MicroRNA-29a activates a multi-component growth and invasion program in glioblastoma.

BACKGROUND: Glioblastoma is a malignant brain tumor characterized by rapid growth, diffuse invasion and therapeutic resistance. We recently used microRNA expression profiles to subclassify glioblastoma into five genetically and clinically distinct subclasses, and showed that microRNAs both define and contribute to the phenotypes of these subclasses. Here we show that miR-29a activates a multi-faceted growth and invasion program that promotes glioblastoma aggressiveness. METHODS: microRNA expression profiles from 197 glioblastomas were analyzed to identify the candidate miRNAs that are correlated to glioblastoma aggressiveness. The candidate miRNA, miR-29a, was further studied in vitro and in vivo. RESULTS: Members of the miR-29 subfamily display increased expression in the two glioblastoma subclasses with the worst prognoses (astrocytic and neural). We observed that miR-29a is among the microRNAs that are most positively-correlated with PTEN copy number in glioblastoma, and that miR-29a promotes glioblastoma growth and invasion in part by targeting PTEN. In PTEN-deficient glioblastoma cells, however, miR-29a nevertheless activates AKT by downregulating the metastasis suppressor, EphB3. In addition, miR-29a robustly promotes invasion in PTEN-deficient glioblastoma cells by repressing translation of the Sox4 transcription factor, and this upregulates the invasion-promoting protein, HIC5. Indeed, we identified Sox4 as the most anti-correlated predicted target of miR-29a in glioblastoma. Importantly, inhibition of endogenous miR-29a decreases glioblastoma growth and invasion in vitro and in vivo, and increased miR-29a expression in glioblastoma specimens correlates with decreased patient survival. CONCLUSIONS: Taken together, these data identify miR-29a as a master regulator of glioblastoma growth and invasion.

Imaging of human mesenchymal stromal cells: homing to human brain tumors.

Human mesenchymal stromal cells (hMSC) can be used as a drug delivery vehicle for the treatment of GBM. However, tracking the migration and distribution of these transplanted cells is necessary to interpret therapeutic efficacy. We compared three labeling techniques for their ability to track the migration of transplanted hMSC in an orthotopic mouse xenograft model. hMSC were labeled with three different imaging tags (fluorescence, luciferase or ferumoxide) for imaging by fluorescence, bioluminescence or magnetic resonance imaging (MRI), respectively. hMSC were labeled for all imaging modalities without the use of transfection agents. The labeling efficacy of the tags was confirmed, followed by in vitro and in vivo migration assays to track hMSC migration towards U87 glioma cells. Our results confirmed that the labeled hMSC retained their migratory ability in vitro, similar to unlabeled hMSC. In addition, labeled hMSC migrated towards the U87 tumor site, demonstrating their retention of tumor tropism. hMSC tumor tropism was confirmed by all three imaging modalities; however, MRI provides both real time assessment and the high resolution needed for clinical studies. Our findings suggest that ferumoxide labeling of hMSC is feasible, does not alter their migratory ability and allows detection by MRI. Non invasive tracking of transplanted therapeutic hMSC in the brain will allow further development of human cell based therapies.

Cerebrospinal fluid biomarkers in idiopathic normal pressure hydrocephalus.

The diagnosis of idiopathic normal pressure hydrocephalus (iNPH) is still challenging. Alzheimer's disease (AD), along with vascular dementia, the most important differential diagnosis for iNPH, has several potential cerebrospinal fluid (CSF) biomarkers which might help in the selection of patients for shunt treatment. The aim of this study was to compare a battery of CSF biomarkers including well-known AD-related proteins with CSF from patients with suspected iNPH collected from the external lumbar drainage test (ELD). A total of 35 patients with suspected iNPH patients were evaluated with ELD. CSF was collected in the beginning of the test, and the concentrations of total tau, ptau(181), Aß(42), NFL, TNF-α, TGFß1, and VEGF were analysed by ELISA. Twenty-six patients had a positive ELD result-that is, their gait symptoms improved; 9 patients had negative ELD. The levels of all analyzed CSF biomarkers were similar between the groups and none of them predicted the ELD result in these patients. Contrary to expectations lumbar CSF TNF-α concentration was low in iNPH patients.

SNAI2/Slug promotes growth and invasion in human gliomas.

