprobed for actin to control for protein loading. C, four primary human GBM cultures were analyzed for phospho-Tyr705 STAT-3, phospho-Ser727 STAT-3, STAT-3, and PIAS3 protein expression by immunoblotting. Resected human GBM tissues were cultured, as described in Materials and Methods, and lysates were collected for analysis. Anti-actin antibody was used to control for protein loading. D, SNB-19, U251-MG, and primary human GBM cells were incubated in the presence or absence of MG-132 (10 Amol/L)forupto6h,andthencell lysates were assessed by immunoblotting for PIAS3 expression. Immunoblots were reprobed for actin. Data are representative of three independent experiments. Fold induction is calculated based on densitometry measurements, normalized to actin, and compared with the untreated sample.

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tures expressed PIAS3 and had nondetectable to low constitu- tive STAT-3 activation (Fig. 2C), which is in contrast to the GBM tissues. These data suggest that regulation of the PIAS3 protein differs between GBM tissues and the in vitro systems tested, including cultures established from resected GBM tissues. To further examine the in vitro expression pattern of PIAS3, primary murine astrocytes, murine microglia, and rat cortical neurons were examined (Supplementary Fig. S1). In all central nervous system cell types, expression of PIAS3 was detected. Collectively, these data indicate that in vitro, human glioblas- toma cell lines, as well as primary glial and neuronal cultures, show constitutive PIAS3 expression. The discrepancy of PIAS3 protein expression patterns between primary GBM tissues and cell lines suggested that the absence of PIAS3 in vivo might be due to degradation. We tested this hypothesis using the SNB-19 and U251-MG cell lines as well as primary human GBM cultures. Treatment with MG-132, a classic inhibitor of the proteasome, resulted in an accumu- lation of the PIAS3 protein (Fig. 2D), which increased over time in U251-MG cells. These results showed that expression of PIAS3 in vitro is enhanced by inhibition of the proteasome, suggesting that ubiquitin-mediated degradation may promote the loss of PIAS3 in vivo. Fig. 3. OSM activates STAT-3 in human glioma cells.A, U251-MG cells were STAT-3 is activated byOSM in U251-MG cells. Having incubated in the presence or absence of OSM (50 ng/mL) for up to 6 h and cell illustrated the activation of STAT-3 and loss of PIAS3 in vivo,the 705 lysates were assessed by immunoblotting with phospho-Tyr STAT-3 antibody. activation status of this signaling pathway was analyzed in vitro. Blots were reprobed for total STAT-3 and actin. Representative of four experiments. B, U251-MG cells were transiently transfected with the 3xLy6epZLuc-TK (STAT-3) In untreated U251-MG cells, STAT-3 had little to no basal luciferase construct (500 ng), either alone or with pcDNA3 and/or STAT-3F tyrosine phosphorylation. Treatment with OSM induced (0-500 ng), and then recovered overnight before treatment with medium or OSM for 16 h. Lysates were analyzed in triplicate for luciferase activity and normalized to STAT-3 tyrosine phosphorylation at 30 min, which persisted protein levels. Columns, mean of one of three independent experiments; bars, SD. to 6 h (Fig. 3A). Comparable results were observed using IL-6/ *, P V 0.002, compared with medium only condition; **, P V 0.05, compared with sIL-6R (Fig. 2A). Other GBM cell lines, including U87-MG, OSM plus pcDNA3 (500 ng) condition. CH235-MG, and D54-MG, were tested for STAT-3 activation, and similar results were observed (data not shown). A measure activated STAT-3 (see Fig. 1A). Samples 9, 10, 11, 17, 19, 20, of the transcriptional activity of STAT-3 is its ability to activate a 21, and 22 lacked PIAS3 expression and had high levels of STAT-3–driven promoter. On treatment with OSM, U251-MG activated STAT-3, except for sample 21 (see Fig. 1A). These cells showed a 35-fold increase in STAT-3 promoter activity results suggest that a lack of PIAS3 in GBMs may contribute (Fig. 3B), which was inhibited by a dominant-negative STAT-3 to constitutive STAT-3 activation. construct, STAT-3F (Fig. 3B). Collectively, these data showed Tissues were examined using immunohistochemistry, and that basal levels of STAT-3 activation in glioblastoma cells were sections were analyzed by a neuropathologist blinded to the minimal. Importantly, activation was observed on OSM or IL- antibodies used. Strong nuclear staining for PIAS3 was 6/sIL-6R treatment, which resembled the pattern observed in observed in two control brain specimens (Fig. 1D, c and f, human GBM tissues. arrowheads). Positive staining appeared mainly in neurons, with siRNA-mediated inhibition of PIAS3 increases prolifera- weaker staining in astrocytes and endothelial cells. Human tion. To mimic the observed in vivo loss of PIAS3, siRNA GBM (n = 3) tissues had little to no positive reactivity for PIAS3 was used to inhibit PIAS3 expression in U87-MG cells. These (Fig. 1D, i, l, and o). These data indicate that loss of PIAS3 cells were selected as they were the most amenable to siRNA protein expression in GBM samples is frequent. knockdown. Transient transfection using PIAS3 siRNA showed The PIAS3 protein is constitutivelyexpressed in vitro. Because specific inhibition of PIAS3 mRNA at 24 h (Fig. 4A). PIAS3 the expression levels of PIAS3 were substantially different protein levels decreased over time in culture (Fig. 4B, lanes 1, 3, between control and GBM samples, we examined the expres- and 5); however, PIAS3 knockdown was detected at 48 and sion of PIAS3 in human glioblastoma cells. Constitutive 72 h (lanes 4 and 6). STAT-3 activation in the PIAS3 knockdown expression of PIAS3 was observed in U251-MG cells (Fig. 2A). cells was examined, and minimal to no enhancement of basal IL-6/sIL-6R and OSM treatment induced STAT-3 tyrosine tyrosine-phosphorylated STAT-3 was observed (data not phosphorylation but had little effect on PIAS3 expression shown), indicating that in cells with reduced PIAS3 expression, (Fig. 2A; data not shown). Comparable results were obtained levels of phosphorylated STAT-3 remain relatively unchanged. with U87-MG cells (data not shown). Whole-cell extracts Although activation of STAT-3 is measured by tyrosine and from four additional glioblastoma lines, SNB-19, M059K-MG, serine phosphorylation, one functional indicator of STAT-3 U138-MG, and U118-MG, showed constitutive, although transcriptional activation is cell proliferation (30). We exam- varying, levels of PIAS3 expression, which were unaffected ined this variable using the [3H]TdR incorporation assay. by the presence of serum (Fig. 2B). Primary human GBM cul- Knockdown of PIAS3 resulted in a significant increase in

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further explore the effects of PIAS3 on the cell cycle, the expression of p21, the cyclin-dependent kinase inhibitor, was evaluated. Expression of the p21 protein was enhanced by f2- fold on PIAS3 ectopic expression (Fig. 5D). Collectively, these data indicated that PIAS3 overexpression attenuated STAT-3 transcriptional activity, inhibited the expression of survivin and Bcl-xL, and reduced the proliferation of glioma cells. The inhibition of cell proliferation may be due to enhanced expression of p21, which regulates the cell cycle at the transition from G1 to S phase. PIAS3 overexpression inhibits SOCS-3, and SOCS-3 expression is elevated in human GBM tissues. Unlike the constitutively expressed PIAS proteins, SOCS proteins are cytoplasmic, inducibly expressed proteins that act in a negative feedback loop to inhibit JAK activation. SOCS-3 is not only an endogenous inhibitor of STAT-3 (32) but also a STAT-3 transcriptional target (28). Recent investigations have revealed a role for SOCS-3 in cancer cells (33, 34). Ectopic PIAS3 expression repressed OSM activation of the SOCS-3 promoter in a dose-dependent manner (Fig. 6A). In addition, levels of OSM- induced SOCS-3 mRNA were significantly inhibited on PIAS3 overexpression (Fig. 6B). These results indicated that over- expression of PIAS3 can reduce STAT-3 transcriptional activity and inhibit the expression of the STAT-3 target gene SOCS-3. Given the observation that activated STAT-3 levels were elevated, and PIAS3 