Potential Of Glioblastoma Stem Cells Research Articles

REST-DRD2 mechanism impacts glioblastoma stem cell-mediated tumorigenesis.

Glioblastoma (GBM) is a lethal, heterogeneous human brain tumor, with regulatory mechanisms that have yet to be fully characterized. Previous studies have indicated that the transcriptional repressor REST (repressor element-1 silencing transcription factor) regulates the oncogenic potential of GBM stem cells (GSCs) based on level of expression. However, how REST performs its regulatory role is not well understood. We examined 2 independent high REST (HR) GSC lines using genome-wide assays, biochemical validations, gene knockdown analysis, and mouse tumor models. We analyzed in-house patient tumors and patient data present in The Cancer Genome Atlas (TCGA). Genome-wide transcriptome and DNA-binding analyses suggested the dopamine receptor D2 (DRD2) gene, a dominant regulator of neurotransmitter signaling, as a direct target of REST. Biochemical analyses and mouse intracranial tumor models using knockdown of REST and double knockdown of REST and DRD2 validated this target and suggested that DRD2 is a downstream target of REST regulating tumorigenesis, at least in part, through controlling invasion and apoptosis. Further, TCGA GBM data support the presence of the REST-DRD2 axis and reveal that high REST/low DRD2 (HRLD) and low REST/high DRD2 (LRHD) tumors are specific subtypes, are molecularly different from the known GBM subtypes, and represent functional groups with distinctive patterns of enrichment of gene sets and biological pathways. The inverse HRLD/LRHD expression pattern is also seen in in-house GBM tumors. These findings suggest that REST regulates neurotransmitter signaling pathways through DRD2 in HR-GSCs to impact tumorigenesis. They further suggest that the REST-DRD2 mechanism forms distinct subtypes of GBM.

Abstract 1113: Mir-194-3p suppresses tumorigenic potential of glioblastoma stem cells by targeting the canonical NF-kB pathway

Abstract Glioblastoma (GBM) is the most common tumor of the central nervous system, with its deadly nature owing to high rates of invasion and angiogenesis. A key element in its pathogenesis is a small subpopulation of neoplastic cells called glioma stem cells (GSCs) that are responsible for tumor initiation, maintenance and relapse. Approaches to target GSCs have mostly proved futile due to their chemo- and radio-resistance potential. Given that microRNAs carry a potential to affect both normal and stem cells, we employed a microRNA-based targeted approach to identify the subset of microRNAs that could distinctively affect the stemness of GSCs. Our study tests the hypothesis that GSCs will show an altered miRNA expression upon their differentiation, and their profiling may lead to identification of specific microRNAs that regulate the process. Our initial efforts resulted in identification of hsa-miR-194-3p as one of the potential candidates, which was consistently downregulated in multiple GSCs that we investigated. Stable cell lines overexpressing hsa-miR-194-3p showed a decreased proliferation rate and reduced sphere-forming ability. In silico analysis revealed TAB2, an upstream activator of NF-κB pathway to be the potential target of hsa-miR-194-3p, which was confirmed by luciferase assay and knockdown approaches. The regulatory role of hsa-miR-194-3p was substantiated by its ability to inhibit the NF-κB pathway and the corresponding downstream targets including IL-6 and STAT3. In vivo studies revealed a reduced tumor forming ability when the cell lines overexpressing hsa-miR-194-3p were injected intracranially in NOD/SCID mice, compared with the non-target control. Our results indicate that hsa-miR-194-3p blocked the canonical NF-κB signaling and negatively affected tumor formation. We have carried out this study this in a unique model system expecting it to uncover another layer of complexity that may add a new facet to meet the challenge imposed by the cancer stem cell paradigm. In conclusion, we accentuate the anti-tumorigenic role of hsa-miR-194-3p as a connecting link to both stemness and NF-κB signaling. Funding sources: This work was supported by R01CA108633 (To AC), 1RC2CA148190 (To AC) U10CA180850-01 (To AC), 1R01CA169368 (To AC) from the National Cancer Institute (NCI), Brain Tumor Funders Collaborative Grant (To AC), Ohio State University Comprehensive Cancer Center Award (To AC). Citation Format: Rajbir Singh, Kamalakannan Palanichamy, John R. Jacob, Arnab Chakravarti. Mir-194-3p suppresses tumorigenic potential of glioblastoma stem cells by targeting the canonical NF-kB pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1113.

