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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Oncogene. Author manuscript; available in PMC Mar 4, 2010.
Published in final edited form as:
PMCID: PMC2832310
NIHMSID: NIHMS106129

The progenitor cell marker NG2/MPG promotes chemoresistance by activation of integrin-dependent PI3K/Akt signalling

Abstract

Chemoresistance represents a major problem in the treatment of many malignancies. Overcoming this obstacle will require improved understanding of the mechanisms responsible for the phenomenon. The progenitor cell marker NG2/MPG is aberrantly expressed by various tumors, but its role in cell death signalling and its potential as a therapeutic target is largely unexplored. We have assessed cytotoxic drug-induced cell death in glioblastoma (GBM) spheroids from fifteen patients, as well as in five cancer cell lines that differ with respect to NG2/MPG expression. The tumors were treated with doxorubicin, etoposide, carboplatin, temodal, cisplatin and TNFα. High NG2/MPG expression correlated with multi-drug resistance mediated by increased activation of α3β1 integrin/PI3K signalling and their downstream targets, promoting cell survival. NG2/MPG knockdown with shRNAs incorporated into lentiviral vectors attenuated β1 integrin signalling revealing potent anti-tumor effects and further sensitized neoplastic cells to cytotoxic treatment in vitro and in vivo. Thus, as a novel regulator of the anti-apoptotic response, NG2/MPG may represent an effective therapeutic target in several cancer subtypes.

Keywords: Apoptosis, Chemoresistance, Integrin, NG2/MPG

INTRODUCTION

Malignant brain tumors belong to the subgroup of cancers with the poorest prognosis, and the introduction of new chemotherapy regimens has only marginally improved survival. Attempts to improve survival must include strategies that identify and target molecules that confer chemoresistance.

NG2 is a transmembrane chondroitin sulfate proteoglycan that is expressed by progenitor cells in several types of tissues (Stallcup, 2002), including oligodendrocyte progenitors in the CNS (Belachew et al, 2003; Nishiyama et al., 1996b). NG2 is the rat homologue of the human melanoma proteoglycan (MPG), also known as the high molecular weight melanoma associated antigen (Campoli et al., 2004; Pluschke et al., 1996). NG2/MPG is over-expressed by several tumor types that fail to respond to conventional chemotherapy, including glioblastomas, most melanomas, and some leukemias (Behm et al., 1996; Chekenya et al., 2002a; Li et al., 2003; Mauvieux et al., 1999; Schrappe et al., 1991; Shoshan et al., 1999; Smith et al., 1996).

NG2/MPG potentiates cell motility (Burg et al., 1997; Eisenmann et al., 1999; Fang et al., 1999; Makagiansar et al., 2004; Makagiansar et al., 2007; Stallcup & Dahlin-Huppe, 2001) and modulates responses to growth factors (Goretzki et al., 1999; Grako et al., 1999; Grako & Stallcup, 1995; Nishiyama et al., 1996a), processes that are critical for the proliferation and migration of both immature progenitor and tumor cells. NG2/MPG expression increases melanoma growth and metastasis (Burg et al., 1998), and enhances glioma growth and angiogenesis (Chekenya et al., 2002b). The expression of NG2/MPG in childhood acute myeloid leukemic (AML) blasts has also been shown to correlate with poor clinical outcome (Hilden et al., 1997; Smith et al, 1996). The role of NG2/MPG in regulating cell death signalling is largely unexplored. We show here that by activating integrins and downstream PI3K/Akt signalling, NG2/MPG promotes tumor resistance to cytotoxic agents.

