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53BP1 depletion causes PARP inhibitor resistance in ATM-deficient breast cancer cells

View Article: PubMed Central - PubMed

ABSTRACT

Background: Mutations in DNA damage response factors BRCA1 and BRCA2 confer sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors in breast and ovarian cancers. BRCA1/BRCA2-defective tumors can exhibit resistance to PARP inhibitors via multiple mechanisms, one of which involves loss of 53BP1. Deficiency in the DNA damage response factor ataxia-telangiectasia mutated (ATM) can also sensitize tumors to PARP inhibitors, raising the question of whether the presence or absence of 53BP1 can predict sensitivity of ATM-deficient breast cancer to these inhibitors.

Methods: Cytotoxicity of PARP inhibitor and ATM inhibitor in breast cancer cell lines was assessed by MTS, colony formation and apoptosis assays. ShRNA lentiviral vectors were used to knockdown 53BP1 expression in breast cancer cell lines. Phospho-ATM and 53BP1 protein expressions were determined in human breast cancer tissues by immunohistochemistry (IHC).

Results: We show that inhibiting ATM increased cytotoxicity of PARP inhibitor in triple-negative and non-triple-negative breast cancer cell lines, and depleting the cells of 53BP1 reduced this cytotoxicity. Inhibiting ATM abrogated homologous recombination induced by PARP inhibitor, and down-regulating 53BP1 partially reversed this effect. Further, overall survival was significantly better in triple-negative breast cancer patients with lower levels of phospho-ATM and tended to be better in patients with negative 53BP1.

Conclusion: These results suggest that 53BP1 may be a predictor of PARP inhibitor resistance in patients with ATM-deficient tumors.

Electronic supplementary material: The online version of this article (doi:10.1186/s12885-016-2754-7) contains supplementary material, which is available to authorized users.

No MeSH data available.


Treatment of Olarparib, Ku-55933 or their combination induced γ-H2AX foci formation A, B, CAL-51 cells (a) and MCF-7 cells (b) were treated with 0, 5, 10 μM Olaparib with or wihout 10 μM KU55933 respectively for 48 h and analyzed by immunofluorescence (IF) with γH2AX antibody. DAPI was used to visualize cell nuclei. Representative pictures (left) and quantitative data (right) of the γ-H2AX foci. c, CAL-51 cells (left) and MCF-7 cells (right) were treated with 0, 10 μM Olaparib with or wihout 10 μM KU55933 respectively 48 h and analyzed by Western Blot assay (WB) with γH2AX antibody. All experiments were performed at least three times and data were statistically analyzed by two-tail t-test. ***p < 0.001. Error bars indicate S.E.M
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Fig2: Treatment of Olarparib, Ku-55933 or their combination induced γ-H2AX foci formation A, B, CAL-51 cells (a) and MCF-7 cells (b) were treated with 0, 5, 10 μM Olaparib with or wihout 10 μM KU55933 respectively for 48 h and analyzed by immunofluorescence (IF) with γH2AX antibody. DAPI was used to visualize cell nuclei. Representative pictures (left) and quantitative data (right) of the γ-H2AX foci. c, CAL-51 cells (left) and MCF-7 cells (right) were treated with 0, 10 μM Olaparib with or wihout 10 μM KU55933 respectively 48 h and analyzed by Western Blot assay (WB) with γH2AX antibody. All experiments were performed at least three times and data were statistically analyzed by two-tail t-test. ***p < 0.001. Error bars indicate S.E.M

Mentions: An early cellular response to DSBs is rapid phosphorylation of H2AX at Ser139, forming γ-H2AX foci [26]. H2AX phosphorylation plays a key role in signalling and initiating the repair of DSBs, and the numbers of γ-H2AX foci correlate closely with the number of DSBs [27]. Treating breast cancer cell lines with both Olaparib and KU55933 led to significantly more γ-H2AX foci formation than treating them with either inhibitor alone based on fluorescence microscopy (Fig. 2a-b). These findings were further confirmed by Western Blot assay (Fig. 2c). This provides further evidence that that Olaparib and Ku-55933 synthetically induce cellular DSBs.Fig. 2


