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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.


KU55933 reduced Olaparib induced ATM-Chk2 pathway activation and Rad51 up-regulation while knockdown 53BP1 supressed these effects. a, Western blot assay showed the protein level of Rad51, Chk2, phospho-Chk2 (Thr68), ATM and phosphor-ATM (Ser1981) in CAL-51 and MCF-7 cells after treatment with 10 μM Olaparib, 10 μM KU55933 and their combination respectively for 48 h. b, Western Blot assay showed the protein level of Rad51, Chk2, phospho-Chk2 (Thr68), ATM and phosphor-ATM (Ser1981) in CAL-51-sh53BP1, MCF-7-sh53BP1 cells and their control-transfected cells after synergistic treatment with 10 μM Olaparib and 10 μM KU55933. All experiments were performed at least three times
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Fig5: KU55933 reduced Olaparib induced ATM-Chk2 pathway activation and Rad51 up-regulation while knockdown 53BP1 supressed these effects. a, Western blot assay showed the protein level of Rad51, Chk2, phospho-Chk2 (Thr68), ATM and phosphor-ATM (Ser1981) in CAL-51 and MCF-7 cells after treatment with 10 μM Olaparib, 10 μM KU55933 and their combination respectively for 48 h. b, Western Blot assay showed the protein level of Rad51, Chk2, phospho-Chk2 (Thr68), ATM and phosphor-ATM (Ser1981) in CAL-51-sh53BP1, MCF-7-sh53BP1 cells and their control-transfected cells after synergistic treatment with 10 μM Olaparib and 10 μM KU55933. All experiments were performed at least three times

Mentions: It is well accepted that ATM, Chk2 and Rad51 are critical regulators of HR DNA repair pathway. We sought to investigate the protein levels of these factors under the treatment with Olaparib, KU55933 or both. As is shown in Fig. 5a, Olaparib induced ATM phosphorylation and activated its downstream effector Chk2, and subsequently up-regulated Rad51. Adding KU55933 inhibited the activation of ATM and Chk2 and down-regulated Rad51. Taken together, these observations suggest that Olaparib activated the HR pathway and adding KU55933 inhibited it.Fig. 5


53BP1 depletion causes PARP inhibitor resistance in ATM-deficient breast cancer cells
KU55933 reduced Olaparib induced ATM-Chk2 pathway activation and Rad51 up-regulation while knockdown 53BP1 supressed these effects. a, Western blot assay showed the protein level of Rad51, Chk2, phospho-Chk2 (Thr68), ATM and phosphor-ATM (Ser1981) in CAL-51 and MCF-7 cells after treatment with 10 μM Olaparib, 10 μM KU55933 and their combination respectively for 48 h. b, Western Blot assay showed the protein level of Rad51, Chk2, phospho-Chk2 (Thr68), ATM and phosphor-ATM (Ser1981) in CAL-51-sh53BP1, MCF-7-sh53BP1 cells and their control-transfected cells after synergistic treatment with 10 μM Olaparib and 10 μM KU55933. All experiments were performed at least three times
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: KU55933 reduced Olaparib induced ATM-Chk2 pathway activation and Rad51 up-regulation while knockdown 53BP1 supressed these effects. a, Western blot assay showed the protein level of Rad51, Chk2, phospho-Chk2 (Thr68), ATM and phosphor-ATM (Ser1981) in CAL-51 and MCF-7 cells after treatment with 10 μM Olaparib, 10 μM KU55933 and their combination respectively for 48 h. b, Western Blot assay showed the protein level of Rad51, Chk2, phospho-Chk2 (Thr68), ATM and phosphor-ATM (Ser1981) in CAL-51-sh53BP1, MCF-7-sh53BP1 cells and their control-transfected cells after synergistic treatment with 10 μM Olaparib and 10 μM KU55933. All experiments were performed at least three times
Mentions: It is well accepted that ATM, Chk2 and Rad51 are critical regulators of HR DNA repair pathway. We sought to investigate the protein levels of these factors under the treatment with Olaparib, KU55933 or both. As is shown in Fig. 5a, Olaparib induced ATM phosphorylation and activated its downstream effector Chk2, and subsequently up-regulated Rad51. Adding KU55933 inhibited the activation of ATM and Chk2 and down-regulated Rad51. Taken together, these observations suggest that Olaparib activated the HR pathway and adding KU55933 inhibited it.Fig. 5

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.