Background
Nasopharyngeal carcinoma (NPC) is a form of epithelial cancer with high occurrence rates in Southeast Asia and southern China, where its incidence is approximately 25 to 50 cases per 100,000 individuals [
1]. Radiotherapy has been used as the primary treatment of nasopharyngeal carcinoma [
2], and a majority of NPC patients can be cured when diagnosed and treated at an early disease stage. However, approximately 20 % of NPC patients suffer from local recurrence after treatment [
3], and radiosensitivity is widely perceived as one of the major obstacles for radiotherapy.
Investigators have made many efforts to understand the DNA damage response(DDR). In this process, factors gathered to sites of DNA damage within minutes of irradiation [
4‐
6], and initiated a phosphorylation signaling cascade. Firstly, DNA damage induce ATM/ATR phosphorylation on S139 of histone H2AX, then directly recruits MDC1 through its BRCT domains, and the phosphorylation of MDC1 leads to the recruitment of an ubiquitin ligase RNF8/UBC13 to damage sites. The subsequent ubiqutination events on the damaged chromatin create docking sites for DNA repair proteins to accumulate at DNA double strand breaks(DSBs) [
7‐
11]. Following the resolution of DNA damage, repair proteins dissociate from DSBs, thus alleviating cell cycle checkpoint responses and allowing resumption of cell proliferation.
BRCA1-BRCA2-containing complex(BRCC), a novel multiprotein complex composed of BRCA1, BARD1, BRCA2, RAD51, BRCC3 and BRCC45 in addition to other proteins [
12], was reported to participate in the DNA damage reponse illustrated above [
13‐
15]. As one of the subunits of BRCC, BRCC3 functions to counteract Ubc13-RNF8 activity to provide a balanced level of ubiquitin near DNA lesions, which is essential for the recruitment and dissociation of DNA repair proteins [
15,
16]. Knocking down BRCC3 expression impairs the DNA repair pathway [
15,
17], resulting in the disruption of G2/M checkpoint arrest [
12] and increased cell apoptosis [
17]. Furthermore, BRCC3 is overexpressed in the vast majority of breast tumors [
12]. Taken together, this suggests that BRCC3 is accountable for cell radioresistance and has potential clinical relevance in breast cancer. Thus, we hypothesize that BRCC3 plays a similar role in NPC radioresistance and accounts for the poor prognosis of NPC patients.
To study the clinical application value of BRCC3, we determined the relationship between the BRCC3 expression level and nasopharyngeal carcinoma patient survival. Moreover, we investigated the contribution of BRCC3 to radiation resistance in HNE1 and CNE2R cells, two human nasopharyngeal carcinoma cell lines that expressed a high level of BRCC3 and exhibited resistance to radiation.
Methods
Immunohistochemical staining (IHC)
Tissue samples
This study was conducted on a total of 100 paraffin-embedded NPC tissue samples obtained from patients who were histologically and clinically diagnosed at the Sun Yat-Sen University Cancer Center, China, between 1994 and 1999. Patient consent and approval from the Institute Research Ethics Committee was obtained prior to the use of these clinical materials for research purposes. The clinical characteristics of the patients are shown in Table
1.
Table 1
Correlation between the clinicopathologic features and expression of BRCC3
Gender | | | | | |
Male | 77 | 41 | 36 | 1.030 | 0.310a
|
Female | 23 | 15 | 8 |
Age | | | | | |
<45 | 47 | 27 | 20 | 0.075 | 0.784 a
|
≥45 | 53 | 29 | 24 |
Histological classification | | | | | |
Type II | 8 | 5 | 3 | 0.149 | 0.699 b
|
Type III | 92 | 51 | 41 |
Clinical stage | | | | | |
I-II | 56 | 25 | 31 | 6.662 | 0.010 a * |
III-IV | 44 | 31 | 13 |
T | | | | | |
T1-T2 | 66 | 39 | 27 | 0.753 | 0.386 a
|
T3-T4 | 34 | 17 | 17 |
N | | | | | |
N0 | 60 | 39 | 21 | 4.931 | 0.026 a * |
N1-N3 | 40 | 17 | 23 |
M | | | | | |
M0 | 83 | 52 | 31 | 8.764 | 0.003 a * |
M1 | 17 | 4 | 13 |
Relapse | | | | | |
Yes | 14 | 8 | 6 | 0.009 | 0.926 a
|
No | 86 | 48 | 38 |
Skull-based invasion | | | | | |
Yes | 20 | 11 | 9 | 0.010 | 0.920 a
|
No | 80 | 45 | 35 |
Immunohistochemical staining (IHC)
IHC staining was performed using the Dako Envision system (Dako, Carpinteria, CA) following the manufacturer’s recommended protocols. All paraffin-embedded specimens were cut into 4-μm sections and baked for 2 h at 65 °C. All sections were deparaffinized with xylenes and rehydrated with graded ethanol to distilled water and then submerged in EDTA antigen retrieval buffer (pH 8.0) and microwaved for antigen retrieval. After being treated with 0.3 % H2O2 for 15 min and normal goat serum for 30 min, the sections were incubated with a BRCC3 antibody (1:200; Pierce Biotechnology; PA5-20426) overnight at 4 °C. After washing, the sections were incubated with a biotinylated anti-rabbit secondary antibody (Zymed) followed by further incubation with streptavidin-horseradish peroxidase (Zymed) at 37 °C for 30 min. For the color reaction, diaminobenzidine (DAB) was used.
