Autocrine regulation of glioblastoma cell-cycle progression, viability ...

[Cell Cycle 7:16, 2553-2561; 15 August 2008]; ©2008 Landes Bioscience

Report

Autocrine regulation of glioblastoma cell cycle progression, viability and radioresistance through the VEGF-VEGFR2 (KDR) interplay Petra Knizetova,1-3,* Jiri Ehrmann,1 Alice Hlobilkova,1 Iveta Vancova,4 Ondrej Kalita,5 Zdenek Kolar1 and Jiri Bartek2,3 1Laboratory

of Molecular Pathology; Institute of Pathology; Faculty of Medicine; Palacky University; Olomouc, Czech Republic; 2Institute of Cancer Biology and Centre for Genotoxic Stress Research; Danish Cancer Society; Copenhagen, Denmark; 3Laboratory of Genome Integrity; Palacky University; Olomouc, Czech Republic; 4Institute of Virology; Slovak Academy of Sciences; Bratislava, Slovak Republic; 5Department of Neurosurgery; University Hospital; Palacky University; Olomouc, Czech Republic

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Abbreviations: VEGF, vascular endothelial growth factor; GBM, glioblastoma multiforme; VEGFR, vascular endothelial growth factor receptor; KDR, kinase-insert domain receptor; Flt-1, fms-related tyrosine kinase-1; LGA, low grade astrocytoma; HGA, high grade astrocytoma; IR, ionizing radiation; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; PLC, phospholipase C; PKC, protein kinase C; PI, propidium iodide; ELISA, enzyme linked immunosorbent assay

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Introduction

Interactions between tumor cells and their microenvironment are critically important in the biology of cancer, and include growth factor signaling in paracrine and autocrine manners.1-5 Beside its pro-angiogenic function, VEGF also increases vascular permeability and VEGF overexpression occurs in a variety of tumors including highly vascularized and infiltrative astrocytomas (grade III/IV), where VEGF expression correlates with poor prognosis.6,7 The bulk of current knowledge about the biology of VEGF and VEGFRs derives from studies of endothelial cells and VEGF’s paracrine effects when secreted by diverse tumor cell types. Emerging evidence, however, suggests co-expression of VEGF and VEGFRs in some types of tumors and highlights the importance of understanding the potential autocrine loop-signaling of VEGF within a tumor mass. Moreover, it has been observed that serum VEGF levels are elevated in subsets of patients with malignant tumors after ionizing radiation (IR) therapy.8 These findings suggest that the VEGF-mediated paracrine signaling between brain tumor mass and its vasculature, and possibly autocrine effects of VEGF on tumor growth and cell viability, may represent a plausible target for a novel strategy to sensitize malignant astrocytomas to radiation treatment. Despite the well established role of angiogenesis and tumor spread in malignant astrocytomas, however, very little is known about the autocrine effect(s) of VEGF in human astrocytoma biology and clinical behavior. To address this issue, the present study was designed to assess the potential autocrine role of VEGF signaling in astrocytomas, using a range of molecular and cell biology approaches to examine a panel of human astrocytoma (mainly GBM) cell lines, complemented by immunohistochemical analyses of VEGF and its two receptors, VEGFR1 (Flt-1) and VEGFR2 (KDR), in a series of clinical specimens from low- and high-grade human astrocytomas. As described below, our results not only support such plausible autocrine regulation of astrocytomas by VEGF, but also provide novel insights into the critical VEGF receptor type and the downstream intracellular signaling pathways involved. Finally, our study shows an

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Vascular endothelial growth factor (VEGF) plays a crucial role in angiogenesis and progression of malignant brain tumors. Given the significance of tumor microenvironment in general, and the established role of paracrine VEGF signaling in glioblastoma (GBM) biology in particular, we explored the potential autocrine control of human astrocytoma behavior by VEGF. Using a range of cell and molecular biology approaches to study a panel of astrocytoma (grade III and IV/GBM)-derived cell lines and a series of clinical specimens from low- and high-grade astrocytomas, we show that co-expression of VEGF and VEGF receptors (VEGFRs) occurs commonly in astrocytoma cells. We found VEGF secretion and VEGF-induced biological effects (modulation of cell cycle progression and enhanced viability of glioblastoma cells) to function in an autocrine manner. Morevover, we demonstrated that the autocrine VEGF signaling is mediated via VEGFR2 (KDR), and involves co-activation of the c-Raf/MAPK, PI3K/Akt and PLC/PKC pathways. Blockade of VEGFR2 by the selective inhibitor (SU1498) abrogated the VEGF-mediated enhancement of astrocytoma cell growth and viability under unperturbed culture conditions. In addition, such interference with VEGF-VEGFR2 signaling potentiated the ionizing radiation-induced tumor cell death. In clinical specimens, both VEGFRs and VEGF were co-expressed in astroglial tumor cells, and higher VEGF expression correlated with tumor progression, thereby supporting the relevance of functional VEGFVEGFR signaling in vivo. Overall, our results are consistent with a potential autocrine role of the VEGF-VEGFR2 (KDR) interplay as a factor contributing to malignant astrocytoma growth and radioresistance, thereby supporting the candidacy of this signaling cascade as a therapeutic target, possibly in combination with radiotherapy.

