Nu Zhang

Clinical significance of sphingosine kinase-1 expression in human astrocytomas progression and overall patient survival.

Purpose: To characterize the expression of sphingosine kinase-1 (SPHK1) in human astrocytomas and to investigate the association between SPHK1 expression and progression of astrocytomas. Experimental Design: The expression of SPHK1 in normal human astrocytes, astrocytoma cell lines, and four pairs of matched astrocytoma tissues and their adjacent normal brain tissues were detected by quantitative reverse transcription-PCR and Western blot. In addition, SPHK1 protein expression was examined in 243 cases of histologically characterized astrocytomas by immunohistochemistry. Statistical analyses were applied to test for prognostic and diagnostic associations. Results: SPHK1 in astrocytoma cell lines was elevated at both mRNA and protein levels, and the SPHK1 mRNA and protein were significantly up-regulated by up to 6.8- and 40-fold, respectively, in primary astrocytomas compared with those in the adjacent noncancerous brain tissues. Immunohistochemical analysis showed that 100 of 243 (41.2%) paraffin-embedded archival astrocytoma biopsies exhibited high expression of SPHK1. Statistical analysis suggested that the up-regulation of SPHK1 was significantly correlated with the histologic grade of astrocytoma (P = 0.000) and that patients with high SPHK1 level exhibited shorter survival time (P < 0.001). Multivariate analysis revealed that SPHK1 up-regulation might be an independent prognostic indicator for the survival of patients with astrocytoma. Conclusions: SPHK1 might represent a novel and useful prognostic marker for astrocytoma and play a role during the development and progression of the disease.

Sp1 is upregulated in human glioma, promotes MMP-2-mediated cell invasion and predicts poor clinical outcome.

Sp1, the first identified transcription factor, has been reported to be associated with the development and progression of various human cancer types. However, the clinical significance and biological role of Sp1 in glioma are less well understood. In this study, we found that the expression of Sp1 was markedly elevated in glioma cell lines and tissues. Immunohistochemistry analysis revealed that the vast majority of 222 paraffin‐embedded archival glioma specimens tested displayed positive Sp1 expression, and 58.6% exhibited high‐level Sp1 expression. Statistical analysis suggested that the high Sp1 expression was correlated strongly with the WHO grading (p < 0.001) and survival status (p < 0.001) of glioma patients. Patients with lower Sp1 expression had better overall survival than those with higher Sp1 expression. Multivariate analysis suggested that Sp1 expression might be an independent prognostic indicator of the survival of patients with glioma. Furthermore, overexpression of Sp1 in glioma cells was found to increase their invasiveness, and in contrast, silencing Sp1 by siRNA caused an inhibition of cell invasion. Moreover, we demonstrated that the up‐regulation of Sp1 could increase activity and expression of MMP‐2. Collectively, our data suggest that Sp1 might represent a valuable prognostic marker for glioma and is involved in modulation of tumor invasion.

