MCF7 (cat. no. HTB-22™) and Vero cells (cat. no. CCL-81™) were cultured at 37°C in a humidified incubator with 5% CO₂ in Eagle's Minimum Essential Medium (cat. no. 30-2003) with 10% FBS for <4 weeks. T47D (cat. no. HTB-133™) cells were cultured at 37°C in a humidified incubator with 5% CO₂ in RPMI-1640 (cat. no. 30-2001) with 10% FBS (all from American Tissue Culture Collection) for <4 weeks.

Doxorubicin-resistant (DR) MCF7 cells were generated by incubating MCF7 cells in medium with 0.25 µM doxorubicin for 2 weeks, followed by 0.5 and 1 µM doxorubicin for 2 weeks at each concentration. IR-resistant (IRR) MCF7 cells were generated by treating MCF7 cells with 1-Gy IR twice in one week, followed by exposure to 2-Gy IR twice a week for two weeks.

MV-Edm was harvested as previously described (23). Attenuated measles virus vaccines were obtained from National Biotec Group Co., Ltd., China. The virus was propagated in Vero cells (cat. no. CCL-81™; ATCC) with an MOI 0.01 at 37°C for 2 h. The medium was then replaced, and the cells were maintained at 32°C for virus propagation. At 3 days post-infection, the infected cells were scraped into 1 ml Opti-MEM (cat. no. 51985091; Thermo Fisher Scientific, Inc.). The viral particles were harvested from Vero cells by snap-freezing in liquid nitrogen and thawing in a water bath at 37°C for 5 cycles. A 50% tissue culture infective dose titer was calculated from the 50% endpoint dilution assay in Vero cells (24). Viral infection was performed as previously described (25). Briefly, MCF7 and T47D cells were seeded at a density of 3×10⁵ cells/well in triplicate in 6-well plates with 2 ml complete media. After 24 h, the cells were incubated with 0–0.5 MOI virus for 24 h at 37°C. The cells were trypsinized and harvested for further experiments.

MCF7 and T47D cells were seeded at a density of 5×10³ cells/well in triplicate in 96-well plates with 100 µl complete media. After 24 h at 37°C, the cells were treated with doxorubicin for 48 h at 37°C, and the cell viability was determined using MTT assay. Briefly, 10 µl MTT (Sigma-Aldrich; Merck KGaA) solution was added to each well, and the plates were incubated for 3 h at 37°C. DMSO was subsequently added to each well (100 µl/well) to dissolve the formazan crystals, and the absorbance was measured at 490 nm using an Epoch™ 2 Microplate spectrophotometer (BioTek Instruments, Inc.).

A total of 10 µg of NHEJ or HR reporter plasmid was linearized using NheI and purified using the QIAquick Gel Extraction kit (cat. no. 28704; Qiagen, Inc.) according to the manufacturer's instructions. The purified plasmid (1 µg) was transfected into MCF7 and T47D cells (3×10⁵ cells/ml; 2 ml) using Lipofectamine^® 3,000 (cat. no. L3000015; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol and incubated at 37°C for 48 h. Stably transfected cells were selected by incubating in medium with 1 mg/ml geneticin for 2 weeks and subsequently stored in liquid nitrogen until further use. To measure NHEJ efficiency, stably transfected cells were seeded at 3×10⁵ cells/ml in a 6-well plate and cultured for 24 h. For the NHEJ or HR assay, 2 µg I-SceI plasmid (cat. no. 26477; Addgene, Inc.) was transfected into MCF7 and T47D cells (3×10⁵ cells/ml; 2 ml) using Lipofectamine^® 3,000 to recognize the I-SceI restriction enzyme site and generate DSB. After incubation at 37°C for 48 h, positive cells expressing green fluorescent protein (GFP), which indicated successful DSB repair, were measured using a FACSCelesta flow cytometer (BD Biosciences) and analyzed using FlowJo software (V.10; FlowJo, LLC).