BACKGROUND: Numerous factors that contribute to malignant glioma invasion have been identified, but the upstream genes coordinating this process are poorly known. METHODS: To identify genes controlling glioma invasion, we used genome-wide mRNA expression profiles of primary human glioblastomas to develop an expression-based rank ordering of 30 transcription factors that have previously been implicated in the regulation of invasion and metastasis in cancer. RESULTS: Using this approach, we identified the oncogenic transcriptional repressor, SNAI2/Slug, among the upper tenth percentile of invasion-related transcription factors overexpressed in glioblastomas. SNAI2 mRNA expression correlated with histologic grade and invasive phenotype in primary human glioma specimens, and was induced by EGF receptor activation in human glioblastoma cells. Overexpression of SNAI2/Slug increased glioblastoma cell proliferation and invasion in vitro and promoted angiogenesis and glioblastoma growth in vivo. Importantly, knockdown of endogenous SNAI2/Slug in glioblastoma cells decreased invasion and increased survival in a mouse intracranial human glioblastoma transplantation model. CONCLUSION: This genome-scale approach has thus identified SNAI2/Slug as a regulator of growth and invasion in human gliomas.

Novel local drug delivery system using thermoreversible gel in combination with polymeric microspheres or liposomes.

BACKGROUND: The purpose of our study was to evaluate the application of thermoreversible gelation polymer (TGP) as a local drug delivery system for malignant glioma. MATERIALS AND METHODS: Polymeric microspheres or liposomes loaded with doxorubicin (sphere-dox or lipo-dox) were combined with TGP to provide continuous drug delivery of doxorubicin (dox) for kinetic release studies and cell viability assays on glioma cell lines in vitro. For in vivo studies, TGP loaded with dox alone (TGP-dox) was combined with sphere-dox or lipo-dox. Their antitumor effects on subcutaneous human glioma xenografts were evaluated in nude mice. RESULTS: In vitro, TGP combined with sphere-dox or lipo-dox released dox for up to 30 days. In vivo, TGP-dox combined with sphere-dox or lipo-dox inhibited subcutaneous glioma tumor growth until day 32 and day 38, respectively. CONCLUSION: TGP in combination with microspheres or liposomes successfully prolonged the release of dox and its antitumor effects.

Translational modulation of proteins expressed from bicistronic vectors.

Bicistronic vectors are useful tools for exogenous expression of two gene products from a single promoter element; however, reduced expression of protein from the second cistron compared with the first cistron is a common limitation to this approach. To overcome this limitation, we explored use of dihydrofolate reductase (DHFR) complementary DNA encoded in bicistronic vectors to induce a second protein of interest by methotrexate (MTX) treatment. Previous studies have demonstrated that levels of DHFR protein and DHFR fusion protein can be induced translationally following MTX treatment of cells. We demonstrated that in response to MTX treatment, DHFR partner protein in a bicistronic construct is induced for longer periods of time when compared with endogenous DHFR and DHFR fusion protein, in vitro and in vivo. Using rapamycin pretreatment followed by MTX treatment, we also devised a strategy to modulate levels of two proteins expressed from a bicistronic construct in a cap-independent manner. To our knowledge, this is the first report demonstrating that levels of proteins in DHFR-based bicistronic constructs can be induced and modulated using MTX and rapamycin treatment.

Human bone marrow-derived mesenchymal stromal cells expressing S-TRAIL as a cellular delivery vehicle for human glioma therapy.

Glioblastoma is among the most aggressive and treatment resistant of all human cancers. Conventional therapeutic approaches are unsuccessful because of diffuse infiltrative invasion of glioma tumor cells into normal brain parenchyma. Stem cell-based therapies provide a promising approach for the treatment of malignant gliomas because of their migratory ability to invasive tumor cells. Our therapeutic strategy was to use human bone marrow-derived mesenchymal stromal cells (hMSCs) as a cellular vehicle for the targeted delivery and local production of the biologic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) at the glioma tumor site. hMSCs were transduced with a lentivirus expressing secretable TRAIL (S-TRAIL) and mCherry (red fluorescent protein). Our results clearly demonstrate the retention of tumor tropic ability of hMSC S-TRAIL cells by in vitro and in vivo migration assays. In vitro assays confirmed the expression, release, and biological activity of S-TRAIL produced by hMSC S-TRAIL cells. For the in vivo assessment of therapeutic efficacy, hMSCs were injected ipsilateral to an established intracranial glioma tumor in a mouse xenograft model. Genetically engineered hMSC S-TRAIL cells were effective in inhibiting intracranial U87 glioma tumor growth (81.6%) in vivo and resulted in significantly longer animal survival. Immunohistochemical studies demonstrated significant, eight fold greater tumor cell apoptosis in the hMSC S-TRAIL-treated group than in controls. Our study demonstrates the therapeutic efficacy of hMSC S-TRAIL cells and confirms that hMSCs can serve as a powerful cell-based delivery vehicle for the site-specific release of therapeutic proteins.

