Glioblastoma Radioresistance Research Articles

Abstract 1122: MST4 phosphorylation of ATG4B regulates autophagic activity, tumorigenicity, and radio resistance in glioblastoma

Abstract Autophagy-mediated intracellular catabolism sustains the rapid growth of established tumors and tumors in response to multiple stresses including genotoxic/cytotoxic therapies. Here we identify and validate ATG4B, a key regulator that stimulates autophagic process by promoting autophagosome through reversible modification of ATG8 as a novel substrate of mammalian sterile20-like kinase (STK) 26/MST4 that is less known for its function in cellular process (none in autophagy) and unknown authentic substrates in cancer. We show that MST4 phosphorylates ATG4B at serine residue 383, which stimulates ATG4B activity and increases autophagic flux. Inhibition of MST4 or ATG4B activities suppresses autophagic process and the tumorigenicity of glioblastoma (GBM) cells. Furthermore, radiation induces MST4 expression, ATG4B phosphorylation and autophagy. Inhibiting ATG4B in combination with radiotherapy in treating mice with intracranial GBM tumor xenografts markedly slows tumor growth and provides significant survival benefit to animal subjects. This study not only describes a novel regulatory mechanism by which the MST4-ATG4B axis accelerates autophagic process, regulates GBM tumorigenicity, and responses to radiotherapy (RT), but also explores imminent clinical utility of combination of ATG4B inhibition with RT to suppress orthotopic GBM tumor xenografts. This study should prove generalizable to other types of cancer and have positive impacts in advancing our knowledge of cancer biology and designing new cancer treatments. Citation Format: Tianzhi Huang, Chung Kwon Kim, Angel A. Alvarez, Rajendra P Pangeni, Xuechao Wan, xiao song, Taiping shi, Yongyong Yongyong, Namratha Sastry, Craig Horbinski, Songjian Lu, Roger Stupp, John Kessler, Ryo Nishikawa, Ichiro Nakano, Erik Sulman, Xinghua Lu, Charles David James, Xiao-Ming Yin, Bo Hu, Shi-Yuan Cheng. MST4 phosphorylation of ATG4B regulates autophagic activity, tumorigenicity, and radio resistance in glioblastoma [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 1122.

Molecular Markers of Therapy-Resistant Glioblastoma and Potential Strategy to Combat Resistance.

Glioblastoma (GBM) is the most common primary malignant tumor of the central nervous system. With its overall dismal prognosis (the median survival is 14 months), GBMs demonstrate a resounding resilience against all current treatment modalities. The absence of a major progress in the treatment of GBM maybe a result of our poor understanding of both GBM tumor biology and the mechanisms underlying the acquirement of treatment resistance in recurrent GBMs. A comprehensive understanding of these markers is mandatory for the development of treatments against therapy-resistant GBMs. This review also provides an overview of a novel marker called acid ceramidase and its implication in the development of radioresistant GBMs. Multiple signaling pathways were found altered in radioresistant GBMs. Given these global alterations of multiple signaling pathways found in radioresistant GBMs, an effective treatment for radioresistant GBMs may require a cocktail containing multiple agents targeting multiple cancer-inducing pathways in order to have a chance to make a substantial impact on improving the overall GBM survival.

Ibrutinib inactivates BMX-STAT3 in glioma stem cells to impair malignant growth and radioresistance.

