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. 2017 Sep 26;20(4):484–493. doi: 10.1093/neuonc/nox178

CUX1 stimulates APE1 enzymatic activity and increases the resistance of glioblastoma cells to the mono-alkylating agent temozolomide

Simran Kaur 1,2, Zubaidah M Ramdzan 1, Marie-Christine Guiot 3,5, Li Li 1, Lam Leduy 1, Dindial Ramotar 6, Siham Sabri 4, Bassam Abdulkarim 4, Alain Nepveu 1,2,4,✉
PMCID: PMC5909652  PMID: 29036362

Abstract

Background

Cut Like homeobox 1 (CUX1), which encodes an auxiliary factor in base excision repair, resides on 7q22.1, the most frequently and highly amplified chromosomal region in glioblastomas. The resistance of glioblastoma cells to the mono-alkylating agent temozolomide is determined to some extent by the activity of apurinic/apyrimidinic endonuclease 1 (APE1).

Methods

To monitor the effect of CUX1 and its CUT domains on APE1 activity, DNA repair assays were performed with purified proteins and cell extracts. CUX1 protein expression was analyzed by immunohistochemistry using a tumor microarray of 150 glioblastoma samples. The effect of CUX1 knockdown and overexpression on the resistance of glioblastoma cell lines to temozolomide was investigated.

Results

We show that CUT domains stimulate APE1 activity. In agreement with these findings, CUX1 knockdown causes an increase in the number of abasic sites in genomic DNA and a decrease in APE1 activity as measured in cell extracts. Conversely, ectopic CUX1 expression increases APE1 activity and lowers the number of abasic sites. Having established that CUX1 is expressed at high levels in most glioblastomas, we next show that the resistance of glioblastoma cells to temozolomide and to a combined treatment of temozolomide and ionizing radiation is reduced following CUX1 knockdown, but increased by overexpression of CUX1 or a short protein containing only 2 CUT domains, which is active in DNA repair but devoid of transcriptional activity.

Conclusion

These findings indicate that CUX1 expression level impacts on the response of glioblastoma cells to treatment and identifies the CUT domains as potential therapeutic targets.

Keywords: APE1 endonuclease, CUT domains, temozolomide resistance

Importance of the study

The region encompassing 7q21.13 to 7q22.1 is the most frequently and highly amplified chromosomal region in glioblastomas. In this study, we show that the CUX1 gene, which resides on 7q22.1, codes for a protein that functions as an auxiliary factor in base excision repair and is highly expressed in the vast majority of glioblastomas. We establish that the CUT domains of CUX1 stimulate the endonuclease activity of APE1 and accelerate the repair of DNA damage caused by the mono-alkylating agent temozolomide. We demonstrate that resistance to temozolomide is affected by CUX1 expression levels, in both O6-methylguanine-DNA methyltransferase (MGMT) low-expressing and MGMT high-expressing glioblastoma cells. Moreover, a recombinant protein containing only CUT domains 1 and 2 is sufficient to accelerate DNA repair and increase resistance to temozolomide. These results identify the CUT domains as potential therapeutic targets.

Base excision repair (BER) is the pathway that repairs altered bases, apurinic/apyrimidinic (AP) sites, and single-strand breaks. In response to a deaminated, an alkylated, or an oxidized base, the pathway is initiated by a DNA glycosylase that recognizes a specific base lesion and cleaves the N-glycosylic bond linking the altered base to the DNA backbone to produce an AP site.1,2 In mammals, AP sites are rapidly recognized by AP endonuclease 1 (APE1), which incises the DNA backbone 5ʹ to the AP site, thereby generating a 3ʹ-OH and 5ʹ-deoxyribose phosphate.3 APE1 expression is elevated in many cancers and has been associated with poor response to chemotherapy.4 In particular, APE1 expression was found to be elevated in gliomas, medulloblastomas, and primitive neuroectodermal tumors, and higher APE1 activity was shown to contribute to resistance to mono-alkylating agents.5–8 Conversely, reduction in APE1 activity through various manipulations can increase cancer cell sensitivity to the mono-alkylating agent temozolomide (TMZ).4,9–12

