Role of WW domain proteins WWOX in development,prognosis, and treatment response of glioma

Glioblastoma multiforme (GBM) is the most aggressive and malignant brain tumor. Delicate microenvironment and lineage heterogeneity of GBM cells including infiltration, hypoxia, angiogenesis, and stemness make them highly resistant to current conventional therapies, with an average life expectancy for GBM patients of less than 15 months. Poor response to cytotoxic agents of GBM cells remains the major challenge of GBM treatment. Resistance of GBM to clinical treatment is a result of genomic alternation and deregulated signaling pathways, such as p53 mutation and apoptosis signaling blockage, providing cancer cells more opportunities for survival rather than cell death. WW domain-containing oxidoreductase (WWOX) is a tumor suppressor gene, commonly downregulated in various types of tumors, including GBM. It has been found that the reintroduction of WWOX induced p53-mutant GBM cells to undergo apoptosis, but not in p53 wild-type GBM cells, indicating WWOX is likely to reopen apoptosis pathways in a p53-independent manner in GBM. Identifying the crucial target modulated by WWOX deficiency provides a potential therapeutic target for GBM treatment. Here, we have reviewed the literatures about the role of WWOX in development, signaling pathway, prognosis, and treatment response in malignant glioma.     

LncRNA RP1-86C11.7 exacerbates the glioma progression and oncogenicity by hsa-miR-144-3p/TFRC signaling.

Glioblastoma (GBM) remains the most common and malignant tumor of the human central nervous system. Increasing evidence has highlighted that tumor cells with high transferrin receptor (TFRC) expression show advantages in growth. Long noncoding RNAs (lncRNAs) are related to glioma progression by mediating microRNAs (miRNAs). However, the underlying mechanism among TFRC, miRNA and lncRNA in GBM is limited. In the current study, we identified a new lncRNA-induced signaling mechanism that regulates the TFRC levels in GBM. The TFRC level was higher in glioma cell lines, and elevated TFRC expression promoted the proliferation and survival of glioma cells. Further study showed that hsa-miR-144a-3p bound to the 3′-UTR of TFRC mRNA and inhibited its expression, preventing the malignant properties of glioma cells, such as proliferation and survival. We also found that the lncRNA RP1-86C11.7 sponges hsa-miR-144-3p to suppress its protective role in glioma. RP1-86C11.7 overexpression in glioma cells elevated TFRC expression, increased the intracellular free iron level, and deteriorated oncogenicity, with a significant reduction in hsa-miR-144-3p. By contrast, silencing RP1-86C11.7 upregulated the hsa-miR-144-3p level, resulting in decreased TFRC expression and repressed glioma progression. However, the effect of silencing RP1-86C11.7 was reversed with simultaneous hsa-miR-144-3p inhibitor treatment: the TFRC level, intracellular iron level and proliferation in glioma cells increased. Mechanistically, our data indicated that RP1-86C11.7 exacerbates the malignant behavior of glioma through the hsa-miR-144-3p/TFRC axis. RP1-86C11.7 may be a potential biomarker or target to treat glioma in the future.     

LncRNA RP1-86C11.7 exacerbates the glioma progression and oncogenicity by hsa-miR-144-3p/TFRC signaling.

Glioblastoma (GBM) remains the most common and malignant tumor of the human central nervous system. Increasing evidence has highlighted that tumor cells with high transferrin receptor (TFRC) expression show advantages in growth. Long noncoding RNAs (lncRNAs) are related to glioma progression by mediating microRNAs (miRNAs). However, the underlying mechanism among TFRC, miRNA and lncRNA in GBM is limited. In the current study, we identified a new lncRNA-induced signaling mechanism that regulates the TFRC levels in GBM. The TFRC level was higher in glioma cell lines, and elevated TFRC expression promoted the proliferation and survival of glioma cells. Further study showed that hsa-miR-144a-3p bound to the 3′-UTR of TFRC mRNA and inhibited its expression, preventing the malignant properties of glioma cells, such as proliferation and survival. We also found that the lncRNA RP1-86C11.7 sponges hsa-miR-144-3p to suppress its protective role in glioma. RP1-86C11.7 overexpression in glioma cells elevated TFRC expression, increased the intracellular free iron level, and deteriorated oncogenicity, with a significant reduction in hsa-miR-144-3p. By contrast, silencing RP1-86C11.7 upregulated the hsa-miR-144-3p level, resulting in decreased TFRC expression and repressed glioma progression. However, the effect of silencing RP1-86C11.7 was reversed with simultaneous hsa-miR-144-3p inhibitor treatment: the TFRC level, intracellular iron level and proliferation in glioma cells increased. Mechanistically, our data indicated that RP1-86C11.7 exacerbates the malignant behavior of glioma through the hsa-miR-144-3p/TFRC axis. RP1-86C11.7 may be a potential biomarker or target to treat glioma in the future.     

