A Trojan-Horse-Like Biomimetic Nano-NK to Elicit an Immunostimulatory Tumor Microenvironment for Enhanced GBM Chemo-Immunotherapy.

Although the chemo- and immuno-therapies have obtained good responses for several solid tumors, including those with brain metastasis, their clinical efficacy in glioblastoma (GBM) is disappointing. The lack of safe and effective delivery systems across the blood-brain barrier (BBB) and the immunosuppressive tumor microenvironment (TME) are two main hurdles for GBM therapy. Herein, a Trojan-horse-like nanoparticle system is designed, which encapsulates biocompatible PLGA-coated temozolomide (TMZ) and IL-15 nanoparticles (NPs) with cRGD-decorated NK cell membrane (R-NKm@NP), to elicit the immunostimulatory TME for GBM chemo-immunotherapy. Taking advantage of the outer NK cell membrane cooperating with cRGD, the R-NKm@NPs effectively traversed across the BBB and targeted GBM. In addition, the R-NKm@NPs exhibited good antitumor ability and prolonged the median survival of GBM-bearing mice. Notably, after R-NKm@NPs treatment, the locally released TMZ and IL-15 synergistically stimulated the proliferation and activation of NK cells, leading to the maturation of dendritic cells and infiltration of CD8+ cytotoxic T cells, eliciting an immunostimulatory TME. Lastly, the R-NKm@NPs not only effectively prolonged the metabolic cycling time of the drugs in vivo, but also has no noticeable side effects. This study may offer valuable insights for developing biomimetic nanoparticles to potentiate GBM chemo- and immuno-therapies in the future.

Preclinical evaluation of Mito-LND, a targeting mitochondrial metabolism inhibitor, for glioblastoma treatment.

BACKGROUND: Glioblastoma (GBM) is a brain tumor with the highest level of malignancy and the worst prognosis in the central nervous system. Mitochondrial metabolism plays a vital role in the occurrence and development of cancer, which provides critical substances to support tumor anabolism. Mito-LND is a novel small-molecule inhibitor that can selectively inhibit the energy metabolism of tumor cells. However, the therapeutic effect of Mito-LND on GBM remains unclear. METHODS: The present study evaluated the inhibitory effect of Mito-LND on the growth of GBM cells and elucidated its potential mechanism. RESULTS: The results showed that Mito-LND could inhibit the survival, proliferation and colony formation of GBM cells. Moreover, Mito-LND induced cell cycle arrest and apoptosis. Mechanistically, Mito-LND inhibited the activity of mitochondrial respiratory chain complex I and reduced mitochondrial membrane potential, thus promoting ROS generation. Importantly, Mito-LND could inhibit the malignant proliferation of GBM by blocking the Raf/MEK/ERK signaling pathway. In vivo experiments showed that Mito-LND inhibited the growth of GBM xenografts in mice and significantly prolonged the survival time of tumor-bearing mice. CONCLUSION: Taken together, the current findings support that targeting mitochondrial metabolism may be as a potential and promising strategy for GBM therapy, which will lay the theoretical foundation for further clinical trials on Mito-LND in the future.

Dual blockade of EGFR and PI3K signaling pathways offers a therapeutic strategy for glioblastoma.