Decreased levels of UMP kinase as a mechanism of fluoropyrimidine resistance.

5-Fluorouracil (5-FU) continues to be widely used for treatment of gastrointestinal cancers. Because many tumors show primary or acquired resistance, it is important to understand the molecular basis underlying the mechanism of resistance to 5-FU. In addition to its effect on thymidylate synthase inhibition and DNA synthesis, 5-FU may also influence RNA metabolism. Our previous studies revealed that colorectal cancer cells resistant to bolus 5-FU (HCT-8/4hFU) showed significantly decreased incorporation of the drug into RNA. Resistance to bolus 5-FU was associated with lower expression of UMP kinase (UMPK), an enzyme that plays an important role in the activation of 5-FU to 5-FUTP and its incorporation into RNA. Activities of other 5-FU-metabolizing enzymes (e.g., thymidine kinase, uridine phosphorylase, thymidine phosphorylase, and orotate phosphoribosyltransferase) remained unchanged between sensitive and resistant cell lines. Herein, we show that UMPK down-regulation in 5-FU-sensitive cells (HCT-8/P) induces resistance to bolus 5-FU treatment. Moreover, HCT-8/4hFU cells are even more cross-resistant to treatment with 5-fluorouridine, consistent with the current understanding of 5-fluorouridine as a RNA-directed drug. Importantly, colorectal cancer hepatic metastases isolated from patients clinically resistant to weekly bolus 5-FU/leucovorin treatment exhibited decreased mRNA expression of UMPK but not thymidylate synthase or dihydropyrimidine dehydrogenase compared with tumor samples of patients not previously exposed to 5-FU. Our findings provide new insights into the mechanisms of acquired resistance to 5-FU in colorectal cancer and implicate UMPK as an important mechanism of clinical resistance to pulse 5-FU treatment in some patients.

Local delivery of poly lactic-co-glycolic acid microspheres containing imatinib mesylate inhibits intracranial xenograft glioma growth.

PURPOSE: In an effort to develop new therapeutic strategies to treat malignant gliomas, we have designed poly (lactic-co-glycolic) acid (PLGA) microparticles that deliver imatinib mesylate, a small molecule tyrosine kinase inhibitor. The local continuous release of imatinib mesylate at the tumor site overcomes many obstacles associated with systemic delivery. EXPERIMENTAL DESIGN: Polymeric microspheres were prepared from various compositions of PLGA and loaded with imatinib mesylate. Imatinib release profiles, biological activity, and effect on PDGFR-B phosphorylation were confirmed in vitro. The therapeutic efficacy of imatinib microspheres was examined in two s.c. and orthotopic human glioblastoma xenograft models. RESULTS: A single local injection of PLGA microspheres loaded with a low concentration of imatinib mesylate led to 88% and 79% reduction in s.c. human (U87-MG) and murine (GL261) glioma tumors, respectively. PLGA-imatinib mesylate administered intracranially led to a 79% reduction in U87MG tumor volume. Immunohistochemical analysis showed a marked decrease in proliferation indices and tumor vessel density in the s.c. model and induction of apoptosis in an intracranial model. CONCLUSION: This is the first study to show the therapeutic efficacy of the local delivery of imatinib mesylate using a polymeric delivery system.

In vivo fate and therapeutic efficacy of PF-4/CTF microspheres in an orthotopic human glioblastoma model.

The correlation between glioma grade and angiogenesis suggests that antiangiogenic therapies are potentially therapeutically effective for these tumors. However, to achieve tumor suppression, antiangiogenic therapies need to be administered daily using high systemic quantities. We designed a biodegradable polymeric device that overcomes those barriers by providing sustained local delivery of a C-terminal fragment of platelet factor 4 (PF-4/CTF), an antiangiogenic agent. Fluorescent-labeled microspheres composed of poly lactic-coglycolic acid (PLGA) were loaded with rhodamine-labeled PF-4/CTF and formulated to release their contents over time. Fluorescent labeling enabled the correlation between the in vitro to the in vivo kinetic and release studies. PF-4/CTF microspheres were injected into established intracranial human glioma tumors in nude mice. Noninvasive magnetic resonance imaging (MRI) was used to assess the therapeutic response. Tumor size, microvessel density, proliferation, and apoptosis rate were measured by histological analysis. Intracranially, the microspheres were located throughout the tumor bed and continuously released PF-4/CTF during the entire experimental period. MRI and histological studies showed that a single injection of microspheres containing PF-4/CTF caused a 65.2% and 72% reduction in tumor volume, respectively, with a significant decrease in angiogenesis and an increase in apoptosis. Our data demonstrate that polymeric microspheres are an effective therapeutic approach for delivering antiangiogenic agents that result in the inhibition of glioma tumor growth.

