Glioblastoma Multiforme Invasion Research Articles

Long non-coding RNA plasmacytoma variant translocation 1 (PVT1) promotes glioblastoma multiforme progression via regulating miR-1301-3p/TMBIM6 axis.

To explore whether plasmacytoma variant translocation 1 (PVT1) could regulate glioblastoma multiforme (GBM) progression via microRNA-1301-3p (miR-1301-3p) and transmembrane BAX inhibitor motif containing 6 (TMBIM6) axis. Expression patterns of PVT1 and RMBIM6 in GBM patients were analyzed using GEPIA, an online gene expression analysis tool. Levels of PVT1 in GBM cells and normal cells were analyzed with quantitative real-time PCR method. Cell Counting Kit-8 (CCK-8), transwell invasion assay, and flow cytometry assay were applied to detect cell viability and apoptosis. Connections of PVT1 or TMBIM6 with miR-1301-3p were validated with bioinformatic tool and luciferase activity reporter assay. PVT1 was significantly expressed in GBM tissues and cells. PVT1 promotes GBM cell proliferation and invasion but inhibits apoptosis in vitro. TMBIM6 was significantly expressed in GBM tissues. The knockdown of TMBIM6 reversed the stimulation effects of PVT1 on GBM cell malignancy behaviors with miR-1301-3p as a bridge. Collectively, we showed PVT1 elevated TMBIM6 expression mediated by miR-1301-3p and thus to promote GBM progression.

MiR-4286 is Involved in Connections Between IGF-1 and TGF-β Signaling for the Mesenchymal Transition and Invasion by Glioblastomas.

The insulin-like growth factor (IGF)-1 and transforming growth factor (TGF)-β signal pathways are both recognized as important in regulating cancer prognosis, such as the epithelial-to-mesenchymal transition (EMT) and cell invasion. However, cross-talk between these two signal pathways in glioblastoma multiforme (GBM) is still unclear. In the present study, by analyzing data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GSE) 4412, GBM patients with higher IGF-1 levels exhibited poorer survival. Genes positively correlated with IGF-1 were enriched in EMT and TGF-β signal pathways. IGF-1 treatment enhanced mesenchymal marker expressions and GBM cell invasion. A significant positive correlation was observed for IGF-1 with TGF-β1 (TGFB1) or TGF-β receptor 2 (TGFBR2), both of which participate in TGF-β signaling and are risk genes in the GBM process. IGF-1 stimulation promoted both TGFB1 and TGFBR2 expressions. LY2157299, a TGF-β signaling inhibitor, attenuated IGF-1-enhanced GBM cell invasion and mesenchymal transition. By analyzing IGF-1-regulated microRNA (miR) profiles, miR-4286 was found to be significantly downregulated in IGF-1-treated cells and could be targeted to both TGFB1 and TGFBR2. Overexpression of miR-4286 significantly attenuated expressions of the IGF-1-mediated mesenchymal markers, TGFB1 and TGFBR2. Using kinase inhibitors, only U0126 treatment showed an inhibitory effect on IGF-1-reduced miR-4286 and IGF-1-induced TGFB1/TGFBR2 expressions, suggesting that MEK/ERK signaling is involved in the IGF-1/miR-4286/TGF-β signaling axis. Finally, our results suggested that miR-4286 might act as a tumor suppressive microRNA in inhibiting IGF-1-enhanced GBM cell invasion. In conclusion, IGF-1 is connected to TGF-β signaling in regulating the mesenchymal transition and cell invasion of GBM through inhibition of miR-4286. Our findings provide new directions and mechanisms for exploring GBM progression.

LncRNA HOXA-AS3 promotes the malignancy of glioblastoma through regulating miR-455-5p/USP3 axis.

