Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jan 7;111(1):409-14.
doi: 10.1073/pnas.1314469111. Epub 2013 Dec 23.

mTOR target NDRG1 confers MGMT-dependent resistance to alkylating chemotherapy

Markus Weiler  1 , Jonas Blaes, Stefan Pusch, Felix Sahm, Marcus Czabanka, Sebastian Luger, Lukas Bunse, Gergely Solecki, Viktoria Eichwald, Manfred Jugold, Sibylle Hodecker, Matthias Osswald, Christoph Meisner, Thomas Hielscher, Petra Rübmann, Philipp-Niklas Pfenning, Michael Ronellenfitsch, Tore Kempf, Martina Schnölzer, Amir Abdollahi, Florian Lang, Martin Bendszus, Andreas von Deimling, Frank Winkler, Michael Weller, Peter Vajkoczy, Michael Platten, Wolfgang Wick
Affiliations

mTOR target NDRG1 confers MGMT-dependent resistance to alkylating chemotherapy

Markus Weiler et al. Proc Natl Acad Sci U S A. .

Erratum in

Abstract

A hypoxic microenvironment induces resistance to alkylating agents by activating targets in the mammalian target of rapamycin (mTOR) pathway. The molecular mechanisms involved in this mTOR-mediated hypoxia-induced chemoresistance, however, are unclear. Here we identify the mTOR target N-myc downstream regulated gene 1 (NDRG1) as a key determinant of resistance toward alkylating chemotherapy, driven by hypoxia but also by therapeutic measures such as irradiation, corticosteroids, and chronic exposure to alkylating agents via distinct molecular routes involving hypoxia-inducible factor (HIF)-1alpha, p53, and the mTOR complex 2 (mTORC2)/serum glucocorticoid-induced protein kinase 1 (SGK1) pathway. Resistance toward alkylating chemotherapy but not radiotherapy was dependent on NDRG1 expression and activity. In posttreatment tumor tissue of patients with malignant gliomas, NDRG1 was induced and predictive of poor response to alkylating chemotherapy. On a molecular level, NDRG1 bound and stabilized methyltransferases, chiefly O(6)-methylguanine-DNA methyltransferase (MGMT), a key enzyme for resistance to alkylating agents in glioblastoma patients. In patients with glioblastoma, MGMT promoter methylation in tumor tissue was not more predictive for response to alkylating chemotherapy in patients who received concomitant corticosteroids.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
NDRG1 is a hypoxia-associated chemoresistance marker in glioma. (A) Immunoblot analyses for NDRG1 of lysates prepared from glioma cells exposed to 1% O2 (H) or 21% O2 (N) for the indicated intervals. α-tubulin served as a loading control. (B) qRT-PCR analysis of NDRG isoforms exposed to 1% O2 (mean ± SD, n = 3, **P < 0.01, ***P < 0.005). (C) NDRG1 staining in WHO °II (n = 46), WHO °III (n = 57), and WHO °IV (n = 81) gliomas presented as number of NDRG1+ cells per field (mean ± SD). Representative images of scattered NDRG1+ cells (Left), increased numbers of NDRG1+ cells (Center), and perinecrotic NDRG1+ cells (Right) are depicted by the specific red staining. (D) Cell cycle distributions and mean G2/M-arrest of TMZ-treated glioma cells relative to DMSO- (vehicle-) treated cells dependent on the NDRG1 status. TMZ concentrations used were 10 µM for U87MG, 40 µM for T269, 300 µM for T325, and 300 µM for T98G, and the medium was changed every 24 h with addition of fresh TMZ. (Upper) Lentiviral knockdown in U87MG, T269, and T325 GIC. (Lower) NDRG1 overexpression in U87MG and T98G cells. (E) Proliferation of TMZ/vehicle-treated U87MG cells overexpressing NDRG1 or control in RTCA. (F) MRI-determined tumor volumes of intracranially implanted U87MG gliomas overexpressing NDRG1 or control vector. TMZ was given on days 10–15 as described in Materials and Methods (*P < 0.05 versus control, t test, n = 6).
Fig. 2.
Fig. 2.
NDRG1 is induced by glioblastoma therapy and serves as a negative prognostic factor. (A) Representative tissues of 19 patients before and at recurrence after radiochemotherapy with TMZ were scored for the number of NDRG1-positive cells (mean ± SD). (B) Representative perivascular tumor region from 20 relapsed (after radiochemotherapy) glioblastoma tissue samples. (C) Correlation of NDRG1 levels and PFS (Upper) or OS (Lower) of glioblastoma patients of the UKT-05 trial. (D) NDRG1 expression relative to patient survival in glioblastoma (REMBRANDT). (E) Correlation of NDRG1 levels and PFS of patients with anaplastic gliomas of the NOA-04 trial separated for treatment.
Fig. 3.
Fig. 3.