Abstract 1986: CEBPD promotes stemness in the pathogenesis of gliomas

Abstract Glioblastoma multiforme (GBM) is the most common and lethal type of brain tumor with a median survival of 12-15 months despite optimal therapy. Nowadays, an increasing number of studies has indicated the connection between malignancy and stemness. The molecular mechanisms controlling the stem cell-like properties and tumorigenic potential of glioblastoma stem cells (GSCs) remain less investigated. CCAAT/Enhancer binding protein delta (CEBPD) is a C/EBP family of transcription factors. In this study, CEBPD was highly expressed in GBM specimens and GBM patients with low levels of CEBPD show a significantly better overall survival. The transcription factor OCT4 is known to play an important role in maintaining stem cell self-renewal within the central nervous system (CNS) and this activity is present in gliomas as well. Further, our data showed that CEBPD contributes to stem-like characteristics and promoted cancer cells toward stem cell traits through direct activation of OCT4 gene expression. In conclusion, this study highlights the involvement of CEBPD in enchaining stem-cell property in GBM. Citation Format: Shao-Ming Wang, Chiung-Yuan Ko, Ju-Ming Wang. CEBPD promotes stemness in the pathogenesis of gliomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1986.

Isoflurane enhances the malignant potential of glioblastoma stem cells by promoting their viability, mobility in vitro and migratory capacity in vivo

BackgroundIsoflurane is one of the most common general anaesthetics used during surgical procedures, including tumour resection. However, the effects of isoflurane on the viability and migration capacity of cancer cells, specifically in the context of brain cancer cells, remain unclear. Therefore, the aim of this study was to evaluate the influence that isoflurane has on the function of glioblastoma stem cells (GCSs) in regards to cell proliferation, survival and migration. MethodU251-GSCs were exposed to isoflurane at clinically relevant concentrations and incubation times. The effects on proliferation, survival and migration capacities of the cells were evaluated in vitro. The potential risk was assessed in mice by intracranial injection of U251-GSCs pretreated with isoflurane. Furthermore, the average tumour volume and migration distance of U251-GSCs from the tumour centre were calculated. ResultsExposure of U251-GSCs to 1.2% isoflurane for 6 h resulted in increased proliferation (P<0.05) and decreased apoptosis rate (P<0.05) when compared with the control group. In addition, isoflurane exposure caused increased migration capacity in vitro (P<0.05) and the distance migrated was increased in vivo (P<0.05). ConclusionClinically relevant concentrations and incubation times of isoflurane could promote the viability and mobility of U251-GSCs, suggesting this general anaesthetic may have detrimental effects in glioblastoma by facilitating its growth and migration.

Abstract 3014: Hyaluronan-mediated motility receptor maintains stemness and tumorigenic potential of glioblastoma stem cells

Abstract Glioblastoma (GBM) stem cells (GSCs) are a subpopulation of tumor cells that display stem-like characteristics (stemness) and play unique roles in tumor propagation, therapeutic resistance and tumor recurrence. It is becoming increasingly important to identify novel therapeutic targets in GSC for anti-GBM therapies. Here, we demonstrate that hyaluronan-mediated motility receptor (HMMR) is hyper-expressed in GBM tumors and supports the self-renewal and tumorigenic potential of GSCs. HMMR silencing impairs GSC self-renewal and inhibits the expression of GSC markers and regulators. HMMR silencing suppresses GSC-derived tumor growth and extends the survival of mice bearing GSC xenografts. HMMR overexpression promotes GSC self-renewal and intracranial tumor propagation. In human GBM tumor specimens, HMMR expression is positively correlated with the expression of cell-stemness-associated markers and regulators. Our findings identify HMMR as a potential therapeutic target for inhibiting GSCs, suggesting therapeutic applications against GBM. Citation Format: Jessica Tilghman, John Laterra, Mingyao Ying. Hyaluronan-mediated motility receptor maintains stemness and tumorigenic potential of glioblastoma stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3014. doi:10.1158/1538-7445.AM2014-3014

The zinc finger transcription factor ZFX is required for maintaining the tumorigenic potential of glioblastoma stem cells.