MiR-125a-3p Regulates Glioma Apoptosis and Invasion by Regulating Nrg1

The current study was designed to examine the functional role and mechanism of miR-125a-3p in glioma development. Quantitative RT-PCR was used to evaluate miR-125a-3p expression in 60 glioma cases of different malignant grades. Then, the clinic pathologic significance of miR-125a-3p expression was determined in combination with the prognosis of the patients. In addition, the effects and mechanisms of miR-125a-3p on the proliferation, apoptosis and invasion of glioma cells were further investigated. The results showed that the expression of miR-125a-3p was decreased significantly in most malignant glioma samples relative to normal brain tissues and glioma tissues of low-malignant degree. Further kaplan-meier survival analysis showed that the lower expression of miR-125a-3p was associated with a poor prognosis of GBM patients. Functional analysis showed that the reintroduction of miR-125a-3p into glioblastoma cell lines induces markedly the apoptosis and suppresses the proliferation and migration of glioblastoma cells in vitro and in vivo. Luciferase assay and Western blot analysis revealed that Nrg1 is a direct target of miR-125a-3p. Furthermore, an increased expression of Nrg1 could reverse the effects of overexpression of miR-125a-3p on the proliferation, apoptosis and migration of glioblastoma cells. These findings suggest that miR-125a-3p performed an important role in glioma development mediated by directly regulating the expression of Nrg1. This study also provides a potential target for diagnosis and treatment of malignant glioma.     

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.     

Chromosomal aberration arises during somatic reprogramming to pluripotent stem cells

Recurrence of Glioblastoma multiforme (GBM) seems to be the rule despite combination therapies. Cell invasion and cell proliferation are major reasons for recurrence of GBM. And insulin-like growth factor binding protein 5 (IGFBP5) is the most conserved of the IGFBPs and is frequently dysregulated in cancers and metastatic tissues. By studying the human glioma tissues, we find that IGFBP5 expression associate to the histopathological classification and highly expressed in GBM. Using IGFBP5 mutants we demonstrate that knockdown of IGFBP5 inhibited cell invasion, whereas promoting cell proliferation in GBM cells. Mechanistically, we observed that promoting GBM cell proliferation by inhibiting IGFBP5 was associated with stimulating Akt (Protein kinase B) phosphorylation. However, IGFBP5 promote GBM cell invasion was related to the epithelial-to-mesenchymal transition (EMT). Furthermore, the Chinese Glioma Genome Altas (CGGA) database show that IGFBP5 is significantly increased in recurrent glioma and it predicted worse survival. The obtained results indicate that IGFBP5 has two sides in GBM—inhibiting cell proliferation but promoting cell invasion.     

Human Cytomegalovirus Gene Expression in Long-Term Infected Glioma Stem Cells

The most common adult primary brain tumor, glioblastoma (GBM), is characterized by fifteen months median patient survival and has no clear etiology. We and others have identified the presence of human cytomegalovirus (HCMV) gene products endogenously expressed in GBM tissue and primary cells, with a subset of viral genes being consistently expressed in most samples. Among these viral genes, several have important oncomodulatory properties, regulating tumor stemness, proliferation, immune evasion, invasion and angiogenesis. These findings lead us to hypothesize that a specific HCMV gene signature may be associated with GBM pathogenesis. To investigate this hypothesis, we used glioma cell lines and primary glioma stem-like cells (GSC) infected with clinical and laboratory HCMV strains and measured relative viral gene expression levels along several time points up to 15 weeks post-infection. While HCMV gene expression was detected in several infected glioma lines through week 5 post-infection, only HCMV-infected GSC expressed viral gene products 15 weeks post-infection. Efficiency of infection across time was higher in GSC compared to cell lines. Importantly, HCMV-infected GSC outlived their uninfected counterparts, and this extended survival was paralleled by increased tumorsphere frequency and upregulation of stemness regulators, such as SOX2, p-STAT3, and BMX (a novel HCMV target identified in this study). Interleukin 6 (IL-6) treatment significantly upregulated HCMV gene expression in long-term infected glioma cultures, suggesting that pro-inflammatory signaling in the tumor milieu may further augment HCMV gene expression and subsequent tumor progression driven by viral-induced cellular signaling. Together, our data support a critical role for long-term, low-level HCMV infection in promoting survival, stemness, and proliferation of GSC that could significantly contribute to GBM pathogenesis.     