levels were diminished in GBMs, we examined the expression of SOCS-3 in vivo. Enhanced levels of the SOCS-3 protein were observed in GBM tissues compared with controls (Fig. 6C), with variability in expression levels. SOCS-3 protein expression was observed in 16 of 18 GBM tissues, whereas only 1 of 8 control samples expressed SOCS-3 (data not shown). These data showed that SOCS-3, a STAT-3 transcriptional target gene, was elevated at the protein level in Fig. 4. PIAS3 inhibition increases proliferation of U87-MG cells. A, U87-MG cells were transfected using increasing amounts of transfection reagent and 100 nmol GBM tissues. PIAS3 siPOOL reagent. Twenty-four hours after transfection, RNA was collected PIAS3 inhibits transcriptional activation of the NF-kB– and reverse transcribed to cDNA. Semiquantitative PCR was done to analyze levels MMP-9 of PIAS3 and GAPDH mRNA. B, U87-MG cells were transfected using 2.5 ALof regulated gene . PIAS3 is classically defined as an transfection reagent plus either 100 nmol nontarget siRNAor 100 nmol PIAS3 inhibitor of the JAK-STAT pathway, but its expression has siPOOL reagent.Twenty-four, 48, and 72 h after transfection, lysates were assessed recently been shown to modulate several signal transduction for PIAS3 protein by immunoblotting. Blots were reprobed for actin as loading and specificity controls. C, U87-MG cells were transfected as described above with pathways, many of which are implicated in glioma cell biology, either nontarget siRNA( Control) or PIAS3 siRNA. Twenty-four hours after such as the NF-nB and transforming growth factor-h pathways 3 transfection, cells were incubated in serum-free medium containing 1 ACi [ H]TdR (16). To test the effect of PIAS3 on the NF-nB pathway, we for16 h, and then cell proliferation was determined using the [3H]TdRincorporation assay. Columns, mean of two experiments; bars, SD.Values are expressed as a examined transcriptional activation of the MMP-9 promoter, as percentage of the control. *, P V 0.02, compared with control condition. we have previously shown that MMP-9 gene expression is dependent on the NF-nB signaling pathway (35). We observed proliferation of U87-MG cells (Fig. 4C), indicating that in the that ectopic expression of PIAS3 in U251-MG glioma cells absence of PIAS3, GBM cell proliferation is enhanced. inhibited phorbol 12-myristate 13-acetate–induced activation PIAS3 overexpression inhibits STAT-3–mediated gene expres- of the MMP-9 promoter in a dose-dependent manner (Supple- sion and cell proliferation. As a complement to PIAS3 mentary Fig. S2). These results confirm that PIAS3 expression knockdown, the effects of PIAS3 overexpression were modulates the transcriptional activation of NF-nB target genes examined. Ectopic expression of PIAS3 in U251-MG cells and describe this effect on the MMP-9 gene for the first time in caused a significant dose-dependent inhibition of OSM- human glioma cells. induced STAT-3 transcriptional activity (Fig. 5A). STAT-3 in GBM cells is important for transcriptional induction of Discussion expression of antiapoptotic genes, including Bcl-xL and survivin (10, 31). Basal levels of both survivin and Bcl-xL The role of PIAS3 in human disease states is largely unclear. were inhibited by PIAS3 overexpression (Fig. 5B). These Although PIAS3 expression has been shown to correlate with results suggest that PIAS3 regulates the expression of genes malignancy (17, 36), other reports describe deficient PIAS3 important in cell survival. expression in various human cancers (18). PIAS3 function has To test the functional significance of PIAS3 overexpression, been examined in vitro using transformed cell lines. Infection of cell proliferation was examined. Ectopic expression of PIAS3 prostate cancer cells with adenovirus encoding PIAS3 increased resulted in f80% reduction of cell proliferation (Fig. 5C). To apoptosis (19). Furthermore, PIAS3 overexpression prevented

