BUB1 regulates non-homologous end joining pathway to mediate radioresistance in triple-negative breast cancer

Abstract

Background: Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer subtype often treated with radiotherapy (RT). Due to its intrinsic heterogeneity and lack of effective targets, it is crucial to identify novel molecular targets that would increase RT efficacy. Here we demonstrate the role of BUB1 (cell cycle Ser/Thr kinase) in TNBC radioresistance and offer a novel strategy to improve TNBC treatment.

Methods: Gene expression analysis was performed to look at genes upregulated in TNBC patient samples compared to other subtypes. Cell proliferation and clonogenic survivals assays determined the IC₅₀ of BUB1 inhibitor (BAY1816032) and radiation enhancement ratio (rER) with pharmacologic and genomic BUB1 inhibition. Mammary fat pad xenografts experiments were performed in CB17/SCID. The mechanism through which BUB1 inhibitor sensitizes TNBC cells to radiotherapy was delineated by γ-H2AX foci assays, BLRR, Immunoblotting, qPCR, CHX chase, and cell fractionation assays.

Results: BUB1 is overexpressed in BC and its expression is considerably elevated in TNBC with poor survival outcomes. Pharmacological or genomic ablation of BUB1 sensitized multiple TNBC cell lines to cell killing by radiation, although breast epithelial cells showed no radiosensitization with BUB1 inhibition. Kinase function of BUB1 is mainly accountable for this radiosensitization phenotype. BUB1 ablation also led to radiosensitization in TNBC tumor xenografts with significantly increased tumor growth delay and overall survival. Mechanistically, BUB1 ablation inhibited the repair of radiation-induced DNA double strand breaks (DSBs). BUB1 ablation stabilized phospho-DNAPKcs (S2056) following RT such that half-lives could not be estimated. In contrast, RT alone caused BUB1 stabilization, but pre-treatment with BUB1 inhibitor prevented stabilization (t_1/2, ~8 h). Nuclear and chromatin-enriched fractionations illustrated an increase in recruitment of phospho- and total-DNAPK, and KAP1 to chromatin indicating that BUB1 is indispensable in the activation and recruitment of non-homologous end joining (NHEJ) proteins to DSBs. Additionally, BUB1 staining of TNBC tissue microarrays demonstrated significant correlation of BUB1 protein expression with tumor grade.

Conclusions: BUB1 ablation sensitizes TNBC cell lines and xenografts to RT and BUB1 mediated radiosensitization may occur through NHEJ. Together, these results highlight BUB1 as a novel molecular target for radiosensitization in women with TNBC.

Keywords: BUB1; DNA damage response; DNAPK; NHEJ; Radiation sensitization; TNBC.

Fig. 1

BUB1 is highly expressed in breast cancer compared to normal, non-malignant breast tissue and is associated with triple-negative and basal-like breast cancers. A-B, BUB1 expression is significantly increased in breast tumors compared to normal breast tissue. C, BUB1 expression is strongly associated with the PAM50-defined basal-like subtype of breast cancer and (D), is also significantly elevated in TNBC. E, BUB1 expression is significantly increased in basal-like breast cancer cell lines. F, BUB1 expression strongly correlates with metastatic potential to the lungs and liver as measured by CAM assay in vivo. All CAM assays performed at least in triplicate. G, Kaplan-Meier survival plot demonstrate that high BUB1 levels are associated with worse overall survival in breast cancer patients (data from Hatzis et al, JAMA 2011). H, On multivariable analysis, BUB1 expression discriminates overall survival with high sensitivity and specificity (AUC: 0.68, <0.01). I, Raw data that was used for the analysis of the receiver operating characteristic curve (ROC). z statistic 3.631, ***P= 0.0003, ^aDeLong et al., 1988 [31], ^bBinomial exact

Fig. 2

Effect of BUB1 inhibitor on cell proliferation in TNBC cell lines. BAY1816032 is cytotoxic to cells at low micromolar range (A) SUM159, IC₅₀: 2.90 μM; (B) MDA-MB-231, IC₅₀: 2.10 μM; (C) MDA-MB-468, IC₅₀: 2.59 μM; (D) BT-549 IC₅₀: 1.59 μM; (E) T-47D, IC₅₀: 3.9μM; (F) MCF10A, IC₅₀: 18 μM. G BUB1 protein expression in cell lines by immunoblotting; gray scale values of BUB1 are normalized over Actin for each cell line. Pharmacological inhibition of BUB1 induces radiosensitivity in TNBC cell lines: (H) SUM159, (I) MDA-MB-231, (J) MDA-MB-468, (K) BT-549, (L) T-47D, and (M) MCF10A. P-values were defined as * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001

Fig. 3

Effect of BUB1 genomic depletion on cell survival and radiation sensitivity. Transient transfection of BUB1 siRNA (20, 60 and 100 nM) or control siRNA (100 nM) measured cell viability using alamarBlue assay in (A) SUM159, (B) MDA-MB-231, (C) MDA-MB-468, (D) BT-549, and (E) T-47D. Effect of siRNA-mediated BUB1 depletion on radiosensitization was measured in these cell lines. Transient BUB1 siRNA transfection led to moderate radiosensitization with rER 1.0 to 1.2 in (F) SUM159, and (G) MDA-MB-231; After silencing of BUB1, BUB1-WT re-expression rescues the radiosensitization phenotype while BUB1-KD does not in (H) SUM159 and (I) MDA-MB-231. Genomic depletion of BUB1 by CRISPR/Cas9 leads to radiosensitization in (J) SUM159 and (K) MDA-MB-231 cells; Re-expression of BUB1-WT rescues the radiosensitization phenotype in BUB1 CRISPR KO (L) SUM159 and (M) MDA-MB-231 cells but BUB1-KD does not in. P-values were defined as * P≤0.05, ** P≤0.01, and *** P≤0.001