RESULTS

NG2/MPG expressing glioma cells are resistant to drug induced apoptosis

Since TNFα is a potent and well-characterized inducer of cell death, we compared its ability to induce apoptosis in NG2/MPG-negative U251 glioma cells (U251-Wt) and U251 cells transfected with NG2/MPG (U251-NG2/MPG). Morphological analyses revealed more frequent nuclear condensation and DNA fragmentation in the U251-Wt cells, indicating that they were significantly more sensitive to TNFα than the U251-NG2/MPG cells (Fig. 1A and B, p< 0.024). Since TNFα also stimulates protein synthesis-dependent cell survival (Beg & Baltimore, 1996) we investigated whether protein synthesis inhibition with cycloheximide (CHX) increased sensitivity to TNFα. TNFα increased apoptosis in both U251-Wt and U251-NG2/MPG cells (Fig 1A and B), however, the extent of cell death in CHX-treated U251-NG2/MPG cells remained less than in U251-Wt cells. While these data support the existence of NG2/MPG-mediated survival signals that are protein synthesis independent, they do not rule out a component that is protein synthesis dependent. The requirement for caspases in TNFα induced apoptosis was confirmed by the dramatic cell death inhibition for both U251-NG2/MPG and U251-Wt (Fig 1A and B) cells in the presence of zVAD. We also tested the effects of other drugs known to induce cell death (Fig. 1C). U251-NG2/MPG cells were less sensitive than U251-Wt cells to Etoposide and Vincristine. In the latter case the difference was small but still statistically significant. Our assessment of apoptosis was confirmed by flow cytometric analyses of DNA content during cell cycle progression (Fig ID). TNFα induced apoptosis in U251-Wt cells resulted in a large increase in the sub-G1 population, whereas this population was markedly smaller in the U251-NG2/MPG cells (Fig 1D).

Figure 1
NG2/MPG expressing tumor cells are more resistant than NG2/MPG negative cells to TNFα and chemotherapy induced apoptosis

To assess the functional significance of NG2/MPG, we used rat sequence specific siRNA2 to reduce the proteoglycan’s expression in the U251-NG2 cells that overexpressed the rat orthologue, (Fig 2A and B), and human specific siRNA3 in U87 cells that endogenously expressed the human homologue, (Fig 2A and C). These siRNAs reduced NG2/MPG protein levels in a dose dependent manner and at low concentrations (10–25nM) (Fig 2D and F). The effect of siRNA treatment on NG2 mRNA levels was confirmed by attenuation of transcripts by approximately 75% in siRNA2-treated U251-NG2 cells (Fig 2E). In contrast, a mutant siRNA2 with 4 consecutive mismatched nucleotides did not perturb NG2 mRNA levels, demonstrating that the NG2 siRNA knockdown was both sequence and target specific.

Figure 2
siRNA-mediated suppression of NG2/MPG gene expression

Next, we investigated the effect of NG2/MPG knock down on TNFα induced apoptosis. As shown in Fig 3A, the sensitivity of U251-NG2/MPG cells to TNFα treatment was significantly increased when NG2/MPG expression was reduced by treatment with siRNA2 (ANOVA F11.65, df= 2, p=0.0015). In contrast, U251-NG2/MPG cells transfected with control siRNA2, maintained their resistance to apoptosis. Intriguingly, siRNA knockdown sensitized U251-NG2/MPG cells to apoptosis beyond the levels seen in U251-Wt cells, indicating that the proteoglyan positive cells may be highly dependent on NG2/MPG mediated survival signals. NG2/MPG-dependent apoptosis resistance was also seen with U87 and A172 cells that endogenously express the proteoglycan (Fig 3B). These cells are highly resistant to TNFα-induced apoptosis, as are U87 and A172 cells transfected with control shRNA In contrast, their sensitivity to TNFα was significantly increased by transfection with the shRNA3 species that effectively knocks down human NG2/MPG expression (Fig 3B, immunoblots). U87wt cells and cells transfected with control shRNA were also resistant to clinically relevant chemotherapy drugs, but became sensitised upon shRNA mediated NG2/MPG knock down (Fig 3C). Flow cytometric analyses of DNA content during cell cycle progression also reflected the Etoposide-induced increase in apoptosis in U87 cells treated with NG2/MPG shRNAs, as visualised by the increased sub-G1 fraction in the DNA histograms. In addition, Etoposide induced greater G2/M phase arrest in the NG2/MPG shRNA knock down cells (Fig 3E).

Figure 3
SiRNA mediated suppression of NG2/MPG expression abrogates resistance to TNFα

Similar results were also obtained with A375 melanoma cells that endogenously express human NG2/MPG (Fig 3D, and immunoblot). Both Wt and control shRNA-transfected A375 cells were resistant to TNFα Doxorubicin, and Cisplatin, drugs commonly used for treatment of melanomas (Fig 3D). However, shRNA 3-transfected cells were significantly sensitized to these cytostatic agents (ANOVA F12.01, df= 8, p<0.0001). These findings establish NG2/MPG-mediated chemoresistance as a general phenomenon associated with several tumor types.

