Inhibiting the Redox Function of APE1 Suppresses Cervical Cancer Metastasis via Disengagement of ZEB1 from E-cadherin in EMT

Inhibiting the Redox Function of APE1 Suppresses Cervical Cancer Metastasis via Disengagement of ZEB1 from E-cadherin in EMT | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Inhibiting the Redox Function of APE1 Suppresses Cervical Cancer Metastasis via Disengagement of ZEB1 from E-cadherin in EMT qing li, Zhi-Wei Zhou, Wei Duan, Cheng-Yuan Qian, Shu-Nan Wang, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-152454/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 2 You are reading this latest preprint version Abstract Background: Metastasis is a major challenge in cervical cancer treatment. Previous studies have shown that the dual functional protein apurinic/apyrimidinic endonuclease 1 (APE1) promotes tumor metastasis and is overexpressed in cervical cancer. However, the biological role and mechanism of APE1 in cervical cancer metastasis have rarely been studied. Methods: We used gene set enrichment analysis (GSEA) to determine the APE1-related signaling pathways in cervical cancer. To investigate the role and mechanism of APE1 in cervical cancer metastasis, invasion, immunohistochemistry, immunofluorescence, western blotting, secondary structure prediction, coimmunoprecipitation, luciferase reporter, and electrophoretic mobility shift assays were performed. The inhibitory effects of the APE1 redox function inhibitor APX3330 on cervical cancer metastasis were evaluated using animal models. Results: Clinical data showed that high expression of APE1 was associated with lymph node metastasis in cervical cancer patients. GSEA results showed that APE1 was associated with epithelial to mesenchymal transition (EMT) in cervical cancer. Ectopic expression of APE1 promoted EMT and invasion of cervical cancer cells, whereas inhibition of APE1 suppressed EMT and invasion of cervical cancer cells in a redox function-dependent manner. Notably, APE1 redox function inhibitor APX3330 treatment dramatically suppressed cervical cancer cell lymph node and distant metastasis in vivo . Furthermore, we found that APE1 enhanced the interaction between ZEB1 and the E-cadherin promoter by binding to ZEB1, thereby suppressing the expression of E-cadherin, a negative regulator of EMT. Conclusion: Our findings help to elucidate the role played by APE1 in cervical cancer metastasis and targeting APE1 redox function may be a novel strategy for inhibiting cervical cancer metastasis. Cancer Biology Oncology APE1 EMT E-cadherin ZEB1 cervical cancer metastasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Cervical cancer is the third most common cancer and the second leading cause of cancer-related death in women worldwide 1 , 2 . The major challenge in cervical cancer treatment is metastasis because most of the mortality associated with cervical cancer is caused by metastasis 3 . Unfortunately, no effective therapeutic strategies exist for preventing or inhibiting cervical cancer metastasis, in part because the mechanisms that underlie metastasis are incompletely understood. Growing evidence illustrates that epithelial-mesenchymal transition (EMT) plays a key role in tumor metastasis 4 , 5 , 6 , 7 . EMT is a process in which epithelial tumor cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties 8 . Notably, the clinical data show that EMT is closely related to a poor prognosis in cervical cancer patients 9 , 10 . E-cadherin is a homotypic cell-to-cell adhesion molecule ubiquitously expressed on epithelial cells 11 . However, E-cadherin is frequently downregulated in cervical cancer 12 , and downregulation of E-cadherin is sufficient to induce EMT in tumor cells and promote tumor cell metastasis 13 , 14 . Importantly, clinical data show that low expression of E-cadherin is closely associated with metastasis and a poor prognosis in cervical cancer patients 15 , 16 . In cancer, one cause of downregulation of E-cadherin is abnormal overexpression of zinc finger E-box-binding homeobox 1 (ZEB1). ZEB1 is a transcriptional repressor that inhibits E-cadherin expression at the transcriptional level by binding to the E-cadherin promoter 17 . According to Chen et al., ZEB1 is not expressed in normal cervical epithelial cells but is expressed in most invasive cervical carcinomas, and the ZEB1 expression level is strongly associated with lymph node metastasis in cervical cancer patients 7 . Consistent with this, ZEB1 silencing inhibits cervical cancer cell EMT and metastasis 18 , suggesting that ZEB1 is a potential therapeutic target for EMT-induced cervical cancer metastasis treatment. Unfortunately, no specific ZEB1 inhibitors are available. Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1) functions both as a redox regulator of transcription factor activation and as part of the DNA damage response 19 . Notably, recent studies showed that upregulated APE1 inhibits E-cadherin expression and stimulates EMT and metastasis in non-small-cell lung cancer 20 , 21 . In addition, Wei et al. reported that upregulated APE1 was closely associated with lymph node metastasis in gastric cancer 22 . These findings suggest that APE1 may be involved in the regulation of E-cadherin-mediated EMT and tumor metastasis, but the mechanism is unclear. Aberrantly upregulated expression of APE1 has also been detected in cervical cancer 23 , but the effects of APE1 on cervical cancer metastasis have not been studied. In this study, we found that high expression of APE1 was closely associated with EMT and lymph node metastasis in cervical cancer patients. Ectopic expression of APE1 inhibited E-cadherin expression and stimulated EMT and invasion in cervical cancer cells. In contrast, inhibition of APE1 redox function significantly suppressed lymph node and distant metastasis of cervical cancer cells in vivo . Furthermore, we found that APE1 inhibited E-cadherin expression in a redox-dependent manner by enhancing the interaction between ZEB1 and the E-cadherin promoter by directly binding to ZEB1. In summary, our findings provide new insights into the underlying mechanism of metastasis in cervical cancer and provide a potential therapeutic target for metastatic cervical cancer therapy. Materials And Methods Cell culture and human specimens HeLa and SiHa cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco`s modified Eagle`s medium with 10% fetal bovine serum (HyClone, Logan, UT). Human samples were obtained from patients with cervical cancer by biopsy or surgery at Daping Hospital and Research Institute of Surgery. This study was approved by the Ethics Committee of Daping Hospital, Army Medical University. We obtained consent to publish from the participants (or legal parents or guardians for children) to report individual patient data. Plasmid constructs The ZEB1 ORF was amplified by PCR from human cDNAs and inserted into the BamH I and Hind III sites of the pcDNA3.1-flag vector for expression in mammalian cells. The E-cadherin promoter region from − 670 to + 92 was amplified using primers with restriction enzyme sites Bgl II or Hind III at each end and inserted into the upstream region of the firefly luciferase gene of the pGL3-Basic vector (Promega, Madison, WI, USA) 24 . APE1 (NM_001641.4) and ZEB1 (NM_001323642.2) expression vectors were obtained from Shanghai GeneChem Co., Ltd. (Shanghai, China). All primer sequences used in this study are given in Table S1. Immunohistochemistry (IHC), immunofluorescence (IF), and western blot For western blotting, cells were lysed in RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich, Saint Louis, MO, USA), and the protein concentration of the lysate was measured using a Bradford kit (Bio-Rad, Hercules, CA, USA). Equal amounts of proteins (30 µg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membrane was blocked for 1 hour in Tris-buffered saline with Tween 20 (TBST) containing 5% skim milk at room temperature (RT), and immunoblotting was performed by incubating the membranes overnight with their corresponding primary antibodies in 5% skim milk at 4°C. The membrane was washed with TBST and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at RT. After washing, the proteins were visualized with an enhanced chemiluminescence detection kit (Thermo Fisher Scientific) in accordance with the manufacturer’s recommendations. For IHC, the tumor tissues were fixed in 10% buffered formalin (Sigma-Aldrich), embedded in paraffin, and sectioned at 4 µM. The tissue sections were deparaffinized in xylene, rehydrated through an alcohol gradient, washed and incubated in 0.3% hydrogen peroxide (AppliChem, Darmstadt, Germany) for 15 min. After washing, the tissue sections were blocked with 5% bovine serum albumin in PBS for 1 hour. Then, primary antibodies were applied to the tissue sections overnight at 4°C. The following day, the tissue sections were washed and incubated with secondary HRP-conjugated antibodies for 1 hour at RT and counterstained with Mayer's hematoxylin (Dako, Carpinteria, CA, USA) for 10 seconds. Coverslips were mounted using Permount (Thermo Fisher Scientific). A panel of pathologists reviewed the IHC staining and scored it as follows: score 0, no staining positive tumor cells; score 1, staining positive tumor cells less than 10% of the total tumor cells; score 2, staining positive tumor cells more than 10% of the total tumor cells, but less than 50%; and score 3, staining positive tumor cells more than 50% of the total tumor cells. Scores of 0 and 1 were defined as low expression, and scores of 2 and 3 were defined as high expression. For IF, cells were grown on coverslips and transfected with the indicated oligonucleotides. After 72 hours of transfection, the cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.3% Triton X-100 for 10 min. The cells were washed with PBS and incubated with 3.5% bovine serum albumin (BSA) for 1 hour followed by incubation with the primary antibody in 3.5% BSA for 1 hour at RT. The cells were washed with PBS and incubated with FITC-conjugated secondary antibody