PTEN Loss Compromises Homologous Recombination Repair in Astrocytes: Implications for Glioblastoma Therapy with Temozolomide or Poly(ADP-Ribose) Polymerase Inhibitors (original) (raw)

Therapeutics, Targets, and Chemical Biology| June 30 2010

Authors' Affiliations: Departments of 1Radiation Oncology and 2Neurology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas

Search for other works by this author on:

Cristel V. Camacho;

Authors' Affiliations: Departments of 1Radiation Oncology and 2Neurology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas

Search for other works by this author on:

Bipasha Mukherjee;

Authors' Affiliations: Departments of 1Radiation Oncology and 2Neurology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas

Search for other works by this author on:

Brandon Hahm;

Authors' Affiliations: Departments of 1Radiation Oncology and 2Neurology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas

Search for other works by this author on:

Nozomi Tomimatsu;

Authors' Affiliations: Departments of 1Radiation Oncology and 2Neurology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas

Search for other works by this author on:

Robert M. Bachoo;

Authors' Affiliations: Departments of 1Radiation Oncology and 2Neurology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas

Search for other works by this author on:

Sandeep Burma

Authors' Affiliations: Departments of 1Radiation Oncology and 2Neurology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas

Corresponding Author: Sandeep Burma, Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, NC7.214E, Dallas, TX 75390. Phone: 214-648-7440; Fax: 214-648-5995; E-mail: Sandeep.Burma@UTSouthwestern.edu.

Search for other works by this author on:

Received: November 25 2009

Revision Received: April 13 2010

Accepted: April 28 2010

Online ISSN: 1538-7445

Print ISSN: 0008-5472

2010

Cancer Res (2010) 70 (13): 5457–5464.

Article history

Received:

November 25 2009

Revision Received:

April 13 2010

Split-Screen
Views Icon Views
Open the PDF for in another window
Tools Icon Tools
Search Site
Article Versions Icon Versions
- Version of Record June 30 2010
- Proof June 8 2010

Abstract

Glioblastomas (GBM) are lethal brain tumors that are highly resistant to therapy. The only meaningful improvement in therapeutic response came from use of the SN1-type alkylating agent temozolomide in combination with ionizing radiation. However, no genetic markers that might predict a better response to DNA alkylating agents have been identified in GBMs, except for loss of _O_6-methylguanine-DNA methyltransferase via promoter methylation. In this study, using genetically defined primary murine astrocytes as well as human glioma lines, we show that loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) confers sensitivity to _N_-methyl-_N_′-nitro-_N_-nitrosoguanidine (MNNG), a functional analogue of temozolomide. We find that MNNG induces replication-associated DNA double-strand breaks (DSB), which are inefficiently repaired in PTEN-deficient astrocytes and trigger apoptosis. Mechanistically, this is because PTEN-null astrocytes are compromised in homologous recombination (HR), which is important for the repair of replication-associated DSBs. Our results suggest that reduced levels of Rad51 paralogs in PTEN-null astrocytes might underlie the HR deficiency of these cells. Importantly, the HR deficiency of PTEN-null cells renders them sensitive to the poly(ADP-ribose) polymerase (PARP) inhibitor ABT-888 due to synthetic lethality. In sum, our results tentatively suggest that patients with PTEN-null GBMs (about 36%) may especially benefit from treatment with DNA alkylating agents such as temozolomide. Significantly, our results also provide a rational basis for treating the subgroup of patients who are PTEN deficient with PARP inhibitors in addition to the current treatment regimen of radiation and temozolomide. Cancer Res; 70(13); 5457–64. ©2010 AACR.

Introduction

Despite considerable work in recent years elucidating the molecular underpinnings of glioblastoma (GBM), the most deadly of brain cancers, little progress has been made in improving clinical outcomes. The most significant breakthrough in patient response to date emerged from the use of the SN1-type alkylating agent temozolomide in combination with ionizing radiation (IR), which increased the overall median survival from approximately 12 to 15 months (1–3). However, no genetic markers that might predict a better response to DNA alkylating agents have been identified for GBMs except for _O_6-methylguanine-DNA methyltransferase (MGMT) promoter methylation (3, 4). The past year has seen unprecedented advances in the genomic analyses of adult GBM tumors by the Cancer Genome Atlas Network and other groups which reveal that these tumors have radically altered genomes with many mutations, gene copy number gains and losses, and methylation changes (5–7). Among the myriad of genetic alterations that populate the GBM genomic landscape, five genetic changes dominate: loss of Ink4a, Arf, p53, or phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and amplification of epidermal growth factor receptor (EGFR). How these genetic aberrations confer therapeutic resistance remains unclear. Understanding the contribution of these lesions, singly and in combination, to GBM therapy resistance along with the underlying mechanism(s) will be of paramount importance in developing more effective therapeutic modalities.