The NG2/MPG proteoglycan interacts with the α3β1 integrin to mediate apoptosis resistance

Integrin signalling plays a key role in regulating cell death and survival (Damiano et al., 1999; Downward, 2004; Frisch & Ruoslahti, 1997; Khwaja et al, 1997; Kumar, 1998; Wewer et al., 1997). Since NG2/MPG has been shown to interact with β1 integrins (Burg et al., 1998; Eisenmann et al., 1999; Fukushi et al., 2004; Yang et al., 2004), we investigated whether β1 integrin signalling could be involved in NG2/MPG-mediated resistance to cell death. U251-Wt and U251-NG2/MPG cells were immunostained with the HUTS-21 mAb that recognizes an activation-dependent epitope in the human β1 integrin subunit (Lenter et al, 1993; Luque et al, 1996). While activated β1 integrin was barely detectable on U251-Wt cells (Fig 4A, top left), strong labelling was observed in the U251-NG2/MPG cells (Fig 4A top right and 4B top left). Both cell lines exhibited comparable levels of total β1 integrin (Fig 4A and 4B, bottom panels), indicating the involvement of NG2/MPG in β1 activation rather than β expression. Equivalent results were also obtained for A375 melanoma cells (data not shown). To provide evidence for a functional role of β1 integrin signalling in NG2/MPG-mediated apoptosis resistance, we tested the effect of two β1 integrin function blocking antibodies on TNFα-induced apoptosis in U251-NG2/MPG cells. Both antibodies partially abolished the apoptosis resistance in U251-NG2/MPG cells, p = 0.029, Fig 4C, right-hand panel. The Ha2/5 antibody had a small, non-significant effect in U251-Wt cells, perhaps due to blocking low levels of NG2/MPG-independent integrin β1 activation. Next we asked whether activation of β1 integrin via an NG2/MPG-independent mechanism could rescue U251 Wt cells from TNFα-induced apoptosis. Treating U251-Wt cells with the β1 activating antibody TS2/16 (Arroyo et al., 1992; Hemler et al., 1984), significantly protected the U251-Wt cells from apoptosis (p<0.001, Fig 4C, left panel). Similarly, while U87wt cells and U87 cells transfected with control shRNA expressed equal levels of activated β1 integrin subunit, NG2/MPG knock down with NG2 shRNA cells attenuated levels of activated β1 (Fig 4D), further demonstrating the connection between NG2/MPG and integrin activation. Similar results were obtained with A375 cells (supplemenraty Fig 1).

Figure 4
Apoptosis resistance in NG2/MPG cells is mediated by β1 integrin signaling

α3 integrin subunit levels were also comparable in U251-Wt and U251-NG2/MPG (Fig 4E), while α2, α4, and α6 subunits were barely detectable (data not shown). Thus, the α3β1 heterodimer is the predominant integrin in the U251 cells, but only displays the activated conformation in NG2/MPG expressing cells. In order to provide evidence for a physical interaction between NG2/MPG and α3β1, we performed co-immunoprecipitation studies. The α3 subunit was detected in NG2/MPG immunoprecipitates prepared from both U251-NG2/MPG (Fig 4F) and U87 cells (Fig 4H) with two independent NG2/MPG antibodies but not with control immunoglobulins, demonstrating the specificity of the assays. The same two NG2/MPG antibodies did not immunoprecipitate α3 from the U251-Wt cells (Fig 4G), demonstrating the requirement for NG2/MPG in the co-immunoprecipitation. We were unable to demonstrate co-immunoprecipitation of NG2/MPG using antibodies against α3 integrin. Isotype specific negative controls and a monoclonal α3 integrin blocking antibody had no effect on NG2/MPG-mediated apoptosis resistance (data not shown). Taken together, these findings indicate the existence of an interaction between NG2/MPG and α3β1 integrin that promotes anti-apoptotic signalling via activated β1 integrin.

NG2/MPG-α3β1-mediated resistance to TNFα-induced apoptosis requires PI3K/Akt signalling

Since PI3K is a well-known promoter of cell survival acting downstream of β1 integrin, we studied the effect of the irreversible PI3K inhibitor wortmannin on TNFα-induced apoptosis in the U251-NG2/MPG and U251-Wt glioma cells (Fig 3A). Wortmannin dramatically reduced the cytoprotective effect of NG2/MPG in U251-NG2/MPG cells treated with control siRNA as well as untreated cells (ANOVA F 45.27, df=1, p<0.002). In contrast, the effect was much smaller in U251-Wt or U251-NG2/MPG siRNA knockdown cells already exhibiting high levels of cell death. These results indicate that NG2/MPG expression protects cells from TNFα-induced cell death via a mechanism involving the PI3K/Akt survival pathway.

