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Glioblastoma (GBM) is the most aggressive brain cancer and its relapse after surgery, chemo and radiotherapy appears to be led by GBM stem cells (GSCs). Also, tumor networking and intercellular communication play a major role in driving GBM therapy-resistance. Tunneling Nanotubes (TNTs), thin membranous open-ended channels connecting distant cells, have been observed in several types of cancer, where they emerge to drive a more malignant phenotype. Here, we investigated whether GBM cells are capable to intercommunicate by TNTs. Two GBM stem-like cells (GSLCs) were obtained from the external and infiltrative zone of one GBM from one patient. We show, for the first time, that both GSLCs, grown in classical 2D culture and in 3D-tumor organoids, formed functional TNTs which allowed mitochondria transfer. In the organoid model, recapitulative of several tumor's features, we observed the formation of a network between cells constituted of both Tumor Microtubes (TMs), previously observed in vivo, and TNTs. In addition, the two GSLCs exhibited different responses to irradiation in terms of TNT induction and mitochondria transfer, although the correlation with the disease progression and therapy-resistance needs to be further addressed. Thus, TNT-based communication is active in different GSLCs derived from the external tumoral areas associated to GBM relapse, and we propose that they participate together with TMs in tumor networking.
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Neoplasias Encefálicas/metabolismo , Comunicação Celular , Extensões da Superfície Celular/metabolismo , Glioblastoma/metabolismo , Mitocôndrias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Organoides/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Extensões da Superfície Celular/patologia , Células Cultivadas , Progressão da Doença , Proteína GAP-43/metabolismo , Glioblastoma/patologia , Humanos , Mitocôndrias/patologia , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos da radiação , Organoides/patologia , Radiação , Recidiva , Imagem com Lapso de TempoRESUMO
BACKGROUND: Etoposide dosing is based on body surface area. We evaluated if further dose individualization would be required for high dose (HD) etoposide within the TI-CE (taxol, ifosfamide, carboplatin, and etoposide) protocol. METHODS: Eighty-eight patients received 400 mg/m2/day of etoposide as a 1-hour IV infusion on 3 consecutive days over 3 cycles as part of a phase II trial evaluating efficacy of therapeutic drug monitoring (TDM) of carboplatin in the TI-CE HD protocol. Pharmacokinetic (PK) data were analyzed using population PK model on NONMEM to quantify inter- and intra-individual variabilities. Relationship between etoposide exposure and pharmacodynamic (PD) endpoints, and between selected genetic polymorphisms and tumor response or toxicity were evaluated. RESULTS: The inter-patient, inter- and intra-cycle variabilities of clearance were 16%, 9% and 0.1%, respectively. The PK-PD relationship was not significant despite a trend toward higher etoposide exposure in patients responding to treatment. A significant correlation was found between exposure and extended neutropenia at cycle 3. A significant association between UGT1A1*28 polymorphism and late neutropenia was observed but needs further evaluation. CONCLUSIONS: The present study suggests that neither a priori dose individualization nor dose adaptation using TDM is required validating body surface area dosing of etoposide in the TI-CE protocol.
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Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/farmacocinética , Etoposídeo/farmacologia , Etoposídeo/farmacocinética , Neoplasias Embrionárias de Células Germinativas/tratamento farmacológico , Farmacogenética , Adulto , Antineoplásicos/administração & dosagem , Antineoplásicos Alquilantes/administração & dosagem , Antineoplásicos Fitogênicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica , Carboplatina/administração & dosagem , Monitoramento de Medicamentos , Etoposídeo/administração & dosagem , Feminino , Genótipo , Humanos , Ifosfamida/administração & dosagem , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Paclitaxel/administração & dosagem , Testes Farmacogenômicos , Adulto JovemRESUMO
GSCs play an important role in GBM recurrence. Understanding the resistance mechanisms in these cells is therefore crucial for radiation therapy optimization. In this study, using patient-derived GSCs, we demonstrate that GDF15, a cytokine belonging to the TGF-ß superfamily, is regulated by irradiation (IR) and the transcription factor WWTR1/TAZ. Blocking WWTR1/TAZ using specific siRNAs significantly reduces GDF15 basal expression and reverses the upregulation of this cytokine induced by IR. Furthermore, we demonstrate that GDF15 plays an important role in GSC radioresistance. Targeting GDF15 expression by siRNA in GSCs expressing high levels of GDF15 sensitizes the cells to IR. In addition, we also found that GDF15 expression is critical for GSC spheroid formation, as GDF15 knockdown significantly reduces the number of GSC neurospheres. This study suggests that GDF15 targeting in combination with radiotherapy may be a feasible approach in patients with GBM.
