CHAF1B induces radioresistance by promoting DNA damage repair in nasopharyngeal carcinoma

Biomedicine & Pharmacotherapy 123 (2020) 109748

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CHAF1B induces radioresistance by promoting DNA damage repair in nasopharyngeal carcinoma

T

Muping Dia,b,1, Meng Wanga,1, Jingjing Miaoa,b, Boyu Chena,b, Huageng Huanga,b, Chuyong Lina, Yunting Jiana, Yue Lia, Ying Ouyanga, Xiangfu Chena, Lin Wanga,b,*, Chong Zhaoa,b,* a Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China b Department of Nasopharyngeal Carcinoma, Sun Yat-sen University Cancer Center, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Guangzhou 510060, China

A R T I C LE I N FO

A B S T R A C T

Keywords: CHAF1B Nasopharyngeal carcinoma DNA damage repair Radioresistance DNA-PK pathway

Background: Radiotherapy is the main treatment for nasopharyngeal carcinoma (NPC); however radioresistance restricts its efficacy. Therefore, new molecular regulators are required to improve the radiosensitivity of NPC. Chromatin assembly factor 1 subunit B (CHAF1B) plays a role in DNA synthesis and repair, and participates in the progression of various malignancies. However, the expression and function of CHAF1B in NPC is unclear. Methods: The expression of CHAF1B was determined using real-time PCR and western blotting. CHAF1B expression in 160 human NPC tissue samples was evaluated using immunochemistry (IHC). The correlations between CHAF1B expression and NPC clinicopathological features were determined. The effect of CHAF1B on the radiosensitivity of NPC cells was detected using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and colony formation assays. Apoptosis rates were analyzed using flow cytometry. A nude mouse subcutaneous xenograft model and living fluorescence imaging were applied to evaluate tumor regression in vivo. The molecular mechanisms of radioresistance were confirmed by bioinformatics analysis and detection of phosphorylated H2A histone family member X (γH2AX) foci. Results: Significantly increased CHAF1B levels were observed in NPC tissues, which correlated positively with radioresistance and poor prognosis. In addition, CHAF1B was upregulated in radioresistant NPC cell lines. Overexpression of CHAF1B reduced, while silencing of CHAF1B enhanced, the radiosensitivity of NPC cells in vitro and in vivo. Mechanistically, CHAF1B inhibited NPC cell apoptosis by promoting DNA damage repair. Finally, the DNA-dependent protein kinase (DNA-PK) pathway was observed to be essential for CHAF1B promotion of DNA damage repair-mediated radioresistance. Conclusion: The results suggested CHAF1B enhances radioresistance by promoting DNA damage repair and inhibiting cell apoptosis, in a DNA-PK pathway-dependent manner. CHAF1B may serve as a novel factor for predicting radiorsensitivity. Besides, DNA-dependent protein kinase inhibitor could serve as a radiosensitizer for patients with NPC and high CHAF1B expression.

1. Introduction Among head and neck malignancies, nasopharyngeal carcinoma (NPC) is one of the most common in southern China, occurring in 30–80/100,000 population annually [1]. Radiotherapy is the primary treatment for NPC. In the past decade, with the widespread application of concurrent chemoradiotherapy and the progress of radiotherapy technology, the survival rate of patients with NPC has been improved

significantly [2]. However, a high proportion of patients still experience local recurrence or distant metastasis due to radioresistance [1,3–6]. Therefore, studying the molecular mechanisms that mediate NPC radioresistance could provide more effective strategies for clinical treatment and to improve the clinical outcome of NPC. The factors that affect radiosensitivity include proliferation, apoptosis, cell cycle, and DNA damage repair [7–9]. In particular, recent studies have shown that the ability to repair damaged DNA is closely

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Corresponding authors at: Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Department of Nasopharyngeal Carcinoma, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Guangzhou 510060, China. E-mail addresses: [email protected] (L. Wang), [email protected] (C. Zhao). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2019.109748 Received 6 September 2019; Received in revised form 11 November 2019; Accepted 29 November 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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related to tumor cell radiosensitivity [10]. Radiotherapy delivers ionizing radiation, which damages DNA and activates intracellular death pathways, ultimately leading to tumor cell death. Yet at the same time, damaged DNA is recognized by the repair system, which recruits certain proteins to initiate the DNA damage repair pathway. However, the molecules that regulate DNA damage repair remain to be investigated. The chromatin assembly factor 1 (CAF-1) complex comprises p150, p60, and p48 subunits, which participates in DNA synthesis and the subsequent nucleosomes assembly [11]. Chromatin assembly factor 1B (CHAF1B) is the p60 subunit of CAF-1, which is a central facilitator in chromatin assembly after DNA synthesis and repair [12]. Recent studies have revealed the intimate relationship between CHAF1B and cell proliferation [13,14]. Overexpression of CHAF1B is closely connected with poor prognosis in several solid tumor types, including high-grade gliomas, prostatic cancer, breast cancer, salivary gland tumors, and melanoma [15–20]. Meanwhile, CHAF1B also repairs the damaged DNA induced by ultraviolet radiation via the nucleotide excision repair system [21]. Recently, CHAF1B was reported to play an important role in the regulation of cell cycle arrest and cell apoptosis in lung cancer [22]. Nevertheless, the expression and function of CHAF1B in NPC remain unknown. In the present study, we found that CHAF1B is significantly upregulated in NPC and is closely connected with radioresistance. Overexpression of CHAF1B reduced, while silencing of CHAF1B enhanced, the radiosensitivity of NPC cells in vitro and in vivo. Mechanistically, CHAF1B inhibits apoptosis by promoting DNA damage repair, dependent on the DNA-dependent protein kinase (DNA-PK) pathway, ultimately leading to radioresistance in NPC. Taken together, CHAF1B might represent a useful biomarker for NPC sensitivity, and DNA-dependent protein kinase inhibitor (DNA-PKi) could serve as a radiosensitizer for patients with NPC and high CHAF1B expression.

