Promotes Glioma Cell Migration Research Articles

Methylation-induced downregulation and tumor-suppressive role of microRNA-98 in glioma through targeting Sal-like protein 4.

MicroRNAs (miRs) have been found to play key roles in various human cancers, but the detailed regulatory mechanism of miR-98 in glioma remains largely unknown. The findings of the present study demonstrated that miR-98 was frequently downregulated in glioma tissues and cell lines (U87, U251, U373 and SHG44), and the decreased miR-98 levels were associated with DNA methylation. Treatment with 5-Aza-20-deoxycytidine, a DNA methyltransferase inhibitor, significantly increased the expression of miR-98 in glioma cells. Moreover, both miR-98 downregulation and methylation were significantly associated with a more aggressive tumor phenotype in glioma, as well as shorter survival time of glioma patients. Restoration of miR-98 expression caused a marked decrease in the migration and invasion of U87 cells, but did not affect cell proliferation. Sal-like protein 4 (SALL4) was further identified as a novel target gene of miR-98, and its protein expression was negatively regulated by miR-98 in U87 cells. Restoration of SALL4 expression reversed the suppressive effects of miR-98 on the migration and invasion of U87 cells. Furthermore, SALL4 was significantly upregulated in glioma tissues and cell lines, and an inverse correlation between miR-98 and SALL4 expression in glioma tissues was identified. In addition, the increased expression of SALL4 was significantly associated with glioma progression. Taken together, these data demonstrated that downregulation of miR-98, induced by methylation, promotes glioma cell migration and invasion via targeting SALL4. Therefore, miR-98 may become a potential therapeutic candidate for glioma.

RNA-binding Protein UNR Promotes Glioma Cell Migration and Regulates the Expression of Ribosomal Protein L9.

Objective To investigate the role of RNA binding protein─upstream-of-N-Ras (UNR) in the development of glioma and its molecular mechanism.Methods First, bioinformatics analysis of CGGA database was performed to detect UNR expression level and prognosis of patients with glioma. Western blot and real-time PCR were used to detect UNR expression level in glioma cell lines and tissues. Next, UNR siRNAs were transfected in glioma cells, and MTS assay and scratch wound-healing assay were used to detect changes in cell proliferation and migration. Then, the candidate UNR target mRNAs were identified by analyzing the sequencing data of UNR iCLIP-seq, RNA sequencing and ribosome profiling databases of human melanoma. RNA immunoprecipitation and biotin pull-down assays were used to identify the UNR target mRNAs in glioma cells. Finally, western blot was used to detect the effect of UNR knockdown on ribosomal protein L9 (RPL9) and RPL9 protein expression level in glioma cell lines. RPL9 siRNA was transfected in A172 and T98G and the expression of vimentin in the cells was detected with western blot.Results Bioinformatics analysis showed that UNR mRNA expression level was significantly higher in high-grade glioma [Grade 2 (n=126), Grade 3 (n=51), Grade 4 (n=128), P<0.001]. UNR high expression levels were associated with poor prognosis (P=0.0177). UNR had high expression level in glioma cell lines and patient samples compared with normal cell lines and normal brain samples (P<0.01). Knockdown of UNR inhibited glioma cells migration (P<0.05), but did not inhibit glioma cells growth in three glioma cell lines. UNR binded the 3' untranslated region (UTR) of PTEN and RPL9 mRNAs. RPL9 protein was significantly highly expressed in most glioma cell lines (n=9) and knockdown of UNR resulted in a downregulation of RPL9 protein expression. Epithelial-mesenchymal transition (EMT)-related marker─vimentin was positively regulated by RPL9.Conclusions UNR could bind to the 3'UTR of PTEN and RPL9 in glioma cell lines, therefore promoting glioma cell migration and regulating the expression of RPL9. Here, we establish a link between UNR and RPL9 protein, which will provide new ideas for the further study of glioma.

Effects of syndecan-1 on the expression of syntenin and the migration of U251 glioma cells.

