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.     

Conversion of differentiated cancer cells into cancer stem-like cells in a glioblastoma model after primary chemotherapy

Glioblastoma multiforme patients have a poor prognosis due to therapeutic resistance and tumor relapse. It has been suggested that gliomas are driven by a rare subset of tumor cells known as glioma stem cells (GSCs). This hypothesis states that only a few GSCs are able to divide, differentiate, and initiate a new tumor. It has also been shown that this subpopulation is more resistant to conventional therapies than its differentiated counterpart. In order to understand glioma recurrence post therapy, we investigated the behavior of GSCs after primary chemotherapy. We first show that exposure of patient-derived as well as established glioma cell lines to therapeutic doses of temozolomide (TMZ), the most commonly used antiglioma chemotherapy, consistently increases the GSC pool over time both in vitro and in vivo. Secondly, lineage-tracing analysis of the expanded GSC pool suggests that such amplification is a result of a phenotypic shift in the non-GSC population to a GSC-like state in the presence of TMZ. The newly converted GSC population expresses markers associated with pluripotency and stemness, such as CD133, SOX2, Oct4, and Nestin. Furthermore, we show that intracranial implantation of the newly converted GSCs in nude mice results in a more efficient grafting and invasive phenotype. Taken together, these findings provide the first evidence that glioma cells exposed to chemotherapeutic agents are able to interconvert between non-GSCs and GSCs, thereby replenishing the original tumor population, leading to a more infiltrative phenotype and enhanced chemoresistance. This may represent a potential mechanism for therapeutic relapse.Glioblastoma multiforme (GBM) is a heterogeneous, highly invasive brain tumor, which is treated with a multimodal approach that includes surgery followed by radio- and chemotherapy.¹ Temozolomide (TMZ) is currently the best chemotherapeutic drug available on the market against malignant glioma because of its ability to cross the blood–brain barrier (BBB). Even after such an aggressive therapeutic intervention, disease relapse is inevitable due to GBM''s infiltrative nature and ability to resist conventional therapies.^{2, 3} Thus, understanding the mechanisms of therapeutic escape and disease recurrence is crucial for developing more effective treatments against GBM.GBMs are among the first solid tumors in which the discovery of stem-like tumor-initiating cells has suggested the existence of a hierarchical model of tumorigenesis. Such a dogma proposes that a distinct population of tumor cells, referred to as glioma stem cells (GSCs), are not only responsible for driving tumor growth, but also represent a population that can survive intensive oncological therapies and give rise to recurrent malignancies.^{4, 5} In the clinical setting, the presence of CD133+ GSCs correlates with a shorter overall survival as well as reduced progression-free survival and is considered a critical target for successful antiglioma therapies.⁶The inability of conventional treatments, such as radio- and chemotherapies, to exterminate all infiltrative tumor foci is considered one of the main causes of therapeutic failure and malignant recurrence in GBM. Although the radio-resistance properties of glioma cells are fairly well established, the underlying molecular mechanisms of chemoresistance have been addressed only in a few studies.^{7, 8} On the basis of this, we set to investigate the biology of GSCs following TMZ therapy both in vitro and in vivo. We observed significant expansion of different GSC subpopulations after exposure to TMZ at the plasma (50 μM) and cerebral spinal fluid (CSF; 5 μM) concentrations detected in GBM patients.^{9, 10, 11, 12} This expansion arises from the high degree of plasticity that exists within glioma cell populations. After long-term exposure to therapeutic concentrations of TMZ, differentiated tumor cells convert into glioma stem-like cells. These newly formed GSCs acquire phenotypic and functional characteristics similar to those of native GSCs. Once implanted orthotopically in the animal brain, these newly converted GSCs demonstrate a very invasive characteristic similar to that of parental GSCs. In light of these findings, we propose that TMZ may induce specific changes in the tumor microenvironment, which facilitate a GSC-specific ‘niche'', thereby providing the necessary contextual signals to initiate the interconversion between differentiated tumor cells and GSCs. Therefore, such cellular plasticity represents a new mechanism for therapeutic resistance in GBM, and understanding this may allow us to optimize TMZ-based antiglioma chemotherapy.     