Our objective was to determine the molecular mechanisms by which lncRNA HOXA‐AS3 regulates the biological behaviour of glioblastoma multiforme (GBM). We used an lncRNA microarray assay to identify GBM‐related lncRNA expression profiles. Qrt‐PCR was used to survey the levels of expression of long non‐coding RNA (lncRNA) HOXA‐AS3 and the target gene. Dual‐luciferase reporter assays were used to investigate the interaction of lncRNA HOXA‐AS3, the target gene and miRNA. Western blot analysis was used to examine the expression of USP3 and epithelial‐mesenchymal transition (EMT) genes. The MTT assay, transwell assay and wound healing assay were used to analyse the effects of lncRNA HOXA‐AS3 on GBM cell viability, mobility and invasiveness, respectively. Our results showed that lncRNA HOXA‐AS3 was significantly up‐regulated in GBM cells and could promote GBM cell proliferation, invasion and migration in vitro and in vivo. HOXA‐AS was found to be associated with poor survival prognosis in glioma patients. The dual‐luciferase reporter assay also revealed that lncRNA HOXA‐AS3 acts as a mir‐455‐5p sponge by up‐regulating USP3 expression to promote GBM progression. Western blot analysis showed that lncRNA HOXA‐AS3 could up‐regulate EMT‐related gene expression in GBM. Experiments showed mir‐455‐5p could rescue the effect of lncRNA HOXA‐AS3 on cell proliferation and invasion. The newly identified HOXA‐AS3/mir‐455‐5p/USP3 pathway offers important clues to understanding the key mechanisms underlying the action of lncRNA HOXA‐AS3 in glioblastoma.

Rhoj Is a Novel Target for Progression and Invasion of Glioblastoma by Impairing Cytoskeleton Dynamics.

Rho GTPase family members were identified as critical regulators of cell morphology, actin cytoskeleton organization, cell movement, and cell cycle and also contributed to tumor progression, which have been implicated in various types of cancer metastasis and growth. Here, we firstly reported the dysregulation of Rhoj in glioblastoma multiforme (GBM) and aimed to investigate the role and mechanism of Rhoj in GBM. We analyzed the expression of 21 Rho GTPases family members and validated the expression of Rhoj in GBM by immunohistochemistry. We further investigated the role and mechanism of Rhoj in GBM both in vitro and in vivo. We observed that Rhoj is significantly overexpressed in GBM and associated with patients' survival. However, the role and underlying molecular mechanism of Rhoj in GBM are still unclear. We demonstrated that transcription factor c-Jun regulated the expression of Rhoj, and Rhoj interacted with moesin to promote GBM cell proliferation and migration by potentiating the activation of Rac1/PAK pathway and cytoskeletal dynamics. Rhoj may promote migration and invasion of GBM cells by regulating epithelial-mesenchymal transition (EMT)-like process. In conclusion, the Rhoj/Rac1/PAK signaling mediates invasion and progression of GBM and is a potential therapeutic target for GBM treatment. Rhoj may also be a promising biomarker for GBM diagnosis and prognosis.

MiR-1271 regulates glioblastoma cell proliferation and invasion by directly targeting the CAMKK2 gene

This study explored the regulatory role of microRNA-1271 (miR-1271) in glioblastoma multiforme (GBM) proliferation and invasion via calcium/calmodulin-dependent protein kinase 2 (CaMKK2). MiR-1271 and CaMKK2 expression were quantified in normal human astrocyte cells, GBM cell lines, and low- and high-grade glioma tissues. MKI67 expression in GBM cells was measured using quantitative real-time polymerase chain reaction. The target relationship between miR-1271 and the CAMKK2 gene was confirmed using the luciferase reporter assay. MTT and Transwell assays were used to analyze the role of miR-1271 and CAMKK2 in cell proliferation and invasion. Finally, CaMKK2 expression and AKT phosphorylation were detected by western blotting. MiR-1271 was significantly downregulated in high-grade glioma tissues and GBM cell lines. Conversely, CAMKK2 mRNA expression was upregulated in high-grade glioma tissues and GBM cell lines. We observed that miR-1271 directly targeted the 3′-untranslated region of CAMKK2, indicating an inverse relationship with miR-1271. Overexpressing miR-1271 inhibited GBM cell proliferation and invasion, whereas inhibiting miR-1271 increased cell proliferation and invasion. Silencing CAMKK2 expression also inhibited GBM cell proliferation and invasion. Furthermore, overexpressing miR-1271 inhibited AKT phosphorylation and MKI67 mRNA expression by targeting CAMKK2. These results indicate that miR-1271 regulates GBM cell proliferation and invasion, and that these effects involve directly targeting the CAMKK2 gene. Therefore, miR-1271 may serve as a new therapeutic target for developing GBM treatments.