Clin Cancer Res 2008;14(15) August 1, 2008 4700 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. Loss of PIAS3 in GBMs growth of prostate tumor xenografts in nude mice (19). quickly degraded by the ubiquitin-proteasome system, releasing Overexpression of PIAS3 in lung cancer cells led to inhibition constraints on STAT-3 signal transduction. MG-132 treatment of cell growth, increased susceptibility to chemotherapeutic of primary GBM cells and glioma cell lines induced an agents, and suppression of AKT activation (20). Additionally, appreciable accumulation of the PIAS3 protein in vitro (Fig. PIAS3 overexpression in COS-7 cells suppressed gene induction 2D), supporting the notion that PIAS3 is rapidly degraded in by v-src–activated STAT-3 (37). GBM tissues. PIAS3 protein stability was shown to be regulated This study examined PIAS3 expression in human GBM by nitric oxide, which promoted its association with an E3 tissues. The striking absence of PIAS3 protein in GBM tissues and subsequent degradation (38). Nitric oxide compared with control tissues was observed by both immuno- metabolism is closely associated with glioma progression, and blotting and immunohistochemistry (Fig. 1C and D) and the responsible for nitric oxide production has been confirmed by two PIAS3 antibodies targeting different PIAS3 detected in human glioma tissues (39). The loss of PIAS3 epitopes. Our results contrast those of Wang and Banerjee (17) protein, either by rapid degradation or another mechanism, who detected PIAS3 in GBM specimens. However, they may allow activated STAT-3 to sustain transcription of genes, examined a very small number of GBM tissues and used a which control proliferation and the cell cycle. Proteins different PIAS3 antibody, which may account for the differ- including p53 and p27Kip-1 are destabilized and degraded by ences observed. There were no differences between control and ubiquitination in a variety of cancers (40). The loss of PIAS3 GBM tissue expression of PIAS3 mRNA (Fig. 1B); thus, we protein provides a mechanism to promote aberrant STAT-3 postulated that the differences seen at the protein level were signal transduction in GBM tissues, which is a downstream most likely due to posttranslational changes to PIAS3. In the target of mutated epidermal growth factor receptor, vascular GBM tissue microenvironment, it is possible that PIAS3 was endothelial growth factor receptor, and the IL-6 cytokine family

Fig. 5. Overexpression of PIAS3 inhibits STAT-3 transcriptional activity, STAT-3 ^ regulated gene expression, and cell proliferation. A, U251-MG cells were transiently transfected with the STAT-3 luciferase construct (500 ng), either alone or with pcDNA3 and/or PIAS3-pcDNA3 (0-500 ng), and recovered overnight before treatment with medium or OSM for 16 h. Lysates were analyzed in triplicate for luciferase activity and normalized to protein levels. Columns, mean of one of three independent experiments; bars, SD. *, P V 0.002, compared with medium only; **, P V 0.05, compared with OSM treatment. B, U251-MG cells were transiently transfected with pcDNA3 (0-500 ng) or PIAS3-pcDNA3 (0-500 ng).Twenty-four hours after transfection, total RNA was collected and reverse transcribed to cDNA. Semiquantitative PCR was done to analyze levels of survivin, Bcl-xL,andGAPDH mRNA. Data are representative of two independent experiments. Fold induction is calculated based on densitometry measurements, normalized to GAPDH, and compared with the untreated sample. C, U251-MG cells were transiently transfected with pcDNA3 or PIAS3-pcDNA3 (200 ng).Twenty-four hours after transfection, cells were incubated in serum-free medium containing 1 ACi [3H]TdRfor 16 h, and then cell proliferation was determined using the [3H]TdR incorporation assay. Columns, mean of two experiments; bars, SD.Values are expressed as a percentage of the pcDNA3 control. *, P V 0.0002, compared with pcDNA3 condition. D, U251-MG cells were transiently transfected with pcDNA3 (0-500 ng) or PIAS3-pcDNA3 (0-500 ng).Twenty-four hours after transfection, cell lysates were harvested and analyzed for p21expression by immunoblotting. Immunoblots were reprobed with actin. Data are representative of three independent experiments. Fold induction is calculated based on densitometry measurements, normalized to actin, and compared with the pcDNAsample.

