Background
Cervical cancer is one of the leading causes of cancer related-death in women worldwide [
1,
2]. Radiotherapy, alone or in combination with chemotherapy, is the mainstream for the cure of cervical cancer [
3‐
5]. Nearly 80% cervical cancer patients received radiation therapy in the process of treatment. However, overall survival was still heavily hampered by radioresistance [
6‐
8]. Thus, discovering and identifying the novel targets of tumorigenesis and radioresistance is of importance for cervical cancer treatment.
Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of telomerase whichcan maintain telomeric integrity. hTERT is activated in most cancer cells but not in most normal cells [
9]. As a repair response to radiation-induced DNA damage, an increased activity of hTERT was found in human colon carcinoma, lymphoma, and myeloma cells [
10‐
13]. Previous studies have also found that hTERT could accelerate the process of EMT and stemness, which then lead to treatment resistance, metastasis, and recurrence [
14]. hTERT is regulated at multiple molecular levels, among which, transcriptional modulation is the most important [
15]. Identification of novel cellular factors, which modulate hTERT in transcriptional process, deserves comprehensive research. In this study, we have first identified high-mobility group box 3 (HMGB3) as a new regulator which specifically bind to hTERT promoter and transcriptionally activate hTERT expression in cervical cancer radioresistant cells.
The high-mobility group (HMG) super family is the most abundant nonhistone proteins in the eukaryotic nucleus [
16]. HMG-Box family plays a significant role in DNA replication, transcription, recombination and repair. HMGB1 and 2 can bind to DNA without sequence specificity, and promote the formation of nucleoprotein complex. They can also directly interact with DNA-binding proteins and then affect transcription. HMGB3 has 80% identity with HMGB 1, 2 and may share some functions with them [
17]. Previous studies revealed that HMGB3could promote tumor development and maintain dedifferentiation in urinary bladder cancer, oesophageal squamous cancer, gastric cancer, non-small cell lung cancer, breast cancer and hematopathy [
18‐
21]. Nevertheless, there has not yet been a report about the biological function of HMGB3 in regulating cervical cancer radioresistance.
In this study, we examined the regulation of HMGB3 on cervical cancer cell proliferation and apoptosisafter exposing to radiation, and also explored its underlying mechanism including of DNA damage pathways. Moreover, we demonstrated that HMGB3 induced radioresistance by transcriptionally activating hTERT. Finally, we also analyzed the relationship between HMGB3/hTERT signalling axis and clinical outcome in the cervical cancer patients. Our findings demonstrate that targeting the HMGB3/hTERT axis may be a potential promising for the treatment of cervical cancer.
Methods
Patients and tissue samples
This study was approved by Ethics Committee of The Second Affiliated Hospital of Dalian Medical University. Fifty three patients diagnosed with cervical squamous cell carcinoma were recruited. Before the end of the statistics, all the patients did not undergo surgical resection, and all the patients received standard concurrent chemoradiotherapy. All patients signed informed consents before being enrolled into research and we comply with the Declaration of Helsinki when conducting the study. Cervical cancer tissue microarrays containing 119 patients were purchased from OutdoBiotech (Shanghai, China). The tumor tissue samples (tumor and adjacent tissue) were took from patients who had not undergone anti-tumor therapy since diagnosis, with all of the information frompatients authenticated.
Cell lines and cell culture
The human cervical cancer cell lines HeLa, SiHa, C33A, DoTc2, HeLa S3, and CaSki were obtained from American Type Culture Collection (ATCC, VA). HeLa, SiHa, and C33A cells were specifically cultured in Eagle’s minimal essential medium (EMEM) containing 10% foetal bovine serum (FBS), and DoTc2, HeLa S3, and CaSki cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS. All the cells were cultured in a humidified atmosphere with 5% CO2 at 37 °C.
The following human cervical cancer cell lines were plated in 6 cm dishes: HeLa, SiHa, C33A, DoTc2, HeLa S3, and CaSki. After 24 h, the cells reached exponential growth phase (75% density, 1 × 106 cells) and were then exposed to 15 sequential of 2 Gy/day for a total dose of 30 Gy. The subclones from the survival populations display distinct resistance to radiotherapy (here named as RR SiHa and RR HeLa) (there were no survival in the other four cervical cancer cell lines after 30 Gy radiation).
Antibodies
Anti-hTERT was purchased from Millipore. Caspase-3, Caspase-9, PARP and γH2AX antibodies were purchased from Cell Signaling Technology (MA, USA). GAPDH, β-actin, HMGB3 and antibodies were purchased from Proteintech (Wuhan, China). H3K4me3 antibody was purchased from Absin Bioscience (China).
