Background
Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death worldwide [
1]. Liver section and transplantation comprise the first-line treatment strategy for patients with early-staged HCC. However, most of HCC patients are diagnosed at advanced stage and are ineligible for curative ablative therapies. Radiation therapy (RT) is emerging as a local ablative non-invasive treatment approach in patients with HCC. It has been reported that RT is a feasible and well-tolerated treatment for HCC patients, but confers limited tumor control benefits due to intrinsic and therapy-induced radioresistance in HCC cells [
2‐
8].
The effectiveness of RT mostly relies on lethal damage to cancer cells by inducing double-strand DNA breaks (DSBs), which then activates DNA damage response (DDR) signaling pathway, which is composed of DNA damage repair and cell cycle checkpoint regulation [
9,
10]. In response to ionizing radiation (IR)-induced DSBs, ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related protein (ATM/ATR) kinases are recruited to DSB lesion sites and become activated, which then induces phosphorylation of the histone variant H2AX (γH2AX), followed by monoubiquitination and polyubiquitination of H2AX/γH2AX. As a further consequence, DNA repair-related proteins gather at the damaged lesions and complete DNA repair [
11,
12]. Meanwhile, CHK1/2 are activated by ATM/ATR to trigger cell cycle arrest and provide sufficient time for DNA repair [
13,
14]. It is well known that dysregulation of the key factors involved in DDR causes altered radiosensitivity in cancer cells [
15‐
17]. For example, Wu et al. revealed that H2AX was required for the recruitment of ATM to DNA damage foci and H2AX deficiency enhanced radiosensitivity [
15]. Wang et al. demonstrated that MYC promoted radioresistance of nasopharyngeal carcinoma cells through transcriptional activation of CHK1 and CHK2 [
17]. However, the means by which DNA repair and cell cycle control are integrated in the DDR is largely unknown. Thus, identifying the potential key DDR-regulatory factors and understanding the underlying mechanism involved could provide a theoretical basis for HCC radioresistance.
Ubiquitin-conjugating enzyme E2T (UBE2T) is an oncogene, widely reported to be upregulated in multiple types of cancers [
18‐
21]. It is well documented that UBE2T is involved in HCC cell cycle modulation. Moreover, UBE2T was shown to display radio-sensitization effect on osteosarcoma and lung cancer cells. These reports shed light on the possibility that UBE2T might serve as a regulator of HCC radioresistance and DDR signal transduction cascade. As a member of the E2 family, which participates in conjugating ubiquitin to the substrates, UBE2T plays an important role in varied pathological processes in a E2-enzyme dependent manner. For example, UBE2T promotes breast cancer proliferation via polyubiquitinating and degrading BRCA1 [
21]. UBE2T enhances DNA crosslinking-induced damage repair by monoubiquitinating FANCD2 [
22,
23]. In this study, we explored its functional role in HCC radioresistance and the underlying mechanism.
Herein, we found that UBE2T plays a crucial role in HCC radioresistance by integrating DNA repair and cell cycle checkpoint activation, and facilitating DDR. The mechanistic study revealed that UBE2T increased H2AX monoubiquitination upon IR exposure, and then maintained CHK1 activation and promoted G2/M arrest, thus resulting in increased DDR and HCC radioresistance. Importantly, clinical evidence revealed that a correlation between UBE2T level with the response of HCC patients to RT. In summary, our findings contribute to the better understanding of HCC radioresistance and imply that targeting UBE2T might represent a therapeutic strategy for HCC radiation therapy in the future.
Methods
Cell culture and transfection
MHCC-97H, Huh7 and HEK-293 T cells were purchased from the Cell Bank of Type Culture Collection (CBTCC, Chinese Academy of Sciences, Shanghai, China). Cells were cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) at 37 °C incubators with 5% CO2. All transfections were conducted using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instruction.