Glioblastoma (GBM) is the most lethal primary brain tumor and is highly resistant to current treatments. GBM harbors glioma stem cells (GSCs) that not only initiate and maintain malignant growth but also promote therapeutic resistance including radioresistance. Thus, targeting GSCs is critical for overcoming the resistance to improve GBM treatment. Because the bone marrow and X-linked (BMX) nonreceptor tyrosine kinase is preferentially up-regulated in GSCs relative to nonstem tumor cells and the BMX-mediated activation of the signal transducer and activator of transcription 3 (STAT3) is required for maintaining GSC self-renewal and tumorigenic potential, pharmacological inhibition of BMX may suppress GBM growth and reduce therapeutic resistance. We demonstrate that BMX inhibition by ibrutinib potently disrupts GSCs, suppresses GBM malignant growth, and effectively combines with radiotherapy. Ibrutinib markedly disrupts the BMX-mediated STAT3 activation in GSCs but shows minimal effect on neural progenitor cells (NPCs) lacking BMX expression. Mechanistically, BMX bypasses the suppressor of cytokine signaling 3 (SOCS3)-mediated inhibition of Janus kinase 2 (JAK2), whereas NPCs dampen the JAK2-mediated STAT3 activation via the negative regulation by SOCS3, providing a molecular basis for targeting BMX by ibrutinib to specifically eliminate GSCs while preserving NPCs. Our preclinical data suggest that repurposing ibrutinib for targeting GSCs could effectively control GBM tumor growth both as monotherapy and as adjuvant with conventional therapies.

Identification of radiation responsive genes and transcriptome profiling via complete RNA sequencing in a stable radioresistant U87 glioblastoma model.

The absence of major progress in the treatment of glioblastoma (GBM) is partly attributable to our poor understanding of both GBM tumor biology and the acquirement of treatment resistance in recurrent GBMs. Recurrent GBMs are characterized by their resistance to radiation. In this study, we used an established stable U87 radioresistant GBM model and total RNA sequencing to shed light on global mRNA expression changes following irradiation. We identified many genes, the expressions of which were altered in our radioresistant GBM model, that have never before been reported to be associated with the development of radioresistant GBM and should be concertedly further investigated to understand their roles in radioresistance. These genes were enriched in various biological processes such as inflammatory response, cell migration, positive regulation of epithelial to mesenchymal transition, angiogenesis, apoptosis, positive regulation of T-cell migration, positive regulation of macrophage chemotaxis, T-cell antigen processing and presentation, and microglial cell activation involved in immune response genes. These findings furnish crucial information for elucidating the molecular mechanisms associated with radioresistance in GBM. Therapeutically, with the global alterations of multiple biological pathways observed in irradiated GBM cells, an effective GBM therapy may require a cocktail carrying multiple agents targeting multiple implicated pathways in order to have a chance at making a substantial impact on improving the overall GBM survival.

ATM inhibition prevents interleukin-6 from contributing to the proliferation of glioblastoma cells after ionizing radiation.

Glioblastoma (GBM) is a highly fatal disease with a 5year survival rate of less than 22%. One of the most effective treatment regimens to date is the use of radiotherapy which induces lethal DNA double-strand breaks to prevent tumour growth. However, recurrence occurs in the majority of patients and is in-part a result of robust radioresistance mechanisms. In this study, we demonstrate that the multifunctional cytokine, interleukin-6 (IL-6), confers a growth advantage in GBM cells but does not have the same effect on normal neural progenitor cells. Further analysis showed IL-6 can promote radioresistance in GBM cells when exposed to ionising radiation. Ablation of the Ataxia-telangiectasia mutated serine/threonine kinase that is recruited and activated by DNA double-strand breaksreverses the effect of radioresistance andre-sensitised GBM to DNA damagethus leading to increase cell death.Our finding suggests targeting the signaling cascade of DNA damage response is a potential therapeutic approach to circumvent IL-6 from promoting radioresistance in GBM.

CDC7-dependent transcriptional regulation of RAD54L is essential for tumorigenicity and radio-resistance of glioblastoma

Accumulating evidence indicates that cell division cycle 7-related protein kinase(CDC7) plays an essential role in tumor cells and it could induces cell proliferation and could be related to prognosis in multiple types of cancer. However, the biological role and molecular mechanism of CDC7 in GBM still remains unclear. In this study, we identified that CDC7 expression was enriched in glioblastoma (GBM) tumors and was functionally required for tumor proliferation and its expression was associated to poor prognosis in GBM patients. Mechanically, CDC7 induced radio resistance in GBM cells and CDC7 knock down increased cell apoptosis when combined with radiotherapy. Moreover, CDC7 regulated The DNA repair/recombination protein 54L (RAD54L) expression via regulation of RAD54L promoter activity. Therapeutically, we found that CDC7 inhibitor attenuated tumor growth both in vitro and in vivo. Collectively, CDC7 promotes proliferation, induces radio resistance in GBM, and could become a potential therapeutic target for GBM.

