MRE11 deficiency increases sensitivity to poly(ADP-ribose) polymerase inhibition in microsatellite unstable colorectal cancers - PubMed (original) (raw)

MRE11 deficiency increases sensitivity to poly(ADP-ribose) polymerase inhibition in microsatellite unstable colorectal cancers

Eduardo Vilar et al. Cancer Res. 2011.

Abstract

Microsatellite instability (MSI) is displayed by approximately 15% of colorectal cancers (CRC). Defective DNA mismatch repair generates mutations at repetitive DNA sequences such as those located in the double strand break (DSB) repair gene MRE11. We assessed the mutational status of MRE11 in a panel of 17 CRC cell lines and 46 primary tumors and found a strong correlation with MSI status in both cell lines and tumors. Therefore, we hypothesized that deficiency in MRE11 may sensitize CRC cells to poly(ADP-ribose) polymerase (PARP-1) inhibition based on the concept of synthetic lethality. We further assessed the activity of the PARP-1 inhibitor, ABT-888, in CRC cell lines and observed preferential cytotoxicity in those MSI cell lines harboring mutations in MRE11 compared with both wild-type cell lines and microsatellite stable (MSS) cell lines. A significant correlation between MRE11 expression levels and cytotoxicity to ABT-888 at 10 μM was observed (R² = 0.915, P < 0.001). Using two experimental approaches, including short hairpin RNA knocking down MRE11 in the wild-type and MSS cell line SW-480 and a second cell line model transfected with mutant MRE11, we experimentally tried to confirm the role of MRE11 in conferring sensitivity to PARP-1 inhibition. Both models led to changes in proliferation in response to ABT-888 at different concentrations, and a drug-response effect was not observed, suggesting a possible contribution of additional genes. We conclude that MSI colorectal tumors deficient in DSB repair secondary to mutation in MRE11 show a higher sensitivity to PARP-1 inhibition. Further clinical investigation of PARP-1 inhibitors is warranted in MSI CRCs.

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Figures

Figure 1

Figure 1

A) a polyT(11) tract located at the Intron4 is the target for mutations in MSI tumors. Shortening in one or more nucleotides causes changes in the splicing can induce complete skipping of exon 5 and protein truncation. Two sets of primers were designed to measure the levels of the wild type (wt) and the mutant transcript (mut) of MRE11; B), C) and D) levels of expression of the wt and mut transcript of MRE11, as well as PARP-1 assessed by qRT-PCR in 10 CRC cell lines.

Figure 2

Figure 2

Schematic representation of the homologous recombination pathway hsa03440 retrieved from the KEGG database. In red are those genes upregulated and in blue those downregulated. Note that not all of the proteins acting in this pathway are shown in this figure; B) Probe sets from hsa03440 significantly deregulated in MSI compared to MSS tumors and their corresponding fold-changes;

Figure 3

Figure 3

Rad51 foci were analyzed under baseline conditions (B, t=0) and 18 hours after irradiation (IR, t=18). Percentages of foci-positive cells at each time point are presented. Error bars represent standard errors of the mean for two independent observations. Asterisks designate statistically significant differences in percent positivity at baseline vs. 18 hours post-irradiation (_P-_value<0.05). Representative images are presented for both markers and time points.

Figure 4

Figure 4

A) and B) Comparison of cytotoxicity at 10 μM and IC50 in biallelic mutants and wild-type plus monoallelic mutants; C) Correlation between expression levels of the MRE11 mutant transcript and growth inhibition at 10 μM of ABT-888; D) Cell cycle changes after treatment with two different concentrations of ABT-888. Note that cell cycle changes were more pronounced in biallelic than monoallelic mutants and wild type cells.

Figure 5

Figure 5

A) Levels of MRE11 wild type transcript in transfected cells with a shRNA plasmid against MRE11 and in the derivative cell line SM1.3 that has been transfected with an expression construct for Δ5-7MRE11 lacking exons 5–7. Levels of expression were normalized to mock cells and to the parental cell line SW480/SN3, respectively. B) Proliferation after treatment with ABT-888. Note a difference in proliferation between SW-480 transfected with shRNA anti-MRE11 plasmid and SW-480 transfected with a mock plasmid at 50 μM and also between SM1.3 and its parental cell line at 10 μM. C) Expression levels of the wild type transcript of MRE11 compared to levels expressed in the original panel of cell lines.

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