Proteolysis of the matricellular protein hevin by matrix metalloproteinase-3 produces a SPARC-like fragment (SLF) associated with neovasculature in a murine glioma model.

The matricellular SPARC-family member hevin (Sparc-like 1/SPARCL-1/SC1/Mast9) contributes to neural development and alters tumor progression in a range of mammalian models. Based on sequence similarity, we hypothesized that proteolytic digestion of hevin would result in SPARC-like fragments (SLF) that affect the activity and/or location of these proteins. Incubation of hevin with matrix metalloproteinase-3 (MMP-3), a protease known to cleave SPARC, produced a limited number of peptides. Sequencing revealed the major proteolytic products to be SPARC-like in primary structure. In gliomas implanted into murine brain, a SLF was associated with SPARC in the neovasculature but not with hevin, the latter prominent in the astrocytes encompassed by infiltrating tumor. In this model of invasive glioma that involves MMP-3 activity, host-derived SLF was not observed in the extracellular matrix adjacent to tumor cells. In contrast, it occurred with its homolog SPARC in the angiogenic response to the tumor. We conclude that MMP-3-derived SLF is a marker of neovessels in glioma, where it could influence the activity of SPARC.

Gliosarcoma stem cells undergo glial and mesenchymal differentiation in vivo.

Cancer stem cells (CSCs) are characterized by their self-renewing potential and by their ability to differentiate and phenocopy the original tumor in orthotopic xenografts. Long-term propagation of glioblastoma (GBM) cells in serum-containing medium results in loss of the CSCs and outgrowth of cells genetically and biologically divergent from the parental tumors. In contrast, the use of a neurosphere assay, a serum-free culture for selection, and propagation of central nervous system-derived stem cells allows the selection of a subpopulation containing CSCs. Gliosarcoma (GS), a morphological variant comprising approximately 2% of GBMs, present a biphasic growth pattern, composed of glial and metaplastic mesenchymal components. To assess whether the neurosphere assay would allow the amplification of a subpopulation of cells with "gliosarcoma stem cell" properties, capable of propagating both components of this malignancy, we have generated neurospheres and serum cultures from primary GS and GBM surgical specimens. Neurosphere cultures from GBM and GS samples expressed neural stem cell markers Sox2, Musashi1, and Nestin. In contrast to the GBM neurosphere lines, the GS neurospheres were negative for the stem cell marker CD133. All neurosphere lines generated high-grade invasive orthotopic tumor xenografts, with histological features strikingly similar to the parental tumors, demonstrating that these cultures indeed are enriched in CSCs. Remarkably, low-passage GS serum cultures retained the expression of stem cell markers, the ability to form neurospheres, and tumorigenicity. The GS experimental tumors phenocopied the parental tumor, exhibiting biphasic glial and mesenchymal components, constituting a clinically relevant model to investigate mesenchymal differentiation in GBMs.

Radiation sensitization of glioblastoma by cilengitide has unanticipated schedule-dependency.

We investigated whether cilengitide could amplify the antitumor effects of radiotherapy in an orthotopic rat glioma xenograft model. Cilengitide is a specific inhibitor of alphav series integrins, and acts as an antiangiogenic. U251 human glioma cells express alphavbeta3 and alphavbeta5 integrins. We used in vitro assays of adhesion and growth of tumor and endothelial cells to evaluate cytotoxicity and the potential for cilengitide to enhance radiation toxicity. Treatment was then evaluated in an orthotopic model to evaluate synergy with therapeutic radiation in vivo. In vitro, cilengitide blocked cell adhesion, but did not influence the effects of radiation on U251 cells; cilengitide strongly amplified radiation effects on endothelial cell survival. In vivo, radiotherapy prolonged the survival of U251 tumor-bearing rats from 50 to over 110 days. Cotreatment with cilengitide and radiation dramatically amplified the effects of radiation, producing survival over 200 days and triggering an enhanced apoptotic response and suppression of tumor growth by histology at necropsy. Signaling pathways activated in the tumor included NFkappab, a documented mediator of cellular response to radiation. Because cilengitide has a short plasma half-life (t((1/2)) approximately 20 min), antiangiogenic scheduling typically uses daily injections. We found that a single dose of cilengitide (4 mg/kg) given between 4 and 12 hr prior to radiation was sufficient to produce the same effect. Our results demonstrate that blockade of alphav integrins mediates an unanticipated rapid potentiation of radiation, and suggests possible clinical translation for glioma therapy.

HSP27 mediates SPARC-induced changes in glioma morphology, migration, and invasion.

