Heterogeneous Nuclear Ribonucleoprotein C1/C2 Controls the Metastatic Potential of Glioblastoma by Regulating PDCD4

MicroRNAs (miRNAs) have been implicated in the pathogenesis and progression of brain tumors. miR-21 is one of the most highly overexpressed miRNAs in glioblastoma multiforme (GBM), and its level of expression correlates with the tumor grade. Programmed cell death 4 (PDCD4) is a well-known miR-21 target and is frequently downregulated in glioblastomas in accordance with increased miR-21 expression. Downregulation of miR-21 or overexpression of PDCD4 can inhibit metastasis. Here, we investigate the role of heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC) in the metastatic potential of the glioblastoma cell line T98G. hnRNPC bound directly to primary miR-21 (pri-miR-21) and promoted miR-21 expression in T98G cells. Silencing of hnRNPC lowered miR-21 levels, in turn increasing the expression of PDCD4, suppressing Akt and p70S6K activation, and inhibiting migratory and invasive activities. Silencing of hnRNPC reduced cell proliferation and enhanced etoposide-induced apoptosis. In support of a role for hnRNPC in the invasiveness of GBM, highly aggressive U87MG cells showed higher hnRNPC expression levels and hnRNPC abundance in tissue arrays and also showed elevated levels as a function of brain tumor grade. Taken together, our data indicate that hnRNPC controls the aggressiveness of GBM cells through the regulation of PDCD4, underscoring the potential usefulness of hnRNPC as a prognostic and therapeutic marker of GBM.     

RECK‐mediated inhibition of glioma migration and invasion

RECK is an anti‐tumoral gene whose activity has been associated with its inhibitory effects regulating MMP‐2, MMP‐9, and MT1‐MMP. RECK level decreases as gliobastoma progresses, varying from less invasive grade II gliomas to very invasive human glioblastoma multiforme (GBM). Since RECK expression and glioma invasiveness show an inverse correlation, the aim of the present study is to investigate whether RECK expression would inhibit glioma invasive behavior. We conducted this study to explore forced RECK expression in the highly invasive T98G human GBM cell line. Expression levels as well as protein levels of RECK, MMP‐2, MMP‐9, and MT1‐MMP were assessed by qPCR and immunoblotting in T98G/RECK+ cells. The invasion and migration capacity of RECK+ cells was inhibited in transwell and wound assays. Dramatic cytoskeleton modifications were observed in the T98G/RECK+ cells, when compared to control cells, such as the abundance of stress fibers (contractile actin–myosin II bundles) and alteration of lamellipodia. T98G/RECK+ cells also displayed phosphorylated focal adhesion kinase (P‐FAK) in mature focal adhesions associated with stress fibers; whereas P‐FAK in control cells was mostly associated with immature focal complexes. Interestingly, the RECK protein was predominantly localized at the leading edge of migrating cells, associated with membrane ruffles. Unexpectedly, introduced expression of RECK effectively inhibited the invasive process through rearrangement of actin filaments, promoting a decrease in migratory ability. This work has associated RECK tumor‐suppressing activity with the inhibition of motility and invasion in this GBM model, which are two glioma characteristics responsible for the inefficiency of current available treatments. J. Cell. Biochem. 110: 52–61, 2010. © 2010 Wiley‐Liss, Inc.     

The effects of cytomegalovirus on human immunodeficiency virus replication in brain-derived cells correlate with permissiveness of the cells for each virus.