Novel Role of FBXW7 Circular RNA in Repressing Glioma Tumorigenesis

Abstract Background Circular RNAs (circRNAs) are RNA transcripts that are widespread in the eukaryotic genome. Recent evidence indicates that circRNAs play important roles in tissue development, gene regulation, and carcinogenesis. However, whether circRNAs encode functional proteins remains elusive, although translation of several circRNAs was recently reported. Methods CircRNA deep sequencing was performed by using 10 pathologically diagnosed glioblastoma samples and their paired adjacent normal brain tissues. Northern blotting, Sanger sequencing, antibody, and liquid chromatograph Tandem Mass Spectrometer were used to confirm the existence of circ-FBXW7 and its encoded protein in in two cell lines. Lentivirus-transfected stable U251 and U373 cells were used to assess the biological functions of the novel protein in vitro and in vivo (five mice per group). Clinical implications of circ-FBXW7 were assessed in 38 pathologically diagnosed glioblastoma samples and their paired periphery normal brain tissues by using quantitative polymerase chain reaction (two-sided log-rank test). Results Circ-FBXW7 is abundantly expressed in the normal human brain (reads per kilobase per million mapped reads [RPKM] = 9.31). The spanning junction open reading frame in circ-FBXW7 driven by internal ribosome entry site encodes a novel 21-kDa protein, which we termed FBXW7-185aa. Upregulation of FBXW7-185aa in cancer cells inhibited proliferation and cell cycle acceleration, while knockdown of FBXW7-185aa promoted malignant phenotypes in vitro and in vivo. FBXW7-185aa reduced the half-life of c-Myc by antagonizing USP28-induced c-Myc stabilization. Moreover, circ-FBXW7 and FBXW7-185aa levels were reduced in glioblastoma clinical samples compared with their paired tumor-adjacent tissues (P < .001). Circ-FBXW7 expression positively associated with glioblastoma patient overall survival (P = .03). Conclusions Endogenous circRNA encodes a functional protein in human cells, and circ-FBXW7 and FBXW7-185aa have potential prognostic implications in brain cancer.

Phosphoglycerate dehydrogenase induces glioma cells proliferation and invasion by stabilizing forkhead box M1

Phosphoglycerate dehydrogenase (PHGDH) is the first enzyme branching from glycolysis in the three-step serine biosynthetic pathway. Recent evidence has shown that PHGDH is amplified in human breast cancer and melanoma and plays a key role in cancer metabolism. However, PHGDH expression in glioma and a potential non-metabolic role in tumorigenesis have not been reported. We analyzed PHGDH levels in specimens from glioma patients and found that PHGDH, although negative in normal brain tissues, was highly expressed in astrocytic tumors and increasingly expressed in more aggressive cancer types. Inhibition of PHGDH expression in glioma cells downregulated the expression of VEGF, MMP-2, CHK2 and cyclin D1 and reduced glioma cell proliferation, invasion and tumorigenicity in vitro and in vivo. Interestingly, we found that the oncogenic transcription factor FOXM1 was also downregulated in PHDGH-silenced glioma cells. Using LC/LC MS analysis, we identified PHGDH as a novel binding partner of FOXM1. PHGDH interacted with and stabilized FOXM1 at the protein level, promoting the proliferation, invasion and tumorigenicity of glioma cells. Our data identified PHGDH as a potential prognostic marker of glial brain tumors and identified a non-metabolic role for PHGDH in glioma tumorigenesis, providing a novel angle of targeting the PHGDH–FOXM1 axis in future brain tumor therapy.

AURKA Governs Self-Renewal Capacity in Glioma-Initiating Cells via Stabilization/Activation of β-catenin/Wnt Signaling

Glioma-initiating cells (GIC), which are characterized by their self-renewal capacity and tumorigenicity, were recently identified as a highly tumorigenic subpopulation of glioblastoma multiforme and are considered responsible for glioblastoma recurrence and chemo/radiation resistance. Previously, it was revealed that Wnt signaling activation is critical to the self-renewal of GICs. However, the molecular mechanism underlying the high expression of β-catenin, the key transcription factor of the Wnt signaling pathway, remains elusive. In this investigation, it was determined that aurora kinase A (AURKA) regulates the self-renewal and tumorigenicity of GICs by stabilizing β-catenin. In GICs, AURKA directly interacts with AXIN and disrupts the AXIN/GSK3β/β-catenin destruction complex and stabilizes β-catenin, thereby activating Wnt signaling to promote self-renewal. Stable knockdown of AURKA destabilizes β-catenin by increasing phosphorylated β-catenin bound to AXIN and suppresses Wnt signaling, which inhibits the ability of GICs to self-renew. This effect is rescued by expression of an AURKA kinase dead mutant, D274A, which lacks the ability to phosphorylate GSK3β, indicating that stabilization of β-catenin by AURKA in GICs is independent from phosphorylation of GSK3β. Functional experiments confirm that inhibition of AUKRA in GICs could suppress their “stemness,” self-renewal ability, and tumorigenicity both in vitro and in vivo, and these effects could be rescued by stabilized β-catenin mutant. These findings indicate that AURKA competes away the binding of AXIN from β-catenin, induces β-catenin stabilization, and activates Wnt signaling in GICs. Implications: AURKA kinase inhibition could effectively attenuate Wnt signaling, thereby inhibiting the self-renewal and tumorigenicity of GICs, and may be a novel target for glioblastoma treatment strategies. Mol Cancer Res; 11(9); 1101–11. ©2013 AACR.

