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TheprogenitorcellmarkerNG2/MPGpromoteschemoresistancebyactivationofintegrin-dependentPI3K/Aktsignaling

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

M Chekenya1, C Krakstad2,*, A Svendsen1,*, IA Netland1, V Staalesen3, B.B Tysnes1, FSelheim2, J Wang1, P.Ø Sakariassen1, T Sandal2, P.E Lønning4, T Flatmark5, P.Ø Enger1,6,R Bjerkvig1,7, M Sioud8,†, and WB Stallcup9,†1Norlux Neuro-Oncology, Department of Biomedicine, University of Bergen2Department of Biomedicine, University of Bergen3Department of Molecular Biology, University of Bergen4Department of Oncology, Haukeland University Hospital5Department of Biomedicine, Section of Biochemistry and Molecular Biology, University of Bergen6Department of Neurosurgery, Haukeland University Hospital7Norlux Neuro-Oncology, CPP Santé, Luxemburg8Department of Immunology, Molecular Medicine Group, The Radium Hospital, N-0310 Oslo,Norway9Burnham Institute for Medical Research, Cancer Research Center, La Jolla, CA 92037

AbstractChemoresistance represents a major problem in the treatment of many malignancies. Overcomingthis obstacle will require improved understanding of the mechanisms responsible for thephenomenon. The progenitor cell marker NG2/MPG is aberrantly expressed by various tumors, butits role in cell death signalling and its potential as a therapeutic target is largely unexplored. We haveassessed 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 weretreated with doxorubicin, etoposide, carboplatin, temodal, cisplatin and TNFα. High NG2/MPGexpression correlated with multi-drug resistance mediated by increased activation of α3β1 integrin/PI3K signalling and their downstream targets, promoting cell survival. NG2/MPG knockdown withshRNAs 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 therapeutictarget in several cancer subtypes.

KeywordsApoptosis; Chemoresistance; Integrin; NG2/MPG

Corresponding Author: Martha Chekenya, PhD, DPhil, Norlux Neuro-Oncology Group, Department of Biomedicine, University ofBergen, Jonas Lies vei 91, N-5009 Bergen, Norway, E-Mail: martha.chekenya@biomed.uib.no, Telephone: 47-55586358, Fax:47-55586360.*Contributed equally.†Co-senior author

NIH Public AccessAuthor ManuscriptOncogene. Author manuscript; available in PMC 2010 March 4.

Published in final edited form as:Oncogene. 2008 September 4; 27(39): 5182–5194. doi:10.1038/onc.2008.157.

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INTRODUCTIONMalignant brain tumors belong to the subgroup of cancers with the poorest prognosis, and theintroduction of new chemotherapy regimens has only marginally improved survival. Attemptsto improve survival must include strategies that identify and target molecules that conferchemoresistance.

NG2 is a transmembrane chondroitin sulfate proteoglycan that is expressed by progenitor cellsin 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 humanmelanoma proteoglycan (MPG), also known as the high molecular weight melanomaassociated antigen (Campoli et al., 2004; Pluschke et al., 1996). NG2/MPG is over-expressedby several tumor types that fail to respond to conventional chemotherapy, includingglioblastomas, 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; Smithet 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 andmigration of both immature progenitor and tumor cells. NG2/MPG expression increasesmelanoma growth and metastasis (Burg et al., 1998), and enhances glioma growth andangiogenesis (Chekenya et al., 2002b). The expression of NG2/MPG in childhood acutemyeloid 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 signallingis largely unexplored. We show here that by activating integrins and downstream PI3K/Aktsignalling, NG2/MPG promotes tumor resistance to cytotoxic agents.

RESULTSNG2/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 abilityto induce apoptosis in NG2/MPG-negative U251 glioma cells (U251-Wt) and U251 cellstransfected with NG2/MPG (U251-NG2/MPG). Morphological analyses revealed morefrequent nuclear condensation and DNA fragmentation in the U251-Wt cells, indicating thatthey were significantly more sensitive to TNFα than the U251-NG2/MPG cells (Fig. 1A andB, 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 ofNG2/MPG-mediated survival signals that are protein synthesis independent, they do not ruleout a component that is protein synthesis dependent. The requirement for caspases in TNFαinduced 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 effectsof other drugs known to induce cell death (Fig. 1C). U251-NG2/MPG cells were less sensitivethan U251-Wt cells to Etoposide and Vincristine. In the latter case the difference was smallbut still statistically significant. Our assessment of apoptosis was confirmed by flow cytometricanalyses of DNA content during cell cycle progression (Fig ID). TNFα induced apoptosis inU251-Wt cells resulted in a large increase in the sub-G1 population, whereas this populationwas markedly smaller in the U251-NG2/MPG cells (Fig 1D).

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To assess the functional significance of NG2/MPG, we used rat sequence specific siRNA2 toreduce the proteoglycan’s expression in the U251-NG2 cells that overexpressed the ratorthologue, (Fig 2A and B), and human specific siRNA3 in U87 cells that endogenouslyexpressed the human homologue, (Fig 2A and C). These siRNAs reduced NG2/MPG proteinlevels in a dose dependent manner and at low concentrations (10–25nM) (Fig 2D and F). Theeffect of siRNA treatment on NG2 mRNA levels was confirmed by attenuation of transcriptsby approximately 75% in siRNA2-treated U251-NG2 cells (Fig 2E). In contrast, a mutantsiRNA2 with 4 consecutive mismatched nucleotides did not perturb NG2 mRNA levels,demonstrating that the NG2 siRNA knockdown was both sequence and target specific.