BACKGROUND: Glioblastoma multiforme (GBM) is a devastating disease that lacks effective drugs for targeted therapy. Previously, we found that the third-generation epidermal growth factor receptor (EGFR) inhibitor AZD-9291 persistently blocked the activation of the ERK pathway but had no inhibitory effect on the phosphoinositide 3-kinase (PI3K)/Akt pathway. Given that the PI3K inhibitor GDC-0084 is being evaluated in phase I/II clinical trials of GBM treatment, we hypothesized that combined inhibition of the EGFR/ERK and PI3K/Akt pathways may have a synergistic effect in the treatment of GBM. METHODS: The synergistic effects of cotreatment with AZD-9291 and GDC-0084 were validated using cell viability assays in GBM and primary GBM cell lines. Moreover, the underlying inhibitory mechanisms were assessed through colony formation, EdU proliferation, and cell cycle assays, as well as RNA-seq analyses and western blot. The therapeutic effects of the drug combination on tumor growth and survival were investigated in mice bearing tumors using subcutaneously or intracranially injected LN229 xenografts. RESULTS: Combined treatment with AZD-9291 and GDC-0084 synergistically inhibited the proliferation and clonogenic survival, as well as induced cell cycle arrest of GBM cells and primary GBM cells, compared to monotherapy. Moreover, AZD-9291 plus GDC-0084 combination therapy significantly inhibited the growth of subcutaneous tumors and orthotopic brain tumor xenografts, thus prolonging the survival of tumor-bearing mice. More importantly, the combination of AZD-9291 and GDC-0084 simultaneously blocked the activation of the EGFR/MEK/ERK and PI3K/AKT/mTOR signaling pathways, thereby exerting significant antitumor activity. CONCLUSION: Our findings demonstrate that the combined blockade of the EGFR/MEK/ERK and PI3K/AKT/mTOR pathways is more effective against GBM than inhibition of each pathway alone, both in vitro and in vivo. Our results suggest that AZD-9291 combined with GDC-0084 may be considered as a potential treatment strategy in future clinical trials. Video Abstract.

The value of texture analysis in peritumoral edema of differentiating diagnosis between glioblastoma and primary brain lymphoma.

OBJECTIVE: To evaluate the value of texture analysis of routine MRI image in peritumoral edema of differentiating diagnosis between glioblastoma (GBM) and primary brain lymphoma (PBL). METHODS: The MRI imaging data of 22 patients with glioblastoma and 21 patients with PBL who were hospitalized in our hospital from January 2010 to October 2018 were selected. All the patients were pathologically diagnosed as glioblastoma or PBL, and MRI plain scan and enhanced examination were performed before operation. FireVoxel software was used to delineate the region of interest (ROI) on the most obvious level of peritumoral edema based on T1WI enhancement. Texture parameters were extracted and compared between glioblastoma and PBL. RESULTS: In the glioblastoma group, the inhomogeneity, kurtosis and entropy texture parameters were statistically different from those in the PBL group. The entropy parameter area under the curve (AUC) (0.903) was significantly better than the kurtosis parameter AUC (0.859) and the inhomogeneity parameter AUC (0.729). When the entropy parameter Cut-off point = 3.883, the sensitivity, specificity and accuracy of glioblastoma and PBL were 85.7, 86.4 and 86.0%, respectively, by differential diagnosis. CONCLUSION: Texture analysis of tumor peritumoral edema provided quantifiable information, which might be a new method for differentiating glioblastoma from PBL.

Csnk1a1 inhibition modulates the inflammatory secretome and enhances response to radiotherapy in glioma.

Glioblastoma multiforme (GBM), a fatal brain tumour with no available targeted therapies, has a poor prognosis. At present, radiotherapy is one of the main methods to treat glioma, but it leads to an obvious increase in inflammatory factors in the tumour microenvironment, especially IL-6 and CXCL1, which plays a role in tumour to resistance radiotherapy and tumorigenesis. Casein kinase 1 alpha 1 (CK1α) (encoded on chromosome 5q by Csnk1a1) is considered an attractive target for Tp53 wild-type acute myeloid leukaemia (AML) treatment. In this study, we evaluated the anti-tumour effect of Csnk1a1 suppression in GBM cells in vitro and in vivo. We found that down-regulation of Csnk1a1 or inhibition by D4476, a Csnk1a1 inhibitor, reduced GBM cell proliferation efficiently in both Tp53 wild-type and Tp53-mutant GBM cells. On the contrary, overexpression of Csnk1a1 promoted cell proliferation and colony formation. Csnk1a1 inhibition improved the sensitivity to radiotherapy. Furthermore, down-regulation of Csnk1a1 reduced the production and secretion of pro-inflammatory factors. In the preclinical GBM model, treatment with D4476 significantly inhibited the increase in pro-inflammatory factors caused by radiotherapy and improved radiotherapy sensitivity, thus inhibiting tumour growth and prolonging animal survival time. These results suggest targeting Csnk1a1 exert an anti-tumour role as an inhibitor of inflammatory factors, providing a new strategy for the treatment of glioma.

