Supplementary Materialscells-08-01258-s001

Supplementary Materialscells-08-01258-s001. of WIP1 may boost sensitivity of BRCA1-proficient cancer cells to olaparib. gene and its expression is increasing towards the G2 phase of the cell cycle [30,31,32]. WIP1 terminates the DNA damage response by dephosphorylation of H2AX, ATM pS1981 and KAP1 pS824 and promotes release from the cell cycle checkpoint by dephosphorylation of p53 pS15 [30,33,34,35,36,37]. locus is amplified in about 10% of breast cancers, in medulloblastoma and ovary cancer [38,39,40]. Importantly, amplifications occur mostly in tumors harboring wild-type p53 [38,41]. Activity of WIP1 can be specifically inhibited by a small-molecule compound GSK2830371 and WIP1 was proposed as perspective pharmacological target particularly in p53-proficient cancers [42,43,44,45,46]. Here we report a novel role of WIP1 in DSB repair through HR. We find that WIP1 stably interacts with BRCA1-BARD1 complex and inhibition of WIP1 delays recruitment of BRCA1 to DSBs. Consistent with WIP1 function in HR, inhibition of WIP1 leads to accumulation of DNA damage in S/G2 cells and sensitizes cancer cells to olaparib. Thus, inhibition of WIP1 may promote efficiency of PARP inhibitors in tumors with normal BRCA1 function. 2. Results 2.1. WIP1 Promotes DSB Repair by Homologous Recombination WIP1 phosphatase was shown to counteract ATM kinase activity at chromatin to terminate DNA damage response and to facilitate recovery form the G2 checkpoint [30,34,35]. In addition, overexpression of WIP1 affects DSB repair efficiency through dephosphorylation of H2AX leading to disruption of DDR signaling [30,47]. To evaluate the role of WIP1 in more physiological condition we used different established cell based reporter assays together with a recently described specific WIP1 inhibitor GSK2830371 [42,44]. To this end we generated stable Traffic light reporter cell lines in U2OS and RPE that allowed us to analyze the overall repair efficiency as well as the ratio of repair efficiency by homologous recombination (GFP+) and non-homologous end joining (RFP+) (Figure S1A) [48]. As expected, inhibition of DNA-PK increased the HR/NHEJ ratio reflecting its essential role in NHEJ (Figure S1B). Conversely, inhibition of ATM decreased the HR/NHEJ ratio which is consistent with involvement of ATM in mediating DNA resection (Figure S1B) [49]. Interestingly, inhibition of WIP1 lowered DSB repair efficiency by homologous recombination while NHEJ was not affected and thus decreased the HR/NHEJ ratio in two independent clones of both U2OS and RPE cells (Figure 1ACD). To further confirm this phenotype, we used established U2OS DR-GFP and E5J reporter cell lines and consistently CCG-63808 we observed decreased HR efficiency after inhibition of WIP1 (Figure S1C) [50]. Open in a separate window Figure 1 Inhibition of WIP1 impairs homologous recombination (HR). (A) Traffic light reporter assay in U2OS cells. Two independent stable cell lines (clones #10 and #12) were transfected with ISceI together with CCG-63808 BFP-donor vector with or without pretreatment with 1 M WIP1i. Efficiency of repair was analyzed 3 days after transfection by FACS. Plotted is mean of normalized ratio of GFP+/RFP+ cells. Bars indicate SD, n 3. Statistical significance evaluated by two-tailed 0.05; *** 0.001). (F) Cell survival of parental U2OS and two independent U2OS-WIP1-KO cell lines treated with indicated doses of camptothecin with or without combined treatment with WIP1 inhibitor was evaluated after 7 days using CCG-63808 resazurin viability assay. Plotted is mean and SD, n 3. ARPC2 Statistical significance evaluated by two-way ANOVA (* 0.05; *** 0.001). (G) Cell survival after irradiation of parental RPE and RPE-WIP1-KO cell lines assayed as in E. (H) Cell survival of parental RPE and RPE-WIP1-KO cell lines with treated with camptothecin and analyzed as.