Next, we investigated the effect of NG2/MPG knock down on TNFα induced apoptosis. Asshown in Fig 3A, the sensitivity of U251-NG2/MPG cells to TNFα treatment was significantlyincreased when NG2/MPG expression was reduced by treatment with siRNA2 (ANOVAF11.65, 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 theproteoglyan 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 thatendogenously 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, theirsensitivity to TNFα was significantly increased by transfection with the shRNA3 species thateffectively knocks down human NG2/MPG expression (Fig 3B, immunoblots). U87wt cellsand cells transfected with control shRNA were also resistant to clinically relevantchemotherapy drugs, but became sensitised upon shRNA mediated NG2/MPG knock down(Fig 3C). Flow cytometric analyses of DNA content during cell cycle progression also reflectedthe Etoposide-induced increase in apoptosis in U87 cells treated with NG2/MPG shRNAs, asvisualised by the increased sub-G1 fraction in the DNA histograms. In addition, Etoposideinduced greater G2/M phase arrest in the NG2/MPG shRNA knock down cells (Fig 3E).

Similar results were also obtained with A375 melanoma cells that endogenously express humanNG2/MPG (Fig 3D, and immunoblot). Both Wt and control shRNA-transfected A375 cellswere resistant to TNFα Doxorubicin, and Cisplatin, drugs commonly used for treatment ofmelanomas (Fig 3D). However, shRNA 3-transfected cells were significantly sensitized tothese 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 resistanceIntegrin 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; Weweret 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 whetherβ1 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 recognizesan activation-dependent epitope in the human β1 integrin subunit (Lenter et al, 1993; Luqueet al, 1996). While activated β1 integrin was barely detectable on U251-Wt cells (Fig 4A, topleft), strong labelling was observed in the U251-NG2/MPG cells (Fig 4A top right and 4B topleft). Both cell lines exhibited comparable levels of total β1 integrin (Fig 4A and 4B, bottompanels), 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 provideevidence for a functional role of β1 integrin signalling in NG2/MPG-mediated apoptosisresistance, 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

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resistance in U251-NG2/MPG cells, p = 0.029, Fig 4C, right-hand panel. The Ha2/5 antibodyhad a small, non-significant effect in U251-Wt cells, perhaps due to blocking low levels ofNG2/MPG-independent integrin β1 activation. Next we asked whether activation of β1 integrinvia an NG2/MPG-independent mechanism could rescue U251 Wt cells from TNFα-inducedapoptosis. 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 shRNAexpressed equal levels of activated β1 integrin subunit, NG2/MPG knock down with NG2shRNA cells attenuated levels of activated β1 (Fig 4D), further demonstrating the connectionbetween NG2/MPG and integrin activation. Similar results were obtained with A375 cells(supplemenraty Fig 1).

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 α3β1heterodimer is the predominant integrin in the U251 cells, but only displays the activatedconformation in NG2/MPG expressing cells. In order to provide evidence for a physicalinteraction between NG2/MPG and α3β1, we performed co-immunoprecipitation studies. Theα3 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 withcontrol immunoglobulins, demonstrating the specificity of the assays. The same two NG2/MPG antibodies did not immunoprecipitate α3 from the U251-Wt cells (Fig 4G), demonstratingthe requirement for NG2/MPG in the co-immunoprecipitation. We were unable to demonstrateco-immunoprecipitation of NG2/MPG using antibodies against α3 integrin. Isotype specificnegative 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 theexistence of an interaction between NG2/MPG and α3β1 integrin that promotes anti-apoptoticsignalling via activated β1 integrin.

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

Since PI3K is a well-known promoter of cell survival acting downstream of β1 integrin, westudied the effect of the irreversible PI3K inhibitor wortmannin on TNFα-induced apoptosisin the U251-NG2/MPG and U251-Wt glioma cells (Fig 3A). Wortmannin dramatically reducedthe cytoprotective effect of NG2/MPG in U251-NG2/MPG cells treated with control siRNAas well as untreated cells (ANOVA F 45.27, df=1, p<0.002). In contrast, the effect was muchsmaller in U251-Wt or U251-NG2/MPG siRNA knockdown cells already exhibiting highlevels of cell death. These results indicate that NG2/MPG expression protects cells fromTNFα-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 apoptosisresistant U251-NG2/MPG cells, (t 4.262, df= 4; p =0.01), (Fig 5A), but TNFα increased PIP3levels 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/MPGcells compared to U251-Wt cells (supplementary Fig 2) adding further support for activationof PI3K/Akt as downstream targets of NG2/MPG dependent α3β1 integrin signaling. Thesedata suggest that the NG2/MPG-positive cells may be primed for drug resistance.

Similar to U251-NG2/MPG cells, constitutively high level of S473 phospho-Akt were alsodemonstrated in U87 (Fig 5B), A172 (Fig 5C) and A375 cells (Fig 5D) that endogenouslyexpress NG2/MPG. Transfection of these cells with NG2/MPG siRNA3, but not control

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siRNA, attenuated levels of S473 phospho-Akt (Fig 5B–D), substantiating the link betweenNG2/MPG expression and increased PI3K/Akt signalling.

NG2/MPG promotes resistance to TNFα and enhances tumor growth in vivoTo determine whether NG2/MPG-induced chemoresistance affects tumor growth, U87 cellswere infected with lentiviruses expressing GFP and either control shRNAs (U87LVcontrolshRNA), or NG2/MPG shRNAs (U87LVNG2/MPG shRNA). 96 hours after a single exposureto the lentivirus-encoding NG2/MPG shRNAs, 65% of the cells expressed GFP compared touninfected cells (Fig 6A and B). Western blot analyses confirmed that while both the U87parental and U87LVcontrol shRNA cells contained high levels of NG2/MPG protein,successful down-regulation of the NG2/MPG protein was achieved in the U87LVNG2/MPGshRNA cells (Fig 6C). The knockdown of NG2/MPG was associated with significant tumorgrowth inhibition in vivo (ANOVA, F20.92 df=3; p = 0.0004, Fig 6D and E).