RNA was extracted from MCF7 and T47D cells using the RNeasy Mini Kit (cat. no. 74104; Qiagen GmbH) and was reverse-transcribed using the iScript™ RT Supermix (Bio-Rad Laboratories, Inc.) according to the manufacturer's instructions. Subsequently the iQ™ SYBR^® Green Supermix (Bio-Rad Laboratories, Inc.) was used for qPCR to detect the mRNA levels of the NHEJ factors according to the manufacturer's instructions using a Real-Time PCR system (Eppendorf Thermal Cycler Eco; Eppendorf) (26). The thermocycling conditions included initial denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 10 sec and annealing and extension at 60°C for 50 sec. The relative expression levels were quantified using the 2^−∆∆Cq method (27).

The following primers were used: β-actin forward, 5′-ACCAACTGGGACGACATGGAG-3′ and reverse, 5′-GTGAGGATCTTCATGAGGTAGTC-3′; 70 kDa subunit of Ku antigen (Ku70) forward, 5′-ATGGCAACTCCAGAGCAGGTG-3′ and reverse, 5′-AGTGCTTGGTGAGGGCTTCCA-3′; 86 kDa subunit of Ku antigen (Ku80) forward, 5′-TGACTTCCTGGATGCACTAATCGT-3′ and reverse, 5′-TTGGAGCCAATGGTCAGTCG-3′; catalytic subunit of a nuclear DNA-dependent serine/threonine protein kinase (DNA-PKcs) forward, 5′-CCAAGTCCAACACCAAGTAGCCACCCA-3′ and reverse, 5′-CCGCCATGCCGCCGAGTCCC-3′; X-ray repair cross-complementing 4 (XRCC4) forward, 5′-CCCTCACAGAAACACAACTCA-3′ and reverse, 5′-CAAGGAGGTGGCCACTAGTT-3; XRCC4-like factor (XLF) forward, 5′-ACAAGGTCTAATGCACCCCA-3′ and reverse, 5′-GGGTTGCAGCCTTAGAAAAGT-3′; DNA ligase IV forward, 5′-CACCTTGCGTTTTCCACGAA-3′ and reverse, 5′-CAGATGCCTTCCCCCTAAGTTG-3′; and p53-binding protein 1 (53BP1) forward, 5′-CCAGCACCAACAAGAGC-3′ and reverse, 5′-GGATGCCTGGTACTGTTTGG-3′.

MCF7 and T47D cells were pelleted and resuspend in hypotonic buffer (20 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂). The cells were lysed by adding 10% NP40 and vortexing. The cell lysate was centrifuged for 15 min at 1,200 × g at 4°C, and the supernatant (cytoplasmic fraction) was removed. The nuclear pellet was lysed in lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 2 mM Na3VO4, 1% Triton X-100, 1 mM EDTA, 0.1% SDS, 0.5% deoxycholate, 1 mM NaF, 10% glycerol) with 1X protease cocktail (Roche Diagnostics) for 30 min on ice with vortexing and centrifuged at 14,000 × g at 4°C, following which the protein concentration of the supernatant (nuclear fraction) was determined using Bradford assay. The samples (20 µg/lane) were separated using SDS-PAGE and transferred to PVDF membranes (Roche Diagnostics). After blocking with 3% BSA in 1X PBS with 0.1% Tween-20, the membrane was incubated with primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The signals were detected using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.), and the proteins were visualized using the ChemiDoc MP imaging system (Bio-Rad Laboratories, Inc.) and analyzed using ImageJ version 1.51 software (National Institutes of Health). The following antibodies (1:1,000 dilution) were used: Anti-Ku70 (cat. no. 4588; Cell Signaling Technology, Inc.), anti-Ku80 (Cell Signaling Technology, Inc.), anti-DNA-PKcs (cat. no. 38168; Cell Signaling Technology, Inc.), anti-XLF (cat. no. 2854; Cell Signaling Technology, Inc.), anti-XRCC4 (cat. no. sc-136124; Santa Cruz Biotechnology, Inc.), anti-DNA ligase IV (cat. no. 14649; Cell Signaling Technology, Inc.), anti-53BP1 (cat. no. 4937; Cell Signaling Technology, Inc.), anti-lamin B1 (cat. no. 65986; Abcam) and anti-origin recognition complex subunit 2 (ORC2; cat. no. 4736; Cell Signaling Technology, Inc.).

