Radioresistance Of Glioma Stem Cells Research Articles

MicroRNA-17-5p and MicroRNA-130b-3p Promote Radioresistance of Glioma Stem Cells by Targeting PTEN/AKT/HIF-1α Pathway-Controlled Phosphopentose Metabolism

Background. The radioresistance of glioma stem cells (GSCs) is related to some microRNAs (miRs) generated by radiation. This study aimed to investigate the effects of miR-17-5p and miR-130b-3p on the radiosensitivity of GSCs. Methods. miR-17-5p and miR-130b-3p expressions in SU3 and SU3-5R cells were determined. SU3 cells transfected with miR-17-5p or miR-130b-3p mimics or inhibitors were used to determine cell viability after irradiation as well as to examine changes of supernatant glucose, intracellular glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6-PGDH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), reduced glutathione (GSH), glutathione peroxidase (GSH-Px), phosphatase and tensin homolog (PTEN), hypoxia-inducible factor-1α (HIF-1α), glucose transporter (GLUT)-1/3, protein kinase B (AKT), and p-AKT levels. The target gene of the two miRs was verified by luciferase reporter gene assay. Results. miR-17-5p and miR-130b-3p expressions in the radiation-resistant SU3-5R cells or 8 Gy irradiation-treated SU3 cells were high. After transfection of SU3 cells with miR-17-5p or miR-130b-3p mimics, cell viability, intracellular HIF-1α, GLUT-1/3, AKT, and p-AKT protein expressions, and intracellular G6PDH, 6-PGDH, NADPH, GSH-Px, and GSH levels were high, whereas intracellular PTEN expression and supernatant glucose were low. The opposite effects were also observed in the two miR inhibitors-transfected SU3 cells. Further study confirmed that miR-17-5p or miR-130b-3p could directly bind with the PTEN. Conclusion. Radiation-induced miR-17-5p and miR-130b-3p might cause the radioresistance of GSCs, and the mechanisms were associated with the enhancement of antioxidant production, which was from the increments of AKT/HIF-1α signaling pathway-controlled glucose transmembrane transport and phosphopentose metabolism by targeting PTEN.

SPI1-mediated MIR222HG transcription promotes proneural-to-mesenchymal transition of glioma stem cells and immunosuppressive polarization of macrophages

Background: Glioma stem cells (GSCs) are a key factor in glioblastoma (GBM) development and treatment resistance. GSCs can be divided into the mesenchymal (MES) and proneural (PN) subtypes, and these two subtypes of GSCs can undergo interconversion under certain conditions. MES GSCs have higher malignancy and radioresistance and are closely associated with an immunosuppressive microenvironment. Long noncoding RNAs (lncRNAs) play a broad role in GBM, while the role of GSCs subtype remains unknown. Methods: We performed RNA sequencing to explore the lncRNA expression profile in MES- and PN-subtype GBM tissues. The biological function of a host gene-MIR222HG-in GBM development was confirmed in vitro and in vivo. Specifically, RNA sequencing, RNA pulldown, mass spectrometry, RIP, ChIP, luciferase reporter assays and Co-IP were performed. Results: MIR222HG, the expression of which can be induced by SPI1, has high levels in MES GBM tissues. Functionally, we demonstrated that MIR222HG promotes the MES transition and radioresistance in GSCs in vivo and in vitro. Mechanistically, MIR222HG can bind to the YWHAE/HDAC5 complex to promote the MES transition of GSCs through H4 deacetylation. Moreover, cotranscribed miR221 and miR222 can be delivered to macrophages via exosomes to target SOCS3, causing immunosuppressive polarization. Finally, PLX-4720 sensitivity is associated with SPI1 expression and acts on MES GSCs to enhance radiosensitivity. Conclusions: This study demonstrates that targeting SPI1 to block transcription of the MIR222HG cluster helps to reduce radioresistance and combat the immunosuppressive microenvironment in GBM. PLX-4720 is a potential GBM drug and radiosensitizer.