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Glioma is the most common primary brain tumor in dogs and predominantly affects brachycephalic breeds. Diagnosis relies on CT or MRI imaging, and the proposed treatments include surgical resection, chemotherapy, and radiotherapy depending on the tumor's location. Canine glioma from domestic dogs could be used as a more powerful model to study radiotherapy for human glioma than the murine model. Indeed, (i) contrary to mice, immunocompetent dogs develop spontaneous glioma, (ii) the canine brain structure is closer to human than mice, and (iii) domestic dogs are exposed to the same environmental factors than humans. Moreover, imaging techniques and radiation therapy used in human medicine can be applied to dogs, facilitating the direct transposition of results. The objective of this study is to fully characterize 5 canine glioma cell lines and to evaluate their intrinsic radiosensitivity. Canine cell lines present numerous analogies between the data obtained during this study on different glioma cell lines in dogs. Cell morphology is identical, such as doubling time, clonality test and karyotype. Immunohistochemical study of surface proteins, directly on cell lines and after stereotaxic injection in mice also reveals close similarity. Radiosensitivity profile of canine glial cells present high profile of radioresistance.
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Despite maximally safe resection of the magnetic resonance imaging (MRI)-defined contrast-enhanced (CE) central tumor area and chemoradiotherapy, most patients with glioblastoma (GBM) relapse within a year in peritumoral FLAIR regions. Magnetic resonance spectroscopy imaging (MRSI) can discriminate metabolic tumor areas with higher recurrence potential as CNI+ regions (choline/N-acetyl-aspartate index >2) can predict relapse sites. As relapses are mainly imputed to glioblastoma stem-like cells (GSCs), CNI+ areas might be GSC enriched. In this prospective trial, 16 patients with GBM underwent MRSI/MRI before surgery/chemoradiotherapy to investigate GSC content in CNI-/+ biopsies from CE/FLAIR. Biopsy and derived-GSC characterization revealed a FLAIR/CNI+ sample enrichment in GSC and in gene signatures related to stemness, DNA repair, adhesion/migration, and mitochondrial bioenergetics. FLAIR/CNI+ samples generate GSC-enriched neurospheres faster than FLAIR/CNI-. Parameters assessing biopsy GSC content and time-to-neurosphere formation in FLAIR/CNI+ were associated with worse patient outcome. Preoperative MRI/MRSI would certainly allow better resection and targeting of FLAIR/CNI+ areas, as their GSC enrichment can predict worse outcomes.
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Neoplasias Encefálicas , Glioblastoma , Humanos , Neoplasias Encefálicas/patologia , Glioblastoma/metabolismo , Imageamento por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/métodos , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/metabolismo , Estudos Prospectivos , RecidivaRESUMO
Systematic recurrence of glioblastoma (GB) despite surgery and chemo-radiotherapy is due to GB stem cells (GBSC), which are particularly invasive and radioresistant. Therefore, there is a need to identify new factors that might be targeted to decrease GBSC invasive capabilities as well as radioresistance. Patient-derived GBSC were used in this study to demonstrate a higher expression of the glycoprotein M6a (GPM6A) in invasive GBSC compared to non-invasive cells. In 3D invasion assays performed on primary neurospheres of GBSC, we showed that blocking GPM6A expression by siRNA significantly reduced cell invasion. We also demonstrated a high correlation of GPM6A with the oncogenic protein tyrosine phosphatase, PTPRZ1, which regulates GPM6A expression and cell invasion. The results of our study also show that GPM6A and PTPRZ1 are crucial for GBSC sphere formation. Finally, we demonstrated that targeting GPM6A or PTPRZ1 in GBSC increases the radiosensitivity of GBSC. Our results suggest that blocking GPM6A or PTPRZ1 could represent an interesting approach in the treatment of glioblastoma since it would simultaneously target proliferation, invasion, and radioresistance.