Glioma is the most frequently occuring primary brain tumor. Syndecan-1 (SDC1) expression is related to poor prognosis of numerous human malignancies including glioma. Syndecan binding protein (SDCBP) is an important partner for SDC1. The present study investigated whether SDC1 and SDCBP are expressed in glioma and their functions on glioma cell migration. An immunohistochemical assay revealed that SDC1 and SDCBP were expressed and were positively related to malignant level of glioma (SDC1, rs=0.576, P=0.001; SDCBP, rs=0.661, P<0.001). Moreover, the protein levels of SDC1 were positively correlated with those of SDCBP in glioma tissues (rs=0.628, P=0.001). In U251 glioma cells, protein levels of SDC1 and SDCBP were both upregulated in U251 cells with SDC1 overexpression, while downregulated with SDC1 knockdown. Transwell assay and scratch-wound healing assay showed that SDC1 overexpression significantly increased U251 cell migration, while SDC1 knockdown had the opposite effects. Rac1 activity, signal transducer and activator of transcription 3 (STAT3) phosphorylation, as well as expression of matrix metalloproteinase 2 (MMP2) and MMP9 was significantly increased by SDC1 overexpression, while was decreased by SDC1 knockdown. In conclusion, SDC1 overexpression upregulated SDCBP expression, and promoted glioma cell migration via Rac1 activation.

IGF2 mRNA binding protein 3 (IMP3) promotes glioma cell migration by enhancing the translation of RELA/p65

The diffusely infiltrative nature of glioblastoma (GBM) makes them highly recurrent. IGF2 mRNA-binding protein 3 (IMP3), a GBM upregulated RNA binding protein, promotes glioma cell migration. An integrative bioinformatics analysis identified p65 (RELA), a subunit of NF-κB heterodimer as a target and an important mediator of IMP3 promoted glioma cell migration. IMP3 increased p65 protein levels without any change in p65 transcript levels, but promoted its polysome association. RIP-PCR demonstrated the binding of IMP3 to p65 transcript. UV crosslinking experiments with in vitro transcribed RNA confirmed the specific and direct binding of IMP3 to sites on p65 3′UTR. Further, IMP3 induced luciferase activity from p65 3′UTR reporter carrying wild type sites but not mutated sites. Exogenous overexpression of p65 from a 3′UTR-less construct rescued the reduced migration of glioma cells in IMP3 silenced condition. In addition, IMP3 silencing inhibited glioma stem-like cell maintenance and migration. The exogenous overexpression of 3′UTR-less p65 significantly alleviated the inhibition of neurosphere formation observed in IMP3 silenced glioma stem-like cells. Further, we show that IMP3 is transcriptionally activated by NF-κB pathway indicating the presence of a positive feedback loop between IMP3 and p65. This study establishes p65 as a novel target of IMP3 in increasing glioma cell migration and underscores the significance of IMP3-p65 feedback loop for therapeutic targeting in GBM.

MicroRNA‑21 promotes migration and invasion of glioma cells via activation of Sox2 and β‑catenin signaling.

The expression of microRNA 21 (miR-21) has been reported to be upregulated in various types of cancer, including malignant gliomas. However, its functions and mechanisms in glioma remain to be fully elucidated. The present study established miRNA-21 overexpression and knockdown cell lines using SRY-box 2 (Sox2) small interfering RNA (siRNA) to knockdown expression and Sox2 cDNA was cloned into pcDNA 3.1 mammalian expression vector for ectopic expression. BIO and XAV-939 were used for β-catenin signaling activation and knockdown, respectively. Transwell chambers were used to assay the capacity of cells to migrate. The present study determined that increased expression of miR-21 significantly promoted the migration and invasion of glioma cells, which was accompanied by an upregulated expression of the Sox2 protein. Sox2 overexpression also promoted glioma cell migration and invasion, whereas Sox2 siRNA markedly reduced the miR-21-enhanced migration and invasion of glioma cells, indicating Sox2 may act as a crucial mediator of miR-21 function. Furthermore, miR-21 also upregulated the protein expression level of β-catenin, whereas anti-miR-21 and Sox2 knockdown significantly reduced β-catenin expression. BIO, a β-catenin specific agonist, enhanced migration and invasion of glioma cells. XAV-939, an inhibitor of β-catenin signaling, markedly inhibited the migration and invasion of glioma cells, suggesting that β-catenin may be associated with miR-21- and Sox2-induced invasion of glioma cells. Notably, BIO restored the migration and invasion potential of glioma cells, which were inhibited by Sox2 siRNA and anti-miR-21. These findings indicated that β-catenin may be an important downstream mediator of miR-21 and Sox2. Therefore, the present study identified the miR-21/Sox2/β-catenin signaling pathway, which may regulate the migration and invasion of human glioma cells.