Human cytomegalovirus (HCMV) is commonly found in the brains of patients with AIDS and in some cases can be detected in the same cells as can human immunodeficiency virus type 1 (HIV-1). In this study, we analyzed the patterns of replication of HIV-1 and HCMV in singly infected cells and the effects of dual infection in human brain-derived cell lines of three different origins: neuroblastoma cell lines SK-N-MC and SY5Y; astrocytoma/glioblastoma cell lines U373-MG and Hs 683; and undifferentiated glioblastoma cell lines A172 and T98G. To bypass the restriction at the adsorption/penetration step in these CD4-negative cells, we used HIV-1 (amphotropic retrovirus) pseudotypes. These HIV-1 pseudotypes infected the majority of the cells in the cultures and expressed high levels of HIV-1 gene products in all except the SY5Y cells. The cell lines differed in the ability to support HCMV infection, but coinfection with HIV-1 had no effect on HCMV replication. The A172 cells were completely nonpermissive for HCMV gene expression, while HCMV replication in the singly infected T98G and SK-N-MC cell lines was restricted at the level of some early gene products. This resulted in complete and partial inhibition, respectively, of viral DNA synthesis. Dual infection of the A172, T98G, and SK-N-MC cells had no effect on HIV-1 replication. The other three cell lines, U373-MG, Hs 683, and SY5Y, were fully permissive for HCMV replication. In the U373-MG and Hs 683 cells, HCMV markedly inhibited the synthesis of HIV-1 gene products. In contrast, a transient stimulation of HIV-1 production followed by a repression was observed in the dually infected SY5Y cells. We conclude from these results that under conditions in which both HIV-1 and HCMV can undergo fully permissive infection, HCMV can repress HIV-1 gene expression. In cells in which HCMV replication is limited but HIV-1 replicates well, there is no effect on HIV-1 gene expression. However, activation of HIV-1, at least transiently, may occur in cells in which HIV-1 gene expression is limited. These studies suggest that a threshold level of some HIV-1 gene product(s) may obscure activation or promote repression of HIV replication by HCMV.     

Profiling and comparative analysis of glycoproteins in Hs578BST and Hs578T and investigation of prolyl 4-Hydroxylase alpha polypeptide II expression and influence in breast cancer cells

To identify potential cancer related glycoproteins in breast cancer cells, we enriched N-linked glycoproteins by lentil lectin from the human breast cancer cell line Hs578T and the normal breast cell line Hs578BST for proteomic comparison. Glycoproteins were separated and compared by two-dimensional electrophoresis. Twenty-four glycoproteins were identified that expressed remarkably differently, among which nine were involved in the progress of collagen synthesis. Prolyl 4-hydroxylase alpha polypeptide II (P4HA2) expression and influence in breast cancer was further investigated. Immunohistochemistry revealed that P4HA2 was upregulated in breast tumor cells compared with its adjacent normal tissues. Moreover, overexpression and RNA interference of P4HA2 showed that P4HA2 expression suppressed cell proliferation and migration in Hs578T in vitro.     

Changes in the expression of proteins associated with aerobic glycolysis and cell migration are involved in tumorigenic ability of two glioma cell lines

ABSTRACT: BACKGROUND: The most frequent and malignant brain cancer is glioblastoma multiforme (GBM). In gliomas, tumor progression and poor prognosis are associated with the tumorigenic ability of the cells. U87MG cells (wild-type p53) are known to be tumorigenic in nude mice, but T98G cells (mutant p53) are not tumorigenic. We investigated the proteomic profiling of these two cell lines in order to gain new insights into the mechanisms that may be involved in tumorigenesis. RESULTS: We found 24 differentially expressed proteins between T98G and U87MG cells. Gene Ontology supports the notion that over-representation of differentially expressed proteins is involved in glycolysis, cell migration and stress oxidative response. Among those associated with the glycolysis pathway, TPIS and LDHB are up-regulated in U87MG cells. Measurement of glucose consumption and lactate production suggests that glycolysis is more effective in U87MG cells. On the other hand, G6PD expression was 3-fold higher in T98G cells and this may indicate a shift to the pentose-phosphate pathway. Moreover, GRP78 expression was also three-fold higher in T98G than in U87MG cells. Under thapsigargin treatment both cell lines showed increased GRP78 expression and the effect of this agent was inversely correlated to cell migration. Quantitative RT-PCR and immunohistochemistry of GRP78 in patient samples indicated a higher level of expression of GRP78 in grade IV tumors compared to grade I and non-neoplastic tissues, respectively. CONCLUSIONS: Taken together, these results suggest an important role of proteins involved in key functions such as glycolysis and cell migration that may explain the difference in tumorigenic ability between these two glioma cell lines and that may be extrapolated to the differential aggressiveness of glioma tumors.     