mTORC2 is a master regulator of NDRG1. (A) Immunoblot of siRNA-treated U87MG cells targeting RAPTOR or RICTOR (Top). Assessment of NDRG1 phosphorylation at T346 in siRICTOR transfected U87MG_LV-NDRG1 cells (Bottom Left) and ΔG2/M after treatment with TMZ (Bottom Right). NDRG1 mRNA expression 48 h after siRNA-mediated knockdown of RICTOR in U87MG and T98G cells (Middle). (B) SGK1 mRNA expression relative to GAPDH in T98G 72 h after treatment with dexamethasone (DEX; Left) and phosphorylation status of NDRG1 24–72 h after DEX treatment (Right). (C) Progression-free survival of the NOA-08 cohort patients differentiated according to treatment [radiotherapy (RT) versus TMZ] and steroid use. (D) (Upper) Timeline depicting course of animal experiment including dates of MRI measurements and treatment period. (Lower) Tumor volumes on postoperative day 24. The left segment shows a comparison of mean tumor volumes relative to average tumor volumes of methylcellulose group. The right segment shows representative MRI pictures of respective treatment groups. (E) Phosphorylation status of NDRG1 at T346 24 and 48 h after treatment with the SGK1 inhibitor EMD638683 (Left) and TMZ-mediated shift of G2/M-phase in U87MG cells treated with EMD638683 relative to DMSO (vehicle) (Right; *P < 0.05 for the effect of LV-NDRG1, +P < 0.05 for the effect of EMD).
Fig. 4.
Fig. 4.
NDRG1 interacts with the DNA repair proteins MGMT, APEX1, and PNKP and promotes MGMT-mediated protection from TMZ. (A) Split Ubiquitin Screen for interaction partners of NDRG1 (NNMT, Nicotinamide N-methyl transferase). (B) Validation of the interaction of NDRG1 with MGMT, PNKP, and APEX1 via coimmunoprecipitation using HEK293T lysates and Flag-/Myc-tagged constructs of NDRG1, MGMT, PNKP, and APEX1, respectively. (C) Cell cycle and proliferation analysis of siRNA-treated U87MG and T98G cells targeting APEX1 and PNKP. Experiments were performed three times with one representative example shown. (D) Cell cycle analysis after siRNA-mediated knockdown of MGMT in T98G cells in response to 300 µM (regular dose) or 20 µM TMZ. (E) (Upper) BiFC assay with NDRG1. HEK293T cells cotransfected with NDRG1 in pGW-myc-LC151 and bJun in pGW-HA-LN-151 as negative control (1, 3) or with MGMT in pGW-HA-LN-151 (2, 4). The overlay with the DAPI stain shows a clear nuclear localization of the NDRG1 interaction with MGMT (2, 4). (Lower) PLA with NDRG1 and MGMT. Parental T98G cells exposed to 1% O2 for 72 h are analyzed for interaction of NDRG1 and MGMT (n = 3). Relevant controls are depicted in SI Appendix, Fig. S9B. (F) (Top) Schematic overview on the location of SGK1 target residues within the C terminus of the NDRG1 protein. (Middle) BiFC assay with genetically modified versions of NDRG1 resulting in amino acid substitutions. HEK293 cells cotransfected with wt-NDRG1 (Left), T346V-T356V-NDRG1 (Center), or T328V-S330A-T346V-T356V-NDRG1 (Right). (Bottom) Quantification of interaction between MGMT and the three NDRG1 versions depicted as mean fluorescence intensity.
Fig. 5.
Fig. 5.
NDRG1 stabilizes MGMT. (A) Time-dependent abundance of MGMT, pNDRG1T346, and NDRG1 proteins in T98G cells (Co, control transfected cells; ND, cells lentivirally overexpressing NDRG1) after treatment with actinomycin D (Upper) or TMZ (Lower). (B) Immunofluorescent staining of O6-methylguanine of TMZ-treated T98G LV-Co and LV-NDRG1 cells (Upper) and quantification of relative O6-methylguanine content depicted as log-transformed fluorescence for three independent replications (Lower). (C) Progression-free survival of the NOA-08 cohort patients differentiated according to MGMT promoter methylation status (+, methylated; −, unmethylated) and steroid use in the temozolomide treatment group. (D) Schematic overview of the signaling cascade with iatrogenic and microenvironmental activating factors (left side) and options for therapeutic intervention (right side).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Sarkaria JN, et al. Mechanisms of chemoresistance to alkylating agents in malignant glioma. Clin Cancer Res. 2008;14(10):2900–2908. - PMC - PubMed
    1. Dunn GP, et al. Emerging insights into the molecular and cellular basis of glioblastoma. Genes Dev. 2012;26(8):756–784. - PMC - PubMed
    1. Amberger-Murphy V. Hypoxia helps glioma to fight therapy. Curr Cancer Drug Targets. 2009;9(3):381–390. - PubMed
    1. Harris AL. Hypoxia—A key regulatory factor in tumour growth. Nat Rev Cancer. 2002;2(1):38–47. - PubMed
    1. Winkler F, et al. Kinetics of vascular normalization by VEGFR2 blockade governs brain tumor response to radiation: Role of oxygenation, angiopoietin-1, and matrix metalloproteinases. Cancer Cell. 2004;6(6):553–563. - PubMed

Publication types

MeSH terms

Substances