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Key words: astrocytoma, glioblastoma, vascular endothelial growth factor, cell cycle, autocrine signaling, ionizing radiation, cell survival

*Correspondence to: Petra Knizetova; Institute of Cancer Biology and Centre for Genotoxic Stress Research; Danish Cancer Society; Strandboulevarden 49; Copenhagen DK-2100 Denmark; Tel: +45.35257788; Fax: +45.35257721; Email: [email protected] Submitted: 06/11/08; Accepted: 06/12/08 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/6442 www.landesbioscience.com

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VEGF in glioblastoma biology

anti-tumor activity of a small molecule capable of inhibiting the VEGF signaling cascade in astrocytoma cell lines, and combined experiments with IR support a scientific rationale for effective use of a targeted anti-VEGF(R) therapy in combination with a DNA-damaging treatment such as IR.

Results

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Co-expression of VEGF and its two receptors in human astrocytoma cell lines. To explore possible signal transduction pathways for VEGF, expression of VEGF and VEGFRs: Flt-1 (VEGFR1) and KDR (VEGFR2) (Fig. 1A) was analyzed using RT-PCR in a panel of eight human astrocytoma/GBM cell lines. VEGF, Flt-1 and KDR mRNAs were expressed in all the cell lines tested (Fig. 1B). Endothelial cells (HUVEC) known to express both Flt-1 and KDR and SY-5Y neuroblastoma cells known to lack expression of VEGFRs served as positive and negative controls, respectively.9,10 The RT-PCR analysis of HUVEC cells yielded products of the expected size. The VEGF primers were designed within exon 2 and the 3' untranslated region of the VEGF cDNA, thereby allowing amplification of all known VEGF splice variants in a single reaction.11 We were able to amplify cDNAs for three VEGF isoforms: VEGF121, VEGF165 and VEGF189, which are known to be expressed in astrocy- Figure 1. RT-PCR analysis of VEGF, Flt-1 and KDR mRNA expression in astrocytoma cell lines. (A) toma cell lines, with the VEGF121 isoform A schematic model of astroglial tumor cell membrane receptors of the VEGF tyrosine kinase receptor family: Flt-1 (VEGFR1) and KDR (VEGFR2), each of which consists of seven Ig-like domains, a being expressed at the highest level (Fig. 1C). transmembrane region and an intracellular tyrosine kinase binding domain. VEGF may stimulate VEGF signaling in astrocytoma cells is receptor dimerization and auto-phophorylation in an autocrine-loop mode. The VEGF homologue mediated via VEGFR2 (KDR). To gain a PlGF-1 is regarded as a selective activator of Flt-1. The small molecule inhibitor SU1498 blocks the better insight into the functional significance of kinase activity of KDR and thereby may prevent the biological impact of the putative autocrine VEGF VEGF-VEGFR co-expression, we first sought signaling. (B) As indicated by names, eight human astrocytoma cell lines were analyzed by RT-PCR to assess the cellular effects of VEGF signaling to assess expression of VEGF and the Flt-1 and KDR receptors. HUVEC cells and the neuroblastoma cell line SY-5Y served as positive (VEGF and VEGFRs expression) and negative (VEGFRs expression) by Western blot analysis of the phosphorylation controls, respectively. GAPDH is a housekeeping gene used here as a positive control for the mRNA status of key components of the three major quality and RT-PCR reactions. (C) More detailed image of a gel with the PCR fragments representing signaling pathways implicated in cell growth, the three different VEGF isoforms expressed in A172 cells: VEGF189, VEGF165 and VEGF121, with proliferation, migration and survival: the PI3-K/ VEGF121 as the most abundant form. Akt, c-Raf/MAPK and PLCγ1/PKC cascades, respectively. VEGF121 (20 ng/ml) added to cultured human A172 respectively. While VEGF121 is capable of stimulating both VEGF glioblastoma cells stimulated phosphorylation of c-Raf (Ser338), receptors, PlGF-1 is a known selective ligand for VEGFR1 only, MAPK (Thr202/Tyr204), Akt (Thr308) and PLCγ1 (Tyr783) in a and SU1498 represents a highly specific VEGFR2 tyrosine kinase time-dependent manner (Fig. 2A), reaching the maximum at 30–60 domain inhibitor.12,13 To judge the effects of these different treatminutes of treatment. In addition, these results were not restricted to ments on intracellular VEGF signaling in glioblastoma cells, selected A172 cells, as similar data were obtained when the same treatment markers including phospho-Akt (Ser473) and phospho-p44/42with VEGF121 was applied to another astroglial tumor cell line, MAPK (Thr202/Tyr204) were monitored by Western blotting. As U251MG (data not shown). The observed responses were transient, shown in Figure 2B, VEGF121 but not PlGF-1 was able to induce and by 12 hours of VEGF121 stimulation, the degree of phosphory- phosphorylation of Akt/PKB and p44/42 MAPK when either ligand lations in the majority of the monitored proteins returned to basal was applied at an identical concentration (20 ng/ml). Interestingly, levels (Fig. 2A). pre-treatment of the glioblastoma cells with the VEGFR2 tyrosine Next, we analyzed the ability of two ligands of VEGFRs’: PlGF-1 kinase inhibitor SU1498 (30 μM, applied one hour before VEGF and VEGF121, and the small molecule inhibitor of VEGFR2/ treatment) abrogated the otherwise pronounced stimulatory effect KDR: SU1498, to stimulate or inhibit functional VEGF signaling, of VEGF121 on Akt (Ser473) and p44/42-MAPK (Thr202/Tyr204) 2554