IKBIP,a novel glioblastoma biomarker,maintains abnormal proliferation of tumor cells by inhibiting the ubiquitination and degradation of CDK4

Sustained proliferative signaling is a crucial hallmark and therapeutic target in glioblastoma (GBM); however, new intrinsic regulators and their underlying mechanisms remain to be elucidated. In this study, I kappa B kinase interacting protein (IKBIP) was identified to be correlated with the progression of GBM by analysis of The Cancer Genome Atlas (TCGA) data. TCGA database analysis indicated that higher IKBIP expression was associated with high tumor grade and poor prognosis in GBM patients, and these correlations were subsequently validated in clinical samples. IKBIP knockdown induced G1/S arrest by blocking the Cyclin D1/CDK4/CDK6/CDK2 pathway. Our results showed that IKBIP may bind directly to CDK4, a key cell cycle checkpoint protein, and prevent its ubiquitination-mediated degradation in GBM cells. An in vivo study confirmed that IKBIP knockdown strongly suppressed cell proliferation and tumor growth and prolonged survival in a mouse xenograft model established with human GBM cells. In conclusion, IKBIP functions as a novel driver of GBM by binding and stabilizing the CDK4 protein. IKBIP could be a potential therapeutic target in GBM.     

The Jak2 Small Molecule Inhibitor,G6, Reduces the Tumorigenic Potential of T98G Glioblastoma Cells In Vitro and In Vivo

Glioblastoma multiforme (GBM) is the most common and the most aggressive form of primary brain tumor. Jak2 is a non-receptor tyrosine kinase that is involved in proliferative signaling through its association with various cell surface receptors. Hyperactive Jak2 signaling has been implicated in numerous hematological disorders as well as in various solid tumors including GBM. Our lab has developed a Jak2 small molecule inhibitor known as G6. It exhibits potent efficacy in vitro and in several in vivo models of Jak2-mediated hematological disease. Here, we hypothesized that G6 would inhibit the pathogenic growth of GBM cells expressing hyperactive Jak2. To test this, we screened several GBM cell lines and found that T98G cells express readily detectable levels of active Jak2. We found that G6 treatment of these cells reduced the phosphorylation of Jak2 and STAT3, in a dose-dependent manner. In addition, G6 treatment reduced the migratory potential, invasive potential, clonogenic growth potential, and overall viability of these cells. The effect of G6 was due to its direct suppression of Jak2 function and not via off-target kinases, as these effects were recapitulated in T98G cells that received Jak2 specific shRNA. G6 also significantly increased the levels of caspase-dependent apoptosis in T98G cells, when compared to cells that were treated with vehicle control. Lastly, when T98G cells were injected into nude mice, G6 treatment significantly reduced tumor volume and this was concomitant with significantly decreased levels of phospho-Jak2 and phospho-STAT3 within the tumors themselves. Furthermore, tumors harvested from mice that received G6 had significantly less vimentin protein levels when compared to tumors from mice that received vehicle control solution. Overall, these combined in vitro and in vivo results indicate that G6 may be a viable therapeutic option against GBM exhibiting hyperactivation of Jak2.     

Downregulation of the RECK-tumor and metastasis suppressor gene in glioma invasiveness

Invasive behavior is the pathological hallmark of malignant gliomas, being responsible for the failure of surgery, radiation, and chemotherapy. Matrix metalloproteinases (MMPs) are essential for proper ECM remodeling and invasion. The tumor and metastasis suppressor RECK protein regulates at least three members of the MMPs family: MMP-2, MMP-9, and MT1-MMP. In order to mimic the in vivo invasion process, A172 and T98G, respectively, non-invasive and invasive human glioblastoma cell lines, were cultured onto uncoated (control) or type I collagen gel-coated surface, and maintained for up to 7 days to allow establishment of the invasive process. We show that the collagen substrate causes decreased growth rates and morphological alterations correlated with the invasive phenotype. Electronic transmission microscopy of T98G cells revealed membrane invaginations resembling podosomes, which are typically found in cells in the process of crossing tissue boundaries, since they constitute sites of ECM degradation. Real time PCR revealed higher RECK mRNA expression in A172 cells, when compared to T98G cells and, also, in samples obtained from cultures where the invasive process was fully established. Interestingly, the collagen substrate increases RECK expression in A172 cells and the same tendency is displayed by T98G cells. MMPs-2 and -9 displayed higher levels of expression and activity in T98G cells, and their activities are also upregulated by collagen. Therefore, we suggest that: (1) RECK downregulation is critical for the invasiveness process displayed by T98G cells; (2) type 1 collagen could be employed to modulate RECK expression in glioblastoma cell lines. Since a positive correlation between RECK expression and patients survival has been noted in several types of tumors, our results may contribute to elucidate the complex mechanisms of malignant gliomas invasiveness.     