The cancer stem cell niche(s): The crosstalk between glioma stem cells and their microenvironment

Background

The initiation and progression of various types of tumors, including glioma, are driven by a population of cells with stem cell properties. Glioma stem cells (GSCs) are located in specialized microenvironments (niches) within tumors. These niches represent the hallmarks of malignant gliomas (vascular proliferations, hypoxia/necrosis) and bear analogy to the microenvironments in which physiological stem cells in the brain are found.

Scope of the review

Here we review the progress that has been made towards uncovering the function of the perivascular and the hypoxic niche and the molecular pathways that control the properties of GSCs within them. We propose models of how the different niches and GSC pools in them interact with each other.

Major conclusions

GSCs are not merely passive residents of their niches, but actively contribute to the shaping of the niches through a complex crosstalk with different components of the microenvironment. For example, GSCs play a dominant role in promoting new blood vessel formation through a variety of mechanisms, including the hypoxia dependent stimulation of angiogenesis, recruitment of endothelial progenitor cells and direct transdifferentiation into endothelial cells. Recent work has also revealed that GSCs can recruit and modulate the function of various immune cells to suppress anti-tumor immune responses and to foster tumor-promoting inflammation, which in turn could support the maintenance of GSCs.

General significance

These findings underscore the central role of the GSC microenvironment in driving glioma progression making the GSC niche a prime therapeutic target for the design of therapies aimed at eradicating GSCs.This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Cell quiescence correlates with enhanced glioblastoma cell invasion and cytotoxic resistance

Glioblastoma (GBM) tumor cells exhibit drug resistance and are highly infiltrative. GBM stem cells (GSCs), which have low proliferative capacity are thought to be one of the sources of resistant cells which result in relapse/recurrence. However, the molecular mechanisms regulating quiescent-specific tumor cell biology are not well understood. Using human GBM cell lines and patient-derived GBM cells, Oregon Green dye retention was used to identify and isolate the slow-cycling, quiescent-like cell subpopulation from the more proliferative cells in culture. Sensitivity of cell subpopulations to temozolomide and radiation, as well as the migration and invasive potential were measured. Differential expression analysis following RNAseq identified genes enriched in the quiescent cell subpopulation. Orthotopic transplantation of cells into mice was used to compare the in vivo malignancy and invasive capacity of the cells. Proliferative quiescence correlated with better TMZ resistance and enhanced cell invasion, in vitro and in vivo. RNAseq expression analysis identified genes involved in the regulation cell invasion/migration and a three-gene signature, TGFBI, IGFBP3, CHI3L1, overexpressed in quiescent cells which correlates with poor GBM patient survival.     

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.     

Serum-Induced Differentiation of Glioblastoma Neurospheres Leads to Enhanced Migration/Invasion Capacity That Is Associated with Increased MMP9

Glioblastoma (GBM) is a highly infiltrative brain tumor in which cells with properties of stem cells, called glioblastoma stem cells (GSCs), have been identified. In general, the dominant view is that GSCs are responsible for the initiation, progression, invasion and recurrence of this tumor. In this study, we addressed the question whether the differentiation status of GBM cells is associated with their invasive capacity. For this, several primary GBM cell lines were used, cultured either as neurospheres known to enrich for GSCs or in medium supplemented with 10% FCS that promotes differentiation. The differentiation state of the cells was confirmed by determining the expression of stem cell and differentiation markers. The migration/invasion potential of these cells was tested using in vitro assays and intracranial mouse models. Interestingly, we found that serum-induced differentiation enhanced the invasive potential of GBM cells, which was associated with enhanced MMP9 expression. Chemical inhibition of MMP9 significantly reduced the invasive potential of differentiated cells in vitro. Furthermore, the serum-differentiated cells could revert back to an undifferentiated/stem cell state that were able to form neurospheres, although with a reduced efficiency as compared to non-differentiated counterparts. We propose a model in which activation of the differentiation program in GBM cells enhances their infiltrative potential and that depending on microenvironmental cues a significant portion of these cells are able to revert back to an undifferentiated state with enhanced tumorigenic potential. Thus, effective therapy should target both GSCs and differentiated offspring and targeting of differentiation-associated pathways may offer therapeutic opportunities to reduce invasive growth of GBM.     

