53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers

Abstract

Germ-line mutations in breast cancer 1, early onset (BRCA1) result in predisposition to breast and ovarian cancer. BRCA1-mutated tumors show genomic instability, mainly as a consequence of impaired recombinatorial DNA repair. Here we identify p53-binding protein 1 (53BP1) as an essential factor for sustaining the growth arrest induced by Brca1 deletion. Depletion of 53BP1 abrogates the ATM-dependent checkpoint response and G2 cell-cycle arrest triggered by the accumulation of DNA breaks in Brca1-deleted cells. This effect of 53BP1 is specific to BRCA1 function, as 53BP1 depletion did not alleviate proliferation arrest or checkpoint responses in Brca2-deleted cells. Notably, loss of 53BP1 partially restores the homologous-recombination defect of Brca1-deleted cells and reverts their hypersensitivity to DNA-damaging agents. We find reduced 53BP1 expression in subsets of sporadic triple-negative and BRCA-associated breast cancers, indicating the potential clinical implications of our findings.

Figure 1

Inactivation of 53BP1 rescues proliferation defects and drug sensitivity of Brca1 null ES cells. (a) Schematic overview of mutant alleles in R26^CreERT2;Brca1^SCo/Δ and R26^CreERT2;Brca1^Δ/Δ ES cells. Before 4-hydroxytamoxifen (4OHT) mediated induction of the CreERT2 recombinase, R26^CreERT2;Brca1^SCo/Δ cells are BRCA1 proficient and puromycin sensitive. Addition of 4OHT leads to CreERT2-mediated deletion of Brca1 exons 5–6, resulting in BRCA1 inactivation and concomitant expression of puromycin from the PGK promoter, thereby enabling selection of BRCA1-deficient R26^CreERT2;Brca1^Δ/Δ ES cells. (b) Western blot analysis of 53BP1 expression in R26^CreERT2;Brca1^SCo/Δ ES cells non transduced or transduced with two independent lentiviral shRNA vectors against 53bp1, after treatment with 4OHT to delete the Brca1^SCo allele. (c) Crystal violet staining of untransduced R26^CreERT2;Brca1^SCo/Δ ES cells treated with 4OHT and stably transduced with lentiviral vectors expressing a control non-targeting shRNA (NT) or two independent shRNAs against 53bp1. (d–e) Susceptibility of R26^CreERT2;Brca1^SCo/Δ ES cells untreated or treated with 4OHT to DNA cross-linking agents cisplatin (d) or mitomycin C (e). Cell viability was measured after 4 days. Mean ± s.d. is shown from three independent measurements.

Figure 1

Inactivation of 53BP1 rescues proliferation defects and drug sensitivity of Brca1 null ES cells. (a) Schematic overview of mutant alleles in R26^CreERT2;Brca1^SCo/Δ and R26^CreERT2;Brca1^Δ/Δ ES cells. Before 4-hydroxytamoxifen (4OHT) mediated induction of the CreERT2 recombinase, R26^CreERT2;Brca1^SCo/Δ cells are BRCA1 proficient and puromycin sensitive. Addition of 4OHT leads to CreERT2-mediated deletion of Brca1 exons 5–6, resulting in BRCA1 inactivation and concomitant expression of puromycin from the PGK promoter, thereby enabling selection of BRCA1-deficient R26^CreERT2;Brca1^Δ/Δ ES cells. (b) Western blot analysis of 53BP1 expression in R26^CreERT2;Brca1^SCo/Δ ES cells non transduced or transduced with two independent lentiviral shRNA vectors against 53bp1, after treatment with 4OHT to delete the Brca1^SCo allele. (c) Crystal violet staining of untransduced R26^CreERT2;Brca1^SCo/Δ ES cells treated with 4OHT and stably transduced with lentiviral vectors expressing a control non-targeting shRNA (NT) or two independent shRNAs against 53bp1. (d–e) Susceptibility of R26^CreERT2;Brca1^SCo/Δ ES cells untreated or treated with 4OHT to DNA cross-linking agents cisplatin (d) or mitomycin C (e). Cell viability was measured after 4 days. Mean ± s.d. is shown from three independent measurements.

Figure 2

53BP1 depletion rescues cell cycle defects of Brca1 null ES cells. (a) Western blot analysis of 53BP1 and p53 expression in R26^CreERT2;Brca1^SCo/Δ ES cells stably tranduced with lentiviral shRNA vectors against 53bp1 or p53. Samples were taken before or at 4 and 9 days after 4-hydroxytamoxifen (4OHT) induced deletion of Brca1. (b) Flow cytometry profiles of R26^CreERT2;Brca1^SCo/Δ ES cells stably transduced with non-targeting shRNA lentiviruses or shRNA vectors against 53bp1 and p53. Shown are percentages of cells in G1 and G2 before or nine days after 4OHT-induced Brca1 deletion by CreERT2. Mean ± s.d. is shown from two experiments.

Figure 3

53BP1 depletion abrogates CHK2-mediated DNA damage responses and rescues proliferation defects in Brca1 null but not Brca2 null MEFs. (a) Western blot analysis of cell extracts from Brca1^SCo/Δ MEFs infected with retroviruses expressing self-deleting Cre recombinase (+Cre) or empty vector (−Cre), together with retroviruses expressing 53BP1 or GFP control shRNAs. SMC1 and tubulin were used as loading controls. NSB: non-specific band. (b) Quantification of chromatid and chromosome break frequency in metaphase spreads prepared from cells treated as in (a). At least 100 metaphases were scored for each sample. Shown are average number of events per metaphase ± s.d. (c) Proliferation curves of Brca1^SCo/Δ or Brca2^F/Δ MEFs infected with retroviruses expressing self-deleting Cre recombinase (+Cre) or empty vector (−Cre), together with retroviruses expressing p53, 53BP1 or GFP control shRNAs. (d) Western blot analysis of cell extracts from Brca2^F/Δ MEFs treated as in (c). Tubulin was used as a loading control.

