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

Glioblastoma tumors are characterized by their invasiveness and resistance to therapies. The transcription factor signal transducer and activator of transcription 3 (STAT3) was recently identified as a master transcriptional regulator in the mesenchymal subtype of glioblastoma (GBM), which has generated an increased interest in targeting STAT3. We have evaluated more closely the mechanism of action of one particular STAT3 inhibitor, JSI-124 (cucurbitacin I). In this study, we confirmed that JSI-124 inhibits both constitutive and stimulus-induced Janus kinase 2 (JAK2) and STAT3 phosphorylation, and decreases cell proliferation while inducing apoptosis in cultured GBM cells. However, we discovered that before the inhibition of STAT3, JSI-124 activates the nuclear factor-kB (NF-kB) pathway, via NF-kB p65 phosphorylation and nuclear translocation. In addition, JSI-124 treatment induces the expression of IL-6, IL-8, and suppressor of cytokine signaling (SOCS3) mRNA, which leads to a corresponding increase in IL-6, IL-8, and SOCS3 protein expression. Moreover, the NF-kB–driven SOCS3 expression acts as a negative regulator of STAT3, abrogating any subsequent STAT3 activation and provides a mechanism of STAT3 inhibition after JSI-124 treatment. Chromatin immunoprecipitation analysis confirms that NF-kB p65 in addition to other activating cofactors are found at the promoters of IL-6, IL-8, and SOCS3 after JSI-124 treatment. Using pharmacological inhibition of NF-kB and inducible knockdown of NF-kB p65, we found that JSI-124–induced expression of IL-6, IL-8, and SOCS3 was significantly inhibited, showing an NF-kB–dependent mechanism. Our data indicate that although JSI-124 may show potential antitumor effects through inhibition of STAT3, other off-target proinflammatory pathways are activated, emphasizing that more careful and thorough preclinical investigations must be implemented to prevent potential harmful effects. Mol Cancer Res; 11(5); 494–505. 2013 AACR. Introduction Glioblastoma (GBM) is the most common malignant tumor of the central nervous system and remains a challenging disease to treat because of the high rate of recurrence and resistance to current treatments (1, 2). Recent efforts have been made elucidating genetic classifications of GBM tumors with the hopes of developingmore effective therapies (3, 4). The Cancer Genome Atlas (TCGA) has determined that GBM tumors can be genetically divided into 4 subtypes: classical, mesenchymal, proneural, and neural (5). Of these subtypes, mesenchymal tumors have been associated with the 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 of transcription (STAT), have been linked to this deadly subtype (5–7). The NF-kB family consists of 5 members (p65/RelA, RelB, c-Rel, NF-kB1/p50, and NF-kB/p52; ref. 8). The predominant form of NF-kB is a dimer composed of p65 and p50 proteins and under basal conditions is found in an inactive form in the cytosol through interactions with the inhibitor of NF-kB (IkB) proteins, specifically IkBa. In response to stimuli such asTNF-a, the upstreamkinase, IkB Kinase (IKK), phosphorylates IkBa, which leads to ubiquitination and degradation of IkBa (8, 9). The NF-kB p65/ p50 dimer then translocates into the nucleus where it becomes phosphorylated, binds to DNA, and induces the expression of genes involved in inflammation such as IL-8 and 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 are responsible for phosphorylating 7 STAT proteins (STAT1-5a, -5b, and -6). Cytokines of the interleukin (IL)-6 family Authors' Affiliation: Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). Corresponding Author: Braden C. McFarland, Department of Cell, Developmental 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: bdcox@uab.edu doi: 10.1158/1541-7786.MCR-12-0528 2013 American Association for Cancer Research. Molecular Cancer Research Mol Cancer Res; 11(5) May 2013 494 on December 16, 2021. © 2013 American Association for Cancer Research. mcr.aacrjournals. org Downloaded from Published OnlineFirst February 5, 2013; DOI: 10.1158/1541-7786.MCR-12-0528