Clonogenic survival assay
When reached exponential growth phase, the cervical cancer cell were trypsinized, counted and seeded different numbers into 60 mm culture dishes according to different radiation doses: 0 Gy (200 cells), 2 Gy (500 cells), 4 Gy (500 cells), 6 Gy (1000 cells), 8 Gy (2000 cells),10 Gy (2000 cells). After 24 h incubation, the cells adhere to the dishes and were performed radiation treatment. For 10–14 days at 37 °C with 5% CO2, the cells were fixed with methanol: glacial: acetic(1:1:8) for 10 min, and stained with 0.1% crystal violet for 30 min. Calculate the clone formation rates and survival fractions, and the obtained values were analyzed by Prism 8 software to fit the single-hit multi-target model (Y = 1-(1-exp(−n*X))^m).
Comet assay
Briefly, cells were harvested following the indicated treatments and were mixed with low-melting-point agarose. The samples were then immersed into the fresh lysis buffer (10 mM Tris-HCl, 2.5 M NaCl, 100 mM EDTA, 1% Triton X-100, and 10% DMSO) for 1 h at 4 °C and neutralized for 15 min. Then the slides were subjected to electrophoresis at 21 V for 20 min under alkaline conditions. Slides were stained with ethidium bromide solution (20 μg/ml) and photoed using a fluorescence microscope (Leica DMI4000B). We analysed 100 individual cells from each group by Comet Assay Software (CaspLab). The tail moment served as a quantitative measure of DNA damage..
High-throughput mRNA-sequence and data analysis
Cells were harvested following the indicated treatments and resuspended into RIzolreagent(Invitrogen). RNA sample quantification, qualification, library preparation and subsequent RNA sequencing were performed by Novogene Co., LTD (Beijing, China). EdgeR R package (3.12.1) was used to analyse the differential expression of the samples. Corrected P-values of 0.05 and absolute fold-changes of 2 were set as the threshold for significantly different expression.
siRNA design and transfection
The HMGB3-specific siRNAs (siRNA1, F 5′-GGAAGUGAUCAUCUCCGAUTT-3′, R 5′-AUCGGAGAUGAUCACUUCCTT-3′; siRNA2, F 5′-GGUCUUCGCCUUGAUUCAUTT-3′, R 5′- AUGAAUAAGGCGAAGACCTT-3′) and negative control siRNA were purchased from GenePharma (Shanghai, China). Transient transfection was performed by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturers’ protocols. Briefly, cervical cancer cells were plated into 96-well plates or six-well plates. The cells were transfected with siRNA 1, siRNA 2, or negative control (1–2 μg) for 6–8 h and then replaced with the fresh medium. After 12 h, the cells were exposed to radiation. At 24 h, the cells were harvested for different analysis.
Plasmid vectors and transfection
HMGB3-targeting shRNAs (sh1, sh2, sh3) and negtive control shRNA plasmids were purchased from GeneCopoeia. The lentivirus mediated vectors were transfected using Lenti-Pac HIV expression packaging kit according to the manufacturer’s instructions (GeneCopoeia). Briefly, 1.3–1.5 × 10 6 293 T cells were plated into one 10 cm culture dish and were transfected with 2.5 μg shRNA expression plasmids and 5.0μllenti-Pac HIV mix usingLipofectamine 3000 (Invitrogen). After 7-8 h, the mix was replaced with fresh complete medium. The viral supernatants were collected after 48 h and used for lentiviral infection obtaining stably transduced cells.
MTT assay
The cells were seeded in 96-well plates (2000 cells/well) and then transfected with HMGB3 specific siRNAs. After 24 h, the cells were treated with different dose of DDP, Vin and TAX. Forty-eight hours later, add MTT reagent and incubate for 3 h. Then, MTT was replaced with 150 μl of dimethyl sulfoxide (DMSO). The OD value was measured at 490 nm.
Immunofluorescence
Cells were planted in six well plate with coverslips and treatment as indicated. The coverslips were fixed and were then immersed into 0.2% Triton X-100 for 3 min. 10% bovine serum albumin was used for 30 min to eliminate the nonspecific binding. The cells were then incubated with the primary antibodies (1:200) overnight at 4 °C. After washing with PBS, the cells were incubated with corresponding secondary antibody labelled with fluoresce for 1 h at room temperature. The nucleus was stained with DAPI. The immunofluorescence was analysed by confocal laser scanning microscope (Leica).
Dual luciferase assay
The promoter of hTERT (− 1655 to + 40) was cut into 4 segments and each segment was inserted into a luciferase reporter vector pGL3. The cervical cancer cells were seeded into a 6-well plate and transfected with HMGB3 siRNA or a negative control for 6–7 h. At 24 h, the cells were transfected with the hTERT reporter or a pRL-TK control plasmid. After 48 h, the luciferase activity was measured according to the kit Dual-Luciferase® Reporter Assay System (Promega, Cat: E1910).