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 treatedwith 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 furtherretarded, indicating that they had been sensitized to TNFα treatment. Immunoblot analyses oftumor 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 achievedin 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 Aktremained largely unchanged across the groups (Fig 6F), levels of phosphorylated Akt wereattenuated in the U87LV NG2/MPG shRNA tumors (Fig 6F). Using TUNEL staining, we wereable to document increased tumor cell death after NG2/MPG knockdown, which was furtherpotentiated by TNFα treatment, as indicated by TUNEL positive cells (supplementary Fig 2).

NG2/MPG expression is associated with chemoresistance in human GBMTo validate the physiological relevance of our observations in human tissues, GBM biopsyspheroids derived from tumors with varying NG2/MPG levels (Fig 7A and Table I) wereexamined for chemosensitivity to doxorubicin, Etoposide and Carboplatin (Fig 7B). Spheroidsfrom glioblastomas with high NG2/MPG expression, as determined by bothimmunohistochemistry and qPCR, were resistant to Doxorubicin, Etoposide and Carboplatin(Fig 7B). In contrast, the GBM samples with low NG2/MPG exhibited greater sensitivity tothese agents. These findings strongly link NG2/MPG expression to chemoresistance in gliomasamples.

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

We provide evidence via co-immunoprecipitation and immunoblotting studies that themechanism responsible for these effects involves complexing of NG2/MPG with α3β1 integrinon the cell surface, with subsequent activation of the PI3K/Akt signalling pathway. Whilelevels 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/MPGcompared to U251-Wt cells. Furthermore, inhibition of integrin-mediated signalling using β1

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function blocking antibodies attenuated apoptosis resistance in the NG2/MPG expressing cells,identifying α3β1 integrin as an important mediator of NG2/MPG -dependent survivalsignalling in cancer cells. Our results indicate that NG2/MPG-mediated α3β1 activation resultsin survival signals transduced via stimulation of PI3K/Akt signalling, a pathway widelyrecognized 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-dependentincrease 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 varyaccording to the cell type and the nature of the environmental stimuli received by the cell. Forexample, under inflammatory conditions, NG2 can promote anoikis in fibroblasts by opposingfibronectin-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 theproteoglycan´s effect on chemosensitivity. From a therapeutic point of view, it was equallyinformative to study the effects of downregulating NG2/MPG in tumor cell lines thatconstitutively express the proteoglycan. Together, these studies demonstrated thatheterologous expression of NG2/MPG confers resistance to apoptosis in cells that are normallyNG2/MPG -negative, while downregulation of endogenous NG2/MPG expression leads toincreased sensitivity to TNFα-induced apoptosis. In addition, the relevance of these findingswas validated in human GBM biopsy spheroids where low NG2/MPG expression correlatedwith chemosensitivity to doxorubicin, etoposide and carboplatin. In contrast, tumours withhigh levels of NG2/HMP were more resistant to, and in some cases were even stimulated tohigher metabolic activity, by this cytotoxic treatment. This increased metabolic activity maybe 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 thatNG2/MPG expressing cells can be sensitised to various drugs after knockdown of proteoglycanexpression.

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

The in vivo studies reveal two distinct effects of NG2/MPG on tumor progression. First, thedifferential effects of TNFα on U87 tumors transfected with control or NG2/MPG shRNAsdemonstrate the protective effect of NG2/MPG against TNFα -induced apoptosis. Furthercomparisons 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 deliveredshRNAs) led to marked reduction of tumor growth rates even in the absence of TNFα. Thiseffect 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 factorsignalling (Goretzki et al., 1999; Grako et al, 1999). NG2/MPG also stimulates angiogenesis,by sequestering angiostatin and neutralizing its inhibitory effects on angiogenesis (Chekenyaet al., 2002b; Goretzki et al., 2000).

In conclusion, we have applied a variety of experimental conditions to both in vitro and invivo model systems, all of which establish NG2/MPG as a mediator of multi-drug resistancein the tumors examined. The mechanisms responsible for these effects involve increased

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survival signals that counteract cell death. Future work will be directed at elucidating in moredepth the genetic or epigenetic mechanisms involved in the NG2/MPG inducedchemoresistance. This involvement of NG2/MPG in multiple aspects of tumor biology makesthe proteoglycan an attractive candidate for future therapies against cancer.

MATERIALS AND METHODSCell 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/mlTNFα for 15 min with or without 30 min pre-treatment with the PI3K inhibitor Wortmannin(Sigma). The chemotherapy agents Vincristine, Etoposide, Temodal, Doxorubicin, Cisplatinand Carboplatin were also used. In some experiments cells were pre-treated for 20 min withthe irreversible caspase inhibitor zVAD-FMK (BIOMOL, Plymouth Meeting, PA). Cells werealso 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 functionblocking monoclonal antibody AIIB2 (a gift from M. Bissell) at 37°C for 2hr prior to treatmentwith TNFα. Prior to analysis of apoptosis, cells were fixed in 2% (v/v) glutaraldehydecontaining 1µg/ml Hoechst 33342 (Sigma).

Scoring of apoptosisApoptosis was determined by morphological changes in Hoechst stained cell nuclei using lightand UV microscopy as previously described (Sandal et al., 2002). Apoptosis (sub G1population) was also assessed by flow cytometric analysis of DNA content in cells stained withpropidium iodide using ModFit LTTM version 3.0 software. For fluorescence, goat anti-mousequantum dots 565 (Invitrogen, Carlsbad, CA) were used. Negative controls were secondaryantibody only.