Evaluation of Radioresponse and Radiosensitizers in Glioblastoma Organotypic Cultures.

Glioblastoma (GBM), a deadly primary brain malignancy, manifests pronounced radioresistance. Identifying agents that improve the sensitivity of tumor tissue to radiotherapy is critical for improving patient outcomes. The response to ionizing radiation is regulated by both cell-intrinsic and -extrinsic mechanisms. In particular, the tumor microenvironment is known to promote radioresistance in GBM. Therefore, model systems used to test radiosensitizing agents need to take into account the tumor microenvironment. We recently showed that GBM explant cultures represent an adaptable ex vivo platform for rapid and personalized testing of radiosensitizers. These explants preserve the cellular composition and tissue architecture of parental patient tumors and therefore capture the microenvironmental context that critically determines the response to radiotherapy. This chapter focuses on the detailed protocol for testing candidate radiosensitizing agents in GBM explants.

RNF138-mediated ubiquitination of rpS3 is required for resistance of glioblastoma cells to radiation-induced apoptosis

An interaction between ribosomal protein S3 (rpS3) and nuclear factor kappa B or macrophage migration inhibitory factor in non-small-cell lung cancer is responsible for radioresistance. However, the role of rpS3 in glioblastoma (GBM) has not been investigated to date. Here we found that in irradiated GBM cells, rpS3 translocated into the nucleus and was subsequently ubiquitinated by ring finger protein 138 (RNF138). Ubiquitin-dependent degradation of rpS3 consequently led to radioresistance in GBM cells. To elucidate the apoptotic role of rpS3, we analyzed the interactome of rpS3 in ΔRNF138 GBM cells. Nuclear rpS3 interacted with DNA damage inducible transcript 3 (DDIT3), leading to DDIT3-induced apoptosis in irradiated ΔRNF138 GBM cells. These results were confirmed using in vivo orthotopic xenograft models and GBM patient tissues. This study aims to clarify the role of RNF138 in GBM cells and demonstrate that rpS3 may be a promising substrate of RNF138 for the induction of GBM radioresistance, indicating RNF138 as a potential target for GBM therapy.

Serine/Threonine Kinase CHEK1-Dependent Transcriptional Regulation of RAD54L Promotes Proliferation and Radio Resistance in Glioblastoma

Accumulating evidence indicates that Checkpoint kinase 1 (CHEK1) plays an essential role in tumor cells and that it could induce cell proliferation and could be related to prognosis in multiple types of cancer. However, the biological role and molecular mechanism of CHEK1 in GBM still remain unclear. In this study, we identified that CHEK1 expression was enriched in glioblastoma (GBM) tumors and was functionally required for tumor proliferation and that its expression was associated to poor prognosis in GBM patients. Mechanically, CHEK1 induced radio resistance in GBM cells, and CHEK1 knockdown increased cell apoptosis when combined with radiotherapy via regulation of the DNA repair/recombination protein 54L (RAD54L) expression. Therapeutically, we found that CHEK1 inhibitor attenuated tumor growth both in vitro and in vivo. Collectively, CHEK1 promotes proliferation, induces radio resistance in GBM, and could become a potential therapeutic target for GBM.

MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma

SummaryATG4B stimulates autophagy by promoting autophagosome formation through reversible modification of ATG8. We identify ATG4B as a substrate of mammalian sterile20-like kinase (STK) 26/MST4. MST4 phosphorylates ATG4B at serine residue 383, which stimulates ATG4B activity and increases autophagic flux. Inhibition of MST4 or ATG4B activities using genetic approaches or an inhibitor of ATG4B suppresses autophagy and the tumorigenicity of glioblastoma (GBM) cells. Furthermore, radiation induces MST4 expression, ATG4B phosphorylation, and autophagy. Inhibiting ATG4B in combination with radiotherapy in treating mice with intracranial GBM xenograft markedly slows tumor growth and provides a significant survival benefit. Our work describes an MST4-ATG4B signaling axis that influences GBM autophagy and malignancy, and whose therapeutic targeting enhances the anti-tumor effects of radiotherapy.

Overexpressed miR-183 promoted glioblastoma radioresistance via down-regulating LRIG1

BackgroundGlioma is the most common cause of cancer-related death. Therapy based on radiation seemed to effectively, while the radioresistance of several glioblastoma cells abolished the therapy. Thus, to employ the potential mechanism underlying the radioresistance is essential for glioma treatment. MethodsRadioresistant cells were constructed using the X-ray radiation. Cell viability and apoptosis were detect using CCK-8 and Annexin-V/propidium iodide (PI), respectively. Real-time PCR and western blot were performed to determine gene expression. Luciferase reporter assay was carried out to detect the relationship between miR-183 and LRIG1. Mice xenotransplant model of glioma was established to detect the role of miR-183 in vivo. ResultsThe expression of miR-183 was increased, while LRIG1 was decreased in resistant tissues rather than in sensitive tissues. The expression of LRIG1 was lower in radioresistant gliblastoma cell line U251R rather than in normal glioblastoma cell line U251. Overexpressed miR-183 suppressed cell apoptosis in radioresistance U251R cells (U251R). MiR-183 targets LRIG1 to regulate its expression. U251R cells transfected miR-183 inhibitor promoted the expression of LRIG1, and decreased the expression of EFGR and p-Akt, while U251R cells co-transfected with shRNA-LRIG1 abolished the effects of miR-183 knockdown. U251 cells transfected with miR-183 mimic decreased the expression of LRIG1, and promoted the expression of EFGR and p-Akt, while cells co-transfected with pcDNA-LRIG1 abolished the effects of miR-183 overexpression. In vivo experiments demonstrated that miR-183 inhibitor suppressed tumor growth, while miR-183 mimic promoted tumor growth. ConclusionMiR-183 overexpression promoted radioresistance of glioblastoma via down-regulating LRIG1 and increasing the activity of EFGR/Akt.

GPR56/ADGRG1 Inhibits Mesenchymal Differentiation and Radioresistance in Glioblastoma

A mesenchymal transition occurs both during the natural evolution of glioblastoma (GBM) and in response to therapy. Here, we report that the adhesion G-protein-coupled receptor, GPR56/ADGRG1, inhibits GBM mesenchymal differentiation and radioresistance. GPR56 is enriched in proneural and classical GBMs and is lost during their transition toward a mesenchymal subtype. GPR56 loss of function promotes mesenchymal differentiation and radioresistance of glioma initiating cells both invitro and invivo. Accordingly, a low GPR56-associated signature isprognostic of a poor outcome in GBM patients even within non-G-CIMP GBMs. Mechanistically, we reveal GPR56 as an inhibitor of the nuclear factor kappaB (NF-κB) signaling pathway, thereby providing the rationale by whichthis receptor prevents mesenchymal differentiation and radioresistance. A pan-cancer analysis suggests that GPR56 might be an inhibitor of the mesenchymal transition across multiple tumor types beyond GBM.

The FOXM1/BUB1B signaling pathway is essential for the tumorigenicity and radioresistance of glioblastoma.