Abstract LB-343: Integrative functional genomic analysis identifies epigenetically regulated fibromodulin as an essential gene for glioma cell migration

Abstract Glioblastoma (GBM), the grade IV astrocytoma, is the most malignant form of glioma with a median survival of 15 months approximately. In spite of all the progress in the fields of treatments, the prognosis of GBM still remains very poor. One of the major reasons why GBM is resistant to various therapeutic modalities is its diffused nature which makes it difficult to accurately identify the tumor margin by MRI. This makes the complete tumor resection impossible. Thus, it is important to study the molecules regulating cell migration and invasion for better understanding and developing targeted therapies in GBM. In this study, we present an integrated genomic and epigenomic strategy utilizing genome-wide DNA methylation and transcriptome profile data to ascertain epigenetically regulated genes implicated in cancer development and invasive progression. This analysis identified Fibromodulin (FMOD) as a GBM upregulated gene due to the loss of promoter methylation. FMOD, a member of small leucine-rich repeat proteoglycan, is an extracellular matrix protein and its C-terminus is known to interact with collagen fibrils aiding in the proper formation of extracellular matrix. Secreted FMOD (full length), not ΔFMOD (lacks C-terminal domain that interacts with collagen), promotes glioma cell migration through its ability to induce ilamentous actin stress fiber formation. A 9-mer synthetic peptide (either wild type or mutant) against FMOD C-terminal domain was used to compete with full length FMOD to assess the effect on migration phenotype. We observed that the wild type 9-mer peptide was able to decrease FMOD-induced migration completely but not the mutant peptide thereby indicating that the secreted FMOD induces migration by its interaction with collagen fibrils, a ligand for integrins. siRNA and small molecule inhibitor-based studies identified that FMOD-induced glioma cell migration is dependent on Integrin-FAK-Src-Rho-ROCK signaling pathway. ChIP-PCR experiments revealed that TGF-β1 regulates FMOD expression through epigenetic remodeling of FMOD promoter that involved demethylation and gain of active histone marks with a simultaneous loss of DNMT3A and EZH2 occupancy, but with an enrichment of SMAD2 and CBP. FMOD silencing inhibited the TGF-β1 mediated glioma cell migration significantly. In univariate and multivariate cox regression analysis, both FMOD promoter methylation and transcript levels predicted prognosis in GBM. To summarize, we found secreted FMOD as an important regulator of glioma cell migration downstream of TGF-β1 pathway and forms a potential basis for therapeutic intervention in GBM. Citation Format: Baisakhi Mondal. Integrative functional genomic analysis identifies epigenetically regulated fibromodulin as an essential gene for glioma cell migration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-343.

Integrative functional genomic analysis identifies epigenetically regulated fibromodulin as an essential gene for glioma cell migration.

An integrative functional genomics study of multiple forms of data are vital for discovering molecular drivers of cancer development and progression. Here, we present an integrated genomic strategy utilizing DNA methylation and transcriptome profile data to discover epigenetically regulated genes implicated in cancer development and invasive progression. More specifically, this analysis identified fibromodulin (FMOD) as a glioblastoma (GBM) upregulated gene because of the loss of promoter methylation. Secreted FMOD promotes glioma cell migration through its ability to induce filamentous actin stress fiber formation. Treatment with cytochalasin D, an actin polymerization inhibitor, significantly reduced the FMOD-induced glioma cell migration. Small interfering RNA and small molecule inhibitor-based studies identified that FMOD-induced glioma cell migration is dependent on integrin-FAK-Src-Rho-ROCK signaling pathway. FMOD lacking C-terminus LRR11 domain (ΔFMOD), which does not bind collagen type I, failed to induce integrin and promote glioma cell migration. Further, FMOD-induced integrin activation and migration was abrogated by a 9-mer wild-type peptide from the FMOD C-terminus. However, the same peptide with mutation in two residues essential for FMOD interaction with collagen type I failed to compete with FMOD, thus signifying the importance of collagen type I-FMOD interaction in integrin activation. Chromatin immunoprecipitation-PCR experiments revealed that transforming growth factor beta-1 (TGF-β1) regulates FMOD expression through epigenetic remodeling of FMOD promoter that involved demethylation and gain of active histone marks with a simultaneous loss of DNMT3A and EZH2 occupancy, but enrichment of Sma- and Mad-related protein-2 (SMAD2) and CBP. FMOD silencing inhibited the TGF-β1-mediated glioma cell migration significantly. In univariate and multivariate Cox regression analysis, both FMOD promoter methylation and transcript levels predicted prognosis in GBM. Thus, this study identified several epigenetically regulated alterations responsible for cancer development and progression. Specifically, we found that secreted FMOD as an important regulator of glioma cell migration downstream of TGF-β1 pathway and forms a potential basis for therapeutic intervention in GBM.