Continuous intracranial administration of suberoylanilide hydroxamic acid (SAHA) inhibits tumor growth in an orthotopic glioma model.

OBJECT: Current treatments for malignant gliomas produce only a modest increase in survival time. New therapeutic approaches are desperately needed. Suberoylanilide hydroxamic acid (SAHA) is an effective inhibitor of the growth of many solid and hematological malignancies. Nevertheless, very few studies have investigated the effects of SAHA on glial tumors. The present study was designed to investigate the therapeutic effects of the intracranial local delivery of SAHA in an orthotopic glioma model. METHODS: The antiproliferative effect of SAHA was examined in six glioblastoma and one endothelial cell lines in vitro. In addition, one glioblastoma cell line (U87MG) used in in vivo short term (14 days) and survival studies in an orthotopic human glioma athymic mice model. Tumor volume, apoptosis rate, microvessel density, and proliferation index were determined by immunohistochemistry. RESULTS: SAHA treatment inhibited the growth of all cell lines in concentrations ranging from 1 microM to 30 microM. For short-term studies, histological analysis showed an 80% reduction of tumor volume in the treatment group (P < 0.001). This reduction in tumor volume was associated with a significant increase in the apoptosis rate (31.9%, P < 0.001), a significant decrease in the proliferation (36.8%, P < 0.001) and angiogenesis rates (30%, P < 0.05). For survival studies, the mean survival time was 22 days in the control group, whereas it was 42 days in the treatment group. CONCLUSIONS: These results suggest that local delivery with SAHA inhibits intracranial glioma growth in vitro and in vivo. SAHA is a promising candidate for further preclinical and clinical studies on glial tumors.

Differential gene expression associated with migration of mesenchymal stem cells to conditioned medium from tumor cells or bone marrow cells.

Distinct signals that guide migration of mesenchymal stem cells (MSCs) to specific in vivo targets remain unknown. We have used rat MSCs to investigate the molecular mechanisms involved in such migration. Rat MSCs were shown to migrate to tumor microenvironment in vivo, and an in vitro migration assay was used under defined conditions to permit further mechanistic investigations. We hypothesized that distinct molecular signals are involved in the homing of MSCs to tumor sites and bone marrow. To test this hypothesis, gene expression profiles of MSCs exposed in vitro to conditioned medium (CM) from either tumor cells or bone marrow were compared. Analysis of the microarray gene expression data revealed that 104 transcripts were upregulated in rat MSCs exposed to CM from C85 human colorectal cancer cells for 24 hours versus control medium. A subset of 12 transcripts were found to be upregulated in rat MSCs that were exposed to tumor cell CM but downregulated when MSCs were exposed to bone marrow CM and included CXCL-12 (stromal cell-derived factor-1 [SDF-1]), CXCL-2, CINC-2, endothelial cell specific molecule-1, fibroblast growth factor-7, nuclear factor-kappaB p105, and thrombomodulin. Exposure to tumor cell CM enhanced migration of MSCs and correlated with increased SDF-1 protein production. Moreover, knockdown of SDF-1 expression in MSCs inhibited migration of these cells to CM from tumor cells, but not bone marrow cells, confirming the importance of SDF-1 expression by MSCs in this differential migration. These results suggest that increased SDF-1 production by MSCs acts in an autocrine manner and is required for migratory responses to tumor cells.

Human neural stem cells target experimental intracranial medulloblastoma and deliver a therapeutic gene leading to tumor regression.

