Human Cancer Biology Loss of Protein Inhibitors of Activated STAT-3 Expression in Glioblastoma MultiformeTumors: Implications for STAT-3 Activation and Gene 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 cytokines, controlling prolifera- tion, gene expression, 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 genes, 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 ligand 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 cytokine 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 Clin Cancer Res 2008;14(15) August 1, 2008 4694 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. Loss of PIAS3 in GBMs 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