Accumulating evidence indicates that mitotic checkpoint serine/threonine kinase B (BUB1B) plays a critical role in multiple types of cancer. However, the biological function and molecular regulatory mechanism of BUB1B in glioblastoma (GBM) remain unclear. In the present study, we identified that BUB1B expression was enriched in GBM tumors and was functionally required for tumor proliferation both in vitro and in vivo. Clinically, BUB1B expression was associated with poor prognosis in GBM patients and BUB1B-dependent radioresistance in GBM was decreased by targeting BUB1B via shRNAs. Mechanistically, forkhead box protein M1 (FOXM1) transcriptionally regulated BUB1B expression by binding to and then activating the BUB1B promoter. Therapeutically, we found that FOXM1 inhibitor attenuated tumorigenesis and radioresistance of GBM both in vitro and in vivo. Altogether, BUB1B promotes tumor proliferation and induces radioresistance in GBM, indicating that BUB1B could be a potential therapeutic target for GBM.

TRPM8 is required for survival and radioresistance of glioblastoma cells.

TRPM8 is a Ca2+-permeable nonselective cation channel belonging to the melastatin sub-group of the transient receptor potential (TRP) family. TRPM8 is aberrantly overexpressed in a variety of tumor entities including glioblastoma multiforme where it reportedly contributes to tumor invasion. The present study aimed to disclose further functions of TRPM8 in glioma biology in particular upon cell injury by ionizing radiation. To this end, TCGA data base was queried to expose the TRPM8 mRNA abundance in human glioblastoma specimens and immunoblotting was performed to analyze the TRPM8 protein abundance in primary cultures of human glioblastoma. Moreover, human glioblastoma cell lines were irradiated with 6 MV photons and TRPM8 channels were targeted pharmacologically or by RNA interference. TRPM8 abundance, Ca2+ signaling and resulting K+ channel activity, chemotaxis, cell migration, clonogenic survival, DNA repair, apoptotic cell death, and cell cycle control were determined by qRT-PCR, fura-2 Ca2+ imaging, patch-clamp recording, transfilter migration assay, wound healing assay, colony formation assay, immunohistology, flow cytometry, and immunoblotting. As a result, human glioblastoma upregulates TRPM8 channels to variable extent. TRPM8 inhibition or knockdown slowed down cell migration and chemotaxis, attenuated DNA repair and clonogenic survival, triggered apoptotic cell death, impaired cell cycle and radiosensitized glioblastoma cells. Mechanistically, ionizing radiation activated and upregulated TRPM8-mediated Ca2+ signaling that interfered with cell cycle control probably via CaMKII, cdc25C and cdc2. Combined, our data suggest that TRPM8 channels contribute to spreading, survival and radioresistance of human glioblastoma and, therefore, might represent a promising target in future anti-glioblastoma therapy.

Abstract 937: Plasticity in heterogenous cancer stem cells promotes glioblastoma radioresistance

Abstract Some cancers including Glioblastoma (GBM) have hierarchical cellular architecture with cancer stem cells (CSCs) as a minor tumorigenic subpopulation. While CSCs have been touted to contribute to initiate recurrence, post-surgical therapy-induced phenotypic changes in CSCs remain elusive. Here we report that treatment of GBM cells with ionizing radiation (IR) promotes a wound-healing mesenchymal signature, accompanied by conversion of naïve CSCs expressing CD133 to post-treatment more radioresistant CSCs expressing the GPI-anchored, TGFb-inhibitory protein CD109 in an ATM, NF-kB, CEBP-b dependent manner. In post-IR GBM, these therapy-escaped CD133-/CD109+ GBM cells, but not the rest of the tumor, uniquely retain clonogenic, tumorigenic, and multi-potent capacities both in vitro and in vivo. CD109 silencing unexpectedly eliminates cancer stemness via controlling YAP/TAZ as the downstream target and this CD109-YAP/TAZ signaling axis is evolutionally conserved between drosophila and human brain tumors. Clinically, both CD133 and CD109 are independently associated with poorer prognosis of GBM patients, while the poorest prognosis group of GBM expressed both CD133 and CD109 at higher rates. These data indicate a possibility of the post-IR CSC plasticity and subsequent heterogeneity, and the activation of the CD109-YAP/TAZ signaling axis as previously unidentified therapeutic targets for post-IR recurrent GBM. Citation Format: Mutsuko Minata, Alessandra Audia, Junfeng Shi, Songjian Lu, Marat Pavlyukov, Yancey Gillespie Gillespie, Krishna Bhat, Ichiro Nakano. Plasticity in heterogenous cancer stem cells promotes glioblastoma radioresistance [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 937. doi:10.1158/1538-7445.AM2017-937

Radioresistance of mesenchymal glioblastoma initiating cells correlates with patient outcome and is associated with activation of inflammatory program.