Sensitivity of human glioblastoma biopsy spheroids to doxorubicinTo assess viability/metabolic activity in NG2/MPG positive (n=8) and negative (n=7) humanglioma 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) dependingon 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 usedas described by the manufacturer (Promega, Madison, WI). Untreated spheroids were used ascontrols. 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 ethicalboard at Haukeland University Hospital, Bergen, Norway, approved the collection of tumortissue. The patients gave their informed consent to specimen collection.

Real-time qPCRThe snap frozen GBM tissue was crushed in liquid nitrogen, total RNA extracted and cDNAreverse transcribed using iScript™ (Bio-Rad Laboratories, Hercules, CA), with iQ SYBRGreen in the Real time qPCR using iCycler™ Thermal Cycler fitted with iCycler™ OpticalModule (Bio-Rad Laboratories). Primers directed against 18S RNA were used as an internalcontrol. 3 adult normal brain cDNAs used as reference were either prepared from cerebralcortical tissue after radical resection and verified by a neuropathologist, or were purchasedfrom Ambion (Austin, Tx).

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siRNA synthesissiRNAs were transcribed in vitro using chemically synthesised DNA oligonucleotides and T7RNA polymerase. The following siRNAs were designed: siRNA25’´GUAGAUCAAUACCCUACACUU-3’ (positions 970 to 989) in the rat NG2/MPG cDNA.A non–functional mutant version of siRNA2 with four consecutive nucleotide mismatches wasused as a control (5’GUAGAUCAAUUGGGUACACUU-3’). The expression of human NG2/MPG was targeted with siRNA3 (5’GUGGACCAGUACCCUACGG-3’), corresponding toHomo sapiens melanoma-associated chondroitin sulfate proteoglycan (cspg4).

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

Western blottingProteins 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, supplementedwith Complete Protease Inhibitor (Roche Molecular Biochemicals) and 0.02U ofchondroitinase ABC (Sigma). Rabbit anti-total Akt and anti-phospho Akt (Ser 473) (diluted1:1000) (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-probedwith 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 glycerophosphoinositidesU251-Wt and U251-NG2/MPG glioma and cells treated with 50ng/ml TNFα for 15 min werepropagated in the presence of [32P] Pi to produce [32P] labelled phospholipids. Extraction andthin-layer chromatography of polyphosphoinositides were performed as described previously(Tysnes et al., 1985).

AnimalsTwenty-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 U87MGtumor cells stably expressing LVNG2/MPG shRNAs and LVCtr shRNAs lentiviruses into thehind 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), U87MGLVNG2/MPG siRNA + TNFα (n=6). 48 hrs after tumor implantation, some animals weretreated with intraperitoneal injections (IP) of 150µg/kg rhTNFα, (Peprotech LTD, UK), oncea day for 7 days. The animals were anaesthetised with 1–2% isoflurane in a 70%/30% N2/O2mix and all procedures were in accordance with protocols approved by The National AnimalResearch Authority (Oslo, Norway). Tumor growth was determined by measuring twoperpendicular 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.

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Statistical AnalysesData 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).

AcknowledgmentsThis work was supported by research grants from The Norwegian Cancer Society, The Bergen translational ResearchGroup, Familien Blix Fond, The University of Bergen, and a RO1 CA95287 grant from the National Institutes ofHealth. The work was also supported by the Sixth EU Framework Programme and Helse-Vest. We wish to thankNarve 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.

ReferencesArroyo AG, Sanchez-Mateos P, Campanero MR, Martin-Padura I, Dejana E, Sanchez-Madrid F.

Regulation of the VLA integrin-ligand interactions through the beta 1 subunit. J Cell Biol1992;117:659–670. [PubMed: 1374069]

Beg AA, Baltimore D. An essential role for NF-kappaB in preventing TNF-alpha-induced cell death.Science 1996;274:782–784. [PubMed: 8864118]

Behm FG, Smith FO, Raimondi SC, Pui CH, Bernstein ID. Human homologue of the rat chondroitinsulfate proteoglycan, NG2, detected by monoclonal antibody 7.1, identifies childhood acutelymphoblastic leukemias with t(4;11)(q21;q23) or t(11;19)(q23;p13) and MLL gene rearrangements.Blood 1996;87:1134–1139. [PubMed: 8562939]

Belachew S, Chittajallu R, Aguirre AA, Yuan X, Kirby M, Anderson S, Gallo V. Postnatal NG2proteoglycan-expressing progenitor cells are intrinsically multipotent and generate functional neurons.J Cell Biol 2003;161:169–186. [PubMed: 12682089]

Burg MA, Grako KA, Stallcup WB. Expression of the NG2 proteoglycan enhances the growth andmetastatic properties of melanoma cells. J Cell Physiol 1998;177:299–312. [PubMed: 9766527]

Burg MA, Nishiyama A, Stallcup WB. A central segment of the NG2 proteoglycan is critical for theability of glioma cells to bind and migrate toward type VI collagen. Exp Cell Res 1997;235:254–264.[PubMed: 9281375]

Campoli MR, Chang CC, Kageshita T, Wang X, McCarthy JB, Ferrone S. Human high molecular weight-melanoma-associated antigen (HMW-MAA): a melanoma cell surface chondroitin sulfateproteoglycan (MSCP) with biological and clinical significance. Crit Rev Immunol 2004;24:267–296.[PubMed: 15588226]

Cantrell DA. Phosphoinositide 3-kinase signalling pathways. J Cell Sci 2001;114:1439–1445. [PubMed:11282020]

Chekenya M, Enger PO, Thorsen F, Tysnes BB, Al-Sarraj S, Read TA, Furmanek T, Mahesparan R,Levine JM, Butt AM, Pilkington GJ, Bjerkvig R. The glial precursor proteoglycan, NG2, is expressedon tumour neovasculature by vascular pericytes in human malignant brain tumours. Neuropathol ApplNeurobiol 2002a;28:367–380. [PubMed: 12366818]

