Prognostic significance of telomere maintenance mechanisms in pediatric high-grade gliomas.

Children with high-grade glioma, including diffuse intrinsic pontine glioma (DIPG), have a poor prognosis despite multimodal therapy. Identifying novel therapeutic targets is critical to improve their outcome. We evaluated prognostic roles of telomere maintenance mechanisms in children with HGG, including DIPG. A multi-institutional retrospective study was conducted involving 50 flash-frozen HGG (35 non-brainstem; 15 DIPG) tumors from 45 children (30 non-brainstem; 15 DIPG). Telomerase activity, expression of hTERT mRNA (encoding telomerase catalytic component) and TERC (telomerase RNA template) and alternative lengthening of telomeres (ALT) mechanism were assayed. Cox Proportional Hazard regression analyses assessed association of clinical and pathological variables, TERC and hTERT levels, telomerase activity, and ALT use with progression-free or overall survival (OS). High TERC and hTERT expression was detected in 13/28 non-brainstem HGG samples as compared to non-neoplastic controls. High TERC and hTERT expression was identified in 13/15 and 11/15 DIPG samples, respectively, compared to controls. Evidence of ALT was noted in 3/11 DIPG and 10/19 non-brainstem HGG specimens. ALT and telomerase use were identified in 4/19 non-brainstem HGG and 2/11 DIPG specimens. In multivariable analyses, increased TERC and hTERT levels were associated with worse OS in patients with non-brainstem HGG, after controlling for tumor grade or resection extent. Children with HGG and DIPG, have increased hTERT and TERC expression. In children with non-brainstem HGG, increased TERC and hTERT expression levels are associated with a worse OS, making telomerase a promising potential therapeutic target in pediatric HGG.

Deletion of the SPARC acidic domain or EGF-like module reduces SPARC-induced migration and signaling through p38 MAPK/HSP27 in glioma.

We previously demonstrated that secreted protein acidic and rich in cysteine (SPARC) increases heat shock protein 27 (HSP27) expression and phosphorylation and promotes glioma cell migration through the p38 mitogen-activated protein kinase (MAPK)/HSP27 signaling pathway. As different regions of the SPARC protein mediate different SPARC functions, elucidating which SPARC domains regulate HSP27 expression, signaling and migration might provide potential therapeutic strategies to target these functions. To investigate the roles of specific domains, we used an SPARC-green fluorescent protein (GFP) fusion protein and constructs of SPARC-GFP with deletions of either the acidic domain (ΔAcidic) or the epidermal growth factor (EGF)-like module (ΔEGF). GFP, SPARC-GFP and the two deletion mutants were expressed in U87MG glioma cells. Characterization of the derived stable clones by confocal imaging and western blotting suggests proper folding, processing and secretion of the deletion constructs. Uptake of the constructs by naive cells suggests enhanced internalization of ΔAcidic and reduced internalization of ΔEGF. Wound and transwell migration assays and western blot analysis confirm our previous results and indicate that ΔAcidic reduces SPARC-induced migration and p38 MAPK/HSP27 signaling and ΔEGF decreases SPARC-induced migration and dramatically decreases the expression and phosphorylation of HSP27 but is poorly internalized. Loss of the EGF-like module suppresses the enhanced HSP27 protein stability conferred by SPARC. In conclusion, deletions of the acidic domain and EGF-like module have differential effects on cell surface binding and HSP27 protein stability; however, both regions regulate SPARC-induced migration and signaling through HSP27. Our data link the domains of SPARC with different functions and suggest one or both of the constructs as potential therapeutic agents to inhibit SPARC-induced migration.

Inhibition of HSP27 alone or in combination with pAKT inhibition as therapeutic approaches to target SPARC-induced glioma cell survival.

