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Synthetic lethal targeting of PTEN mutant cells with PARP inhibitors - PubMed

Synthetic lethal targeting of PTEN mutant cells with PARP inhibitors

Ana M Mendes-Pereira et al. EMBO Mol Med. 2009 Sep.

Abstract

The tumour suppressor gene, phosphatase and tensin homolog (PTEN), is one of the most commonly mutated genes in human cancers. Recent evidence suggests that PTEN is important for the maintenance of genome stability. Here, we show that PTEN deficiency causes a homologous recombination (HR) defect in human tumour cells. The HR deficiency caused by PTEN deficiency, sensitizes tumour cells to potent inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), both in vitro and in vivo. PARP inhibitors are now showing considerable promise in the clinic, specifically in patients with mutations in either of the breast cancer susceptibility genes BRCA1 or BRCA2. The data we present here now suggests that the clinical assessment of PARP inhibitors should be extended beyond those with BRCA mutations to a larger group of patients with PTEN mutant tumours.

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Figures

Figure 1
Figure 1. PTEN deficiency causes an impairment of DNA repair by HR

  1. PTEN deficiency causes a reduction in RAD51 expression. Total cell lysates from isogenic HCT116 colorectal tumour cells were immunoblotted for PTEN and RAD51. Detection of β Tubulin is shown as a loading control. Lysates from parental HCT116 cells were used (HCT116) along with two individually derived PTEN−/− lines (KO35 and KO22) and also a HCT116-derived line bearing a random integration of the PTEN targeting construct (neo124). Lysates from PTEN+/− HCT116 cells are also shown (Lee et al, 2007).

  2. PTEN deficiency causes a reduction in radiation-induced nuclear RAD51 focus formation. Cells were exposed to 10 Gy γ-irradiation and nuclear RAD51 foci quantified 8 h later by confocal microscopy (Farmer et al, 2005). Bar chart shows the average number of cells with >5 foci per nucleus. Error bars represent three standard deviations of the mean. * p values versus +IR HCT116 PTEN+/+<0.05 (Student's t-test). All other comparisons returned non-significant (>0.05) p values.

  3. PTEN deficiency causes a reduction in HR as measured using a synthetic HR substrate. As a measure of HR activity, a reporter plasmid-based assay was used comprising two defective copies of GFP, where one serves as a template to restore an induced DSB in the other. HR between the two GFP coding sequences results in an intact GFP coding sequence and cellular fluorescence (Saeki et al, 2006). Error bars represent three standard deviations of the mean. * p values versus HCT116 PTEN+/+<0.05 (Student's t-test).

Figure 2
Figure 2. PTEN deficiency sensitizes tumour cells to agents that target HR

A, B. PTEN deficiency sensitizes cells to drug-like PARP inhibitors. Survival curves for HCT116 cells exposed to the PARP inhibitors KU0058948 (Farmer et al, 2005) or KU0059436 (Evers et al, ; Fong et al, 2008). Cells were plated in six-well plates and treated for 15 days, after which SFs were estimated. For both PARP inhibitors, PTEN−/− SFs were significantly different to both PTEN+/+ and PTEN+/− cells (p<0.05 two-way analysis of variance (ANOVA)). No other comparisons returned significant p values. C. PTEN deficiency sensitizes cells to cisplatin (p<0.05 PTEN−/− lines versus PTEN+/+ cells, two-way ANOVA). D. PTEN deficiency does not confer sensitization to taxol.

Figure 3
Figure 3. PARP inhibitor sensitivity in human tumour cells

  1. Western blot showing PTEN expression in a panel of human tumour lines. PTEN mutant lines are shown in red and PTEN wild type lines in black. PTEN genotypes are as follows: DU145 (prostate), PTEN wild type; MCF7 (breast), PTEN wild type; PC3 (prostate) p.R55fs*1 homozygous; MDAMB468 (breast), p.A72fs*5 homozygous; A172 (glioma), p.R55fs*1 homozygous; UM-UC3 (bladder), p.M1*404del homozygous; RPMI-7951 (melanoma) p.null, c.1-79del79 homozygous; HCC70 (breast), p.F90fs*9 homozygous (see Table S1 of Supporting Information for complete genotypes).

  2. PARPi sensitivity in the same cell line panel. For each PTEN null line, survival was significantly different than in DU145 and MCF7 cells (p<0.05, two-way ANOVA) (see Fig S3 of Supporting Information for analysis of an enlarged cell line panel).

  3. Cisplatin sensitivity in the same cell line panel. With the exception of A172, survival of each PTEN null line at 1 µM was significantly different than in DU145 and MCF7 cells (p<0.05, two-way ANOVA).

Figure 4
Figure 4. PARP inhibitor sensitivity in human tumour cells

A, B. Wild type and catalytically inactive PTEN, both restore PARP inhibitor resistance, as does expression of RAD51; SFs in PC3 cells infected with PTEN wild type, PTEN p.C124S or RAD51 cDNA expression constructs was significantly different than in PC3 + empty vector (p < 0.05, two-way ANOVA). PC3 cells infected with p.K289E cDNA expression construct did not cause significant resistance. PTEN null PC3 cells were infected with cDNA expression constructs as shown and exposed to PARP inhibitor. Western blot and survival curves are shown.

Figure 5
Figure 5. In vivo efficacy of Olaparib in PTEN deficient xenografts

A, B. The clinical PARP inhibitor KU0059436/Olaparib (Evers et al, ; Fong et al, 2008) suppresses growth in PTEN−/− subcutaneous xenografts (A), but not in PTEN+/+ xenografts (B). PTEN deficient or proficient HCT116 cells were each mixed 2:1 in matrigel and then injected into either bilateral flank of 6–8 week old female athymic nude mice. After two days recovery, mice were randomized into treatment and control groups, (10 animals per cohort, 20 in total) and drug dosing initiated. A treatment regime similar to that known to elicit Brca2 selectivity (Farmer et al, 2005) was used. For five consecutive days, single daily intraperitoneal doses of KU0059436 (or vehicle) were administered at a dose of 15 mg/kg in HBC (Farmer et al, 2005). This was followed by two consecutive days of no treatment after which the same treatment cycle was continued until the end of the study. Tumour volumes were measured every four days from the initiation of drug dosing and the results expressed as fold increase in tumour volume relative to that at the first drug administration. Tumour volume vehicle treated vs. drug treated, p = 0.04 for PTEN−/− xenografts and p = 0.44 for PTEN+/+ xenografts (two-way repeated measures ANOVA).

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