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to cell membrane throughout the washing period and then being released over time). Furthermore, this experiment also indicates that the autocrine loop signaling of VEGF is mediated via the VEGFR2 (KDR) receptor. Our results show that astrocytoma cells are capable of responding to VEGF stimulation by enhanced expression and secretion of VEGF into their extracellular environment, a notion consistent with the potential autocrine role of VEGF. VEGF stimulates cell growth of glioblastoma cell lines. To examine the biological impact of VEGF stimulation on human GBM cell models, we treated the cell lines A172 and U251MG with increasing concentrations of VEGF (10, 20, 40 and 80 ng/ ml) and measured MTT absorbance in time-course experiments, as an index of cell growth/proliferation, as employed in previous studies of VEGF-induced astrocytoma cell proliferation.15-18 As early as 6–12 hours after addition of VEGF121 to culture medium, MTT absorbance was significantly increased (by 19–35% for A172 and 14–79% for U251MG, p < 0.001 for any of the four VEGF concentrations used) compared with the mock-treated control cells. Despite some modestly higher increases of MTT absorbance at the higher versus lower concentrations of added VEGF, such concentration-dependence was not statistically significant. Unlike exposure to VEGF, treatment of the same GBM cell lines with the VEGFR1-selective ligand PlGF-1 did not induce any detectable increase of the MTT absorbance (negative data, not shown). Importantly, selective blockade of the VEGFR2 (KDR) receptor by SU1498 pre-treatment prevented the VEGF-dependent increase of cell growth as judged from the decreased MTT absorbance at 6, 12, 24 and 48 hours after VEGF addition (p < 0.001 at all time-points, data not shown). To examine whether the above effects of VEGF on cell growth/ viability were accompanied by increased entry into S phase and DNA synthesis, incorporation of EdU (equivalent of BrdU, after 30-minute pulse-labeling with EdU at 6, 12 and 24 hours after VEGF addition) in the two serum-starved GBM cell lines was assessed. As shown

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phosphorylation levels (Fig. 2B). Collectively, these results strongly indicate that the key receptor through which the signaling pathways are triggered by VEGF in our GBM cell model is KDR (VEGFR2). Exogenous VEGF121 stimulates endogenous VEGF mRNA levels and VEGF secretion in cultured glioblastoma cells. If an autocrine loop of VEGF signaling should indeed operate in astrocytomas, another strong prediction would be that VEGF itself should be secreted by the tumor cells, and possibly regulate also the expression of endogenous VEGF production. The latter effect has been reported for endothelial cells,14 but there is no information on VEGF-mediated regulation of VEGF expression in astrocytomas. To test these conceptually important issues, we first set to determine whether recombinant VEGF121 is able to induce alterations of endogenous VEGF expression levels in the GBM cell line A172, as assessed by semi-quantitative RT-PCR. Compared to a mock-treated control sample, VEGF mRNA levels increased by 6–12 hours of treatment in the VEGF-exposed cells (Fig. 3A), and this effect persisted for the next 24–48 hours. To investigate whether the observed increase in VEGF mRNA is accompanied by enhanced VEGF protein secretion, we used ELISA to examine the effect of exogenous VEGF121 (40 ng/ml) on VEGF secretion by the A172 cells (Fig. 3B). Under serum-free conditions, when cells were washed 2 hours after VEGF stimulation, a significant secretion of VEGF, increasing over the time, was detected. Importantly, this effect was durable, as documented by elevated VEGF secretion for up to 48 hours after the 2-hour pulse-stimulation by VEGF121. In addition, when the signaling of the added exogenous VEGF121 into the cells was blocked by addition of the VEGFR2 kinase inhibitor SU1498 (30 μM, for 1 hour), the VEGFdependent increase of the secreted endogenous VEGF was inhibited (Fig. 3C). This critical result documents that the extracellular VEGF, which we detected at increasing amounts with time after washing out the initially added exogenous VEGF, was indeed secreted (rather than representing a fraction of the exogenous VEGF still adherent

