F25P preproinsulin abrogates the secretion of pro-growth factors from EGFRvIII cells and suppresses tumor growth in an EGFRvIII/wt heterogenic model.

Extensive heterogeneity is a defining hallmark of glioblastoma multiforme (GBM) at the cellular and molecular levels. EGFRvIII, the most common EGFR mutant, is expressed in 24-67% of cases and strongly indicates a poor survival prognosis. By co-expressing EGFRvIII and EGFRwt, we established an EGFRvIII/wt heterogenic model. Using this approach, we confirmed that a mixture of EGFRvIII and EGFRwt at a certain ratio could clearly enhance tumor growth in vitro and in vivo compared with EGFRwt cells, thereby indicating that EGFRvIII cells promote tumor growth. Furthermore, we demonstrated that the EGFRvIII cells could support the growth of EGFRwt cells by secreting growth factors, thus acting as the principal source for maintaining tumor survival. F25P preproinsulin effectively reduced the concentrations of EGF, VEGF, and MMP-9 in the blood of tumor-bearing mice by competitively inhibiting the endoplasmic reticulum signal peptidase and increased the overall survival in orthotopic models. Taken together, our results provided an effective therapy of F25P preproinsulin in the EGFRvIII/wt heterogenic model.

BASI, a potent small molecular inhibitor, inhibits glioblastoma progression by targeting microRNA-mediated ß-catenin signaling.

BACKGROUND AND AIMS: The nuclear localization of ß-catenin, a mediator of canonical Wnt signaling, has been indicated in a variety of cancers and is frequently related to tumor progression and metastasis. Therefore, targeting ß-catenin is an attractive therapeutic strategy for cancers. METHODS: Herein, we identified a natural, small molecule inhibitor of ß-catenin signaling, BASI, and evaluated its therapeutic efficacy both in vitro and in orthotopic mouse models of glioma. RESULTS: BASI significantly suppressed proliferation and invasion and induced apoptosis in glioblastoma cells and resulted in the remarkable attenuation of orthotopic tumor growth in vivo. Furthermore, we found that BASI altered the expression of several microRNAs, which mediated the posttranscriptional silencing of ß-catenin expression either directly or indirectly through a von Hippel-Lindau (VHL)-mediated ß-catenin degradation pattern. CONCLUSIONS: Taken together, our findings offer preclinical validation of BASI as a promising new type of ß-catenin inhibitor with a mechanism of inhibition that has broad potential for the improved treatment of glioblastoma.

MicroRNA-566 activates EGFR signaling and its inhibition sensitizes glioblastoma cells to nimotuzumab.

BACKGROUND: Epidermal growth factor receptor (EGFR) is amplified in 40% of human glioblastomas. However, most glioblastoma patients respond poorly to anti-EGFR therapy. MicroRNAs can function as either oncogenes or tumor suppressor genes, and have been shown to play an important role in cancer cell proliferation, invasion and apoptosis. Whether microRNAs can impact the therapeutic effects of EGFR inhibitors in glioblastoma is unknown. METHODS: miR-566 expression levels were detected in glioma cell lines, using real-time quantitative RT-PCR (qRT-PCR). Luciferase reporter assays and Western blots were used to validate VHL as a direct target gene of miR-566. Cell proliferation, invasion, cell cycle distribution and apoptosis were also examined to confirm whether miR-566 inhibition could sensitize anti-EGFR therapy. RESULTS: In this study, we demonstrated that miR-566 is up-regulated in human glioma cell lines and inhibition of miR-566 decreased the activity of the EGFR pathway. Lentiviral mediated inhibition of miR-566 in glioblastoma cell lines significantly inhibited cell proliferation and invasion and led to cell cycle arrest in the G0/G1 phase. In addition, we identified von Hippel-Lindau (VHL) as a novel functional target of miR-566. VHL regulates the formation of the ß-catenin/hypoxia-inducible factors-1α complex under miR-566 regulation. CONCLUSIONS: miR-566 activated EGFR signaling and its inhibition sensitized glioblastoma cells to anti-EGFR therapy.

Blockage of a miR-21/EGFR regulatory feedback loop augments anti-EGFR therapy in glioblastomas.

