RESUMO
To clarify whether differential compartmentalization of Survivin impacts temozolomide (TMZ)-triggered end points, we established a well-defined glioblastoma cell model in vitro (LN229 and A172) and in vivo, distinguishing between its nuclear and cytoplasmic localization. Expression of nuclear export sequence (NES)-mutated Survivin (SurvNESmut-GFP) led to impaired colony formation upon TMZ. This was not due to enhanced cell death but rather due to increased senescence. Nuclear-trapped Survivin reduced homologous recombination (HR)-mediated double-strand break (DSB) repair, as evaluated by γH2AX foci formation and qPCR-based HR assay leading to pronounced induction of chromosome aberrations. Opposite, clones, expressing free-shuttling cytoplasmic but not nuclear-trapped Survivin, could repair TMZ-induced DSBs and evaded senescence. Mass spectrometry-based interactomics revealed, however, no direct interaction of Survivin with any of the repair factors. The improved TMZ-triggered HR activity in Surv-GFP was associated with enhanced mRNA and stabilized RAD51 protein expression, opposite to diminished RAD51 expression in SurvNESmut cells. Notably, cytoplasmic Survivin could significantly compensate for the viability under RAD51 knockdown. Differential Survivin localization also resulted in distinctive TMZ-triggered transcriptional pathways, associated with senescence and chromosome instability as shown by global transcriptome analysis. Orthotopic LN229 xenografts, expressing SurvNESmut exhibited diminished growth and increased DNA damage upon TMZ, as manifested by PCNA and γH2AX foci expression, respectively, in brain tissue sections. Consequently, those mice lived longer. Although tumors of high-grade glioma patients expressed majorly nuclear Survivin, they exhibited rarely NES mutations which did not correlate with survival. Based on our in vitro and xenograft data, Survivin nuclear trapping would facilitate glioma response to TMZ.
Assuntos
Núcleo Celular/metabolismo , Senescência Celular , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/tratamento farmacológico , Recombinação Homóloga , Survivina/metabolismo , Temozolomida/farmacologia , Animais , Antineoplásicos Alquilantes/farmacologia , Apoptose , Biomarcadores Tumorais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Núcleo Celular/genética , Proliferação de Células , Dano ao DNA , Reparo do DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Nus , Survivina/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Therapy of malignant glioma relies on treatment with the O6 -methylating agent temozolomide (TMZ) concomitant with ionizing radiation followed by adjuvant TMZ. For the treatment of recurrences, DNA chloroethylating drugs are also used. The main killing lesion induced by these drugs is O6 -alkylguanine. Since this damage is repaired by O6 -methylguanine-DNA methyltransferase (MGMT), the repair enzyme represents a most important factor of drug resistance, limiting the therapy of malignant high-grade gliomas. Although MGMT has been shown to be transcriptionally up-regulated in rodents following genotoxic stress, it is still unclear whether human MGMT is subject to up-regulation. Here, we addressed the question whether MGMT in glioma cells is enhanced following alkylating drugs or ionizing radiation, using promoter assays. We also checked the response of glioma cell lines to dexamethasone. In a series of experiments, we found no evidence that the human MGMT promoter is significantly up-regulated following treatment with TMZ, the chloroethylating agent nimustine or radiation. It was activated, however, by dexamethasone. Using deletion constructs, we further show that the basal level of MGMT is mainly determined by the transcription factor SP1. The high amount of SP1 sites in the MGMT promoter likely prevents transcriptional up-regulation following genotoxic stress by neutralizing inducible signals. The regulation of MGMT by miRNAs plays only a minor role, as shown by DICER knockdown experiments. Since high dose dexamethasone concomitant with temozolomide is frequently used in glioblastoma therapy, induction of the MGMT gene through glucocorticoids in MGMT promoter unmethylated cases might cause further elevation of drug resistance, while radiation and alkylating drugs seem not to induce MGMT at transcriptional level.