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Glioblastoma , Glicoproteínas de Membrana , Proteínas do Tecido Nervoso , Glioblastoma/genética , Glioblastoma/radioterapia , Humanos , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/efeitos da radiação , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/efeitos da radiação , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/efeitos da radiação , Tolerância a Radiação , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a ReceptoresRESUMO
PURPOSE: With current gold standard treatment, which associates maximum safe surgery and chemo-radiation, the large majority of glioblastoma patients relapse within a year in the peritumoral non contrast-enhanced region (NCE). A subpopulation of glioblastoma stem-like cells (GSC) are known to be particularly radio-resistant and aggressive, and are thus suspected to be the cause of these relapses. Previous studies have shown that their distribution is heterogeneous in the NCE compartment, but no study exists on the sensitivity of medical imaging for localizing these cells. In this work, we propose to study the magnetic resonance (MR) signature of these infiltrative cells. METHODS: In the context of a clinical trial on 16 glioblastoma patients, relative Cerebral Blood Volume (rCBV) and Apparent Diffusion Coefficient (ADC) were measured in a preoperative diffusion and perfusion MRI examination. During surgery, two biopsies were extracted using image-guidance in the hyperintensities-FLAIR region. GSC subpopulation was quantified within the biopsies and then cultivated in selective conditions to determine their density and aggressiveness. RESULTS: Low ADC was found to be a good predictor of the time to GSC neurospheres formation in vitro. In addition, GSCs were found in higher concentrations in areas with high rCBV. CONCLUSIONS: This study confirms that GSCs have a critical role for glioblastoma aggressiveness and supports the idea that peritumoral sites with low ADC or high rCBV should be preferably removed when possible during surgery and targeted by radiotherapy.
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Variations in clinical response to tamoxifen (TAM) may be related to polymorphic cytochromes P450 (CYPs) involved in forming its active metabolite endoxifen (ENDO). We developed a population pharmacokinetic (PopPK) model for tamoxifen and six metabolites to determine clinically relevant factors of ENDO exposure. Concentration-time data for TAM and 6 metabolites come from a prospective, multicenter, 3-year follow-up study of adjuvant TAM (20 mg/day) in patients with breast cancer, with plasma samples drawn every 6 months, and genotypes for 63 genetic polymorphisms (PHACS study, NCT01127295). Concentration data for TAM and 6 metabolites from 928 patients (n = 27,433 concentrations) were analyzed simultaneously with a 7-compartment PopPK model. CYP2D6 phenotype (poor metabolizer (PM), intermediate metabolizer (IM), normal metabolizer (NM), and ultra-rapid metabolizer (UM)), CYP3A4*22, CYP2C19*2, and CYP2B6*6 genotypes, concomitant CYP2D6 inhibitors, age, and body weight had a significant impact on TAM metabolism. Formation of ENDO from N-desmethyltamoxifen was decreased by 84% (relative standard error (RSE) = 14%) in PM patients and by 47% (RSE = 9%) in IM patients and increased in UM patients by 27% (RSE = 12%) compared with NM patients. Dose-adjustment simulations support an increase from 20 mg/day to 40 and 80 mg/day in IM patients and PM patients, respectively, to reach ENDO levels similar to those in NM patients. However, when considering Antiestrogenic Activity Score (AAS), a dose increase to 60 mg/day in PM patients seems sufficient. This PopPK model can be used as a tool to predict ENDO levels or AAS according to the patient's CYP2D6 phenotype for TAM dose adaptation.