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Fig. 6. PIAS3 overexpression inhibits SOCS-3 promoter activity and mRNAexpression, and SOCS-3 protein expression is elevated in human GBM tissues. A, U251-MG cells were transiently transfected with the SOCS-3 promoter construct (500 ng), either alone or with pcDNA3 and/or PIAS3-pcDNA3 (0-500 ng), and recovered overnight before treatment with medium or OSM for16 h. Lysates were analyzed in triplicate for luciferase activity and normalized to protein levels. Columns, mean of one of three independent experiments; bars, SD. *, P V 0.002, compared with medium only; **, P V 0.05, compared with OSM treatment. B, U251-MG cells were transiently transfected with pcDNA3 or PIAS3-pcDNA3 (200 ng) and recovered overnight before treatment with either medium or OSM for 1h. Total RNA was isolated. RNAwas reverse transcribed to cDNA, which was analyzed by quantitative real-time PCR. SOCS-3 mRNA expression is represented as a ratio to the housekeeping gene S9. Fold induction is represented as fold over untreated, pcDNA3-transfected condition. Columns, mean of one of two independent experiments; bars, SD. *, P V 0.005, compared with pcDNA3 condition; **, P V 0.05, compared with OSM plus pcDNA3 (500 ng) condition. C, immunoblot of SOCS-3 protein in control brain (n =6)andGBM(n =6) lysates. Blots were probed for actin as a loading control.

(11, 12, 41). The constitutive expression of PIAS3 provides a repression when PIAS3 is overexpressed. Because SOCS-3 unique role for this protein to act as a ‘‘buffer’’ to inhibit promoter activity and mRNA expression were negatively sustained STAT-3 transcriptional activity, and in the absence of affected by PIAS3 overexpression, we examined its expression PIAS3, in conjunction with aberrant growth factor signal in human tissues and discovered SOCS-3 aberrant expression in transduction, activation of STAT-3 and enhanced STAT-3 GBM tissues (Fig. 6C). SOCS-3 is a member of the SOCS transcriptional activity is observed in GBMs. protein family, which is composed of CIS and SOCS-1 to PIAS3 expression was inhibited in astroglioma cells using SOCS-7 (32). SOCS-3 is a cytokine-inducible endogenous siRNA to mimic the in vivo loss observed in GBM tissue. PIAS3 inhibitor of STAT-3 that works in a classic negative feedback knockdown resulted in a significant increase in proliferation of loop (32), but it has recently been described to have a astroglioma cells (Fig. 4C) but did not affect the overall levels of complicated role in cancer cell signaling. A tumor-suppressing phosphorylated STAT-3 (data not shown). These data support function of SOCS-3 was suggested by reports describing the previous findings of Herrmann et al. (42), which show that hypermethylation of SOCS-3, and subsequent loss of expres- PIAS3 expression does not modulate cellular levels of phos- sion, in a variety of cancers (43). This defective expression of phorylated STAT-3 but does influence its ability to activate SOCS-3, along with loss of feedback inhibition of STAT-3 transcription. Accordingly, overexpression of PIAS3 resulted in activation, may contribute to tumor progression. However, significant inhibition of STAT-3 transcriptional activity and other reports revealed that aberrant SOCS-3 expression may down-regulation of survivin and Bcl-xL (Fig. 5A and B). actually promote tumorigenesis. Elevated SOCS-3 expression Attenuation of cell proliferation (Fig. 5C) and enhanced p21 was reported in human breast cancer and melanoma tissues as protein expression (Fig. 5D) was observed in cells that well as primary lymphoma cells (44, 45). Further, growth and ectopically expressed PIAS3. These data support the hypothesis proliferation was markedly induced in cancer cells that