EXTH-09. NEO-ADJUVANT TEMOZOLOMIDE INCREASES THE EFFICACY OF SUBSEQUENT CONCURRENT CHEMORADIATION IN A TRANSGLUTAMINASE-2 DEPENDENT MANNER

Abstract Glioblastoma (GBM) is invariably fatal due to failure of current chemoradiation (Stupp) regimes. Biomarkers such as MGMT have proven to predict response to Temozolomide (TMZ). An equivalent biomarker for radiation (RT) has not yet been identified. Transglutaminase-2 (TGM2) has been implicated in driving radiation resistance; but the mechanism is poorly understood. We have investigated how exposure to neoadjuvant TMZ in glioma stem cells (GSCs) with different levels of TGM2 would affect the response to RT. MATERIALS/METHODS: Primary GSCs lines with different TGM2 levels (high: 1123, 83; low: 528, OPK49) were used to explore the role of TGM2 in RT response and modulation of expression by TMZ in vitro and in-vivo. RESULTS: We showed that TGM2 drives radioresistance in GSCs through restriction of p53 mediated repression of RAD51 expression. We demonstrate that exposure of GSCs to TMZ drives rapid downregulation of TGM2 in vitro and this phenomenon is recapitulated in vivo. Interestingly, we confirm that RT is able to drive reciprocal changes in TGM2 and promotes reactivation of TGM2 in TGM2-high tumours but not TGM2-low tumours. Given these observations, we hypothesized that exposure to neoadjuvant TMZ in TGM2-low tumours would increase the efficacy of subsequent RT in these tumours. Comparison of the effect of standard treatment consisting of 3 weeks of concurrent TMZ and RT (Stupp) to a novel regime (neo-Stupp) consisting of 1 week of neoadjuvant TMZ followed by two weeks of TMZ and hypofractionated RT revealed a superior survival benefit of this novel regime in TGM2-low tumours but not in TGM2-high tumours. Utilization of the TGM2 inhibitor GK921 in combination with neo-Stupp prevented rapid relapse previously observed in TGM2-high tumours. CONCLUSION: We provide evidence that TGM2 is a biomarker of RT response and can be used to tailor chemoradiation protocols to the unique biology of each individual GBM patient.

Upregulation of DNA Metabolism-Related Genes Contributes to Radioresistance of Glioblastoma.

Glioblastomas (GBMs) are the most prevalent brain tumor and exhibit poor prognosis. Radiotherapy is an important strategy for GBMs patients; however, this care remains palliative because of GBMs' radioresistance. Glioma stem cells (GSCs), as a subpopulation residing at the apex of the hierarchy, have been believed to be a pivotal population in radioresistance and recurrence of GBMs. To know the key genes involved in radioresistance of GSCs, the gene expression profiles of GSE54660 and GSE60921 were downloaded from Gene Expression Omnibus for genetic and transcriptomic analysis to identify the potential biomarker genes differentially expressed between GSCs and GBMs. These candidate genes were then filtered by the GSCs gene profile responding to radiation and the radioresistant biomarker genes including DNAJC9, GINS2, STAT1, CHAC2, MT1M, and ZNF226 were screened. The differentially expressed genes in GSCs post-irradiation were submitted to Gene Ontology (GO) for further enrichment analysis and protein-protein interaction (PPI) network analysis. A significant module correlated with GINS2 was finally chosen and a series of genes participating in DNA metabolism were identified. In conclusion, this study propounds a set of novel genes that are differentially expressed in the radioresistant subpopulation within GBMs and could serve as promising therapeutic targets.