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Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Citocromo P-450 CYP2D6/genética , Polimorfismo Genético , Tamoxifeno/farmacocinética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Hormonais/administração & dosagem , Antineoplásicos Hormonais/farmacocinética , Feminino , Humanos , Estudos Longitudinais , Pessoa de Meia-Idade , Modelos Biológicos , Variantes Farmacogenômicos , Tamoxifeno/administração & dosagem , Tamoxifeno/análogos & derivados , Tamoxifeno/metabolismoRESUMO
Recurrence of GBM is thought to be due to GBMSCs, which are particularly chemo-radioresistant and characterized by a high capacity to invade normal brain. Evidence is emerging that modulation of m6A RNA methylation plays an important role in tumor progression. However, the impact of this mRNA modification in GBM is poorly studied. We used patient-derived GBMSCs to demonstrate that high expression of the RNA demethylase, ALKBH5, increases radioresistance by regulating homologous recombination (HR). In cells downregulated for ALKBH5, we observed a decrease in GBMSC survival after irradiation likely due to a defect in DNA-damage repair. Indeed, we observed a decrease in the expression of several genes involved in the HR, including CHK1 and RAD51, as well as a persistence of γ-H2AX staining after IR. We also demonstrated in this study that ALKBH5 contributes to the aggressiveness of GBM by favoring the invasion of GBMSCs. Indeed, GBMSCs deficient for ALKBH5 exhibited a significant reduced invasion capability relative to control cells. Our data suggest that ALKBH5 is an attractive therapeutic target to overcome radioresistance and invasiveness of GBMSCs.
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Hepatotoxicity is an important concern for nearly 40% of the patients treated with trabectedin for advanced soft tissue sarcoma (ASTS). The mechanisms underlying these liver damages have not yet been elucidated but they have been suggested to be related to the production of reactive metabolites. The aim of this pharmacogenetic study was to identify genetic variants of pharmacokinetic genes such as CYP450 and ABC drug transporters that could impair the trabectedin metabolism in hepatocytes. Sixty-three patients with ASTS from the TSAR clinical trial (NCT02672527) were genotyped by next-generation sequencing for 11 genes, and genotype-toxicity association analyses were performed with R package SNPassoc. Among the results, ABCC2 c.1249A allele (rs2273697) and ABCG2 intron variant c.-15994T (rs7699188) were associated with an increased risk of severe cytolysis, whereas ABCC2 c.3563A allele had a protective effect, as well as ABCB1 variants rs2032582 and rs1128503 (p-value < 0.05). Furthermore, CYP3A5*1 rs776746 (c.6986A > G) increased the risk of severe overall hepatotoxicity (p = 0.012, odds ratio (OR) = 5.75), suggesting the implication of metabolites in the hepatotoxicity. However, these results did not remain significant after multiple analysis correction. These findings need to be validated on larger cohorts of patients, with mechanistic studies potentially being able to validate the functional consequences of these variants.
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Glioblastoma (GBM) is the most lethal primary brain tumor in adults and is known to be particularly aggressive and resistant to anti-cancer therapies, mainly due to the presence of GBM stem cells (GBMSC). By in vitro approaches supported by analysis from patients' databases, we determined how α6-integrin and Fibroblast Growth Factor Receptor 1 (FGFR1) work in concert to regulate proliferation and stemness of GBMSC. We showed that α6-integrin regulates the expression of FGFR1 and its target gene Fokhead Box M1 (FOXM1) via the ZEB1/YAP1 transcription complex. These results were in accordance with the positive correlation observed in GBM between α6-integrin expression and its target genes ZEB1/YAP1, FGFR1, and FOXM1 in the databases, TCGA and Rembrandt. In addition, the clinical data demonstrate that GBM patients with high levels of the five genes signature, including α6-integrin, ZEB1/YAP1, FGFR1 and FOXM1, have a significantly shorter overall survival. In vitro, we observed a similar decrease in the expression of stemness-related factors, neurospheres forming capacity, as well as spheroids growth when α6-integrin or FGFR1 was blocked individually with specific siRNA, whereas the combination of both siRNA led to a significantly higher inhibition of spheres formation. These data suggest that co-administration of anti-FGFR1 and anti-α6-integrin could provide an improved therapeutic response in GBMSC.
