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

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 ?M 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.

SUBMITTER: Vilar E

PROVIDER: S-EPMC3407272 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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

Vilar Eduardo E Bartnik Catherine M CM Stenzel Stephanie L SL Raskin Leon L Ahn Jaeil J Moreno Victor V Mukherjee Bhramar B Iniesta Maria D MD Morgan Meredith A MA Rennert Gad G Gruber Stephen B SB

Cancer research 20110207 7

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 t ...[more]

PMID: 21300766

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Project description:We have performed bioinformatic approaches to identify the level of enrichment between gene expression profiles characterizing MSI tumors and gene changes induced in vitro by the PARP-1 inhibitor Phenanthridinone and others using the Connectivity Map tool. In a first step, we have anyzed the expression of 300 colorectal cancers from the MECC study and generated a gene expression signature by microsatellite status. The criteria followed for selection of probe sets and detailed lists to be submitted subsequently to the Connectivity Map have been published previously by us in Clinical Cancer Research in 2009. In a second step, once we observed that deficiency in MRE11 exist among MSI tumors, our interest was focused on assessing if the homologous recombination pathway showed evidence of deregulation in MSI tumors. Therefore, we examined the expression levels of those genes integrated in the KEGG pathway hsa03440 using the previously generated gene expression data set. The dataset generated from our samples included a total of 300 colorectal fresh frozen tumors collected from the MECC study and were analyzed in two batches. The first batch was hybridized to the Affymetrix U133A chip and the second one to U133 Plus 2.0 (Supplementary Table S3). The final list of probe sets defining gene expression of MSI-H compared to MSS tumors was selected based on the strength of multiplicity adjusted P-values (cut-off P-value of < 0.001) and ratio of mean expression values across the two groups (cut-off Fold-change >1.3 and <0.7). It contained 442 upregulated and 480 downregulated probe sets. Then, MSI-H tumors present with changes in gene expression related to the homologous recombination pathway. Therefore, we examined the expression levels of those genes integrated in the KEGG pathway hsa03440 using the same data set. A total of 14 genes out of 30 were significantly differentially expressed in MSI-H compared to MSS tumors (Multiplicity adjusted Benjamini-Hochberg P-value<0.05). MRE11 and RAD50 probe sets showed a lower expression in MSI-H tumors and simultaneously other probe sets such as PARP-1 were significantly upregulated, probably secondary to the deficiency in the MRN complex proteins. This data provides evidence of significant differential expression of the homologous recombination pathway in MSI tumors.

Project description:Inactivation of the DNA mismatch repair pathway manifests as microsatellite instability, an accumulation of mutations that drives carcinogenesis. Here, we determined whether microsatellite instability in acute myeloid leukemia and myelodysplastic syndrome correlated with chromosomal instability and poly (ADP-ribose) polymerase (PARP) inhibitor sensitivity through disruption of DNA repair function. Acute myeloid leukemia cell lines (n=12) and primary cell samples (n=18), and bone marrow mononuclear cells from high-risk myelodysplastic syndrome patients (n=63) were profiled for microsatellite instability using fluorescent fragment polymerase chain reaction. PARP inhibitor sensitivity was performed using cell survival, annexin V staining and cell cycle analysis. Homologous recombination was studied using immunocytochemical analysis. SNP karyotyping was used to study chromosomal instability. RNA silencing, Western blotting and gene expression analysis was used to study the functional consequences of mutations. Acute myeloid leukemia cell lines (4 of 12, 33%) and primary samples (2 of 18, 11%) exhibited microsatellite instability with mono-allelic mutations in CtIP and MRE11. These changes were associated with reduced expression of mismatch repair pathway components, MSH2, MSH6 and MLH1. Both microsatellite instability positive primary acute myeloid leukemia samples and cell lines demonstrated a downregulation of homologous recombination DNA repair conferring marked sensitivity to PARP inhibitors. Similarly, bone marrow mononuclear cells from 11 of 56 (20%) patients with de novo high-risk myelodysplastic syndrome exhibited microsatellite instability. Significantly, all 11 patients with microsatellite instability had cytogenetic abnormalities with 4 of them (36%) possessing a mono-allelic microsatellite mutation in CtIP. Furthermore, 50% reduction in CtIP expression by RNA silencing also down-regulated homologous recombination DNA repair responses conferring PARP inhibitor sensitivity, whilst CtIP differentially regulated the expression of homologous recombination modulating RecQ helicases, WRN and BLM. In conclusion, microsatellite instability dependent mutations in DNA repair genes, CtIP and MRE11 are detected in myeloid malignancies conferring hypersensitivity to PARP inhibitors. Microsatellite instability is significantly correlated with chromosomal instability in myeloid malignancies.

Dataset Information

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

Publications

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

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