BACKGROUND: The current treatment regimen for glioma patients is surgery, followed by radiation therapy plus temozolomide (TMZ), followed by 6 months of adjuvant TMZ. Despite this aggressive treatment regimen, the overall survival of all surgically treated GBM patients remains dismal, and additional or different therapies are required. Depending on the cancer type, SPARC has been proposed both as a therapeutic target and as a therapeutic agent. In glioma, SPARC promotes invasion via upregulation of the p38 MAPK/MAPKAPK2/HSP27 signaling pathway, and promotes tumor cell survival by upregulating pAKT. As HSP27 and AKT interact to regulate the activity of each other, we determined whether inhibition of HSP27 was better than targeting SPARC as a therapeutic approach to inhibit both SPARC-induced glioma cell invasion and survival. RESULTS: Our studies found the following. 1) SPARC increases the expression of tumor cell pro-survival and pro-death protein signaling in balance, and, as a net result, tumor cell survival remains unchanged. 2) Suppressing SPARC increases tumor cell survival, indicating it is not a good therapeutic target. 3) Suppressing HSP27 decreases tumor cell survival in all gliomas, but is more effective in SPARC-expressing tumor cells due to the removal of HSP27 inhibition of SPARC-induced pro-apoptotic signaling. 4) Suppressing total AKT1/2 paradoxically enhanced tumor cell survival, indicating that AKT1 or 2 are poor therapeutic targets. 5) However, inhibiting pAKT suppresses tumor cell survival. 6) Inhibiting both HSP27 and pAKT synergistically decreases tumor cell survival. 7) There appears to be a complex feedback system between SPARC, HSP27, and AKT. 8) This interaction is likely influenced by PTEN status. With respect to chemosensitization, we found the following. 1) SPARC enhances pro-apoptotic signaling in cells exposed to TMZ. 2) Despite this enhanced signaling, SPARC protects cells against TMZ. 3) This protection can be reduced by inhibiting pAKT. 4) Combined inhibition of HSP27 and pAKT is more effective than TMZ treatment alone. CONCLUSIONS: We conclude that inhibition of HSP27 alone, or in combination with pAKT inhibitor IV, may be an effective therapeutic approach to inhibit SPARC-induced glioma cell invasion and survival in SPARC-positive/PTEN-wildtype and SPARC-positive/PTEN-null tumors, respectively.

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.

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.

SPARC upregulates MT1-MMP expression, MMP-2 activation, and the secretion and cleavage of galectin-3 in U87MG glioma cells.

Secreted protein acidic and rich in cysteine (SPARC) is highly expressed in human gliomas and promotes glioma invasion. We have shown by cDNA array analysis that SPARC upregulates membrane type 1-matrix metalloproteinase (MT1-MMP) and matrix metalloproteinase-2 (MMP-2) transcripts. To confirm these findings at the protein level and determine whether SPARC expression correlates with increased MMP activity, we used Western blot to assess the levels of MT1-MMP, and gelatin zymography to assess MMP-2 levels and activity. We also examined the expression, secretion, and cleavage of galectin-3, a target of MT1-MMP and MMP-2. Our data confirm that SPARC upregulates MT1-MMP levels and MMP-2 activity. There was also an increase in secreted galectin-3, as well as an increase in the proteolytically processed form of galectin-3. Previous studies have demonstrated that MT1-MMP, MMP-2, and galectin-3 are increased in gliomas. Our results suggest that their upregulation and activation may be a consequence of increased SPARC expression. These data provide a provisional mechanism whereby SPARC contributes to brain tumor invasion.

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.

Meningiomas: analysis of loss of heterozygosity on chromosome 10 in tumor progression and the delineation of four regions of chromosomal deletion in common with other cancers.