Glioblastoma (GBM) still remains an incurable disease being radiotherapy (RT) the mainstay treatment. Glioblastoma intra-tumoral heterogeneity and Glioblastoma-Initiating Cells (GICs) challenge the design of effective therapies. We investigated GICs and non-GICs response to RT in a paired in-vitro model and addressed molecular programs activated in GICs after RT. Established GICs heterogeneously expressed several GICs markers and displayed a mesenchymal signature. Upon fractionated RT, GICs reported higher radioresistance compared to non-GICs and showed lower α- and β-values, according to the Linear Quadratic Model interpretation of the survival curves. Moreover, a significant correlation was observed between GICs radiosensitivity and patient disease-free survival. Transcriptome analysis of GICs after acquisition of a radioresistant phenotype reported significant activation of Proneural-to-Mesenchymal transition (PMT) and pro-inflammatory pathways, being STAT3 and IL6 the major players. Our findings support a leading role of mesenchymal GICs in defining patient response to RT and provide the grounds for targeted therapies based on the blockade of inflammatory pathways to overcome GBM radioresistance.

P08.36 Radioresistance of glioblastoma stem-like cells is associated with DNA replication stress, which is a promising therapeutic target

Introduction: The inevitability of tumour recurrence in glioblastoma (GBM) patients despite multi-modality treatment consisting of surgery, radiotherapy and chemotherapy, is reflected by a median survival of only 14 months. Tumour recurrence is thought to be driven by a small population of glioblastoma stem-like cells (GSCs) that are resistant to conventional therapies. DNA damage response (DDR) pathways have been shown to be up-regulated in GSCs and implicated in radioresistance and treatment failure. However the precise cause of enhanced DDR signalling in GSCs and the extent to which these signalling networks contribute to therapy resistance remains elusive. The objectives of this study were to investigate the underlying cause of DDR upregulation and treatment resistance in GSCs with a view to identifying novel and promising therapeutic targets. Materials and Methods: A panel of primary patient derived GBM cell lines cultured under conditions to enrich for or deplete the tumour stem cell population (GSC vs bulk respectively) were utilised in order to investigate enhanced GSC DDR under basal conditions and in response to ionising radiation. Confirmatory studies were also performed in cells sorted for the putative GSC marker CD133. The effects of a panel of small molecule DDR inhibitor agents on cell survival in GSC and bulk cells were quantified. Results: GSCs exhibited higher levels of total and activated DDR targets ATR, CHK1, ATM and PARP1 under basal conditions and were radioresistant compared to paired bulk populations. This was not due to increased levels of reactive oxygen species (ROS). Instead, we show that RPA is significantly higher in replicating GSCs and confirm by DNA fibre assays that GSCs and CD133+ cells have increased numbers of stalled replication forks, fewer new origins and slower DNA replication compared to bulk or CD133- populations, demonstrating for the first time that replication stress (RS) is a hallmark of GSCs. We identify increased expression of long neural genes as a likely mechanism for RS and DNA double strand breaks (DSBs) in GSCs and show that their radioresistance is reversed by dual inhibition of key RS and DDR proteins ATR and PARP. Conclusions: This study demonstrates the novel finding that replication stress is a hallmark of GSCs and resonates with recently published studies in neural progenitor cells showing that RS preferentially induces DNA DSB in long neural genes. Taken together, we implicate RS as a driver of enhanced DDR in GSCs and identify novel therapeutics with potential to improve clinical outcomes by overcoming the radioresistance of GBM

New role of osteopontin in DNA repair and impact on human glioblastoma radiosensitivity.

