A Small Interfering RNA Screen of Genes Involved in DNA Repair Identifies Tumor-Specific Radiosensitization by POLQ Knockdown

Gray Institute for Radiation Oncology and Biology, Oxford University, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, United Kingdom.
Cancer Research (Impact Factor: 9.33). 03/2010; 70(7):2984-93. DOI: 10.1158/0008-5472.CAN-09-4040
Source: PubMed


The effectiveness of radiotherapy treatment could be significantly improved if tumor cells could be rendered more sensitive to ionizing radiation (IR) without altering the sensitivity of normal tissues. However, many of the key therapeutically exploitable mechanisms that determine intrinsic tumor radiosensitivity are largely unknown. We have conducted a small interfering RNA (siRNA) screen of 200 genes involved in DNA damage repair aimed at identifying genes whose knockdown increased tumor radiosensitivity. Parallel siRNA screens were conducted in irradiated and unirradiated tumor cells (SQ20B) and irradiated normal tissue cells (MRC5). Using gammaH2AX foci at 24 hours after IR, we identified several genes, such as BRCA2, Lig IV, and XRCC5, whose knockdown is known to cause increased cell radiosensitivity, thereby validating the primary screening end point. In addition, we identified POLQ (DNA polymerase ) as a potential tumor-specific target. Subsequent investigations showed that POLQ knockdown resulted in radiosensitization of a panel of tumor cell lines from different primary sites while having little or no effect on normal tissue cell lines. These findings raise the possibility that POLQ inhibition might be used clinically to cause tumor-specific radiosensitization.

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Available from: Eric J Bernhard
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    • "Interestingly, patients with a more aggressive phenotype of breast cancer (triple negative), also had the highest levels of POLQ expression and lower survival (OR = 4.28; p = 0.0001), regardless of the levels of CYCLIN E and number of positive nodes [23]. These results were then confirmed in an independent cohort by Higgins et al. in 2010 (OR = 5.80; p = 0.001). In addition, fibroblasts transfected with a vector containing POLQ led to replicative stress and chromosomal instability [23]. "
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    ABSTRACT: One of the hallmarks of cancer is the occurrence of high levels of chromosomal rearrangements as a result of inaccurate repair of double-strand breaks (DSB). Germline mutations in BRCA and RAD51 genes, involved in DSB repair, are strongly associated with hereditary breast cancer. Pol theta, a translesional DNA polymerase specialized in the replication of damaged DNA, has been also shown to contribute to DNA synthesis associated to DSB repair. It is noteworthy that POLQ is highly expressed in breast tumors and this expression is able to predict patient outcome. The objective of this study was to analyze genetic variants related to POLQ as new population biomarkers of risk in hereditary (HBC) and sporadic (SBC) breast cancer. We analyzed through case-control study nine SNPs of POLQ in hereditary (HBC) and sporadic (SBC) breast cancer patients using Taqman Real Time PCR assays. Polymorphisms were systematically identified through the NCBI database and are located within exons or promoter regions. We recruited 204 breast cancer patients (101 SBC and 103 HBC) and 212 unaffected controls residing in Southern Brazil. The rs581553 SNP located in the promoter region was strongly associated with HBC (c.-1060A > G; HBC GG = 15, Control TT = 8; OR = 5.67, CI95% = 2.26-14.20; p < 0.0001). Interestingly, 11 of 15 homozygotes for this polymorphism fulfilled criteria for Hereditary Breast and Ovarian Cancer (HBOC) syndrome. Furthermore, 12 of them developed bilateral breast cancer and one had a familial history of bilateral breast cancer. This polymorphism was also associated with bilateral breast cancer in 67 patients (OR = 9.86, CI95% = 3.81-25.54). There was no statistically significant difference of age at breast cancer diagnosis between SNP carriers and non-carriers. Considering that Pol theta is involved in DBS repair, our results suggest that this polymorphism may contribute to the etiology of HBC, particularly in patients with bilateral breast cancer.
    Full-text · Article · Nov 2014 · BMC Cancer
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    • "Thus, this polymerase may have acquired more fundamental role in DNA repair in higher plants. Mouse and human cell lines impaired in DNA pol theta are sensitive to bleomycin and ionizing radiation, implicating its function in some aspects of DSB repair [43], [44]. Studies in Drosophila melanogaster indicated that DNA Pol theta contributes to DSB repair by facilitating microhomology-mediated DNA end-joining [45], which is in accordance with our observation of a 10-fold decrease in the transformation efficiency of the Z16 mutant. "
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    ABSTRACT: While the mechanisms governing DNA damage response and repair are fundamentally conserved, cross-kingdom comparisons indicate that they differ in many aspects due to differences in life-styles and developmental strategies. In photosynthetic organisms these differences have not been fully explored because gene-discovery approaches are mainly based on homology searches with known DDR/DNA repair proteins. Here we performed a forward genetic screen in the green algae Chlamydomonas reinhardtii to identify genes deficient in DDR/DNA repair. We isolated five insertional mutants that were sensitive to various genotoxic insults and two of them exhibited altered efficiency of transgene integration. To identify genomic regions disrupted in these mutants, we established a novel adaptor-ligation strategy for the efficient recovery of the insertion flanking sites. Four mutants harbored deletions that involved known DNA repair factors, DNA Pol zeta, DNA Pol theta, SAE2/COM1, and two neighbouring genes encoding ERCC1 and RAD17. Deletion in the last mutant spanned two Chlamydomonas-specific genes with unknown function, demonstrating the utility of this approach for discovering novel factors involved in genome maintenance.
    Full-text · Article · Aug 2014 · PLoS ONE
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    • "Accumulating evidence suggests a role for POLQ in the repair or tolerance of double-strand breaks. POLQ deficient mouse bone marrow cells (17) and human tumor cells (18) show increased sensitivity to ionizing radiation and to low doses of bleomycin (17), both of which are known to produce double-strand breaks. Knockdown of POLQ in CH12 mouse B lymphoma cells increases their sensitivity to the double-strand break inducer etoposide (19) and chaos-1 mice [these mice have a serine to proline mutation at position 1932 in POLQ (20)] show high levels of micronuclei, indicating increased levels of chromosome breaks (21), both of which suggest a role for POLQ in the repair of double-strand breaks in mammalian cells. "
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    ABSTRACT: The biological role of human DNA polymerase θ (POLQ) is not yet clearly defined, but it has been proposed to participate in several cellular processes based on its translesion synthesis capabilities. POLQ is a low-fidelity polymerase capable of efficient bypass of blocking lesions such as abasic sites and thymine glycols as well as extension of mismatched primer termini. Here, we show that POLQ possesses a DNA polymerase activity that appears to be template independent and allows efficient extension of single-stranded DNA as well as duplex DNA with either protruding or multiply mismatched 3′-OH termini. We hypothesize that this DNA synthesis activity is related to the proposed role for POLQ in the repair or tolerance of double-strand breaks.
    Full-text · Article · Dec 2011 · Nucleic Acids Research
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