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Glioblastomas (GB) are malignant brain tumors with poor prognosis despite treatment with surgery and radio/chemotherapy. These tumors are defined by an important cellular heterogeneity and notably contain a subpopulation of GB-initiating cells (GIC), which contribute to tumor aggressiveness, resistance, and recurrence. Some integrins are specifically expressed by GICs and could be actionable targets to improve GB treatment. Here, integrin ß8 (ITGB8) was identified as a potential selective target in this highly tumorigenic GIC subpopulation. Using several patient-derived primocultures, it was demonstrated that ITGB8 is overexpressed in GICs compared with their differentiated progeny. Furthermore, ITGB8 is also overexpressed in GB, and its overexpression is correlated with poor prognosis and with the expression of several other classic stem cell markers. Moreover, inhibiting ITGB8 diminished several main GIC characteristics and features, including self-renewal ability, stemness, migration potential, and tumor formation capacity. Blockade of ITGB8 significantly impaired GIC cell viability via apoptosis induction. Finally, the combination of radiotherapy and ITGB8 targeting radiosensitized GICs through postmitotic cell death. IMPLICATIONS: This study identifies ITGB8 as a new selective marker for GICs and as a promising therapeutic target in combination with chemo/radiotherapy for the treatment of highly aggressive brain tumors.
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Neoplasias Encefálicas/genética , Glioblastoma/genética , Cadeias beta de Integrinas/metabolismo , Radiossensibilizantes/uso terapêutico , Animais , Diferenciação Celular , Humanos , Camundongos , Camundongos Nus , Radiossensibilizantes/farmacologia , TransfecçãoRESUMO
In addition to the effect of cytochrome P450 (CYP) 2D6 genetic polymorphisms, the metabolism of tamoxifen may be impacted by other factors with possible consequences on therapeutic outcome (efficacy and toxicity). This analysis focused on the pharmacokinetic (PK)-pharmacogenetic evaluation of tamoxifen in 730 patients with adjuvant breast cancer included in a prospective multicenter study. Plasma concentrations of tamoxifen and six major metabolites, the genotype for 63 single-nucleotide polymorphisms, and comedications were obtained 6 months after treatment initiation. Plasma concentrations of endoxifen were significantly associated with CYP2D6 diplotype (P < 0.0001), CYP3A4*22 genotype (P = 0.0003), and concomitant intake of potent CYP2D6 inhibitors (P < 0.001). Comparison of endoxifen levels showed that the CYP2D6 phenotype classification could be improved by grouping intermediate metabolizer (IM)/IM and IM/poor metabolizer diplotype into IM phenotype for future use in tamoxifen therapy optimization. Finally, the multivariable regression analysis showed that formation of tamoxifen metabolites was independently impacted by CYP2D6 diplotype and CYP3A4*22, CYP2C19*2, and CYP2B6*6 genetic polymorphisms.
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Antineoplásicos Hormonais/administração & dosagem , Antineoplásicos Hormonais/farmacocinética , Neoplasias da Mama/tratamento farmacológico , Sistema Enzimático do Citocromo P-450/genética , Tamoxifeno/administração & dosagem , Tamoxifeno/farmacocinética , Adulto , Idoso , Antineoplásicos Hormonais/uso terapêutico , Citocromo P-450 CYP2B6/genética , Inibidores do Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP3A/genética , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Farmacogenética , Polimorfismo de Nucleotídeo Único , Estudos Prospectivos , Tamoxifeno/análogos & derivados , Tamoxifeno/sangue , Tamoxifeno/uso terapêuticoRESUMO
Integrins are extracellular matrix receptors involved in tumour invasion and angiogenesis. Although there is evidence that inhibiting integrins might enhance the efficiency of radiotherapy, little is known about the exact mechanisms involved in the integrin-dependent modulation of tumor radiosensitivity. The purpose of this study was to investigate the role of alphavbeta3 and alphavbeta5 integrins in glioblastoma cell radioresistance and overall to decipher the downstream biological pathways. We first demonstrated that silencing alphavbeta3 and alphavbeta5 integrins with specific siRNAs significantly reduced the survival after irradiation of 2 glioblastoma cell lines: U87 and SF763. We then showed that integrin activity and integrin signalling pathways controlled the glioma cell radiosensitivity. This regulation of glioma cell response to ionising radiation was mediated through the integrin-linked kinase, ILK, and the small GTPase, RhoB, by two mechanisms. The first one, independent of ILK, consists in the regulation of the intracellular level of RhoB by alphavbeta3 or alphavbeta5 integrin. The second pathway involved in cell radiosensitivity consists in RhoB activation by ionising radiation through ILK. Furthermore, we demonstrated that the alphavbeta3/alphavbeta5 integrins/ILK/RhoB pathway controlled the glioma cells radiosensitivity by regulating radiation-induced mitotic cell death. This work identifies a new biological pathway controlling glioblastoma cells radioresistance, activated from the membrane through alphavbeta3 and/or alphavbeta5 integrins via ILK and RhoB. Our results are clues that downstream effectors of alphavbeta3 and alphavbeta5 integrins as ILK and RhoB might also be promising candidate targets for improving the efficiency of radiotherapy and thus the clinical outcome of patients with glioblastoma.