Evaluation of CD98 light chain-LAT1 as a potential marker of cancer stem-like cells in glioblastoma

ObjectiveGlioma stem cells (GSCs) are a minority population of glioma cells that regarded as the cause of tumor formation and recurrence. Identifying new molecular strategies targeting GSCs must be urgently developed to treat glioblastoma. In this study, one of CD98 light chain-L type amino acid transporter 1 (LAT1) was found as a potential GSC marker. LAT1 served as EAA transporter has been shown to be closely related with tumor invasion, metastasis, angiogenesis, and radiosensitivity.MethodsLAT1⁺ and LAT1^? glioma cells were sorted by flow cytometry. Cellular immunofluorescence, sphere-formation arrays, and in vitro limiting dilution experiments were used to identify cell stemness. Differentiated glioma stem cells were cultured, and the expressions of β-tubulinIII, GFAP, and LAT1 were detected by Western blot. Nude mouse models were constructed to observe tumor formation and metastasis in nude mice.ResultsLAT1⁺ glioma cells were testified a small percentage of all cells and selected as the subsequent sorting marker. LAT1⁺ cells were separated from U87 and U251 cells could express high level of stem cell markers, and possessed GSC properties including self-renewal ability and multi-directional differentiation potential. But LAT1^? cells did not have these characteristics. In addition, LAT1⁺ cells were able to generate tumors in vivo, tumor size of LAT1⁺ cells formed were much bigger than that of LAT1^? cells.ConclusionOur study, including molecular, cell, vitro and vivo experiments, has shown that LAT1⁺ cells possess GSC properties, and present for the first time that LAT1 can be used as a new marker for GSCs screening.     

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.     

Fasting Enhances the Response of Glioma to Chemo- and Radiotherapy

Background

Glioma, including anaplastic astrocytoma and glioblastoma multiforme (GBM) are among the most commonly diagnosed malignant adult brain tumors. GBM is a highly invasive and angiogenic tumor, resulting in a 12 to 15 months median survival. The treatment of GBM is multimodal and includes surgical resection, followed by adjuvant radio-and chemotherapy. We have previously reported that short-term starvation (STS) enhances the therapeutic index of chemo-treatments by differentially protecting normal cells against and/or sensitizing tumor cells to chemotoxicity.

Methodology and Principal Findings

To test the effect of starvation on glioma cells in vitro, we treated primary mouse glia, murine GL26, rat C6 and human U251, LN229 and A172 glioma cells with Temozolomide in ad lib and STS mimicking conditions. In vivo, mice with subcutaneous or intracranial models of GL26 glioma were starved for 48 hours prior to radio- or chemotherapy and the effects on tumor progression and survival were measured. Starvation-mimicking conditions sensitized murine, rat and human glioma cells, but not primary mixed glia, to chemotherapy. In vivo, starvation for 48 hours, which causes a significant reduction in blood glucose and circulating insulin-like growth factor 1 (IGF-1) levels, sensitized both subcutaneous and intracranial glioma models to radio-and chemotherapy.

Conclusion

Starvation-induced cancer sensitization to radio- or chemotherapy leads to extended survival in the in vivo glioma models tested. These results indicate that fasting and fasting-mimicking interventions could enhance the efficacy of existing cancer treatments against aggressive glioma in patients.     

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy ¹. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM ². This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments ^3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.     

Paradoxical role of β8 integrin on angiogenesis and vasculogenic mimicry in glioblastoma