Glycoproteomic analysis of glioblastoma stem cell differentiation

Cancer stem cells are responsible for tumor formation through self-renewal and differentiation into multiple cell types and thus represent a new therapeutic target for tumors. Glycoproteins play a critical role in determining the fates of stem cells such as self-renewal, proliferation, and differentiation. Here we applied a multilectin affinity chromatography and quantitative glycoproteomics approach to analyze alterations of glycoproteins relevant to the differentiation of a glioblastoma-derived stem cell line HSR-GBM1. Three lectins including concanavalin A (Con A), wheat germ agglutinin (WGA), and peanut agglutinin (PNA) were used to capture glycoproteins, followed by LC-MS/MS analysis. A total of 73 and 79 high-confidence (FDR < 0.01) glycoproteins were identified from the undifferentiated and differentiated cells, respectively. Label-free quantitation resulted in the discovery of 18 differentially expressed glycoproteins, wherein 9 proteins are localized in the lysosome. All of these lysosomal glycoproteins were up-regulated after differentiation, where their principal function was hydrolysis of glycosyl residues. Protein-protein interaction and functional analyses revealed the active involvement of lysosomes during the process of glioblastoma stem cell differentiation. This work provides glycoprotein markers to characterize differentiation status of glioblastoma stem cells that may be useful in stem-cell therapy of glioblastoma.     

Gene analysis and dynamics of tumor stem cells in human glioblastoma cells after radiation

Glioblastoma is the most malignant central nervous system tumor. Patients with glioblastoma are treated with a combination of surgery, radiotherapy and chemotherapy; however, this effect is not satisfactory with regard to the prognosis. It is reported that the tumor stem cells affect recurrence, and radio- and chemotherapy resistance of the tumor, and that these cells play an important role in tumorigenesis and tumor progression. Using human glioblastoma cell lines (T98G and A172), irradiated (0, 30, 60 Gy) glioblastoma cells were prepared under the same conditions as clinical therapy. We analyzed cell proliferation rate, side population analysis by fluorescence-activated cell sorting and isolation of CD133⁺ cells, and performed genetic analysis (human stem cells) on these cells. We also investigated the difference in gene expression in the cells after radiation. The stem cell-related genes were highly expressed in the CD133⁺ cells compared with the CD133^? cells, suggesting that the cancer stem cells may be located in these CD133⁺ cells. In the T98G cell line, the cell proliferation rate of 30-Gy irradiated cells was higher than those of non-irradiated cells and 60-Gy irradiated cells. Stem cell-related genes were highly expressed in 30-Gy irradiated CD133⁺ T98G cells. In conclusion, we suggest that CD133⁺ cells may strongly affect tumor proliferation and the resistance against radiation therapy.     

Characterization of cell surface carbohydrates on asexual spores of the water moldSaprolegnia ferax