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Figure 2. Western blot analysis of multiple pathway markers to examine VEGF-KDR-mediated intracellular signaling in the glioblastoma cell line A172. (A) Cells were serum-starved for 24 hours and then stimulated with 20 ng/ml of recombinant VEGF121 as indicated. Activation of multiple signaling pathways was observed, including PI3K/Akt, c-Raf/MAPK and PLCγ1/PKC, as apparent from the appearance of the activatory phosphorylations monitored by phosphospecific antibodies. Mcm7 served as a loading control. (B) Stimulation of A172 cells with PlGF-1 (PI), a VEGFR1-selective ligand (20 ng/ml, for 30 minutes) did not lead to activation (phosphorylation) of p44/42 MAPK Thr202/Tyr204 and Akt Ser473, which were used as markers of intracellular VEGF signaling. Pre-treatment of A172 cells with SU1498 (SU), a VEGFR2-selective kinase inhibitor (30 μM, for 1 hour before VEGF121 stimulation) abrogated the stimulatory effect of VEGF121 (VE; 20 ng/ml, 30 min.) on p44/42 MAPK activation. Mcm7 served as a loading control. Con: mock-treated control.

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in Figure 4A and B, VEGF stimulation led to a significant increase in the proportion of EdU-positive cells in both the A172 and U251MG cell lines, with maximum at 6–12 hours of treatment. Interestingly, pre-treatment of either cell line with the SU1498 inhibitor of VEGFR2/KDR abrogated the VEGFinduced effect, and by 24 hours of incubation the number of proliferating cells (cells incorporating EdU) dropped below even the control levels seen in the VEGF-unstimulated cells (Fig. 4A and B). These results document a positive impact of VEGF on cell growth/metabolic activity and cell cycle progression (S-phase entry) in two astrocytoma models, and suggest that these biological effects are critically dependent on intact signaling through the VEGFR2 (KDR) receptor. VEGF as a radiation-protective factor in glioblastoma cells. IR represents a key therapeutic modality for malignant astrocytomas,19 VEGF is elevated in astroglial tumor cell lines after IR,15,18 and it has recently been proposed that VEGF might protect tumor blood vessels from IR-induced cytotoxicity and thereby possibly contribute to tumor radioresistance in a paracrine manner.20 These facts, and our present data (see above) prompted us to explore a possible role of the autocrine VEGF signaling in the response of malignant astrocytoma models to IR. To this end, GBM cell lines A172 and U251MG were serum starved for 24 hours, treated with SU1498 (30 μM) or left untreated, and 2 hours later the cells were either mock-treated or irradiated with 10 Gy of IR. During the next 24 hours, cells were grown under low-serum [1% (v/v) FBS] conditions and apoptosis was examined 24 hours post-irradiation. The key results from these experiments were the following: (i) Treatment with SU1498 alone undermined the viability of the astrocytoma model cell lines, leading to Figure 3. Levels of endogenous VEGF mRNA and secreted protein in A172 cells enhanced apoptosis in both A172 and U251MG cells (Fig. are increased upon stimulation with recombinant VEGF121. (A) Semi-quantitative 5A and B); (ii) Radiation alone induced pronounced apoptosis RT-PCR analysis of VEGF mRNA expression upon stimulation with exogenous recombinant VEGF (40 ng/ml), determined at 0 hours (Control), 6, 12, 24 of A172 cells, while the U251MG cell line proved resistant and 48 hours after121 stimulation. GAPDH was used as a positive (loading) control to IR (Fig. 5A and B); (iii) Most importatntly, the combined for mRNA expression. (B) VEGF secretion by A172 cells induced by exogenous treatment with SU1498 and IR clearly further potentiated recombinant VEGF121 stimulation for 6, 12, 24 and 48 hours, based on ELISA data cell death, leading to increased apoptosis above the additive from 3 independent experiments. (C) Induction of autocrine VEGF secretion was inhibited using the pharmacological inhibitor of KDR kinase activity: SU1498 (30 values in both cell lines, including the otherwise IR-resistant μM), supporting the notion that autocrine VEGF signaling is mediated by VEGFR2 U251MG cells (Fig. 5A and B). (KDR). Supernatants were analysed by VEGF Elisa (Human VEGF Quantikine Kit, Overall, these results suggest that VEGF can play a ‘neuro- R&D Systems). Results are means ± s.d. protective’ role in astrocytoma cells, at least in vitro, and that inhibition of the VEGF-VEGFR2/KDR signaling cascade sensitizes human GBM cell lines to cytotoxic effects of ionizing In contrast to control human tissues that scored negative (data radiation treatment. not shown), immunoreactivity for VEGF was found in all astroVEGF expression is related to astrocytoma progression in glial tumors irrespective of tumor grade, with variable (commonly clinical specimens. Since our data obtained with the cell culture strong) staining intensity and heterogeneous pattern of positivity models were consistent with a potential autocrine role of VEGF- (Fig. 6). Overall, statistically significant higher VEGF expression was VEGFR2 signaling in regulation of cell cycle and viability of human found among the series of 35 high-grade astrocytomas (HGA) when astrocytomas, we set to search for a biological correlate of such a compared to the 28 specimens from low-grade astrocytomas (LGA, role directly in clinical specimens from human astroglial tumors of p ≤ 0.026; cut off value of the semi-quantitative histoscore values: diverse malignant potential (grade of progression). Two questions 90). On the other hand, despite a modest trend towards higher were addressed by immunohistochemical analysis of VEGF, and the expression of the VEGF receptors in HGA, the observed differences two VEGF receptors on tissue sections: (i) Whether or not VEGF between lesions of different grades were not statistically significant and VEGFRs are coexpressed in such tumors, as would be predicted (VEGFR1/Flt-1: histoscore values 47.1 and 43.4 in HGA and LGA, for an autocrine loop of VEGF signaling; and (ii) Whether there is respectively; and for VEGFR2/KDR: histoscore values 84.2 and 74.3 any correlation between the levels of VEGF and/or VEGFRs and in HGA and LGA, respectively). In summary, these immunohisclinical progression of the tumors. tochemical results confirmed co-expression of VEGF and its cognate 2556