Activation of p53 by nutlin-3a induces apoptosis and cellular senescence in human glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. Despite concerted efforts to improve current therapies and develop novel clinical approaches, patient survival remains poor. As such, increasing attention has focused on developing new therapeutic strategies that specifically target the apoptotic pathway in order to improve treatment responses. Recently, nutlins, small-molecule antagonists of MDM2, have been developed to inhibit p53-MDM2 interaction and activate p53 signaling in cancer cells. Glioma cell lines and primary cultured glioblastoma cells were treated with nutlin-3a. Nutlin-3a induced p53-dependent G1- and G2-M cell cycle arrest and apoptosis in glioma cell lines with normal TP53 status. In addition, nutlin-arrested glioma cells show morphological features of senescence and persistent induction of p21 protein. Furthermore, senescence induced by nutlin-3a might be depending on mTOR pathway activity. In wild-type TP53 primary cultured cells, exposure to nutlin-3a resulted in variable degrees of apoptosis as well as cellular features of senescence. Nutlin-3a-induced apoptosis and senescence were firmly dependent on the presence of functional p53, as revealed by the fact that glioblastoma cells with knockdown p53 with specific siRNA, or cells with mutated or functionally impaired p53 pathway, were completely insensitive to the drug. Finally, we also found that nutlin-3a increased response of glioma cells to radiation therapy. The results provide a basis for the rational use of MDM2 antagonists as a novel treatment option for glioblastoma patients.     

MicroRNA-21 Promotes Glioblastoma Tumorigenesis by Down-regulating Insulin-like Growth Factor-binding Protein-3 (IGFBP3)

Despite advances in surgery, imaging, chemotherapy, and radiation, patients with glioblastoma multiforme (GBM), the most common histological subtype of glioma, have an especially dismal prognosis; >70% of GBM patients die within 2 years of diagnosis. In many human cancers, the microRNA miR-21 is overexpressed, and accumulating evidence indicates that it functions as an oncogene. Here, we report that miR-21 is overexpressed in human GBM cell lines and tumor tissue. Moreover, miR-21 expression in GBM patient samples is inversely correlated with patient survival. Knockdown of miR-21 in GBM cells inhibited cell proliferation in vitro and markedly inhibited tumor formation in vivo. A number of known miR-21 targets have been identified previously. By microarray analysis, we identified and validated insulin-like growth factor (IGF)-binding protein-3 (IGFBP3) as a novel miR-21 target gene. Overexpression of IGFBP3 in glioma cells inhibited cell proliferation in vitro and inhibited tumor formation of glioma xenografts in vivo. The critical role that IGFBP3 plays in miR-21-mediated actions was demonstrated by a rescue experiment, in which IGFBP3 knockdown in miR-21KD glioblastoma cells restored tumorigenesis. Examination of tumors from GBM patients showed that there was an inverse relationship between IGFBP3 and miR-21 expression and that increased IGFBP3 expression correlated with better patient survival. Our results identify IGFBP3 as a novel miR-21 target gene in glioblastoma and suggest that the oncogenic miRNA miR-21 down-regulates the expression of IGFBP3, which acts as a tumor suppressor in human glioblastoma.     

Gastrin-Releasing Peptide Receptor Knockdown Induces Senescence in Glioblastoma Cells

Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor, characterized by excessive cell proliferation, resistance to apoptosis, and invasiveness. Due to resistance to currently available treatment options, the prognosis for patients with GBM is very dismal. The activation of gastrin-releasing peptide receptors (GRPR) stimulates GBM cell proliferation, whereas GRPR antagonists induce antiproliferative effects in in vitro and in vivo experimental models of GBM. However, the role of GRPR in regulating other aspects of GBM cell function related to tumor progression remains poorly understood, and previous studies have not used RNA interference techniques as tools to examine GRPR function in GBM. Here, we found that stable GRPR knockdown by a lentiviral vector using a short hairpin interfering RNA sequence in human A172 GBM cells resulted in increased cell size and altered cell cycle dynamics consistent with cell senescence. These changes were accompanied by increases in the content of p53, p21, and p16, activation of epidermal growth factor receptors (EGFR), and a reduction in p38 content. These results increase our understanding of GRPR regulation of GBM cells and further support that GRPR may be a relevant therapeutic target in GBM.     

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.     

