Cancer Letters

Cancer Letters

Volume 436, 1 November 2018, Pages 10-21
Cancer Letters

Original Articles
Exosomal transfer of miR-151a enhances chemosensitivity to temozolomide in drug-resistant glioblastoma

https://doi.org/10.1016/j.canlet.2018.08.004Get rights and content

Highlights

  • Low miR-151a levels of GBM patients correlate with poor response to TMZ therapy.

  • Overexpression of miR-151a restores TMZ response of resistant GBM cells in vivo.

  • miR-151a could be packaged into exosomes and disseminate TMZ chemosensitivity.

  • miR-151a is a clinical indicator and therapeutic target for GBM TMZ resistance.

Abstract

Chemoresistance blunts the effect of Temozolomide (TMZ) in the treatment of glioblastoma multiforme (GBM). Whether exosomal transfer of miRNAs derived from TMZ-resistant GBM cells could confer TMZ resistance remains to be determined. qPCR was used to determine miR-151a expression in two TMZ-resistant GBM cell lines. The direct targets of miR-151a were identified by microarray assays, bioinformatics and further RNA chromatin immunoprecipitation (RNA-ChIP) assay. We characterized exosomes from TMZ-resistant cell lines, serum and cerebrospinal fluid (CSF) and determined the effect of exosomes from TMZ-resistant cells on recipient GBM cells. miR-151a loss drove the acquisition of TMZ resistance. Restored miR-151a expression sensitized TMZ-resistant GBM cells via inhibiting XRCC4-mediated DNA repair. TMZ-resistant GBM cells conferred TMZ chemoresistance to recipient TMZ-sensitive cells in an exosomal miR-151a loss-dependent manner. Restoration of exosomal miR-151a from donor TMZ-resistant cells abolished the chemoresistance dissemination that was directed by donor TMZ-resistant cells. CSF-derived exosomes contained miRNA signatures reflective of the underlying chemoresistant status of GBMs in terms of miR-151a expression levels. Exosomal miR-151a is not only essentially a less-invasive ‘liquid biopsy’ that might predict chemotherapy response, but also represents a promising therapeutic target for therapy-refractory GBMs.

Introduction

GBM is a WHO grade IV tumor with a dismal prognosis [1]. The current standard of treatment for GBM patients is surgical resection followed by adjuvant radiation therapy and chemotherapy with TMZ [2]. TMZ is used as a first-line therapy in GBM. However, drug resistance limits the durability of the treatment response.

Exosomes, which are adsorption vesicles of ∼50–120 nm in diameter, are implicated in intercellular communication by transporting intracellular cargos, such as proteins and RNAs [3,4]. The transfer of GBM-released extracellular vesicle (EV) miRNAs to microglia could affect target mRNA and its protein levels in microglia [5]. Intratumoral GBM-released EV miRNAs transfer is also found to augment the heterogeneity of GBM stem-like cells [6]. However, the contribution of GBM exosomal miRNAs to TMZ resistance within the heterogeneous intratumoral ecosystem remains to be elucidated.

The monofunctional DNA-alkylating agent, TMZ exerts its cytotoxic function by inducing DNA damage in GBM cells [7,8]. Damaged bases and nucleotides caused by TMZ could ultimately lead to DNA double-strand breaks (DSBs), resulting in GBM cell death via caspase-dependent apoptotic pathways [7,9,10]. Non-homologous end-joining (NHEJ) is the major pathway by which DSBs are repaired [7,9]. As a vital step in NHEJ process, XRCC4 protein can combine with DNA ligase IV to form the DNA ligase IV/XRCC4 heterodimer and this complex in turn covalently joins the broken DNA ends to help cells survive [7,11]. A possible role of NHEJ pathway in TMZ chemosensitivity has also been reported in GBM cells [7]. Previously, our group has demonstrated that polymorphisms of XRCC4 contribute to glioma susceptibility [12]. XRCC4 expression levels are found significantly downregulated in glioma tissue, indicating crucial roles of XRCC4 in brain carcinogenesis [13]. However, it is still unclear how XRCC4 maintains its oncogenic and TMZ chemoresistant properties in GBMs. Here, we explored the contribution of miR-151a/XRCC4 signaling to TMZ resistance and investigated its therapeutic implications for TMZ-resistant GBM patients.

Our results suggest that exosomes derived from resistant GBM cells could confer drug resistance to sensitive GBM cells. We transfected resistant GBM cells with miR-151a to achieve efficient loading of their exosomes with therapeutic miR-151a. Our data provided strong evidences that loading exogenous miR-151a into exosomes abrogated their ability to confer TMZ resistance to recipient cells and promised therapeutic potential in combination with TMZ chemotherapy. Meanwhile, miR-151a expression of CSF-derived exosomes could also be used as surrogate diagnostic markers for biopsy profiling to predict chemotherapy efficacy in GBM patients.

Section snippets

Patients and samples

Fifteen primary GBM and corresponding recurrent GBM samples from patients who received TMZ treatment (each pair was from the same patient) were obtained from the Chinese Glioma Genome Atlas (CGGA) (patients details in Supplementary Table S1). A total of 156 high-grade glioma (HGG) patients (75 GBMs and 81 Grade III gliomas) who underwent TMZ treatment after receiving surgical resection between January 2010 and December 2016 were from CGGA. Patient information is detailed in the online

miR-151a is lowly expressed in recurrent GBM specimens and TMZ-resistant cells

Our previous analysis of 82 primary glioblastomas have identified that the TMZ chemoresistant subtype (TCR subtype) has poor prognosis [18]. Three top downregulated miRNAs in TCR subtype are let-7i, miR-93 and miR-151a [18].

To obtain TMZ-resistant GBM cells, we grafted U251 and primary GBM N3 cells (N3) into nude mice flanks and performed cycles of TMZ treatment along with serial passage in vivo (Fig. 1A). GBM xenografts from the third passage displayed poor response to TMZ treatment (

Discussion

A strengthened DNA repair mechanism can increase the survival of damaged or mutated tumor cells, resulting in resistance and tumor recurrence [22]. NHEJ plays an important role in tumor cell survival through the acute phase of DNA damage [23,24]. XRCC4, a major participator of NHEJ, has previously been reported to be associated with TMZ resistance [[24], [25], [26]]. However, the mechanism by which its expression is controlled and sustained remains unclear. In our study, we found XRCC4 is a

Funding

This work was supported by a grant from the National Natural Science Foundation of China (No. 81402056 and 81472362), National High Technology Research, Development Program of China (863) (No.2012AA02A508) and National Key Research and Development Plan (No. 2016YFC0902500).

Declarations of interest

None.

Conflicts of interest

None.

References (28)

  • M. Goldstein et al.

    The DNA damage response: implications for tumor responses to radiation and chemotherapy

    Annu. Rev. Med.

    (2015)
  • T. Helleday

    Homologous recombination in cancer development, treatment and development of drug resistance

    Carcinogenesis

    (2010)
  • M. Goldstein et al.

    The DNA damage response: implications for tumor responses to radiation and chemotherapy

    Annu. Rev. Med.

    (2015)
  • P. Zhao et al.

    Genetic polymorphisms of DNA double-strand break repair pathway genes and glioma susceptibility

    BMC Canc.

    (2013)
  • Cited by (140)

    • The role of exosomes in glioblastoma treatment

      2023, New Insights into Glioblastoma: Diagnosis, Therapeutics and Theranostics
    View all citing articles on Scopus
    View full text