PURPOSE: Loss of heterozygosity (LOH) of alleles on chromosome 10 has been reported in many cancers, leading to the identification of tumor suppressor genes on this chromosome. Several reports implicate LOH of chromosome 10 alleles in meningioma progression, but the frequency and complexity of the loss have not been well characterized. Furthermore, the location and identity of the putative tumor suppressor genes on this chromosome that contribute to meningioma progression are unknown because the currently characterized tumor suppressor genes do not appear to be involved. Therefore, this study was undertaken to (a) assess the frequency and complexity of LOH in meningioma progression, (b) map the LOH patterns of individual meningiomas to define the smallest regions of shared chromosomal deletion, and (c) compare the identified regions with chromosome 10 deletions in other cancers, and thereby initiate the localization of the putative tumor suppressor genes. EXPERIMENTAL DESIGN: We examined 11 microsatellite dinucleotide repeat loci in 208 meningiomas of all grades using laser capture microdissection and fluorescence-based detection of PCR products. RESULTS: For all markers examined, the incidence of LOH was much higher in all grades than that previously reported, with incidence and complexity of LOH increasing with tumor grade. LOH mapping identified four regions of chromosomal deletion: 10pter-D10S89, D10S109-D10S215, D10S187-D10S209, and D10S169-10qter. These deletions on chromosome 10 are shared with other cancer types. CONCLUSIONS: These results delineate chromosomal locations of putative tumor suppressor genes on chromosome 10 that likely play an early role in meningioma tumorigenesis as well as tumor progression.

Meningiomas: loss of heterozygosity on chromosome 10 and marker-specific correlations with grade, recurrence, and survival.

PURPOSE: In a study of 208 meningiomas, we found a high incidence of loss of heterozygosity (LOH) on chromosome 10 in benign (73.4%), atypical (80.0%), and malignant (86.7%) tumors. A large percentage of the benign and atypical tumors and an increasing percentage of malignant tumors had LOH on multiple loci (43.9%, 45%, and 66.7%, respectively). The high incidence of LOH occurring early in meningioma progression suggests that LOH at individual alleles may serve as a marker of clinically relevant alterations useful for patient diagnosis, the subclassification of tumors, and/or the treatment of patients. EXPERIMENTAL DESIGN: To test this, we examined 208 sporadic and recurrent meningiomas of all grades for correlations between LOH at 11 markers on chromosome 10 and tumor location, histology, and grade and patient race, gender, age, recurrence, and survival. RESULTS: Several significant correlations were found. The data indicate that genetic differences occur not only between tumors of different grade, but also between tumors of the same grade, and therefore may be useful to define genetic subsets with clinical implications. LOH at D10S179 (P = 0.001) or D10S169 (P = 0.004) is most likely present in higher-grade meningiomas and, when present in benign tumors, may signify sampling error or a morphologically benign but biologically aggressive tumor. Furthermore, LOH at D10S209 (P = 0.06) and D10S169 (P = 0.01) may predict shorter survival and/or higher rates of recurrence, respectively, in tumors with benign or malignant histology. CONCLUSIONS: We conclude that these chromosome 10 markers deserve further testing as unfavorable prognostic indicators for meningioma patients.

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.

SPARC affects glioma cell growth differently when grown on brain ECM proteins in vitro under standard versus reduced-serum stress conditions.

Secreted protein acidic and rich in cysteine (SPARC) has a suppressive effect on U87 glioma cell proliferation when assessed in vitro and in vivo using parental U87T2 and U87T2-derived SPARC-transfected clones. Since SPARCinteracts with extracellular matrix (ECM) proteins, we examined the effect of SPARC secretion on proliferation, morphology, and cell density of glioma cells grown in vitro, in the absence and presence of ECM proteins under standard (10% fetal bovine serum [FBSI) and reduced (0.1% FBS) serum stress conditions. Under standard conditions, MTT (3-(4,5-cimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide) growth curves, morphology, and Western blot analyses demonstrated that SPARC had a suppressive and biphasic effect on growth that was not grossly modulated by the ECMs. The SPARC-induced changes in morphology observed at 24 h were not altered by the presence of ECMs. Under reduced-serum stress conditions, Western blot, morphological, and flow cytometric analyses indicated that the SPARC-induced suppressive growth effects were eliminated when the cells were grown on plastic. However, ECM-specific changes in growth were observed, some of which correlated with secreted SPARC levels. These results indicate that the differential effects of SPARC and ECMs on proliferation are dependent on culture conditions. Since the results obtained under standard conditions agree with our in vivo observations, we conclude that the ability of SPARC to suppress proliferation is regulated to a greater degree by the level of SPARC and that this suppressive effect is not influenced by the presence of any of the ECMs examined.