Abstract 1137: ANO1 confers the maintenance of stemness and radioresistance in glioma stem cells by stabilizing EGFRvIII

Abstract Anoctamin-1 (ANO1), also known as TMEM16A, is a Ca2+-activated chloride channel known to mediate a number of physiologic functions such as mediating acute and chronic pain and regulating blood vessels and airway smooth muscle. In particular, ANO1 expression is amplified in a variety of cancers, and overexpression of ANO1 is not favorable for clinical prognosis. Previous studies have reported that ANO1 is associated with tumorigenesis, proliferation, migration, and invasion of cancer cells, but the mechanism is not clear yet. In this study, we examined role of ANO1 in the maintenance of stemness and radio-resistance in glioma stem cells (GSCs). Knockdown of ANO1 with shRNA and treatment of ANO1 specific inhibitors (CaCCAo1 and TAO1) suppressed the expression of Notch intracellular domain, Nestin and EGFRvIII and decreased self-renewal activity and invasion in GSCs. Suppression of ANO1 increased radiation sensitivity by inducing ROS-mediated GSCs death. However, inhibition of chloride channel activity, the main function of ANO1, was found to be unaffected in this event. Interestingly, we found that ANO1 retained stemness by direct binding to EGFRvIII, which is specifically expressed in GSCs, and that the chaperone property of ANO1 function may affect the stability of EGFRvIII. In intracranial mouse model of GSCs, knockdown of ANO1 increased the survival rate of mice by suppressing tumor formation. Taken together, we suggest that ANO1 is a crucial molecule involved in the maintenance of stemness and radiation resistance in GSCs and may be a good therapeutic target for treating glioblastoma. Citation Format: Dae-Hee Lee, Hee-Jin Kim, Jeong-Yub Kim, Jea-Yong Park, Myung-Jin Park. ANO1 confers the maintenance of stemness and radioresistance in glioma stem cells by stabilizing EGFRvIII [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1137.

Regulation of c-Myc and NBS1 by ionizing radiation in glioblastoma cells

Glioblastoma is the most common primary brain tumor presenting the worst prognostic. Radioresistance of these tumors is barrier to better treatment and management. Cellular response to ionizing radiation (IR) depends on various molecular mechanisms, including transcription of DNA damage response genes in response to oncogenes. Previous studies suggested that the radioresistance of glioma stem cells would be associated with c-Myc-dependent upregulation of NBS1. The purpose of this study was to evaluate whether IR and other DNA-damaging agents co-regulate c-Myc and NBS1 in glioblastoma cell lines. We found that c-Myc protein level is downregulated by IR in specific glioblastoma cell lines, while NBS1 protein content was not altered in response to genotoxic agents. Additionally, IR-induced downregulation of c-Myc protein content had no correlation with decreased cell viability. These findings indicate that the functional relationship between c-Myc and NBS1 in the context of genotoxic stress may be genetic background-specific.

HDAC6 inhibition induces glioma stem cells differentiation and enhances cellular radiation sensitivity through the SHH/Gli1 signaling pathway

The existence of small numbers of stem-like cells, called glioma stem cells (GSCs), in human glioblastoma multiforme (GBM) is responsible for recurrence due to resistance to radiotherapy and chemotherapy. Inhibition of histone deacetylase 6 (HDAC6) enhanced radiosensitivity of cancer cells. However, the effect of inhibiting HDAC6 on stemness and radioresistance of GSCs and its molecular mechanism are largely unknown. In the present study, we found that HDAC6 was upregulated in GSCs comparing to non-stem tumor cells. Inhibiting HDAC6 downregulated glioma-associated oncogene homolog 1 (Gli1), Patched (Ptch1 and Ptch2) receptors, components of SHH signal, expression and activity in GSCs. Restraining HDAC6 decreased cell proliferation, induces differentiation and increased apoptosis of GSCs via inactivation of SHH/Gli1 signaling pathway. Moreover, HDAC6 inhibition decreased DNA damage repair capacity of GSCs through degradation of checkpoint kinase (CHK) 1 caused by X-linked inhibitor of apoptosis (XIAP) downregulation, leading to elevated radiosensitivity. Taken together, these findings indicate that HDAC6 inhibition decreased stemness of GSCs and enhanced GSCs radiosensitivity through inactivating SHH/Gli1 pathway. This provides a promising novel drug target to overcome GSCs stemness and radioresistance.