Temozolomide creates DNA damage by adding methyl adducts to N7-guanine (70% total adducts), N3-adenine (9%), and O6-guanine (5%).13 O6-methylguanine (O6-meG) is repaired by O6-methylguanine-DNA methyltransferase (MGMT).14 The genotoxicity and cytotoxicity of O6-meG is mainly due to recognition of O6-meG/thymine (or cytosine) mispairs by the mismatch repair system and induction of futile repair cycles, eventually resulting in cytotoxic double-strand breaks.15 Repair of N-alkyl base lesions in DNA such as N7-methylguanine (N7-MeG) or N3-methyladenine (N3-MeA) is initiated by N-methylpurine-DNA glycosylase (MPG; also termed alkyladenine-DNA glycosylase, or AAG).13 Removal of N-methylpurine by MPG produces an AP site. APE1 introduces a single-strand break to enable repair synthesis by either of 2 subpathways, the short patch BER pathway that utilizes DNA polymerase β (pol β) or the long patch repair pathway that recruits pol β/δ/ε coupled with proliferating cell nuclear antigen and replication factor C.1

CUX1 gene copy number is increased in over 70% of human tumors, and in many cancers higher CUX1 expression is associated with tumor progression and shorter patient survival16–18 (reviewed by Ramdzan and Nepveu19). Genome-wide copy number analysis performed by The Cancer Genome Atlas (TCGA) showed that the chromosomal region including 7q22, where CUX1 resides, is the most frequently and highly amplified in glioblastomas.20CUX1 codes for 2 main protein isoforms commonly referred to as p200 and p110 CUX1. The shorter isoform is produced by proteolytic processing of p200 CUX1 and functions as a transcriptional activator or repressor depending on promoter context.21,22 The p200 CUX1 protein contains a CUT homeodomain and 3 CUT domains, often called Cut repeats.19 Recent studies established that p200 CUX1 functions as an auxiliary factor in BER.23,24 Specifically, CUT domains stimulate both the glycosylase and AP-lyase activities of 8-oxoguanine DNA glycosylase (OGG1).23–27 While a physiological role of p200 CUX1 is obviously to protect cells against mutations caused by oxidative DNA damage, elevated CUX1 expression accelerates the repair of oxidative DNA damage and enables cancer cells that harbor a Ras oncogene to avoid senescence and proliferate in the presence of higher levels of reactive oxygen species (ROS).23 As a by-product of this adaptation, cancer cells overexpressing CUX1 exhibit increased resistance to ionizing radiation.27

The present study was triggered by our questioning whether the stimulation of OGG1 would be sufficient to explain the effect of CUX1 on the repair of oxidative DNA damage. Here, we report that various combinations of CUT domains stimulate the endonuclease activity of APE1. Accordingly, CUX1 expression has a direct impact on AP endonuclease activity in cell extracts and the number of abasic sites in genomic DNA. Moreover, the resistance of glioblastoma cells to TMZ is increased by CUX1 and is reduced following CUX1 knockdown.

Materials and Methods

Bacterial Protein Expression

Purification of histidine (His) or glutathione S-transferase (GST)–tagged fusion proteins containing CUX1 peptides and APE1 was performed as previously described.24,27,28

Cell Culture and Virus Production

T98G and U87 cells were obtained from American Type Culture Collection; U251 from Sigma-Aldrich. All cells were maintained at 37°C, 5% CO2, and atmospheric O2 in Dulbecco’s modified minimum essential medium (Wisent) supplemented with 10% fetal bovine serum (tetracycline-free; Invitrogen) and penicillin/streptomycin (Invitrogen). Production of retroviruses and lentiviruses was previously described.23,27

Immunoblotting

Protein extracts and immunoblotting procedures have been described.25 The following antibodies and dilutions were used: anti-APE1 (1:1000; Santa-Cruz, catalog no. sc-5572), anti-CUX1 861 (1:1000),22 and anti-tubulin (1:15000; Sigma, catalog no. T6557).

In Vitro Binding Assay

Binding assays with GST and His-tagged fusion proteins were performed as before.25,28

Immunoprecipitation

Cells were lysed by sonication in lysis buffer (20 mM Tris, pH 8.0, 150 mM NaCl, and 1% Nonidet P-40) supplemented with a protease inhibitor mixture and centrifuged at 13000 g for 10 min. Endogenous CUX1 protein was immunoprecipitated with anti-CUX1 and protein A Dynabeads (Invitrogen). The samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by immunoblotting with anti-APE1 antibody.