In a previous study, we showed that amplification of EGFRvIII confers radioresistance to GBM-relevant cells and tumors by promoting the repair of radiation-induced DNA double-strand breaks (DSB) by nonhomologous end joining (NHEJ; ref. 8). In this study, using genetically defined primary murine astrocytes as well as human glioma lines, we focused on the role of PTEN in modulating sensitivity to the SN1-type alkylating agent _N_-methyl-_N_′-nitro-_N_-nitrosoguanidine (MNNG; ref. 9). Loss of PTEN is a very prominent event during gliomagenesis, occurring in about 36% of GBMs (5, 6, 10). PTEN is a lipid phosphatase with a canonical role in dampening the phosphatidylinositol 3-kinase (PI3K)/Akt-1 signaling pathway; hence, loss of PTEN has oncogenic consequences during gliomagenesis (11). In addition, it is becoming increasingly clear that PTEN has novel nuclear functions, including transcriptional regulation of the Rad51 gene, whose product is essential for homologous recombination (HR) repair of DNA breaks (12, 13). We report here that loss of PTEN in astrocytes results in increased sensitivity to MNNG. We show that MNNG induces secondary DSBs that are poorly repaired in PTEN-null astrocytes due to compromised HR. The increased sensitivity of PTEN-null astrocytes to MNNG tentatively suggests that patients with PTEN-null GBMs may especially benefit from treatment with temozolomide. More importantly, the HR deficiency of PTEN-null astrocytes opens up the possibility of treating PTEN-deficient GBMs with poly(ADP-ribose) polymerase (PARP) inhibitors that are currently in clinical trials for treating HR-deficient breast and ovarian cancers (14–16).

Materials and Methods

Cell culture

Astrocytes were isolated from 5-day-old pups, as described (17), from littermates of an Ink4a/Arf−/− PTENf/+ × Ink4a/Arf−/− PTENf/+ cross (18, 19). Primary mouse astrocytes were maintained in DMEM containing 10% fetal bovine serum in a humidified 37°C incubator with 5% CO2. The floxed PTEN allele was deleted using an adenovirus expressing Cre. All cells were mycoplasma-free.

Irradiation and drug treatment

A 137Cs source (JL Shepherd and Associates) was used for γ-ray irradiation of cells. MNNG (Sigma) and camptothecin (CPT; Sigma) were dissolved in DMSO and stored at −20°C in aliquots of 100 mmol/L. ABT-888 (Alexis Biochemicals) was dissolved in cell culture grade water and stored at −20°C. MNNG treatments were given as a 1-hour pulse, whereas CPT and ABT-888 were added continuously at the indicated concentrations.

Colony formation assays

Cells were plated in triplicate onto 60-mm dishes (300 cells per dish) and irradiated with graded doses of radiation or treated with increasing concentrations of MNNG, CPT, or ABT-888. Surviving colonies were stained with crystal violet about 7 days later as described (20).

Immunofluorescence staining and Western blot analyses

Immunofluorescence staining of cells and Western blot analyses of whole-cell extracts were done as described (21). Antibodies used were anti-Rad51 (Santa Cruz), anti-γH2AX (Upstate), anti-53BP1 (Cell Signaling), anti-actin (Sigma), anti-Akt, anti–phospho-Akt(Ser473) (Cell Signaling), anti-MGMT (Santa Cruz), rhodamine red–conjugated goat anti-rabbit, and FITC-conjugated goat anti-mouse (Molecular Probes).

Metaphase chromosome preparations and sister chromatid exchange assay

To examine chromosome aberrations, astrocytes were treated with MNNG, and 24 hours later, 1 μg/mL colcemid (Sigma) was added for 3 hours. Metaphase chromosome spreads were then prepared using standard procedures. Aberrations were counted and categorized as breaks or asymmetrical exchanges (triradials and quadriradials). To visualize sister chromatid exchanges (SCE), cells were incubated in the presence of bromodeoxyuridine (BrdUrd) (BD Biosciences; 10 μmol/L) for two cell divisions, after which metaphases were prepared according to the above protocol. For drug treatments, MNNG was added as a 1-hour pulse immediately before BrdUrd, whereas CPT was added concurrently. Aberrations and SCEs were quantified by analyzing 100 metaphase spreads and differences were statistically analyzed as described below.