PAF promotes stemness and radioresistance of glioma stem cells

An integrated genomic and functional analysis to elucidate DNA damage signaling factors promoting self-renewal of glioma stem cells (GSCs) identified proliferating cell nuclear antigen (PCNA)-associated factor (PAF) up-regulation in glioblastoma. PAF is preferentially overexpressed in GSCs. Its depletion impairs maintenance of self-renewal without promoting differentiation and reduces tumor-initiating cell frequency. Combined transcriptomic and metabolomic analyses revealed that PAF supports GSC maintenance, in part, by influencing DNA replication and pyrimidine metabolism pathways. PAF interacts with PCNA and regulates PCNA-associated DNA translesion synthesis (TLS); consequently, PAF depletion in combination with radiation generated fewer tumorspheres compared with radiation alone. Correspondingly, pharmacological impairment of DNA replication and TLS phenocopied the effect of PAF depletion in compromising GSC self-renewal and radioresistance, providing preclinical proof of principle that combined TLS inhibition and radiation therapy may be a viable therapeutic option in the treatment of glioblastoma multiforme (GBM).

MicroRNA-Mediated Modulation of Radioresistance of Glioma Stem Cells

Glioblastoma (GBM) is the most common primary tumor of the central nervous system, with its deadly nature owing to a high rate of invasion and angiogenesis. A key player in the GBM pathogenesis is a distinct subpopulation of tumor cells which are capable of self-renewal, highly angiogenic and possess the potential for extensive proliferation and multi-lineage differentiation. This cell population termed Glioma Stem Cells (GSCs) is highly resistant to the chemo- and radiotherapies and plays an important role in tumor initiation, progression, and recurrence. In response to radiation therapy, GSCs are capable of triggering the activation of multiple signaling pathways including Wnt, Notch, and Hedgehog, and their DNA damage repair system is proactive. Taken together, the efforts to foster therapeutic interventions will prove futile, without first targeting the GSC population. The miRNA network adds a dimension of regulatory control, which serves to maintain pluripotency and reprograms multiple stemness and radioresistance promoting pathways. However, not much information is available that could provide mechanistic insights regarding how specific miRNAs modulate radioresistance pathways. Our initial efforts involving a global screening of microRNA profiles in different GSCs after radiation treatment resulted in identification of multiple microRNAs that were significantly altered in response to radiation. Based on our preliminary data, we selected 7 microRNAs as potential candidates, with each having several targets in Notch, Wnt, PI3K/Akt, and Hedgehog signaling pathways. In order to unfold the role of each of identified microRNAs, we used specific lentiviral constructs to create overexpression/knockdown of each microRNA in GSCs. Using these stable GSC clones, we carried out multiple in vitro studies to analyze the effects of the respective alteration in relation to radioresistance and stemness phenotypes. Other phenotypic studies included growth kinetics, cell proliferation, invasiveness, apoptosis and cell cycle. We also carried out an extensive in silico analysis to identify the potential targets of these microRNAs. This was followed by analyzing various downstream components of the target genes. Additionally, we carried out a context-based assessment of the overall effects to probe if there is an integration of one or more pathways that could lead to radiation sensitivity. Each of the specific miRNA under study was found to modulate radioresistance and/or stemness of the GSCs Our in vitro findings also revealed their roles in regulating stemness and metastatic ability in vivo. Our study provides a basis for understanding the role of miRNAs in radioresistance phenotype and furthermore, it may lead towards another tool to inhibit GSC proliferation and/or survival.

