PTEN loss compromises homologous recombination repair in astrocytes: implications for glioblastoma therapy with temozolomide or poly(ADP-ribose) polymerase inhibitors - PubMed (original) (raw)

PTEN loss compromises homologous recombination repair in astrocytes: implications for glioblastoma therapy with temozolomide or poly(ADP-ribose) polymerase inhibitors

Brian McEllin et al. Cancer Res. 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 S(N)1-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.

Copyright 2010 AACR.

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Figures

Figure 1

Figure 1. PTEN loss sensitizes astrocytes to MNNG

(A) Loss of PTEN and activation of Akt in PTEN+/+ and -/- astrocytes were analyzed by Western blotting with α-PTEN and α-phospho-Akt(ser473) antibodies. (B) 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) Sensitivity of astrocytes to MNNG was quantified by colony formation assays. Please note increased sensitivity of PTEN-null cells to MNNG. Error bars represent standard error of the mean of experiments performed 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 (numbers in red).

Figure 2

Figure 2. MGMT regulation is not affected by PTEN loss

(A) MGMT protein levels in PTEN+/+ and -/- 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 GAPDH levels and expressed as fold change relative to mock-treated PTEN+/+ cells. Error bars represent standard error of the mean.

Figure 3

Figure 3. PTEN loss compromises homologous recombination repair

(A) Induction and repair of DSBs in astrocytes treated with a 1 hour pulse of 5μM MNNG. Cells were co-immunostained for γH2AX (red) and 53BP1 (green) foci at various times post-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. Statistical significance was determined by two-way ANOVA with a Bonferroni post-test: *p<0.05; ***p<0.001. (B) Sensitivity of astrocytes to camptothecin (CPT) was quantified by colony formation assays. Please note increased sensitivity of PTEN-null cells to CPT. (C) To quantify sister chromatid exchanges (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 SCE/metaphase. At least 40 metaphases were counted per treatment. Statistical significance was determined by a two-tailed t test; ***p<0.0001. (D) Metaphase spreads from astrocytes treated with 10μM MNNG were analyzed for chromatid breaks and asymmetric exchanges (1. triradials, 2. quadriradials, and 3. complex exchanges). At least 40 metaphases were counted per treatment and average aberrations per metaphase were plotted. Representative pictures of aberrations are shown. Statistical significance was determined by a two-tailed t test; **p=.0027, *** p=.0004. Error bars represent standard error of the mean for all plots.

Figure 4

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 was 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. Statistical significance was determined by two-way ANOVA with a Bonferroni post-test; ** p<0.01, ***p<0.001. (C) Sensitivity of astrocytes to the PARP inhibitor ABT-888 was quantified by colony formation assays. Please note increased sensitivity of PTEN-null cells to ABT-888. Error bars represent standard error of the mean for all plots.

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