Chekenya M, Hjelstuen M, Enger PO, Thorsen F, Jacob AL, Probst B, Haraldseth O, Pilkington G, ButtA, Levine JM, Bjerkvig R. NG2 proteoglycan promotes angiogenesis-dependent tumor growth inCNS by sequestering angiostatin. Faseb J 2002b;16:586–588. [PubMed: 11919162]

Damiano JS, Cress AE, Hazlehurst LA, Shtil AA, Dalton WS. Cell adhesion mediated drug resistance(CAM-DR): role of integrins and resistance to apoptosis in human myeloma cell lines. Blood1999;93:1658–1667. [PubMed: 10029595]

Downward J. PI 3-kinase, Akt and cell survival. Semin Cell Dev Biol 2004;15:177–182. [PubMed:15209377]

Eisenmann KM, McCarthy JB, Simpson MA, Keely PJ, Guan JL, Tachibana K, Lim L, Manser E, FurchtLT, Iida J. Melanoma chondroitin sulphate proteoglycan regulates cell spreading through Cdc42,Ack-1 and p130cas. Nat Cell Biol 1999;1:507–513. [PubMed: 10587647]

Fang X, Burg MA, Barritt D, Dahlin-Huppe K, Nishiyama A, Stallcup WB. Cytoskeletal reorganizationinduced by engagement of the NG2 proteoglycan leads to cell spreading and migration. Mol BiolCell 1999;10:3373–3387. [PubMed: 10512873]

Chekenya et al. Page 9

Oncogene. Author manuscript; available in PMC 2010 March 4.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Frisch SM, Ruoslahti E. Integrins and anoikis. Curr Opin Cell Biol 1997;9:701–706. [PubMed: 9330874]Fukushi J, Makagiansar IT, Stallcup WB. NG2 proteoglycan promotes endothelial cell motility and

angiogenesis via engagement of galectin-3 and alpha3beta1 integrin. Mol Biol Cell 2004;15:3580–3590. [PubMed: 15181153]

Goretzki L, Burg MA, Grako KA, Stallcup WB. High-affinity binding of basic fibroblast growth factorand platelet-derived growth factor-AA to the core protein of the NG2 proteoglycan. J Biol Chem1999;274:16831–16837. [PubMed: 10358027]

Goretzki L, Lombardo CR, Stallcup WB. Binding of the NG2 proteoglycan to kringle domains modulatesthe functional properties of angiostatin and plasmin(ogen). J Biol Chem 2000;275:28625–28633.[PubMed: 10889192]

Grako KA, Ochiya T, Barritt D, Nishiyama A, Stallcup WB. PDGF (alpha)-receptor is unresponsive toPDGF-AA in aortic smooth muscle cells from the NG2 knockout mouse. J Cell Sci 1999;111(Pt 6):905–915. [PubMed: 10036240]

Grako KA, Stallcup WB. Participation of the NG2 proteoglycan in rat aortic smooth muscle cell responsesto platelet-derived growth factor. Exp Cell Res 1995;221:231–240. [PubMed: 7589250]

Hemler ME, Sanchez-Madrid F, Flotte TJ, Krensky AM, Burakoff SJ, Bhan AK, Springer TA, StromingerJL. Glycoproteins of 210,000 and 130,000 m.w. on activated T cells: cell distribution and antigenicrelation to components on resting cells and T cell lines. J Immunol 1984;132:3011–3018. [PubMed:6327814]

Hilden JM, Smith FO, Frestedt JL, McGlennen R, Howells WB, Sorensen PH, Arthur DC, Woods WG,Buckley J, Bernstein ID, Kersey JH. MLL gene rearrangement, cytogenetic 11q23 abnormalities,and expression of the NG2 molecule in infant acute myeloid leukemia. Blood 1997;89:3801–3805.[PubMed: 9160687]

Joo NE, Watanabe T, Chen C, Chekenya M, Stallcup WB, Kapila YL. NG2, a novel proapoptotic receptor,opposes integrin alpha4 to mediate anoikis through PKCalpha-dependent suppression of FAKphosphorylation. Cell Death Differ. 2008 Feb 22; 2008.

Kumar CC. Signaling by integrin receptors. Oncogene 1998;17:1365–1373. [PubMed: 9779984]Lenter M, Uhlig H, Hamann A, Jeno P, Imhof B, Vestweber D. A monoclonal antibody against an

activation epitope on mouse integrin chain beta 1 blocks adhesion of lymphocytes to the endothelialintegrin alpha 6 beta 1. Proc Natl Acad Sci USA 1993;90:9051–9055. [PubMed: 7692444]

Li Y, Madigan MC, Lai K, Conway RM, Billson FA, Crouch R, Allen BJ. Human uveal melanomaexpresses NG2 immunoreactivity. Br J Ophthalmol 2003;87:629–632. [PubMed: 12714409]

Luque A, Gomez M, Puzon W, Takada Y, Sanchez-Madrid F, Cabanas C. Activated conformations ofvery late activation integrins detected by a group of antibodies (HUTS) specific for a novel regulatoryregion (355–425) of the common beta 1 chain. J Biol Chem 1996;271:11067–11075. [PubMed:8626649]

Makagiansar IT, Williams S, Dahlin-Huppe K, Fukushi JI, Mustelin T, Stallcup WB. Phosphorylationof NG2 proteoglycan by PKC-alpha regulates polarized membrane distribution and cell motility. JBiol Chem 2004;279:55262–55270. [PubMed: 15504744]