Abstract 3892: Delineating the role of ZEB1 loss in the chemo and radioresistance of glioma stem cells

Abstract The most prevalent brain cancer, Glioblastoma Multiforme (GBM) has an average survival of less than 2 years. Despite advances in therapy there is still an overall poor response to treatment which has been attributed to glioma stem cells (GSC’s), a subset of tumor cells that are able to self-renew, inhibit differentiation and are often chemo and radioresistant. The genetics and mechanisms underlying how glioma stem cells are able to repopulate tumors and affect patient survival desperately need to be defined. We are interested in delineating the role that ZEB1 (zinc finger e-box binding homeobox-1) loss has on GSC’s and what role this loss plays in the chemo and radioresistance of GSC’s. Most insights into ZEB1 regard it as tumorigenic and an initiator of EMT (epithelial mesenchymal transition). Surprisingly, in this study, we have shown that in TCGA datasets ZEB1 deletion frequently occurs and this loss actually results in poorer glioma patient survival. Copy number analysis as well as immunohistochemistry on patient samples shows that ZEB1 loss occurs both at the gene and protein level. Stable ZEB1 knockdown in our patient derived glioma stem cell lines results in increased proliferation and a resistance to differentiation. Following chemotherapy as well as radiotherapy, knockdown cell lines show increased cell viability as well as higher levels of secondary neurosphere formation. Decreased apoptosis and changes in cell cycle as well as the DNA damage response pathway were also observed in the knockdown lines following chemo and radiotherapy. Overall, this data indicates that loss of ZEB1 in GSC’s results in enhanced resistance to standard therapy. With these results, we have reason to believe that ZEB1 may be used as a novel therapeutic target to combat the chemo and radioresistance of glioma stem cells. Citation Format: Mecca Madany, Lincoln Edwards, John S. Yu. Delineating the role of ZEB1 loss in the chemo and radioresistance of glioma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3892. doi:10.1158/1538-7445.AM2017-3892

Abstract 3440: MicroRNA-mediated modulation of radioresistance of glioma stem cells

Abstract Glioblastoma (GBM) is the most common primary tumor of the central nervous system, with its deadly nature owing to a high rate of invasion and angiogenesis. A key player in the GBM pathogenesis is a distinct subpopulation of tumor cells which are capable of self-renewal, highly angiogenic and possess the potential for extensive proliferation and multi-lineage differentiation. This cell population termed Glioma Stem Cells (GSCs) is highly resistant to the chemo- and radiotherapies and plays an important role in tumor initiation, progression, and recurrence. In response to radiation therapy, GSCs are capable of triggering the activation of multiple signaling pathways including Wnt, Notch, and Hedgehog, and their DNA damage repair system is proactive. Taken together, the efforts to foster therapeutic interventions will prove futile, without first targeting the GSC population. The miRNA network adds a dimension of regulatory control, which serves to maintain pluripotency and reprograms multiple stemness and radioresistance promoting pathways. However, not much information is available that could provide mechanistic insights regarding how specific miRNAs modulate radioresistance pathways. Our initial efforts involving a global screening of microRNA profiles in different GSCs after radiation treatment resulted in identification of multiple microRNAs that were significantly altered in response to radiation. Based on our preliminary data, we selected 7 microRNAs as potential candidates, with each having several targets in Notch, Wnt, PI3K/Akt, and Hedgehog signaling pathways. In order to unfold the role of each of identified microRNAs, we used specific lentiviral constructs to create overexpression/knockdown of each microRNA in GSCs. Using these stable GSC clones, we carried out multiple in vitro studies to analyze the effects of the respective alteration in relation to radioresistance and stemness phenotypes. Other phenotypic studies included growth kinetics, cell proliferation, invasiveness, apoptosis and cell cycle. We also carried out an extensive in silico analysis to identify the potential targets of these microRNAs. This was followed by analyzing various downstream components of the target genes. Additionally, we carried out a context-based assessment of the overall effects to probe if there is an integration of one or more pathways that could lead to radiation sensitivity. Our in vitro findings also revealed their roles in regulating stemness and metastatic ability in vivo. Lastly, our study provides a basis for understanding the role of miRNAs in radioresistance phenotype and furthermore, it may lead towards another tool to inhibit GSC proliferation and/or survival. Funding: R01CA108633, R01CA169368, RC2CA148190, U10CA180850-01 (NCI), Brain Tumor Funders Collaborative Grant, and The Ohio State University CCC (all to AC). Citation Format: Rajbir Singh, Kamalakannan Palanichamy, Saikh Jaharul Haque, Arnab Chakravarti. MicroRNA-mediated modulation of radioresistance of glioma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3440. doi:10.1158/1538-7445.AM2017-3440