Table 1 Clinicopathological CHAF1B in NPC.

characteristics

Parameters Gender Male Female Age, years ≤ 46 > 46 WHO category II III T classification T1–2 T3–4 N classification N0–1 N2–3 Clinical stage I–II III–IV Recurrence No Yes Metastasis No Yes Vital status Alive Dead Ki67 expression Low High CHAF1B expression Low High

and

tumor

expression

of

Number of cases (%)

130 (81.3) 30 (18.8) 79 (49.4) 81 (50.6) 3 (1.9) 157 (98.1) 25 (15.6) 135 (84.4) 77 (48.1) 83 (51.9) 11 (6.9) 149 (93.1) 143 (89.4) 17 (10.6) 144 (90.0) 16 (10.0) 151 (94.4) 9 (5.6) 69 (43.1) 91 (56.9) 72 (45.0) 88 (55.0)

2. Materials and methods Abbreviations: WHO,World Health Organization, II: differentiated non-keratinized carcinoma; III: undifferentiated non-keratinized carcinoma; T, tumor; N, node; CHAF1B, chromatin assembly factor 1 subunit B.

2.1. Cell culture The State Key Laboratory of Oncology in South China (Sun Yat-sen University Cancer Center, Guangzhou, China) provided the cells lines. The NPC cell lines HK1, C666-1, S18, S26, HONE1, SUNE1, 5-8 F, 610B, CNE1, CNE2, and HNE1 were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, Carlsbad, CA, USA) with 10 % fetal bovine serum(FBS) (HyClone, Logan, UT, USA) and 1 % penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). NP69, a nasopharyngeal epithelial cell line, was cultured in defined keratinocyte serum free medium (KSFM) with epidermal growth factor (EGF) (Invitrogen, Carlsbad, CA, USA). 293 FT cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM) (Gibco, Grand Island, NY, USA) with 10 % fetal bovine serum (FBS). All cell lines were cultured in a humidified incubator in a 5 % CO2 atmosphere at 37 °C.

(RR, 26 cases), with local recurrence or distant metastasis. The patients provide prior consent for the use of these clinical specimens in research and the Institutional Research Ethics Committee provided approval for the study. 2.3. Extraction of RNA, reverse transcription, and quantitative real-time PCR The Trizol reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate total RNA from cells. Random hexamer primers were used to prime cDNA synthesis from 1 μg of RNA. A FastStart Universal SYBR Green Master (ROX; Roche, Toronto, ON, Canada) was used to perform quantitative real-time PCR on a CFX96 Real Time System C1000 Cycler (Bio-Rad Laboratories, Singapore). All reactions were incubated at 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 7 min. Primer sequences were as follows: CHAF1B forward 5′-CCTGGAAAAGCCACTCTTGCTG-3′ and reverse 5′- ACAGAAGCACG GAATCCTCCGA-3′; Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward 5′-AAGGTCATCCCTGAGCTGAA-3′ and reverse 5′-TGACAAAGTGGTCGT TGAGG-3′. The expression of CHAF1B in the samples was normalized to that of GAPDH; these experiments were performed at least in triplicate.

2.2. Patients and tissue samples We obtained paraffin-embedded samples of NPC from 160 patients who were diagnosed clinically and pathologically with non-metastatic NPC between 2011 and 2015 at the Sun Yat-Sen University Cancer Center. Clinical information for the patients is summarized in Table 1. The patients who provided the samples had not received previous chemotherapy or radiotherapy for their disease. The patients received radical radiotherapy alone or chemoradiotherapy. The 7th edition of the Union for International Cancer Control (UICC) staging system was used to stage the samples. The patients were followed up for a median of 41.2 months (range: 5.7–77.27 months). During follow-up, 9/160 (5.6 %) patients died, 17/160 (10.6 %) experienced recurrence, and 16/160 (10 %) experienced distant metastasis. Specimens were classified as radiation sensitive (RS, 134 cases), with no local recurrence or distant metastasis following radiation therapy, or as radiation resistant

2.4. Immunohistochemistry (IHC) Anti-CHAF1B antibody (Sigma, HPA021679, 1:50) was used to perform IHC staining on the 160 paraffin-embedded NPC tissue sections. Two independent pathologists, who were blinded to the clinical 2

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Fig. 1. CHAF1B is upregulated in NPC. (A) Compared with that in normal tissues, the mRNA expression of CHAF1B was significantly upregulated in HNSC tissues, as indicated by the TCGA HNSC dataset. (B) According to the TCGA HNSC dataset, the mRNA expression of CHAF1B mRNA was higher in paired HNSC tissues than that in normal tissues. (C and D) According to the public GEO NPC datasets GSE12452 (C) and GSE13597 (D), the mRNA expression of CHAF1B was significantly higher in NPC tissues compared with that in normal tissues. (E and F) Analysis of CHAF1B expression in human NPC cell lines and an NP69 immortalized nasopharyngeal epithelial cell line using real-time PCR (E) and western blotting (F). ***, P < 0.001. (G and H) Analysis of CHAF1B expression in human NPC tissues and normal tissues using real-time PCR (G) and western blotting (H) analysis. ***, P < 0.001. CHAF1B, Chromatin assembly factor 1 subunit B; NPC, nasopharyngeal carcinoma; HNSC, human head and neck squamous carcinoma; TCGA, The cancer Genome Atlas.

brown); 3, strong staining (brown). The product of the staining intensity score and the proportion of positive cells was used as the staining index (SI). Protein expression was then assessed by determining the SI, with possible scores of 0, 1, 2, 3, 4, 6, 8, 9, and 12. Those samples with an SI ≥ 6 were regarded as high expression, and those with an SI < 6 were regarded as low expression. Heterogeneity

outcome, evaluated and scored the IHC staining results. The scoring system was based on both the proportion of positively stained tumor cells and the staining intensity, as follows: Proportion of stained cells: 0, no positive cells; 1, < 10 % positive cells; 2, 10–35 % positive cells; 3, 35–75 % positive cells; 4, > 75 % positive cells; staining intensity: 0, no staining; 1, weak staining (light yellow); 2, moderate staining (yellow