Imipramine impedes glioma progression by inhibiting YAP as a Hippo pathway independent manner and synergizes with temozolomide.

Patients with malignant glioma often suffered from depression, which leads to an increased risk of detrimental outcomes. Imipramine, an FDA-approved tricyclic antidepressant, has been commonly used to relieve depressive symptoms in the clinic. Recently, imipramine has been reported to participate in the suppression of tumour progression in several human cancers, including prostate cancer, colon cancer and lymphomas. However, the effect of imipramine on malignant glioma is largely unclear. Here, we show that imipramine significantly retarded proliferation of immortalized and primary glioma cells. Mechanistically, imipramine suppressed tumour proliferation by inhibiting yes-associated protein (YAP), a recognized oncogene in glioma, independent of Hippo pathway. In addition to inhibiting YAP transcription, imipramine also promoted the subcellular translocation of YAP from nucleus into cytoplasm. Consistently, imipramine administration significantly reduced orthotopic tumour progression and prolonged survival of tumour-bearing mice. Moreover, exogenous overexpression of YAP partially restored the inhibitory effect of imipramine on glioma progression. Most importantly, compared with imipramine or temozolomide (TMZ) monotherapy, combination therapy with imipramine and TMZ exhibited enhanced inhibitory effect on glioma growth both in vitro and in vivo, suggesting the synergism of both agents. In conclusion, we found that tricyclic antidepressant imipramine impedes glioma progression by inhibiting YAP. In addition, combination therapy with imipramine and TMZ may potentially serve as promising anti-glioma regimens, thus predicting a broad prospect of clinical application.

Multi-responsive nanofibers composite gel for local drug delivery to inhibit recurrence of glioma after operation.

BACKGROUND: The postoperative recurrence of malignant gliomas has presented a clinical conundrum currently. Worse, there is no standard treatment for these recurrent tumours. Therefore, novel promising methods of clinical treatment are urgently needed. METHODS: In this study, we synthesized reactive oxygen species (ROS)-triggered poly(propylene sulfide)60 (PPS60) mixed with matrix metalloproteinases (MMPs)-responsive triglycerol monostearate (T) lipids and TMZ. The mixed solution could self-assemble at 50 â„ƒ to generate hydrogels with MMPs- and ROS-responsiveness. We explored whether the T/PPS + TMZ hydrogel could achieve the MMP- and ROS-responsive delivery of TMZ and exert anti-glioma regrowth effects in vitro and in vivo. These results demonstrated that the T/PPS + TMZ hydrogel significantly improved the curative effect of TMZ to inhibit postsurgical recurrent glioma. RESULTS: The results confirmed the responsive release of TMZ encapsulated in the T/PPS + TMZ hydrogel, and the hydrogel showed excellent performance against glioma in an incomplete glioma operation model, which indicated that the T/PPS + TMZ hydrogel effectively inhibited the growth of recurrent glioma. CONCLUSION: In summary, we successfully developed injectable MMPs- and ROS-responsive hydrogels that could achieve the sustained release of TMZ in the surgical cavity to inhibit local recurrent glioma after surgery.

RIOK2 is negatively regulated by miR-4744 and promotes glioma cell migration/invasion through epithelial-mesenchymal transition.