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several biological effects of VEGF, including radioprotection. The predictions for autocrine signaling of VEGF, that were tested and indeed experimentally validated by our work include the following: (i) VEGF is expressed by astroglial tumor cells in a panel of human cell lines as well as in clinical specimens, as documented at the mRNA and/or protein levels; (ii) The receptors for VEGF are commonly co-expressed by human astroglial tumor cells in both the in vitro and clinical (in vivo) scenarios; (iii) Expression of endogenous VEGF mRNA is enhanced, and secretion of endogenous VEGF into extracellular environment is induced, upon VEGF-mediated stimulation of cultured human GBM cells; (iv) Exposure to physiological concentrations of VEGF elicits a host of biological responses in GBM cells, including effects on cell growth, cell cycle progression and enhanced viability; (v) Increasing abundance of VEGF correlates with progression of human astrocytomas from low- to high-grade tumors. Apart from the evidence for autocrine signaling, our data also provide some insights into the preferential VEGF receptor type and the intracellular pathways involved in the response of astroglial tumor cells to VEGF. Whereas both VEGFR1 (Flt-1) and VEGFR2 (KDR) are expressed in the astroglial tumor cell lines and tumors in vivo, the lack of the VEGFR1-selective ligand PlGF-1 to induce a detectable response, and especially the ability of the selective VEGFR2 inhibitor SU1498 to counteract the VEGF biological effects, indicate a dominant role of VEGFR2 (KDR) in the mechanism of VEGF signaling in our GBM models. Our data do not exclude a possible role of VEGFR1 in subsets of astrocytomas, however the observed prefFigure 4. VEGF121 stimulates cell cycle progression in astrocytoma cell lines in vitro. erential signaling via VEGFR2 suggests that VEGFR2 Two cell lines, A172 (A) and U251MG (B), partially synchronized in G0/G1 phase by (KDR) could represent a promising target for inhibition serum starvation were stimulated by addition of VEGF121 and subjected to flow cytometry analysis at the indicated times, to assess the rate of EdU incorporation (and hence S-phase of astroglial tumor growth. entry/DNA replication) upon 30 min pulse-labeling with EdU. Results are means ± s.d. Extending the current knowledge about the known multifaceted signaling network triggered by VEGF in receptors on astrocytoma tumor cells directly in clinical material, other cell types, particularly in endothelial cells,25-28 our present and showed a positive correlation between VEGF abundance and analysis revealed the involvement of at least three pathways: the c-Raf/MAPK, PI3-K/Akt and PLCγ/PKC cascades, in VEGFastroglial tumor progression in vivo. stimulated astroglial tumor cells. The activation of these pathways Discussion was monitored by induced phosphorylation events on key proteins Malignant astrocytomas and especially glioblastoma multiforme within these signaling cascades: c-Raf/MAPK, PI3-K/Akt and PLCγ, (GBM) are aggressive tumors characterized by rich angiogenesis, the respectively. The kinetics of these phosphorylations was consistent critical role of which is documented by the ability of anti-angiogenic with a prompt multi-pathway response to VEGF in the model A172 inhibitors to decrease tumor growth in vivo.21 In GBM, VEGF- cell line. One exception we observed was the constitutively rather dependent signaling plays a critical role in driving the growth and high level of phosphorylation at Akt Ser473 in our experiments, likely relapse of the disease.22 Previous studies have also suggested that attributable to a loss-of-function mutation of the tumor suppressor overexpression of VEGF in malignant astrocytomas is associated with gene PTEN in the A172 cells.29 PTEN is a strong negative regulator increased vascular density and poor clinical outcome.23,24 However, of the PI3K/Akt pathway, and hence the absence of this regulatory discrepant findings on biological function(s) of VEGF and its contri- effect could result in high basal phospho-Akt Ser473 levels in this cell bution to astrocytoma progression have been reported,15-18 raising line.29 Activated Akt phosphorylates numerous downstream effectors the need to clarify the nature and significance of potential VEGF- to modify important cellular processes and, at least in endothelial cells, the PI3K/Akt axis promotes cell viability, thereby controlling mediated autocrine signaling in astroglial tumors. In the present study, we provide evidence for a VEGF-mediated the balance between survival and cell death.30 Our biochemical and autocrine regulatory loop in human GBM cells, and document cell biology data indicate that in human astrocytoma cells, VEGF www.landesbioscience.com