Makagiansar IT, Williams S, Mustelin T, Stallcup WB. Differential phosphorylation of NG2proteoglycan by ERK and PKCalpha helps balance cell proliferation and migration. J Cell Biol2007;178:155–165. [PubMed: 17591920]

Mauvieux L, Delabesse E, Bourquelot P, Radford-Weiss I, Bennaceur A, Flandrin G, Valensi F,MacIntyre EA. NG2 expression in MLL rearranged acute myeloid leukaemia is restricted tomonoblastic cases. Br J Haematol 1999;107:674–676. [PubMed: 10583275]

Miura M, Miura Y, Padilla-Nash HM, Molinolo AA, Fu B, Patel V, Seo BM, Sonoyama W, Zheng JJ,Baker CC, Chen W, Ried T, Shi S. Accumulated chromosomal instability in murine bone marrowmesenchymal stem cells leads to malignant transformation. Stem Cells 2006;24:1095–1103.[PubMed: 16282438]

Nishiyama A, Lin XH, Giese N, Heldin CH, Stallcup WB. Co-localization of NG2 proteoglycan andPDGF alpha-receptor on O2A progenitor cells in the developing rat brain. J Neurosci Res 1996a;43:299–314. [PubMed: 8714519]

Chekenya et al. Page 10

Oncogene. Author manuscript; available in PMC 2010 March 4.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Nishiyama A, Lin XH, Giese N, Heldin CH, Stallcup WB. Interaction between NG2 proteoglycan andPDGF alpha-receptor on O2A progenitor cells is required for optimal response to PDGF. J NeurosciRes 1996b;43:315–330. [PubMed: 8714520]

Pluschke G, Vanek M, Evans A, Dittmar T, Schmid P, Itin P, Filardo EJ, Reisfeld RA. Molecular cloningof a human melanoma-associated chondroitin sulfate proteoglycan. Proc Natl Acad Sci U S A1996;93:9710–9715. [PubMed: 8790396]

Sandal T, Stapnes C, Kleivdal H, Hedin L, Doskeland SO. A novel, extraneuronal role for cyclin-dependent protein kinase 5 (CDK5): modulation of cAMP-induced apoptosis in rat leukemia cells.J Biol Chem 2002;277:20783–20793. [PubMed: 11909854]

Schrappe M, Klier FG, Spiro RC, Waltz TA, Reisfeld RA, Gladson CL. Correlation of chondroitin sulfateproteoglycan expression on proliferating brain capillary endothelial cells with the malignantphenotype of astroglial cells. Cancer Res 1991;51:4986–4993. [PubMed: 1893386]

Shoshan Y, Nishiyama A, Chang A, Mork S, Barnett GH, Cowell JK, Trapp BD, Staugaitis SM.Expression of oligodendrocyte progenitor cell antigens by gliomas: implications for the histogenesisof brain tumors. Proc Natl Acad Sci U S A 1999;96:10361–10366. [PubMed: 10468613]

Smith FO, Rauch C, Williams DE, March CJ, Arthur D, Hilden J, Lampkin BC, Buckley JD, BuckleyCV, Woods WG, Dinndorf PA, Sorensen P, Kersey J, Hammond D, Bernstein ID. The humanhomologue of rat NG2, a chondroitin sulfate proteoglycan, is not expressed on the cell surface ofnormal hematopoietic cells but is expressed by acute myeloid leukemia blasts from poor-prognosispatients with abnormalities of chromosome band 11q23. Blood 1996;87:1123–1133. [PubMed:8562938]

Stallcup WB. The NG2 proteoglycan: past insights and future prospects. J Neurocytol 2002;31:423–435.[PubMed: 14501214]

Stallcup WB, Dahlin-Huppe K. Chondroitin sulfate and cytoplasmic domain-dependent membranetargeting of the NG2 proteoglycan promotes retraction fiber formation and cell polarization. J CellSci 2001;114:2315–2325. [PubMed: 11493670]

Tan C, Cruet-Hennequart S, Troussard A, Fazli L, Costello P, Sutton K, Wheeler J, Gleave M, SangheraJ, Dedhar S. Regulation of tumor angiogenesis by integrin-linked kinase (ILK). Cancer Cell2004;5:79–90. [PubMed: 14749128]

Tysnes OB, Aarbakke GM, Verhoeven AJ, Holmsen H. Thin-layer chromatography ofpolyphosphoinositides from platelet extracts: interference by an unknown phosphohpid. Thromb Res1985;40:329–338. [PubMed: 3001969]

Vivanco I, Sawyers CL. The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat RevCancer 2002;2:489–501. [PubMed: 12094235]

Wewer UM, Shaw LM, Albrechtsen R, Mercurio AM. The integrin alpha 6 beta 1 promotes the survivalof metastatic human breast carcinoma cells in mice. Am J Pathol 1997;151:1191–1198. [PubMed:9358743]

Yang J, Price MA, Neudauer CL, Wilson C, Ferrone S, Xia H, Iida J, Simpson MA, McCarthy JB.Melanoma chondroitin sulfate proteoglycan enhances FAK and ERK activation by distinctmechanisms. J Cell Biol 2004;165:881–891. [PubMed: 15210734]

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Figure 1. NG2/MPG expressing tumor cells are more resistant than NG2/MPG negative cells toTNFα and chemotherapy induced apoptosis(A) Hoechst stained nuclei from U251-Wt (top panels) and 251-NG2/MPG (bottom panels)cells exposed to 50ng/ml TNFα with or without 1µg/ml CHX for 3 or 6 hrs, magnification×200; scale bar= 60µm.(B) % apoptosis induced by TNFα with or without CHX at 3 hrs in U251-NG2/MPG cells orthe U251-Wt cells, assayed by nuclear fragmentation and DNA condensation as shown in A.The data represent the mean ± S.E.M. from three independent experiments.