PTEN suppresses SPARC-induced pMAPKAPK2 and inhibits SPARC-induced Ser78 HSP27 phosphorylation in glioma.

BACKGROUND: Secreted protein acidic and rich in cysteine (SPARC) is overexpressed in astrocytomas (World Health Organization grades II-IV). We previously demonstrated that SPARC promotes glioma migration and invasion-in part, by activating the P38 mitogen-activated protein kinase (MAPK)-heat shock protein (HSP)27 signaling pathway. The commonly lost tumor suppressor phosphatase and tensin homolog (PTEN) suppresses SPARC-induced migration, which is accompanied by suppression of Shc-Ras-Raf-MEK-ERK1/2 and Akt signaling. As PTEN completely suppresses SPARC-induced migration, we proposed that PTEN must also interfere with SPARC-induced HSP27 signaling. Therefore, this study determined the effects of PTEN expression on SPARC-induced expression and phosphorylation of HSP27. METHODS: Control and SPARC-expressing clones transfected with control- or PTEN-expression plasmids were plated on fibronectin-coated tissue culture plates for 3, 6, 24, and 48 h and then lysed. Equal amounts of protein were subjected to Western blot and densitometric analyses. RESULTS: The results show that SPARC enhances phosphorylated (p)P38 MAPK, phosphorylated MAPK-activated protein kinase 2 (pMAPKAPK2), and serine (Ser)78 HSP27 phosphorylation relative to total HSP27. PTEN suppresses pAkt and pMAPKAPK2, suggesting that PTEN effects are downstream of pP38 MAPK. PTEN suppressed SPARC-induced sustained phosphorylation at Ser78 HSP27. As the level of total HSP27 differed based on the presence of SPARC or PTEN, the ratios of phosphorylation-specific to total HSP27 were examined. The data demonstrate that SPARC-induced phosphorylation at Ser78 remains elevated despite increasing levels of total HSP27. In contrast, PTEN inhibits SPARC-induced increases in Ser78 HSP27 phosphorylation relative to total HSP27. CONCLUSION: These data describe a novel mechanism whereby PTEN inhibits SPARC-induced migration through suppression and differential regulation of pAkt and the P38 MAPK-MAPKAPK2-HSP27 signaling pathway.

MicroRNA-145 is downregulated in glial tumors and regulates glioma cell migration by targeting connective tissue growth factor.

Glioblastomas (GBM), the most common and aggressive type of malignant glioma, are characterized by increased invasion into the surrounding brain tissues. Despite intensive therapeutic strategies, the median survival of GBM patients has remained dismal over the last decades. In this study we examined the expression of miR-145 in glial tumors and its function in glioma cells. Using TCGA analysis and real-time PCR we found that the expression of miR-145/143 cluster was downregulated in astrocytic tumors compared to normal brain specimens and in glioma cells and glioma stem cells (GSCs) compared to normal astrocytes and neural stem cells. Moreover, the low expression of both miR-145 and miR-143 in GBM was correlated with poor patient prognosis. Transfection of glioma cells with miR-145 mimic or transduction with a lentivirus vector expressing pre-miR 145 significantly decreased the migration and invasion of glioma cells. We identified connective tissue growth factor (CTGF) as a novel target of miR-145 in glioma cells; transfection of the cells with this miRNA decreased the expression of CTGF as determined by Western blot analysis and the expression of its 3'-UTR fused to luciferase. Overexpression of a CTGF plasmid lacking the 3'-UTR and administration of recombinant CTGF protein abrogated the inhibitory effect of miR-145 on glioma cell migration. Similarly, we found that silencing of CTGF decreased the migration of glioma cells. CTGF silencing also decreased the expression of SPARC, phospho-FAK and FAK and overexpression of SPARC abrogated the inhibitory effect of CTGF silencing on cell migration. These results demonstrate that miR-145 is downregulated in glial tumors and its low expression in GBM predicts poor patient prognosis. In addition miR-145 regulates glioma cell migration by targeting CTGF which downregulates SPARC expression. Therefore, miR-145 is an attractive therapeutic target for anti-invasive treatment of astrocytic tumors.