Sprouty 4 suppresses glioblastoma invasion by inhibiting ERK phosphorylation and ETS-1-induced matrix metalloproteinase-9.

Glioblastoma multiforme (GBM) is the most malignant glioma with highly aggressive behavior and the worst prognosis. Many efforts have been made to develop new drugs and improve the patient's survival, but the effects are not satisfactory. Here we aimed to evaluate the clinical significance and tumor-repressive function of Sprouty4 (SPRY4) in GBM. In our study, we detected the expression of SPRY4 in 109 GBM patients and 12 pairs of GBM tissues and the corresponding adjacent tissues. χ2 test was applied to analyze the association between SPRY4 expression and the clinicopathological factors. The prognostic significances were evaluated with univariate and multivariate analyses, which were carried out by the Kaplan-Meier method and the Cox-regression proportional hazards model, respectively. With in-vitro experiments, we investigated the tumor-suppressing function of SPRY4 in GBM invasion and investigated the underlying mechanism. SPRY4 mRNAs in GBMs were significantly lower than those in adjacent brain tissues. We demonstrated that SPRY4 expression could predict the favorable prognosis of GBM, and SPRY4 was an independent favorable prognostic factor of GBM. SPRY4 repressed GBM invasion via inhibiting ERK phosphorylation; therefore, suppressing ETS-1-induced MMP9 expression. SPRY4 was an independent favorable prognostic factor of GBM, and it could suppress GBM invasion by ERK-ETS-MMP9 axis. Our results indicated that SPRY4 may be a promising drug target of GBM and SPRY4 detection could stratify patients with low SPRY4 expression who may benefit from anti-FGFR therapy.

The effects of ephrinB2 signaling on proliferation and invasion in glioblastoma multiforme.

The aggressive nature of glioblastoma multiforme (GBM) may be attributed to the dysregulation of pathways driving both proliferation and invasion. EphrinB2, a membrane-bound ligand for some of the Eph receptors, has emerged as a critical target regulating these pathways. In this study, we investigated the role of ephrinB2 in regulating proliferation and invasion in GBM using intracranial and subcutaneous xenograft models. The Cancer Genome Atlas analysis suggested high transcript and low methylation levels of ephrinB2 as poor prognostic indicators in GBM, consistent with its role as an oncogene. EphrinB2 knockdown, however, increased tumor growth, an effect that was reversed by ephrinB2 Fc protein. This was associated with EphB4 receptor activation, consistent with the data showing a significant decrease in tumor growth with ephrinB2 overexpression. Mechanistic analyses showed that ephrinB2 knockdown has anti-invasive but pro-proliferative effects in GBM. EphB4 stimulation following ephrinB2 Fc treatment in ephrinB2 knockdown tumors was shown to impart strong anti-proliferative and anti-invasive effects, which correlated with decrease in PCNA, p-ERK, vimentin, Snail, Fak, and increase in the E-cadherin levels. Overall, our study suggests that ephrinB2 cannot be used as a sole therapeutic target. Concomitant inhibition of ephrinB2 signaling with EphB4 activation is required to achieve maximal therapeutic benefit in GBM.

Largazole is a Brain-Penetrant Class I HDAC Inhibitor with Extended Applicability to Glioblastoma and CNS Diseases.