MicroRNA-130b promotes cell migration and invasion by targeting peroxisome proliferator-activated receptor gamma in human glioma.

MircroRNA-130b (miR-130b) has been recognized as an oncogenic miRNA and is implicated in the initiation and development of human cancers. Deregulation of miR-130b has been reported in several tumors. However, the clinical significance and its underlying role in human glioma are poorly explored. Herein, we found that the expression of miR-130b was significantly up-regulated in glioma tissues as compared with that in normal brain (NB) tissues. Clinical association analysis disclosed that high-expression of miR-130b was evidently associated with advanced tumor stage (grade III+IV) in glioma. Moreover, we disclosed that the high-expression of miR-130b conferred an obviously reduced survival of glioma patients. Multivariate Cox regression analysis showed that miR-130b expression was an independent prognostic indicator for glioma patients. Our gain- or loss-of-function studies showed that miR-130b promoted invasion and migration of glioma cells. Notably, miR-130b regulated peroxisome proliferator-activated receptor gamma (PPARγ) abundance and epithelial-mesenchymal transition (EMT) in glioma cells. Hereby, PPARγ was identified as a functional target of miR-130b in glioma. Furthermore, an inverse correlation between miR-130b and PPARγ expression was observed in glioma tissues. In conclusion, miR-130b is an independent prognostic biomarker for indicating survival of glioma patients and promotes glioma cell migration and invasion by targeting PPARγ.

LIM domain only 2 induces glioma invasion via cytosolic p27(KIP1).

High-grade gliomas are considered the most malignant of brain tumors and have a poor prognosis. In a previous study, we showed that LIM domain only 2 (LMO2) regulates glioma stem cell properties and tumor angiogenesis and gave rise to highly invasive glioma xenografts. Glioma invasion in the surrounding parenchymal tissues is a major hurdle with respect to eliminating glioma by surgery. Invasive glioma cells are considered one of the main culprits for the recurrence of tumors after therapies. In the current study, we focused on determining the molecular mechanism(s) by which LMO2 regulates glioma cell migration and invasion. Forced expression of LMO2 in human U87MG glioma cells led to glioma invasion, as determined by in vivo xenograft assays and enhanced in vitro migration and invasion. LMO2 was associated with increased levels of cytosolic p27(Kip1) protein. LMO2 possibly induced the stabilization and augmented interactions between p27(Kip1) and RhoA. We knocked down the expression of p27(Kip1), which led to a decrease in LMO2-driven glioma cell migration and invasion. Taken together, our findings indicate that LMO2 promotes glioma cell migration and invasion by increasing the levels of cytosolic p27(Kip1).

ATPase inhibitory factor 1 is a potential prognostic marker for the migration and invasion of glioma.

