ATRX/EZH2 complex epigenetically regulates FADD/PARP1 axis, contributing to TMZ resistance in glioma.

Rationale: Glioma is the most common primary malignant brain tumor in adults. Chemoresistance of temozolomide (TMZ), the first-line chemotherapeutic agent, is a major issue in the management of patients with glioma. Alterations of alpha thalassemia/mental retardation syndrome X-linked (ATRX) gene constitute one of the most prevalent genetic abnormalities in gliomas. Therefore, elucidation of the role of ATRX contributing to TMZ resistance in glioma is urgently needed. Methods: We performed the bioinformatics analysis of gene expression, and DNA methylation profiling, as well as RNA and ChIP-seq data sets. CRISPR-Cas9 gene editing system was used to achieve the ATRX knockout in TMZ resistant cells. In vitro and in vivo experiments were carried out to investigate the role of ATRX contributing to TMZ resistance in glioma. Results: We found that ATRX expression was upregulated via DNA demethylation mediated by STAT5b/TET2 complex and strengthened DNA damage repair by stabilizing PARP1 protein in TMZ resistant cells. ATRX elicited PARP1 stabilization by the down-regulating of FADD expression via the H3K27me3 enrichment, which was dependent on ATRX/EZH2 complex in TMZ resistant cells. Magnetic resonance imaging (MRI) revealed that the PARP inhibitor together with TMZ inhibited glioma growth in ATRX wild type TMZ resistant intracranial xenograft models. Conclusions: The present study further illustrated the novel mechanism of the ATRX/PARP1 axis contributing to TMZ resistance. Our results provided substantial new evidence that PARP inhibitor might be a potential adjuvant agent in overcoming ATRX mediated TMZ resistance in glioma.

Han B ，Meng X ，Wu P ，Li Z ，Li S ，Zhang Y ，Zha C ，Ye Q ，Jiang C ，Cai J ，Jiang T ... -  《Theranostics》

Loss of ATRX suppresses ATM dependent DNA damage repair by modulating H3K9me3 to enhance temozolomide sensitivity in glioma.

Mutations in ATRX constitute the most prevalent genetic abnormalities in gliomas. The presence of ATRX mutations in glioma serves as a marker of better prognosis with longer patient survival although the underlying mechanisms are poorly understood. In the present study, we found that ATRX biological function was significantly involved in DNA replication and repair. CRISPR/Cas9-mediated genetic inactivation of ATRX induced inhibition of cell proliferation, invasion and vasculogenic mimicry. In addition, temozolomide (TMZ) treatment induced greater DNA damage and apoptotic changes in ATRX knockout glioma cells. Moreover, we confirmed that ATRX knockout resulted in a failure to trigger ATM phosphorylation and finally restrained the activation of downstream proteins of the ATM pathway. The ATM-associated DNA repair pathway was extensively compromised in ATRX knockout cells owing to decreased histone H3K9me3 availability. Public databases also showed that patients with low ATRX expression exhibited preferable overall survival and profited more from TMZ treatment. These data suggest that ATRX is involved in DNA damage repair by regulating the ATM pathway and might serve as a prognostic maker in predicting TMZ chemosensitivity.

Han B ，Cai J ，Gao W ，Meng X ，Gao F ，Wu P ，Duan C ，Wang R ，Dinislam M ，Lin L ，Kang C ，Jiang C ... -  《-》

EZH2-regulated PARP-1 Expression is a Likely Mechanism for the Chemoresistance of Gliomas to Temozolomide.

Chemoresistance in gliomas accounts for the major cause of tumor progress and recurrence during comprehensive treatment with alkylating agents including temozolomide (TMZ). The oncogenic role of Enhancer of zeste homolog 2 (EZH2) has been identified in many solid malignancies including gliomas, though the accurate effect of EZH2 on chemotherapy resistance of gliomas has been elusive. To elucidate the role of EHZ2 on TMZ resistance of gliomas and the molecular mechanisms. Immunohistochemistry (IHC) and Reverse transcription-quantitative (RT-q) PCR, and western blot assay were performed for expressional analysis. Cell Counting Kit-8 (CCK-8) assay was applied to determine the TMZ sensitivity. EZH2-silencing lentivirus was generated for mechanic study. EZH2 was overexpressed in gliomas both at the transcriptional and protein levels. EZH2 level in glioma cell lines was positively correlated with resistance to TMZ, represented by the 50% inhibition rate (IC50). Moreover, there was increased TMZ sensitivity in EZH2-inhibited glioma cells than in the control cells. Furthermore, we determined that PARP1 was a common molecule among the downregulated DNA repair proteins in both U251 and U87 glioma cell lines after EZH2 inhibition. Specifically, we observed a spontaneous increase of PARP1 expression with TMZ treatment and interestingly, the increase of PARP1 could be also reduced by EZH2 inhibition in the glioma cells. Finally, combined treatment with lentivirus-induced EZH2 inhibition and a PARP1 inhibitor dramatically enhanced TMZ cytotoxicity compared with either one alone. EZH2-PARP-1 signaling axis is possibly responsible for the chemoresistance of gliomas to TMZ. Simultaneously inhibiting these two genes may improve the outcome of TMZ chemotherapy.

Liang Q ，Wang B ，Zhang C ，Song C ，Wang J ，Sun W ，Jiang L ，Lin J ... -  《-》

lncRNA TUG1 inhibits the cancer stem cell‑like properties of temozolomide‑resistant glioma cells by interacting with EZH2.

Cao Y ，Chai W ，Wang Y ，Tang D ，Shao D ，Song H ，Long J ... -  《-》

Regulation of temozolomide resistance in glioma cells via the RIP2/NF-κB/MGMT pathway.

Temozolomide (TMZ) is a first-line chemotherapy drug for the treatment of malignant glioma and resistance to it poses a major challenge. Receptor-interacting protein 2 (RIP2) is associated with the malignant character of cancer cells. However, it remains unclear whether RIP2 is involved in TMZ resistance in glioma. RIP2 expression was inhibited in TMZ-resistant glioma cells and normal glioma cells by using small interfering RNA (siRNA) against RIP2. Plasmid transfection method was used to overexpress RIP2. Cell counting kit-8 assays were performed to evaluate cell viability. Western blotting or immunofluorescence was performed to determine RIP2, NF-κB, and MGMT expression in cells. Flow cytometry was used to investigate cell apoptosis. TMZ-resistant glioma xenograft models were established to evaluate the role of the RIP2/NF-κB/MGMT signaling pathway in drug resistance. We observed that RIP2 expression was upregulated in TMZ-resistant glioma cells, whereas silencing of RIP2 expression enhanced cellular sensitivity to TMZ. Similarly, upon the induction of RIP2 overexpression, glioma cells developed resistance to TMZ. The molecular mechanism underlying the process indicated that RIP2 can activate the NF-κB signaling pathway and upregulate the expression of O6-methylguanine-DNA methyltransferase (MGMT), following which the glioma cells develop drug resistance. In the TMZ-resistant glioma xenograft model, treatment with JSH-23 (an NF-κB inhibitor) and lomeguatrib (an MGMT inhibitor) could enhance the sensitivity of the transplanted tumor to TMZ. We report that the RIP2/NF-κB/MGMT signaling pathway is involved in the regulation of TMZ resistance. Interference with NF-κB or MGMT activity could constitute a novel strategy for the treatment of RIP2-positive TMZ-resistant glioma.

Hu YH ，Jiao BH ，Wang CY ，Wu JL ... -  《-》