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Glioma/metabolismo , Glioma/radioterapia , Integrina alfaVbeta3/metabolismo , Integrinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Radiação Ionizante , Receptores de Vitronectina/metabolismo , Proteína rhoB de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral/efeitos da radiação , Citometria de Fluxo , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Humanos , RNA Interferente Pequeno/metabolismo , Tolerância a Radiação , TransfecçãoRESUMO
Hypoxia is a crucial factor in tumor aggressiveness and resistance to treatment, particularly in glioma. Our previous results have shown that inhibiting the small GTPase RhoB increased oxygenation of U87 human glioblastoma xenografts, in part, by regulating angiogenesis. We investigated here whether RhoB might also control a signaling pathway that would permit glioma cells to adapt to hypoxia. We first showed that silencing RhoB with siRNA induced degradation and inhibition of the transcriptional activity of the hypoxia-inducible factor by the proteasome in U87 hypoxic cells. This RhoB-dependent degradation of hypoxia-inducible factor-1alpha in hypoxic conditions was mediated by the Akt/glycogen synthase kinase-3beta pathway. While investigating how hypoxia could activate this signaling pathway, using the GST-Rhotekin RBD pulldown assay, we showed the early activation of RhoB by reactive oxygen species under hypoxic conditions and, subsequently, its participation in the ensuing cellular adaptation to hypoxia. Overall, therefore, our results have not only highlighted a new signaling pathway for hypoxia controlled by the small GTPase RhoB, but they also strongly implicate RhoB as a potentially important therapeutic target for decreasing tumor hypoxia.
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Glioblastoma/enzimologia , Quinase 3 da Glicogênio Sintase/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteína rhoB de Ligação ao GTP/metabolismo , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Ativação Enzimática , Glioblastoma/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteína rhoB de Ligação ao GTP/antagonistas & inibidoresRESUMO
Despite post-operative radio-chemotherapy, glioblastoma systematically locally recurs. Tumors contacting the periventricular zone (PVZ) show earlier and more distant relapses than tumors not contacting the PVZ. Since glioblastoma stem-like cells (GSCs) have been proposed to play a major role in glioblastoma recurrence, we decided to test whether GSC migration properties could be different according to their anatomical location (PVZ+/PVZ-). For that purpose, we established paired cultures of GSCs from the cortical area (CT) and the PVZ of glioblastoma patient tumors. We demonstrated that PVZ GSCs possess higher migration and invasion capacities than CT GSCs. We highlighted specific transcriptomic profiles in PVZ versus CT populations and identified a down-regulation of the RhoGTPase, RND1 in PVZ GSCs compared to CT GSCs. Overexpression of RND1, dramatically inhibited PVZ GSC migration and conversely, downregulation of RND1 increased CT GSC migration. Additionally, transcriptomic analyses also revealed a down-regulation of RND1 in glioblastoma compared to normal brain. Using the glioblastoma TCGA database, low levels of RND1 were also shown to correlate with a decreased overall survival of patients. Finally, based on signaling pathways activated in patients with low levels of RND1, we identified an RND1 low signature of six genes (MET, LAMC1, ITGA5, COL5A1, COL3A1, COL1A2) that is an independent prognostic factor in glioblastoma. These findings contribute to explain the shorter time to progression of patients with PVZ involvement and, point out genes that establish the RND1 low signature as key targets genes to impede tumor relapse after treatment.