To generate support for this hypothesis, we compared levels of the PI3K product, Ptdlns (3,4,5) P3 in U251-Wt and U251-NG2/MPG cells. Measurement of 32P -radiolabelled Ptdlns (3,4,5) P3 under basal conditions revealed increased synthesis of this phospholipid in the apoptosis resistant U251-NG2/MPG cells, (t 4.262, df= 4; p =0.01), (Fig 5A), but TNFα increased PIP3 levels in both cell types (t 1.024, df= 2; p =0.413), (Fig 5A). Quantitative assessment of Akt (S473) phosphorylation revealed a 2-fold greater baseline phosphorylation in U251-NG2/MPG cells compared to U251-Wt cells (supplementary Fig 2) adding further support for activation of PI3K/Akt as downstream targets of NG2/MPG dependent α3β1 integrin signaling. These data suggest that the NG2/MPG-positive cells may be primed for drug resistance.

Figure 5
NG2/MPG mediates resistance to TNFα via PI3K/Akt signalling in both glioma and melanoma cells

Similar to U251-NG2/MPG cells, constitutively high level of S473 phospho-Akt were also demonstrated in U87 (Fig 5B), A172 (Fig 5C) and A375 cells (Fig 5D) that endogenously express NG2/MPG. Transfection of these cells with NG2/MPG siRNA3, but not control siRNA, attenuated levels of S473 phospho-Akt (Fig 5B–D), substantiating the link between NG2/MPG expression and increased PI3K/Akt signalling.

NG2/MPG promotes resistance to TNFα and enhances tumor growth in vivo

To determine whether NG2/MPG-induced chemoresistance affects tumor growth, U87 cells were infected with lentiviruses expressing GFP and either control shRNAs (U87LVcontrol shRNA), or NG2/MPG shRNAs (U87LVNG2/MPG shRNA). 96 hours after a single exposure to the lentivirus-encoding NG2/MPG shRNAs, 65% of the cells expressed GFP compared to uninfected cells (Fig 6A and B). Western blot analyses confirmed that while both the U87 parental and U87LVcontrol shRNA cells contained high levels of NG2/MPG protein, successful down-regulation of the NG2/MPG protein was achieved in the U87LVNG2/MPG shRNA cells (Fig 6C). The knockdown of NG2/MPG was associated with significant tumor growth inhibition in vivo (ANOVA, F20.92 df=3; p = 0.0004, Fig 6D and E).

Figure 6
NG2/MPG promotes glioblastoma growth in vivo and confers resistance to TNFα treatment

To determine whether knocking down NG2/MPG affects tumor sensitivity to TNFα in vivo, the U87LVcontrol shRNA and U87LVNG2/MPG shRNA tumor bearing mice were treated with TNFα. This treatment had little effect on the growth of U87LVcontrol shRNA tumors (Fig 6D and E). However, the growth of U87LVNG2/MPG shRNA tumors was further retarded, indicating that they had been sensitized to TNFα treatment. Immunoblot analyses of tumor lysates revealed that while U87LVcontrol shRNA tumors had detectable levels of NG2/MPG (Fig 6F, right panels), successful knockdown of NG2/MPG mRNA had been achieved in vivo as indicated by the diminished levels of NG2/MPG protein in the U87MGLV NG2/MPG shRNA tumors (Fig 6F, left panels). Moreover, while levels of β-actin and total Akt remained largely unchanged across the groups (Fig 6F), levels of phosphorylated Akt were attenuated in the U87LV NG2/MPG shRNA tumors (Fig 6F). Using TUNEL staining, we were able to document increased tumor cell death after NG2/MPG knockdown, which was further potentiated by TNFα treatment, as indicated by TUNEL positive cells (supplementary Fig 2).

NG2/MPG expression is associated with chemoresistance in human GBM

To validate the physiological relevance of our observations in human tissues, GBM biopsy spheroids derived from tumors with varying NG2/MPG levels (Fig 7A and Table I) were examined for chemosensitivity to doxorubicin, Etoposide and Carboplatin (Fig 7B). Spheroids from glioblastomas with high NG2/MPG expression, as determined by both immunohistochemistry and qPCR, were resistant to Doxorubicin, Etoposide and Carboplatin (Fig 7B). In contrast, the GBM samples with low NG2/MPG exhibited greater sensitivity to these agents. These findings strongly link NG2/MPG expression to chemoresistance in glioma samples.

