Dysregulated iron metabolism is a hallmark of many cancers, including glioblastoma (GBM). However, its role in tumor progression remains unclear. Herein, we identified coatomer protein complex subunit zeta 1 (COPZ1) as a therapeutic target candidate which significantly dysregulated iron metabolism in GBM cells. Overexpression of COPZ1 was associated with increasing tumor grade and poor prognosis in glioma patients based on analysis of expression data from the publicly available database The Cancer Genome Atlas (P < 0.001). Protein levels of COPZ1 were significantly increased in GBM compared to non-neoplastic brain tissue samples in immunohistochemistry and western blot analysis. SiRNA knockdown of COPZ1 suppressed proliferation of U87MG, U251 and P3#GBM in vitro. Stable expression of a COPZ1 shRNA construct in U87MG inhibited tumor growth in vivo by ~60% relative to controls at day 21 after implantation (P < 0.001). Kaplan–Meier analysis of the survival data demonstrated that the overall survival of tumor bearing animals increased from 20.8 days (control) to 27.8 days (knockdown, P < 0.05). COPZ1 knockdown also led to the increase in nuclear receptor coactivator 4 (NCOA4), resulting in the degradation of ferritin, and a subsequent increase in the intracellular levels of ferrous iron and ultimately ferroptosis. These data demonstrate that COPZ1 is a critical mediator in iron metabolism. The COPZ1/NCOA4/FTH1 axis is therefore a novel therapeutic target for the treatment of human GBM.

Astrocytes have important physiological roles in CNS homeostasis and serve as a bridge between the CNS and immune system. IL-17 and IL-6 are important in many CNS disorders characterized by neuroinflammation. We examined the role of IL-17 on the IL-6 signaling cascade in primary astrocytes. IL-17 functioned in a synergistic manner with IL-6 to induce IL-6 expression in astrocytes. The synergistic effect involved numerous signaling pathways including NF-κB, JNK MAPK, and p38 MAPK. The NF-κB pathway inhibitor BAY-11, JNK inhibitor JNKi II, and p38 inhibitor SB203580 suppressed the synergistic effect of IL-6 and IL-17 on IL-6 expression. IL-17 synergized with IL-6 to enhance the recruitment of activated NF-κB p65, c-Fos, c-Jun, and the histone acetyltransferases CREB-binding protein and p300 to the IL-6 promoter in vivo to induce IL-6 transcription. This was accompanied by enhanced acetylation of histones H3 and H4 on the IL-6 promoter. Moreover, we elucidated an important role for suppressor of cytokine signaling (SOCS) 3 in IL-17 enhancement of IL-6 signaling in astrocytes. SOCS3 small interfering RNA knockdown and SOCS3 deletion in astrocytes augmented the synergistic effect of IL-6 and IL-17 due to an enhancement of activation of the NF-κB and MAPK pathways. These results indicate that astrocytes can serve as a target of Th17 cells and IL-17 in the CNS, and SOCS3 participates in IL-17 functions in the CNS as a negative feedback regulator.

Berberine induces autophagy in glioblastoma by targeting the AMPK/mTOR/ULK1-pathway

NF-κB signaling plays a critical role in tumor growth and treatment resistance in GBM as in many other cancers. However, the molecular mechanisms underlying high, constitutive NF-κB activity in GBM remains to be elucidated. Here, we screened a panel of tripartite motif (TRIM) family proteins and identified TRIM22 as a potential activator of NF-κB using an NF-κB driven luciferase reporter construct in GBM cell lines. Knockout ofTRIM22using Cas9-sgRNAs led to reduced GBM cell proliferation, whileTRIM22overexpression enhanced proliferation of cell populations, in vitro and in an orthotopic xenograft model. However, two TRIM22 mutants, one with a critical RING-finger domain deletion and the other with amino acid changes at two active sites of RING E3 ligase (C15/18A), were both unable to promote GBM cell proliferation over controls, thus implicating E3 ligase activity in the growth-promoting properties of TRIM22. Co-immunoprecipitations demonstrated that TRIM22 bound a negative regulator of NF-κB, NF-κB inhibitor alpha (IκBα), and accelerated its degradation by inducing K48-linked ubiquitination. TRIM22 also formed a complex with the NF-κB upstream regulator IKKγ and promoted K63-linked ubiquitination, which led to the phosphorylation of both IKKα/β and IκBα. Expression of a non-phosphorylation mutant, srIκBα, inhibited the growth-promoting properties of TRIM22 in GBM cell lines. Finally, TRIM22 was increased in a cohort of primary GBM samples on a tissue microarray, and high expression of TRIM22 correlated with other clinical parameters associated with progressive gliomas, such as wild-type IDH1 status. In summary, our study revealed that TRIM22 activated NF-κB signaling through posttranslational modification of two critical regulators of NF-κB signaling in GBM cells.