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Fig. 2. Upregulation of CHAF1B is associated with radioresistance and poor prognosis in NPC. (A) CHAF1B staining shown as representative images. Staining was scored as negative 0, weak +1, moderate +2, and strong +3. The magnified inset area is shown in the bottom row. Bracketed values show the proportion of each staining score. (B) The number of cases with each staining intensity is shown. (C) T/N classified clinical specimens and their associated CHAF1B expression levels. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) CHAF1B expression levels in the radioresistant and radiosensitive NPC tissues. (E) Five-year PFS and 5-year LRFS for NPC analyzed using Kaplan-Meier analysis and stratified by low and high CHAF1B levels. (5-year PFS: hazard ratio [HR] 4.073; 95 % CI 1.534–10.816; P < 0.01; by log-rank test) (5-year LRFS: hazard ratio [HR] 4.517; 95 % CI 1.297–15.738; P < 0.05; by log-rank test). (F) Evaluation of the significance of the association between the CHAF1B expression signature and 5-year PFS and 5-year LRFS using Multivariate Cox regression analysis in the presence of other clinical variables. CHAF1B, Chromatin assembly factor 1 subunit B; NPC, nasopharyngeal carcinoma; S, radiosensitiviy; R, radioresistance; PFS, progression-free survival; LRFS, local recurrence-free survival.

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2.5. Gene expression profiling and analysis

Table 2 Correlation between CHAF1B and clinicopathological characteristics of patients with NPC.

Publicly available human head and neck squamous carcinoma (HNSC) datasets comprising Gene expression Omnibus (GEO) datasets, including GSE12452 and GSE13597, in the NCBI database (https:// www.ncbi.nlm.nih.gov/geo/) and The Cancer Genome Atlas (TCGA) (https://portal.gdc.cancer.gov/projects/TCGA-HNSC) were used for gene expression profiling and analysis. Samples from the GEO and TCGA datasets were classified into high or low-CHAF1B expression groups using the median of CHAF1B mRNA expression. Gene Set Enrichment Analysis (GSEA) is a computational method that assesses whether a defined gene set shows statistically significant and concordant differences between two biological states (e.g.,phenotypes). GSEA 2.0.9 (http://www.broadinstitute.org/gsea/) was used to perform the GSEA [23].

CHAF1B expression Characteristic

Total

Low

High

P values

Gender

Male Female

130 30

68 (52.3) 4 (13.3)

62 (47.7) 26 (86.7)

< 0.001

Age, years

≤ 46 > 46

79 81

36 (45.6) 36 (44.4)

43 (54.4) 45 (55.6)

0.886

WHO category

II III

3 157

3 (100.0) 69 (43.9)

0 (0) 88 (56.1)

0.178

T classification

T1–2 T3–4

25 135

6 (100.0) 5 (11.9)

0 (0) 37 (88.1)

< 0.001

N classification

N0–1 N2–3

77 83

49 (63.6) 23 (27.7)

28 (36.4) 60 (72.3)

< 0.001

Clinical stage

I–II III–IV

11 149

10 (90.9) 62 (41.6)

1 (9.1) 87 (58.4)

0.004

Recurrence

No Yes

143 17

69 (48.3) 3 (17.6)

74 (51.7) 14 (82.4)

0.016

Metastasis

No Yes

144 16

70 (48.6) 2 (12.5)

74 (51.4) 14 (87.5)

0.006

Disease progression

No Yes

134 26

67 (50.0) 5 (19.2)

67 (50.0) 21 (80.8)

0.004

Vital status

Alive Dead Low High

151 9 69 91

72 (47.7) 0 (0) 39 (56.5) 33 (36.3)

79 (52.3) 9 (100.0) 30(43.5) 58 (63.7)

0.014

Ki67 expression

2.6. Plasmids, virus constructs, and retroviral infection of target cells Polymerase chain reaction was used to amplify the human CHAF1B cDNA, which was then cloned into vector pMSCV-puro-retro. A short hairpin RNA (shRNA) oligonucleotide targeting the CHAF1B mRNA was cloned into vector pSuper-puro to generate pSuper-puro-CHAF1BshRNA, which was used to knockdown endogenous CHAF1B expression. The Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) was used to transfect the plasmids into cells, according to the manufacturer’s instructions. The target cells were infected using retroviruses generated by pMSCV-puro-CHAF1B and pSuper-puro-CHAF1B-shRNA for 72 h. Puromycin at 0.6 μg/mL was used to select stable cell lines expressing CHAF1B and CHAF1B-shRNA for 9 days.

0.0044

2.7. Western blotting analysis Western blotting was performed according to a previously described method [24]. The primary antibodies and the concentrations used were as follows: Anti-CHAF1B antibody (Sigma, HPA021679; 1:500), antiγH2AX antibody (CST, #80312; 1:1000), anti-BCL2 apoptosis regulator (BCL2) antibody (Proteintech, 1:1000; 12789-1-AP), anti-BCL2 associated X, apoptosis regulator (BAX) antibody (Proteintech, 1:1000; 50599-2-Ig), anti-cleaved caspase-3 antibody (CST, 9664; 1:1000), and anti-α-tubulin antibody (Sigma, T9026, 1:4000). The secondary antibodies comprised horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit antibodies (GE Healthcare).

Table 3 Univariate and multivariate analysis of factors associated with PFS and LRFS in 160 patients with NPC. Characteristics

PFS CHAF1B expression (high) Age (> 46 years) Clinical stage (III–IV) T classification (T3–4) N classification (N2–3) LRFS CHAF1B expression (high) Age (> 46 years) Clinical stage (III–IV) T classification (T3–4) N classification (N2–3)

Univariate analysis

Multivariate analysis

HR (95 % CI)

P values

HR (95 % CI)

P values

4.073 (1.534–10.816) 1.994 (0.902–4.405) 1.917 (0.260–14.166) 0.608 (0.228–1.617) 5.519 (2.070–14.720)

0.005

3.997 (1.303–12.258) 1.954 (0.859–4.444) 1.122 (0.093–13.530) 0.335 (0.096–1.165) 3.536 (1.150–10.867)

0.015

4.517 (1.297–15.738) 0.816 (0.310–2.145) 1.147 (1.52–8.652) 0.651 (0.187–2.268) 4.168 (1.356–12.810)

0.088 0.523 0.319 0.001

0.018 0.680 0.894 0.500 0.013

4.629 (1.095–19.577) 1.347 (0.487–3.729) 0.985 (0.059–16.477) 0.274 (0.047–1.579) 0.353 (0.095–1.318)

0.110 0.928

2.8. Radiation treatment 0.086

For in vitro irradiation, cells were exposed to X-rays using an X-ray irradiator RS2000 (1.1 Gy/min, 160 kV; RAD SOURCE, USA). For in vivo irradiation, the mice were anesthetized, placed in a box, and fixed in place. The subcutaneous tumor was situated in the center of the irradiation field. Only the tumor was exposed to irradiation and lead was used to shield the other parts of the mouse.