Single-Cell Gel Electrophoresis

Cells at ~80% confluence were treated with 100 µM TMZ for 60 min. After treatment, cells were allowed to recover at 37°C in fresh medium for the indicated periods of time before collection. Comet assays were carried out as previously described.25

Cell Viability Assays

[Methyl-14C]thymidine incorporation was conducted as described.25,27 Tumor cell sensitivity was assessed using the alamarBlue viability assay (ThermoFisher). Five days after exposure to TMZ (Schering Plough), alamarBlue was added. After 1 to 4 h, fluorescence was measured at 560 and 590 nm. Each treatment group was repeated in triplicate. Results were normalized to the zero-dose treatment to determine the cell viability at each dose.

In Vitro APE1 Assay

Endonuclease reactions with bacterially purified proteins were conducted using 0.08 nM APE1 (New England Biolabs) and 50 nM bovine serum albumin (BSA) or the indicated proteins unless otherwise indicated and 1 pmol of 32P-radiolabeled double-stranded oligonucleotides containing a tetrahydrofuran site or an abasic site generated by uracil-DNA glycosylase (UDG)/MPG and performed as described.29

In Vitro Fluorogenic APE1 Endonuclease Assay

The fluorogenic assay was performed with a 50 nM double-stranded molecular beacon probe as originally described.30 Endonuclease reactions were conducted using a 50 nM probe, 0.08 nM APE1 (New England Biolabs), and various concentrations of BSA or the indicated bacterially purified His-tagged proteins in buffer containing 10 mM Tris (pH 7.5), 25 mM NaCl, 1 mM MgCl2, 5% glycerol, 1 mM dithiothreitol, and 2 ng poly(deoxyinosinic-deoxycytidylic) acid [poly(dI-dC)]). Reactions with cell extracts were performed with a 50 nM probe, 1 µg of nuclear extract, 50 mM Tris–HCl (pH 7.1), 1 mM EDTA, 115 mM KCl, and 50 ng of poly(dI-dC). Reactions were incubated at 37°C, and fluorescence data were collected on a Realplex machine (Eppendorf Mastercycler) equipped with standard optics (excitation filter, 494 nm; emission filter, 520 nm).

Immunohistochemistry

Immunohistochemistry was performed on tissue microarrays (TMAs) of glioblastomas, diagnosed and centrally reviewed in a single institution.31 Ethical approval was obtained from the McGill University Health Centre Research Ethics Board (12-018 [#2442] GEN). The TMA slides contained triplicate core tissue samples (1 mm in diameter) from each tumor. Four-micron-thick sections were cut and stained using a Benchmark XT immunostainer (Ventana). The Ventana staining protocol included a pretreatment with Cell Conditioner 1 for 40 minutes, followed by incubation with the primary antibodies at 37°C for 60 minutes. All the antibodies were diluted in the Ventana dilution buffer as followed: CUX 861 1:100 and CUX 1300 1:100.22 Incubation was followed by detection using the Optiview DAB IHC detection kit (Ventana). Nuclear and cytoplasmic CUX1 immunoreactivities were scored by a neuropathologist (M-C.G.).

Abasic Site Quantification

DNA extraction was performed with a DNA extraction kit (Qiagen). Aldehyde-reactive probe labeling and quantification of abasic sites were performed with an AP sites assay kit (Dojindo Molecular Technologies).

Clonogenic Assay

For clonogenic survival assays, exponentially growing cells were seeded at a density of 300 cells per 6-well plate in triplicate and treated with the indicated concentrations of TMZ. After 10–14 days of incubation, cells were washed with phosphate-buffered saline, fixed with cold methanol for 20 min, then stained with 0.1% crystal violet (Acros Organics) in 20% methanol for 30 min. The number of colonies with 50 cells or more was counted and then normalized to the zero-dose plating efficiency to determine the clonogenic efficiency at each dose.