Glioblastomas are highly lethal brain tumors containing tumor-propagating glioma stem cells (GSCs). The molecular mechanisms underlying the maintenance of the GSC phenotype are not fully defined. Here we demonstrate that the zinc finger and X-linked transcription factor (ZFX) maintains GSC self-renewal and tumorigenic potential by upregulating c-Myc expression. ZFX is differentially expressed in GSCs relative to non-stem glioma cells and neural progenitor cells. Disrupting ZFX by shRNA reduced c-Myc expression and potently inhibited GSC self-renewal and tumor growth. Ectopic expression of c-Myc to its endogenous level rescued the effects caused by ZFX disruption, supporting that ZFX controls GSC properties through c-Myc. Furthermore, ZFX binds to a specific sequence (GGGCCCCG) on the human c-Myc promoter to upregulate c-Myc expression. These data demonstrate that ZFX functions as a critical upstream regulator of c-Myc and plays essential roles in the maintenance of the GSC phenotype. This study also supports that c-Myc is a dominant driver linking self-renewal to malignancy.

Tumor Angiogenesis: A New Source of Pericytes

Glioblastoma stem cells have been reported to directly contribute to the tumor vasculature by endothelial cell differentiation. Interestingly, a recent study demonstrates that glioblastoma stem cells preferentially differentiate into vascular pericytes to support vasculature function and tumor growth.

A Tumorigenic MLL-Homeobox Network in Human Glioblastoma Stem Cells

Glioblastoma growth is driven by cancer cells that have stem cell properties, but molecular determinants of their tumorigenic behavior are poorly defined. In cancer, altered activity of the epigenetic modifiers Polycomb and Trithorax complexes may contribute to the neoplastic phenotype. Here, we provide the first mechanistic insights into the role of the Trithorax protein mixed lineage leukemia (MLL) in maintaining cancer stem cell characteristics in human glioblastoma. We found that MLL directly activates the Homeobox gene HOXA10. In turn, HOXA10 activates a downstream Homeobox network and other genes previously characterized for their role in tumorigenesis. The MLL-Homeobox axis we identified significantly contributes to the tumorigenic potential of glioblastoma stem cells. Our studies suggest a role for MLL in contributing to the epigenetic heterogeneity between tumor-initiating and non-tumor-initiating cells in glioblastoma.

Abstract 3476: Hyaluronan-mediated motility receptor as a novel target for inhibiting glioblastoma stem cells

Abstract Introduction: Glioblastoma stem cells (GSCs) are a subpopulation of tumor cells that promote tumor invasion and angiogenesis, therapeutic resistance and tumor recurrence. Here, we study the function of hyaluronan-mediated motility receptor (HMMR) in human GSCs. HMMR is hyper-expressed in human glioblastoma samples relative to lower-grade glioma and non-neoplastic specimens. Hyper-expression of HMMR also correlates with poor survival in glioma patients. HMMR is a multifunctional oncogenic protein that plays essential roles in tumorgenesis. Extracellular HMMR forms a complex with CD44 that upon binding to hyaluronan activates intracellular signaling pathways that regulate tumor cell survival, proliferation and invasion. Intracellular HMMR associates with microtubules, interacts with the mitotic spindle and contributes to tumor progression by promoting genomic instability. HMMR and CD44 are two ubiquitous receptors for hyaluronan, which is a prominent component of the microenvironment in most malignant tumors. CD44 has been identified as a cancer stem cell marker and CD44 directly regulates cancer stem cells in a variety of cancers including glioblastoma. Critical evidence regarding the function of HMMR in actual glioblastoma tumors and in the context of tumor-initiating GSCs is still lacking. Results: We found that HMMR is ubiquitously expressed in a panel of human glioblastoma-derived neurosphere cultures that are enriched for GSCs. Targeting HMMR by lentivirus-mediated shRNA delivery potently disrupted in vitro self-renewal of GSCs and inhibited the expression of key GSC markers, such as CD133, SOX2 and Olig2. HMMR knockdown also blocked the in vivo tumorigenic potential of GSCs in mouse orthotopic xenograft model. Notably, disruption of HMMR in GSCs attenuated the activity of Notch signaling, which supports the cancer stem cell hierarchy in glioblastoma and various other cancer types. Significance: This research identifies HMMR as a novel therapeutic target in the treatment of glioblastoma by depleting the tumor-initiating GSCs. It also introduces a novel hypothesis regarding the mechanistic crosstalk between HMMR-mediated signaling and Notch signaling pathway. Understanding this crosstalk between HMMR and Notch signaling will have a significant impact on current efforts to target Notch driven mechanisms for GBM therapy. Overall, these studies will lay an essential foundation for the development of HMMR-targeting strategies, such as HMMR inhibitors or monoclonal antibodies, as new therapeutic approaches targeting glioblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3476. doi:1538-7445.AM2012-3476