over- that PIAS3 influences the expression of genes that control cell expressed SOCS-3 (33). SOCS-3 overexpression was attributed proliferation and survival. Thus, the PIAS3 protein exhibits to constitutive STAT-3 activation in various cancers (46). several antitumorigenic functional properties important for Interestingly, human GBM tissues were recently reported to restraining GBM cell growth and gene expression. constitutively express SOCS-3, which correlated with enhanced Although SOCS-3 is a negative regulator of the STAT-3 cell growth/survival and radioresistance (34). Because SOCS-3 signaling pathway, it is also a STAT-3 transcriptional target. is a transcriptional target of STAT-3, the parallel, elevated Overexpression of the PIAS3 protein caused a significant expression of both activated STAT-3 and SOCS-3 proteins in reduction in SOCS-3 promoter activity and SOCS-3 mRNA GBM is plausible and may contribute to the antiapoptotic and expression (Fig. 6A and B), showing SOCS-3 transcriptional radioresistant phenotype of GBMs.

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In many cancers, including GBMs, mechanisms to regulate with AKT in vitro, suppressing its phosphorylation and STAT-3 activity have failed, thus enabling activated STAT-3 to activation (20). PIAS proteins were also implicated in either function as a tumor promoter (30). Interestingly, recent data positively or negatively regulating SMAD transcriptional activ- showed that STAT-3 served conflicting roles, acting either as a ity, which mediate transforming growth factor-h biological tumor suppressor or as a promoter depending on the genetic activities (16). The aberrant activation of the NF-nB, phospha- profile of the tumor (47), furthering the customized, clinical tidylinositol 3-kinase, and transforming growth factor-h path- use of STAT-3 inhibitors as potential therapeutics. Abrogation ways has been correlated with poor prognosis in patients with of STAT-3 activity by a variety of inhibitors, such as AG490 GBMs (12, 29, 50). Thus, the loss of PIAS3 expression in GBMs and WP1066, inhibited GBM cell growth, promoted apoptosis, has implications for multiple signaling pathways contributing and suppressed expression of antiapoptotic genes (3, 10, 48). to gliomagenesis. The loss of PIAS3 expression observed in GBMs may be a contributing factor to the STAT-3 transcriptional activation Disclosure of Potential Conflicts of Interest observed in this cancer. More recently, PIAS3 was shown to regulate other signaling pathways, including the NF-nB, No potential conflicts of interest were disclosed. phosphatidylinositol 3-kinase/AKT, and transforming growth h n factor- pathways (16, 20). PIAS3 interacted with NF- B p65 Acknowledgments and repressed its transcriptional activity, thereby functioning as a negative regulator of NF-nB (49). Supporting this finding, we We thank Dr. Susan Nozell for the PIAS3-pcDNA3 construct and technical assis- showed that transcriptional activation of the NF-nB–regulated tance, Dr. Hongwei Qin for the SOCS-3 promoter construct and helpful advice, Dr. gene MMP-9 is inhibited by PIAS3 overexpression in glioma Sun-Jung Lee for assistance in manuscript preparation, Dr. Kevin Roth and Cecelia Latham of the UABNeuroscience Core Facility (P30 NS-47466) for technical assis- cells (Supplementary Fig. S2), linking PIAS3 expression to tance and discussions, Drs. Hirano, Hibi, and Nakajima (Osaka University Medical inhibition of NF-nB signaling for the first time in glioma cell School, Osaka, Japan) for kindly providing the STAT-3F construct, and Dr. D. Boyd biology. Furthermore, others have shown that PIAS3 interacted for the MMP-9 luciferase promoter construct.

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