Glioblastoma (GBM) represents the most aggressive and common solid human brain tumor. We have recently demonstrated the importance of osteopontin (OPN) in the acquisition/maintenance of stemness characters and tumorigenicity of glioma initiating cells. Consultation of publicly available TCGA database indicated that high OPN expression correlated with poor survival in GBM patients. In this study, we explored the role of OPN in GBM radioresistance using an OPN-depletion strategy in U87-MG, U87-MG vIII and U251-MG human GBM cell lines. Clonogenic experiments showed that OPN-depleted GBM cells were sensitized to irradiation. In comet assays, these cells displayed higher amounts of unrepaired DNA fragments post-irradiation when compared to control. We next evaluated the phosphorylation of key markers of DNA double-strand break repair pathway. Activating phosphorylation of H2AX, ATM and 53BP1 was significantly decreased in OPN-deficient cells. The addition of recombinant OPN prior to irradiation rescued phospho-H2AX foci formation thus establishing a new link between DNA repair and OPN expression in GBM cells. Finally, OPN knockdown improved mice survival and induced a significant reduction of heterotopic human GBM xenograft when combined with radiotherapy. This study reveals a new function of OPN in DNA damage repair process post-irradiation thus further confirming its major role in GBM aggressive disease.

FGFR1 Induces Glioblastoma Radioresistance through the PLCγ/Hif1α Pathway.

FGF2 signaling in glioblastoma induces resistance to radiotherapy, so targeting FGF2/FGFR pathways might offer a rational strategy for tumor radiosensitization. To investigate this possibility, we evaluated a specific role for FGFR1 in glioblastoma radioresistance as modeled by U87 and LN18 glioblastomas in mouse xenograft models. Silencing FGFR1 decreased radioresistance in a manner associated with radiation-induced centrosome overduplication and mitotic cell death. Inhibiting PLCγ (PLCG1), a downstream effector signaling molecule for FGFR1, was sufficient to produce similar effects, arguing that PLCγ is an essential mediator of FGFR1-induced radioresistance. FGFR1 silencing also reduced expression of HIF1α, which in addition to its roles in hypoxic responses exerts an independent effect on radioresistance. Finally, FGFR1 silencing delayed the growth of irradiated tumor xenografts, in a manner that was associated with reduced HIF1α levels but not blood vessel alterations. Taken together, our results offer a preclinical proof of concept that FGFR1 targeting can degrade radioresistance in glioblastoma, a widespread problem in this tumor, prompting clinical investigations of the use of FGFR1 inhibitors for radiosensitization. Cancer Res; 76(10); 3036-44. ©2016 AACR.

MET inhibition overcomes radiation resistance of glioblastoma stem-like cells.

Glioblastoma (GBM) contains stem‐like cells (GSCs) known to be resistant to ionizing radiation and thus responsible for therapeutic failure and rapidly lethal tumor recurrence. It is known that GSC radioresistance relies on efficient activation of the DNA damage response, but the mechanisms linking this response with the stem status are still unclear. Here, we show that the MET receptor kinase, a functional marker of GSCs, is specifically expressed in a subset of radioresistant GSCs and overexpressed in human GBM recurring after radiotherapy. We elucidate that MET promotes GSC radioresistance through a novel mechanism, relying on AKT activity and leading to (i) sustained activation of Aurora kinase A, ATM kinase, and the downstream effectors of DNA repair, and (ii) phosphorylation and cytoplasmic retention of p21, which is associated with anti‐apoptotic functions. We show that MET pharmacological inhibition causes DNA damage accumulation in irradiated GSCs and their depletion in vitro and in GBMs generated by GSC xenotransplantation. Preclinical evidence is thus provided that MET inhibitors can radiosensitize tumors and convert GSC‐positive selection, induced by radiotherapy, into GSC eradication.