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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.


Related in: MedlinePlus

ATM inhibition enhanced Olaparib induced apoptosis in MCF-7 and CAL-51 cells a, b, DAPI staining indicated the nuclear morphological changes of CAL-51 cells (a) and MCF-7 cells (b). Cells were treated with 0, 10 μM Olaparib with or wihout 10 μM KU55933 respectively for 48 h. Left, representative pictures; right, quantitative data. c, d, Annexin V/PI staining and FACS analysis revealed cell apoptosis of CAL-51 cells (c) and MCF-7 cells (d). Cells were treated with 0, 10 μM Olaparib with or wihout 10 μM KU55933 respectively for 48 h. Left, representative pictures; right, quantitative data. e, Western Blots showed that caspase 3, caspase 8, caspase 9 and their cleaved forms were increased after treatment with 10 μM Olaparib or 10 μM KU55933 or their combination in CAL-51 cells and MCF-7 cells. All experiments were performed at least three times and data were statistically analyzed by two-tail t-test.*p < 0.05, **p < 0.01. Error bars indicate S.E.M
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Fig3: ATM inhibition enhanced Olaparib induced apoptosis in MCF-7 and CAL-51 cells a, b, DAPI staining indicated the nuclear morphological changes of CAL-51 cells (a) and MCF-7 cells (b). Cells were treated with 0, 10 μM Olaparib with or wihout 10 μM KU55933 respectively for 48 h. Left, representative pictures; right, quantitative data. c, d, Annexin V/PI staining and FACS analysis revealed cell apoptosis of CAL-51 cells (c) and MCF-7 cells (d). Cells were treated with 0, 10 μM Olaparib with or wihout 10 μM KU55933 respectively for 48 h. Left, representative pictures; right, quantitative data. e, Western Blots showed that caspase 3, caspase 8, caspase 9 and their cleaved forms were increased after treatment with 10 μM Olaparib or 10 μM KU55933 or their combination in CAL-51 cells and MCF-7 cells. All experiments were performed at least three times and data were statistically analyzed by two-tail t-test.*p < 0.05, **p < 0.01. Error bars indicate S.E.M

Mentions: Since the experiments above indicated that Olaparib and KU55933 increased DSB formation, we wanted to know whether they induced changes in nuclear morphology. Such changes could reflect DSB-induced chromosome rearrangements or mutations, which can lead to apoptosis or death [23]. Treating MCF-7 and CAL-51 cells for 48 h with 10 μM Olaparib, 10 μM KU55933 or both led to nuclei with chromatin condensation and apoptotic bodies detectable by DAPI fluorescence (Fig. 3a-b), with the combination treatment causing more severe changes in nuclear morphology than either inhibitor alone. Similarly, combination treatment caused significantly greater apoptosis than Olaparib alone, based on Annexin V/PI staining followed by fluorescence-activated cell sorting (Fig. 3c-d). In addition, Western blotting experiments showed that combination treatment, as well as KU55933 treatment on its own, up-regulated expression of apoptotic caspases 3, 8, and 9, and their cleaved forms and it stimulated PARP-1 cleavage (Fig. 3e). These results suggest that Olarparib and KU55933 activate apoptosis pathways.Fig. 3


53BP1 depletion causes PARP inhibitor resistance in ATM-deficient breast cancer cells
ATM inhibition enhanced Olaparib induced apoptosis in MCF-7 and CAL-51 cells a, b, DAPI staining indicated the nuclear morphological changes of CAL-51 cells (a) and MCF-7 cells (b). Cells were treated with 0, 10 μM Olaparib with or wihout 10 μM KU55933 respectively for 48 h. Left, representative pictures; right, quantitative data. c, d, Annexin V/PI staining and FACS analysis revealed cell apoptosis of CAL-51 cells (c) and MCF-7 cells (d). Cells were treated with 0, 10 μM Olaparib with or wihout 10 μM KU55933 respectively for 48 h. Left, representative pictures; right, quantitative data. e, Western Blots showed that caspase 3, caspase 8, caspase 9 and their cleaved forms were increased after treatment with 10 μM Olaparib or 10 μM KU55933 or their combination in CAL-51 cells and MCF-7 cells. All experiments were performed at least three times and data were statistically analyzed by two-tail t-test.*p < 0.05, **p < 0.01. 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

Fig3: ATM inhibition enhanced Olaparib induced apoptosis in MCF-7 and CAL-51 cells a, b, DAPI staining indicated the nuclear morphological changes of CAL-51 cells (a) and MCF-7 cells (b). Cells were treated with 0, 10 μM Olaparib with or wihout 10 μM KU55933 respectively for 48 h. Left, representative pictures; right, quantitative data. c, d, Annexin V/PI staining and FACS analysis revealed cell apoptosis of CAL-51 cells (c) and MCF-7 cells (d). Cells were treated with 0, 10 μM Olaparib with or wihout 10 μM KU55933 respectively for 48 h. Left, representative pictures; right, quantitative data. e, Western Blots showed that caspase 3, caspase 8, caspase 9 and their cleaved forms were increased after treatment with 10 μM Olaparib or 10 μM KU55933 or their combination in CAL-51 cells and MCF-7 cells. All experiments were performed at least three times and data were statistically analyzed by two-tail t-test.*p < 0.05, **p < 0.01. Error bars indicate S.E.M
Mentions: Since the experiments above indicated that Olaparib and KU55933 increased DSB formation, we wanted to know whether they induced changes in nuclear morphology. Such changes could reflect DSB-induced chromosome rearrangements or mutations, which can lead to apoptosis or death [23]. Treating MCF-7 and CAL-51 cells for 48 h with 10 μM Olaparib, 10 μM KU55933 or both led to nuclei with chromatin condensation and apoptotic bodies detectable by DAPI fluorescence (Fig. 3a-b), with the combination treatment causing more severe changes in nuclear morphology than either inhibitor alone. Similarly, combination treatment caused significantly greater apoptosis than Olaparib alone, based on Annexin V/PI staining followed by fluorescence-activated cell sorting (Fig. 3c-d). In addition, Western blotting experiments showed that combination treatment, as well as KU55933 treatment on its own, up-regulated expression of apoptotic caspases 3, 8, and 9, and their cleaved forms and it stimulated PARP-1 cleavage (Fig. 3e). These results suggest that Olarparib and KU55933 activate apoptosis pathways.Fig. 3

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.


Related in: MedlinePlus