Chromatin immunoprecipitation assay
ChIP assays were performed as previously described [
9]. Firstly, the cells were treatment with indication and then fixed with 1% formaldehyde for 15 min at room temperature. 10% 1.25 M glycine was then added into for 5 min to prevent excessive cross linking. Scraped, collected, and sonicated the samples on ice to cleave the DNA into 100–1000 bp fragments. Then, part of the cell lysate was incubated with protein A/G agarose beads (Santa Cruz Biotechnology) and HMGB3 or IgG antibody. Part of the cell lysate was used for the DNA input. The mixture was washed and reverse cross-linked at 65 °C for 12 h. The DNA was extracted by phenol/chloroform and used as a template to PCR. The primers: F 5′- TTTCCCACCCTTTCTCGACG-3′; and R 5′-CAGCGGAGAGAGGTCGAATC − 3′.
Pulldown assay
The biotin-labelled hTERT promoter probe corresponding to − 1645 to + 40 was synthesized by Sigma (Sigma, USA) (F 5′-GACACACTAACTGCACCCAT-3′ and R 5′-ACGCAGCGCTGCCTGAAACT-3′). Nucleoprotein extract (400 μg) mixed with 4 μg of hTERT promoter probe and 45 μl of streptavidin-agarose beads (Sigma, USA) and incubated at room temperature to pull down the DNA-protein complexes. After 2 h, the complex was collected by centrifugation, and boiled at 100 °C for further analysis.
Xenograft models studies
Animal study has been approved by Animal Care and Ethics Committee of Dalian Medical University. Female nude mice (Balb/c) were obtained from Beijing Vital River Laboratory Animal Technology (Beijing, China). Mice were randomly grouped. Cervical cancer cells were implanted subcutaneously in left armpit. The mice were placed on the plate after anesthesia and was irradiated as the “Irradiation” described. After 2 weeks, mice were sacrificed, and tumours were removed. The tumour tissue was stored with 4% formaldehyde or liquid nitrogen for further analysis.
Immunohistochemical staining
The tissue was immersed in paraffin and sliced. The mice tissue slides and human cervical cancer tissue microarray was processed as follows: dewaxing, rehydration, antigen repair, blocking. After that, the slides were incubated with the primary antibodies overnight, incubated with the HRP-labeled second antibody, developed with DAB and then counterstained with hematoxylin. Staining intensity was defined as: 0 points are (−), 2–3 points are(±), 4–5 points are (+), and 6–7 points are (++). In the final evaluation,(+) (++) was judged to be positive.
Irradiation
Irradiation of cell lines was performedby an X-RAD320 instrument using an X-ray generator (dose rate: 0.40 Gy/min, target distance: 50 cm, Precision X-Ray Inc., North Branford, CT, USA).Animal irradiations were performed with6MV X-ray (2 Gy/day, a rate of 1.32 Gy/min with 320 keV (peak), 6 mA, filtered with 2 mm Al, radiated every 2 days, 12Gy totally).
Statistical analysis
Each experiment was repeated 3 times under the same conditions. Student’s t tests were used to compare the continuous variables in both test and control groups. Cumulative survival probability was calculated by Kaplan-Meier analysis. Cox-regression model was performed for multivariate analysis, hazard radios (HRs) and 95% confidence intervals (CIs). All the data in the research were analysed using SPSS 20 software (Inc., Chicago, IL). A 2-sided p-value 0.05 was defined to be significant.
Discussion
Radioresistance is commonly recognized as the crucial bottleneck for the cure of cervical cancer [
6,
28]. Therefore, it is urgent to discover and identify the exact mechanism of radiation resistance and potential targets. In this study, we have identified HMGB3/hTERT signaling axis as a new target for cervical cancer radioresistance.
The HMGB proteins are ubiquitous chromatin-associated DNA binding proteins in mammals, which acts as a DNA chaperone in transcription, replication, recombination and repair [
29,
30].An inverse relationship between HMGB expression levels and overall survival has been reported in many cancer types [
31‐
33]. HMGB proteins have 80% homology, so they may have some of the same functions [
17]. HMGB1, the most studied member of the HMGB protein family, has pleiotropic roles in cells. HMGB1 could activate the ERK1/2, MAPK, NF-kB and Akt signaling pathways by binding to the receptor for advanced glycation end products (RAGE), leading to the reprogramming of cancer cells. Also, upregulation of HMGB1 was found to contribute to radioresistance in squamous cell carcinoma by promoting chromatin modification and increasing the phosphorylation of CHK1 to activate DNA damage responses (DDR) [
33]. HMGB1 is a very important proinflammatory cytokine and can stimulate the immune system to protect against infections. Loss of the pro-inflammatory cytokine functions of HMGB1 may increase the risk of infection and lead to autophagy deficiency, contributing to inflammation [
34,
35]. HMGB3 may be more suitable as a target for cancer treatment because of the high expression and non-proinflammatory effect. To date, there is no report on the relationship between HMGB3 and radiotherapy resistance. In our study, we showed that HMGB3 was highly expressed in cervical cancer radioresistant cells and HMGB3 knockdown significantly enhanced the susceptibility of cervical cancer cells to radiation in vivo and in vitro. An inverse relationship between HMGB3 expression and radiotherapy response was also found. Our findings suggest that HMGB3 may be a new target for cervical cancer radiosensitization.