Secreted protein acidic and rich in cysteine (SPARC) regulates cell-extracellular matrix interactions that influence cell adhesion and migration. We have demonstrated that SPARC is highly expressed in human gliomas, and it promotes brain tumor invasion in vitro and in vivo. To further our understanding regarding SPARC function in glioma migration, we transfected SPARC-green fluorescent protein (GFP) and control GFP vectors into U87MG cells, and assessed the effects of SPARC on cell morphology, migration, and invasion after 24 h. The expression of SPARC was associated with elongated cell morphology, and increased migration and invasion. The effects of SPARC on downstream signaling were assessed from 0 to 6 h and 24 h. SPARC increased the levels of total and phosphorylated HSP27; the latter was preceded by activation of p38 MAPK and inhibited by the p38 MAPK inhibitor SB203580. Augmented expression of SPARC was correlated with increased levels of HSP27 mRNA. In a panel of glioma cell lines, increasing levels of SPARC correlated with increasing total and phosphorylated HSP27. SPARC and HSP27 were colocalized to invading cells in vivo. Inhibition of HSP27 mRNA reversed the SPARC-induced changes in cell morphology, migration, and invasion in vitro. These data indicate that HSP27, a protein that regulates actin polymerization, cell contraction, and migration, is a novel downstream effector of SPARC-regulated cell morphology and migration. As such, it is a potential therapeutic target to inhibit SPARC-induced glioma invasion.

SPARC-induced increase in glioma matrix and decrease in vascularity are associated with reduced VEGF expression and secretion.

Glioblastomas are heterogeneous tumors displaying regions of necrosis, proliferation, angiogenesis, apoptosis and invasion. SPARC, a matricellular protein that negatively regulates angiogenesis and cell proliferation, but enhances cell deadhesion from matrix, is upregulated in gliomas (Grades II-IV). We previously demonstrated that SPARC promotes invasion while concomitantly decreasing tumor growth, in part by decreasing proliferation of the tumor cells. In other cancer types, SPARC has been shown to influence tumor growth by altering matrix production, and by decreasing angiogenesis via interfering with the VEGF-VEGFR1 signaling pathway. We therefore examined whether the SPARC-induced decrease in glioma tumor growth was also, in part, due to alterations in matrix and/or decreased vascularity, and assessed SPARC-VEGF interactions. The data demonstrate that SPARC upregulates glioma matrix, collagen I is a constituent of the matrix and SPARC promotes collagen fibrillogenesis. Furthermore, SPARC suppressed glioma vascularity, and this was accompanied by decreased VEGF expression and secretion, which was, in part, due to reduced VEGF165 transcript abundance. These data indicate that SPARC modulates glioma growth by altering the tumor microenvironment and by suppressing tumor vascularity through suppression of VEGF expression and secretion. These experiments implicate a novel mechanism, whereby SPARC regulates VEGF function by limiting the available growth factor. Because SPARC is considered to be a therapeutic target for gliomas, a further understanding of its complex signaling mechanisms is important, as targeting SPARC to decrease invasion could undesirably lead to the growth of more vascular and proliferative tumors.

GRP78 is overexpressed in glioblastomas and regulates glioma cell growth and apoptosis.

We characterized the expression and function of the endoplasmic reticulum protein GRP78 in glial tumors. GRP78 is highly expressed in glioblastomas but not in oligodendrogliomas, and its expression is inversely correlated with median patient survival. Overexpression of GRP78 in glioma cells decreases caspase 7 activation and renders the cells resistant to etoposide- and cisplatin-induced apoptosis, whereas silencing of GRP78 decreases cell growth and sensitizes glioma cells to etoposide, cisplatin, and gamma-radiation. Thus, GRP78 contributes to the increased apoptosis resistance and growth of glioma cells and may provide a target for enhancing the therapeutic responsiveness of these tumors.

Influence of ionizing radiation on the course of kindled epileptogenesis.