Results

CUT Domains Stimulate APE1 Endonuclease Activity In Vitro

We first tested the effect of CUT domains on APE1 endonuclease activity using purified proteins and a radiolabeled probe containing the abasic site mimic, tetrahydrofuran. As a control, the reaction was performed in the presence of BSA (Fig. 1B, lanes 2 and 8). The recombinant proteins containing CUT domains 1 and 2 (C1C2) or CUT domains 2 and 3 plus the CUT homeodomain (C2C3HD) both stimulated the endonuclease activity of APE1 (Fig. 1B, lanes 1 and 7). Importantly, no incision was observed in the reaction containing the C2C3HD protein alone (Fig. 1B, lanes 4–6). To confirm and extend these results, incision assays were then performed with probes containing an AP site generated by UDG (Fig. 1C). In these assays, in addition to BSA we also tested another control, homeobox B3, which was purified from bacterial cells using the same protocol as for the CUT domains (Fig. 1C, lanes 1 and 2). The C1C2 protein containing 2 CUT domains also stimulated APE1 activity with the UDG substrate, while CUT domain 1 (C1) and CUT domain 2 (C2) separately had a lesser effect (Fig. 1C, lanes 6, 8, and 10). The Coomassie Blue stain analysis of the recombinant CUX1 proteins used for these assays is shown in Supplementary Figure S1A. Identical results to those shown in Fig. 1C were obtained with a probe containing an AP site generated by MPG (Supplementary Figure S1B). As an alternative assay, we employed deoxyoligonucleotides containing a carboxyfluorescein (FAM)-fluorophore in close proximity to a Dabcyl quencher and a tetrahydrofuran site at position 6 (Fig. 1D).30 Incision by APE1 at this position causes the release of a short FAM-linked oligonucleotide that emits fluorescence. We observed that the C1C2 and C2C3HD recombinant proteins stimulate APE1 endonuclease activity, as compared with the control reactions with either BSA or no other protein (Fig. 1D). Additional experiments established that APE1 endonuclease activity increased with the concentration of C1C2, C2C3HD, or C3HD recombinant proteins (Supplementary Figure S1C–F). Interaction between CUX1 and APE1 in cells as well as in vitro was confirmed by immunoprecipitation and pull-down assays (Supplementary Figure S2). In agreement with our in vitro data, CUX1 knockdown in HCT116 cells was found to cause a decrease in APE1 endonuclease activity in cell extracts (Supplementary Figure S3B), which is associated with an increase in the number of abasic sites (Supplementary Figure S3C).

Fig. 1.

Fig. 1

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CUT domains stimulate APE1 endonuclease activity in vitro. (A) Schematic of CUX1 recombinant proteins used in APE1 endonuclease assays. (B, C) APE1 endonuclease assays were carried out using purified APE1, in the presence of purified CUX1 recombinant proteins or controls (BSA or homeobox B3) at 50 nM or as indicated, and a radiolabeled probe containing an abasic site produced in 2 different ways: as a tetrahydrofuran (B) or through removal of a uracil residue by UDG (C). (D) Schematic of the APE1 assay that utilizes a molecular beacon probe containing a tetrahydrofuran site. The APE1 assay was carried out with purified APE1 in the presence of nothing, 50 nM BSA, or purified CUX1 recombinant proteins (C2C3HD, C1C2, and C3HD), as indicated.

CUX1 Is Highly Expressed in Glioblastomas

Previous studies established that a decrease in APE1 activity can increase cancer cell sensitivity to the mono-alkylating agent TMZ, which is part of the standard-of-care treatment for glioblastomas.4,9–12 At the time of the present study, TCGA had provisional data from RNA sequencing on approximately 113 samples showing a shorter survival among patients with high CUX1 mRNA expression (Supplementary Figure S4A). These results concurred with those from the Repository for Molecular Brain Neoplasia Data (REMBRANDT) using expression profiling analysis on microarray with one valid CUX1 probe (Supplementary Figure S4B). In light of the scarcity of data, we analyzed CUX1 expression in a retrospective series of 150 adult glioblastomas, using immunohistochemistry with 2 well-characterized CUX1 antibodies (Fig. 1A, ab 861 and 1300).22,32 Examples of results obtained with the CUX1-861 antibody are shown in Fig. 2. While we observe no signal in the white matter, we observe a weak signal in neurons of the cortex, in agreement with previous studies.33 The analysis established that CUX1 is expressed in a majority of glioblastoma cells in over 90% of glioblastoma samples (Fig. 2 and Supplementary Table S1). Importantly, there is a very good correspondence between the results obtained with the CUX1-861 and 1300 antibodies (Supplementary Figure S5 and Supplementary Table S1). Using a combined score based on the proportion of tumor cells showing CUX1 expression and the intensity of the immunohistochemical signal, more than 80% of glioblastomas exhibit a score of ≥4 on a scale of 0 to 6 (Supplementary Table S2). These results establish that CUX1 proteins are highly expressed in glioblastomas.