Glioblastoma multiforme (GBM) is the most aggressive and highly vascularized brain tumor with poor prognosis. Endothelial cell-dependent angiogenesis and tumor cell-dependent Vasculogenic mimicry (VM) synergistically contribute to glioma vascularization and progression. However, the mechanism underlying GBM vascularization remains unclear. In this study, GBM stem cells (GSCs) were divided into high and low β8 integrin (ITGB8) subpopulations. Co-culture assays followed by Cell Counting Kit-8 (CCK-8), migration, Matrigel tube formation, and sprouting assays were conducted to assess the proliferative, migratory and angiogenic capacity of GBM cells and human brain microvascular endothelial cells (hBMECs). An intracranial glioma model was constructed to assess the effect of ITGB8 on tumor vascularization in vivo. Our results indicated that ITGB8 expression was elevated in GSCs and positively associated with stem cell markers in glioma tissues, and could be induced by hypoxia and p38 activation. ITGB8 in GSCs inhibited the angiogenesis of hBMECs in vitro, while it promoted the ability of network formation and expression of VM-related proteins. The orthotopic GBM model showed that ITGB8 contributed to decreased angiogenesis, meanwhile enhanced invasiveness and VM formation. Mechanistic studies indicated that ITGB8-TGFβ1 axis modulates VM and epithelial-mesenchymal transition (EMT) process via Smad2/3-RhoA signaling. Together, our findings demonstrated a differential role for ITGB8 in the regulation of angiogenesis and VM formation in GBM, and suggest that pharmacological inhibition of ITGB8 may represent a promising therapeutic strategy for treatment of GBM.Subject terms: Cancer stem cells, CNS cancer     

Lyn Facilitates Glioblastoma Cell Survival under Conditions of Nutrient Deprivation by Promoting Autophagy

Members of the Src family kinases (SFK) can modulate diverse cellular processes, including division, death and survival, but their role in autophagy has been minimally explored. Here, we investigated the roles of Lyn, a SFK, in promoting the survival of human glioblastoma tumor (GBM) cells in vitro and in vivo using lentiviral vector-mediated expression of constitutively-active Lyn (CA-Lyn) or dominant-negative Lyn (DN-Lyn). Expression of either CA-Lyn or DN-Lyn had no effect on the survival of U87 GBM cells grown under nutrient-rich conditions. In contrast, under nutrient-deprived conditions (absence of supplementation with L-glutamine, which is essential for growth of GBM cells, and FBS) CA-Lyn expression enhanced survival and promoted autophagy as well as inhibiting cell death and promoting proliferation. Expression of DN-Lyn promoted cell death. In the nutrient-deprived GBM cells, CA-Lyn expression enhanced AMPK activity and reduced the levels of pS6 kinase whereas DN-Lyn enhanced the levels of pS6 kinase. Similar results were obtained in vitro using another cultured GBM cell line and primary glioma stem cells. On propagation of the transduced GBM cells in the brains of nude mice, the CA-Lyn xenografts formed larger tumors than control cells and autophagosomes were detectable in the tumor cells. The DN-Lyn xenografts formed smaller tumors and contained more apoptotic cells. Our findings suggest that on nutrient deprivation in vitro Lyn acts to enhance the survival of GBM cells by promoting autophagy and proliferation as well as inhibiting cell death, and Lyn promotes the same effects in vivo in xenograft tumors. As the levels of Lyn protein or its activity are elevated in several cancers these findings may be of broad relevance to cancer biology.     

Rewiring of purine metabolism in response to acidosis stress in glioma stem cells

Glioma stem cells (GSCs) contribute to therapy resistance and poor outcomes for glioma patients. A significant feature of GSCs is their ability to grow in an acidic microenvironment. However, the mechanism underlying the rewiring of their metabolism in low pH remains elusive. Here, using metabolomics and metabolic flux approaches, we cultured GSCs at pH 6.8 and pH 7.4 and found that cells cultured in low pH exhibited increased de novo purine nucleotide biosynthesis activity. The overexpression of glucose-6-phosphate dehydrogenase, encoded by G6PD or H6PD, supports the metabolic dependency of GSCs on nucleotides when cultured under acidic conditions, by enhancing the pentose phosphate pathway (PPP). The high level of reduced glutathione (GSH) under acidic conditions also causes demand for the PPP to provide NADPH. Taken together, upregulation of G6PD/H6PD in the PPP plays an important role in acidic-driven purine metabolic reprogramming and confers a predilection toward glioma progression. Our findings indicate that targeting G6PD/H6PD, which are closely related to glioma patient survival, may serve as a promising therapeutic target for improved glioblastoma therapeutics. An integrated metabolomics and metabolic flux analysis, as well as considering microenvironment and cancer stem cells, provide a precise insight into understanding cancer metabolic reprogramming.