Several clinical and experimental reports suggest that low-dose irradiation of an established epileptic focus can reduce the occurrence of spontaneous seizures. Conversely, some recent reports suggest that under some conditions low-dose irradiation may have disinhibitory effects on seizure expression. Here, we have investigated mechanistic aspects of this phenomenon in the kindling model of epilepsy by applying focal irradiation at various points during kindling development. Rats were kindled to stage 5 by afterdischarge-threshold electrostimulation of the left amygdala. Treatment groups were irradiated using a collimated X-ray beam (18 MV) either prior to kindling, at kindling stage 3, or at kindling stage 5, by exposure of the left amygdala to a single-fraction central-axis dose of 25 Gy. Generalized seizure thresholds (GSTs) were subsequently assayed at weekly intervals for 10 weeks and at monthly intervals for an additional 3 months, along with the severity of the evoked seizures. Irradiation produced no significant effects on seizure threshold, but did produce persistent changes in seizure severity which varied as a function of the timing of irradiation. Relative to sham irradiated controls, the occurrence of stage 6 seizures was significantly increased by irradiation prior to kindling, but was unaffected by irradiation at kindling stage 3, and significantly reduced by irradiation at kindling stage 5. Quantitative immunohistochemical assays for neuron and astrocyte densities within the amygdala and hippocampus revealed only subtle changes in neuronal density within the dentate granule cell layer. These results are discussed in relation to mechanisms of seizure- and radiation-induced plasticity.

Secreted protein acidic and rich in cysteine promotes glioma invasion and delays tumor growth in vivo.

Secreted protein acidic and rich in cysteine (SPARC) is highly expressed in human astrocytomas, grades II-IV. We demonstrated previously that SPARC promotes invasion in vitro using the U87MG-derived clone U87T2 and U87T2-derived SPARC-transfected clones, A2b2, A2bi, and C2a4, in the spheroid confrontation assay. Additional in vitro studies demonstrated that SPARC delays growth, increases attachment, and modulates migration of tumor cells in extracellular matrix-specific and concentration-dependent manners. Therefore, we propose that SPARC functionally contributes to brain tumor invasion and delays tumor growth in vivo, and that the effects of SPARC are related to the level of SPARC secreted into the extracellular matrix. To test these hypotheses, we stereotactically injected these clones into nude rat brains (six animals were injected per clone). Animals were sacrificed on day 7 to assess growth and invasion for all clones at the same time in tumor development. To determine whether SPARC delayed but did not inhibit growth, rats were injected with U87T2 or clone A2b2, and the animals were sacrificed on days 9 (U87T2) and 20 (A2b2), when the animals demonstrated neurological deficit. Brains were removed, fixed, photographed, paraffin embedded, and sectioned. Sections were then serially stained with H&E for morphological assessment of invasion and to measure tumor volume, immunohistochemically stained to visualize SPARC, subjected to in situ hybridization with the human AluII DNA-binding probe to identify human cells, and immunohistochemically stained with MIB-1 to measure proliferation index. The results demonstrate that SPARC promotes invasion in vivo at day 7. Both the low (A2bi) and the high (A2b2) SPARC-secreting clones produced invasive tumors, invading with fingerlike projections and satellite masses into adjacent brain, as well as along the corpus collosum. The intermediate SPARC secreting clone (C2a4) primarily migrated as a bulk tumor along the corpus collosum. SPARC significantly decreased tumor growth at day 7, as measured both by adjusted MIB-1 proliferation indices (U87T2 = 95.3 +/- 1.4 versus A2bi = 73.4 +/- 4.0, A2b2 = 30.8 +/- 6.7 and C2a4 = 15.7 +/- 13.0) and tumor volumes (U87T2 = 13.4 +/- 0.6 mm(3) versus A2bi = 4.5 +/- 0.6 mm(3), A2b2 = 1.1 +/- 0.1 mm(3), and C2a4 = 0.4 +/- 0.1 mm(3)). Furthermore, SPARC delayed but did not inhibit tumor growth. The patterns of invasion and the extent of growth delay correlated with the level of SPARC expression. We propose that the ability of SPARC to promote invasion depends on the level of its secretion and the resultant modulation of the level of adherence and motility induced. This demonstration that SPARC functionally contributes to brain tumor invasion in vivo suggests that SPARC is a candidate therapeutic target for the design of therapies directed toward inhibition of the invasive phenotype.

Loss of Sparc in p53-null Astrocytes Promotes Macrophage Activation and Phagocytosis Resulting in Decreased Tumor Size and Tumor Cell Survival.

Both the induction of SPARC expression and the loss of the p53 tumor suppressor gene are changes that occur early in glioma development. Both SPARC and p53 regulate glioma cell survival by inverse effects on apoptotic signaling. Therefore, during glioma formation, the upregulation of SPARC may cooperate with the loss of p53 to enhance cell survival. This study determined whether the loss of Sparc in astrocytes that are null for p53 would result in reduced cell survival and tumor formation and increased tumor immunogenicity in an in vivo xenograft brain tumor model. In vitro, the loss of Sparc in p53-null astrocytes resulted in an increase in cell proliferation, but a loss of tumorigenicity. At 7 days after intracranial implantation, Sparc-null tumors had decreased tumor cell survival, proliferation and reduced tumor size. The loss of Sparc promoted microglia/macrophage activation and phagocytosis of tumor cells. Our results indicate that the loss of p53 by deletion/mutation in the early stages of glioma formation may cooperate with the induction of SPARC to potentiate cancer cell survival and escape from immune surveillance.