0.028

0.037 0.566 0.991 0.147

2.9. Assessing cell proliferation

0.121

The 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma) assay was used to determine cell viability. All groups of cells were seeded in 96-well plates at 2 × 103 cells/well and cultured at 37 °C with 5 % CO2. The cells were then irradiated and incubated for 1–6 days. At defined time points, the MTT solution (10 μL at 5 mg/ml) was added to each well and incubated continued for 4 h at 37 °C. The medium was then removed and DMSO (150 μL; Sigma) was added. A Microplate Reader (Bio-Tek EPOCH2, USA) then used to record the absorbance at 490 nm.

HR, hazard ratio; CI, confidence interval; PFS, progression-free survival; LRFS, local recurrence-free survival.

was measured using the log-rank test with respect to 5-year progression-free survival (PFS) and 5-year local recurrence-free survival (LRFS).

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Fig. 3. CHAF1B induces radioresistance in NPC. (A) The expression levels of CHAF1B were demonstrated using western blotting and real-time PCR in 5−8 F, 5–8 F-IR, SUNE1, SUNE1-IR, HK1 and HK1-IR cells. The data is shown as the mean ± SE. ***, P < 0.001. (B) Western blotting analysis of CHAF1B in 5−8 F cells that stably overexpress CHAF1B, and in SUNE1 cells that are stably silenced for CHAF1B. α-Tubulin was used as a loading control. (C and D) The cell growth curves of 5–8 F-vector, 5–8 F-CHAF1B, SUNE1-scramble, and SUNE1-shCHAF1B cells after 4 Gy irradiation treatment. The data shown are the mean ± SE from three independent experiments. *, P < 0.05. (E and F) At 10–14 days after treatment using a single dose of 0, 2, 4, 6, or 8 Gy IR, the colony formation abilities of 5–8 Fvector, 5–8 F-CHAF1B, SUNE1-scramble, and SUNE1-shCHAF1B cells were evaluated. (G and H) Survival curve of 5–8 F-vector, 5–8 F-CHAF1B, SUNE1-scramble, and SUNE1-shCHAF1B cells. A comparison of the number of colonies formed in each treatment group with that formed by the untreated control (0 Gy) was used to calculate the surviving fraction. CHAF1B, Chromatin assembly factor 1 subunit B; NPC, nasopharyngeal carcinoma; IR, radioresistance.

violet were used to fix and stain the cells. Colonies were counted if they comprised at least 50 cells. The following calculation was used to determine the survival fraction (SF): Number of colonies / (number of cells seeded × plating efficiency),where the plating efficiency (PE) was the number of control colonies obtained/number of control cells seeded. A dose survival curve was calculated and fitted with a linear-

2.10. Colony formation assay For attachment, cells were seeded in 6-well plates and cultured at 37 °C for 24 h, before being irradiated with 0, 2, 4, 6, or 8 Gy using Xrays. Ten to fourteen days later, the plates were washed with phosphate-buffered saline (PBS), and 4 % formaldehyde and 0.05 % crystal 6

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Fig. 4. CHAF1B suppresses radiation-induced apoptosis in NPC. (A and C) Representative results of a cell apoptosis assay by flow cytometry in 5–8 F-vector, 5–8 F-CHAF1B, SUNE1-scramble, and SUNE1-shCHAF1B cells treated with IR (0 and 6 Gy). (B and D) Percentage of apoptotic cells quantification. Data represent the mean ± SE from three independent experiments. *P < 0.05. CHAF1B, Chromatin assembly factor 1 subunit B; NPC, nasopharyngeal carcinoma.

(1:1000) were incubated with the cells for 1 h. The cells were then washed three times for 10 min each. DAPI (4′,6-diamidino-2-phenylindole; Vector Laboratories, Burlingame, CA, USA) was then used to stain the nuclei. A fluorescence microscope (Olympus) was used to determine the number of γH2AX foci.

quadratic model using GraphPad Prism 5.0 Software (GraphPad Software Inc., La Jolla, CA, USA). 2.11. Analysis of apoptosis The cells were irradiated with 0 and 6 Gy of X-rays and harvested after culture for 72 h. An AnnexinV-fluorescein isothiocyanate (FITC) and propidium iodide (PI) Apoptosis Kit (Nanjing KeyGen Biotech, Nanjing, China) was used to analyze apoptosis, following the manufacturer’s instructions. Flow cytometry was used to detect apoptotic cells using a FACSort system (BD Biosciences), the results of which were analyzed using CellQuest software.