Clinical cohort
Thirty fresh HCC tissue specimens and the matched non-tumors tissues, which were obtained from at least 2 cm away from the tumor border and were proved to lack tumor cells by microscopy, were randomly collected from patients undergoing hepatectomy at the Nanfang Hospital affiliated to the Southern Medical University (Guangzhou, China). Another 133 HCC tissue specimens for IHC who had undergone surgery were collected at the same bank from January 2006 to July 2012. Each sample was histologically and clinically diagnosed at the Nanfang Hospital. All the patients were followed up for 5 years. Clinical information about the patients is described in detail in. Table
S1–2. The disease-free survival (DFS) was defined as the interval between the date of diagnosis and the date of relapse, death, or the last observation point. The overall survival (OS) was defined as the interval between diagnosis and death or the last follow-up examination.
Cells (400–20,000/well) were plated in 6-well plates and treated with different doses of IR (2–8 Gy). The cells were cultured for 14 days, then fixed in methanol, and stained with crystal violet. The colonies (with > 50 cells) visible to the eyes were counted. The plating efficiency (PE) was defined as the number of formed colonies/the number of seeded cells × 100%. The survival fraction is the colonies at one IR dose divided by the number of colonies with a correction for the PE. All related data were analyzed in GraphPad Prism 7 software, and survival curves of the clone formation assays were calculated using the single-hit multi-targeted model (y = 1-(1-exp(−k*x))ˆN).
In vivo mice model
Nude mice (4–6 weeks old) were obtained from the Southern Medical University Animal Center. Ectopic tumors were established by subcutaneous injection of MHCC-97H cells (1 × 107). Tumor volumes were calculated using a standard formula: length × width2/2. IR (6 Gy/day × 3 days) was administered to mice when tumor volume reached 100 mm3. Mice were divided into 4 groups (n > 8/group): control, UBE2T overexpression, control + IR and UBE2T overexpression + IR. For MK-8776 experiment, once tumor reached 300 mm3, mice were injected by MK-8776 (50 mg/kg; Sellect) intraperitoneally for 3 days. Tumors were monitored for 3 weeks. The study was approved by the Animal Care Committee of Southern Medical University.
RNAi targeting sequence
SiRNAs were synthesized by GenePharma (Suzhou, China). Cells were transfected with the indicated siRNA using Lipofectamine® RNAiMAX (Invitrogen, USA) according to the manufacturer’s instructions. The sequences of siRNAs were as follows:
-
UBE2T (5′-GCUGACAUAUCCUCAGAAUTT-3′),
-
CHK1 (5′-GCAGUGAAGAUUGUAGAUATT-3′),
-
H2AX(5′-TGCTGCGGAAGGGCCACTA-3′),
-
RNF8(5′-GCTAGAGAATGAGCTCCAA-3′),
-
TRIM21 (5′-GCTGCAGGAGGTGATAATT-3′),
-
BMI1(5′-ATGAAGAGAAGAAGGGATT-3′),
-
RING2(5′-GGCUAGAGCUUGAUAAUAATT-3′).
Constructs
The full-length cDNA of UBE2T, H2AX, RNF8 and TRIM21 were subcloned into N-terminal pFLAG-CMV expression vector, respectively. The mutant constructs of UBE2T and H2AX were generated using KOD -Plus- Mutagenesis Kit (Toyobo, Japan). The constructs were confirmed by DNA sequencing. Lentivirus and adenovirus were purchased from GeneChem Company (Shanghai, China) and Vigene Corporation (Shandong, China).
Immunoblotting
Cells were harvested, lysed in RIPA buffer containing protease inhibitors (MCE, Shanghai, China). Cell extracts were separated on 10% SDS-PAGE gels, transferred to PVDF membranes (Millipore), blocked with Tris-buffered saline/Tween 20 (TBST) containing 5% skim milk for 1 h at room temperature, and then probed with the indicated primary antibody at 4 °C overnight. After three washes, the membranes were incubated with a 1:5000 dilution of HRP-conjugated secondary antibodies for 1 h at room temperature. The immunoblots were detected using ECL (Thermo) according to the manufacturer’s protocol.