Summary In asexual reproduction of the water mold,Saprolegnia ferax, four distinct and sequentially produced spores are involved in dispersal, two of which are motile and two of which are nonmotile. Composition of cell surface glycoproteins may be important in dispersal strategies for each of these stages. Binding patterns of fluorescently labelled lectins were investigated to identify differences in glycoproteins of asexually produced dispersal stages. The pattern of lectin binding to zoospores was diverse. FITC-Con A bound to surfaces of zoospores and membranes of the water expulsion vacuole system, indicating the prescence of mannosyl and glucosyl residues. In zoospores incubated for more than 30 min in FITC-WGA and FITC-GS II. which bind N-acetyl glucosamine, fluorescence was sometimes localized in peripheral, intracellular patches. In shorter incubations, secondary zoospores bound these lectins along the groove region where K-bodies were located. Surfaces of cystospores typically bound FITC-WGA, but not FITC-GS II. FITC-GS II, however, bound to empty cystospore walls, probably because reactive sugars were available at the inner surface of the wall. Germ tubes emerging from cystospores bound labelled WGA and GS II, but not Con A. The same lectin binding pattern was found along discharge papilla of primary cystospores, indicating that modifications in cystospore walls associated with direct germination and zoospore discharge were similar. Thus, glycoproteins involved in early establishment of the hyphal system differ from those forming the cell surface of cystospores. Differences in the binding pattern of lectins to zoospores and cystospores highlight differences between cell surface carbohydrates of motile and nonmotile asexual stages.Abbreviations BPA lectin fromBauhinia purpurea - C₁ primary cystospore - C₂ secondary cystospore - Con A concanavalin A, lectin fromCanavalia ensiformis - DBA lectin fromDolichos biflorus - DIC Nomarski differential interference contrast optics - DS dilute salts - FITC fluorescein isothiocyanate - FUC fucose - Gal galactose - GalNAc N-acetyl galactosamine - Glc glucose - GlcNAc N-acetyl glucosamine - GS I Griffonia simplicifolia lectin I - GS II G. simplicifolia lectin II - Man mannose - MPA lectin fromMaclura pomifera - PC phase contrast optics - PNA lectin fromArachis hypogaea - SBA soybean agglutinin, lectin fromGlycine max - UEA-1 lectin fromUlex europaeus - WGA wheat germ agglutinin fromTriticum vulgare - WV water expulsion vacuole     

A survey of lectin binding in Paramecium

To better understand the general distribution of glycoproteins and the distribution of specific glycoprotein-bound sugar residues in Paramecium, a survey of the binding pattern of selected lectins was carried out in P. tetraurelia, P. caudatum, and P. multimicronucleatum. Lectins studied were concanavalin A (Con A), Griffonia simplicifolia agglutinins I and II (GS I and GS II), wheat germ agglutinin (WGA), Ulex europaeus (UEA I), peanut agglutinin (PNA), Ricinis communis toxin (RCA60) and agglutinin (RCA120), soybean agglutinin (SBA), Bauhinia purpurea agglutinin (BPA), Dolichos biflorus agglutinin (DBA), and Maclura pomifera agglutinin (MPA). Those giving the most distinctive patterns were Con A, GS II, WGA, UEA I, and PNA. No significant differences were found between the three species. Concanavalin A, a mannose/glucose-binding lectin, diffusely labeled the cell surface and cytoplasm and, unexpectedly, the nuclear envelopes. Events of nuclear division, and nuclear size and number were thus revealed. Both WGA and GS II, which are N-acetylglucosamine-binding lectins, labeled trichocyst tips, the cell surface, and the oral region, revealing stages of stomatogenesis. The lectin WGA, in addition, labeled the compartments of the phagosome-lysosome system. The lectin PNA, an N-acetyl galactosamine/galactose-binding protein, was very specific for digestive vacuoles. Finally, UEA I, a fucose-binding lectin, brightly labeled trichocysts, both their tips and body outlines. We conclude that a judicious choice of lectins can be used to localize glycoproteins and specific sugar residues as well as to study certain events of nuclear division, cellular morphogenesis, trichocyst discharge, and events in the digestive cycle of Paramecium.     

Preferential in situ CD4+CD56+ T cell activation and expansion within human glioblastoma