Statistical analyses

P values for experiments were calculated using GraphPad Prism. SCE and chromosome aberration data were analyzed by a two-tailed t test, whereas quantitative real-time PCR (qRT-PCR) data and repair kinetics were analyzed using two-way ANOVA.

Additional methods

Results

For this study, primary astrocytes were generated from Ink4a/Arf−/−PTEN+/+ or Ink4a/Arf−/−PTENf/f transgenic littermates. Once in culture, floxed PTEN alleles were deleted by adenoviral expression of Cre recombinase, generating a set of matched astrocytes with the following genotypes: Ink4a/Arf−/−PTEN+/+ and Ink4a/Arf−/−PTEN−/−. Because primary mouse astrocytes senesce within a couple of passages after extraction, the Ink4a/Arf−/− background ensures that the astrocytes are immortal and provides a tumor-suppressor background that is very relevant to GBMs (5, 6). PTEN deletion on adenovirus infection was confirmed by Western blotting (Fig. 1A), as well as by PCR analysis (Supplementary Fig. S1). As expected, PTEN loss strongly activated PI3K signaling as evidenced by increased levels of phosphorylated Akt-1 (phospho-Ser473; ref. 11). In accordance with previous reports (22, 23), we found that loss of PTEN resulted in increased resistance to IR as assayed by colony survival (Fig. 1B). In contrast, PTEN loss resulted in sensitization to MNNG (Fig. 1C). Flow cytometric analyses revealed a significant increase in the sub-G0 population in MNNG-treated PTEN-deficient cultures, indicating that the sensitivity of these astrocytes to MNNG was due to an increase in cell death (Fig. 1D).

Figure 1.

$Figure 1. PTEN loss sensitizes astrocytes to MNNG. A, loss of PTEN and activation of Akt in PTEN+/+ and PTEN−/− astrocytes were analyzed by Western blotting with α-PTEN and α-phospho-Akt(ser473) antibodies. B, the radiation survival of astrocytes was quantified by colony formation assays. The fraction of surviving colonies (y-axis) was plotted against corresponding radiation dose (x-axis). C, the sensitivity of astrocytes to MNNG was quantified by colony formation assays. Note increased sensitivity of PTEN-null cells to MNNG. Bars, SEM of experiments done three or more times. D, induction of cell death by MNNG was assessed by quantifying the sub-G0 population in MNNG-treated cultures by flow cytometry (percentages in red).$

PTEN loss sensitizes astrocytes to MNNG. A, loss of PTEN and activation of Akt in PTEN+/+ and PTEN−/− astrocytes were analyzed by Western blotting with α-PTEN and α-phospho-Akt(ser473) antibodies. B, the radiation survival of astrocytes was quantified by colony formation assays. The fraction of surviving colonies (_y_-axis) was plotted against corresponding radiation dose (_x_-axis). C, the sensitivity of astrocytes to MNNG was quantified by colony formation assays. Note increased sensitivity of PTEN-null cells to MNNG. Bars, SEM of experiments done three or more times. D, induction of cell death by MNNG was assessed by quantifying the sub-G0 population in MNNG-treated cultures by flow cytometry (percentages in red).

Figure 1.

Close modal

Whereas the cytoplasmic role of PTEN in squelching the PI3K/Akt-1 pathway is an established concept, recent reports clearly indicate novel nuclear functions for this protein, including roles in transcription regulation (11–13). The sensitivity of PTEN-null cells to MNNG was probably not due to hyperactivation of Akt-1 because astrocytes expressing constitutively active myristylated Akt-1 (8) did not display an increased sensitivity to MNNG compared with parental cells (Supplementary Fig. S2). Therefore, it is plausible that the sensitivity to MNNG observed in PTEN−/− astrocytes was due to loss of a nuclear function of PTEN.