Effects of kindling and irradiation on neuronal density in the rat dentate gyrus.

Low-dose radiosurgery is presently in use as a treatment modality for focal epilepsy, but the mechanisms underlying the associated changes in seizure expression are poorly understood. We investigated whether total and parvalbumin expressing (PV+) neuronal densities within the hippocampus and amygdala are affected by analogous focal irradiation in amygdala-kindled rats. Adult rats were kindled by electrical stimulation through 10 stage 5 seizures. The kindled amygdala was then focally irradiated at 18 or 25 Gy, and generalized seizure thresholds were subsequently monitored for approximately 6 months. Histological and immunohistochemical assays of total and PV+ neuronal densities were performed bilaterally throughout the hippocampus and within the basolateral amygdala. PV+ neuronal densities were unaffected by kindling or irradiation in these regions. Kindling selectively reduced neuronal densities in the dentate granule cell layer, and medial CA3 pyramidal cell layer. Irradiation at 25 Gy, but not at 18 Gy, prevented or reversed this kindling-associated reduction in density.

Optimization of high grade glioma cell culture from surgical specimens for use in clinically relevant animal models and 3D immunochemistry.

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor. Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Sox2 promotes malignancy in glioblastoma by regulating plasticity and astrocytic differentiation.

The high-mobility group-box transcription factor sex-determining region Y-box 2 (Sox2) is essential for the maintenance of stem cells from early development to adult tissues. Sox2 can reprogram differentiated cells into pluripotent cells in concert with other factors and is overexpressed in various cancers. In glioblastoma (GBM), Sox2 is a marker of cancer stemlike cells (CSCs) in neurosphere cultures and is associated with the proneural molecular subtype. Here, we report that Sox2 expression pattern in GBM tumors and patient-derived mouse xenografts is not restricted to a small percentage of cells and is coexpressed with various lineage markers, suggesting that its expression extends beyond CSCs to encompass more differentiated neoplastic cells across molecular subtypes. Employing a CSC derived from a patient with GBM and isogenic differentiated cell model, we show that Sox2 knockdown in the differentiated state abolished dedifferentiation and acquisition of CSC phenotype. Furthermore, Sox2 deficiency specifically impaired the astrocytic component of a biphasic gliosarcoma xenograft model while allowing the formation of tumors with sarcomatous phenotype. The expression of genes associated with stem cells and malignancy were commonly downregulated in both CSCs and serum-differentiated cells on Sox2 knockdown. Genes previously shown to be associated with pluripontency and CSCs were only affected in the CSC state, whereas embryonic stem cell self-renewal genes and cytokine signaling were downregulated, and the Wnt pathway activated in differentiated Sox2-deficient cells. Our results indicate that Sox2 regulates the expression of key genes and pathways involved in GBM malignancy, in both cancer stemlike and differentiated cells, and maintains plasticity for bidirectional conversion between the two states, with significant clinical implications.

TRAIL conjugated to nanoparticles exhibits increased anti-tumor activities in glioma cells and glioma stem cells in vitro and in vivo.

Glioblastomas (GBM) are characterized by resistance to chemotherapy and radiotherapy, and therefore, alternative therapeutic approaches are needed. TRAIL induces apoptosis in cancer but not in normal cells and is considered to be a promising anti-tumor agent. However, its short in vivo half-life and lack of efficient administration modes are serious impediments to its therapeutic efficacy. Nanoparticles (NP) have been used as effective delivery tools for various anticancer drugs. TRAIL was conjugated to magnetic ferric oxide NP by binding the TRAIL primary amino groups to activated double bonds on the surface of the NP. The effect of NP-TRAIL was examined on the apoptosis of glioma cells and self-renewal of glioma stem cells (GSCs). In addition, the ability of the NP-TRAIL to track U251 cell-derived glioma xenografts and to affect cell apoptosis, tumor volume, and survival among xenografted rats was also examined. Conjugation of TRAIL to NP increased its apoptotic activity against different human glioma cells and GSCs, as compared with free recombinant TRAIL. Combined treatment with NP-TRAIL and γ-radiation or bortezomib sensitized TRAIL-resistant GSCs to NP-TRAIL. Using rhodamine-labeled NP and U251 glioma cell-derived xenografts, we demonstrated that the NP-TRAIL were found in the tumor site and induced a significant increase in glioma cell apoptosis, a decrease in tumor volume, and increased animal survival. In summary, conjugation of TRAIL to NP increased its apoptotic activity both in vitro and in vivo. Therefore, NP-TRAIL represents a targeted anticancer agent with more efficient action for the treatment of GBM and the eradication of GSCs.