Adenosine triphosphatase inhibitory factor 1 (IF1) has previously been considered to be a driving oncogene in human cancers. Several studies have revealed that IF1 overexpression is present in a variety of tumor types and promotes tumor growth and metastasis. The present study aimed to investigate the clinical significance of IF1 in glioma and the role of IF1 in cell migration and invasion. The mRNA and protein expression of IF1 in glioma tissues was found to be significantly increased compared with the expression in normal brain tissues (P<0.05). The presence of IF1 expression was significantly associated with an advanced clinical stage in glioma (P<0.05). Furthermore, the presence of IF1 expression was found to be associated with a reduced overall survival rate of glioma patients (P<0.05). Multivariate Cox regression analysis indicated that IF1 was an independent factor for predicting the overall survival rate of patients with glioma (P<0.05). IF1 knockdown also significantly reduced the number of migratory and invasive U251 and U87 cells (P<0.05). In addition, IF1 knockdown inhibited the expression of nuclear factor-κB (NF-κB) and Snai1, and led to increased E-cadherin expression and reduced vimentin expression. In conclusion, the presence of IF1 expression is associated with poor clinicopathological features in glioma. IF1 expression is an independent prognostic marker for predicting the overall survival rate of patients with glioma. Mechanistically, IF1 may promote glioma cell migration and invasion through the NF-κB/Snai1 axis.

RTVP-1 promotes mesenchymal transformation of glioma via a STAT-3/IL-6-dependent positive feedback loop.

Glioblastomas (GBMs), the most aggressive primary brain tumors, exhibit increased invasiveness and resistance to anti-tumor treatments. We explored the role of RTVP-1, a glioma-associated protein that promotes glioma cell migration, in the mesenchymal transformation of GBM. Analysis of The Cancer Genome Atlas (TCGA) demonstrated that RTVP-1 expression was higher in mesenchymal GBM and predicted tumor recurrence and poor clinical outcome. ChiP analysis revealed that the RTVP-1 promoter binds STAT3 and C/EBPβ, two master transcription factors that regulate mesenchymal transformation of GBM. In addition, IL-6 induced RTVP-1 expression in a STAT3-dependent manner. RTVP-1 increased the migration and mesenchymal transformation of glioma cells. Similarly, overexpression of RTVP-1 in human neural stem cells induced mesenchymal differentiation, whereas silencing of RTVP-1 in glioma stem cells (GSCs) decreased the mesenchymal transformation and stemness of these cells. Silencing of RTVP-1 also increased the survival of mice bearing GSC-derived xenografts. Using gene array analysis of RTVP-1 silenced glioma cells we identified IL-6 as a mediator of RTVP-1 effects on the mesenchymal transformation and migration of GSCs, therefore acting in a positive feedback loop by upregulating RTVP-1 expression via the STAT3 pathway. Collectively, these results implicate RTVP-1 as a novel prognostic marker and therapeutic target in GBM.

Nrdp1-mediated ErbB3 degradation inhibits glioma cell migration and invasion by reducing cytoplasmic localization of p27(Kip1).

We previously reported that loss of Nrdp1 contributes to human glioma progression by reducing apoptosis. However, the role of Nrdp1 in glioma migration and invasion has not been investigated. Here, we report that ErbB3, a substrate of Nrdp1, is undetectable in normal brain tissues and grade II/III glioma tissues, but is abundant in a certain percentage of grade IV glioma tissues and is associated with the loss of Nrdp1. This suggests that Nrdp1 may be involved in glioma migration and invasion by regulating ErbB3. Thus, the role of Nrdp1/ErbB3 signaling in glioma cell migration and invasion was investigated using Nrdp1 loss- and gain-of-function. The results show that down-regulation of Nrdp1 by use of short hairpin RNA promoted glioma cell migration and invasion. In contrast, overexpression of Nrdp1 significantly inhibited glioma cell migration and invasion. Further investigation on molecular targets revealed that Nrdp1 decreased the level of ErbB3, which resulted in decreasing p-AKT thereby reducing cytoplasmic p27(Kip1). Taken together, these findings suggest that Nrdp1-mediated ErbB3 degradation suppresses glioma migration and invasion and that loss of Nrdp1 may amplify ErbB3 signaling to contribute to glioma migration and invasion. These findings suggest that Nrdp1 may be a target for glioma therapy.