We used the intensity and extent of the staining to assess BRCC3. The entire tissue section was observed to assign scores. The staining intensity was scored as 0 (no staining), 1 (weak staining exhibited as light yellow), 2 (moderate staining exhibited as yellow brown), or 3 (strong staining exhibited as brown). The extent of staining was scored as 0 (0 %), 1 (1 to 25 %), 2 (26 to 50 %), 3 (51 to75%), or 4 (76 to 100 %), according to the percentages of the positive staining areas in relation to the whole carcinoma area or the entire section for the normal samples. The product of the intensity and extent scores was used as the final staining score (0 to 12) for BRCC3. For the purpose of statistical evaluation, tumors having a final staining score of <4 were grouped into low BRCC3 expression, and those with scores ≥4 were grouped into high BRCC3 expression [
18].
Cell culture, siRNA transfection, and IR
We received the identified CNE2 and CNE2R cells (tested by analysis of the DNA microsatellite short tandem repeats) from Professor Hui-ling Yang (Department of Pathophysiology, Zhongshan School of Medicine, Sun Yat-Sen University) [
19,
20]. Other human NPC cell lines (CNE1, SUNE1, SUNE2, HNE1, HONE1) were maintained in Sun Yat-Sen University Cancer Center [
21,
22] and were not authenticated prior to this study. All of the NPC cell lines were maintained in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10 % fetal bovine serum (FBS; Hyclone, Logan, UT), penicillin (100 units/ml), and streptomycin (100 units/ml) at 37 °C in a humidified 5 % CO2 incubator.
For the BRCC3 depletion studies, NPC cells were plated at a density of 3 × 10
5 cells/cm
2. After reaching 30 % to 40 % confluence, cells were transfected with siRNA using riboFECT™ CP and OPTI-MEM I reduced serum medium (Invitrogen/Life Technologies, Inc., Carlsbad, CA) according to the manufacturer’s protocol. The siRNA duplexes with the following sense and antisense sequences were used, siRNA1: 5′-GAGGAAGGACCGAGUAGAAdTdT (sense) and 5′-UUCUACUCG-GUCCUUCCUCdTdT (antisense); siRNA2: 5′-AACAUCAACAUGUGAAGGCdTdT(sense) and 5′-GCCUUCACAUGUUGAUGUUdTdT(antisense) [
13,
17]. All of the siRNA duplexes were synthesized by RIBOBIO. Twenty-four hours after transfection, the cells were irradiated using a R2000 X-ray irradiator (1.1 Gy/min, 160 kV, 25 mA, 0.3 mm copper filters).
Cells were transfected with the indicated siRNAs, and 24 h later, they were trypsinized, seeded into 6-well plates at different densities(100, 200, 10
3, 10
4cells for 0Gy, 2Gy, 4Gy, 6Gy groups separately) and X-ray irradiated at defined doses. After 7–14 days of incubation, the cultures were fixed and stained with Giemsa. Colonies with more than 50 cells were scored as survivors. The plating efficiency (PE) was calculated by dividing the number of colonies counted by the number of cells plated. The surviving fractions (SF) were then calculated by dividing the PE by the PE of the non-irradiated control [
15,
23,
24]. Sensitization enhancement ratio(SER) was calculated by division of SF2(surviving fractions at 2Gy). Clonogenic survival curves were compared through the extra sum-of-squares F test in GraphPad Prism [
25].