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Radiotherapy is the cornerstone of glioblastoma (GBM) standard treatment. However, radioresistance of cancer cells leads to an inevitable recurrence. In the present study, we showed that blocking α6-integrin in cells derived from GBM biopsy specimens cultured as neurospheres, sensitized cells to radiation. In cells downregulated for α6-integrin expression, we observed a decrease in cell survival after irradiation and an increase in radio-induced cell death. We also demonstrated that inhibition of α6-integrin expression affects DNA damage checkpoint and repair. Indeed, we observed a persistence of γ-H2AX staining after IR and the abrogation of the DNA damage-induced G2/M checkpoint, likely through the downregulation of the checkpoint kinase CHK1 and its downstream target Cdc25c. We also showed that α6-integrin contributes to GBM radioresistance by controlling the expression of the transcriptional network ZEB1/OLIG2/SOX2. Finally, the clinical data from TCGA and Rembrandt databases demonstrate that GBM patients with high levels of the five genes signature, including α6-integrin and its targets, CHK1, ZEB1, OLIG2 and SOX2, have a significantly shorter overall survival. Our study suggest that α6-integrin is an attractive therapeutic target to overcome radioresistance of GBM cancer cells.
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Neoplasias Encefálicas/genética , Dano ao DNA/genética , Glioblastoma/genética , Cadeias alfa de Integrinas/genética , Tolerância a Radiação/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Divisão Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Quinase 1 do Ponto de Checagem/genética , Regulação para Baixo/genética , Fase G2/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Fator de Transcrição 2 de Oligodendrócitos/genética , Fatores de Transcrição SOXB1/genética , Transcrição Gênica/genética , Fosfatases cdc25/genéticaRESUMO
A high percentage of advanced rectal cancers are resistant to radiation. Therefore, increasing the efficacy of radiotherapy by targeting factors involved in radioresistance seems to be an attractive strategy. Here we demonstrated that the pro-hormone progastrin (PG), known to be over-expressed in CRC, and recognized as a pro-oncogenic factor, is a radioresistance factor that can be targeted to sensitize resistant rectal cancers to radiations. First, we observed an increase in PG mRNA expression under irradiation. Our results also demonstrated that down-regulating PG mRNA expression using a shRNA strategy, significantly increases the sensitivity to irradiation (IR) in a clonogenic assay of different colorectal cancer cell lines. We also showed that the combination of PG gene down-regulation and IR strongly inhibits tumours progression in vivo. Then, we demonstrated that targeting PG gene radiosensitizes cancer cells by increasing radio-induced apoptosis shown by an increase in annexin V positive cells, caspases activation and PARP cleavage. We also observed the up-regulation of the pro-apoptotic pathway, JNK and the induction of the expression of pro-apoptotic factors such as BIM. In addition, we demonstrated in this study that inhibition of PG gene expression enhances radiation-induced DNA damage. Our data also suggest that, in addition to increase radio-induced apoptosis, targeting PG gene also leads to the inhibition of the survival pathways, AKT and ERK induced by IR. Taken together, our results highlight the role of PG in radioresistance and provide a preclinical proof of concept that PG represents an attractive target for sensitizing resistant rectal tumours to irradiation. .
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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.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fosfolipase C gama/metabolismo , Tolerância a Radiação , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Apoptose/efeitos da radiação , Western Blotting , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/radioterapia , Proliferação de Células/efeitos da radiação , Centrossomo/efeitos da radiação , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Camundongos Nus , Mitose/efeitos da radiação , Fosfolipase C gama/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We previously demonstrated in vitro that inhibiting the biological pathways of the small GTPase Rho radiosensitizes the human glioma U87 cell line. The aim of this study was to determine if Rho might be involved in the control of in vivo radiosensitivity altogether by controlling cellular radioresistance and by modifying tumor microenvironment. We demonstrate here that the in vivo induction of the dominant negative of Rho, RhoBN19, in U87 xenografts induces a significant decrease of tumor cell survival after irradiation more important than the one we previously observed in vitro. This in vivo increased effect of RhoBN19 expression is due to the improvement of the tumor oxygenation associated with a significant decrease of the vessel density and of the metalloproteinase 2 (MMP2) expression. Moreover, in vitro RhoBN19 expression in U87 cells leads to the inhibition of MMP2 activity. Our results demonstrate for the first time that inhibiting Rho pathways modifies the in vivo radiosensitivity of human glioma cells by controlling intrinsic radioresistance, hypoxia and angiogenesis. These data strongly suggest that Rho should be a major determinant of cellular resistance to ionizing radiation.