Abstract 2879: Dj-1 confers maintenance of stemness and radioresistance by regulating EGFRvIII expression in glioma stem cells

Abstract DJ-1 is involved in many key cellular processes including cell proliferation, transcriptional regulation, differentiation, oxidative stress protection, and mitochondrial function maintenance. Deregulation of DJ-1 has been implicated in the pathogenesis of many human diseases such as Parkinson's disease, amyotrophic lateral sclerosis, infertility, and cancer. DJ-1 was initially identified as an oncogene product involved in human tumorigenesis in numerous cancer types. Alteration in expression of DJ-1 is associated with tumorigenesis in several types of cancers. Herein, we aimed to clarify the effect of DJ-1 on stemness and radioresistance of glioma stem cells (GSCs). Firstly, we found that the reduction in expression of DJ-1 in brain tumor tissues when compared with tumor adjacent normal brain tissues, as indicated by reduced DJ-1 staining. Knock-down of DJ-1 inhibited self-renewal activity of GSCs in vitro as evidenced by neurosphere formation, limiting dilution, and soft agar clonogenic assays. DJ-1 knock-down suppressed maintenance of stemness by suppressing the expression of Notch intracellular domain, Sox2, and phosphorylated STAT3 proteins. Intriguingly, DJ-1 knock-down inhibited the expression of EGFRvIII in GSCs which might be the cause of suppression of stemness pathways. In addition, knockdown of DJ-1 induced apoptosis, reduced cell invasiveness, and sensitized GSCs to ionizing radiation. Based on these data, we propose that DJ-1 could be considered as a crucial diagnostics and therapeutics target for malignant glioma. Note: This abstract was not presented at the meeting. Citation Format: Myung-Jin Park, Jeong-Yub Kim, Hee-Jin Kim, Ji-Soo Kim, Jeong-Chul Kim, Dea-Hee Lee, Yoo-Jin Na. Dj-1 confers maintenance of stemness and radioresistance by regulating EGFRvIII expression in glioma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2879. doi:10.1158/1538-7445.AM2017-2879

OS3.2 Delta-aminolevulinic acid can decrease the radioresistance of glioma stem cells with mesenchymal phenotypes in vitro and in vivo

OS3.2 Delta-aminolevulinic acid can decrease the radioresistance of glioma stem cells with mesenchymal phenotypes in vitro and in vivo

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.

Serine/Threonine Kinase MLK4 Determines Mesenchymal Identity in Glioma Stem Cells in an NF-κB-dependent Manner

Activation of nuclear factor κB (NF-κB) induces mesenchymal (MES) transdifferentiation and radioresistance in glioma stem cells (GSCs), but molecular mechanisms for NF-κB activation in GSCs are currently unknown. Here, we report that mixed lineage kinase 4 (MLK4) is overexpressed in MES but not proneural (PN) GSCs. Silencing MLK4 suppresses self-renewal, motility, tumorigenesis, and radioresistance of MES GSCs via a loss of the MES signature. MLK4 binds and phosphorylates the NF-κB regulator IKKα, leading to activation of NF-κB signaling in GSCs. MLK4 expression is inversely correlated with patient prognosis in MES, but not PN high-grade gliomas. Collectively, our results uncover MLK4 as an upstream regulator of NF-κB signaling and a potential molecular target for the MES subtype of glioblastomas.