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Enzimas Reparadoras do DNA/genética , Glucocorticoides/farmacologia , O(6)-Metilguanina-DNA Metiltransferase/genética , Fator de Transcrição Sp1/genética , Temozolomida/farmacologia , Enzimas Reparadoras do DNA/efeitos dos fármacos , Enzimas Reparadoras do DNA/efeitos da radiação , Dexametasona/farmacologia , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/efeitos da radiação , Técnicas de Silenciamento de Genes , Humanos , O(6)-Metilguanina-DNA Metiltransferase/efeitos dos fármacos , O(6)-Metilguanina-DNA Metiltransferase/efeitos da radiação , Regiões Promotoras Genéticas/genética , RNA Mensageiro/farmacologia , Fator de Transcrição Sp1/efeitos dos fármacos , Fator de Transcrição Sp1/efeitos da radiação , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/efeitos da radiaçãoRESUMO
XAF1 (X-linked inhibitor of apoptosis (XIAP)-associated factor 1) is a tumor suppressor that counteracts the anti-apoptotic effects of XIAP and can sensitize cells to cell death triggering events. XAF1 knockdown abrogated the temozolomide (TMZ)-induced G2-arrest and prevented TMZ-induced apoptosis in the glioblastoma (GB) cell line LN229. Promoter methylation of XAF1 was found to be inversely correlated with mRNA expression in GB cells. We analyzed XAF1 methylation in a panel of 16 GB cell lines and 80 patients with first-diagnosed WHO grade III/IV high-grade gliomas using methylation-sensitive high-resolution melt (MS-HRM) analysis. In those patients, XAF1 promoter methylation was strongly associated with enhanced progression free and overall survival. Interestingly, XAF1 promoter methylation was strictly correlated with the occurrence of IDH1 mutations, indicating a causal link to the IDH1 mutant phenotype. XAF1 methylation was observed in 18 grade III tumors all of which showed heterozygous mutations in the IDH1 gene. 17 harbored a mutation leading to an arginine > histidine (R132H) and one carried a mutation causing an arginine > glycine (R132G) substitution. Furthermore, six out of six recurrent and IDH1 mutated grade III tumors also showed XAF1 promoter methylation. The data demonstrate that XAF1 promoter methylation determined by MS-HRM is a robust and precise indicator of IDH1 mutations in grade III gliomas. It is useful for complementing the immunohistochemistry-based detection of mutant IDH, uncovering rare 2-HG-producing IDH1 and potentially IDH2 mutations. The MS-HRM-based detection of XAF1 methylation could therefore be a reliable tool in assisting the sub-classification of high-grade gliomas.
Assuntos
Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/mortalidade , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Isocitrato Desidrogenase/genética , Mutação/genética , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal , Idoso , Apoptose , Proteínas Reguladoras de Apoptose , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Proliferação de Células , Metilação de DNA , Feminino , Inativação Gênica , Glioma/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Gradação de Tumores , Proteínas de Neoplasias/genética , Prognóstico , Regiões Promotoras Genéticas , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
The transcription factors NF-κB and p53 as well as their crosstalk determine the fate of tumor cells upon therapeutic interventions. Replicative stress and cytokines promote signaling cascades that lead to the co-regulation of p53 and NF-κB. Consequently, nuclear p53/NF-κB signaling complexes activate NF-κB-dependent survival genes. The 18 histone deacetylases (HDACs) are epigenetic modulators that fall into four classes (I-IV). Inhibitors of histone deacetylases (HDACi) become increasingly appreciated as anti-cancer agents. Based on their effects on p53 and NF-κB, we addressed whether clinically relevant HDACi affect the NF-κB/p53 crosstalk. The chemotherapeutics hydroxyurea, etoposide, and fludarabine halt cell cycle progression, induce DNA damage, and lead to DNA fragmentation. These agents co-induce p53 and NF-κB-dependent gene expression in cell lines from breast and colon cancer and in primary chronic lymphatic leukemia (CLL) cells. Using specific HDACi, we find that the class I subgroup of HDACs, but not the class IIb deacetylase HDAC6, are required for the hydroxyurea-induced crosstalk between p53 and NF-κB. HDACi decrease the basal and stress-induced expression of p53 and block NF-κB-regulated gene expression. We further show that class I HDACi induce senescence in pancreatic cancer cells with mutant p53.