RIOK2 is a member of RIO (right open reading frame) kinase family. Recent studies have revealed the involvement of RIO kinases in glioma cell growth and expansion. However, the role and mechanism of RIOK2 in glioma cell migration and invasion remain unclear. Wound healing assay, Transwell assay and real-time quantitative PCR (qRT-PCR) detection of matrix metalloproteinases (MMPs) were used to evaluate the migration/invasion of glioma cells. Western blot and qRT-PCR were employed to measure the expression of epithelial-mesenchymal transition (EMT) markers. Dual luciferase reporter assay was performed to determine the binding between RIOK2 and miR-4744. In addition, RIOK2 and miR-4744 levels were quantified by qRT-PCR and/or immunohistochemistry in glioma tissues. Transfection of RIOK2 siRNAs significantly inhibited glioma cell migration and invasion and down-regulated the expression of MMPs (MMP2 and MMP9) and mesenchymal markers (N-cadherin, ß-catenin, Twist1, fibronectin, ZEB-1) in glioma cells. Overexpression of RIOK2 showed the opposite effects. MiR-4744 directly bound to the 3'-untranslated region of RIOK2 and negatively regulated the expression of RIOK2. Up-regulation of miR-4744 inhibited the migration and invasion of glioma cells. Overexpression of RIOK2 could reverse the effects of miR-4744 up-regulation on the migration, invasion and EMT process in glioma cells. Moreover, RIOK2 was high, while miR-4744 was low in glioma tissues, and a negative correlation was found between them. These results suggest that RIOK2 is post-transcriptionally targeted by miR-4744, the low miR-4744 and high RIOK2 levels in glioma may contribute to tumour cell infiltration through promoting the EMT.

MALT1 is a potential therapeutic target in glioblastoma and plays a crucial role in EGFR-induced NF-κB activation.

Glioblastoma multiforme (GBM) is the most common malignant tumour in the adult brain and hard to treat. Nuclear factor κB (NF-κB) signalling has a crucial role in the tumorigenesis of GBM. EGFR signalling is an important driver of NF-κB activation in GBM; however, the correlation between EGFR and the NF-κB pathway remains unclear. In this study, we investigated the role of mucosa-associated lymphoma antigen 1 (MALT1) in glioma progression and evaluated the anti-tumour activity and effectiveness of MI-2, a MALT1 inhibitor in a pre-clinical GBM model. We identified a paracaspase MALT1 that is involved in EGFR-induced NF-kB activation in GBM. MALT1 deficiency or inhibition significantly affected the proliferation, survival, migration and invasion of GBM cells both in vitro and in vivo. Moreover, MALT1 inhibition caused G1 cell cycle arrest by regulating multiple cell cycle-associated proteins. Mechanistically, MALTI inhibition blocks the degradation of IκBα and prevents the nuclear accumulation of the NF-κB p65 subunit in GBM cells. This study found that MALT1, a key signal transduction cascade, can mediate EGFR-induced NF-kB activation in GBM and may be potentially used as a novel therapeutic target for GBM.

GOLPH3 silencing inhibits adhesion of glioma U251 cells by regulating ITGB1 degradation under serum starvation.

GOLPH3, an oncoprotein, plays crucial roles in tumor etiology. Compelling evidences have demonstrated that GOLPH3 contributes to regulate tumor cell growth, migration and invasion under normal nutrient condition. However, the oncogenic activity of GOLPH3 under serum starvation remains largely unknown. In this study, we reported that GOLPH3 depletion led to marked reduction in adhesion of glioma U251 cells, particularly under serum deprivation. We found that silencing of GOLPH3 expression reduced the protein amount of ITGB1 only in serum-free medium. Further insights into the mechanism between GOLPH3 and ITGB1, we applied proteasome or lysosome inhibitor to block the degradation of ITGB1, and identified GOLPH3 silencing can prompt ITGB1 lysosomal degradation under serum starvation. Finally, we found the reductions in glioma cell adhesion and ITGB1 protein amount could be rescued by ITGB1 overexpression. Taken together, these results show that GOLPH3 contributes to the adhesion of glioma cells by regulating the lysosomal degradation of ITGB1 under serum starvation.