Mesenchymal stem cells deliver synthetic microRNA mimics to glioma cells and glioma stem cells and inhibit their cell migration and self-renewal.

MicroRNAs (miRNAs) have emerged as potential cancer therapeutics; however, their clinical use is hindered by lack of effective delivery mechanisms to tumor sites. Mesenchymal stem cells (MSCs) have been shown to migrate to experimental glioma and to exert anti-tumor effects by delivering cytotoxic compounds. Here, we examined the ability of MSCs derived from bone marrow, adipose tissue, placenta and umbilical cord to deliver synthetic miRNA mimics to glioma cells and glioma stem cells (GSCs). We examined the delivery of miR-124 and miR-145 mimics as glioma cells and GSCs express very low levels of these miRNAs. Using fluorescently labeled miRNA mimics and in situ hybridization, we demonstrated that all the MSCs examined delivered miR-124 and miR-145 mimics to co-cultured glioma cells and GSCs via gap junction- dependent and independent processes. The delivered miR-124 and miR-145 mimics significantly decreased the luciferase activity of their respected reporter target genes, SCP-1 and Sox2, and decreased the migration of glioma cells and the self-renewal of GSCs. Moreover, MSCs delivered Cy3-miR-124 mimic to glioma xenografts when administered intracranially. These results suggest that MSCs can deliver synthetic exogenous miRNA mimics to glioma cells and GSCs and may provide an efficient route of therapeutic miRNA delivery in vivo.

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.

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.

Controlling integrin specificity and stem cell differentiation in 2D and 3D environments through regulation of fibronectin domain stability.

The extracellular matrix (ECM) exerts powerful control over many cellular phenomena, including stem cell differentiation. As such, design and modulation of ECM analogs to ligate specific integrin is a promising approach to control cellular processes in vitro and in vivo for regenerative medicine strategies. Although fibronectin (FN), a crucial ECM protein in tissue development and repair, and its RGD peptide are widely used for cell adhesion, the promiscuity with which they engage integrins leads to difficulty in control of receptor-specific interactions. Recent simulations of force-mediated unfolding of FN domains and sequences analysis of human versus mouse FN suggest that the structural stability of the FN's central cell-binding domains (FN III9-10) affects its integrin specificity. Through production of FN III9-10 variants with variable stabilities, we obtained ligands that present different specificities for the integrin alpha(5)beta(1) and that can be covalently linked into fibrin matrices. Here, we demonstrate the capacity of alpha(5)beta(1) integrin-specific engagement to influence human mesenchymal stem cell (MSC) behavior in 2D and 3D environments. Our data indicate that alpha(5)beta(1) has an important role in the control of MSC osteogenic differentiation. FN fragments with increased specificity for alpha(5)beta(1) versus alpha(v)beta(3) results in significantly enhanced osteogenic differentiation of MSCs in 2D and in a clinically relevant 3D fibrin matrix system, although attachment/spreading and proliferation were comparable with that on full-length FN. This work shows how integrin-dependant cellular interactions with the ECM can be engineered to control stem cell fate, within a system appropriate for both 3D cell culture and tissue engineering.