Elevated invasive potential of glioblastoma stem cells

Glioblastomas (GBMs) are the most lethal and common types of primary brain tumors. The hallmark of GBMs is their highly infiltrative nature. The cellular and molecular mechanisms underlying the aggressive cancer invasion in GBMs are poorly understood. GBM displays remarkable cellular heterogeneity and hierarchy containing self-renewing glioblastoma stem cells (GSCs). Whether GSCs are more invasive than non-stem tumor cells and contribute to the invasive phenotype in GBMs has not been determined. Here we provide experimental evidence supporting that GSCs derived from GBM surgical specimens or xenografts display greater invasive potential in vitro and in vivo than matched non-stem tumor cells. Furthermore, we identified several invasion-associated proteins that were differentially expressed in GSCs relative to non-stem tumor cells. One of such proteins is L1CAM, a cell surface molecule shown to be critical to maintain GSC tumorigenic potential in our previous study. Immunohistochemical staining showed that L1CAM is highly expressed in a population of cancer cells in the invasive fronts of primary GBMs. Collectively, these data demonstrate the invasive nature of GSCs, suggesting that disrupting GSCs through a specific target such as L1CAM may reduce GBM cancer invasion and tumor recurrence.     

Cancer stem cells in glioblastoma — molecular signaling and therapeutic targeting

Glioblastomas (GBMs) are highly lethal primary brain tumors. Despite current therapeutic advances in other solid cancers, the treatment of these malignant gliomas remains essentially palliative. GBMs are extremely resistant to conventional radiation and chemotherapies. We and others have demonstrated that a highly tumorigenic subpopulation of cancer cells called GBM stem cells (GSCs) promotes therapeutic resistance. We also found that GSCs stimulate tumor angiogenesis by expressing elevated levels of VEGF and contribute to tumor growth, which has been translated into a useful therapeutic strategy in the treatment of recurrent or progressive GBMs. Furthermore, stem cell-like cancer cells (cancer stem cells) have been shown to promote metastasis. Although GBMs rarely metastasize beyond the central nervous system, these highly infiltrative cancers often invade into normal brain tissues preventing surgical resection, and GSCs display an aggressive invasive phenotype. These studies suggest that targeting GSCs may effectively reduce tumor recurrence and significantly improve GBM treatment. Recent studies indicate that cancer stem cells share core signaling pathways with normal somatic or embryonic stem cells, but also display critical distinctions that provide important clues into useful therapeutic targets. In this review, we summarize the current understanding and advances in glioma stem cell research, and discuss potential targeting strategies for future development of anti-GSC therapies.     

Rosuvastatin reduces microglia in the brain of wild type and ApoE knockout mice on a high cholesterol diet; implications for prevention of stroke and AD

IGFBP2 is overexpressed in the most common brain tumor, glioblastoma (GBM), and its expression is inversely correlated to GBM patient survival. Previous reports have demonstrated a role for IGFBP2 in glioma cell invasion and astrocytoma development. However, the function of IGFBP2 in the restricted, self-renewing, and tumorigenic GBM cell population comprised of tumor-initiating stem cells has yet to be determined. Herein we demonstrate that IGFBP2 is overexpressed within the stem cell compartment of GBMs and is integral for the clonal expansion and proliferative properties of glioma stem cells (GSCs). In addition, IGFBP2 inhibition reduced Akt-dependent GSC genotoxic and drug resistance. These results suggest that IGFBP2 is a selective malignant factor that may contribute significantly to GBM pathogenesis by enriching for GSCs and mediating their survival. Given the current dearth of selective molecular targets against GSCs, we anticipate our results to be of high therapeutic relevance in combating the rapid and lethal course of GBM.     

Identification of Global DNA Methylation Signatures in Glioblastoma-Derived Cancer Stem Cells

Glioblastoma(GBM)is the most common and most aggressive primary brain tumor in adults.The existence of a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy is one proposed mechanism leading to the resilient behavior of tumor cells and poor prognosis.In this study,we performed an in-depth analysis of the DNA methylation landscape in GBMderived cancer stem cells(GSCs).Parallel comparisons of primary tumors and GSC lines derived from these tumors with normal controls(a neural stem cell(NSC)line and normal brain tissue)identified groups of hyper- and hypomethylated genes that display a trend of either increasing or decreasing methylation levels in the order of controls,primary GBMs,and their counterpart GSC lines,respectively.Interestingly,concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT,AJAP1 and PTPRN2.These unique DNA methylation signatures were also found in primary GBM-derived xenograft tumors indicating that they are not tissue culture-related epigenetic changes.Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down-regulated in GBMs such as SPINT2,NEFM and PENK.Forced re-expression of SPINT2 reduced glioma cell proliferative capacity,anchorage independent growth,cell motility,and tumor sphere formation in vitro.The results from this study demonstrate that GSCs possess unique epigenetic signatures that may play important roles in the pathogenesis of GBM.     

