Stemness Of Glioma Stem Cells Research Articles

Novel KDM1A inhibitors induce differentiation and apoptosis of glioma stem cells via unfolded protein response pathway.

Glioma stem cells (GSCs) have a central role in glioblastoma (GBM) development and chemo/radiation resistance, and their elimination is critical for the development of efficient therapeutic strategies. Recently, we showed that lysine demethylase KDM1A is overexpressed in GBM. In the present study, we determined whether KDM1A modulates GSCs stemness and differentiation and tested the utility of two novel KDM1A-specific inhibitors (NCL-1 and NCD-38) to promote differentiation and apoptosis of GSCs. The efficacy of KDM1A targeting drugs was tested on purified GSCs isolated from established and patient-derived GBMs using both in vitro assays and in vivo orthotopic preclinical models. Our results suggested that KDM1A is highly expressed in GSCs and knockdown of KDM1A using shRNA-reduced GSCs stemness and induced the differentiation. Pharmacological inhibition of KDM1A using NCL-1 and NCD-38 significantly reduced the cell viability, neurosphere formation and induced apoptosis of GSCs with little effect on differentiated cells. In preclinical studies using orthotopic models, NCL-1 and NCD-38 significantly reduced GSCs-driven tumor progression and improved mice survival. RNA-sequencing analysis showed that KDM1A inhibitors modulate several pathways related to stemness, differentiation and apoptosis. Mechanistic studies showed that KDM1A inhibitors induce activation of the unfolded protein response (UPR) pathway. These results strongly suggest that selective targeting of KDM1A using NCL-1 and NCD-38 is a promising therapeutic strategy for elimination of GSCs.

Mesenchymal stem cells enhance the oncolytic effect of Newcastle disease virus in glioma cells and glioma stem cells via the secretion of TRAIL.

BackgroundNewcastle disease virus (NDV) is an avian paramyxovirus, which selectively exerts oncolytic effects in cancer cells. Mesenchymal stem cells (MSCs) have been reported to affect tumor growth and deliver anti-tumor agents to experimental glioblastoma (GBM). Here, we explored the effects of NDV-infected MSCs derived from different sources, on glioma cells and glioma stem cells (GSCs) and the mechanisms involved in their effects.MethodsThe glioma cell lines (A172 and U87) and primary GSCs that were generated from GBM tumors were used in this study. MSCs derived from bone marrow, adipose tissue or umbilical cord were infected with NDV (MTH-68/H). The ability of these cells to deliver the virus to glioma cell lines and GSCs and the effects of NDV-infected MSCs on cell death and on the stemness and self-renewal of GSCs were examined. The mechanisms involved in the cytotoxic effects of the NDV-infected MSCs and their influence on the radiation sensitivity of GSCs were examined as well.ResultsNDV induced a dose-dependent cell death in glioma cells and a low level of apoptosis and inhibition of self-renewal in GSCs. MSCs derived from bone marrow, adipose and umbilical cord that were infected with NDV delivered the virus to co-cultured glioma cells and GSCs. Conditioned medium of NDV-infected MSCs induced higher level of apoptosis in the tumor cells compared with the apoptosis induced by their direct infection with similar virus titers. These results suggest that factor(s) secreted by the infected MSCs sensitized the glioma cells to the cytotoxic effects of NDV. We identified TRAIL as a mediator of the cytotoxic effects of the infected MSCs and demonstrated that TRAIL synergized with NDV in the induction of cell death in glioma cells and GSCs. Moreover, conditioned medium of infected MSCs enhanced the sensitivity of GSCs to γ-radiation.ConclusionsNDV-infected umbilical cord-derived MSCs may provide a novel effective therapeutic approach for targeting GSCs and GBM and for sensitizing these tumors to γ-radiation.

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.

MiR-146b-5p overexpression attenuates stemness and radioresistance of glioma stem cells by targeting HuR/lincRNA-p21/β-catenin pathway.