2.13. In vivo tumor xenograft model The Experimental Animal Center of Sun Yat-sen University (Guangzhou, China) provided male BALB/c nude mice (4–5 weeks old), which were reared in barrier facilities with a 12 h light/dark cycle. All experimental procedures were approved by The Institutional Animal Care and Use Committee of Sun Yat-sen University. NPC cells (1 × 106) were injected subcutaneously into both the right and left front flank regions of the mice. When the xenograft volumes reached approximately 50–100 mm3, the xenografts were irradiated with a dose of 2 Gy per day for 6 days. The tumor length (L) and width (W) were measured every 2 days, and the tumor volume (in mm3) was calculated as: Volume = 1/2 × L × W2. In the end, the tumors were observed using the IVIS Lumina II with Living Image Software(Caliper). After isoflurane induction, the mice were injected with luciferin (150 mg/kg) in the intraperitoneal cavity and placed onto the imaging platform, dorsal side up. Images were taken about 5 min post luciferin injection. Then the mice were excised, and weighed. Serial 6.0 μm sections were cut

2.12. The detection of γH2AX foci Cells were exposed to 6 Gy of irradiation and cultured for the indicated times to allow the repair of DNA damage after irradiation. At 0, 1, and 12 h after irradiation, 4 % paraformaldehyde treatment for 30 min was used to fix the cells, which were then permeabilized in 0.5 % Triton X-100 for 15 min. Non-specific binding was blocked by incubation in 5 % bovine serum albumin (BSA) for 30 min. The cells were then incubated with anti-γH2AX antibodies (CST, D7T2V; 1:100) overnight at 4 °C. Then, Alexa Fluor 594-conjugated goat anti-mouse and rhodamine red-conjugated goat anti-mouse secondary antibodies 7

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Fig. 5. CHAF1B compels NPC cells to resist radiotherapy in a xenograft model. (A) Representative luciferase signal images of the tumor bearing mice 24 days after radiation treatment. (B) The growth curves of xenografts with radiation treatment. (C) Images of excised tumors. (D) Tumor weight at 24 days after treatment. *P < 0.05. (E) Apoptotic cells were visualized by TUNEL staining (red) and counterstained with DAPI (blue). The right panel represented the corresponding apoptotic index in the tumor sections. CHAF1B, Chromatin assembly factor 1 subunit B; NPC, nasopharyngeal carcinoma.

the Spearman-rank correlation test. A Cox regression model was used to perform multivariate statistical analysis. Data are presented as the mean ± SD. Statistical significance was considered at a P value < 0.05.

and subjected to TUNEL staining. 2.14. Statistical analyses SPSS version 19.0 (IBM Corp., Armonk, NY, USA) was used to perform the statistical analyses. The statistical tests used to analyze the data included the χ2 test, log-rank test, Student’s t test (two-tailed), and 8

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(caption on next page)

3. Results

CHAF1B expression in human HNSC from the publicly available database, The Cancer Genome Atlas (TCGA). The analysis revealed that CHAF1B was significantly upregulated in HNSC tissues compared with that in normal controls (Fig. 1A). Similarly, CHAF1B expression was dramatically increased in paired HNSC tissues compared with that in

3.1. CHAF1B is upregulated in NPC To investigate the role of CHAF1B in NPC, we first analyzed 9

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Fig. 6. The DNA-PK pathway is crucial for CHAF1B-promoted DNA repair. (A) In samples with high CHAF1B expression, DNA repair gene modules REACTOME DNA REPAIR and REACTOME DOUBLE STRAND BREAK REPAIR were significantly enriched according to GSEA of GSE12452. (B) Immunofluorescence staining for γ-H2AX foci at different times after 6 Gy irradiation. The data shown are the mean ± SE from three independent experiments. *, P < 0.05. (C) GSEA of GSE12452 showing significant enrichment of the DNA-PK pathway (PID_DNA_PK_PATHWAY) in samples with high expression of CHAF1B. ES, enrichment score. NES, normalized enrichment score. FDR, false discovery rate. (D) Analysis of γ-H2AX foci in 5–8 F-vector cells and 5–8 F-CHAF1B cells combined with DNA-PKi at different times after 6 Gy irradiation. The data shown are the mean ± SE from three independent experiments. *P < 0.05. (E) Western blotting assays of the levels of γH2AX, BCL2, BAX and cleaved-caspase-3 in 5–8 F-vector cells, 5–8 F-CHAF1B cells and 5–8 F-CHAF1B cells combined with DNA-PKi at 12 h after 6 Gy irradiation. (F) Proposed model. When radiotherapy results in DSBs, KU70/KU80 proteins immediately localize to the DSBs, followed by recruitment of DNA-PKcs. Subsequently, the DNA-PK complex recruits DNA damage repair associated proteins, including CHAF1B, to the free DNA ends, initiating the non-homologous end joining process. Upregulation of CHAF1B inhibites radiation-activated BAX/BCL2/caspase-3 apoptosis pathway by accelerating the repair of DNA damage, ultimately leading to radioresistance in NPC. DNA-PKi could serve as a radiosensitizer for patients with NPC patients and high CHAF1B expression. CHAF1B, Chromatin assembly factor 1 subunit B; NPC, nasopharyngeal carcinoma; GSEA, gene set enrichment analysis; ES, enrichment score; NES, normalized enrichment score; FDR, false discovery rate; γ-H2AX, phosphorylated H2A histone family member X; DNA-PK, DNA-dependent protein kinase; DNA-PKcs, DNA-PK catalytic subunit; DNA-PKi, DNA-PK inhibitor; BCL2, BCL2 apoptosis regulator; BAX, BCL2 associated X, apoptosis regulator; DSBs, double strand breaks; KU70, lupus Ku autoantigen protein 70; KU80, lupus Ku autoantigen protein 80.

has a low basal expression of CHAF1B. Meanwhile, we silenced endogenous CHAF1B expression in the SUNE1 cell line, which has high basal expression of CHAF1B, using an shRNA (Fig. 3B). Next, we used MTT cell growth assays to measure the growth potential of NPC cells. In the absence of irradiation treatment, there was little difference in cell viability between cells with upregulated or silenced CHAF1B and the controls (Fig. 3C and D). By contrast, under 4 Gy irradiation treatment, the cell viability of cells upregulated for CHAF1B was higher than that of the control (Fig. 3C). The inhibition of cell viability induced by radiation was increased by CHAF1B silencing (Fig. 3D). Consistently, colony formation assays showed that overexpression of CHAF1B significantly reduced the radiosensitivity of the 5-8 F cell line (Fig. 3E and G). Silencing of CHAF1B significantly enhanced the radiosensitivity of the SUNE1 cell line (Fig. 3F and H). Taken together, these data supported the notion that CHAF1B induces radioresistance in NPC.