The Expression of Connexins and SOX2 Reflects the Plasticity of Glioma Stem-Like Cells

Glioblastoma (GBM) is the most malignant primary brain tumor, with an average survival rate of 15 months. GBM is highly refractory to therapy, and such unresponsiveness is due, primarily, but not exclusively, to the glioma stem-like cells (GSCs). This subpopulation express stem-like cell markers and is responsible for the heterogeneity of GBM, generating multiple differentiated cell phenotypes. However, how GBMs maintain the balance between stem and non-stem populations is still poorly understood. We investigated the GBM ability to interconvert between stem and non-stem states through the evaluation of the expression of specific stem cell markers as well as cell communication proteins. We evaluated the molecular and phenotypic characteristics of GSCs derived from differentiated GBM cell lines by comparing their stem-like cell properties and expression of connexins. We showed that non-GSCs as well as GSCs can undergo successive cycles of gain and loss of stem properties, demonstrating a bidirectional cellular plasticity model that is accompanied by changes on connexins expression. Our findings indicate that the interconversion between non-GSCs and GSCs can be modulated by extracellular factors culminating on differential expression of stem-like cell markers and cell-cell communication proteins. Ultimately, we observed that stem markers are mostly expressed on GBMs rather than on low-grade astrocytomas, suggesting that the presence of GSCs is a feature of high-grade gliomas. Together, our data demonstrate the utmost importance of the understanding of stem cell plasticity properties in a way to a step closer to new strategic approaches to potentially eliminate GSCs and, hopefully, prevent tumor recurrence.     

Multi-Kinase Inhibitor C1 Triggers Mitotic Catastrophe of Glioma Stem Cells Mainly through MELK Kinase Inhibition

Glioblastoma multiforme (GBM) is a highly lethal brain tumor. Due to resistance to current therapies, patient prognosis remains poor and development of novel and effective GBM therapy is crucial. Glioma stem cells (GSCs) have gained attention as a therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Previously, we identified that the mitotic kinase maternal embryonic leucine-zipper kinase (MELK) is highly expressed in GBM tissues, specifically in GSCs, and its expression is inversely correlated with the post-surgical survival period of GBM patients. In addition, patient-derived GSCs depend on MELK for their survival and growth both in vitro and in vivo. Here, we demonstrate evidence that the role of MELK in the GSC survival is specifically dependent on its kinase activity. With in silico structure-based analysis for protein-compound interaction, we identified the small molecule Compound 1 (C1) is predicted to bind to the kinase-active site of MELK protein. Elimination of MELK kinase activity was confirmed by in vitro kinase assay in nano-molar concentrations. When patient-derived GSCs were treated with C1, they underwent mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed with shRNA-mediated MELK knockdown. In addition, C1 treatment strongly induced tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuated growth of mouse intracranial tumors derived from GSCs in a dose-dependent manner. Lastly, C1 treatment sensitizes GSCs to radiation treatment. Collectively, these data indicate that targeting MELK kinase activity is a promising approach to attenuate GBM growth by eliminating GSCs in tumors.     

Increased microglia/macrophage gene expression in a subset of adult and pediatric astrocytomas

Glioblastoma (GBM) is a highly malignant brain tumor with a dismal prognosis. Gene expression profiling of GBM has revealed clinically relevant tumor subtypes, and this provides exciting opportunities to better understand disease pathogenesis. Results from an increasing number of studies demonstrate a role for the immune response in cancer progression, yet it is unclear how the immune response differs across tumor subtypes and how it affects outcome. Utilizing gene expression data from The Cancer Genome Atlas Project and the Gene Expression Omnibus database, we demonstrate an enrichment of immune response-related gene expression in the mesenchymal subtype of adult GBM (n = 173) and pediatric high-grade gliomas (n = 53). In an independent cohort of pediatric astrocytomas (n = 24) from UCSF, we stratified tumors into subtypes and confirmed these findings. Using novel immune cell-specific gene signatures we demonstrate selective enrichment of microglia/macrophage-related genes in adult and pediatric GBM tumors of the mesenchymal subtype. Furthermore, immunostaining of adult GBM tumors showed significantly higher cell numbers of microglia/macrophages in mesenchymal versus non-mesenchymal tumors (p = 0.04). Interestingly, adult GBM tumors with the shortest survival had significant enrichment of microglia/macrophage-related genes but this was not true for pediatric GBMs. Consistent with an association with poor outcome, immune response-related genes were highly represented in an adult poor prognosis gene signature, with the expression of genes related to macrophage recruitment and activation being most strongly associated with survival (p<0.05) using CoxBoost multivariate modeling. Using a microglia/macrophage high gene signature derived from quantification of tumor-infiltrating cells in adult GBM, we identified enrichment of genes characteristic of CD4 T cells, granulocytes, and microglia/macrophages (n = 573). These studies support a role for the immune response, particularly the microglia/macrophage response, in the biology of an important subset of GBM. Identification of this subset may be important for future therapeutic stratification.     