Fig. 2.

Fig. 2

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CUX1 protein expression is high in glioblastomas. Immunohistochemical (IHC) staining of CUX1 was carried out on a tissue microarray of glioblastoma samples using the CUX1 antibody 861. A combined IHC score was assigned to each stained sample. The combined IHC score, from 0 to 6, was obtained by adding the percentage score (%, 0 to 3) and the intensity score (0 to 3). The percentage score indicates the proportion of cells showing a positive signal (0, 0%; 1, <10%; 2, 10% to 50%; 3, 51% to 100%). The intensity score indicates the intensity of the IHC signal, from no signal to +++ (0, 0; 1, +; 2, ++; 3, +++). A representative image for each combined score is shown. (A) Normal white matter, (B) normal cortex, (C) score 0 (negative), (D) score 2 (++, <10%), (E) score 3 (+, >50), (F) score 4 (++, <50%), (G) score 5 (++, >50%), (H) score 6 (+++, 100%). The “+” indicates intensity of staining and the percentage indicates the proportion of cells stained. Magnification of images is 400x. Bar scale is 60 microns.

CUX1 Knockdown in T98GMGMT-high Glioblastoma Cells Causes a Decrease in APE1 Activity and a Decrease in the Resistance to Temozolomide

We tested the effect of CUX1 knockdown on T98G glioblastoma cells that express high levels of the MGMT enzyme and therefore are sensitive only to high concentrations of TMZ.34CUX1 knockdown in T98GMGMT-high cells did not affect APE1 expression (Fig. 3A) but caused a decrease in APE1 activity in cell extracts (Fig. 3B) and a concomitant increase in the number of abasic sites in genomic DNA upon treatment with TMZ (Fig. 3C). In agreement with these results, CUX1 knockdown caused a modest decrease in the proliferation potential of T98GMGMT-high cells treated with 10, 20, and 50 µM TMZ (Fig. 3D). Additional experiments in U87 glioblastoma cells, which express low levels of the MGMT enzyme, confirmed that CUX1 knockdown sensitizes these cells to low concentrations of TMZ (Supplementary Figure S6D, E). Moreover, CUX1 knockdown also sensitized cells to methyl methansulfonate (MMS), a chemical whose toxicity is specifically driven by N7-methylguanine lesions35 (Supplementary Figure S7A, C).

Fig. 3.

Fig. 3

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CUX1 knockdown in T98GMGMT-high glioblastoma cells causes a decrease in APE1 activity and a decrease in the resistance to TMZ. Lentivirus expressing a doxycycline-inducible short hairpin (sh)RNA against CUX1 was introduced in T98G glioblastoma cells. Cells were cultured in the presence (shCUX1) or absence (control) of doxycycline for 4 days. (A) Immunoblotting analysis with the indicated antibodies. (B) APE1 activity in cell extracts was measured using a fluorophore-based probe carrying an AP site as in Figure 1D. (C) Control and CUX1 knockdown cells were either untreated or treated with 1000 µM TMZ for 24 hours. Genomic DNA was isolated from cells and the numbers of AP sites in genomic DNA were quantified. (D) Cells were treated with the indicated concentrations of TMZ. Cell numbers were counted on day 5.

CUX1 Overexpression in T98GMGMT-high Increases APE1 Activity and Resistance to Temozolomide

To investigate whether higher CUX1 expression would increase resistance to TMZ, we established populations of T98G glioblastoma cells stably carrying either a retroviral vector expressing p200 CUX1 or an empty vector (Fig. 4A). Ectopic expression of p200 CUX1 increased APE1 activity in cell extracts (Fig. 4B) and reduced the number of abasic sites in genomic DNA after treatment with TMZ (Fig. 4C). In accordance with these findings, p200 CUX1 increased the resistance of T98G glioblastoma cells to TMZ, as assessed by cell counting and clonogenic efficiency (Fig. 4D, E). Similar observations were made when p200 CUX1 was expressed in U251MGMT-low glioblastoma cells (Supplementary Figure S8A–F). Moreover, p200 CUX1 also increased the resistance to MMS (Supplementary Figure S7B, D) and counteracted the effect of CUX1 knockdown (Supplementary Figure S8G).