Real-time RT-PCR
Total RNA from different NPC cell lines were extracted with Trizol reagent (Invitrogen, Carlsbad,CA), then the first-strand complementary DNA (cDNA) was synthesized with 1 μg of total RNA. Realtime-PCR was performed in triplicate by using the Absolute blue SYBER green Rox mix (Thermo Scientific). PCR reaction and data collection were conducted with an ABI PRISM 7900HT sequence detection system For normalization, GAPDH was used as endogenous control. The primer sequences are sense 5′-AATTTCTCCAGAGCAGCTGTCTG, anti-sense 5′-CATGGC TTGTGTGCGAACAT for BRCC3, and sense 5′-CTCCTCCTGTTCGACAGTCAG C-3′, Antisense 5′- CCCAATACGACCAAATCCGTT-3′ for GAPDH.
Western blot analysis
Western blot analysis was performed as described previously [
18]. Briefly, total cellular proteins were extracted with lysis buffer. The protein concentration was determined by BCA Protein Assay Kit (Beyotime Biotechnology). Equal amounts of protein samples were loaded and separated by SDS-PAGE gels, electrophoretically transferred to polyvinylidene difluoride membranes(Millipore). The membrane was probed with primary antibody 4 °C overnight and corresponding secondary antibody, The enhanced chemiluminescence was conducted according to the manufacturer’s suggested protocols(Amersham Pharmacia Biotech, Piscataway, NJ). Antibodies: BRCC3 (1:1000; Pierce Biotechnology; PA5-20426), CyclinB1 (1:2000; Cell Signaling; #4135), GAPDH (1:5000; Bioworld Technology, Inc.; BSAP0063).
Immunofluorescence analysis
Cells were transfected with the indicated siRNAs and were trypsinized 24 h after transfection, seeded on glass coverslips in a 24-well plate for 24 h, and irradiated at the indicated doses. γ-H2AX immunofluorescence analysis was carried out as described previously [
24]. At 0 h, 0.5 h, 6 h, 12 h, and 24 h post irradiation, cells were fixed with 4 % paraformaldehyde (15 min, AppliChem, Darmstadt, Germany) at room temperature (RT) and permeabilized by the addition of 0.25 % Triton-X 100 in PBS for 15 min, followed by blocking in 5 % bovine serum albumin (BSA) in PBS for 30 min. Next, cells were incubated with Phospho-Histone H2AX (Ser139) (20E3) rabbit mAb (1:1000; Cell Signal Technology; #9718S), followed by the appropriate Alexa 488-conjugated (green; Molecular Probes) secondary antibodies Subsequently, nuclei were counterstained with DAPI solution (Invitrogen) and coverslips were mounted with Vectashield (Vector Laboratories, Peterborough, UK). Images were taken using an Olympus confocal imaging system (Olympus FV100) for the quantification of γH2AX foci formation. At least three independent experiments were performed for each data point.
Cell cycle analysis
Cell cycle analysis was performed as described previously [
26]. Briefly, cells were harvested 24 h post-radiation by trypsinization and washed with phosphate-buffered saline (PBS). For cell cycle analysis, the cells were fixed with 70 % ethanol at -20 °C overnight. On the following day, the fixed cells were washed with PBS, treated with RNase A (50 μg/ml) in PBS at 37 °C for 20 min, and then mixed with propidium iodide (PI, 50 μg/ml) for 30 min in the dark. The stained cells were analyzed with fluorescence-activated cell sorting (FACS) by flow cytometry (FACSCalibur, Becton Dickinson, Bedford, MA). The cell-cycle profile was analyzed using the ModFit software (Becton Dickinson). At least 10,000 cells in each sample were analyzed to obtain a measurable signal.
Statistical analysis
The Student t-test or Chi-square test was used to compare the differences as appropriate. Survival analysis was conducted by the Kaplan-Meier method and the log-rank test. Multivariate analysis with Cox proportional hazards model was employed to investigate independent prognostic factors. A P-value of < 0.05 was considered statistically significant. Statistical analysis was executed by the SPSS software package (version 16.0, SPSS Inc) or GraphPad prism 5.
Discussion
In this study, we explored the effect of BRCC3 on NPC patient survival and NPC cellular radiosensitivity. The key findings of this work are described in the following: First, it indicates a negative correlation between BRCC3 expression and NPC patient survival (Fig.
2); therefore, BRCC3 may be a potential prognostic biomarker for NPC. Second, it demonstrates that the expression of BRCC3 was upregulated in both inherent (CNE1, HNE1) and induced (CNE2R) radioresistant cell lines (Fig.
3), which suggests that BRCC3 might be a critical factor in the NPC radioresistance. Third, it reveals that knockdown of BRCC3 expression through the use of siRNAs sensitizes HNE1 and CNE2R cells to ionizing radiation, impairs γH2AX foci absorption and induces G2/M cell cycle arrest (Figs.