Figure 7
Chemosensitivity of human GBMs expressing different levels of NG2/MPG
Table I
NG2/MPG expression correlates with chemoresistance in GBM biopsies

Discussion

The present study demonstrates a novel role for NG2/MPG in mediating protection from apoptosis induced by TNFα and by other cytotoxic drugs with different modes of action. This effect is seen in a panel of cancer cell lines, as well as in biopsy material from brain tumor patients. Furthermore, knockdown of NG2/MPG sensitized malignant cells to chemotherapy and suppressed the growth rates of gliomas in vivo.

We provide evidence via co-immunoprecipitation and immunoblotting studies that the mechanism responsible for these effects involves complexing of NG2/MPG with α3β1 integrin on the cell surface, with subsequent activation of the PI3K/Akt signalling pathway. While levels of total β1 and α3 integrin subunits were unchanged in both U251-Wt and U251-NG2/MPG cells, increased levels of activated β1 integrin were observed in U251-NG2/MPG compared to U251-Wt cells. Furthermore, inhibition of integrin-mediated signalling using β1 function blocking antibodies attenuated apoptosis resistance in the NG2/MPG expressing cells, identifying α3β1 integrin as an important mediator of NG2/MPG -dependent survival signalling in cancer cells. Our results indicate that NG2/MPG-mediated α3β1 activation results in survival signals transduced via stimulation of PI3K/Akt signalling, a pathway widely recognized as a mediator of cell survival signalling (Cantrell, 2001; Vivanco & Sawyers, 2002). It is likely that this same PI3K/Akt signalling cascade mediates the integrin-dependent increase in cell motility that occurs in response to NG2/MPG expression (Fukushi et al, 2004; Makagiansar et al., 2007). Integrin-dependent effects of NG2 on apoptosis may vary according to the cell type and the nature of the environmental stimuli received by the cell. For example, under inflammatory conditions, NG2 can promote anoikis in fibroblasts by opposing fibronectin-stimulated α4β1 integrin signaling via a PKCα-dependent mechanism (Joo et al, 2008).

Our initial experiments utilized U251 cells overexpressing NG2/MPG in order to assess the proteoglycan´s effect on chemosensitivity. From a therapeutic point of view, it was equally informative to study the effects of downregulating NG2/MPG in tumor cell lines that constitutively express the proteoglycan. Together, these studies demonstrated that heterologous expression of NG2/MPG confers resistance to apoptosis in cells that are normally NG2/MPG -negative, while downregulation of endogenous NG2/MPG expression leads to increased sensitivity to TNFα-induced apoptosis. In addition, the relevance of these findings was validated in human GBM biopsy spheroids where low NG2/MPG expression correlated with chemosensitivity to doxorubicin, etoposide and carboplatin. In contrast, tumours with high levels of NG2/HMP were more resistant to, and in some cases were even stimulated to higher metabolic activity, by this cytotoxic treatment. This increased metabolic activity may be a result of energy consuming processes such as drug efflux or active DNA repair processes. The chemotherapeutic drugs used in this study serve as proof of principle to demonstrate that NG2/MPG expressing cells can be sensitised to various drugs after knockdown of proteoglycan expression.

We have confirmed that the U251-Wt and U251- NG2/MPG cell lines carry identical alterations in PTEN and p53 (supplementary figure 3 and 4, respectively), while these genes were differentially altered in the endogenously expressing cells (supplementary Table I). This demonstrates that differences in apoptosis resistance between these lines are likely to be due to the effects of NG2/MPG rather than to different genetic backgrounds. The over-expression studies and the siRNA knockdown studies clearly demonstrate the cause-effect relationship between the presence of the proteoglycan and resistance to apoptosis.

The in vivo studies reveal two distinct effects of NG2/MPG on tumor progression. First, the differential effects of TNFα on U87 tumors transfected with control or NG2/MPG shRNAs demonstrate the protective effect of NG2/MPG against TNFα -induced apoptosis. Further comparisons of these tumors revealed higher levels of phospho-Akt in the NG2/MPG -expressing tumors, indicative of increased PI3K/Akt signalling in support of the in vitro data. Second, stable downregulation of NG2/MPG in U87 (mediated by lentivirally delivered shRNAs) led to marked reduction of tumor growth rates even in the absence of TNFα. This effect may be due to NG2/MPG ´s role in functions that are unrelated to apoptosis. For example, NG2/MPG potentiates cell proliferation, possibly as a result of its participation in growth factor signalling (Goretzki et al., 1999; Grako et al, 1999). NG2/MPG also stimulates angiogenesis, by sequestering angiostatin and neutralizing its inhibitory effects on angiogenesis (Chekenya et al., 2002b; Goretzki et al., 2000).