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(C) % cell death in U251-NG2/MPG and U251-Wt glioma cells treated with 0.5 and 10 µMetoposide, or vincristine for 72 hrs and assayed by MTT. Data represent the mean ± S.E.M ofthree independent experiments.(D) Effect of TNFα. (50ng/ml).. treatment for 6hr on apotosis of U251-Wt and U251-NG2/MPG cells, as determined by flow cytometric DNA analysis after staining with propidiumiodide. In addition to illustrating the cell cycle distribution from G1 to G2-M, the DNA profilesquantify the appearance of apoptotic cells having less than G1 DNA content (Sub-Gl, blue).The histograms are representative of 3 independent experiments.

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Figure 2. siRNA-mediated suppression of NG2/MPG gene expression(A) Analysis of NG2/MPG expression in U251-Wt, U251-NG2, and U87MG cells by flowcytometry and immunoblotting with anti-NG2 antibodies. The arrowhead indicates the 300kDa NG2/MPG core protein.(B) NG2/MPG expression in U251-NG2 cells (green), (panel (i)), propidium iodide nuclearcounterstain, magnification ×630, scale bar=10µm. NG2/MPG expression (panel (ii), red) andknockdown with siRNA2 in U251-NG2 cells (panel (iii), red), Dapi nuclear counterstain(panels (iv) and (v), magnification ×200, scale bar=60µm.(C) NG2/MPG expression (green) in U87MG cells, (panel (i)), Dapi nuclear counterstain,magnification ×630, scale bar=10µm. NG2/MPG knockdown (panel (ii), red) with siRNA3 in

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U87MG cells (panel (iii), red), Dapi nuclear counterstain (panels (iv) and (v), magnification×200, scale bar=60µm.(D) Dose dependent knockdown of NG2/MPG expression in U251-NG2/MPG cells bysiRNA2 after 48 hrs. Protein expression was determined by immunoblot analysis. (*)proteolytic products. The membranes were stripped and re-probed with β-actin antibodies(E) Northern blotting of NG2 mRNA transcripts after 48 hr treatment with siRNA2 or mutantsiRNA.(F) Dose dependent knockdown of NG2/MPG expression in U87 cells by siRNA3 after 48 hrs.Protein expression was determined by immunoblot analysis.

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Figure 3. SiRNA mediated suppression of NG2/MPG expression abrogates resistance to TNFα(A) %. apoptosis in U251-Wt cells or U251-NG2/MPG cells transfected with 25nM siRNA orcontrol siRNA for 48 hrs. Cells were treated with or without (+/−) TNFα after 20 min pre-incubation with wortmannin (100ng/ml), and assayed by nuclear fragmentation and DNAcondensation The data represent the mean ± S.E.M. from three independent experiments.(B) %. apoptosis in U87MG and A172 cells transfected with 25nM siRNA or control siRNAfor 48 hrs. Cells were either untreated or treated with 50ng/ml of TNFα for 6 hrs, and assayedby nuclear fragmentation and DNA condensation. The data represent the mean ± S.E.M. fromthree independent experiments.

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(C) %. Cell death in U87MG cells transfected with control siRNA or NG2/MPG siRNA andtreated with 50µM of temodal, carboplatin and etoposide for 72 hrs. Cells were assayed byMTT. The data represent the mean ± S.E.M. from three independent experiments.(D) % Apoptosis in A375 melanoma cells transfected with NG2/MPG shRNA or controlshRNA. Cells were treated with 50ng/ml of TNFα for 6 hrs or with Doxorubicin (3µM), andCisplatin (5µM) for 72 hrs assayed by nuclear fragmentation and DNA condensation. The datarepresent the mean ± S.E.M. from three independent experiments.(E) Effect of Etoposide (0.5 µM) on cell cycle distribution of control U87wt and U87 cellstransduced with control shRNA or NG2/MPG shRNA as determined by flow cytometricanalysis conducted after staining with propidium iodide. The profiles indicate cell cycledistribution from G1 to G2-M, as well as the appearance of apoptotic cells in the sub-G1fraction, (blue). Histograms are representative of two independent experiments.

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Figure 4. Apoptosis resistance in NG2/MPG cells is mediated by β1 integrin signaling(A) Activated β1 integrin (HUTS-21, green, top panels) or total β1 integrin TS2/16, green,bottom panels) in U251-Wt (left) and U251-NG2/MPG (right), Magnification ×400, scale bar60µm.(B) Activated β1 (HUTS-21, green, top left); total β1 (TS2/16, green, bottom left) subunits inU251-Wt and U251-NG2/MPG cells determined by flow cytometry. % values reflect the totalfluorescence intensity relative to an arbitrary window setting. Controls profiles were obtainedby using secondary antibody alone.(C) Left panel: % Apoptosis in U251-Wt cells pre-incubated with β1 activating antibodyTS2/16 (10µg/ml) for 30 min or with CD29 blocking antibody Ha2/5 for 2 hrs prior to 6 hrtreatment with 50ng/ml TNFα. Right panel: % apoptosis in U251-NG2/MPG cells pre-incubated with β1 function blocking antibodies (CD29 Ha2/5 or AIIB2) for 2 hrs prior to

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treatment with 50ng/ml TNFα Apoptosis was assayed by nuclear fragmentation and DNAcondensation. Each bar represents the mean ± S.E.M. of three independent experiments.(D) Activated β1 integrin levels in U87Wt, U87 ctrl siRNA and U87 NG2/MPG siRNA cellsdetermined by HUTS-21 flow cytometry. % values reflect the total fluorescence intensityrelative to an arbitrary window setting. Controls profiles were obtained by using secondaryantibody alone.(E) α3 integrin subunits in U251-Wt and U251-NG2/MPG cells determined by flow cytometry.% values reflect the total fluorescence intensity relative to an arbitrary window setting. Controlsprofiles were obtained by using secondary antibody alone.(F) Detergent extracts of U251-NG2/MPG cells or (G) U251Wt cells or (H) U87 cellsimmunoprecipitated with two different anti-NG2/MPG antibodies (ectodomain and D2) andblotted with anti-NG2/MPG antibody (top row of bands) and anti-α3 antibody (bottom row ofbands). Crude extracts (far left) and α3 immunoprecipitates (far right) were loaded as positivecontrols. Control rabbit and mouse immunoglobulin did not immunoprecipitate either protein.Arrowheads denote positions of 200 and 116 kDa molecular weight markers, respectively.