Fig. 4.

Fig. 4

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CUX1 overexpression in T98G (MGMThigh) glioblastoma cells increases APE1 activity and resistance to TMZ. T98G glioblastoma cells were stably infected with a lentivirus expressing p200 CUX1 or nothing (vector). (A) Immunoblotting analysis with the indicated antibodies. (B) APE1 activity in cell extracts was measured using a fluorophore-based probe carrying an AP site as in Fig. 1D. (C) Control and CUX1-overexpressing cells were either untreated or treated with 1000 µM TMZ for 24 hours. Genomic DNA was isolated from cells and the numbers of AP sites in genomic DNA were quantified. (D, E) Cells were treated with the indicated concentrations of TMZ. (D) Cell numbers were counted on day 5. (E) On day 1, cells were submitted to a clonogenic assay.

CUT Domains Are Sufficient to Increase Resistance to Temozolomide

To ascertain that the DNA repair function of CUX1 is involved in the resistance to TMZ, we expressed a recombinant protein containing the CUT domains 1 and 2 fused to a nuclear localization signal, C1C2-NLS (Fig. 5A, B). The NLS peptide is necessary for nuclear localization in the absence of the CUT homeodomain. Importantly, the C1C2-NLS protein was previously shown in 2 different cell types to be devoid of any transcription activation potential.23,24 Ectopic expression of C1C2-NLS enhanced APE1 activity detected in cell extracts (Fig. 5C) and increased the clonogenic efficiency of U251MGMT-low glioblastoma cells treated with TMZ (Fig. 5D). Moreover, C1C2-NLS counteracted the effect of CUX1 knockdown on resistance to MMS (Supplementary Figure S7C) and TMZ (Supplementary Figure S8H).

Fig. 5.

Fig. 5

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CUT domains are sufficient to increase resistance to TMZ. (A) Diagrammatic representation of the expression vector expressing the CUT domains 1 and 2 fused to a nuclear localization signal (C1C2-NLS). (B) U251 glioblastoma cells were infected with lentiviruses expressing either lacZ or C1C2-NLS. Nuclear extracts were analyzed by immunoblotting using the indicated antibodies. (C) APE1 activity in U251 cell extracts was measured using a fluorophore-based probe carrying an AP site as in Fig. 1D. (D) U251 cells were treated with the indicated concentrations of TMZ and were submitted to a clonogenic assay. The lower panel shows the actual crystal violet stained cells.

CUX1 Expression Impacts Sensitivity to Combined Treatment

Standard of care for glioblastomas involves combined treatment with TMZ and radiation. We observed that resistance to combined treatment, consisting of ionizing radiation (2 Gy) and increasing concentrations of TMZ, is reduced by CUX1 knockdown (Fig. 6A) but increased by ectopic expression of either the full-length p200 CUX1 protein (Fig. 6B) or the smaller C1C2-NLS protein (Fig. 6C). Altogether, these results establish that the role of CUX1 as an auxiliary factor in BER is an important determinant of the resistance of glioblastoma cells to treatment.

Fig. 6.

Fig. 6

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CUX1 expression impacts sensitivity to combined treatment. (A) Lentivirus expressing a doxycycline-inducible short hairpin (sh)RNA against CUX1 was introduced in U251 glioblastoma cells. Cells were cultured in the presence (shCUX1) or absence (control) of doxycycline. On day 4, cells were submitted or not to ionizing radiation (2 Gy) and treated with increasing concentrations of TMZ. Six days after treatment, cell viability was measured with the alamar blue assay. (B) U251 glioblastoma cells were stably infected with a lentivirus expressing p200 CUX1 or nothing (vector). Cells were submitted or not to ionizing radiation (2 Gy) and treated with increasing concentrations of TMZ before being submitted to a clonogenic assay. (C) U251 glioblastoma cells were infected with lentiviruses expressing either lacZ or C1C2-NLS. Cells were submitted or not to ionizing radiation (2 Gy) and treated with increasing concentrations of TMZ. On day 6, cell viability was measured with the alamar blue assay.