A novel NF-κB/MMP-3 signal pathway involves in the aggressivity of glioma promoted by Bmi-1

Matrix metalloproteinase 3 (MMP-3) is implicated in the pathogenesis and progression of glioma. However, whether MMP-3 participates in the regulation of metastasis and its mechanisms in glioma is mostly unknown. In the present study, glioma cells were stably transfected with Bmi-1 small interfering RNA (siRNA) to knockdown off Bmi-1 or were transiently transfected with Bmi-1 complementary DNA (cDNA) to upregulate the Bmi-1 level and to evaluate their effects on invasion and expression analysis for molecules involved in invasion. Knockdown of Bmi-1 dramatically reduced a nuclear factor kappa B (NF-κB) and MMP-3 expression and activity in T98G cells. When the T98G cells were upregulated in the Bmi-1 levels, the T98G cells were treated with 10μM BAY 11-7028 to inhibit the NF-κB activity. The invasion induced by upregulation of Bmi-1 was severely abolished by BAY 11-7028 in Bmi-1 overexpression cells. The T98G cell metastatic potential was increased by overexpression of Bmi-1; completely at the same time, the NF-κB activity and MMP-3 expression was also increased. Taken together, these findings suggest that Bmi-1 promotes glioma cell migration and invasion via NF-κB-mediated upregulation of MMP-3.

IL-1β microenvironment promotes proliferation, migration, and invasion of human glioma cells.

Among the primary brain tumors, glioblastoma is the most common and severe. Glioblastoma have poor prognosis because of their highly diffusive growth pattern and invasion into surrounding brain tissue. Human glioblastoma cells overexpress interleukin-1β (IL-1β) and also the levels of IL-1β in U87MG glioma cells are significantly higher than in U373, T98G, A172 glioma cell lines. Malignant tumors are characterized by unlimited proliferation, migration and invasion. This study examines the effect of IL-1β microenvironment on proliferation, migration and invasion in human glioma cell line U87MG that expresses wild-type p53 protein, and U251MG which expresses mutant p53. Proliferation was investigated by MTT assay, migration by wound-healing migration assay and invasion by in vitro transwell Matrigel invasion assay. An IL-1β microenvironment significantly increased migration and invasion of both wild-type and mutant p53 expressing glioma cells, but significantly increased proliferation only in U87MG glioma cells. These effects were inhibited by IL-1 receptor antagonist (IL-1Ra), thus giving evidence that an IL-1β milieu promotes glioma cell migration, invasion, and proliferation.

Abstract 1644: Microglia promote glioma cell migration and dispersal by activating the PLCγ1 and Pyk2 signaling cascade.

Abstract Glioblastoma multiforme is one of the most aggressive and fatal brain cancers mostly because of the highly invasive nature of glioma cells. The ability of glioma cells to disperse to healthy brain stroma makes surgical and chemotherapeutic treatments mostly ineffective. Microglia infiltrate most gliomas and release factors, which favor tumor growth and dispersal. We hypothesize that microglia residing within the tumor stimulate glioma cell migration and dispersal through the phospholipase C (PLC)γ1 and proline rich tyrosine kinase 2 (Pyk2) signaling cascade. In the present study, we investigated rodent C6 and GL261 as well as human glioma cell lines with varying levels of invasiveness: U-87MG, A172, and HS683. Using a standard invasion assay, we demonstrated that the presence of rodent or immortalized human microglia in the bottom compartment of Boyden chambers activated invasion of all glioma cell types. Knock-down of PLCγ1 and Pyk2 proteins using siRNA reversed the stimulatory effect of microglia on glioma invasion in both cell lines. Pharmacological blockers of PLC (U73872 1nM) and Pyk2/focal adhesion kinase (FAK) (PF562271 20nM) also completely eliminated the ability of microglia to stimulate glioma cell migration in a wound healing assay. Using Western blot, we found upregulation of phosphorylated Tyr579/580 Pyk2 protein in glioma cells treated with microglia conditioned medium. Taken together, these data indicate that microglial cells activate glioma cell migration/dispersal through the pro-migratory Pyk2 and PLCγ1 signaling pathway in glioma cells. This research was made possible by NIH grant numbers: G11 HD052352, G12 RR03035, 8G12MD007583-27, U54 NS039408, UCC Pilot Project Program and Title V PPOHA grant number P031M105050 from the US Dept. of Education. Citation Format: Kimberleve Rolon-Reyes, Miguel Mendez, David E. Rivera-Aponte, Serguei N. Skatchkov, Misty J. Eaton, Lilia Kucheryavykh. Microglia promote glioma cell migration and dispersal by activating the PLCγ1 and Pyk2 signaling cascade. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1644. doi:10.1158/1538-7445.AM2013-1644