Toxicity from SN1-type alkylating agents results mainly from a specific type of DNA lesion, methylation of the O6 position of guanine (O6meG; ref. 24), which can be reversed by the suicide repair enzyme MGMT (25). It has been previously reported that MGMT promoter silencing by methylation corresponds to a better therapeutic response to temozolomide (3, 4). Therefore, we investigated whether a decrease in MGMT levels due to PTEN loss might underlie the sensitivity of these cell lines to MNNG. However, Western blot analyses of basal MGMT protein levels did not show a significant difference between PTEN+/+ and PTEN−/− astrocytes (Fig. 2A). MGMT transcription is induced in response to various DNA-damaging agents, including MNNG (24, 26). However, qRT-PCR analyses revealed that both lines were capable of inducing MGMT transcription on MNNG treatment (Fig. 2B). These data strongly suggest that the sensitivity of PTEN-null cells to MNNG was not due to attenuation of MNNG transcript or protein levels.

Figure 2.

MGMT regulation is not affected by PTEN loss. A, MGMT protein levels in PTEN+/+ and PTEN−/− astrocytes were analyzed by Western blotting with α-MGMT antibody. B, MGMT transcript levels in mock-treated and MNNG-treated astrocytes were quantified by qRT-PCR. Values were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels and expressed as fold change relative to mock-treated PTEN+/+ cells. Bars, SEM.

Figure 2.

Close modal

The cytotoxicity of O6meG lesions is attributed to the recognition of O6-meG/C or O6-meG/T mispairs by the mismatch repair system (27), with two opposing models proposed: (a) DNA damage signal transduction by the mismatch repair complex engaged at the mismatch sites directly triggering apoptosis (direct signaling model) or (b) reiterative and futile repair attempts by mismatch repair resulting in single-strand and double-strand DNA breaks (futile cycle model; ref. 28). Because DNA DSBs are the most lethal of DNA lesions, we investigated whether such breaks were induced in MNNG-treated astrocytes and whether these breaks were more persistent in PTEN-deficient cells. We analyzed the formation and dissolution of γH2AX and 53BP1 foci on pulse treatment with MNNG (8, 20), with these foci being bona fide surrogate markers for DSBs (29, 30). Interestingly, MNNG induced equivalent levels of DSBs in both lines; however, PTEN-deficient astrocytes exhibited higher levels of DSBs at 24 hours posttreatment, indicating a deficiency in repair of these breaks (Fig. 3A).

Figure 3.

PTEN loss compromises HR repair. A, induction and repair of DSBs in astrocytes pulsed for 1 h with 5 μmol/L MNNG. Cells were co-immunostained for γH2AX (red) and 53BP1 (green) foci at various times after MNNG treatment. Representative pictures are shown. Foci were scored at the indicated times (average of 100 nuclei) and, after subtracting background (number of foci in untreated nuclei), average foci per nucleus was plotted against time. *, P < 0.05; ***, P < 0.001 (two-way ANOVA with a Bonferroni posttest). B, the sensitivity of astrocytes to CPT was quantified by colony formation assays. Note increased sensitivity of PTEN-null cells to CPT. C, to quantify SCEs, metaphase spreads were prepared from astrocytes treated with MNNG or CPT as indicated. Reciprocal exchange events (see arrows, inset) were counted and plotted as average number of SCEs per metaphase. Approximately 100 metaphases were counted per treatment. ***, P < 0.0001 (two-tailed t test). D, metaphase spreads from astrocytes treated with 10 μmol/L MNNG were analyzed for chromatid breaks and asymmetrical exchanges (1, triradials; 2, quadriradials; and 3, complex exchanges). Approximately 100 metaphases were counted per treatment and average aberrations per metaphase were plotted. Representative pictures of aberrations are shown. **, P = 0.0027; ***, P = 0.0004 (two-tailed t test). Bars, SEM (for all plots).

Figure 3.

Close modal

DSBs are repaired by NHEJ or HR in mammalian cells. Whereas NHEJ is operative in all phases of the cell cycle, HR is limited to S/G2 and is particularly important for resolving replication-associated breaks (31). MNNG-induced breaks are presumed to occur in the S/G2 phases because DNA replication is required for mispairing, and this is borne out by our observations indicating that MNNG-induced H2AX phosphorylation occurs only in S/G2 cells (Supplementary Fig. S3). Therefore, it is likely that these breaks may be resolved by HR rather than by NHEJ. Indeed, we observed no further sensitization on treating these cells with NU7026, a potent inhibitor of the major NHEJ repair enzyme DNA-PKcs (ref. 32; Supplementary Fig. S4), thereby implicating HR in repair. In support of this idea, a recent report showed that MNNG induces DSBs and that cells defective in HR (XRCC2 and Brca2 mutants), but not cells defective in NHEJ (Ku80 and DNA-PKcs mutants), were sensitive to MNNG, similar to our PTEN-null cells (33).