DNA damage was induced using 2-Gy of IR or 0.5 µM doxorubicin, and the cells were allowed to recover for 2 h to allow the DNA repair mechanism to assemble. Subsequently, MCF7 cells were lysed in buffer A (50 mM HEPES-KOH, pH 8.0, 150 mM NaCl, 10% glycerol, 3 mM MgCl₂, 1 mM EGTA, 1% NP-40, 1 mM DTT) with 1X protease inhibitor cocktail (Roche Diagnostics) on ice for 20 min and centrifuged at 1,200 × g at 4°C for 3 min. The cell pellets were collected and lysed in buffer B (10 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1 mM EGTA) with 1X protease inhibitor cocktail and centrifuged at 1,200 × g at 4°C for 3 min. The sediment was resuspended in buffer C (50 mM Tris-HCl, pH 8, 20 mM NaCl, 1 mM MgCl₂, 0.1% SDS, 1% NP-40) supplemented with 1X protease inhibitor cocktail and denatured with SDS loading buffer.

A 50% DNA cellulose (Invitrogen; Thermo Fisher Scientific, Inc.) mixture was washed in washing buffer [50 mM HEPES, pH 8, 100 mM potassium acetate, 0.5 mM magnesium acetate, 1 mM ATP, 1 mM DTT, 0.1 mg/ml bovine serum albumin (Sigma-Aldrich; Merck KGaA)] three times. MCF7 and T47D cells (3×10⁵ cells/ml; 2 ml) were cultured in the presence or absence of 0.5 multiplicity of infection (MOI) MV-Edm for 24 h and lysed using RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 25 mM Tris, pH 7.4) supplemented with 1X protease inhibitor cocktail. The 53BP1 protein was immunoprecipitated from the cell extract using an anti-53BP1 antibody (cat. no. 4937; Cell Signaling Technology, Inc.) followed by A/G protein binding beads (Thermo Fisher Scientific, Inc.). A total of 0.1 µg 53BP1 was added to 20 µl of the DNA cellulose mixture. The reactions were rotated at room temperature for 3 h, and the DNA cellulose was centrifuged at 1,000 × g for 1 min and was washed twice with washing buffer. The pellet was resuspended in 20 µl 2X SDS buffer, and DNA-bound 53BP1 was analyzed using western blot analysis. The non-biotinylated DNA-bound 53BP1 was used as negative control.

A pool of 3 plasmids, each encoding the Cas9 coding gene and ligase IV-specific 20 nt guide RNA (cat. no. sc-401372; Santa Cruz Biotechnology, Inc.), targeting the 5′ constitutive exon within the ligase IV gene, was transfected into MCF7-DR and MCF7-IRR cells using Lipofectamine^® 3,000 and selected as previously described (28). Cells were trypsinized and seeded in 96-well plate at densities of 100, 300 and 500 cells/ml and incubated for 14 days at 37°C. Single clones were selected, expanded and screened for DNA ligase IV expression using western blot analysis (29). The CRISPR/Cas9-Ctr plasmid (cat. no. sc-418922; Santa Crus Biotechnology, Inc.), which encoded a single scrambled gRNA sequence, was used as negative control. For each knockout, two clones (KO1 and KO2) were selected for further experiments.

Caspase-3/7 activity was measured using the Caspase-Glo^® 3/7 Assay System according to the manufacturer's protocol (Promega Corporation). MCF7-IRR, MCF7-DR, T47D-IRR and T47D-DR cells (1×10⁴ cells/well in 96-well plate) were treated with MV-Edm for 24 h, followed by exposure to 2-Gy IR or 0.1 µM doxorubicin. Subsequently, 100 µl Caspase-Glo^® 3/7 reagent was added in each well and gently mixed with the cells, and incubated at room temperature for 1.5 h before caspase-3/7 activity was measured using a luminometer (490 nm excitation, 570 nm emission) (FLUOstar Optima).