4 and
5), supporting a role for BRCC3 in cellular responsiveness to ionizing radiation, DNA repair and G2/M checkpoint progression.
In addition to the effect of BRCC3 on patient overall survival, interestingly, the present study discovered an unforeseen correlation between BRCC3 expression and metastasis (Table
1). A previous study demonstrated that BRCC3 gene knockdown caused a decline in the migration and invasion capabilities of glioma cells [
31]. Furthermore, high expression of DNA repair pathways was associated with metastasis in melanoma patients, and BRCA1 showed a higher expression level in metastatic patients [
32]. Accordingly, in bladder carcinoma [
33] and breast cancer [
34,
35] studies, the majority of the significant repair genes were overexpressed in the primary tumors that are going to metastasize. The proportion of significant repair genes that are overexpressed in cancers that will metastasize reaches 90 % in melanomas, 82 % in bladder carcinomas, 80 % in the van’t Veer breast cancer and 58 % in the breast cancers from Wang’s study [
35]. Consequently, the correlation between BRCC3 expression and metastasis in NPC patients is not surprising. The current study revealed the relationship between BRCC3 and NPC metastasis. Further research is necessary to determine the correlation.
In addition to the pertinent literature indicating the involvement of BRCC3 in the DNA damage response, the two important reasons attracting our attention to the contribution of BRCC3 to NPC radioresistance is the high BRCC3 level in 3-year LRRFS and the overexpression of BRCC3 in radioresistant NPC cell lines. The BRCC3 level is upregulated in both inherent NPC cells (CNE1, HNE1) and radiation induced resistant CNE2R cells (Fig.
3). Additionally, the knockdown of BRCC3 expression sensitized the HNE1 and CNE2R cells to ionizing radiation (Fig.
4). This indicated that BRCC3 may be a potential therapeutic target for not just the treatment-naive patients, but for patients who suffered a relapse after standardized treatment.
Although a variety of studies revealed that the depletion of BRCC3 results in enhanced radiosensitivity in vitro, the mechanisms of the increased radiosensitivity caused by BRCC3 depletion have not yet been established and could be partially due to its interaction with BRCA1. BRCC3 directly interacts with BRCA1 at the region encompassing amino acids 502 to 1054 [
12], which falls within the BRCA1 DNA-binding domain (amino acids 452-1079). BRCC3 deficiency inhibits BRCA1 focus formation and disrupts the IR-induced phosphorylation of BRCA1 [
17] that may contribute to BRCA1-dependent DSB repair. Another mechanism lies in the BRCC3 de-ubiquitinating activity, which plays an important role for recruitment events in the repair of DSBs. BRCC3 disruption has been reported to be associated with increased 53BP1 and γH2AX foci accumulation after IR [
15]. This is consistent with our result indicating that BRCC3 knockdown increased the persistence of γH2AX foci positive cells 24 h after irradiation (Fig.
4d,e), which indicated a delay of the DNA damage repair.
Regulation of the cell cycle may be another important mechanism for radiosensitization, as supported by our results that BRCC3 disruption increased cell-cycle arrest in nasopharyngeal carcinoma cell lines (Fig.
5a,
b). The Cdk1-cyclin B complex is the main target molecule of the G2/M cell cycle checkpoint [
36,
37]. The results of our study showed that BRCC3 knockdown upregulated cyclinB1 expression after irradiation compared to the control cells (Fig.
5c). In addition to BRCC3, the knockdown of other BRCC complex subunits, such as BRCA1, BRE, RAP80 and NBA1, also induced G2/M arrest [
12,
38,
39], suggesting that the integrity of this complex is vital for the G2/M transition. However, the subunits may also play a separate role in the G2/M cell cycle regulation. Cdk1 binds with RAP80 protein and mediates its phosphorylation at an evolutionarily conserved Ser-677 residue, which is important for RAP80 functional sensitivity to IR and G2/M checkpoint control [
38]. Nevertheless, further studies are needed to determine the exact mechanism for the G2/M arrest induced by BRCC3.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YX contributed to the study design and to the sample collection. ZT conducted the experimental work. BX, YT, CC, HC, ZL, GL, CJ and WY performed the chart reviews for clinical data, follow-up and data collection and established the clinical database. ZT and CQ did the statistical analysis and had access to the raw data. WH and GF provided the infrastructural support for this study. The manuscript was drafted by ZT, edited by YX and MZ, and submitted for comments to all the authors. All authors approved the final version of the manuscript.