Heparanase expression is associated with histone modifications in glioblastoma.

In this study we investigated epigenetic modifications such as DNA methylation, histone acetylation and histone methylation in the regulation of heparanase expression in glioblastoma. We found that heparanase promoters are differentially methylated among three glioblastoma cell lines; however, all these cells expressed baseline levels of heparanase. 5-Aza-2'-deoxycytidine (5-Aza-dC), a DNA methyltransferase inhibitor, revoked heparanase expression in all the examined cells. Trichostatin A (TSA), a histone deacetylase inhibitor, activated heparanase expression in promoter unmethylated LN229 and T98G cells but not in promoter methylated U251n cells. To identify the mechanisms of heparanase induction by 5-Aza-dC, heparanase expression-related transcription factors were examined. No detected transcription factors (EGR1, Ets1, GABPα and Sp1) were found to be induced either by 5-Aza-dC or TSA. Furthermore, we found that 5-Aza-dC increased acetylation of histone H3 and di-methylation of histone H3 lysine K4 (H3K4me2) in LN229 and T98G cells. The increased histone acetylation and H3K4me2 were also observed in heparanase-expressing tumor tissues by immunohistochemistry staining. Additionally, we found that nuclear factor κB (NFκB) p65 but not NFκB p50 was correlated with heparanase expression, which could be expressed both by neoplastic cells and angiogenesis-related neovessel cells. However, we did not observe any regulatory mechanism between heparanase and NFκB p65 via transient transfection of their cDNA in T98G and U251n cells. We concluded that heparanase expression is associated with histone modifications and promoter DNA methylation plays a role in the control of gene silencing. Overexpression of both heparanase and NFκB p65 may be the result of excessive histone modifications.

Cilengitide induces autophagy-mediated cell death in glioma cells.

We studied the effect of the integrin inhibitor cilengitide in glioma cells. Cilengitide induced cell detachment and decreased cell viability, and induction of autophagy followed by cell apoptosis. In addition, cilengitide decreased the cell renewal of glioma stem-like cells (GSCs). Inhibition of autophagy decreased the cytotoxic effect of cilengitide. Pretreatment of glioma cells and GSCs with cilengitide prior to γ-irradiation resulted in a larger increase in autophagy and a more significant decrease in cell survival. We found that cilengitide induced autophagy collectively in glioma cells, xenografts, and GSCs, which contributed to its cytotoxic effects and sensitized these cells to γ-radiation.

PTEN augments SPARC suppression of proliferation and inhibits SPARC-induced migration by suppressing SHC-RAF-ERK and AKT signaling.

SPARC (secreted protein acidic and rich in cysteine) is expressed in all grades of astrocytoma, including glioblastoma (GBM). SPARC suppresses glioma growth but promotes migration and invasion by mediating integrin and growth factor receptor-regulated kinases and their downstream effectors. PTEN (phosphatase and tensin homolog deleted on chromosome 10), which is commonly lost in primary GBMs, negatively regulates proliferation and migration by inhibiting some of the same SPARC-mediated signaling pathways. This study determined whether PTEN reconstitution in PTEN-mutant, SPARC-expressing U87MG cells could further suppress proliferation and tumor growth but inhibit migration and invasion in SPARC-expressing cells in vitro and in vivo, and thereby prolong survival in animals with xenograft tumors. In vitro, PTEN reduced proliferation and migration in both SPARC-expressing and control cells, with a greater suppression in SPARC-expressing cells. PTEN reconstitution suppressed AKT activation in SPARC-expressing and control cells but suppressed the SHC-RAF-ERK signaling pathway only in SPARC-expressing cells. Importantly, coexpression of SPARC and PTEN resulted in the smallest, least proliferative tumors with reduced invasive capacity and longer animal survival. Furthermore, direct inhibition of the AKT and SHC-RAF-ERK signaling pathways suppressed the proliferation and migration of SPARC-expressing cells in vitro. These findings demonstrate that PTEN reconstitution or inhibition of signaling pathways that are activated by the loss of PTEN provide potential therapeutic strategies to inhibit SPARC-induced invasion while enhancing the negative effect of SPARC on tumor growth.