Long non-coding RNAs play critical roles in tumour progression. Through analysis of publicly available genomic datasets, we found that MIR22HG, the host gene of microRNAs miR-22-3p and miR-22-5p, is ranked among the most dysregulated long non-coding RNAs in glioblastoma. The main purpose of this work was to determine the impact of MIR22HG on glioblastoma growth and invasion and to elucidate its mechanistic function. The MIR22HG/miR-22 axis was highly expressed in glioblastoma as well as in glioma stem-like cells compared to normal neural stem cells. In glioblastoma, increased expression of MIR22HG is associated with poor prognosis. Through a number of functional studies, we show that MIR22HG silencing inhibits the Wnt/β-catenin signalling pathway through loss of miR-22-3p and -5p. This leads to attenuated cell proliferation, invasion and in vivo tumour growth. We further show that two genes, SFRP2 and PCDH15, are direct targets of miR-22-3p and -5p and inhibit Wnt signalling in glioblastoma. Finally, based on the 3D structure of the pre-miR-22, we identified a specific small-molecule inhibitor, AC1L6JTK, that inhibits the enzyme Dicer to block processing of pre-miR-22 into mature miR-22. AC1L6JTK treatment caused an inhibition of tumour growth in vivo. Our findings show that MIR22HG is a critical inducer of the Wnt/β-catenin signalling pathway, and that its targeting may represent a novel therapeutic strategy in glioblastoma patients.

Flavokawain B (FKB), a natural kava chalcone, displays potent antitumor activity in various types of cancer. The mechanism of action, however, remains unclear. Here, we evaluated the efficacy of FKB in the treatment of human glioblastoma multiforme (GBM) as well as the molecular basis for its inhibitory effects in cancer. Approximately 60% of GBM cells became senescent after treatment with FKB as assessed in the senescence-associated (SA)-GLB1/SA-β-galactosidase assay. The cellular process of autophagy potentially contributed to the establishment of senescence. Transmission electron microscopy revealed the formation of autophagic vesicles under FKB treatment, and MAP1LC3B (microtubule associated protein 1 light chain 3 beta)-II was increased. Transfection of ATG5 or ATG7 small interfering RNAs (siRNAs) inhibited FKB-induced autophagy in U251 cells. Western blot revealed that molecular components of the endoplasmic reticulum stress pathway were activated, including ATF4 (activating transcription factor 4) and DDIT3 (DNA damage inducible transcript 3), while levels of TRIB3 (tribbles pseudokinase 3) increased. In addition, based on the phosphorylation status, the AKT-MTOR-RPS6KB1 pathway was inhibited, which induced autophagy in GBM cells. Inhibition of autophagy by autophagy inhibitors 3-methyladenine and chloroquine or knockdown of ATG5 or ATG7 caused FKB-treated U251 cells to switch from senescence to apoptosis. Finally, knockdown of ATG5 or treatment with chloroquine in combination with FKB, significantly inhibited tumor growth in vivo. Our results demonstrated that FKB induced protective autophagy through the ATF4-DDIT3-TRIB3-AKT-MTOR-RPS6KB1 signaling pathway in GBM cells, indicating that the combination treatment of FKB with autophagy inhibitors may potentially be an effective therapeutic strategy for GBM. Abbreviations: 3-MA: 3-methyladenine; 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATF4: activating transcription factor 4; ATG: autophagy related; CASP3: caspase 3; CCK-8: cell counting kit-8; CDKN1A: cyclin-dependent kinase inhibitor 1A; CQ: chloroquine; DDIT3: DNA damage inducible transcript 3; DMEM: Dulbecco’s modified Eagle’s medium; EIF2A: eukaryotic translation initiation factor 2A; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; FKB: flavokawain B; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GBM: glioblastoma multiforme; GFP: green fluorescent protein; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PARP1: poly(ADP-ribose) polymerase; 1RPS6KB1: ribosomal protein S6 kinase B1; SA-GLB1: senescence-associated galactosidase beta 1; siRNA: short interfering RNA; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TRIB3: tribbles pseudokinase 3; TUNEL: deoxynucleotidyl transferase-mediated dUTP nick-end labeling