Data are presented as the mean ± standard error of the mean from three experimental repeats. GraphPad Prism v7.0 software (GraphPad Software, Inc.) was used to plot the graphs and analyze the data. Student's t-test was used for comparisons between two groups. One-way or two-way ANOVA with the Bonferroni post hoc test was used to compare multiple groups. P<0.05 was considered to indicate a statistically significant difference.

To determine the cell viability inhibition induced by MV-Edm, the MCF7 and T47D cell lines were infected with 0, 0.1 and 0.5 MOI MV-Edm. As presented in Fig. 1A and B, the viral infection significantly inhibited the viability of the two breast cancer cell lines compared with that of uninfected cells.

To investigate which DSB repair pathway was affected by MV, MCF7 and T47D cells were treated with MV-Edm and analyzed using NHEJ and HR efficiency assays. These reporter assays integrate inducible DSB in chromosomal DNA, and the successful repair of DSB results in GFP expression. NHEJ and HR efficiency was measured using flow cytometry, and the results revealed that 0.5 MOI MV-Edm decreased NHEJ efficiency to ~30% of that observed in control groups in the MCF7 and T47D cell lines (Fig. 1C and D). The viral infection increased HR efficiency by 10–20% compared with the respective control groups (Fig. 1E and F). These results suggested that impaired NHEJ may be a mechanism of inhibition of cell viability induced by MV-Edm in breast cancer cells.

To elucidate how MV-Edm affected NHEJ efficiency, the mRNA expression levels of NHEJ-associated factors were investigated, including Ku70, Ku80, DNA-PKcs, XRCC4 and XLF, as well as the pathway factor 53BP1, which favors NHEJ (30). In both breast cancer cell lines, 0.5 MOI MV-Edm reduced the mRNA expression levels of 53BP1, but not those of other NHEJ factors (Fig. 2A and B). Western blot analysis demonstrated that the protein expression level of 53BP1 was decreased in MV-Edm-infected T47D and MCF7 cells (Fig. 2C), indicating that MV-Edm may impair NHEJ efficiency by downregulating 53BP1.

Impaired 53BP1 mRNA and protein expression by MV-Edm infection results in decreased NHEJ efficiency, which may be due to the activation of an unfavored pathway. To determine whether MV-Edm impedes the NHEJ pathway in cells, in which NHEJ was previously incorporated for DSB repair, DNA damage was induced using 2-Gy IR, and the cells were allowed to recover for 2 h to allow the DNA repair mechanism to assemble. The chromatin binding activity of the NHEJ-associated factors was determined using a chromatin fractionation assay, and the results demonstrated that 0.5 MOI MV-Edm treated cells exhibited a notable decrease in chromatin binding of DNA-PKcs, XRCC4, XLF and ligase IV in MCF7 cells compared with that in the control cells (Fig. 3A). A similar result was also observed in doxorubicin-treated MCF7 cells (Fig. 3B).

To determine whether MV-Edm affected the DNA binding activity of 53BP1, biotinylated DNA was used to pull down the 53BP1 immunoprecipitate from MV-Edm-infected and control cells. As presented in Fig. 3C and D, MV-Edm did not affect the 53BP1-DNA interaction in MCF7 cells after IR and doxorubicin treatment. These observations suggested that MV-Edm may induce NHEJ defects in breast cancer cells by reducing NHEJ incorporation during DSB repair by downregulating 53BP1, as well as disrupting the NHEJ protein complex during NHEJ.

As abnormal DNA repair contributes to radio- and chemoresistance in various types of cancer, we hypothesized that MV-Edm, which significantly impairs NHEJ in breast cancer cells, may inhibit cell viability in treatment-resistant cells. First, MCF7-IRR, T47D-IRR, MCF7-DR and T47D-DR cell lines were established (Fig. S1). Cells resistant to IR or doxorubicin exhibited a ~40–60% increase in NHEJ efficiency compared with that in the respective parental cells (P<0.001; Fig. S2).