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triggers multiple interacting pathways and, as discussed in the next section below, such signaling translates into cell fate decisions about cell cycle progression and cell survival. From a biological perspective, our experiments showed VEGFinduced effects in terms of promoted cell growth and cell cycle progression of the two model glioblastoma cell lines, as monitored by the enhanced metabolic activity (MTT test) and accelerated S-phase entry (EdU incorporation during DNA replication), respectively. While these effects were significant, they were relatively modest and transient. In contrast, the arguably most noticeable biological effect was the modulation of cell viability, both in unperturbed growth conditions and even more so after exposure to ionizing radiation. Our experiments with blocking the VEGF-VEGFR2 (KDR) signaling via a small molecule chemical inhibitor SU1498 indicate that the two GBM cell lines tested, A172 and U251MG, both require ongoing VEGF-mediated signaling for optimal viability. This is particularly noteworthy in the case of the p53-mutant U251MG cells that proved to be radioresistant, while responding by enhanced apoptosis when their VEGFR2 (KDR)-mediated signaling was blocked under standard growth conditions. These results with the GBM cell lines were cell-type specific, since analogous exposure of normal human fibroblasts to SU1498 did not cause any cytotoxicity (our unpublished data), further indicating that interference with VEGF signaling could be a promising strategy for treatment of astrocytomas including GBM. Ionizing radiation is a mainstream treatment modality for malignant astrocytomas, and the inherent or progressively acquired resistance to such DNA-damaging treatment is a major cause of overall treatment failure and dismal prognosis of these brain malignancies. In this regard, we believe that our present data on the cooperative effects of IR and concomitant blocking of VEGFR2 signaling is potentially very relevant to astrocytoma behavior and response to therapy. Importantly, the concomitant treatment with IR and SU1498 resulted in a synergistic, rather than just additive or overlapping effect, indicating that VEGF signaling provides some kind of radioprotective function to astrocytoma cells, and this can be overcome by blocking the VEGFR2 signaling axis (Fig. 7).

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Figure 5. Blockage of VEGFR2 (KDR) activity potentiates pro-apoptotic effects of ionizing radiation in glioblastoma cells. GBM cells A172 (A) and U251MG (B) were plated at 70% density, serum-starved for 24 hours and treated as indicated. The percentage of cells undergoing apoptosis was estimated by Anexin V staining as described in Materials and Methods. Representative flow cytometry histograms (the peak of apoptotic cells marked by arrow) and corresponding graphs show the extent of apoptosis for cells that were: non-irradiated but treated with SU1498 (30 μM), only irradiated (10 Gy), or SU1498-treated and irradiated (30 μM, 10 Gy). The experiment was performed 2 times in duplicate (100.000 cells per sample). Results are means ± s.d.

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Figure 6. Immunohistochemical detection of VEGF, Flt-1 and KDR in clinical specimens of human astroglial tumors. Representative examples of immunoperoxidase staining for the indicated proteins on paraffin sections, on low-grade (LGA) versus high-grade (HGA) tumors. Original magnification: x200.

Considered within the context of astrocytoma biology and response to DNA damaging treatments such as IR, it will now be important to pinpoint the nature of such radioprotective effect of VEGF signaling, and pursue the tempting possibility to exploit the observed synergistic effect for a more efficient, combined targeted therapy in a preclinical setting. As to the underlying mechanisms, one might speculate that the VEGF-triggered pathways not only affect the cell death machinery,20 but could also impinge on the DNA damage response pathways, either by modulating the checkpoint responses or efficiency of DNA repair, both of which are critical for the biological outcome and are often deregulated in human cancer.31-34 Another conceptually important issue is whether the so-called cancer stem cells, recently identified also in malignant astrocytomas,5,35-40 might

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Figure 7. Model of autocrine regulation of VEGF signaling in human astrocytomas. We propose that regulation and biological effects of VEGF in VEGFR-expressing astrocytomas including GBM operate, at least in part, in an autocrine manner. Signaling is mediated by VEGFR2 (KDR) and activates multiple intracellular pathways, as indicated. Beside stimulation of cell growth/proliferation, the pool of VEGF secreted by astrocytoma cells within the stromal niche creates a “pro-survival microenvironment” that enhances the viability of the tumor cells and protects them against IR-induced apoptosis. Autocrine (*) VEGF intracellular signaling as well as its biological effects may be inhibited by pre-treatment with the selective KDR tyrosine kinase domain inhibitor, SU1498.

