The acquisition of drug resistance is a persistent clinical problem limiting the successful treatment of human cancers, including glioblastoma (GBM). However, the molecular mechanisms by which initially chemoresponsive tumors develop therapeutic resistance remain poorly understood. In this study, we report that Pol k, an error-prone polymerase that participates in translesion DNA synthesis, was significantly upregulated in GBM cell lines and tumor tissues following temozolomide treatment. Overexpression of Pol k in temozolomide-sensitive GBM cells conferred resistance to temozolomide, whereas its inhibition markedly sensitized resistant cells to temozolomide in vitro and in orthotopic xenograft mouse models. Mechanistically, depletion of Pol k disrupted homologous recombination (HR)-mediated repair and restart of stalled replication forks, impaired the activation of ATR-Chk1 signaling, and delayed cell-cycle re-entry and progression.Further investigation of the relationship between Pol k and temozolomide revealed that Pol k inactivation facilitated temozolomide-induced Rad17 ubiquitination and proteasomal degradation, subsequently silencing ATR-Chk1 signaling and leading to defective HR repair and the reversal of temozolomide resistance. Moreover, overexpression of Rad17 in Pol k-depleted GBM cells restored HR efficiency, promoted the clearance of temozolomide-induced DNA breaks, and desensitized cells to the cytotoxic effects of temozolomide observed in the absence of Pol k. Finally, we found that Pol k overexpression correlated with poor prognosis in GBM patients undergoing temozolomide therapy. Collectively, our findings identify a potential mechanism by which GBM cells develop resistance to temozolomide and suggest that targeting the DNA damage tolerance pathway may be beneficial for overcoming resistance. Cancer Res; 76(8); 2340-53. Ó2016 AACR.
Resistance to temozolomide poses a major clinical challenge in glioblastoma multiforme treatment, and the mechanisms underlying the development of temozolomide resistance remain poorly understood. Enhanced DNA repair and mutagenesis can allow tumour cells to survive, contributing to resistance and tumour recurrence. Here, using recurrent temozolomide-refractory glioblastoma specimens, temozolomide-resistant cells, and resistant-xenograft models, we report that loss of miR-29c via c-Myc drives the acquisition of temozolomide resistance through enhancement of REV3L-mediated DNA repair and mutagenesis in glioblastoma. Importantly, disruption of c-Myc/miR-29c/REV3L signalling may have dual anticancer effects, sensitizing the resistant tumours to therapy as well as preventing the emergence of acquired temozolomide resistance. Our findings suggest a rationale for targeting the c-Myc/miR-29c/REV3L signalling pathway as a promising therapeutic approach for glioblastoma, even in recurrent, treatment-refractory settings.
PurposeMicroRNAs (miRNAs) have been shown to be involved in the initiation and progression of glioma. However, the underlying molecular mechanisms are still unclear.MethodsWe performed microarray analysis to evaluate miRNA expression levels in 158 glioma tissue samples, and examined miR-1231 levels in glioma samples and healthy brain tissues using qRT-PCR. In vitro analyses were performed using miR-1231 mimics, inhibitors, and siRNA targeting EGFR. We used flow cytometry, CCK-8 assays, and colony formation assays to examine glioma proliferation and cell cycle analysis. A dual luciferase reporter assay was performed to examine miR-1231 regulation of EGFR, and the effect of upregulated miR-1231 was investigated in a subcutaneous GBM model.ResultsWe found that miR-1231 expression was decreased in human glioma tissues and negatively correlated with EGFR levels. Moreover, the downregulation of miR-1231 negatively correlated with the clinical stage of human glioma patients. miR-1231 overexpression dramatically downregulated glioma cell proliferation, and suppressed tumor growth in a nude mouse model. Bioinformatics prediction and a luciferase assay confirmed EGFR as a direct target of miR-1231. EGFR overexpression abrogated the suppressive effect of miR-1231 on the PI3K/AKT pathway and G1 arrest.ConclusionsTaken together, these results demonstrated that EGFR is a direct target of miR-1231. Our findings suggest that the miR-1231/EGFR axis may be a helpful future diagnostic target for malignant glioma.Electronic supplementary materialThe online version of this article (10.1007/s11060-018-2903-8) contains supplementary material, which is available to authorized users.
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