53BP1 depletion causes PARP inhibitor resistance in ATM-deficient breast cancer cells
Treatment of Olarparib, Ku-55933 or their combination induced γ-H2AX foci formation A, B, CAL-51 cells (a) and MCF-7 cells (b) were treated with 0, 5, 10 μM Olaparib with or wihout 10 μM KU55933 respectively for 48 h and analyzed by immunofluorescence (IF) with γH2AX antibody. DAPI was used to visualize cell nuclei. Representative pictures (left) and quantitative data (right) of the γ-H2AX foci. c, CAL-51 cells (left) and MCF-7 cells (right) were treated with 0, 10 μM Olaparib with or wihout 10 μM KU55933 respectively 48 h and analyzed by Western Blot assay (WB) with γH2AX antibody. All experiments were performed at least three times and data were statistically analyzed by two-tail t-test. ***p < 0.001. Error bars indicate S.E.M
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5017014&req=5

Fig2: Treatment of Olarparib, Ku-55933 or their combination induced γ-H2AX foci formation A, B, CAL-51 cells (a) and MCF-7 cells (b) were treated with 0, 5, 10 μM Olaparib with or wihout 10 μM KU55933 respectively for 48 h and analyzed by immunofluorescence (IF) with γH2AX antibody. DAPI was used to visualize cell nuclei. Representative pictures (left) and quantitative data (right) of the γ-H2AX foci. c, CAL-51 cells (left) and MCF-7 cells (right) were treated with 0, 10 μM Olaparib with or wihout 10 μM KU55933 respectively 48 h and analyzed by Western Blot assay (WB) with γH2AX antibody. All experiments were performed at least three times and data were statistically analyzed by two-tail t-test. ***p < 0.001. Error bars indicate S.E.M
Mentions: An early cellular response to DSBs is rapid phosphorylation of H2AX at Ser139, forming γ-H2AX foci [26]. H2AX phosphorylation plays a key role in signalling and initiating the repair of DSBs, and the numbers of γ-H2AX foci correlate closely with the number of DSBs [27]. Treating breast cancer cell lines with both Olaparib and KU55933 led to significantly more γ-H2AX foci formation than treating them with either inhibitor alone based on fluorescence microscopy (Fig. 2a-b). These findings were further confirmed by Western Blot assay (Fig. 2c). This provides further evidence that that Olaparib and Ku-55933 synthetically induce cellular DSBs.Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

Background: Mutations in DNA damage response factors BRCA1 and BRCA2 confer sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors in breast and ovarian cancers. BRCA1/BRCA2-defective tumors can exhibit resistance to PARP inhibitors via multiple mechanisms, one of which involves loss of 53BP1. Deficiency in the DNA damage response factor ataxia-telangiectasia mutated (ATM) can also sensitize tumors to PARP inhibitors, raising the question of whether the presence or absence of 53BP1 can predict sensitivity of ATM-deficient breast cancer to these inhibitors.

Methods: Cytotoxicity of PARP inhibitor and ATM inhibitor in breast cancer cell lines was assessed by MTS, colony formation and apoptosis assays. ShRNA lentiviral vectors were used to knockdown 53BP1 expression in breast cancer cell lines. Phospho-ATM and 53BP1 protein expressions were determined in human breast cancer tissues by immunohistochemistry (IHC).

Results: We show that inhibiting ATM increased cytotoxicity of PARP inhibitor in triple-negative and non-triple-negative breast cancer cell lines, and depleting the cells of 53BP1 reduced this cytotoxicity. Inhibiting ATM abrogated homologous recombination induced by PARP inhibitor, and down-regulating 53BP1 partially reversed this effect. Further, overall survival was significantly better in triple-negative breast cancer patients with lower levels of phospho-ATM and tended to be better in patients with negative 53BP1.

Conclusion: These results suggest that 53BP1 may be a predictor of PARP inhibitor resistance in patients with ATM-deficient tumors.

Electronic supplementary material: The online version of this article (doi:10.1186/s12885-016-2754-7) contains supplementary material, which is available to authorized users.

No MeSH data available.