Epidermal growth factor receptors (EGFR) expression is frequently amplified in human glioblastoma cells. Nimotuzumab, a monoclonal antibody (mAb) against EGFR, has been used globally in clinics as an anti-cancer agent. It is largely unknown whether the blockade of miR-21, a microRNA that is upregulated in glioma cells, could amplify the effects of nimotuzumab. Herein, we have demonstrated that miR-21 directly targets von Hippel-Lindau (VHL) and peroxisome-proliferator-activated receptor α (PPARα) and that miR-21 regulates EGFR/AKT signaling through VHL/ß-catenin and the PPARα/AP-1 axis. Further, the expression of miR-21 is regulated by EGFR via the activation of ß-catenin and AP-1. These data indicate that a feedback loop exists between miR-21 and EGFR. We also show that the combination of nimotuzumab and an inhibitor of miR-21 is superior to single-agent therapy. These results clarify a novel association between miR-21 and EGFR in the regulation of cancer cell progression.

HOTAIR, a cell cycle-associated long noncoding RNA and a strong predictor of survival, is preferentially expressed in classical and mesenchymal glioma.

BACKGROUND: Long noncoding RNA Hox transcript antisense intergenic RNA (HOTAIR) has been characterized as a negative prognostic factor in breast and colon cancer patients. The clinical significance and function of HOTAIR in glioma remains unclear. METHODS: We analyzed the clinical significance of HOTAIR in 3 different glioma cohorts with gene expression data, including correlation with tumor grade, prognosis, and molecular subtype. The function of HOTAIR in glioma was explored by performing gene set enrichment analysis and in vitro and in vivo experiments. RESULTS: HOTAIR expression was closely associated with glioma grade and poor prognosis. Multivariate Cox regression analysis revealed that HOTAIR was an independent prognostic factor in glioblastoma multiforme patients. HOTAIR expression correlated with glioma molecular subtype, including those of The Cancer Genome Atlas. HOTAIR was preferentially expressed in the classical and mesenchymal subtypes compared with the neural and proneural subtypes. A gene set enrichment analysis designed to show gene set differences between patients with high and low HOTAIR expression indicated that HOTAIR expression was associated with gene sets involved in cell cycle progression. HOTAIR reduction induced colony formation suppression, cell cycle G0/G1 arrest, and orthotopic tumor growth inhibition. CONCLUSION: Our data establish that HOTAIR is an important long noncoding RNA that primarily serves as a prognostic factor for glioma patient survival, as well as a biomarker for identifying glioma molecular subtypes, a critical regulator of cell cycle progression.

Aspirin-/TMZ-coloaded microspheres exert synergistic antiglioma efficacy via inhibition of ß-catenin transactivation.

BACKGROUND AND AIMS: Currently temozolomide (TMZ) as a potent agent is widely used to treat the glioblastoma multiforme (GBM), whereas recurrence due to intrinsic or acquired therapeutic resistance often occurs. Combination chemotherapy with TMZ may be a promising therapeutic strategy to improve treatment efficacy. METHODS: Aspirin, TMZ, and aspirin-/TMZ-coloaded poly (L-lactide-co-glycolide) (PLGA) microspheres were prepared by spray drying, and cytotoxicities of glioblastoma cells were measured. RESULTS: Aspirin microsphere treatment induced slight apoptosis and modestly inhibited proliferation of LN229 and U87 cells in vitro and in vivo through inhibition of ß-catenin transactivation. However, aspirin-/TMZ-coloaded microspheres presented synergistic antitumor efficacy compared with single TMZ-loaded microspheres. Aspirin/TMZ microspheres induced more apoptosis and repressed proliferation of LN229 and U87 cells. Corresponding to inhibition of ß-catenin signaling, ß-catenin/TCF4 transcriptional activity and STAT3 luciferase activity were strongly suppressed, and downstream targets expression was decreased. Furthermore, aspirin/TMZ microsphere intratumoral injection downregulated the expression of ß-catenin, TCF4, pAKT, pSTAT3, and PCNA and delayed tumor growth in nude mice harboring subcutaneous LN229 xenografts. CONCLUSIONS: Aspirin sensitized TMZ chemotherapy efficacy through inhibition of ß-catenin transactivation; furthermore, the coloaded microspheres achieved a sustained release action to reduce the TMZ dosage, offering the potential for improved treatment of glioblastomas.