Reversible inhibitor of CRM1 sensitizes glioblastoma cells to radiation by blocking the NF-κB signaling pathway.

BACKGROUND: Activation of nuclear factor-kappa B (NF-κΒ) through DNA damage is one of the causes of tumor cell resistance to radiotherapy. Chromosome region 1 (CRM1) regulates tumor cell proliferation, drug resistance, and radiation resistance by regulating the nuclear-cytoplasmic translocation of important tumor suppressor proteins or proto-oncoproteins. A large number of studies have reported that inhibition of CRM1 suppresses the activation of NF-κΒ. Thus, we hypothesize that the reversible CRM1 inhibitor S109 may induce radiosensitivity in glioblastoma (GBM) by regulating the NF-κΒ signaling pathway. METHODS: This study utilized the cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), and colony formation assay to evaluate the effect of S109 combined with radiotherapy on the proliferation and survival of GBM cells. The therapeutic efficacy of S109 combined with radiotherapy was evaluated in vivo to explore the therapeutic mechanism of S109-induced GBM radiosensitization. RESULTS: We found that S109 combined with radiotherapy significantly inhibited GBM cell proliferation and colony formation. By regulating the levels of multiple cell cycle- and apoptosis-related proteins, the combination therapy induced G1 cell cycle arrest in GBM cells. In vivo studies showed that S109 combined with radiotherapy significantly inhibited the growth of intracranial GBM and prolonged survival. Importantly, we found that S109 combined with radiotherapy promoted the nuclear accumulation of IκΒα, and inhibited phosphorylation of p65 and the transcriptional activation of NF-κΒ. CONCLUSION: Our findings provide a new therapeutic regimen for improving GBM radiosensitivity as well as a scientific basis for further clinical trials to evaluate this combination therapy.

CAPON Is a Critical Protein in Synaptic Molecular Networks in the Prefrontal Cortex of Mood Disorder Patients and Contributes to Depression-Like Behavior in a Mouse Model.

Aberrant regulation and activity of synaptic proteins may cause synaptic pathology in the prefrontal cortex (PFC) of mood disorder patients. Carboxy-terminal PDZ ligand of NOS1 (CAPON) is a critical scaffold protein linked to synaptic proteins like nitric oxide synthase 1, synapsins. We hypothesized that CAPON is altered together with its interacting synaptic proteins in the PFC in mood disorder patients and may contribute to depression-like behaviors in mice subjected to chronic unpredictable mild stress (CUMS). Here, we found that CAPON-immunoreactivity (ir) was significantly increased in the dorsolateral PFC (DLPFC) and anterior cingulate cortex in major depressive disorder (MDD), which was accompanied by an upregulation of spinophilin-ir and a downregulation of synapsin-ir. The increases in CAPON and spinophilin and the decrease in synapsin in the DLPFC of MDD patients were also seen in the PFC of CUMS mice. CAPON-ir positively correlated with spinophilin-ir (but not with synapsin-ir) in mood disorder patients. CAPON colocalized with spinophilin in the DLPFC of MDD patients and interacted with spinophilin in human brain. Viral-mediated CAPON downregulation in the medial PFC notably reversed the depression-like behaviors in the CUMS mice. These data suggest that CAPON may contribute to aspects of depressive behavior, possibly as an interacting protein for spinophilin in the PFC.

Inhibition of chromosomal region maintenance 1 suppresses the migration and invasion of glioma cells via inactivation of the STAT3/MMP2 signaling pathway.