Largazole is a potent class I selective histone deacetylase inhibitor prodrug with anticancer activity against solid tumors in preclinical models. Largazole possesses in vitro activity against glioblastoma multiforme (GBM) cells and sufficiently crosses the blood-brain barrier based on measurement of the active species, largazole thiol, to achieve therapeutically relevant concentrations in the mouse brain. The effective dose resulted in pronounced functional responses on the transcript level based on RNA sequencing and quantitative polymerase chain reaction after reverse transcription (RT-qPCR), revealing desirable expression changes of genes related to neuroprotection, including Bdnf and Pax6 upregulation, extending the applicability of largazole to the treatment of brain cancer and neurodegenerative disorders. The largazole-induced modulation of Pax6 unifies both activities, since Pax6 expression suppresses GBM proliferation and invasion and inversely correlates with GBM tumor grade, while it is also implicated in neurogenesis, neuronal plasticity, and cognitive ability. Our results suggest that largazole could be repurposed for diseases of the brain.

SRC Kinase in Glioblastoma News from an Old Acquaintance.

Glioblastoma multiforme (GBM) is one of the most recalcitrant brain tumors characterized by a tumor microenvironment (TME) that strongly supports GBM growth, aggressiveness, invasiveness, and resistance to therapy. Importantly, a common feature of GBM is the aberrant activation of receptor tyrosine kinases (RTKs) and of their downstream signaling cascade, including the non-receptor tyrosine kinase SRC. SRC is a central downstream intermediate of many RTKs, which triggers the phosphorylation of many substrates, therefore, promoting the regulation of a wide range of different pathways involved in cell survival, adhesion, proliferation, motility, and angiogenesis. In addition to the aforementioned pathways, SRC constitutive activity promotes and sustains inflammation and metabolic reprogramming concurring with TME development, therefore, actively sustaining tumor growth. Here, we aim to provide an updated picture of the molecular pathways that link SRC to these events in GBM. In addition, SRC targeting strategies are discussed in order to highlight strengths and weaknesses of SRC inhibitors in GBM management, focusing our attention on their potentialities in combination with conventional therapeutic approaches (i.e., temozolomide) to ameliorate therapy effectiveness.

MicroRNA-128-3p Enhances the Chemosensitivity of Temozolomide in Glioblastoma by Targeting c-Met and EMT

Temozolomide is a first line anti-tumor drug used for the treatment of patients with Glioblastoma multiforme (GBM). However, the drug resistance to temozolomide limits its clinical application. Therefore, novel strategies to overcome chemoresistance are desperately needed for improved treatment of human GBM. Recent studies have demonstrated that miRNAs are closely related to resistance to cancer chemotherapy. This study aimed to further validate the biological role of miR-128-3p and to investigate whether miR-128-3p can enhance the chemosensitivity of glioblastoma to temozolomide (TMZ) and the underlying mechanisms. The effects of miR-128-3p and TMZ on the proliferation of glioblastoma cells were investigated by cell counting kit-8 (cck8). Transwell and intracerebral invasion assays were applied to determine the effects of the combination of miR-128-3p and TMZ on the invasion and migration of glioblastoma in vitro and in vivo. Flow cytometry was used to detect apoptosis in each group, and immunofluorescence was used to determine the expression levels of EMT-related proteins. RT-PCR and Western-blot were applied to detect EMT-transformed proteins (c-Met, PDGFRα, Notch1, and Slug) and EMT phenotype-associated proteins (Vim, CD44, and E-cadherin) at both mRNA and protein levels. Based on the microRNA.org database, we predicted the target genes of miR-128-3p. The target-relationship between miR-128-3p and c-Met and PDGFRα was verified by dual luciferase reporter gene. The tumor volume, weight and the expression levels of the proteins described above were measured in subcutaneously transplanted tumor model in nude mice. We found that the expression of miR-128-3p was down-regulated in glioblastoma tissue samples and cell lines. miR-128-3p suppressed the proliferation, migration, and invasion of GBM both in vitro and in vivo; miR-128-3p enhanced the therapeutic effect of TMZ via inhibition of proliferation, invasion and migration of glioblastoma cells and induction of apoptosis. Overexpression of miR-128-3p down-regulated the expression levels of EMT-transformed proteins (c-Met, PDGFRα, Notch1 and Slug) to enhance the effect of TMZ. In addition, we found that miR-128-3p targeted and bound c-Met. More importantly, the upregulation of c‐Met significantly prompted U87 and U251 cell proliferation. This effect could be abolished when c‐Met was silenced. The investigation in tumor bearing nude mice showed that miR-128-3p in combination with TMZ reduced tumor volume and the invasion extent, and increased the sensitivity of glioblastoma to TMZ. miR-128-3p is capable of enhancing the sensitivity of glioblastoma to TMZ through regulating c-Met/EMT.