To determine whether MV-Edm affected NHEJ efficiency in resistant cell lines, the IRR and DR cell lines were infected with 0.5 MOI MV-Edm, and the NHEJ efficiency compared with that in the control cells was investigated. As presented in Fig. 4A-D, MV-Edm significantly decreased NHEJ efficiency in the IRR and DR cell lines. Subsequently, cell viability was measured in the resistant cell lines infected with MV-Edm following IR or doxorubicin treatment. The results demonstrated that MV-Edm significantly inhibited cell viability in MCF7-IRR cells; in particular, 0.5 MOI MV-Edm decreased cell survival by 4.36-fold at 2 Gy (Fig. 4E) compared with 0 MOI MV-Edm treatment. In addition, MCF7-DR cells were re-sensitized to doxorubicin by MV-Edm infection, and cell survival decreased by 4.56-fold at 0.2 µM compared with 0 MOI MV-Edm treatment (Fig. 4F). Similar re-sensitization to IR and doxorubicin by MV-Edm was observed in T47D-IRR cells by 3.70-fold at 2 Gy and in T47D-DR cells 3.61-fold at 0.2 µM compared with 0 MOI MV-Edm treatment (Fig. 4G and H). To further elucidate the mechanism underlying IR- and doxorubicin-induced cell death, the caspase-3/7 activity assay was used to determine apoptosis following IR or doxorubicin treatment in the absence or presence of MV-Edm. As presented in Fig. S3, combination treatment with MV-Edm and IR or doxorubicin induced a significant dose-dependent increase of caspase-3/7 activation in resistant cells. These results suggested that MV-Edm may serve a promising role as a re-sensitizing agent for radio- or chemotherapy.

To determine whether MV-Edm may overcome IR or doxorubicin resistance by impeding the NHEJ pathway, NHEJ-deficient cells were constructed by knocking down its key factor, ligase IV. Using a commercially available CRISPR/cas9 plasmid with a ligase IV guiding sequence, ligase IV-deficient MCF7-IRR and MCF7-DR cells [MCF7-IRR-knockout (KO)1, MCF7-IRR-KO2, MCF7-DR-KO1 and MCF7-DR-KO2] were generated. Western blot analysis of DNA ligase IV demonstrated that there was no detectable ligase IV protein expression in the KO cells compared with that in the parental cell lines transfected with CRISPR/cas9-Ctr (Fig. 5A and B).

To verify the ligase IV deficient cell lines, NHEJ efficiency was compared between the ligase IV-KO and the control cell lines. MCF7-IRR-KO1 and MCF7-IRR-KO2 exhibited near complete loss of NHEJ compared with that in the MCF7-IRR-Ctr cells (Fig. 5C). As demonstrated in Fig. 5D, MCF7-DR-KO1 and MCF7-DR-KO2 were also NHEJ-deficient. Therefore, NHEJ-deficient radio- and chemoresistant cells were successfully constructed to elucidate the re-sensitizing mechanism of MV-Edm.

To verify the role of the NHEJ pathway in the re-sensitization to IR or doxorubicin induced by MV-Edm, the cell viability in MCF7-IRR-Ctr and MCF7-IRR-KO cells was investigated. As presented in Fig. 5E, following infection with 0.5 MOI MV-Edm, both MCF7-IRR-KO cell lines exhibited a significant increase in IR resistance compared with that in the MCF7-IRR-Ctr cells. These results suggested that the effects of MV-Edm on IR resistance in breast cancer cells required efficient NHEJ.

Subsequently, MCF7-DR-Ctr, MCF7-DR-KO1 and MCF7-DR-KO2 cell lines were infected with 0.5 MOI MV-Edm, and cell viability was measured following doxorubicin treatment. Consistent with the results presented in Fig. 5E, MCF7-DR-KO cells were more resistant to doxorubicin compared with MCF7-DR-Ctr cells (Fig. 5F). Thus, these results demonstrated that the MV-Edm-induced re-sensitization to IR and doxorubicin was achieved by inhibition of NHEJ.