normal controls (Fig. 1B). Subsequently, we analyzed CHAF1B expression using public data from GEO (GSE12452 and GSE13597). Interestingly, the results showed the same tendency in NPC tissues (Fig. 1C and D). Moreover, real-time PCR and western blotting assays showed that CHAF1B was upregulated in all eleven NPC cell lines compared with that in the immortalized nasopharyngeal epithelial cell line (Fig. 1E and F). CHAF1B expression was further validated in freshly collected nasopharyngeal epithelial tissues and NPC tissues. Both the mRNA and protein levels of CHAF1B were consistently and markedly upregulated in NPC tissues compared with those in nasopharyngeal epithelial tissues (Fig. 1G and H). These results suggested that CHAF1B expression is substantially increased in NPC. 3.2. Upregulation of CHAF1B is associated with radioresistance and poor prognosis in NPC

3.4. CHAF1B suppresses radiation-induced apoptosis in NPC

Next, IHC staining of 160 archived NPC tissues was used to assess the clinical significance of high CHAF1B expression (Table 1). Fig. 2A shows the staining intensities of CHAF1B expression (0–3). Fig. 2B shows the number of cases with all staining intensities. Subsequent analysis revealed higher CHAF1B expression in advanced malignant tumors (clinical stage III–IV, T2–4, and N1–3) compared with that in early stage tumors (clinical stage I, T1, and N0) (Fig. 2C). Moreover, there was no association of age or histological type with CHAF1B expression; however, CHAF1B expression correlated closely with gender (P < 0.001),T (P < 0.001), and N classification (P < 0.001), clinical stage (P = 0.004), recurrence (P = 0.016), distant metastasis (P = 0.006), disease progression (P = 0.004), and vital status (P = 0.014) (Table 2). Besides, the radiosensitivity of patients with high expression of CHAF1B was weaker than that with low CHAF1B (50.0 %, 67/134 vs. 80.8 % 21/26; P = 0.004; Fig. 2D). Importantly, Kaplan–Meier survival curves and log-rank tests revealed that patients with high CHAF1B expression had a shorter PFS (P < 0.01) and LRFS (P < 0.05) (Fig. 2E). In addition, CHAF1B expression was identified as an independent prognostic factor for 5-year PFS and 5-year LRFS in NPC upon multivariate Cox regression analysis (Fig. 2F and Table 3). Thus, overexpression of CHAF1B might contribute to NPC progression and radioresistance, which result in poor clinical outcome.

Apoptosis is a critical cellular response to radiotherapy; therefore, we examined the apoptosis rate of NPC cells after irradiation treatment using flow cytometry. CHAF1B overexpression significantly reduced radiation-induced apoptosis in 5-8 F cells compared with that of 5-8Fvector control cells. In the 5-8 F groups, the apoptosis rates of the vector, CHAF1B overexpression, 6 Gy irradiation treatment, and the combination of CHAF1B overexpression and 6 Gy irradiation treatment groups were 4.06 ± 0.35, 4.13 ± 0.33, 25.34 ± 1.04, and 17.40 ± 1.14 %, respectively (Fig. 4A and B). In addition, CHAF1B silencing markedly increased cell apoptosis in SUNE1 cells compared with that in SUNE1-scramble-transfected cells. In the SUNE1 groups, the apoptosis rates of the scramble, shCHAF1B, 6 Gy irradiation treatment, and the combination of shCHAF1B and 6 Gy irradiation treatment groups were 2.64 ± 0.31, 3.47 ± 0.32, 12.37 ± 1.14, and 19.48 ± 0.95 %, respectively (Fig. 4C and D). Therefore, CHAF1B suppresses radiation-induced apoptosis in NPC. 3.5. CHAF1B induces NPC cell resistance to radiotherapy in a xenograft model We used a xenograft mouse model to further verify whether CHAF1B could promote NPC radioresistance in vivo. Control cells and CHAF1B overexpressing or silenced cells were injected subcutaneously and separately into the left and right front flank regions of the mice. On the 24th day after exposure to irradiation, the IVIS imagining system displayed that the CHAF1B upregulated group had stronger fluorescence signal than the control group. By contrast, knockdown of CHAF1B significantly decreased the fluorescence signal (Fig. 5A). Consistently, CHAF1B overexpressing tumors grew faster than those in the control group after exposure to irradiation, whereas CHAF1B knockdown in SUNE1 cells exhibited contrasting results (Fig. 5B). The tumors were then excised, imaged, and weighed. The tumors in the CHAF1B

3.3. CHAF1B induces radioresistance in NPC Cell lines 5-8 F, SUNE1, and HK1 were irradiated with X-ray doses of 2 Gy every other day for 40 days, resulting in 20 total irradiation treatments. The presence of surviving cells identified radioresistant cell lines (5-8F-IR, SUNE1-IR, and HK1-IR). Increased mRNA and protein levels of CHAF1B were observed in the radioresistant cell lines compared with that in the maternal cells (Fig. 3A). Thus the above results suggested that CHAF1B might contribute to radioresistance. To further investigate the effect of CHAF1B on the radiosensitivity of NPC cells, we stably overexpressed CHAF1B in 5-8 F cell line, which 10

