Luyue Chen

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.

AC1MMYR2, an Inhibitor of Dicer-Mediated Biogenesis of Oncomir miR-21, Reverses Epithelial–Mesenchymal Transition and Suppresses Tumor Growth and Progression

The extensive involvement of miRNAs in cancer pathobiology has opened avenues for drug development based on oncomir inhibition. Dicer is the core enzyme in miRNA processing that cleaves the terminal loop of precursor microRNAs (pre-miRNAs) to generate mature miRNA duplexes. Using the three-dimensional structure of the Dicer binding site on the pre-miR-21 oncomir, we conducted an in silico high-throughput screen for small molecules that block miR-21 maturation. By this method, we identified a specific small-molecule inhibitor of miR-21, termed AC1MMYR2, which blocked the ability of Dicer to process pre-miR-21 to mature miR-21. AC1MMYR2 upregulated expression of PTEN, PDCD4, and RECK and reversed epithelial-mesenchymal transition via the induction of E-cadherin expression and the downregulation of mesenchymal markers, thereby suppressing proliferation, survival, and invasion in glioblastoma, breast cancer, and gastric cancer cells. As a single agent in vivo, AC1MMYR2 repressed tumor growth, invasiveness, and metastasis, increasing overall host survival with no observable tissue cytotoxicity in orthotopic models. Our results offer a novel, high-throughput method to screen for small-molecule inhibitors of miRNA maturation, presenting AC1MMYR2 as a broadly useful candidate antitumor drug.

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/β-catenin and the PPARα/AP-1 axis. Further, the expression of miR-21 is regulated by EGFR via the activation of β-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.

Nuclear Translocation of β-catenin is Essential for Glioma Cell Survival

Identification of molecular pathways that are essential for cancer cell survival is vital for understanding the underlying biology, as well as to design effective cancer therapeutics. β-catenin, a multifunctional oncogenic protein, participates in cell development. Its multifaceted functions primarily lie to the subcellular distribution. The present study demonstrated that β-catenin accumulated in the nucleus to a greater extent in high-grade gliomas compared with low-grade gliomas. In addition, nuclear localization correlated with a worse prognosis for patients, as determined by immunohistochemical analysis of 74 glioma samples. Nuclear expression of β-catenin was down-regulated in LN229 and U87 glioma cells by a small molecule inhibitor of β-catenin/TCF4 signaling, demonstrating strongly inhibited β-catenin/TCF4 transcriptional activity and STAT3 luciferase activity, as well as decreased mRNA and protein levels of nuclear β-catenin, TCF4, EGFR, AKT1, AKT2 and STAT3. Furthermore, repressed nuclear translocation of β-catenin resulted in inhibition of proliferation and invasiveness, and also induced apoptosis of glioma cells. Similar results were also observed in vivo; intratumoral injection of such small molecule inhibitor downregulated expression of nuclear β-catenin, TCF4, and components of the EGFR pathway, and also delayed tumor growth in nude mice harboring subcutaneous U87 xenografts. Results from the present study provided evidence that nuclear accumulation of β-catenin participated in malignant progression of gliomas and implicated poor prognosis, highlighting it as a potential therapeutic target for gliomas.

EZH2 is a negative prognostic factor and exhibits pro-oncogenic activity in glioblastoma

The identification of single or less genes based on mRNA expression as clinical diagnostic markers for glioblastoma (GBM) remains a challenge. Recent data have shown the potential oncogenic role and prognostic significance of EZH2 in several human cancers. However, the clinical signature and further mechanisms of EZH2 function in gliomagenesis are still poorly understood. In this study, we found that increased EZH2 expression was associated with tumor grade. High expression of EZH2 in GBM was determined to be a strong and independent predictor of short overall survival. Further, we screened EZH2 targets and associated genes in GBM. Repression of EZH2 induced cell cycle arrest and inhibited tumor growth in vivo. This event represents a positive feedback loop with β-catenin/TCF4 and STAT3 signaling. Taken together, EZH2 could be an independent prognostic factor and potential therapeutic target for GBM.

ICAT inhibits glioblastoma cell proliferation by suppressing Wnt/β-catenin activity

Inhibitor of β-catenin and T-cell factor (ICAT) is a key component of Wnt/β-catenin signaling. ICAT blocks the formation of the β-catenin/TCF complex and has been demonstrated to be involved in embryonic development and carcinogenesis. As an inhibitor of canonical Wnt signaling, ICAT was presumed to be a tumor-suppressor gene. However, the ICAT functions in human glioma remain unknown. In this study, we evaluated the expression of ICAT in 305 human glioma tissues and found that negative ICAT expression correlated with higher grade glioma and poor survival in patients with glioma. Then we transfected glioma cells with ICAT plasmid. Western blotting showed an increased ICAT protein expression level in glioma cells. MTT assay, flow cytometry and cell invasion assay were used to detect cell proliferation, cell cycle distribution, apoptosis and invasion. Our studies confirmed that ICAT inhibits glioma cell proliferation and invasion, and it induces cell apoptosis and cell cycle progression arrest. Besides, ICAT slowed down tumor growth in a glioblastoma xenograft model. Therefore, our study demonstrates that ICAT may serve as a tumor-suppressor in human glioma suggesting a promising direction for targeting therapy in glioma.

Aspirin-/TMZ-coloaded Microspheres Exert Synergistic Antiglioma Efficacy via Inhibition of β-catenin Transactivation

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.

BASI, A Potent Small Molecular Inhibitor, Inhibits Glioblastoma Progression by Targeting microRNA-mediated β-Catenin Signaling

The nuclear localization of β‐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 β‐catenin is an attractive therapeutic strategy for cancers.

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.

Abstract 235: SNORD76, a box C/D snoRNA, acts as a tumor suppressor in glioblastoma

Background Gliomblastoma (GBM) is associated with disproportionately high morbidity and mortality, reflecting the need to develop new diagnostic and therapeutic targets for this disease. Recently, accumulating evidence has suggested that small nucleolar RNAs (snoRNAs) are gaining prominence and are more actively involved in tumorigenesis than previously thought. However, no report concerning the implication of snoRNAs in glioma has been published to date. Methods The expression of SNORD76 and its host gene, GAS5, in glioma patients were evaluated by quantitative PCR. Flow cytometry was used to analyze to cell cycle distribution after SNORD76 over-expression. After SNORD76 over-expression in GBM cell lines, cell growth and anti-tumor effect were investigated in vitro and in vivo. Results SNORD76 expression was selectively decreased in glioblastoma (WHO grade IV) compared with low grades glioma (WHO grade II and III). Forcibly expressed SNORD76 inhibited proliferation and anchorage-independent growth of glioma cells. Flow cytometry demonstrated that enforced SNORD76 expression arrested the cells at S phase of the cell cycle. Western blot Cell cycle-associated proteins cyclin A1, cyclin B1 and p107 were down-regulated after SNORD76 over-expression, while the expression of Rb and pRb increased. The growth of orthotopic tumors was significantly inhibited by forced expression of SNORD76. Conclusion In summary, engineered restoration of SNORD76 caused marked repression of glioma growth in vitro and in vivo. The tumor suppression could be achieved at least partially through inducing S phase arrest in an Rb-associated manner. Thus, SNORD76 may act as a potential tumor suppressive gene in the development and progression of glioblastoma and may be a potential specific glioblastoma biomarker. Citation Format: Luyue chen, Lei Han, kailiang zhang, jianwei wei, peiyu pu, jianning zhang, chunsheng kang. SNORD76, a box C/D snoRNA, acts as a tumor suppressor in glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 235. doi:10.1158/1538-7445.AM2015-235