Cells deficient in various HR components show a decrease in the number of SCEs after treatment with DNA-damaging agents (34), especially agents that induce replication-associated DSBs such as CPT (35). Also, HR-deficient cells are sensitive to CPT (35), and we found that PTEN-deficient astrocytes were more sensitive to this drug compared with their PTEN-proficient counterparts (Fig. 3B). We quantified the number of SCEs in PTEN+/+ and PTEN−/− astrocytes after treatment with CPT or MNNG to determine the relative HR proficiencies of these lines. A statistically significant reduction in SCE events was observed in PTEN−/− astrocytes relative to PTEN+/+ astrocytes, indicating a defect in HR (Fig. 3C). Consequently, PTEN-null cells surviving MNNG treatment exhibited greater numbers of chromosome breaks and radial chromosomes (Fig. 3D), similar to that seen in HR-deficient cells, particularly those deficient in Brca1 or Brca2 (36). These aberrations are indicative of a diminished capacity to repair MNNG-induced DSBs by error-free HR and subsequent repair of these lesions by error-prone pathways such as NHEJ.

Interestingly, Shen and colleagues recently showed that PTEN is important for maintaining basal levels of transcription of the Rad51 gene in mouse embryonic fibroblasts (12), providing a potential mechanism to explain the reduced HR capability of PTEN-null astrocytes. However, no significant changes in Rad51 protein or mRNA levels in mouse astrocytes on PTEN loss were noted (Fig. 4A and B). Whereas Rad51 forms a presynaptic nucleofilament that is critical for HR, ancillary proteins such as BRCA1, BRCA2, Rad52, and the Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3) facilitate multiple steps during the repair process (37). Because there are numerous recent reports of PTEN acting as a transcriptional regulator (13), we screened several of these “recombination mediators” for expression changes on PTEN loss by qRT-PCR and observed decreases in the transcript levels of Rad51B, Rad51C, and Rad51D (Fig. 4B). As these proteins are known to exist in complexes facilitating Rad51 nucleofilament formation (37), it is plausible that reduced levels of these proteins could result in attenuated HR on PTEN loss.

Figure 4.

PTEN-null astrocytes express lower levels of Rad51 paralogs and are sensitive to PARP inhibitors. A, Rad51 levels in PTEN+/+ and PTEN−/− astrocytes were analyzed by Western blotting with α-Rad51 antibody. B, transcript levels of critical HR genes were analyzed by qRT-PCR. Values were normalized to GAPDH levels and expressed as fold change relative to PTEN+/+ astrocytes. **, P < 0.01; ***, P < 0.001 (two-way ANOVA with a Bonferroni posttest). C, the sensitivity of astrocytes to the PARP inhibitor ABT-888 was quantified by colony formation assays. Note increased sensitivity of PTEN-null cells to ABT-888. Bars, SEM (for all plots).

Figure 4.

Close modal

A very important prediction from the observed deficiency in HR is that PTEN-null astrocytes should be sensitive to PARP inhibitors that induce replication-associated DSBs. This phenomenon of “synthetic lethality” was originally identified in the context of BRCA1 and BRCA2 mutations in breast cancer (14, 15), and PARP inhibitors are now in clinical trials for treating HR-deficient breast and ovarian cancers (16). We found that PTEN-null cells were significantly more sensitive to the PARP inhibitor ABT-888 (38) compared with PTEN-proficient cells (Fig. 4C). The sensitivity to ABT-888 is consistent with a HR deficiency in PTEN-null cells, and suggests that it might be logical to treat PTEN-deficient GBMs with PARP inhibitors in the future.