Chromosomal region maintenance 1 (CRM1) is associated with an adverse prognosis in glioma. We previously reported that CRM1 inhibition suppressed glioma cell proliferation both in vitro and in vivo. In this study, we investigated the role of CRM1 in the migration and invasion of glioma cells. S109, a novel reversible selective inhibitor of CRM1, was used to treat Human glioma U87 and U251 cells. Cell migration and invasion were evaluated by wound-healing and transwell invasion assays. The results showed that S109 significantly inhibited the migration and invasion of U87 and U251 cells. However, mutation of Cys528 in CRM1 abolished the inhibitory activity of S109 in glioma cells. Furthermore, we found that S109 treatment decreased the expression level and activity of MMP2 and reduced the level of phosphorylated STAT3 but not total STAT3. Therefore, the inhibition of migration and invasion induced by S109 may be associated with the downregulation of MMP2 activity and expression, and inactivation of the STAT3 signaling pathway. These results support our previous conclusion that inhibition of CRM1 is an attractive strategy for the treatment of glioma.

FRK suppresses human glioma growth by inhibiting ITGB1/FAK signaling.

Fyn-related kinase (FRK), a member of the Src-related tyrosine kinase family, functions as a tumor suppressor in several malignancies. We previously showed that FRK overexpression inhibited the growth of glioma cells. However, it is unknown whether FRK is equally effective against intracranial glioma in vivo, and the mechanism by which FRK influences glioma cell growth remains unclear. In this study, we found that tumor volume was reduced by about one-third in mice with FRK overexpression, which showed improved survival relative to controls. Immunofluorescence analysis revealed that FRK overexpression inhibited glioma cell proliferation and induced their apoptosis. Importantly, in vitro we further found that FRK decreased the expression of integrin subunit ß1 (ITGB1) at both the mRNA and protein levels. FRK also inhibited transactivation by ITGB1, resulting in the suppression of its target proteins AKT and focal adhesion kinase (FAK). ITGB1 overexpression promoted glioma cell growth and partially reduced FRK-induced growth suppression. These results indicate that FRK inhibits human glioma growth via regulating ITGB1/FAK signaling and provide a potential therapeutic target for the treatment of glioma.

SWAP-70 promotes glioblastoma cellular migration and invasion by regulating the expression of CD44s.

BACKGROUND: Switch-associated protein 70 (SWAP-70) is a guanine nucleotide exchange factor that is involved in cytoskeletal rearrangement and regulation of migration and invasion of malignant tumors. However, the mechanism by which SWAP-70 regulates the migration and invasion of glioblastoma (GB) cells has not been fully elucidated. METHODS: This study used an online database to analyze the relationship between SWAP-70 expression and prognosis in GB patients. The in vitro wound healing assay and transwell invasion assay were used to determine the role of SWAP-70 in GB cell migration and invasion as well as the underlying mechanism. RESULTS: We found that patients with high SWAP-70 expression in the GB had a poor prognosis. Downregulation of SWAP-70 inhibited GB cell migration and invasion, whereas SWAP-70 overexpression had an opposite effect. Interestingly, SWAP-70 expression was positively correlated with the expression of the standard form of CD44 (CD44s) in GB tissues. Downregulation of SWAP-70 also reduced CD44s protein expression, whereas SWAP-70 overexpression enhanced CD44s protein expression. However, downregulation of SWAP-70 expression did not affect the mRNA expression of CD44s. Reversal experiments showed that overexpressing CD44s in cell lines with downregulated SWAP-70 partially abolished the inhibitory effects of downregulated SWAP-70 on GB cell migration and invasion. CONCLUSIONS: These results suggest that SWAP-70 may promote GB cell migration and invasion by regulating the expression of CD44s. SWAP-70 may serve as a new biomarker and a potential therapeutic target for GB.

Delivery of Doxorubicin from Hyaluronic Acid-Modified Glutathione-Responsive Ferrocene Micelles for Combination Cancer Therapy.