Discussion

The standard-of-care treatment for brain tumors involves the combination of radiation and TMZ.36,37 It is generally accepted that cytotoxicity caused by TMZ results mostly from the production of O6-meG, which is repaired by MGMT, failing which cell death occurs through futile repair cycles by the mismatch repair system.14,15 Indeed, improved survival following concurrent TMZ and radiotherapy is more frequent in patients with glioblastomas that exhibit promoter methylation of the MGMT gene and consequently express less MGMT.38,39 However, this relationship is not universally observed: Less than half of these patients benefited from standard of care, whereas ~20% of patients with glioblastomas lacking promoter methylation displayed superior outcome.37,38 These findings indicate that response to standard of care is determined not only by MGMT and mismatch repair status, but also by additional factors, including most likely the capacity of glioblastoma cells to repair the other DNA lesions produced by TMZ. N-methylpurines are ~15-fold more abundant but are considered less cytotoxic because they are repaired efficiently by BER. However, MPG was shown to promote resistance to mono-alkylating agents, thus establishing that part of the toxicity results from the presence of N-methylpurine residues in DNA.40,41 Yet, for MPG to protect against cytotoxicity, the AP site that it generates upon removal of an N-methylpurine must be processed by the next enzyme in the BER pathway, APE1. In agreement with this notion, elevated APE1 expression in cancer cells has been associated with poor response to mono-alkylating agents.5–8 Moreover, various approaches to reduce APE1 activity were shown to increase cancer cell sensitivity to TMZ.4,9–12 Our findings that CUT domains from the CUX1 protein stimulate APE1 activity led us to test the effect of CUX1 expression on the response of glioblastoma cells to TMZ. The resistance to TMZ was reduced by CUX1 downregulation, whereas it was increased by ectopic CUX1 expression, both in MGMT-high and MGMT-low glioblastoma cell lines (Fig. 3, 4; Supplementary Figures S6 and S8). Importantly, a recombinant protein that contains CUT domains 1 and 2 fused to a nuclear localization signal, C1C2-NLS, was able to stimulate APE1 activity and increase the resistance to TMZ and to the combination of TMZ and ionizing radiation (Figs. 5C, D and 6C). As the C1C2-NLS protein was previously shown to be devoid of transcription activation potential, these results demonstrate that the DNA repair activity of CUT domains contributes to the capacity of cancer cells to resist treatment.23,24 Altogether, our results identify CUT domains as potential therapeutic targets. Interestingly, as CUX1 is a non-essential gene in normal cells, as shown from lethality screens in human cells and the viability of Cux1−/− knockout mice, a CUT domain inhibitor would be expected to have a desirable therapeutic window.42,43

The finding that CUX1 is overexpressed in most glioblastomas concurs well with the fact that the CUX1 gene is located on the highly amplified 7q22 region.20 Frequent amplification of a specific chromosomal region is indicative of the presence of drivers of oncogenesis whose higher expression confers selective clonal growth advantage. We have previously shown that cancer cells with high levels of ROS are acutely dependent on the DNA repair function of p200 CUX1.23,27 As many glioblastoma tumors show activation of the epidermal growth factor receptor pathway, it is likely that high p200 CUX1 expression is selected in glioblastomas to adapt to high ROS levels and the resulting oxidative DNA damage, as previously shown in Ras-transformed cells.23 As a by-product of this adaptation, we show in the present study that high CUX1 expression in glioblastomas confers resistance to treatment (Figs. 4, 6 and Supplementary Figure S8). Importantly, the stimulation of APE1 by the CUT domains of CUX1 and its implication in the resistance to TMZ and ionizing radiation identify the CUT domains as potential targets for future therapeutic interventions to enhance the efficacy of treatment.

Supplementary Material

Supplementary material is available at Neuro-Oncology online.

Funding

This research was supported by grant #702996 from the Cancer Research Society to A.N. In addition, S.K. was supported by the 2015 Karassik PhD Fellowship Award.

Conflict of interest statement. None declared.

Supplementary Material

Supplemental_Material
Click here for additional data file. (4.7MB, docx)

Acknowledgments

We are grateful to Dr Gianluca Tell, from the University of Udine, for the gift of the pGEX-3X-APE1 plasmid. We thank Priya Aneja, Caroline Donovan, Fanny Lo, and Ranjana Pal for excellent technical assistance.

References

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