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A172, Gli-06, SF126, U373MG, U251MG), neuroblastoma cell line (SY-5Y) and HUVEC cells was extracted using TRIZOL® (Invitrogen) and reverse transcribed using human-specific primers for VEGF, Flt-1, KDR and primers for GAPDH used as a RNA quality control. Primers for VEGF, Flt-1 and KDR were designed, and conditions for RT-PCR reactions set as published previously.11 Semi-quantitative RT-PCR analysis. Total RNA (600 ng) was isolated from the cell line A172, serum-starved for 24 hours, then either left unstimulated or stimulated with recombinant VEGF121 (20 ng/ml) for 6, 12, 24 and 48 hours, followed by semi-quantitative RT-PCR analysis for VEGF mRNA expression using GAPDH as a house-keeping gene control. ELISA. Cells were serum-starved for 24 hours, either pre-treated by the SU1498 inhibitor (30 μM; from Calbiochem) for one hour, followed by stimulation with 40 ng/ml of VEGF121, or incubated in medium containing 40 ng/ml of VEGF121 only. Negative control cells were left untreated. Following 2 hours of stimulation, cells were washed twice with PBS and supplemented with serum-free medium and allowed to secrete VEGF for 6, 12, 24 and 48 hours. Secretion of VEGF into cell culture supernatants was measured by Human VEGF Quantikine Kit (R&D Systems) according to manufacturer’s instructions. All tests were performed 3 times in triplicate. Western blot analysis. The 6 cm dishes were seeded with 300.000 A172 cells in standard medium (see above) and cultured for 24 hours at 37°C in humidified atmosphere with 5% CO2. Following 24 hours of starvation, cells were stimulated for 0.5, 1, 6 and 12 hours with 20 ng/ml of recombinant VEGF121 (Sigma) or 20 ng/ ml PlGF-1 (Chemicon). When using SU1498 (KDR inhibitor, 30 μM, Calbiochem), cells were pre-incubated with the inhibitor for 1 hour before stimulation with VEGF121. Dishes with un-treated and treated cells were washed twice with ice-cold PBS and exposed to 120 μl of lysis buffer [containing 150 mM NaCl, 1% (v/v) NP-40, 50 mM Tris, pH 8.0, 0.5 mM EDTA, protease inhibitor cocktail tablets Complete (Roche Diagnostics)] at time points 0.5 h, 1 h, 6 h and 12 h after VEGF addition. The lysate was centrifuged (15,000 rpm) for 30 minutes at 4°C, and the supernatant was kept and stored at -80°C until use. Total protein content per sample was analyzed by Bradford protein assay (Bio-Rad, Hercules, CA). Protein loading buffer 4x concentrated (200 mM Tris-HCL, pH 7.8, 400 mM DTT, 8% SDS, 0.4% bromophenol blue, and 40% glycerol) was added to each sample and boiled at 95°C for 4 minutes. Equal amounts (40 μg) of total protein extracted from cells were subjected to 10% SDS-PAGE. Then proteins were transferred using semidry Trans-Blot SD cell (Bio-Rad) onto a nitrocellulose membrane Hybond-ECL (Amersham Biosciences). Membranes were blocked in 5% (w/v) fat milk in PBS containing 0.1% Tween-20 and incubated with primary antibodies (phospho-Akt Ser473, phospho-c-Raf Ser338, phospho-p44/42 MAPK Thr202/Tyr204, phospho-PLCγ1 Tyr783, Cell Signaling Technology) overnight at 4°C. Finally, the membranes were rinsed with PBS containing 0.1% Tween-20 for 45 minutes and incubated with secondary antibodies (anti-mouse/ anti-rabbit IgG, HRP-linked antibody, Cell Signaling Technology) at room temperature for 45 minutes, washed in PBS with 0.1% Tween-20 for 45 minutes, and the reaction visualized using the ECL solution (Amersham Biosciences). Click-iT™ EdU alexa fluor 488 cell proliferation assay. Following 24 hours of starvation in serum-free medium, cells were incubated with VEGF121 (40 ng/ml) or pre-treated with SU1498 (30 μM) or

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be targeted by such combined treatment. Among other features, these astrocytoma stem-like cells are particularly resistant to IR, and they have been shown to respond favorably to inhibition of DNA damage checkpoint kinases.41 One possibility, supported by the accumulating evidence in the field,5,41-42 as well as by our present data on the VEGF-VEGFR2 (KDR) signaling, would be to apply a standard DNA damaging modality such as IR or chemotherapy, and concomitantly block the DNA damage checkpoints or repair, and inhibit the VEGF-VEGFR signaling, thereby eliminating the major pro-survival mechanisms in malignant astrocytomas.