MicroRNA-542-3p Suppresses Tumor Cell Invasion via Targeting AKT Pathway in Human Astrocytoma

Background: MicroRNAs play important roles in regulating AKT pathway. Results: miR-542-3p suppresses glioblastoma cell invasion through directly inhibiting AKT1, ILK, and PIK3R1. Conclusion: miR-542-3p down-regulation contributes to aberrant activation of the AKT signaling, and miR-542-3p acts as a negative regulator in astrocytoma progression. Significance: Learning how miRNAs participate in AKT pathway is crucial for understanding its regulators and cancer therapy. The molecular mechanism underlying constitutive activation of AKT signaling, which plays essential roles in astrocytoma progression, is not fully characterized. Increasing numbers of studies have reported that microRNAs are involved in the malignant behavior of astrocytoma cells via directly targeting multiple oncogenes or tumor suppressors. Here, we found that microRNA (miR)-542-3p expression was decreased in glioblastoma cell lines and astrocytoma tissues, and reduced levels of miR-542-3p expression correlated with high histopathological grades and poor prognosis of astrocytoma patients. Exogenous miR-542-3p suppressed glioblastoma cell invasion through not only targeting AKT1 itself but also directly down-regulating its two important upstream regulators, namely, integrin-linked kinase and PIK3R1. Notably, overexpressing miR-542-3p decreased AKT1 phosphorylation and directly and indirectly repressed nuclear translocation and transactivation activity of β-catenin to exert its anti-invasive effect. Furthermore, the miR-542-3p expression level negatively correlated with AKT activity as well as levels of integrin-linked kinase and PIK3R1 in human astrocytoma specimens. These findings suggest that miR-542-3p acts as a negative regulator in astrocytoma progression and that miR-542-3p down-regulation contributes to aberrant activation of AKT signaling, leaving open the possibility that miR-542-3p may be a potential therapeutic target for high grade astrocytoma.

Transcriptional factor specificity protein 1 (SP1) promotes the proliferation of glioma cells by up-regulating midkine (MDK)

Midkine (MDK) expression is associated with the proliferation of many cancers, including glioma. SP1 directly up-regulates the expression of MDK, and the SP1-MDK axis cooperates in glioma tumorigenesis.

A novel protein encoded by the circular form of the SHPRH gene suppresses glioma tumorigenesis

Circular RNAs (circRNAs) are recognized as functional non-coding transcripts in eukaryotic cells. Recent evidence has indicated that even though circRNAs are generally expressed at low levels, they may be involved in many physiological or pathological processes, such as gene regulation, tissue development and carcinogenesis. Although the ‘microRNA sponge’ function is well characterized, most circRNAs do not contain perfect trapping sites for microRNAs, which suggests the possibility that circRNAs have functions that have not yet been defined. In this study, we show that a circRNA containing an open reading frame (ORF) driven by the internal ribosome entry site (IRES) can translate a functional protein. The circular form of the SNF2 histone linker PHD RING helicase (SHPRH) gene encodes a novel protein that we termed SHPRH-146aa. Circular SHPRH (circ-SHPRH) uses overlapping genetic codes to generate a ‘UGA’ stop codon, which results in the translation of the 17 kDa SHPRH-146aa. Both circ-SHPRH and SHPRH-146aa are abundantly expressed in normal human brains and are down-regulated in glioblastoma. The overexpression of SHPRH-146aa in U251 and U373 glioblastoma cells reduces their malignant behavior and tumorigenicity in vitro and in vivo. Mechanistically, SHPRH-146aa protects full-length SHPRH from degradation by the ubiquitin proteasome. Stabilized SHPRH sequentially ubiquitinates proliferating cell nuclear antigen (PCNA) as an E3 ligase, leading to inhibited cell proliferation and tumorigenicity. Our findings provide a novel perspective regarding circRNA function in physiological and pathological processes. Specifically, SHPRH-146aa generated from overlapping genetic codes of circ-SHPRH is a tumor suppressor in human glioblastoma.