A combination of different chemotherapy approaches can obtain the best response for many cancers. However, the greatest challenge is the development of a nanoparticle formulation that can encapsulate different chemotherapeutic agents to achieve the proper synergetic chemotherapy for the tumor. Here, amphiphilic ferrocenium-tetradecyl (Fe-C14) was constructed to form cationic micelles in an aqueous solution via self-assembly. Then, it was coated by hyaluronic acid (HA) through electrostatic interactions to generate HA-Fe-C14 micelles. The HA-Fe-C14 micelles were used to deliver doxorubicin (DOX), and it showed that the DOX could be released rapidly under a high-GSH tumor environment. The HA-Fe-C14/DOX micelles were able to accumulate efficiently in tumor and showed significant anticancer effect both in vitro and in vivo. These results suggest that HA-Fe-C14/DOX micelles are a useful drug delivery system that enhances synergic antitumor treatment effects.

Activation of STAT1 by the FRK tyrosine kinase is associated with human glioma growth.

PURPOSE: Glioma is a highly aggressive and lethal brain tumor. Signal transducers and activators of transcription (STAT) pathway are widely implicated in glioma carcinogenesis. Our previous study found that the Fynrelated kinase (FRK) gene, plays as a tumor suppressor in the development and progression of glioma. This study aimed to investigate the role of FRK in the activation pathway of STATs and its effect on the growth of glioma. METHODS: The U251 and U87 cells with stable FRK overexpression were generated by lentivirus technique. The effects of FRK on the related proteins of STAT signaling pathway were detected by western blotting. Coimmunoprecipitation was used to detect the association of FRK and STAT1. The effects of STAT1 on the proliferation of glioma cells were detected by CCK8 or Edu cell proliferation assays. The expressions and correlation of FRK and p-STAT1 in glioma tissues were detectd by western blotting or immunohistochemistry. The effect of FRK on the growth of glioma was investigated in vivo mouse model. RESULTS: The level of p-JAK2 and p-STAT1 increased after FRK overexpression, while they decreased after FRK downregulation both in U251 and U87 cells. However, FRK had no effect on STAT3 phosphorylation. FRK-induced STAT1 activation was not dependent on JAK2. FRK associated with STAT1, induced STAT1 nuclear translocation and regulated the expressions of STAT1-related target genes. STAT1 overexpression suppressed the proliferation of glioma cells. In contrast, STAT1 knockdown by siRNA promoted glioma cell growth. Importantly, down-regulation of STAT1 partially attenuated FRK-induced growth suppression. The clinical sample-based study indicated that the expression of FRK was significantly correlated with the expression of p-STAT1. FRK significantly inhibited glioma tumor growth in vivo. CONCLUSIONS: Our findings highlighted a critical role of FRK in tumor suppression ability through promoting STAT1 activation, and provided a potential therapeutic target for glioma.

Effects of Long Form of CAPON Overexpression on Glioma Cell Proliferation are Dependent on AKT/mTOR/P53 Signaling.

Background: CAPON has two isoforms in human brain: long form of CAPON (CAPON-L) and short form of CAPON (CAPON-S). Recent studies have indicated the involvement of CAPON in tumor cell growth. We aimed to reveal the role of the two CAPON isoforms in the proliferation of glioma cells in this study. Materials and Methods: Lentivirus-mediated stable cell lines with CAPON-L or CAPON-S overexpression were established in U87 and U251 glioma cells. Cell counting kit-8 and colony formation assays were used to evaluate cell proliferation. Western blot analysis of cell cycle-related proteins and flow cytometry were performed to analyze cell cycle progression. Some important molecules of the AKT/mTOR pathway and P53 were also measured by Western blot analysis. Results: Overexpression of CAPON-L showed a significantly inhibitory role in U251 cells, while it exhibited a promoting role in U87 cells. Consistently, overexpressing CAPON-L impeded the cell cycle progression and down-regulated the expression levels of Cyclin D1, CDK4 and CDK6 in U251 cells, whereas it up-regulated the CDK6 level in U87 cells. The overexpression of CAPON-L significantly decreased the phosphorylation and/or total levels of AKT, mTOR and S6 in U251 cells, while it did not affect these signaling molecules in U87 cells, except for a significant increase in the phosphorylation of AKT at Thr-308 site. Transfecting constitutively active AKT (myr-AKT) partially reversed the decreased phosphorylation of AKT and S6 in the CAPON-L-overexpressing U251 cells. In addition, we found a significant decrease in the wild-type P53 level in the CAPON-L-overexpressing U87 cells. The overexpression of CAPON-S also inhibited cell proliferation, blocked cell cycle progression, and decreased the AKT/mTOR pathway activity in U251 cells. Conclusion: The effects of CAPON-L overexpression on glioma cell proliferation are dependent on the AKT/mTOR/P53 activity. The overexpression of CAPON inhibits U251 cell proliferation through the AKT/mTOR signaling pathway, while overexpressing CAPON-L promoted U87 cell proliferation, possibly through down-regulating the P53 level.