GBP2 enhances glioblastoma invasion through Stat3/fibronectin pathway.

Guanylate-binding protein 2 (GBP2) is an interferon-inducible large GTPase which is crucial to the protective immunity against microorganisms. However, its biological function in cancer remains largely unknown. Glioblastoma multiforme (GBM) is the most common and deadly brain tumor in adults. Here we show that GBP2 expression is highly elevated in GBM tumor and cell lines, particularly in those of the mesenchymal subtype. High GBP2 expression is associated with poor prognosis. GBP2 overexpression significantly promotes GBM cell migration and invasion in vitro, and GBP2 silencing by RNA interference exhibits opposite effects. We further show that fibronectin (FN1) is dramatically induced by GBP2 expression at both mRNA and protein levels, and FN1 is essential for GBP2-promoted GBM invasiveness. Inhibition of Stat3 pathway prevents GBP2-promoted FN1 induction and cell invasion. Consistently, GBP2 dramatically promotes GBM tumor growth and invasion in mice and significantly reduces the survival time of the mice with tumor. Taken together, these findings establish the role of GBP2/Stat3/FN1 signaling cascade in GBM invasion and suggest GBP2 may serve as a potential therapeutic target for inhibiting GBM invasion.

Zyxin (ZYX) promotes invasion and acts as a biomarker for aggressive phenotypes of human glioblastoma multiforme

Glioblastoma multiforme (GBM) is characterized by highly invasive growth, which leads to extensive infiltration and makes complete tumor excision difficult. Since cytoskeleton proteins are related to leading processes and cell motility, and through analysis of public GBM databases, we determined that an actin-interacting protein, zyxin (ZYX), may involved in GBM invasion. Our own glioma cohort as well as the cancer genome atlas (TCGA), Rembrandt, and Gravendeel databases consistently showed that increased ZYX expression was related to tumor progression and poor prognosis of glioma patients. In vitro and in vivo experiments further confirmed the oncogenic roles of ZYX and demonstrated the role of ZYX in GBM invasive growth. Moreover, RNA-seq and mass-spectrum data from GBM cells with or without ZYX revealed that stathmin 1 (STMN1) was a potential target of ZYX. Subsequently, we found that both mRNA and protein levels of STMN1 were positively regulated by ZYX. Functionally, STMN1 not only promoted invasion of GBM cells but also rescued the invasion repression caused by ZYX loss. Taken together, our results indicate that high ZYX expression was associated with worse prognosis and highlighted that the ZYX-STMN1 axis might be a potential therapeutic target for GBM.

TLR-4 Signaling vs. Immune Checkpoints, miRNAs Molecules, Cancer Stem Cells, and Wingless-Signaling Interplay in Glioblastoma Multiforme-Future Perspectives.

Toll-like-receptor (TLR) family members were detected in the central nervous system (CNS). TLR occurrence was noticed and widely described in glioblastomamultiforme (GBM) cells. After ligand attachment, TLR-4 reorients domains and dimerizes, activates an intracellular cascade, and promotes further cytoplasmatic signaling. There is evidence pointing at a strong relation between TLR-4 signaling and micro ribonucleic acid (miRNA) expression. The TLR-4/miRNA interplay changes typical signaling and encourages them to be a target for modern immunotherapy. TLR-4 agonists initiate signaling and promote programmed death ligand-1 (PD-1L) expression. Most of those molecules are intensively expressed in the GBM microenvironment, resulting in the autocrine induction of regional immunosuppression. Another potential target for immunotreatment is connected with limited TLR-4 signaling that promotes Wnt/DKK-3/claudine-5 signaling, resulting in a limitation of GBM invasiveness. Interestingly, TLR-4 expression results in bordering proliferative trends in cancer stem cells (CSC) and GBM. All of these potential targets could bring new hope for patients suffering from this incurable disease. Clinical trials concerning TLR-4 signaling inhibition/promotion in many cancers are recruiting patients. There is still a lot to do in the field of GBM immunotherapy.