Antibodies and reagents
The sources of antibodies against the following proteins were as follows: H2AX (D17A3; 7631), γH2AX (Ser139, 20E3; 9718), p-ATR (2853), p-ATM (13050), and p-CHK1 (Ser345, 133D3; 2348) from Cell Signaling Technology; UBE2T (10105–2-AP), CHK1 (25887–1-AP), RNF8 (14112–1-AP), TRIM21 (12108–1-AP), BRCA1 (22362–1-AP), RAD51 (14961–1-AP), BMI1 (10832–1-AP), RING2 (16031–1-AP), ATR (19787–1-AP), ATM (27156–1-AP), HSP70 (10995–1-AP) and LaminB1 (12987–1-AP) from Proteintech Group; H2AX (ab229914) from Abcam; FLAG (F1804) from Sigma-Aldrich; GAPDH (RM2002), HA (RM1004), myc (RM1003) and β-actin (RM2001) from Rui Antibody Biotech. MK-8776 (S2735) was purchased from Sellect. Cycloheximide was purchased from Sigma-Aldrich.
Immunoprecipitation, silver staining and mass spectrometry
Lysates from the cells transfected with indicated plasmids were prepared by incubating the cells in lysis buffer containing protease inhibitor cocktail and phosphatase inhibitor (MCE, Shanghai, China). The protein was immunoprecipitated with primary antibody and the protein complexes were eluted from agarose beads. The elutes were collected and visualized on 12% Tris-Glycine SDS gel, followed by silver staining using a silver staining kit (Thermo Fisher Scientific, USA). The distinct protein bands were retrieved and analyzed by LC-MS/MS.
Comet assay
DSB repair was analyzed by a single-cell gel electrophoresis assay using the Trevigen’s Comet Assay kit (4250–050-K) according to the manufacturer’s instructions. Briefly, cells were collected at the indicated timepoints after 4 Gy IR, immobilized in a bed of low-melting-point agarose on the CometSlides. Cells were lysed, and the remaining nucleoids were subjected to electrophoresis and subsequent staining with SYBR Gold. The presence of comet tails was examined with Olympus BX63 fluorescence microscope. Tail moment was calculated by using CASP software.
Immunofluorescence staining analysis
Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min, and then permeabilized in 0.5% Triton X-100 with 5% BSA in PBS for 20 min. The fixed cells were incubated overnight at 4 °C with primary antibodies against UBE2T, γH2AX, CHK1, RNF8, myc and FLAG, and then with fluorescein isothiocyanate-conjugated goat anti-mouse IgG or Cy3-conjugated goat anti-rabbit IgG (1:100) (Bioworld, China), and mounted with DAPI. An Olympus BX63 fluorescence microscope or a Carl Zeiss LSM880 confocal microscope was used for visualization.
Cell cycle FACS analysis
The cells were washed with PBS and fixed by 70% ethanol at − 20 °C. Then, the cells were washed and incubated for 15 min at 37 °C in propidium iodide. The stained cells were analyzed using cytofluorimeter ATC300 (Bruker, France) equipped with an argon laser tuned at 488 nm.
Chromatin fractionation
Cells were washed in PBS, and resuspended in Buffer A (10 mM Hepes pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 5 mM NaF, 1 mM Na3VO4, 1 mM DTT, and protease inhibitor mixture) containing 0.1% Triton X-100, and incubated on ice for 5 min for permeabilization. The cytosolic fraction was then separated by centrifugation at 4000 rpm for 5 min at 4 °C. The supernatant was discarded, and the nuclei pellet was washed once with Buffer A, and resuspended in Buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, protease inhibitor mixture), and incubated for 30 min on ice. The soluble nuclear fraction was separated by centrifugation at 4500 rpm for 5 min. The chromatin fraction pellet was washed with Buffer B and resuspended in 100 μL sample buffer and sonicated for 10 s before analysis.
Immunohistochemistry staining
Immunohistochemistry (IHC) staining was carried out using a Dako Envision System according to the manufacturer’s protocol. The IHC-stained tissue section was scored by two pathologists blinded to the clinical parameters, respectively. The score of staining intensity was defined as following: 0 (negative), 1 (weak), 2 (medium), 3 (strong). The extent of staining was scored as 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), 4 (76–100%), according to the percentages of the positive staining areas in relation to the entire carcinoma-involved area or the entire section for the normal samples. We regarded the sum of the intensity and extent scores as the final staining score (0–7) for UBE2T. For the purpose of statistical evaluation, tumors with a final staining score of ≥3 were considered to be high expression.