A stem-like subpopulation existed in GBM cells, called glioma stem cells (GSCs), might contribute to cancer invasion, angiogenesis, immune evasion, and therapeutic resistance, providing a rationale to eliminate GSCs population and their supporting niche for successful GBM treatment. LincRNA-p21, a novel regulator of cell proliferation, apoptosis and DNA damage response, is found to be downregulated in several types of tumor. However, little is known about the role of lincRNA-p21 in stemness and radioresistance of GSCs and its regulating mechanisms. In this study, we found that lincRNA-p21 negatively regulated the expression and activity of β-catenin in GSCs. Downregulation of lincRNA-p21 in GSCs was resulted from upregulation of Hu antigen R (HuR) expression caused by miR-146b-5p downregulation. MiR-146b-5p overexpression increased apoptosis and radiosensitivity, decreased cell viability, neurosphere formation capacity and stem cell marker expression, and induced differentiation in GSCs. Moreover, knock-down lincRNA-p21 or HuR and β-catenin overexpression could rescue the phenotypic changes resulted from miR-146b-5p overexpression in GSCs. These findings suggest that targeting the miR-146b-5p/HuR/lincRNA-p21/β-catenin signaling pathway may be valuable therapeutic strategies against glioma.

Inhibition of Fatty Acid Synthase Decreases Expression of Stemness Markers in Glioma Stem Cells.

Cellular metabolic changes, especially to lipid metabolism, have recently been recognized as a hallmark of various cancer cells. However, little is known about the significance of cellular lipid metabolism in the regulation of biological activity of glioma stem cells (GSCs). In this study, we examined the expression and role of fatty acid synthase (FASN), a key lipogenic enzyme, in GSCs. In the de novo lipid synthesis assay, GSCs exhibited higher lipogenesis than differentiated non-GSCs. Western blot and immunocytochemical analyses revealed that FASN is strongly expressed in multiple lines of patient-derived GSCs (G144 and Y10), but its expression was markedly reduced upon differentiation. When GSCs were treated with 20 μM cerulenin, a pharmacological inhibitor of FASN, their proliferation and migration were significantly suppressed and de novo lipogenesis decreased. Furthermore, following cerulenin treatment, expression of the GSC markers nestin, Sox2 and fatty acid binding protein (FABP7), markers of GCSs, decreased while that of glial fibrillary acidic protein (GFAP) expression increased. Taken together, our results indicate that FASN plays a pivotal role in the maintenance of GSC stemness, and FASN-mediated de novo lipid biosynthesis is closely associated with tumor growth and invasion in glioblastoma.

Abstract 951: LY500307 as a novel therapeutic agent for treatment of glioblastoma

Abstract Glioblastoma (GBM) is the most common type of primary brain tumor with poor prognosis. A gender bias exists in the development of GBM with incidence of developing GBM is greater in males compared to females. Epidemiological studies and several lines of evidence suggest tumor suppressive role of female sexual hormone estrogen on brain tumors. However, estrogen as potential therapy for GBM has limited therapeutic application due to safety concerns including breast cancer, uterine cancer, heart disease, and feminization in men. Therefore alternative agents that mimic estrogen effects will have potent utility in treating GBM. Estrogen effects are mediated though its cognate receptors ERα and ERβ and ERβ functions as a tissue-specific tumor suppressor. Recent studies identified a synthetic LY500307 as a selective ERβ agonist (developed by Eli Lilly and Company) with an excellent preclinical profile and is currently in clinical trials for treating Schizophrenia. We recently showed that ERβ is the major ER subtype expressed in GBM. The objective of this study is to determine the therapeutic effects of novel ERβ agonist LY500307 using both in vitro and orthotopic preclinical models and to determine its mechanism(s) of action. In this study, we have tested the therapeutic efficacy of LY500307 using several established as well as patient derived GBM cells. Treatment of LY500307 significantly reduced the proliferation of GBM cells with no activity on normal astrocytes suggesting its tumor specific effects. Overexpression of ERβ reduced proliferation of GBM cells and knockdown of ERβ compromised the treatment effect of LY500307 further suggesting the specificity. RNA sequencing analysis of LY500307 treated and untreated GBM cells revealed several novel pathways related to apoptosis, cell cycle, stem cells and differentiation that were significantly modulated by LY500307. Mechanistic studies revealed that LY500307 mediated apoptosis of GBM involve genes activated by both ERβ- classical as well as AP1 mediated non-classical pathways and also involve p38MAPK and JNK pathways. Since Glioma Stem Cells (GSCs) are implicated in tumor initiation, invasion and therapy resistance of GBM, we also tested the effect of LY500307 on GSCs. LY500307 treatment significantly inhibited the proliferation, neurosphere formation and self-renewal of GSCs and also resulted in the loss of stemness of GSCs and induction of differentiation and apoptosis. Further, LY500307 treatment significantly reduced the tumor growth in in vivo orthotopic GBM models and promoted apoptosis of tumors. Together, our results demonstrate that LY500307 as a potential therapeutic agent for treatment of GBM. Since LY500307 has good blood-brain barrier permeability and less neuronal toxicity, it can be readily transferred to clinical use with current radiation and chemotherapies, thereby providing an additional tool for enhancing survival in GBM patients. Citation Format: Gangadhara R. Sareddy, Aleksandra Gruslova, David A. Cavazos, Rajeshwar R. Tekmal, Andrew J. Brenner, Ratna K. Vadlamudi. LY500307 as a novel therapeutic agent for treatment of glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 951. doi:10.1158/1538-7445.AM2015-951