Forkhead Box M1 positively regulates UBE2C and protects glioma cells from autophagic death

ABSTRACT Ubiquitin-conjugating enzyme E2C (UBE2C) is characterized as a crucial molecule in cancer cell growth that plays an essential role in the development of gliomas, but the detailed mechanisms have not been fully elucidated. In this study, we found that Forkhead box transcription factor M1 (FoxM1) overexpression increased UBE2C expression, whereas FoxM1 suppression inhibited UBE2C expression in glioma cells. In addition, high FoxM1/UBE2C expression was significantly correlated with poor prognosis in glioma. We subsequently demonstrated that UBE2C was a direct transcriptional target of FoxM1, and site-directed mutations markedly down-regulated UBE2C promoter activity. Moreover, UBE2C siRNA (si-UBE2C) significantly induced glioma cell autophagy and increased both mCherry-LC3 punctate fluorescence and LC3B-II/LC3-I expression. Notably, the si-UBE2C-induced decrease in cell viability was markedly inhibited by the autophagy inhibitor bafilomycin A1. The silencing of UBE2C resulted in a distinct inhibition of the PI3K-Akt-mTOR pathway, which functions in the negative modulation of autophagy. Collectively, our findings provide clinical and molecular evidence that FoxM1 promotes glioma progression by enhancing UBE2C transcription and that the inhibition of UBE2C partially induces autophagic glioma cell death. Thus, targeting the FoxM1-UBE2C axis has therapeutic potential in the treatment of gliomas.

The role of SDF-1/CXCR4 in the vasculogenesis and remodeling of cerebral arteriovenous malformation

Background Cerebral arteriovenous malformation (AVM) involves the vasculogenesis of cerebral blood vessels and can cause severe intracranial hemorrhage. Stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, are believed to exert multiple physiological functions including angiogenesis. Thus, we investigated the role of SDF-1/CXCR4 in the vasculogenesis of cerebral AVM. Methods Brain AVM lesions from surgical resections were analyzed for the expression of SDF-1, CXCR4, VEGF-A, and HIF-1 by using immunohistochemical staining. Flow cytometry was used to quantify the level of circulating endothelial progenitor cells (EPCs). Further, in an animal study, chronic cerebral hypoperfusion model rats were analyzed for the expression of SDF-1 and HIF-1. CXCR4 antagonist, AMD3100, was also used to detect its effects on cerebral vasculogenesis and SDF-1 expression. Results Large amounts of CXCR4-positive CD45+ cells were found in brain AVM lesion blood vessel walls, which also have higher SDF-1 expression. Cerebral AVM patients also had higher level of EPCs and SDF-1. In chronic cerebral hypoperfusion rats, SDF-1, HIF-1, and CD45 expressions were elevated. The application of AMD3100 effectively suppressed angiogenesis and infiltration of CXCR4-positive CD45+ cells in hypoperfusion rats compared to controls. Conclusion The SDF-1/CXCR4 axis plays an important role in the vasculogenesis and migration of inflammatory cells in cerebral AVM lesions, possibly via the recruitment of bone marrow EPCs.