In conclusion, we have applied a variety of experimental conditions to both in vitro and in vivo model systems, all of which establish NG2/MPG as a mediator of multi-drug resistance in the tumors examined. The mechanisms responsible for these effects involve increased survival signals that counteract cell death. Future work will be directed at elucidating in more depth the genetic or epigenetic mechanisms involved in the NG2/MPG induced chemoresistance. This involvement of NG2/MPG in multiple aspects of tumor biology makes the proteoglycan an attractive candidate for future therapies against cancer.

MATERIALS AND METHODS

Cell culture

These studies utilized the human glioblastoma multiforme cell lines U251N (U251-Wt), U87, and A172 and the human A375 melanoma (American Type Culture Collection, Rockville, Maryland; ATCC). U251 cells were transfected with the rat NG2/MPG cDNA (U251-NG2/MPG), as previously described (Chekenya et al., 2002b). Cells were exposed to 50ng/ml TNFα for 15 min with or without 30 min pre-treatment with the PI3K inhibitor Wortmannin (Sigma). The chemotherapy agents Vincristine, Etoposide, Temodal, Doxorubicin, Cisplatin and Carboplatin were also used. In some experiments cells were pre-treated for 20 min with the irreversible caspase inhibitor zVAD-FMK (BIOMOL, Plymouth Meeting, PA). Cells were also pre-incubated with 10µg/ml of TS2/16 β1 activating antibody (ATCC), or CD29 (/Ha2/5) β1 function blocking antibody (BD Pharmingen, San Diego, CA), or the β1 integrin function blocking monoclonal antibody AIIB2 (a gift from M. Bissell) at 37°C for 2hr prior to treatment with TNFα. Prior to analysis of apoptosis, cells were fixed in 2% (v/v) glutaraldehyde containing 1µg/ml Hoechst 33342 (Sigma).

Scoring of apoptosis

Apoptosis was determined by morphological changes in Hoechst stained cell nuclei using light and UV microscopy as previously described (Sandal et al., 2002). Apoptosis (sub G1 population) was also assessed by flow cytometric analysis of DNA content in cells stained with propidium iodide using ModFit LTTM version 3.0 software. For fluorescence, goat anti-mouse quantum dots 565 (Invitrogen, Carlsbad, CA) were used. Negative controls were secondary antibody only.

Sensitivity of human glioblastoma biopsy spheroids to doxorubicin

To assess viability/metabolic activity in NG2/MPG positive (n=8) and negative (n=7) human glioma specimens, spheroids of equal sizes (250µm) were treated with 3 µM Doxorubicin (96hr), or 0.5 and 50 µM Etoposide (120hr) or 0.5 and 10 µM Carboplatin (120hr) depending on the nearest IC50 value for the NG2/MPG negative tumors. The 3-(4-5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay was used as described by the manufacturer (Promega, Madison, WI). Untreated spheroids were used as controls. The effects of drugs (% of control) were calculated as absorbance of non-treated cells - absorbance of treated cells)/absorbance of non-treated cells (Miura et al, 2006). The ethical board at Haukeland University Hospital, Bergen, Norway, approved the collection of tumor tissue. The patients gave their informed consent to specimen collection.

Real-time qPCR

The snap frozen GBM tissue was crushed in liquid nitrogen, total RNA extracted and cDNA reverse transcribed using iScript™ (Bio-Rad Laboratories, Hercules, CA), with iQ SYBR Green in the Real time qPCR using iCycler™ Thermal Cycler fitted with iCycler™ Optical Module (Bio-Rad Laboratories). Primers directed against 18S RNA were used as an internal control. 3 adult normal brain cDNAs used as reference were either prepared from cerebral cortical tissue after radical resection and verified by a neuropathologist, or were purchased from Ambion (Austin, Tx).

siRNA synthesis

siRNAs were transcribed in vitro using chemically synthesised DNA oligonucleotides and T7 RNA polymerase. The following siRNAs were designed: siRNA2 5’´GUAGAUCAAUACCCUACACUU-3’ (positions 970 to 989) in the rat NG2/MPG cDNA. A non–functional mutant version of siRNA2 with four consecutive nucleotide mismatches was used as a control (5’GUAGAUCAAUUGGGUACACUU-3’). The expression of human NG2/MPG was targeted with siRNA3 (5’GUGGACCAGUACCCUACGG-3’), corresponding to Homo sapiens melanoma-associated chondroitin sulfate proteoglycan (cspg4).