Abstract 3303: Radioresistance in glioma stem cells driven by Rad51 dependent homologous recombination repair

Abstract A cell population with stem cell like characteristics is thought to underlie treatment resistance and local recurrence in glioma. In this work we investigate the role of the DNA repair protein RAD51 in these cells and the impact of inhibiting homologous recombination repair on radiation resistance in vitro and in vivo. We used a model of inducible differentiation of patient derived glioma cells to investigate repair protein levels, repair foci formation and kinetics in clonogenic, stem-like populations and their differentiated counterparts. We examined co-expression of stem cell markers with RAD51 protein at whole population level using western blotting, immunocytochemistry and RT-PCR in cultured cells and immunohistochemistry in tumor material. Single cell expression was analysed using the Fluidigm C1 platform. We examined the effect of two specific inhibitors of RAD51 (B02, RI-1) on the same cell pairs in vitro and used the γH2AX assay to assess differences in repair kinetics. We used subcutaneous models of glioma to evaluate the effect of one of these agents (RI-1) on tumour growth delay with and without fractionated radiation doses in vivo. Primary glioma stem cells expressed high levels of RAD51 protein and exhibited high numbers of foci per nucleus following radiation exposure. Levels of both protein and repair foci fell after cells were transferred to differentiating conditions (serum+BMP4), as did expression of stem cell markers (NESTIN, SOX2). Single cell analysis showed a highly significant association between SOX2 and RAD51 expression (P< 0.0001). A similar association between RAD51 expression and stem cell marker expression was demonstrated in a series of glioblastoma specimens. When primary glioma cultures were treated with B02, RI-1 or siRNA targeting RAD51 in vitro, significant radiosensitisation was achieved with dose modifying factors ∼1.4 after single radiation doses. Delayed repair of DNA double strand breaks was also apparent, with a higher proportion of γH2AX foci unresolved in treated cells at 24 hours post irradiation. We also demonstrated significantly enhanced tumor growth delay in vivo when RI-1 was combined with fractionated radiation therapy (5Gy x 3) in a subcutaneous model. These data suggest that RAD51 dependent DNA repair by homologous recombination represents a specific target in the stem-like fraction of glioblastoma and inhibiting this pathway is a promising approach to radiosensitisation. Citation Format: Henry King, Helen Payne, Tim Brend, Anjana Patel, Alex Wright, Teklu Englu, Lucy Stead, Heiko Wurdak, Susan C. Short. Radioresistance in glioma stem cells driven by Rad51 dependent homologous recombination repair. [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 3303. doi:10.1158/1538-7445.AM2015-3303

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.

High linear-energy-transfer radiation can overcome radioresistance of glioma stem-like cells to low linear-energy-transfer radiation

Ionizing radiation is applied as the standard treatment for glioblastoma multiforme (GBM). However, radiotherapy remains merely palliative, not curative, because of the existence of glioma stem cells (GSCs), which are regarded as highly radioresistant to low linear-energy-transfer (LET) photons. Here we analyzed whether or not high-LET particles can overcome the radioresistance of GSCs. Glioma stem-like cells (GSLCs) were induced from the GBM cell line A172 in stem cell culture medium. The phenotypes of GSLCs and wild-type cells were confirmed using stem cell markers. These cells were irradiated with 60Co gamma rays or reactor neutron beams. Under neutron-beam irradiation, high-LET proton particles can be produced through elastic scattering or nitrogen capture reaction. Radiosensitivity was assessed by a colony-forming assay, and the DNA double-strand breaks (DSBs) were assessed by a histone gamma-H2AX focus detection assay. In stem cell culture medium, GSLCs could form neurosphere-like cells and express neural stem cell markers (Sox2 and Musashi) abundantly in comparison with their parental cells. GSLCs were significantly more radioresistant to gamma rays than their parental cells, but neutron beams overcame this resistance. There were significantly fewer gamma-H2AX foci in the A172 GSLCs 24 h after irradiation with gamma rays than in their parental cultured cells, while there was no apparent difference following neutron-beam irradiation. High-LET radiation can overcome the radioresistance of GSLCs by producing unrepairable DNA DSBs. High-LET radiation therapy might have the potential to overcome GBM's resistance to X-rays in a clinical setting.