The isogenic murine astrocytes used in this study are ideal for analyzing the effect of a single genetic change (PTEN loss) on MNNG sensitivity. However, in the context of human GBMs, the effect of a single genetic lesion could be modulated by innumerable background genetic changes (5–7). To explore the relevance of our findings in human GBMs, we compared two commonly used glioma lines (U87MG and U251MG) with a normal human astrocyte line (NHA) that had been immortalized by expression of human telomerase catalytic component (hTERT) and human papillomavirus 16 E6/E7 proteins (39). Both glioma lines are PTEN null (40) and were more sensitive to MNNG compared with the NHA line, which has an intact PTEN gene (Supplementary Fig. S5). These results tentatively suggest that PTEN-deficient glioblastoma cells may be more sensitive to DNA alkylating agents compared with PTEN-proficient normal human astrocytes, and this could confer a selective advantage to DNA alkylating agents for the treatment of PTEN-null GBMs. Importantly, siRNA-mediated depletion of PTEN rendered the NHA line sensitive to MNNG as quantified by the colony formation assay (Supplementary Fig. S6). This was possibly due to attenuated HR because we observed a reduced induction of SCEs on PTEN depletion. Interestingly, SCE induction in PTEN-null U87 cells was also reduced compared with the PTEN-proficient NHA line. More importantly, PTEN depletion could also sensitize transformed, gliomagenic human astrocytes (expressing E6, E7, hTERT, H-Ras, and myristylated Akt-1; ref. 41), indicating that PTEN loss might result in sensitivity to DNA alkylating agents in the context of human gliomas. In sum, these results confirm that, as observed in murine astrocytes, PTEN loss plays an important role in modulating the MNNG sensitivity of normal human astrocytes and gliomagenic derivatives.

Discussion

The data presented in this article provide a basis for exciting new therapeutic options for GBMs. Thus far, the only meaningful improvement in GBM therapy came from the addition of temozolomide to radiation, with MGMT promoter methylation associated with a better outcome (3, 42). However, MGMT promoter methylation was also associated with an improvement in survival with radiation alone (4), indicating that this may be a general prognostic marker rather than a specific predictive marker for temozolomide treatment. We show here that PTEN-deficient astrocytes are impaired in HR and, therefore, sensitive to replication-associated DSBs generated by DNA alkylating agents or by PARP inhibitors. This has novel therapeutic implications: GBM patients with PTEN loss (about 36%) may (a) specially benefit from temozolomide treatment and (b) also benefit from the addition of PARP inhibitors to therapeutic regimens. At the time when this paper was in preparation, a report from the Ashworth group showed that PTEN-null cells and tumors are indeed sensitive to PARP inhibitors (43) thereby bolstering the conclusions drawn from our study. Interestingly, a recent report indicates that the acquired resistance of cancer cells to combinatorial treatment with temozolomide and PARP inhibitors is linked to upregulation of HR (44). These results complement our data showing that downregulation of HR due to PTEN loss results in sensitivity to DNA alkylating agents or PARP inhibitors. Both temozolomide and PARP inhibitors are in clinical trials to treat GBMs and have been used in combination to treat other cancers (45). In light of our results and other recent reports, we posit that (a) PARP inhibitors may have a broader applicability outside of Brca1/2-null breast and ovarian cancers and could be used to target other tumors with HR deficiencies due to specific mutations such as PTEN loss, and (b) GBM patients may perhaps be stratified for therapy with temozolomide or PARP inhibitors based not just on their MGMT status but also on their PTEN status.

Although our results with genetically defined models suggest that PTEN-null tumors may be more responsive to temozolomide treatment, it is important to point out the caveat that glioblastomas are genetically very heterogeneous (5, 6, 10). Therefore, it is plausible that treatment with DNA alkylating agents could actually select for resistant clones, ultimately precipitating tumor resurgence and therapeutic failure. This possibility is tentatively indicated by in vitro studies where we find that U87 or U251 clones surviving MNNG treatment are more MNNG resistant (to varying degrees) compared with the parental lines (Supplementary Fig. S7). This possibility is also borne out by the Cancer Genome Atlas Research Network study, which suggests that treatment of _MGMT_-deficient glioblastomas with alkylating agents may result in the selection of mutations in mismatch repair genes, leading to therapy resistance (5). It would be interesting to determine if the HR defect due to PTEN loss may actually increase the likelihood of development of resistant clones due to increased genomic instability.

Although the results described in this article come from proof-of-principle experiments carried out with cells growing in monolayer, it will be important to extend these results to organotypic three-dimensional models to verify whether our conclusions are valid in a more “tumor-like” setting. In preliminary studies, we find that both monolayer cultures as well as “spheroids” of U87 cells [generated by the “hanging-drop” method (46)] are similarly sensitive to MNNG (Supplementary Fig. S8). Eventually, extensive research with preclinical mouse models, especially orthotopic glioblastoma models, will be required to firmly establish the nascent but highly novel concepts formulated by this study.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Prof. David A. Boothman for critically reading the manuscript and Dr. Steve Cho for help with designing qRT-PCR primers.