PURPOSE: Medulloblastoma, a malignant pediatric brain tumor, is incurable in about one third of patients despite multimodal treatments. In addition, current therapies can lead to long-term disabilities. Based on studies of the extensive tropism of neural stem cells (NSC) toward malignant gliomas and the secretion of growth factors common to glioma and medulloblastoma, we hypothesized that NSCs could target medulloblastoma and be used as a cellular therapeutic delivery system. EXPERIMENTAL DESIGN: The migratory ability of HB1.F3 cells (an immortalized, clonal human NSC line) to medulloblastoma was studied both in vitro and in vivo. As proof-of-concept, we used HB1.F3 cells engineered to secrete the prodrug activating enzyme cytosine deaminase. We investigated the potential of human NSCs to deliver a therapeutic gene and reduce tumor growth. RESULTS: The migratory capacity of HB1.F3 cells was confirmed by an in vitro migration assay, and corroborated in vivo by injecting chloromethylbenzamido-Dil-labeled HB1.F3 cells into the hemisphere contralateral to established medulloblastoma in nude mice. In vitro studies showed the therapeutic efficacy of HB1.F3-CD on Daoy cells in coculture experiments. In vitro therapeutic studies were conducted in which animals bearing intracranial medulloblastoma were injected ipsilaterally with HB1.F3-CD cells followed by systemic 5-flourocytosine treatment. Histologic analyses showed that human NSCs migrate to the tumor bed and its boundary, resulting in a 76% reduction of tumor volume in the treatment group (P<0.01). CONCLUSION: These studies show for the first time the potential of human NSCs as an effective delivery system to target and disseminate therapeutic agents to medulloblastoma.

Identification of amino acids required for the functional up-regulation of human dihydrofolate reductase protein in response to antifolate Treatment.

Human dihydrofolate reductase (DHFR) protein levels rapidly increase upon exposure to methotrexate, a potent inhibitor of this enzyme. A model to explain this increase proposes that DHFR inhibits its own translation by binding to its cognate mRNA and that methotrexate disrupts the DHFR protein-mRNA complex allowing its translation to resume. In the present study, Chinese hamster ovary cells lacking DHFR were transfected with wild type and mutants of human DHFR to identify amino acids that are essential for increases in DHFR in response to methotrexate. Glu-30, Leu-22, and Ser-118 were involved in the up-regulation of DHFR protein levels by methotrexate and certain other antifolates. Cells transfected with E30A, L22R, and S118A mutants that did not respond to methotrexate up-regulation had higher basal levels of DHFR, consistent with the model, i.e. lack of feedback regulation of these enzymes. Although cells containing the S118A mutant enzyme had higher levels of DHFR and had catalytic activity similar to that of wild type DHFR, they had the same sensitivity to the cytotoxicity of methotrexate, as were cells with wild type DHFR. This finding provides evidence that the adaptive up-regulation of DHFR by methotrexate contributes to the decreased sensitivity to this drug. Based on these observations, a new model is proposed whereby DHFR exists in two conformations, one bound to DHFR mRNA and the other bound to NADPH. The mutants that are not up-regulated by methotrexate are unable to bind their cognate mRNA.

Role of reporter gene imaging in molecular and cellular biology.

Molecular imaging, including reporter gene methods, provides a unique opportunity to study biology in a living subject, thereby allowing physiological events to be monitored in an intact microenvironment. This review takes a molecular and cell biology perspective on recent studies which utilize reporter gene imaging as a tool to non-invasively monitor specific molecular biology pathways in vivo. Studies in rodent models demonstrate the feasibility of reporter gene imaging to visualize and measure key cellular pathways, such as transcription, translation and protein-protein interactions. The review indicates that molecular imaging is likely to be useful in the translation of molecular biology to medicine and biotechnological applications.

Overexpression of E2F-1 in lung and liver metastases of human colon cancer is associated with gene amplification.

We have shown previously that metastatic tumors of human colorectal cancer in lung as compared to liver have high levels of thymidylate synthase (TS) mRNA expression that correlated with high levels of E2F-1 mRNA expression. We now report that Comparative Genomic Hybridization (CGH) and DNA PCR analyses of lung and liver metastases of human colon cancer show frequent gains in the region of chromosome 20q and have an increase in gene copy number of E2F-1. In as much as TS is transcriptionally regulated by E2F-1, these results provide an explanation for the high levels of TS mRNA noted in some tumor samples.

Tumor regression of B16F10 melanoma in vivo by prevention of neovascularization: study on theophylline.

The aim of this study was to determine the effect of theophylline on neovascularization and tumor regression in murine B16F10 melanoma. Theophylline had no direct toxicity to host and significantly reduced (p < 0.001) tumor volume and neovascularization in B16F10 melanoma implanted murine model. The effect of theophylline on neovascularization was observed distinctly in histologic analysis. This effect is mediated, in part by blocking endothelial cell proliferation, thereby preventing neovascularization of the tumor. Further investigations with theophylline can elucidate the exact mechanism of action which characterize neovascularization activity.