Construction of the shRNA lentiviral vectors and lentiviral infection

Lentiviral particles harboring pRNAt-U6.1 (SD 1257, Genscript, Scotch Plains, NJ), encoding a U6 promoter driving shRNA expression were produced using the Virapower lentiviral packaging mix (Invitrogen, Carlsbad, CA) according to the manufacturers descriptions. Briefly, packaging and transfer vectors were co-transfected into 293FT cells, and viral particles were harvested 48h post-infection. Viral particles were concentrated by ultra-sentrifugation and the infected cells were monitored for GFP expression using flow cytometry. Two weeks post infection the cells were harvested and monitored for GFP fluorescence by flow cytometry.

Western blotting

Proteins from cells or tumor tissue were harvested in ice-cold lysis buffer (10mM K2HPO4, 1mM EDTA (pH 6.8) containing 10mM Chaps, 50 µM NaF, and 0.3µM NaVO3, supplemented with Complete Protease Inhibitor (Roche Molecular Biochemicals) and 0.02U of chondroitinase ABC (Sigma). Rabbit anti-total Akt and anti-phospho Akt (Ser 473) (diluted 1:1000) (Cell Signalling Technology, Beverly, MA), and Rabbit anti-NG2/MPG (553) antibody was used at 1:2000 dilution. For normalization, blots were stripped and re-probed with mouse anti-β-actin (diluted 1:5000, Abeam Limited, Cambridge, UK). Immunoprecipitations were performed as previously described (Fukushi et al., 2004). Immunocyto/histochemistry was performed using standard procedures previously described (Chekenya et al., 2002b).

Extraction and analysis of 3-phosphorylated glycerophosphoinositides

U251-Wt and U251-NG2/MPG glioma and cells treated with 50ng/ml TNFα for 15 min were propagated in the presence of [32P] Pi to produce [32P] labelled phospholipids. Extraction and thin-layer chromatography of polyphosphoinositides were performed as described previously (Tysnes et al., 1985).

Animals

Twenty-four, 5–6 week old, severe combined immunodeficient (NOD-SCID) mice (C.B.-Igh-1b/lcrTac-Prkdc) purchased from Taconic Europe, (Ry, Denmark) received 5×106 U87MG tumor cells stably expressing LVNG2/MPG shRNAs and LVCtr shRNAs lentiviruses into the hind leg. The animals were divided into four groups, U87MGLV-control siRNA (n=6), U87MG LVNG2/MPG siRNA (n=6), U87MG LV control siRNA+ TNFα (n=6), U87MG LVNG2/MPG siRNA + TNFα (n=6). 48 hrs after tumor implantation, some animals were treated with intraperitoneal injections (IP) of 150µg/kg rhTNFα, (Peprotech LTD, UK), once a day for 7 days. The animals were anaesthetised with 1–2% isoflurane in a 70%/30% N2/O2 mix and all procedures were in accordance with protocols approved by The National Animal Research Authority (Oslo, Norway). Tumor growth was determined by measuring two perpendicular diameters. Tumor volumes (V) were calculated using the formula (V)=(a×b×c)/2 that was derived for an ellipsoid (πd3/6)(Tan et al, 2004), where a, b and c are the long axis, short axis and the depth, respectively.

Statistical Analyses

Data were analyzed using the Student’s paired T-test (two-tailed), and One-way (StatViewSE+) or Two-way ANOVA (Graphpad Instat software Inc version 3.05, San Diego, CA).

Acknowledgements

This work was supported by research grants from The Norwegian Cancer Society, The Bergen translational Research Group, Familien Blix Fond, The University of Bergen, and a RO1 CA95287 grant from the National Institutes of Health. The work was also supported by the Sixth EU Framework Programme and Helse-Vest. We wish to thank Narve Brekkå, Tove Johansen, Christine Eriksen, Erna Finsås, and Nina Lied Larsen for their technical assistance. We thank Jesus Planaguma for assistance with his excellent image analysis.

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