AGAP2-AS1 serves as an oncogenic lncRNA and prognostic biomarker in glioblastoma multiforme

Long noncoding RNA (lncRNA) AGAP2 antisense RNA 1 (AGAP2-AS1) has been suggested to function as an oncogenic lncRNA in lung cancer, breast cancer, and anaplastic glioma. However, the expression pattern and molecular mechanism of AGAP2-AS1 in glioblastoma multiforme (GBM) remains unknown. The purpose of this study is to present more evidence about the clinical and biological function of AGAP2-AS1 in GBM. In our results, we found AGAP2-AS1 expression was increased in GBM compared with adjacent normal brain tissues or low-grade glioma tissues, and there was no significantly different between low-grade glioma tissues and normal tissues. Kaplan-Meier survival analysis indicated patients with GBM having high-expression of AGAP2-AS1 had shorter overall survival time than those with low expression of AGAP2-AS1. The loss-of-function studies showed that downregulation of AGAP2-AS1 depressed cell proliferation, migration, and invasion, and promoted cell apoptosis in GBM. In summary, AGAP2-AS1 is a prognostic biomarker for patients with GBM, and functions as an oncogenic lncRNA to modulate GBM cell proliferation, apoptosis, migration, and invasion, which suggests that AGAP2-AS1 is potential therapeutic target for GBM.     

Hairless regulates p53 target genes to exert tumor suppressive functions in glioblastoma

Glioblastoma (GBM) is the most common malignant brain tumor and is associated with a poor prognosis, with most patients living less than a year after diagnosis. Given that GBM nearly always recurs after conventional treatments, there is an urgent need to identify novel molecular targets. Hairless (HR) is a nuclear factor enriched in the skin and has been previously implicated in hair cycling. HR is also highly expressed in the brain, but its significance is unknown. We found that human hairless gene (HR) expression is significantly decreased in all GBM subtypes compared with normal brain tissue and is predictive of prognosis, which suggests that loss of HR expression can contribute to GBM pathogenesis. HR was recently discovered to bind to and regulate p53 responsive elements, and thus we hypothesized that HR may have a tumor suppressive function in GBM by modulating p53 target gene expression. We found that HR indeed regulates p53 target genes, including those implicated in cell cycle progression and apoptosis in the GBM-derived U87 cell line, and restoring HR expression triggered G2/M arrest and apoptosis. An analysis of sequenced genomes from patients with GBM revealed 10 HR somatic mutations in patients with glioma, two of which are located in the histone demethylase domain of HR. These two mutations, P996S and K1004N, were reconstructed and found to have impaired p53 transactivating properties. Collectively, the results of our study suggest that HR has tumor suppressive functions in GBM, which may be clinically relevant and a potential avenue for therapeutic intervention.     

Inhibition of glycolytic metabolism in glioblastoma cells by Pt3glc combinated with PI3K inhibitor via SIRT3-mediated mitochondrial and PI3K/Akt–MAPK pathway

Glioblastoma multiforme (GBM) is the most malignant and aggressive glioma with abnormal expression of genes that mediate glycolytic metabolism and tumor cell growth. Petunidin-3-O-glucoside (Pt3glc) is a kind of anthocyanin in the red grape and derived beverages, representing the most common naturally occurring anthocyanins with a reduced incidence of cancer and heart diseases. In this study, whether Pt3glc could effectively regulate glycolysis to inhibit GBM cell was investigated by using the DBTRG-05MG cell lines. Notably, Pt3glc displayed potent antiproliferative activity and significantly changed the protein levels related to both glycolytic metabolism and GBM cell survival. The expression of the proapoptotic protein Bcl-2-associated X protein was increased with concomitant reduction on the levels of the antiapoptotic protein B-cell lymphoma 2 and caspase-3 activity. Furthermore, the levels of survival signaling proteins, such as protein kinase B (Akt) and phospho-Akt (Scr473), extracellular signal-regulated kinase (ERK) and phospho-ERK, were significantly decreased by Pt3glc in combination with the phosphoinositide 3-kinase (PI3K) inhibitor of LY294002. Most importantly, the levels of Sirtuin 3 (SIRT3) and phosphorylated p53 were also downregulated, indicating that Pt3glc combinated with PI3K inhibitor could induce GBM cell death may act via the SIRT3/p53-mediated mitochondrial and PI3K/Akt-ERK pathways. Our findings thus provide rational evidence that the combination of Pt3glc with PI3K inhibitor, which target alternative pathways in GBM cells, may be a useful adjuvant therapy in glioblastoma treatment.