Glioblastoma multiforme: a perspective on recent findings in human cancer and mouse models

Gliomas are the most frequently occurring primary malignancies in the central nervous system, and glioblastoma multiforme (GBM) is the most common and most aggressive of these tumors. Despite vigorous basic and clinical studies over past decades, the median survival of patients with this disease remains at about one year. Recent studies have suggested that GBMs contain a subpopulation of tumor cells that displays stem cell characteristics and could therefore be responsible for in vivo tumor growth. We will summarize the major oncogenic pathways abnormally regulated in gliomas, and review the recent findings from mouse models that our laboratory as well as others have developed for the study of GBM. The concept of cancer stem cells in GBM and their potential therapeutic importance will also be discussed.     

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers patients only modest benefits, as most patients do not survive more than five years following diagnosis due to glioma relapse ^1,2. The discovery of cancer stem cells in human brain tumors holds promise for having an enormous impact on the development of novel therapeutic strategies for glioma ³. Cancer stem cells are defined by their ability both to self-renew and to differentiate, and are thought to be the only cells in a tumor that have the capacity to initiate new tumors ⁴. Glioma relapse following radiation therapy is thought to arise from resistance of glioma stem cells (GSCs) to therapy ^5-10. In vivo, GSCs are shown to reside in a perivascular niche that is important for maintaining their stem cell-like characteristics ^11-14. Central to the organization of the GSC niche are vascular endothelial cells ¹². Existing evidence suggests that GSCs and their interaction with the vascular endothelial cells are important for tumor development, and identify GSCs and their interaction with endothelial cells as important therapeutic targets for glioma. The presence of GSCs is determined experimentally by their capability to initiate new tumors upon orthotopic transplantation ¹⁵. This is typically achieved by injecting a specific number of GBM cells isolated from human tumors into the brains of severely immuno-deficient mice, or of mouse GBM cells into the brains of congenic host mice. Assays for tumor growth are then performed following sufficient time to allow GSCs among the injected GBM cells to give rise to new tumors-typically several weeks or months. Hence, existing assays do not allow examination of the important pathological process of tumor initiation from single GSCs in vivo. Consequently, essential insights into the specific roles of GSCs and their interaction with the vascular endothelial cells in the early stages of tumor initiation are lacking. Such insights are critical for developing novel therapeutic strategies for glioma, and will have great implications for preventing glioma relapse in patients. Here we have adapted the PoRTS cranial window procedure ¹⁶and in vivo two-photon microscopy to allow visualization of tumor initiation from injected GBM cells in the brain of a live mouse. Our technique will pave the way for future efforts to elucidate the key signaling mechanisms between GSCs and vascular endothelial cells during glioma initiation.     

Glioblastoma stem cells:Molecular characteristics and therapeutic implications

Glioblastoma Multiforme(GBM)is a grade IV astrocytoma,with a median survival of 14.6 mo.Within GBM,stem-like cells,namely glioblastoma stem cells(GSCs),have the ability to self-renew,differentiate into distinct lineages within the tumor and initiate tumor xenografts in immunocompromised animal models.More importantly,GSCs utilize cell-autonomous and tumor microenvironment-mediated mechanisms to overcome current therapeutic approaches.They are,therefore,very important therapeutic targets.Although the functional criteria defining GSCs are well defined,their molecular characteristics,the mechanisms whereby they establish the cellular hierarchy within tumors,and their contribution to tumor heterogeneity are not well understood.This review is aimed at summarizing current findings about GSCs and their therapeutic importance from a molecular and cellular point of view.A better characterization of GSCs is crucial for designing effective GSCtargeted therapies.     

Inhibition of Caveolin-1 Restores Myeloid Cell Function in Human Glioblastoma

Background

Gliomas are the most common primary brain tumor in both children and adults. The prognosis for glioblastoma (GBM), the most common type of malignant glioma, has remained dismal, with median survival a little over one year despite maximal therapy with surgery, chemotherapy, and radiation. Although immunotherapy has become increasingly successful against many systemic tumors, clinical efficacy against brain tumors has been limited. One reason for this is an incomplete understanding of the local immunologic tumor microenvironment, particularly the function of large numbers of infiltrating myeloid derived cells. Monocytes/microglia are myeloid derived immunomodulatory cells, and they represent the predominant infiltrating immune cell population in gliomas. Our group has previously demonstrated using complementary in vitro and in vivo approaches that GBM tumor cells polarize tumor-associated myeloid cells (TAMs) and suppress their immunostimulatory function.