Abstract IA13: The complexity of tumor cell hierarchies

Abstract Cancer research has rediscovered the complexity of tumors through the incorporation of cellular heterogeneity into tumor models with cellular subsets displaying stem cell characteristics. The cancer stem cell hypothesis posits that tumor cells are organized in a hierarchy with cancer stem cells at the apex and functionally defined by the ability to self renew and propagate tumors similar to the parental tumors from which they are derived, cancer stem cells remain controversial due to unresolved relationships with cell of origin, frequency, and defining universal markers but it has been nearly impossible to create absolute rules for even a single cancer type so it is likely that the cancer stem cell phenotype can be acquired through convergent evolution and may be governed by variable rules based on tumor type, grade, course, and therapeutic treatment. Cancer stem cells have been studied in multiple cancer types, most heavily in leukemias, glioblastomas, breast, and colon cancers. Cancer stem cells also yield the non-tumorigenic tumor bulk cells that display a more differentiated phenotype. The ability to prospectively isolate and interrogate cancer stem cells is defining molecular mechanisms responsible for the tumor maintenance and growth. The clinical relevance of cancer stem cells has been supported by cancer stem cell resistance to cytotoxic therapies and promotion of tumor angiogenesis. Cancer stem cells reside in specific functional niches in perivascular and hypoxic niches that may offer the ability to disrupt tumor maintenance and therapeutic resistance through targeting the niche. The cancer stem cell phenotype is regulated by both cell intrinsic and microenvironmental influences that may further increase the complexity of tumor modeling. Notably, cell lines and standard culture conditions used in preclinical studies fail to recapitulate the tumor heterogeneity and do not predict patient responses, supporting the development of improved tumor models. The study of cellular heterogeneity and cancer stem cells has already yielded novel molecular targets and pathways that are amenable to therapeutic targeting, perhaps permitting attenuation of tumor resistance. The conventional pyramidal unidirectional differentiation cascade with cancer stem cells at the apex has been called into question by studies demonstrating plasticity of the cancer stem cell phenotype, thus suggesting that targeting only cancer stem cells will likely fail to cure patients and require simultaneous targeting of cancer stem cells and the bulk tumor. In conclusion although the field of cancer stem cell biology is relatively young, continued elucidation of the tumor hierarchy holds promise for the development of novel patient therapies.

Nonreceptor Tyrosine Kinase BMX Maintains Self-Renewal and Tumorigenic Potential of Glioblastoma Stem Cells by Activating STAT3

Glioblastomas display cellular hierarchies containing tumor-propagating glioblastoma stem cells (GSCs). STAT3 is a critical signaling node in GSC maintenance but molecular mechanisms underlying STAT3 activation in GSCs are poorly defined. Here we demonstrate that the bone marrow X-linked (BMX) nonreceptor tyrosine kinase activates STAT3 signaling to maintain self-renewal and tumorigenic potential of GSCs. BMX is differentially expressed in GSCs relative to nonstem cancer cells and neural progenitors. BMX knockdown potently inhibited STAT3 activation, expression of GSC transcription factors, and growth of GSC-derived intracranial tumors. Constitutively active STAT3 rescued the effects of BMX downregulation, supporting that BMX signals through STAT3 in GSCs. These data demonstrate that BMX represents a GSC therapeutic target and reinforces the importance of STAT3 signaling in stem-like cancer phenotypes.

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.