Fig. 4

BUB1 inhibition sensitizes SUM159 tumor xenografts to radiation (A) Timeline of the experiment; (B) Representative images of tumor growth in different treatment groups; (C) Combination treatment of BAY1816032 + RT reduces tumor volume in vivo; (D, E) Combination treatment increases tumor volume doubling time in Fox Chase SCID mice; Representative images of (F) H&E staining showing structural changes and Ki67 staining (a proliferation marker) revealed a significant reduction in combination treatment of SUM159 xenografts; (G) Ki-67 plot showing decrease in % of positive cells in combination treatment of BUB1i + RT. P-value was defined as **** P≤0.0001

Fig. 5

Tumor xenograft of BUB1 CRISPR KO SUM159 cells are sensitive to irradiation. A Timeline of the experiment; (B) Representative images of tumor growth in different treatment groups; (C) Treatment of BUB1 KO + RT reduces tumor volume in vivo; (D, E) Treatment of BUB1 KO + RT increases tumor volume doubling time in Fox Chase SCID mice. P-value was defined as * P≤0.05

Fig. 6

BUB1 ablation radiosensitize through NHEJ. Representative images of (A) SUM159 and (C) MDA-MB-231 γH2AX foci at 16 h. Original magnification, ×63; Combination treatment of BUB1i and RT leads to delayed resolution of γH2AX foci in (B) SUM159, and (D) MDA-MB-231 cell lines. Inhibition of BUB1 kinase function by BAY1816032, at 1 μM and 10 μM, decreases NHEJ efficiency (V Luc) and increases HR efficiency (G Luc) in (E) SUM159, and (F) MDA-MB-231. Effect of DNAPK inhibitor (NU7441) on cell proliferation in TNBC cell lines. NU7441 is cytotoxic to cells at low nanomolar range (G) SUM159, IC₅₀: 368 nM; (H) MDA-MB-231, IC₅₀: 503 nM; Combination of BAY1816032 and NU7441 does not increase DNAPKcs-mediated radiosensitization in (I) SUM159 (J) MDA-MB-231 cell lines. Inhibition of BUB1 increased transcription of DNA damage genes after radiation. Significant upregulation of H2AFX and downregulation of PRKDC levels in (K) SUM159 and (L) SUM159 BUB1 CRISPR KO cells were observed. P-values were defined as * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001

Fig. 7

BUB1 ablation leads to increased phosphorylation of DNAPKcs, alters chromatin localization of key NHEJ factors and induces apoptotic cell death upon irradiation. A MDA-MB-231 cells were treated with BUB1i or DNAPKi an hour prior to radiation treatment. Cells were harvested 30 minutes post RT (4Gy) and resolved on SDS-PAGE gels and probed with indicated antibodies. B MDA-MB-231 cells were treated as (A) and harvested at 1-, 15-, 30- and 120-minutes post-RT and immunoblotted as specified. C SUM159 (top panel) MDA-MB-231 cells (bottom panel) were treated with cycloheximide followed by BUB1i or DNAPKi and radiation (4Gy). Total protein lysates were made at the indicated time-points and resolved on gels. D BUB1 CRISPR KO SUM159 (left panel) or MDA-MB-231 (right panel) cells were treated with cycloheximide, and radiation and samples were harvested at different time points. E Quantitation of pDNAPKcs protein levels in SUM159 and MDA-MB-231 cells (from 7C and other experiments). F Quantitation of BUB1 protein levels in SUM159 and MDA-MB-231 cells (from above experiments). G Nuclear and chromatin fractions of SUM159 and MDA-MB-231 cells treated with BUB1i, DNAPKi and RT (left) and BUB1 CRISPR KO SUM159 and MDA-MB-231 cells treated with RT (right panels). H Effect of BUB1 inhibitor (red circles) on initial recruitment of YFP-tagged KU80 and YFP-DNAPKcs by laser microirradiation in U2OS cells. I effect of BUB1 inhibition on the accumulation of YFP-KU80 and YFP-DNAPKcs at laser-induced DSBs for up to 120 minutes. J QRT-PCR of BAX, BCL2, PCNA, CASP3 and CASP9 in SUM159 cells treated with BUB1i, DNAPKi and radiation (4 Gy, 72 hours). (I) QRT-PCR of BAX, BCL2, PCNA, CASP3 and CASP9 in SUM159 BUB1 CRISPR cells 72 hours post-irradiation (4 Gy). P-values were defined as * P≤0.05, ** P≤0.01, and *** P≤0.001

Fig. 8

BUB1 is overexpressed in breast tumors. A Representative images of BUB1 staining intensity at 4x and 20x magnifications in breast TMA. B Quantification of BUB1 staining in breast tumor TMA. C Proposed model for a role of BUB1 in mediating radiation induced NHEJ signaling. We propose that radiation induced DNA DSB are repaired efficiently when BUB1 is present (left panel) leading to radiation resistance. In the absence of BUB1 activity or availability, radiation induces hyper phosphorylation of DNAPKcs (Ser2056) and increased binding of NHEJ mediators at the DNA DSB sites (right panel). These NHEJ mediators may not stay on the extended chromatin thus hamper end processing causing radiation-sensitization. P-values were defined as * P≤0.05, ** P≤0.01, *** P≤0.001, and **** P≤0.0001