The CXCL12/CXCR4 axis confers temozolomide resistance to human glioblastoma cells via up-regulation of FOXM1

Glioblastoma multiforme (GBM) is the most common primary malignancy in the adult central nervous, and is characterized by high aggressiveness and a high mortality rate. The high mortality rate is largely due to the development of drug resistance. Temozolomide (TMZ) resistance is considered to be one of the major reasons responsible for GBM therapy failure. CXCL12/CXCR4 has been demonstrated to be involved in cell proliferation, migration, invasion, angiogenesis, and radioresistance in GBM. However, its role in TMZ resistance in GBM is unknown. In this study, we aimed to evaluate the role of CXCL12/CXCR4 in mediating the TMZ resistance to GBM cells and explore the underlying mechanisms. We found that the CXCL12/CXCR4 axis enhanced TMZ resistance in GBM cells. Further study showed that CXCL12/CXCR4 conferred TMZ resistance and promoted the migration and invasion of GBM cells by up-regulating FOXM1. This resistance was partially reversed by suppressing CXCL12/CXCR4 and FOXM1 silencing. Our study revealed the vital role of CXCL12/CXCR4 in mediating the resistance of GBM cells to TMZ, and suggested that targeting CXCL12/CXCR4 axis may attenuate the resistance to TMZ in GBM.

Reciprocal control of ADAM17/EGFR/Akt signaling and miR-145 drives GBM invasiveness.

Glioblastoma multiforme (GBM) is one of the most devastating brain malignancies worldwide and is considered to be incurable. However, the mechanisms underlying its aggressiveness remain unclear. The expression of ADAM17 in tissue samples was detected by immunohistochemistry. Knockdown and rescue experiments were used to demonstrate the regulatory effect of ADAM17 on the invasion ability of GBM cells. Western Blot and qPCR were used to detect the expression of related proteins and RNAs. Moreover, a luciferase reporter assay was performed to verify whether miR-145 directly binds to the 3'-UTR of ADAM17. We revealed that ADAM17 was overexpressed in GBM tissues and correlated positively with poor prognosis. The knockdown of ADAM17 obviously suppressed the invasiveness of GBM cell lines. Furthermore, we found that knockdown of ADAM17 decreased activation of EGFR/Akt/C/EBP-β signaling, and consequently upregulated miR-145 expression in GBM cell lines. Notably, miR-145 directly targeted the ADAM17 3'-UTR and suppressed expression levels of ADAM17. Our findings define an ADAM17/EGFR/miR-145 feedback loop that drives the GBM invasion. Reciprocal regulation between ADAM17 and miR-145 results in aberrant activation of EGFR signaling, suggesting that inhibition of ADAM17 expression can be an ideal therapeutic strategy for the treatment of GBM.

Calpain suppresses cell growth and invasion of glioblastoma multiforme by producing the cleavage of filamin A.