Recent evidence suggests that suppression of the cellular immune response is often attributable to populations of functionally distinct T cells that act to down-regulate Ag-specific effector T cells. Using flow cytometry, we evaluated tumor-infiltrating lymphocytes (TIL) from patients undergoing neurosurgical resection of glioblastoma multiforme (GBM), metastatic lung carcinoma, and meningioma for markers known to be expressed on immunoregulatory T cells. Ex vivo phenotypic characteristics, cellular proliferation, and cytokine expression patterns were compared between T cell subsets found in the PBMC and within TIL from fresh tumor samples. Interestingly, nearly half of all T cells infiltrating GBM specimens were CD56(+) T cells, while much smaller percentages of similar cells were identified within metastatic lung tumors and meningiomas. CD56(+) T cells identified within GBM were not canonical, or "invariant," NKT cells, as they demonstrated diverse TCR expression, a primarily CD4 single-positive phenotype, and lack of CD1d reactivity. The percentage of CD56(+) T cells exhibiting evidence of proliferation within GBM was 3- to 4-fold higher than the proportion of proliferating CD56(-) T cells from these lesions. In addition, direct ex vivo analysis of cytokine expression by TIL from GBM demonstrated significant numbers of IL-4/IL-13 positive cells, cytokines that are integral in the cell-mediated repression of tumor immunity in experimental models. We propose that GBM has a unique capacity to recruit and activate CD4(+)CD56(+) T cells, a population that has not been previously described within human tumors.     

Identification of glycoprotein markers for pancreatic cancer CD24+CD44+ stem-like cells using nano-LC-MS/MS and tissue microarray

Pancreatic adenocarcinoma is characterized by late diagnosis due to lack of early symptoms, extensive metastasis, and high resistance to chemo/radiation therapy. Recently, a subpopulation of cells within pancreatic cancers, termed cancer stem cells (CSCs), has been characterized and postulated to be the drivers for pancreatic cancer and responsible for metastatic spread. Further studies on pancreatic CSCs are therefore of particular importance to identify novel diagnosis markers and therapeutic targets for this dismal disease. Herein, the malignant phenotype of pancreatic cancer stem-like CD24+CD44+ cells was isolated from a human pancreatic carcinoma cell line (PANC-1) and demonstrated 4-fold increased invasion ability compared to CD24-CD44+ cells. Using lectin microarray and nano LC-MS/MS, we identified a differentially expressed set of glycoproteins between these two subpopulations. Lectin microarray analysis revealed that fucose- and galactose-specific lectins, UEA-1 and DBA, respectively, exhibit distinctly strong binding to CD24+CD44+ cells. The glycoproteins extracted by multilectin affinity chromatography were consequently analyzed by LC-MS/MS. Seventeen differentially expressed glycoproteins were identified, including up-regulated Cytokeratin 8/CK8, Integrin β1/CD29, ICAM1/CD54, and Ribophorin 2/RPN2 and down-regulated Aminopeptidase N/CD13. Immunohistochemical analysis of tissue microarrays showed that CD24 was significantly associated with late-stage pancreatic adenocarcinomas, and RPN2 was exclusively coexpressed with CD24 in a small population of CD24-positive cells. However, CD13 expression was dramatically decreased along with tumor progression, preferentially present on the apical membrane of ductal cells and vessels in early stage tumors. Our findings suggest that these glycoproteins may provide potential therapeutic targets and promising prognostic markers for pancreatic cancer.     

T98G: an anchorage-independent human tumor cell line that exhibits stationary phase G1 arrest in vitro.

T98 and T98G are two related cell lines that were derived from a human glioblastoma multiforma tumor. T98G has almost twice as many chromosomes as T98, suggesting that it is a polyploid variant of T98. Three aspects of control of cellular proliferation were studied in T98 and T98G cells in comparison to WI-38 normal human diploid cells. WI-38 cells have the following properties: (1) they can undergo only a limited number of population doublings in vitro; (2) they cannot proliferate without anchorage; and (3) they become arrested in G1 phase under stationary phase conditions. T98 cells differ from normal cells in all three of these properties, as do many other transformed cell lines. However, the derivative of T98, namely T98G, expresses an unique combination of normal and transformed aspects of the control of cellular proliferation. T98G cells are like normal cells in that they become arrested in G1 phase under stationary phase conditions, yet they also exhibit the transformed characteristics of anchorage independence and immortality. Thus, T98G cells demonstrate that transformation to immortality and anchorage independence can exist without concomitant loss of the normal mechanism for G1 arrest in response to stationary phase conditions. This result supports the hypothesis that each of these three aspects of control of cellular proliferation can be altered independently. Partially transformed cell lines, such as T98G, should be useful for sorting out the biochemical changes associated with transformation in each of these aspects.