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human malignancies, acting as an oncogene, and is associated with tumor progression and poor prognosis in, for example, glioma, salivary gland tumors, and melanoma [16–20,26–28]. In addition, CHAF1B was used as an indicator of predictive therapeutic effects in prostate cancer [17]. The results of the present study showed that CHAF1B is markedly upregulated in NPC. In addition, correlation analysis showed that higher CHAF1B protein levels predicted higher TNM stages, weaker radiosensitivity, and shorter PFS and LRFS in patients with NPC. Furthermore, upregulation of CHAF1B increased the viability and clonal formation ability of NPC cells after exposure to radiation. In contrast, silencing of CHAF1B decreased cell viability and clonal formation ability. This was consistent with previous research, in which deletion of CHAF1B resulted in increased sensitivity to ultraviolet radiation of Saccharomyces cerevisiae cells [29]. Our results indicated that CHAF1B has a critical role in cancer development and progression, and might serve as a biomarker to predict the therapeutic effect of radiation in cancer. CHAF1B knockdown promoted programmed cell death in proliferating cells [30]. Recently, CHAF1B knockdown was reported to markedly promote apoptosis via the p53/cBAK/BCL2/caspase-3 pathway in lung cancer [22]. Nevertheless, we did not find a difference in the apoptosis rate between untreated CHAF1B overexpressing cells and control NPC cells. Interestingly, in the case of exposure to radiation, CHAF1B overexpression significantly inhibited radiation-induced apoptosis in NPC cells, whereas CHAF1B knockdown significantly increased radiation-induced apoptosis. This controversial phenomenon may be tissue-specific. The DNA damage response (DDR) signal transduction pathway is complex, and can sense DNA damage and transmit this information to the cell, ultimately affecting the cell’s responses to damaged DNA [31]. In human cells, the CAF-1 complex was recruited to chromatin in response to ultraviolet radiation, which confirmed the importance of CAF-1 in DNA repair [21]. Further research showed that once DNA damage was sensed by the DNA repair system, CAF-1-proliferating cell nuclear antigen (PCNA)-mediated chromatin assembly pathway was immediately activated [32]. However, as a subunit of the CAF-1 complex, the role of CHAF1B in this process is unclear. Recently, a study revealed that DNA double strand breaks (DSBs) accumulated and γH2AX levels increased in cells lacking CHAF1B [30]. In the present study, GSEA and γH2AX foci number analysis demonstrated that CHAF1B could accelerate DNA damage repair after radiation treatment. Moreover, CHAF1B overexpression significantly activated BAX/BCL2/ caspase-3 signaling pathways in NPC cells. Therefore, our observation further supported the role of CHAF1B in DNA damage repair and cell apoptosis. DNA-dependent protein kinase (DNA-PK), a molecular sensor of DNA damage, is involved in DSB repair. In practical terms, lupus Ku autoantigen protein 70 (Ku70) and Ku80 proteins rapidly localize to DSBs, where they recruit and activate the DNA-PK catalytic subunit, DNA-PKcs, to initiate non-homologous end joining (NHEJ) or homologous recombination. During the process, DNA-PKcs recruits proteins related to DNA damage repair [33]. Our research further revealed the close relationship between high CHAF1B expression and the DNA-PK pathway. DNA-PKi application eliminated the acceleration of DNA damage repair and the inhibition of apoptosis induced by the upregulation of CHAF1B after radiation treatment. As one of the two families of kinases responsible for the phosphorylation of CHAF1B, DNA-PK plays an important role in the regulation of CHAF1B, hyperphosphorylation is correlated with CHAF1B displacement from chromatin during mitosis. Protein phosphatase 1 is responsible for dephosphorylation of CHAF1B [34]. All these phosphorylation events and dephosphorylation events might be regulators of CHAF1B activation. And this mechanism regulating CHAF1B that occurs during DSB repair might participate in the CHAF1B induced radioresistance in NPC. This study revealed that CHAF1B’s promotion of DNA damage repair after radiation depends on the DNA-PK pathway.

overexpression group were significantly larger than those in the control group. Conversely, knockdown of CHAF1B had the opposite effect (Fig. 5C). In addition, the weight of tumors was consistent with the above results (Fig. 5D). Furthermore, we performed TUNEL staining of the tumor tissues. As shown in Fig. 5E, tumors formed by CHAF1Boverexpressing NPC cells showed dramatically decreased radiation-induced apoptosis in NPC, as indicated by a lower percentage of TUNELpositive tumor cells as compared with control tumors, whereas tumors formed by CHAF1B-knockdown NPC cells showed increased radiationinduced apoptosis, as indicated by a higher percentage of TUNEL-positive tumor cells as compared with control tumors. Taken together, these results suggested that CHAF1B enhances radioresistance in vivo. 3.6. The DNA-PK pathway is crucial for CHAF1B to promote DNA repair Next, GSEA using the GSE12452 NPC samples was performed to gain a better understanding of the mechanisms underlying CHAF1Binduced NPC radioresistance. DNA repair gene modules (REACTOME DNA REPAIR and REACTOME DOUBLE STRAND BREAK REPAIR) were significantly enriched in samples with high expression of CHAF1B (Fig. 6A). Then, in irradiated NPC cells, we used immunofluorescence staining to assess γH2AX, a sensitive marker of DNA damage [25]. The number of γH2AX foci increased significantly in the 5-8F-vector, 5-8FCHAF1B, SUNE1-scramble, and SUNE1-shCHAF1B cells after 6 Gy irradiation treatment and reached a peak at 1 h. However, the number of γH2AX foci in 5-8F-CHAF1B cells was markedly lower than that in 5-8Fvector cells at 12 h after radiation treatment. Meanwhile, the number of γH2AX foci in SUNE1-shCHAF1B cells was significantly higher than that in SUNE1-scramble cells (Fig. 6B). These results suggested that CHAF1B might accelerate the repair of DNA damage after radiation treatment in NPC. In addition, GSEA also indicated significant enrichment of the DNAPK pathway (PID_DNA_PK_PATHWAY) in samples with high expression of CHAF1B (Fig. 6C). To explore the function of the DNA-PK pathway, the cells overexpressing CHAF1B were pretreated with DNA-PK inhibitor (DNA-PKi) before exposure to irradiation. Remarkably, the acceleration of DNA damage repair by CHAF1B overexpression was eliminated (Fig. 6D). At 12 h post-irradiation, western blotting analysis showed that the protein level of γH2AX was lower in the CHAF1B overexpressing cells compared with that in the controls, which supported the increased DNA damage repair by CHAF1B. Besides, treatment with DNA-PKi counteracted the decrease in the γH2AX level caused by CHAF1B upregulation. In addition, CHAF1B overexpression in NPC cells resulted in increased levels of anti-apoptosis proteins, such as BCL2, as well as decreased levels of pro-apoptosis proteins BAX and cleaved caspase-3. Similarly, the above phenomena could be reversed using DNA-PKi (Fig. 6E). Thus, the DNA-PK pathway is essential for CHAF1B to promote DNA damage repair in NPC. DNA-PKi could be an effective radiosensitizer in NPC cells, and might play a critical role in enhancing radiation-induced cell death, especially for cells with upregulated CHAF1B expression. 4. Discussion Although there have been recent notable advances in diagnosis and treatment, many patients with NPC still suffer from therapeutic failure caused by radioresistance [2]. Thus, exploring targets to enhance radiosensitivity could represent a promising strategy to treat NPC. In the present study, we first investigated the expression and function of CHAF1B in NPC. The results indicated that upregulated expression of CHAF1B induces radioresistance by promoting DNA damage repair, which depends on the DNA-PK pathway (Fig. 6F). On the one hand, CHAF1B might serve as a novel factor to predict radioresistance. On the other hand, DNA-PKi could serve as a radiosensitizer for patients with NPC and high CHAF1B expression. CHAF1B, a subunit of the CAF-1 complex, is overexpressed in many 11

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A limitation of this study is that we have not studied how CHAF1B specifically regulates the downstream DNA damage repair pathway, which deserves further study.