Filamin A is the most widely expressed isoform of filamin in mammalian tissues. It can be hydrolyzed by Calpain, producing a 90-kDa carboxyl-terminal fragment (ABP90). Calpeptin is a chemical inhibitor of Calpain, which can inhibit this effect. It has been shown that ABP90 acts as a transcription factor which is involved in mediating cell signaling. However, the significance of ABP90 and its clinical signature with underlying mechanisms have not been well studied in glioblastoma multiforme (GBM). ABP90 protein was measured in 36 glioma patients by Western blot. Human GBM cell lines U87 and A172 were used to clarify the precise role of ABP90. CCK-8 assay was used to analyze the cell viability. Transwell invasion assay and wound healing assay were used to analyze the migration and invasion. Expression of matrix metalloproteinase 2/tissue inhibitors of metalloproteinase 2 (MMP2/TIMP2) protein was analyzed by Western blot. ABP90 protein expression was lower in GBM tissues. The patients with low ABP90 protein expression had a shorter OS time (p = 0.046). After being treated with Calpain, the expression of ABP90 was upregulated, which led to a decline of cell viability, enhanced the efficacy of temozolomide and restrained the cell invasion. Calpeptin could inhibit the effect. The mechanism might be involved in the balance of MMP2/TIMP2. Our present data suggest that ABP90 expression is a significant prognostic factor and may play an important role in cell viability, chemotherapeutic sensitivity and invasion of GBM.

Development of Pyrazolo[3,4-d]pyrimidine Kinase Inhibitors as Potential Clinical Candidates for Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor. Residual cells at the tumor margin are responsible for up to 85% of GBM recurrences after standard treatment. Despite this evidence, the identification of compounds active on this cell population is still an underexplored field. Herein, starting from the knowledge that kinases are implicated in GBM, we evaluated three in-house pyrazolo[3,4-d]pyrimidines active as Src, Fyn, and SGK1 kinase inhibitors against patient derived cell lines from either the invasive region or contrast-enhanced core of GBM. We identified our Src inhibitor, SI306, as a promising lead compound for eradicating invasive GBM cells. Furthermore, aiming at the development of a feasible oral treatment for GBM, we performed a formulation study using 2D inkjet printing to generate soluble polymer-drug dispersions. Overall, this study led to the identification of a set of polymer-formulated pyrazolo[3,4-d]pyrimidine kinase inhibitors as promising candidates for GBM preclinical efficacy studies.

CBMS-04 SIGNIFICANT ROLE OF HYPOXIA IN THE EXPRESSION AND FUNCTION OF OSTEOPONTIN IN CD44-HIGHLY EXPRESSED GLIOMA STEM-LIKE CELLS IN TUMOR PROGRESSION OF GLIOBLASTOMA

The poor prognosis of glioblastoma multiforme (GBM) may be due to the surviving glioma stem-like cells (GSCs) in the tumor periphery after tumor resection. We demonstrated that CD44-expressed GSCs existed much more in the tumor periphery of high invasive (HI) type GBM than low invasive (LI) type GBM. The HI type was significantly associated with worse outcome, but how GSCs with high CD44 expression relate to tumor progression remains unknown. In this study, we investigated effects of hypoxia on CD44-directed signal pathways, leading to tumor invasion and proliferation in GBM. We focused on the CD44 ligand osteopontin (OPN) because it is known hypoxia affects the interaction of CD44 and OPN which promotes stemness and proliferation of cancer stem cells. We examined mRNA expressions of hypoxia inducible factor (HIF)-1a, HIF-2a, CD44 and OPN in tumor tissues of GBM and investigated effects of hypoxia (1%O2:severe or 5%O2:moderate) on the expression of these molecules using cultured GSCs that were established from tumor tissues showing high CD44 expression in the periphery of GBMs. In addition, we analyzed the effects of OPN on invasive, migratory and proliferative activities of GSCs under the hypoxic conditions. OPN was much higher expressed in the tumor periphery of LI type GBM than HI type GBM. Severe hypoxia significantly increased the expressions of HIF-1a and CD44 but did not OPN. On the other hands, moderate hypoxia promoted the expressions of HIF-2a and OPN. Knockdown of HIF-2a significantly inhibited OPN expression. In addition, the more OPN was expressed in the cultured GSCs under moderate hypoxia, the more the GSCs proliferated and decreased their invasive and migratory activities. In conclusion, GSCs existing in the tumor periphery of GBM can migrate or proliferate by changing CD44-directed signal pathways. Moderate hypoxia promoted HIF-2a/OPN/CD44 pathway, resulting in phenotypic transition to high proliferative tumors.