PHAP1 promotes glioma cell proliferation by regulating the Akt/p27/stathmin pathway.

PHAP1 (Putative HLA-DR-associated protein 1), also termed acidic leucine-rich nuclear phosphoprotein 32A (ANP32A), Phosphoprotein 32 (pp32) or protein phosphatase 2A inhibitor (I1PP2A), is a multifunctional protein aberrantly expressed in multiple types of human cancers. However, its expression pattern and clinical relevance in human glioma remain unknown. In this study, Western blotting and immunohistochemistry analysis demonstrated PHAP1 protein was highly expressed in glioma patients, especially in those with high-grade disease. Publicly available data also revealed high levels of PHAP1 were associated with poor prognosis in glioma patients. The functional studies showed that knock-down of PHAP1 suppressed the proliferation of glioma cells, while overexpression of PHAP1 facilitated it. The iTRAQ proteomic analysis suggested that stathmin might be a potential downstream target of PHAP1. Consistently, PHAP1 knock-down significantly decreased the expression of stathmin, while overexpression of PHAP1 increased it. Also, the upstream negative regulator, p27, expression levels increased upon PHAP1 knock-down and decreased when PHAP1 was overexpressed. As a result, the phosphorylated Akt (S473), an upstream regulator of p27, expression levels decreased upon silencing of PHAP1, but elevated after PHAP1 overexpression. Importantly, we demonstrate the p27 down-regulation, stathmin up-regulation and cell proliferation acceleration induced by PHAP1 overexpression were dependent on Akt activation. In conclusion, the above results suggest that PHAP1 expression is elevated in glioma patients, which may accelerate the proliferation of glioma cells by regulating the Akt/p27/stathmin pathway.

Golgi Phosphoprotein 3 Promotes Wls Recycling and Wnt Secretion in Glioma Progression.

BACKGROUND/AIMS: Golgi phosphoprotein 3 (GOLPH3) plays pro-malignancy roles in several types of cancer. However, the molecular mechanism underlying GOLPH3 promoting tumor progression remains poorly understood. METHODS: The expression of GOLPH3 and Wntless (Wls) in glioma tissues was examined by western blotting and immunohistochemistry. EdU incorporation assay and colony formation assay was used to examine the cell growth ability. The effect of GOLPH3 on Wls recycling, Wnt secretion and ß-catenin activity was detected using western blotting, immunofluorescence, RT-PCR, ELISA or luciferase assay. RESULTS: The protein levels of GOLPH3 and Wls were upregulated and positively correlated with each other in human glioma tissues. The promoting effect of GOLPH3 on glioma cell proliferation was partially mediated by Wls. In addition, GOLPH3 interacted with Wls and GOLPH3 down-regulation drove Wls into lysosome for degradation, inhibiting its recycling to golgi and the plasma membrane. Importantly, GOLPH3 down-regulation inhibited Wnt2b secretion and decreased ß-catenin level and transcription activity. CONCLUSIONS: This study provides a brand new evidence that GOLPH3 promotes glioma cell proliferation by facilitating Wls recycling and Wnt/ß-catenin signaling. Our findings suggest a rationale for targeting the GOLPH3-Wls-Wnt axis as a promising therapeutic approach for glioblastoma.