Activation of the phosphorylation of ATM contributes to radioresistance of glioma stem cells

Ionizing radiation (IR) is currently the most efficient therapy available for malignant glioma. Unfortunately, this strategy is palliative due to the characteristics of radioresistance of malignant glioma. The aim of our study was to compare glioma stem cells (GSCs) with glioma cells (GCs) to determine whether GSCs are responsible for the radioresistance phenotype and to elucidate whether cell cycle checkpoint proteins are responsible for the radioresistance of GSCs. In this study, CD133 (a marker of brain cancer stem cells) and nestin were co-expressed in GSCs isolated from GCs. The percent of CD133+ cells in GSCs and GCs were >80 and <2%, respectively. Significantly more GSCs survived following 2, 4, 6 and 8 Gy IR than GCs. IR kills cancer cells primarily through DNA double-strand breaks (DSBs). The neutral comet assay is often used to intuitively show the level of DSBs. Significantly fewer GSCs showed DNA damage than GCs following 2 Gy IR. This demonstrated that GSCs are more resistant to in vitro radiation than GCs. Furthermore, activated ataxia telangiectasia mutated (ATM) is essential for the activation of downstream effector kinases, such as checkpoint kinase 2 (Chk2) and p53 which mainly contribute to the proper regulation of IR-induced arrest in the G1 phase. DNA damage induced by IR potently initiated activation of phosphorylation of the ATM, p53 and Chk2 checkpoint proteins. Activation of the phosphorylation of these checkpoint proteins was significantly higher in the GSCs compared to GCs. We found that inhibition of ATM activation induced cell cycle checkpoint defects and increased the rate of apoptosis of GSCs following IR. Our results suggest that GSCs were more resistant to radiation compared to GCs due to high expression of phosphorylated cell cycle checkpoint proteins, and inhibition of ATM could significantly reduce the radioresistance of GSCs and GCs. ATM may represent a source of radioresistance in GSCs and a target of improved radiosensitivity of GSCs.

Hypoxia Increases the Expression of Stem-Cell Markers and Promotes Clonogenicity in Glioblastoma Neurospheres

Hypoxia promotes the expansion of non-neoplastic stem and precursor cell populations in the normal brain, and is common in malignant brain tumors. We examined the effects of hypoxia on stem-like cells in glioblastoma (GBM). When GBM-derived neurosphere cultures are grown in 1% oxygen, hypoxia-inducible factor 1alpha (HIF1alpha) protein levels increase dramatically, and mRNA encoding other hypoxic response genes, such as those encoding hypoxia-inducible gene-2, lysyl oxidase, and vascular endothelial growth factor, are induced over 10-fold. Hypoxia increases the stem-like side population over fivefold, and the percentage of cells expressing CD133 threefold or more. Notch pathway ligands and targets are also induced. The rise in the stem-like fraction in GBM following hypoxia is paralleled by a twofold increase in clonogenicity. We believe HIF1alpha plays a causal role in these changes, as when oxygen-stable HIF1alpha is expressed in normoxic glioma cells CD133 is induced. We used digoxin, which has been shown to lower HIF protein levels in vitro and in vivo, to inhibit the hypoxic response. Digoxin suppressed HIF1alpha protein expression, HIF1alpha downstream targets, and slowed tumor growth in vivo. In addition, pretreatment with digoxin reduced GBM flank xenograft engraftment of hypoxic GBM cells, and daily intraperitoneal injections of digoxin were able to significantly inhibit the growth of established subcutaneous glioblastoma xenografts, and suppressed expression of vascular endothelial growth factor.