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Cell culture. Human astroglial tumor (grade III and IV-GBM) cell lines U118MG, U87MG, T98G, A172, Gli-06, SF126, U373MG (the latter three cell lines provided by C.Y. Bree, University of Amsterdam, The Netherlands) and U251MG, and the control HUVEC cells were maintained in Dulbecco’s Eagle’s medium (DMEM, standard medium) supplemented with 10% (v/v) fetal bovine serum, 1.7 mM L-glutamine and penicillin/streptomycin. The neuroblastoma cell line SY-5Y (ATTC) was maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum, 1.7 mM L-glutamine and penicillin/streptomycin. HUVEC cells were provided by J. Prochazkova (Institute of Biophysics, Brno, Czech Republic). Total RNA isolation and RT-PCR analysis. Total RNA (2 μg) from astroglial tumor cell lines (U118MG, U87MG, T98G, www.landesbioscience.com

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Acknowledgements

This work was supported by grants from the Czech Ministry of Education (MSM6198959216), Palacky University (IGUP91110131/39), the Czech Ministry of Health (IGAMZCRNR/8370-3), and the Lundbeck Foundation (R13-A1287). References

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left untreated for 6, 12 and 24 hours. Next, cells were pulse-labeled with 10 μM EdU for 30 min, harvested and analyzed according to manufacturer’s protocol for the Click-iT™ EdU Alexa Fluor 488 Cell Proliferation Kit (Invitrogen). Experiments were performed 3 times in duplicate and analyzed (100.000 cells per sample) using the FACSCalibur flow cytometer and CellQuestPro Software (BD Biosciences). Annexin V-FITC apoptosis staining. Cells were plated at 70% confluence, serum-starved for 24 hours. The next day, cells were treated with 30 μM SU1498 or left untreated for 2 hours in lowserum medium (1% (v/v) FBS). Further, cells were either irradiated using X-ray generator (HF160 [Pantak], Philips Medico; 150 kV, 15 mA, dose-rate 2.18 Gy/min) with 10 Gy of IR or left untreated, harvested 24 hours post-radiation treatment and stained with Annexin V-FITC conjugate according to manufacturer’s instructions (BD Biosciences). Cells that stained positive for Annexin V-FITC were considered as undergoing apoptosis. Experiments were performed 2 times in duplicate and analyzed (100.000 cells per sample) using FACSCalibur flow cytometer and CellQuestPro Software (BD Biosciences). Patients and tissues. A series of 28 WHO grade I and II astrocytomas (LGA) and 35 WHO grade III and IV astrocytomas and glioblastomas, respectively (HGA) were investigated. Tumor samples were obtained from patients who underwent surgery at the Department of Neurosurgery, Faculty of Medicine and Dentistry, Palacky University, Olomouc, between 1989 and 2004. The diagnosis and grading was reviewed independently by two pathologists according to WHO classification.43 Immunohistochemistry. Indirect immunohistochemical detection of VEGF (antibody clone C-1, dilution 1:50, Santa Cruz Biotechnology, INC), Flt-1 (antibody clone C-17, dilution 1:50, Santa Cruz Biotechnology, INC), and KDR (Flk-1, antibody clone A-3, dilution 1:150, Santa Cruz Biotechnology, INC) on formalin (10%, pH 7.4) fixed, paraffin-embedded sections was performed according to standard procedures using a high temperature epitope retrieval technique in citrate buffer (10 mM, pH 6; microwave oven 20 min). Diaminobenzidine (DAB) was used as chromogen. Samples of normal skin fibroblast and vascular endothelial cells of normal mucosa of large intestine were used as markers of negativity of Flt-1/VEGF and KDR, respectively. Non-specific granulation tissue with newly formed vessels was used as a positive control for all antibodies studied (data not shown). Immunohistochemical staining was evaluated by a semi-quantitative method using the histoscore scoring system, which is a multiplication of indexes (scores) obtained for the homogeneity (percentage of positive cells), and the intensity of staining. The latter was scored as weak (1), moderate (2) or strong (3) as described previously.44 The analysis and scoring was performed by two pathologists blinded to the clinical data. Statistical analysis. Matched pair analysis was performed using T test and non-parametric Mann-Whitney test for the analysis of patients’ data. ANOVA (non-parametric Kruskal-Wallis test) and Student t-test were used in statistical evaluation of in vitro experiments (the values are given as the mean ± SD of three independent experiments). In all tests, a value of p ≤ 0.05 was considered as significant.

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