The main mechanism of radiotherapy killing tumor cells is through inducing single and double stranded DNA breaks. The ability of DNA damage detection and repair is closely related to radiosensitivity [
36,
37]. Previous studies have showed that HMGB1, HMGB2 depletion impaired radiation-induced DNA damage repair as indicated by increased γ-H2AX foci [
38,
39]. Also, HMGB1 and HMGB2 facilitate V(D)J recombination by enhance the affinity of the RAG complexes for DNA and increased the cleavage effectiveness [
40,
41]. Inhibition of HMGB3 in cisplatin-resistant ovarian cancer cells resulted in transcriptional downregulation of ATR and CHK1, subsequently attenuating the ATR/CHK1/p-CHK1 DNA damage signalling pathway [
19]. In our study, we found that down-regulation of HMGB3 significantly impaired the radiation-induced DNA damage repair and increased cell apoptosis. There are two main pathways responsible for DNA DSB repair: non-homologous end joining (NHEJ) and homologous recombination (HR). The mechanism by which HMGB3 is involved in DNA DSB repair is sti ll uncertain.Telomeres,a specialized DNA/protein structures, can protect chromosomes from end-to-end fusions and from losing coding sequences. Telomeres maintenance is crucial for the stability of chromosome [
42]. However, previous studies have revealed overactivity of telomerase is strongly associated with occurrence, metastatic potential and CSC phenotype of malignancy. Telomerase overactivity was also considered as a molecular maker of poor prognosis [
43]. hTERT can promote DNA repair and to help cells escape from cell cycle arrest and/or apoptosis, making cancer cells more resistant to chemotherapeutic or radiation therapy [
44‐
46]. Polanska et al. showed that knockout of the HMGB1 gene in mouse embryonic fbroblasts (MEFs) resulted in a decline in telomerase activity and telomere dysfunction, while overexpression of HMGB1 enhanced telomerase activity [
47]. Shaobo et al. showed that downregulation of HMGB1 breaks telomere homeostasis by changing the level of telomere binding proteins, such as TPP1 (PTOP), TRF1 and TRF2 and enhances radiosensitivity in human breast cancer cells [
48]. Our study showed that HMGB3 could bind to hTERT promoter on the region of − 902 to − 321 and induce the expression of hTERT, leading to radioresistance in cervical cancers. Because of the lack of enzymatic function, HMGB proteins facilitate a stable interactions of other transcription factors with their binding sites in DNA and participate in the regulation process [
49]. Whether HMGB3 cooperates or antagonizes with other factors to regulate hTERT needs further study.
The PI3K/Akt signalling cascade plays a crucial role in cell proliferation, growth and apoptosis. It has also been implicated in the cellular response to genotoxic damage. Activated AKT mediates the phosphorylation of hTERT, thereby enhancing telomerase activity [
50]. Activated AKT is also known to stimulate c-Myc and to lead to cytoplasmic retention of BRCA-1. BRCA-1 in turn can negatively regulate hTERT and telomerase activation, and presumably it can support the cell survival strategy [
51]. The regulatory mechanism of hTERT is complex. Whether HMGB3 cooperates or antagonizes with other factors to regulate hTERT needs further study.
A total of 53 cervical cancer patients receiving radiotherapy enrolled in our study. A significant association was found between HMGB3/hTERT expression and radiosensitivity. Patients with higher expression of HMGB3/hTERT, commonly exhibited worse short-term outcomes in response to radiotherapy. In addition, we further explored the relationship between HMGB3/hTERT expression and clinicopathologic features by means of tumor tissue microarray. The patients who had a high HMGB3 expression similarly displayed a higher percentage of high hTERT expression, hinting the potential regulation of hTERT by HMGB3 in cervical cancer. Moreover, HMGB3/hTERT expression was positively correlated to tumor stage, while negatively corelated with long-term survival. The role of HMGB3 and hTERT in cervical cancer should be better to confirm in a larger cohort.
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