MicroRNA-153/Nrf-2/GPx1 pathway regulates radiosensitivity and stemness of glioma stem cells via reactive oxygen species.

Glioma stem cells (GSCs) exhibit stem cell properties and high resistance to radiotherapy. The main aim of our study was to determine the roles of ROS in radioresistance and stemness of GSCs. We found that microRNA (miR)-153 was down-regulated and its target gene nuclear factor-erythroid 2-related factor-2 (Nrf-2) was up-regulated in GSCs compared with that of non-GSCs glioma cells. The enhanced Nrf-2 expression increased glutathione peroxidase 1 (GPx1) transcription and decreased ROS level leading to radioresistance of GSCs. MiR-153 overexpression resulted in increased ROS production and radiosensitization of GSCs. Moreover, miR-153 overexpression led to decreased neurosphere formation capacity and stem cell marker expression, and induced differentiation through ROS-mediated activation of p38 MAPK in GSCs. Nrf-2 overexpression rescued the decreased stemness and radioresistance resulting from miR-153 overexpression in GSCs. In addition, miR-153 overexpression reduced tumorigenic capacity of GSCs and increased survival in mice bearing human GSCs. These findings demonstrated that miR-153 overexpression decreased radioresistance and stemness of GSCs through targeting Nrf-2/GPx1/ROS pathway.

Neural stem cells preferentially migrate to glioma stem cells and reduce their stemness phenotypes.

Glioma stem cells (GSCs), characterized by self-renewal, multi-potentiality and tumorigenicity, are responsible for the tumor propagation, recurrence and resistance to traditional treatments, representing a critical therapeutic target. Neural stem cells (NSCs) possess inherent tropism to brain tumor cells and inhibit their growth. However, there is a limited understanding of the mechanism underlying NSC tropism and the effect of NSC migration on GSC stemness phenotypes. In the present study, we showed that GSCs exhibited enhanced chemotaxis for NSC tropism compared with their differentiated cells. Chemokines secreted by GSCs contributed to the targeted migration of NSCs. Hypoxia enhanced NSC tropism via the upregulated chemokine expression of GSCs, such as VEGF, EGF and bFGF. In vitro migration of NSCs induced GSC differentiation and reduced stem-like phenotypes. Moreover, in vivo data provided direct evidence that transplanted NSCs could migrate to GSCs from either the homolateral or contralateral brain injection site, which prolonged the survival of grafted mice. Taken together, these findings show that NSCs preferentially migrate to GSCs and reduce their stemness phenotypes, raising the intriguing possibility that the targeted migration of NSCs can be applied as a novel therapeutic strategy to target these intractable brain tumors.