Geranylgeranyltransferase I regulates HIF-1α promoting glioblastoma cell migration and invasion

Glioblastoma multiforme is a highly migratory and invasive brain tumor in which hypoxia inducible factor-1α (HIF-1α) plays important roles. However, the underlying mechanisms regulating the action of HIF-1α in glioma cell migration and invasion ability remain unclear. We reported here that HIF-1α was regulated by geranylgeranyltransferase I (GGTI), a protein prenylation transferase, and then promoted glioma cell migration and invasion. The migratory and invasive ability of glioma cells were enhanced by hypoxia treatment but inhibited by down-regulation of HIF-1α. GGTI activity inhibition or GGTI specific β subunit (GGTI β) knocking-down decreased HIF-1α protein level. In addition, down-regulation of GGTI β inhibited migration and invasion of glioma cells under hypoxia, while GGTI β over-expression promoted it. Furthermore, the effect of GGTI β over-expression on cell migration and invasion was abolished by HIF-1α down-regulation. In summary, our study showed, for the first time, that HIF-1α was regulated by protein prenylation transferase GGTI and mediated the effect of GGTI on glioma cell migration and invasion.

Attenuated adenosine-to-inosine editing of microRNA-376a* promotes invasiveness of glioblastoma cells

In the human brain, microRNAs (miRNAs) from the microRNA-376 (miR-376) cluster undergo programmed "seed" sequence modifications by adenosine-to-inosine (A-to-I) editing. Emerging evidence suggests a link between impaired A-to-I editing and cancer, particularly in high-grade gliomas. We hypothesized that disruption of A-to-I editing alters expression of genes regulating glioma tumor phenotypes. By sequencing the miR-376 cluster, we show that the overall miRNA editing frequencies were reduced in human gliomas. Specifically in high-grade gliomas, miR-376a* accumulated entirely in an unedited form. Clinically, a significant correlation was found between accumulation of unedited miR-376a* and the extent of invasive tumor spread as measured by magnetic resonance imaging of patient brains. Using both in vitro and orthotopic xenograft mouse models, we demonstrated that the unedited miR-376a* promoted glioma cell migration and invasion, while the edited miR-376a* suppressed these features. The effects of the unedited miR-376a* were mediated by its sequence-dependent ability to target RAP2A and concomitant inability to target AMFR. Thus, the tumor-dependent introduction of a single base difference in the miR-376a* sequence dramatically alters the selection of its target genes and redirects its function from inhibiting to promoting glioma cell invasion. These findings uncover a new mechanism of miRNA deregulation and identify unedited miR-376a* as a potential therapeutic target in glioblastoma cells.

Abstract 318: Microglia promote glioma cell migration through a Pyk2 signaling pathway

Abstract Glioblastoma multiforme is the most common and most malignant form of glioma. Despite the non-metastatic nature of gliomas, prognosis is poor because tumor cell invasion into surrounding brain leads to recurrence even after radical surgery. Microglia infiltrate most glioma tumors and, therefore, make up an important component of the glioma microenvironment. Microglia may release factors which stimulate signaling pathways and promote glioma cell dispersal and cell invasion. The purpose of the present study was to test the hypothesis that glioma and microglial cells are involved in a reciprocal interaction in the tumor microenvironment where they modulate functions and abilities of each other in order to promote tumor progression and invasion. We hypothesize that microglial cells release soluble factors which promote migration of glioma cells through a Pyk2 signaling pathway. In the present study, we used three different human glioma cell lines with varying levels of invasiveness: A172, U-87MG and HS683, as well as, an immortalized human microglial cell line. We used three experimental groups of glioma cells: group 1 were control glioma cells, group 2 were glioma cells treated with microglia conditioned medium (MCM), and group 3 were glioma cells treated with conditioned medium from microglia that were pretreated with glioma conditioned medium (AMCM). Group 3 simulates the cross-talk between microglia and glioma cells which occurs in the brain. Using a matrigel invasion assay and a wound healing assay, we demonstrated that factors released from microglia significantly increased migration/invasion of glioma cells. The effect was stronger when glioma cells were exposed to microglia activated by glioma conditioned medium (AMCM) to simulate “cross-talk” between glioma and microglial cells as compared to the effect of resting microglia (MCM). To examine potential intracellular pathways that are involved in the process of activation of glioma migration and invasion by microglia, we used a antibody microarray targeting cell signaling proteins. We found upregulation of phosphorylated Tyr579/580 Pyk2 protein in glioma cells treated with MCM and with AMCM. These data were confirmed by Western Blot of rat C6 glioma cells and of A172, U87, HS683 human glioma cell lines demonstrating a common effect of soluble factors released from microglial cells on upregulation of the Pyk2 intracellular pathway in different glioma cell lines. Pyk2 has previously been shown to increase glioma cell migration and invasion. Taken together, these data indicate that microglial cells activate glioma cell migration/dispersal. Furthermore, these interactions activate the pro-migratory-Pyk2 signaling pathway in glioma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 318. doi:1538-7445.AM2012-318