MicroRNA‑432 inhibits the aggressiveness of glioblastoma multiforme by directly targeting IGF‑1R.

MicroRNA‑432 (miR‑432) has been studied in multiple tumors, but the expression status, biological functions and the mechanism of action of miR‑432 in glioblastoma multiforme (GBM) are yet to be elucidated. In the present study, miR‑432 expression in GBM was determined and its clinical significance was evaluated among patients with GBM. The effects on the malignancy of GBM invitro and invivo were examined in detail and the interactions between miR‑432 and insulin‑like growth factor 1 receptor (IGF‑1R) mRNA were then explored. miR‑432 expression in GBM tissue samples and cell lines was measured by reverse transcription‑quantitative (RT‑q)PCR. GBM cell proliferation, apoptosis, migration and invasion invitro and tumor growth invivo were evaluated by a Cell Counting Kit‑8 assay, flow‑cytometric analysis, Transwell migration and invasion assays, and a tumor xenograft experiment, respectively. Bioinformatic analysis followed by a luciferase reporter assay, RT‑qPCR and western blotting was applied to demonstrate that IGF‑1R is a direct target gene of miR‑432 in GBM cells. It was found that miR‑432 is downregulated in GBM tumors and cell lines. miR‑432 under expression obviously correlated with the Karnofsky Performance Status score and shorter overall survival among patients with GBM. Exogenous miR‑432 expression significantly reduced proliferation and induced apoptosis of GBM cells. In addition, miR‑432 overexpression impaired the migratory and invasive abilities of GBM cells invitro and decreased their tumor growth invivo. Furthermore, IGF‑1R was validated as a direct target gene of miR‑432 in GBM cells. IGF‑1R knockdown imitated the tumor‑suppressive actions of miR‑432 overexpression in GBM cells. Rescue experiments proved IGF‑1R downregulation to be essential for the effects of miR‑432 on GBM cells. The results of the present study revealed a tumor‑suppressive role of the miR‑432‑IGF‑1R axis in GBM cells and this axis may have implications for GBM therapy.

MiR-802 inhibits the proliferation, invasion, and epithelial-mesenchymal transition of glioblastoma multiforme cells by directly targeting SIX4.

It is well known that the sine oculis homeobox 4 (SIX4) expression is very relevant to the progression of multiple cancers. Moreover, we found that miR-802 could directly target the SIX4. However, the precise mechanism of miR-802 in glioblastoma multiforme (GBM) is still unknown. The aim of this study is to investigate the roles of miR-802/SIX4 axis in GBM. Here, our results showed that the SIX4 expression was obviously increased in GBM tissues and cell lines, and the miR-802 level was distinctly decreased. What is more, the SIX4 expression was negatively related to the miR-802 level in GBM tissues. Furthermore, increased miR-802 level evidently restrained the proliferation, invasion, and epithelial-mesenchymal transition (EMT) of GBM cells. Next, we confirmed that miR-802 could directly target SIX4 by using luciferase reporter assay. Besides, the knockdown of SIX4 had the similar effects with miR-802 overexpression on GBM cells. The inhibitory effects of miR-802 mimic were partially blocked by SIX4 overexpression. Altogether, the overexpression of miR-802 restrained cell proliferation, invasion, and EMT of GBM cells via the regulation of SIX4. SIGNIFICANCE OF THE STUDY: An elevated expression of SIX4 has been observed in colorectal cancer and nonsmall cell lung cancer. However, the precise roles of SIX4 in GBM have not been elucidated. Our study for the first time demonstrated that SIX4 level was significantly upregulated in GBM. Additionally, the knockdown of SIX4 inhibited cell growth, invasion, and the EMT of GBM. Moreover, our data suggested a significant negative correlation between miR-802 and SIX4 expression in GBM. MiR-802 suppressed GBM cell proliferation, invasion, and EMT by directly targeting SIX4, which suggested important roles for miR-802/SIX4 axis in the GBM pathogenesis and its potential application in cancer therapy.