Public database
Public TCGA (
https://portal.gdc.cancer.gov/) data repositories for liver hepatocellular carcinoma (LIHC) (Cancer Genome Atlas Network, 2014) were used as the sources for the sample data. For the analysis of the LIHC TCGA sets, we used mRNA expression (by RNA sequencing).
Gene set enrichment analysis (GSEA)
The GSEA assay using the TCGA cohort was performed to assess whether UBE2T expression is significantly correlated with certain predefined sets of genes. TC2 (c2.cp.kegg.v7.0.symbols.gmt) from the Molecular Signatures Database (MSigDB) was used as the reference gene set. UBE2T expression was annotated as a high- or low-UBE2T phenotype. A permutation number of 1000 was adopted. Results with a P-value less than 0.05 were regarded significant.
Statistics
Experiments were repeated independently at least three times. Data are shown as mean ± standard deviation (SD). Student’s t-test and one-way ANOVA test were performed for comparing differences. Survival curves were plotted by the Kaplan-Meier method and compared by the log-rank test. The effects of variables on survival were determined by univariate and multivariate Cox proportional hazards model. P < 0.05 was considered significant.
Discussion
Our study suggested a radioresistant function for UBE2T in promoting the DDR and radioresistance in HCC cells. This occurs by UBE2T regulating IR-induced H2AX monoubiquitination, leading to the activation of CHK1 and cell cycle arrest (Fig.
8c). It is well demonstrated that a finely coordinated DNA repair and activation of cell cycle checkpoints, which are the two major components of DDR has evolved to cope with IR-induced DSBs. Abnormal activation of DDR has been reported to be responsible for radioresistance in cancer cells. As for the signaling transduction of DDR, ATM and ATR are well known to have crucial roles in responding to and passaging signaling in both DNA repair and checkpoint arrest pathways. ATM/ATR activates DNA repair by phosphorylating H2AX and recruiting DNA repair-related factors to the DSB lesions, while also activating cell cycle checkpoints by phosphorylating CHK1/CHK2 and initiating cell cycle arrest. Despite numerous studies have revealed the role of dysfunctional DNA repair and checkpoint arrest in tumor radioresistance, the crosstalk between these two pathways and the co-regulatory factors involved were largely unknown. H2AX deficiency was reported to impair the recruitment of DNA damage checkpoint protein 1 (MDC1) to DNA damage sites and G2/M cell cycle arrest, thus increasing radiosensitivity. These reports shed light on the possibility that H2AX is required for cell cycle checkpoint activation. In our study, we revealed a novel functional interaction between H2AX/γH2AX monoubiquitination and cell cycle modulation in the IR-induced DDR, and this process is regulated by UBE2T. As the subsequence, UBE2T facilitated sufficient cell cycle arrest, which provides abundant time for DNA repair, and then promotes the recovery of γH2AX to normal baseline levels.
Accumulating evidence has highlighted the function of the post-translational modification of H2AX. Besides phosphorylated H2AX (γH2AX), ubiquitinated H2AX is emerging as an important transducer of DDR signaling [
31‐
33]. Increasing studies have shown that the monoubiquitination of H2AX plays a central role in regulating the cellular response to DSBs [
34,
35]. A previous study showed that the monoubiquitination of H2AX, in the presence of DSBs, promotes DDR by facilitating the recruitment of MDC1 to DNA damage foci [
15,
36]. Although it is widely known that increased DDR is closely associated with intrinsic radioresistance in cancer cells, there is very little data indicating a role for H2AX monoubiquitination in the radioresistance of HCC cells. As shown here, overexpressing WT H2AX but not monoubiquitination-deficient K119/120R H2AX protected HCC cells from IR in response to DNA damage, compared to that in control cells, which indicated that H2AX monoubiquitination plays an essential role in facilitating DNA damage signal passage and inducing radioresistance in HCC cells. Similar with our data, Wu et al. reported that the restoration of H2AX WT in H2AX
−/− mouse embryonic fibroblasts causes the resistance of the cells to IR compared to that mock restoration, whereas a K119/120R H2AX mutation compromised this effect [
15]. Although this study used a non-mammalian cell system, it provided a rationale for the hypothesis that H2AX monoubiquitination has a critical role in IR sensitivity, and in our study, for the first time, we confirmed this in HCC cells.