Notch1 promotes glioma cell migration and invasion by stimulating β‐catenin and NF‐κB signaling via AKT activation

The Notch signaling pathway has been implicated in both developmental processes and tumorigenesis. Aberrant Notch signaling has been repeatedly demonstrated to facilitate the proliferation and survival of glioma cells by regulating downstream effectors or other signaling pathways. In glioblastoma multiforme specimens from 59 patients, Notch1 was highly expressed in tumor tissues compared with normal brain tissues, and this expression was correlated with elevated AKT phosphorylation and Snail expression. Increased nuclear localization of β-catenin and p50 as well as enhanced IKKα/AKT interaction were also observed in glioma tissues. In U87MG cells, the activation of Notch1 by DLL4 stimulation or by the overexpression of Notch intracellular domain (NICD) resulted in AKT activation and thereby promoted β-catenin activity and NF-κB signaling. Inhibition of EGFR partially blocked the β-catenin and NF-κB signaling stimulated by Notch1 activation. Furthermore, NICD overexpression in U87MG cells led to the upregulated expression of several metastasis-associated molecules, which could be abrogated by the knockdown of either β-catenin or p50. In U87MG and U251 cells, DLL4-induced cellular migration and invasion could be inhibited by either β-catenin or a p50 inhibitor. Collectively, these results indicate that Notch activation could stimulate β-catenin and NF-κB signaling through AKT activation in glioma cells. Thus, Notch activation-stimulated β-catenin and NF-κB signaling synergistically promote the migratory and invasive properties of glioma cells.

Deletion of the SPARC acidic domain or EGF-like module reduces SPARC-induced migration and signaling through p38 MAPK/HSP27 in glioma

We previously demonstrated that secreted protein acidic and rich in cysteine (SPARC) increases heat shock protein 27 (HSP27) expression and phosphorylation and promotes glioma cell migration through the p38 mitogen-activated protein kinase (MAPK)/HSP27 signaling pathway. As different regions of the SPARC protein mediate different SPARC functions, elucidating which SPARC domains regulate HSP27 expression, signaling and migration might provide potential therapeutic strategies to target these functions. To investigate the roles of specific domains, we used an SPARC-green fluorescent protein (GFP) fusion protein and constructs of SPARC-GFP with deletions of either the acidic domain (ΔAcidic) or the epidermal growth factor (EGF)-like module (ΔEGF). GFP, SPARC-GFP and the two deletion mutants were expressed in U87MG glioma cells. Characterization of the derived stable clones by confocal imaging and western blotting suggests proper folding, processing and secretion of the deletion constructs. Uptake of the constructs by naive cells suggests enhanced internalization of ΔAcidic and reduced internalization of ΔEGF. Wound and transwell migration assays and western blot analysis confirm our previous results and indicate that ΔAcidic reduces SPARC-induced migration and p38 MAPK/HSP27 signaling and ΔEGF decreases SPARC-induced migration and dramatically decreases the expression and phosphorylation of HSP27 but is poorly internalized. Loss of the EGF-like module suppresses the enhanced HSP27 protein stability conferred by SPARC. In conclusion, deletions of the acidic domain and EGF-like module have differential effects on cell surface binding and HSP27 protein stability; however, both regions regulate SPARC-induced migration and signaling through HSP27. Our data link the domains of SPARC with different functions and suggest one or both of the constructs as potential therapeutic agents to inhibit SPARC-induced migration.