Grant Support: NASA grants NNA05CS97G and NNX10AE08G (S. Burma), Cancer Prevention and Research Institute of Texas grant RP100644 (S. Burma), the Goldhirsh Foundation (R.M. Bachoo), and National Cancer Institute grant T32CA124334 (C.V. Camacho).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

References

Friedman

Kerby

Calvert

Temozolomide and treatment of malignant glioma

Clin Cancer Res

2000

;

2585

–

Stupp

Mason

van den Bent

, et al.

Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma

N Engl J Med

2005

;

352

987

–

Stupp

Hegi

Mason

, et al.

Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial

Lancet Oncol

2009

;

459

–

Hegi

Diserens

Gorlia

, et al.

MGMT gene silencing and benefit from temozolomide in glioblastoma

N Engl J Med

2005

;

352

997

–

1003

McLendon

Friedman

Bigner

, et al.

Comprehensive genomic characterization defines human glioblastoma genes and core pathways

Nature

2008

;

455

1061

–

Parsons

Jones

Zhang

, et al.

An integrated genomic analysis of human glioblastoma multiforme

Science

2008

;

321

1807

–

Verhaak

Hoadley

Purdom

, et al.

Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1

Cancer Cell

2010

;

–

110

Mukherjee

McEllin

Camacho

, et al.

EGFRvIII and DNA double-strand break repair: a molecular mechanism for radioresistance in glioblastoma

Cancer Res

2009

;

4252

–

Stojic

Brun

Jiricny

Mismatch repair and DNA damage signalling

DNA Repair (Amst)

2004

;

1091

–

101

Furnari

Fenton

Bachoo

, et al.

Malignant astrocytic glioma: genetics, biology, and paths to treatment

Genes Dev

2007

;

2683

–

710

Salmena

Carracedo

Pandolfi

Tenets of PTEN tumor suppression

Cell

2008

;

133

403

–

Shen

Balajee

Wang

, et al.

Essential role for nuclear PTEN in maintaining chromosomal integrity

Cell

2007

;

128

157

–

Yin

Shen

PTEN: a new guardian of the genome

Oncogene

2008

;

5443

–

Bryant

Schultz

Thomas

, et al.

Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase

Nature

2005

;

434

913

–

Farmer

McCabe

Lord

, et al.

Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy

Nature

2005

;

434

917

–

Fong

Boss

Yap

, et al.

Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers

N Engl J Med

2009

;

361

123

–

Bachoo

Maher

Ligon

, et al.

Epidermal growth factor receptor and Ink4a/Arf: convergent mechanisms governing terminal differentiation and transformation along the neural stem cell to astrocyte axis

Cancer Cell

2002

;

269

–

Lesche

Groszer

Gao

, et al.

Cre/loxP-mediated inactivation of the murine Pten tumor suppressor gene

Genesis

2002

;

148

–

Serrano

Lee

Chin

Cordon-Cardo

Beach

DePinho

Role of the INK4a locus in tumor suppression and cell mortality

Cell

1996

;

–

Mukherjee

Camacho

Tomimatsu

Miller

Burma

Modulation of the DNA-damage response to HZE particles by shielding

DNA Repair (Amst)

2008

;

1717

–

Mukherjee

McEllin

Camacho

, et al.

EGFRvIII and DNA Double-strand break repair: a molecular mechanism for radioresistance in glioblastoma

Cancer Res

2009

;

4252

–

Kao

Jiang

Fernandes

Gupta

Maity

Inhibition of phosphatidylinositol-3-OH kinase/Akt signaling impairs DNA repair in glioblastoma cells following ionizing radiation

J Biol Chem

2007

;

282

21206

–

Wick

Furnari

Naumann

Cavenee

Weller

PTEN gene transfer in human malignant glioma: sensitization to irradiation and CD95L-induced apoptosis

Oncogene

1999

;

3936

–

Kaina

Christmann

Naumann

Roos

MGMT: key node in the battle against genotoxicity, carcinogenicity and apoptosis induced by alkylating agents

DNA Repair (Amst)

2007

;

1079

–

Gerson

MGMT: its role in cancer aetiology and cancer therapeutics

Nat Rev Cancer

2004

;

296

–

307

Fritz

Tano

Mitra

Kaina

Inducibility of the DNA repair gene encoding O6-methylguanine-DNA methyltransferase in mammalian cells by DNA-damaging treatments

Mol Cell Biol

1991

;