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5. Conclusion The results of this study suggested that CHAF1B is significantly upregulated in NPC and correlated with a worse outcome. Furthermore, CHAF1B enhances radioresistance by promoting DNA damage repair and inhibiting cell apoptosis, in a DNA-PK pathway-dependent manner. These findings not only increase our understanding of the biological basis of NPC radioresistance, which includes the precise role and molecular mechanism of CHAF1B, but also suggests a novel therapeutic strategy against NPC. Contribution M.D. and M.W. contributed equally to this manuscript. C.Z., C.L. and Y.L. designed the experiments; M.W., Y.O. and M.W. provided research materials and methods; H.H., Y.O. and X.C. conducted experiments; L.W., B.C. and J.M. analyzed data; M.D. and Y.J. wrote the manuscript. All authors read and approved the final manuscript. Declaration of Competing Interest The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China [grant number 81872469]. References [1] M.F. Ji, W. Sheng, W.M. Cheng, M.H. Ng, B.H. Wu, X. Yu, K.R. Wei, F.G. Li, S.F. Lian, P.P. Wang, W. Quan, L. Deng, X.H. Li, X.D. Liu, Y.L. Xie, S.J. Huang, S.X. Ge, S.L. Huang, X.J. Liang, S.M. He, H.W. Huang, S.L. Xia, P.S. Ng, H.L. Chen, S.H. Xie, Q. Liu, M.H. Hong, J. Ma, Y. Yuan, N.S. Xia, J. Zhang, S.M. Cao, Incidence and mortality of nasopharyngeal carcinoma: interim analysis of a cluster randomized controlled screening trial (PRO-NPC-001) in southern China, Ann. Oncol. 30 (10) (2019) 1630–1637. [2] P. Blanchard, A. Lee, S. Marguet, J. Leclercq, W.T. Ng, J. Ma, A.T. Chan, P.Y. Huang, E. Benhamou, G. Zhu, D.T. Chua, Y. Chen, H.Q. Mai, D.L. Kwong, S.L. Cheah, J. Moon, Y. Tung, K.H. Chi, G. Fountzilas, L. Zhang, E.P. Hui, T.X. Lu, J. Bourhis, J.P. Pignon, Chemotherapy and radiotherapy in nasopharyngeal carcinoma: an update of the MAC-NPC meta-analysis, Lancet Oncol. 16 (2015) 645–655. [3] L.L. Tang, Y.P. Chen, Y.P. Mao, Z.X. Wang, R. Guo, L. Chen, L. Tian, A.H. Lin, L. Li, Y. Sun, J. Ma, Validation of the 8th edition of the UICC/AJCC staging system for nasopharyngeal carcinoma from endemic areas in the intensity-modulated radiotherapy era, J. Compr. Canc. Netw. 15 (2017) 913–919. [4] J.J. Pan, W.T. Ng, J.F. Zong, L.L. Chan, B. O’Sullivan, S.J. Lin, H.C. Sze, Y.B. Chen, H.C. Choi, Q.J. Guo, W.K. Kan, Y.P. Xiao, X. Wei, Q.T. Le, C.M. Glastonbury, A.D. Colevas, R.S. Weber, J.P. Shah, A.W. Lee, Proposal for the 8th edition of the AJCC/UICC staging system for nasopharyngeal cancer in the era of intensitymodulated radiotherapy, Cancer 122 (2016) 546–558. [5] C. Qu, Y. Zhao, G. Feng, C. Chen, Y. Tao, S. Zhou, S. Liu, H. Chang, M. Zeng, Y. Xia, RPA3 is a potential marker of prognosis and radioresistance for nasopharyngeal carcinoma, J. Cell. Mol. Med. 21 (2017) 2872–2883. [6] C. Zhao, J. Miao, G. Shen, J. Li, M. Shi, N. Zhang, G. Hu, X. Chen, X. Hu, S. Wu, J. Chen, X. Shao, L. Wang, F. Han, H. Mai, M.L.K. Chua, C. Xie, Anti-epidermal growth factor receptor (EGFR) monoclonal antibody combined with cisplatin and 5fluorouracil in patients with metastatic nasopharyngeal carcinoma after radical radiotherapy: a multicentre, open-label, phase II clinical trial, Ann. Oncol. 30 (2019) 637–643. [7] Y. Wang, W. Chen, J. Lian, H. Zhang, B. Yu, M. Zhang, F. Wei, J. Wu, J. Jiang, Y. Jia, F. Mo, S. Zhang, X. Liang, X. Mou, J. Tang, The lncRNA PVT1 regulates nasopharyngeal carcinoma cell proliferation via activating the KAT2A acetyltransferase and stabilizing HIF-1alpha, Cell Death Differ. (2019). [8] Y. He, Y. Jing, F. Wei, Y. Tang, L. Yang, J. Luo, P. Yang, Q. Ni, J. Pang, Q. Liao, F. Xiong, C. Guo, B. Xiang, X. Li, M. Zhou, Y. Li, W. Xiong, Z. Zeng, G. Li, Long noncoding RNA PVT1 predicts poor prognosis and induces radioresistance by regulating DNA repair and cell apoptosis in nasopharyngeal carcinoma, Cell Death Dis. 9 (2018) 235. [9] Tetrandrine enhances radiosensitivity through the CDC25C/CDK1/cyclin B1

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