Methods and Results

To better understand the mechanisms responsible for this immunosuppression, we used gene expression profiling of stimulated monocytes in the presence or absence of GBM tumor cells. Our analysis identified caveolin-1 (CAV1), a plasma membrane molecule with pleiotropic functions, as significantly up-regulated in monocytes in the presence of GBMs. We validated these findings ex vivo by confirming up-regulation of CAV1 in TAMs isolated from GBMs immediately after surgical resection. Finally, we demonstrate that siRNA inhibition of CAV1 restores myeloid cell function, as measured by TNF-alpha secretion, in the presence of GBMs.

Conclusions

Restoration of TAM function through pharmacologic blockage of CAV1 may facilitate more successful immunotherapeutic strategies directed against a variety of solid human tumors infiltrated by TAMs.     

The Promoter Hypermethylation Status of GATA6, MGMT, and FHIT in Glioblastoma

Glioblastoma (GBM) is an aggressive and lethal cancer, accounting for the majority of primary brain tumors in adults. GBMs are characterized by large and small alterations in genes that control cell growth, apoptosis, angiogenesis, and invasion. Epigenetic alterations also affect the expression of cancer genes, either alone or in combination with genetic mechanisms. The current evidence suggests that hypermethylation of promoter CpG islands is a common epigenetic event in a variety of human cancers. A subset of GBMs is also characterized by a locus-specific and genome-wide decrease in DNA methylation. Epigenetic alterations are important in the molecular pathology of GBM. However, there are very limited data about these epigenetic alterations in GBM. Alterations in promoter methylations are important to understand because histone deacetylases are targets for drugs that are in clinical trial for GBMs. The aim of the current study was to investigate whether the promoter hypermethylation of putative tumor suppressor genes was involved in GBM. We examined the methylation status at the promoter regions of GATA6, MGMT, and FHIT using the methylation-specific polymerase chain reaction in 61 primary GBMs. Our results reveal that there is no promoter hypermethylation of FHIT in the examined GBM tissue specimens. In contrast, the promoter hypermethylation of GATA6 and MGMT was detected in 42.8 and 11.11% of GBMs, respectively. The frequency of MGMT promoter hypermethylation was low in the group of patients we evaluated. In conclusion, our study demonstrates that promoter hypermethylation of MGMT is a common event in GBMs, whereas GATA6 is epigenetically affected in GBMs. Furthermore, inactivation of FHIT by epigenetic mechanisms in GBM may not be associated with brain tumorigenesis.     

Retinoblastoma protein regulates the crosstalk between autophagy and apoptosis,and favors glioblastoma resistance to etoposide

Glioblastomas (GBMs) are devastating tumors of the central nervous system, with a poor prognosis of 1-year survival. This results from a high resistance of GBM tumor cells to current therapeutic options, including etoposide (VP-16). Understanding resistance mechanisms may thus open new therapeutic avenues. VP-16 is a topoisomerase inhibitor that causes replication fork stalling and, ultimately, the formation of DNA double-strand breaks and apoptotic cell death. Autophagy has been identified as a VP-16 treatment resistance mechanism in tumor cells. Retinoblastoma protein (RB) is a classical tumor suppressor owing to its role in G1/S cell cycle checkpoint, but recent data have shown RB participation in many other cellular functions, including, counterintuitively, negative regulation of apoptosis. As GBMs usually display an amplification of the EGFR signaling involving the RB protein pathway, we questioned whether RB might be involved in mechanisms of resistance of GBM cells to VP-16. We observed that RB silencing increased VP-16-induced DNA double-strand breaks and p53 activation. Moreover, RB knockdown increased VP-16-induced apoptosis in GBM cell lines and cancer stem cells, the latter being now recognized essential to resistance to treatments and recurrence. We also showed that VP-16 treatment induced autophagy, and that RB silencing impaired this process by inhibiting the fusion of autophagosomes with lysosomes. Taken together, our data suggest that RB silencing causes a blockage on the VP-16-induced autophagic flux, which is followed by apoptosis in GBM cell lines and in cancer stem cells. Therefore, we show here, for the first time, that RB represents a molecular link between autophagy and apoptosis, and a resistance marker in GBM, a discovery with potential importance for anticancer treatment.