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Figure 5. NG2/MPG mediates resistance to TNFα via PI3K/Akt signalling in both glioma andmelanoma cells(A) Synthesis of 32-P radiolabelled PtdIns (3,4,5) P3 in U251-Wt and U251-NG2/MPG cellstreated with 50ng/ml TNFα for 15 min. Bars represent mean + S.E.M from three independentexperiments.(B) Immunoblot analysis of Akt and phospho- S473 Akt in U87 wt cells or in cells transfectedwith siRNA and mutant-siRNA. Cells were untreated or treated with 50ng/ml TNFα (+/−).(C) Immunoblot detection of Akt and phospho- S473 Akt proteins in untreated A172 cells andA172 cells transfected with control siRNA or NG2/MPG siRNA. β-actin provides an equalloading control. Densitometric quantification of the total Akt (open bars) and P-Akt bands(solid bars).(D) Immunoblot analysis of Akt and phospho- S473 Akt in A375 wt cells and A375 cellstransfected with control siRNA or NG2/MPG siRNA. Cells were treated with 50ng/ml TNFα(+/−) for 15 min Densitometry was used to quantify the decrease in Akt phosphorylationfollowing NG2/MPG knockdown.

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Figure 6. NG2/MPG promotes glioblastoma growth in vivo and confers resistance to TNFαtreatment(A) Expression of the lentiviral shRNA indicated by eGFP fluorescence. Untreated U87glioblastoma cells (left panel,) or transfected with shRNA (right panel), magnification 400×,scale bar 80µm.(B) Flow cytometric analysis reveals eGFP expression in 65% of U87 cells following a singleshRNA lentivirus infection (after 96 hrs).(C) Top: Western blot of NG2/MPG in U87, U87 LVcontrolshRNA and U87LVNG2/MPGcells. β-actin provides a loading control. Bottom: Quantification of signal intensity in arepresentative blot.(D) U87 tumor sizes in the 4 different mouse groups at the end of the experiment.(E) Growth curves showing the effect of NG2/MPG knockdown, and the effect of TNFαtreatment on U87LVcontrol shRNA and U87LVNG2/MPG shRNA tumors. Arrow indicatesend of TNFα treatment at day 7.(F) Western blotting of tumor lysates from U87 LVcontrol shRNA and U87LVNG2/MPGshRNA (treated or untreated with TNFα). Upper panels: NG2/MPG levels in the 4 sets oftumors. Arrowhead denotes NG2/MPG core protein. * indicates proteolytic fragments. Middlepanels: Immunoblot analysis of total Akt and phosphorylated Akt in lysates from the same 4sets of tumors treated with TNFα or vehicle in vivo. Bottom panels: β-actin equal loadingcontrols.

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Figure 7. Chemosensitivity of human GBMs expressing different levels of NG2/MPG(A) Anti-NG2/MPG IHC staining of human GBM tumors. High expressors are shown in theright-hand panels and low expressors are shown on the left. Magnification 400×, scale bar100µm.(B) Chemosensitivity of GBM biopsy spheroids with high and low NG2/MPG expression.Cells were assayed by the MTT method after 96 hr treatment with Doxorubicin, or 120 hrtreatment with Etoposide and Carboplatin. For the histograms, data were pooled from the highand low expressors shown in part A. Data represent the mean ± S.E.M. Results for individualtumors are shown in Table I.

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Table ING2/MPG expression correlates with chemoresistance in GBM biopsies

Tumor numbers correspond to those presented in Fig 7. NG2/MPG levels were determined by real time- qPCR,except in cases marked N.D.(not determined) in which NG2/MPG levels were assessed semi-quantitatively byIHC. High NG2/MPG expression was defined as being greater than that seen in normal brain (i.e greater than1.0 fold change). Low expressors (top of the list) are separated from high expressors (bottom of the list) by aspace. % Viable cells was determined via MTS assays, as absorbance of non-treated cells - absorbance of treatedcells)/absorbance of non-treated cells. Values greater than 100% therefore represent samples, which had increasedmetabolic activity in the presence of the drug. These results were pooled to yield the histograms in Fig 7B.

Tumor number Fold change NG2mRNA, relative to

normal brain

% viable cells after96h Doxorubicin

% viable cellsafter 120hEtoposide

% viable cells after120h Carboplatin

NG2 negative

1 −3.5 56.3

2 −8 56.8

3 −1.1 87.5 47.3

4 −1.1 61.2

5 *N.D 35.4

6 *N.D 83

13 −1.1 71.40 64.20

NG2 positive

7 *1N.D 156

8 1.3 137.4

9 2 159.1

10 4.6 127.4

11 4.3 177 116

12 9.2 89.2 111.3

14 26.6 157.7 163.7

15 43.2 123.1 115.1

*negative on IHC; N.D = not determined by qPCR

*1positive on IHC

∫The tumors are arranged in the Table in ascending order of NG2/MPG expression.

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