Recent studies have illustrated the diversity of factors that regulate H2AX monoubiquitination [
15,
36‐
38]. Among them, BMI1 and RING2, which form an E3 ligase complex, were identified as required for the accumulation of monoubiquitylated γH2AX, which is a prerequisite for γH2AX formation [
15]. The other major E3 ubiquitin ligase, RNF8, was also reported to be responsible for H2AX monoubiquitination [
37,
38]. In our study, we found that the knockdown of RNF8 resulted in a dramatic decrease in H2AX/γH2AX monoubiquitination. However, it should be noted that some earlier reports revealed that the knockdown of BMI1 led to a further reduction in H2AX monoubiquitination in RNF8 KO cells [
39]. While in our study, we showed that UBE2T bound with RNF8, but not BMI1/RING2, and knockdown of RNF8 further decreased H2AX monoubiquitination upon knockdown of BMI1/RING2. These data suggested that RNF8, BMI1 and RING2 are all E3 ligase candidates responsible for H2AX monoubiquitination, but they may exert their roles by binding with different E2-conjugating enzymes.
Our study revealed a novel E2-conjugating enzyme, UBE2T, which interacts with RNF8 to monoubiquitinate H2AX/γH2AX. Several reports have showed that RNF8 can interact with different E2 conjugating enzymes in response to DNA damage, exerting different responses. For example, Ubc13-RNF8/RNF168 forms a K63-linked polyubiquitination chain on H2AX to recruit 53BP1 and BRCA1 to the damage sites [
40,
41]. UBE2C/UBE2S-RNF8 is responsible for Lys11-linkage ubiquitin modification, which plays a role in regulating DNA damage-induced transcription inhibition [
42]. This reflects the intrinsic property of E2 enzymes in synthesizing specific ubiquitin chain that executed different functional roles. While in our case, the novel interaction between UBE2T-RNF8 activates CHK1 and promotes G2/M arrest after IR via H2AX monoubiquitination. This observation was consistent with a previous report that RNF8-depleted cells failed to properly arrest at the G2/M checkpoint upon IR, and H2AX
−/− cells had a partially impaired G2/M checkpoint.
UBE2T was a widely reported oncogene in many types of cancers. Recently, there were several studies demonstrating the radioresistant role of UBE2T. For example, Shen et al. showed that knockdown of UBE2T has radiosensitizing role in osteosarcoma cells without exploring the underlying mechanism [
20]. Yin et al. reported that UBE2T promoted radioresistance and FOXO1 ubiquitination, and FOXO1 reversed radiation resistance in lung cancer cells. (43) However, it was not illustrated that whether UBE2T conferred the radioresistant effect via ubiquitinating FOXO1. While in our study, we demonstrated that UBE2T induced DDR and HCC radioresistance by monoubiquitinating H2AX/γH2AX. Moreover, UBE2T C86A (unable to monoubiquitinate) or H2AX K119/120R (unable to be monoubiquitinated) impaired the radioresistant efficacy of UBE2T. Inconsistent with their findings that UBE2T facilitated G2/M transition, we found that UBE2T led to stronger G2/M arrest upon IR. Although such discrepancy remains unclear, one possible explanation for it may be due to the distinct cancer types we focused.
In our cohort, UBE2T was upregulated in HCC patients and could be used as a prognostic factor, and patients with higher UBE2T levels showed worse responses to RT. Thus, UBE2T could serve as a predictive biomarker for RT response to stratify patients. Due to the significant development of new techniques, RT has emerged as an effective treatment for HCC patients at almost every stage. However, surgery remains the first option for patients with early stage HCC. In our cohort, most of the patients were diagnosed in the early stages of HCC and underwent surgery, followed by receiving RT at metastatic sites. However, we cannot exclude the possibility of heterogeneous alterations during the metastatic process. Collectively, our findings might provide new insights for understanding the DDR and radioresistance in HCC cells, and could contribute to the development of precise treatment strategies for clinical practice in the future. Future study may further screen or develop novel compounds to target UBE2T signaling pathway, which may be a new adjuvant therapy for RT in HCC.
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