4660

–

Jiricny

The multifaceted mismatch-repair system

Nat Rev Mol Cell Biol

2006

;

335

–

Wang

Edelmann

Mismatch repair proteins as sensors of alkylation DNA damage

Cancer Cell

2006

;

417

–

Fernandez-Capetillo

Lee

Nussenzweig

H2AX: the histone guardian of the genome

DNA Repair (Amst)

2004

;

959

–

Mochan

Venere

DiTullio

RA,

Jr.,

Halazonetis

53BP1, an activator of ATM in response to DNA damage

DNA Repair (Amst)

2004

;

945

–

Branzei

Foiani

Regulation of DNA repair throughout the cell cycle

Nat Rev Mol Cell Biol

2008

;

297

–

308

Veuger

Curtin

Richardson

Smith

Durkacz

Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase-1

Cancer Res

2003

;

6008

–

Roos

Nikolova

Quiros

, et al.

Brca2/Xrcc2 dependent HR, but not NHEJ, is required for protection against O6-methylguanine triggered apoptosis, DSBs and chromosomal aberrations by a process leading to SCEs

DNA Repair (Amst)

2009

;

–

Sonoda

Sasaki

Morrison

Yamaguchi-Iwai

Takata

Takeda

Sister chromatid exchanges are mediated by homologous recombination in vertebrate cells

Mol Cell Biol

1999

;

5166

–

Arnaudeau

Lundin

Helleday

DNA double-strand breaks associated with replication forks are predominantly repaired by homologous recombination involving an exchange mechanism in mammalian cells

J Mol Biol

2001

;

307

1235

–

Venkitaraman

Linking the cellular functions of BRCA genes to cancer pathogenesis and treatment

Annu Rev Pathol

2009

;

461

–

San Filippo

Sung

Klein

Mechanism of eukaryotic homologous recombination

Annu Rev Biochem

2008

;

229

–

Donawho

Luo

Penning

, et al.

ABT-888, an orally active poly(ADP-ribose) polymerase inhibitor that potentiates DNA-damaging agents in preclinical tumor models

Clin Cancer Res

2007

;

2728

–

Sonoda

Ozawa

Hirose

, et al.

Formation of intracranial tumors by genetically modified human astrocytes defines four pathways critical in the development of human anaplastic astrocytoma

Cancer Res

2001

;

4956

–

Ishii

Maier

Merlo

, et al.

Frequent co-alterations of TP53, p16/CDKN2A, p14ARF, PTEN tumor suppressor genes in human glioma cell lines

Brain Pathol

1999

;

469

–

Sonoda

Ozawa

Aldape

Deen

Berger

Pieper

Akt pathway activation converts anaplastic astrocytoma to glioblastoma multiforme in a human astrocyte model of glioma

Cancer Res

2001

;

6674

–

Stupp

Dietrich

Ostermann Kraljevic

, et al.

Promising survival for patients with newly diagnosed glioblastoma multiforme treated with concomitant radiation plus temozolomide followed by adjuvant temozolomide

J Clin Oncol

2002

;

1375

–

Mendes-Pereira

Martin

Brough

, et al.

Synthetic lethal targeting of PTEN mutant cells with PARP inhibitors

EMBO Mol Med

2009

;

315

–

Liu

Han

Anderson

, et al.

Acquired resistance to combination treatment with temozolomide and ABT-888 is mediated by both base excision repair and homologous recombination DNA repair pathways

Mol Cancer Res

2009

;

1686

–

Plummer

Inhibition of poly(ADP-ribose) polymerase in cancer

Curr Opin Pharmacol

2006

;

364

–

Del Duca

Werbowetski

Del Maestro

Spheroid preparation from hanging drops: characterization of a model of brain tumor invasion

J Neurooncol

2004

;

295

–

303

2010

Supplementary data

1,083 Views

200 Web of Science

PTEN Loss Compromises Homologous Recombination Repair in Astrocytes: Implications for Glioblastoma Therapy with Temozolomide or Poly(ADP-Ribose) Polymerase Inhibitors (original) (raw)

Abstract

Introduction

Materials and Methods

Cell culture

Irradiation and drug treatment

Colony formation assays

Immunofluorescence staining and Western blot analyses

Metaphase chromosome preparations and sister chromatid exchange assay

Statistical analyses

Additional methods

Results

Discussion

Disclosure of Potential Conflicts of Interest

Acknowledgments

References

Supplementary data

Citing articles via

Email alerts