Figure 3

53BP1 depletion abrogates CHK2-mediated DNA damage responses and rescues proliferation defects in Brca1 null but not Brca2 null MEFs. (a) Western blot analysis of cell extracts from Brca1^SCo/Δ MEFs infected with retroviruses expressing self-deleting Cre recombinase (+Cre) or empty vector (−Cre), together with retroviruses expressing 53BP1 or GFP control shRNAs. SMC1 and tubulin were used as loading controls. NSB: non-specific band. (b) Quantification of chromatid and chromosome break frequency in metaphase spreads prepared from cells treated as in (a). At least 100 metaphases were scored for each sample. Shown are average number of events per metaphase ± s.d. (c) Proliferation curves of Brca1^SCo/Δ or Brca2^F/Δ MEFs infected with retroviruses expressing self-deleting Cre recombinase (+Cre) or empty vector (−Cre), together with retroviruses expressing p53, 53BP1 or GFP control shRNAs. (d) Western blot analysis of cell extracts from Brca2^F/Δ MEFs treated as in (c). Tubulin was used as a loading control.

Figure 4

53BP1 depletion rescues RAD51 foci in Brca1 null cells. (a) RAD51 foci formation in R26^CreERT2;Brca1^SCo/Δ ES cells untreated or treated with 4OHT and transduced with lentiviruses expressing non-targeting shRNAs (NTsh) or shRNAs targeting 53bp1(53BP1sh). Cells were irradiated with 10 Gy, fixed after 6 hours, and stained with anti-RAD51 antibody (red). Nuclei were visualized with DAPI (blue). (b) RAD51 foci formation in Brca1^SCo/Δ MEFs infected with retroviruses expressing self-deleting Cre recombinase (+Cre) or empty vector (−Cre), together with retroviruses expressing 53BP1 or GFP control shRNAs. Cells were irradiated with 10 Gy, fixed after 2 hours, and stained with anti-γH2AX (red) and anti-RAD51 antibodies (green). Nuclei were visualized with DAPI (blue). (c) Quantification of RAD51 foci in R26^CreERT2;Brca1^SCo/Δ ES cells treated as in (a). The percentage of cells with ≥10 Rad51 foci per cell are plotted for each condition. At least 20 nuclei were analyzed for each treatment. (d) Quantification of RAD51 foci in Brca1^SCo/Δ MEFs treated as in (b). The percentage of cells with ≥10 RAD51 foci per cell are plotted for each condition. At least 50 nuclei were analyzed for each treatment.

Figure 4

53BP1 depletion rescues RAD51 foci in Brca1 null cells. (a) RAD51 foci formation in R26^CreERT2;Brca1^SCo/Δ ES cells untreated or treated with 4OHT and transduced with lentiviruses expressing non-targeting shRNAs (NTsh) or shRNAs targeting 53bp1(53BP1sh). Cells were irradiated with 10 Gy, fixed after 6 hours, and stained with anti-RAD51 antibody (red). Nuclei were visualized with DAPI (blue). (b) RAD51 foci formation in Brca1^SCo/Δ MEFs infected with retroviruses expressing self-deleting Cre recombinase (+Cre) or empty vector (−Cre), together with retroviruses expressing 53BP1 or GFP control shRNAs. Cells were irradiated with 10 Gy, fixed after 2 hours, and stained with anti-γH2AX (red) and anti-RAD51 antibodies (green). Nuclei were visualized with DAPI (blue). (c) Quantification of RAD51 foci in R26^CreERT2;Brca1^SCo/Δ ES cells treated as in (a). The percentage of cells with ≥10 Rad51 foci per cell are plotted for each condition. At least 20 nuclei were analyzed for each treatment. (d) Quantification of RAD51 foci in Brca1^SCo/Δ MEFs treated as in (b). The percentage of cells with ≥10 RAD51 foci per cell are plotted for each condition. At least 50 nuclei were analyzed for each treatment.

Figure 5

53BP1 expression is reduced in a subset of human BLBC. (a) Boxplots showing 53BP1 expression levels among breast cancer subtypes. Gene expression array data from 286 early stage breast cancers published by Wang et al. were clustered to classify tumors into basal (BA), HER2-positive (HER2), Luminal A (LA) and Luminal B (LB). The mean expression values of 53BP1 are shown for each subgroup. (b) Boxplots showing 53BP1 expression levels among different breast cancer subtypes defined by robust consensus clustering: normal (NM), basal (BA₁, BA₂), HER2-positive (HER2_I, HER2_NI), and luminal (LA, LB₁, LB₂, LB₃). The data are normalized with mean expression of the combined data being set to 0. Expression of 53BP1 is significantly lower in the BA₁ subtype (p<0.0001 vs. Normal; p<0.0002 vs. BA₂). (c-d) Distant relapse-free survival stratified by 53BP1 protein expression in all breast cancers (c) and in triple-negative (TN) breast cancers (d). Kaplan-Meyer survival curves for distant relapse-free survival are shown for breast cancers that scored positive for 53BP1 staining (black lines) and those that scored negative for 53BP1 staining (red lines).