for 1 hour at RT in the dark. Then, the cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 30 min. Antibodies against APE1, β-actin, E-cadherin, N-cadherin, vimentin, Flag, HA, and GAPDH and secondary antibodies were purchased from Abcam (Cambridge, MA, USA). The anti-ZEB1 antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). Invasion assay Cells were transfected with APE1 construct or siRNA (GeneChem Co., Shanghai, China) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Sequences of the double-stranded siRNAs are antisense (5’-GUCUGGUACGACUGGAGUACC-3’, 5’-UACUCCAGUCGUACCAGACCU-3’) and nonsense (5’-CCAUGAGGUCAGCAUGGUCUG3’, 5’-GACCAUGCUGACCUCAUGGAA-3’) 21 . Nontargeting control siRNA was purchased from Qiagen (Hilden, Germany). After 48 hours of transfection, the cells were subjected to invasion assays. Briefly, 10,000 cells in medium without serum were seeded in the upper wells of invasion chambers (BD Biosciences, San Jose, CA, USA). The lower wells contained the same medium supplemented with 10% fetal bovine serum. After 24 hours, the cells that invaded to the other side of the chamber were fixed with 2.5% glutaraldehyde, stained with 0.1% crystal violet, and counted. To investigate the effects of the APE1 redox inhibitor APX3330 (Selleck, Houston, TX, USA) and the APE1 DNA repair inhibitor APE1 inhibitor III (Merck Millipore, Molsheim, France) on the invasion of cervical cancer cells, cells were pretreated with APE1 inhibitors for 24 hours. After 24 hours, the cells were subjected to invasion assays as described above. Luciferase reporter assay Plasmids and/or nucleotides were transfected into HeLa cells that were transfected with a firefly luciferase reporter construct containing the E-cadherin promoter. The Renilla luciferase plasmid was cotransfected as a transfection control (Promega). The cell extracts were processed 72 hours after transfection, and the luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. The luciferase activity was normalized to the activity of Renilla luciferase. Co-immunoprecipitation HeLa cells were transfected with the indicated plasmids. After 72 hours, the cells were lysed in lysis buffer [1% Nonidet P40, 0.1% SDS, 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 mM DTT and 0.5 mM EDTA containing protease inhibitors], and the protein concentration of the lysate was measured using a Bradford kit (Bio-Rad). Immunoprecipitation was performed using monoclonal antibodies as indicated. Immunocomplexes were collected on protein A/G-agarose beads (Merck Millipore), washed twice with lysis buffer, and then washed twice with wash buffer [10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.2 mM sodium orthovanadate, protease inhibitor cocktail]. The beads were dissolved in SDS loading buffer (Bio-Rad), denatured at 95°C for 10 min, and later subjected to western blot analysis. Secondary structure prediction The sequence of ZEB1 was retrieved from UniProt, and the corresponding secondary structure was generated using Jpred (Jnet version: 2.3.1) ( http://www.compbio.dundee.ac.uk/jpred/ ). Electrophoretic mobility shift assay (EMSA) EMSA was performed using the LightShift chemiluminescence EMSA kit (Thermo Fisher Scientific) according to the manufacturer’s instructions as described previously 25 . Nuclear extracts were isolated from the indicated cells and incubated with 3’-biotin-labeled double-stranded oligonucleotide probes containing consensus sequences for ZEB1 binding sites (Fig. S1), then the samples were separated on a 5% polyacrylamide gel and transferred to a Zeta-Probe GT nylon membrane. The probes were detected by HRP-conjugated streptavidin. The probe sequences are shown in Table S1. Animal experiments All xenograft models were generated using GFP-expressing HeLa cells (HeLa-GFP) in 6-week-old female BALB/c nude mice. For the lymph node metastatic model, HeLa-GFP cells (1×10 6 /50 µl PBS per mouse) were directly injected into the footpad of the mice 26 . For the abdominal cavity metastatic model, HeLa-GFP cells (1×10 6 /200 µl PBS per mouse) were intraperitoneally (IP) injected into the mice. For the lung metastatic model, HeLa-GFP cells (1×10 6 /100 µl PBS per mouse) were injected intravenously into the tail vein of the mice. One week after the cell injection, the mice were randomly divided into two groups. The control group mice were treated with PBS, and the treatment group mice were treated with APX3330 (12.5 mg/kg body weight) by IP injection once every two days. The mice were treated with APX3330 for 3 weeks for the lung and lymph node metastasis experiments, treated with APX3330 for 2 weeks in the abdominal cavity metastasis experiments, and their body weight was measured every 3 days. Tumor metastasis was monitored by an IVIS Spectrum In Vivo Imaging System (Perkin Elmer, Utah, USA) and MRI (7.0-T MRI, Bruker Biospec 70/20USR, Germany). All animal experiments complied with the Daping Hospital, Army Medical University Policy on the Care and Use of Laboratory Animals. Statistical analyses All statistical analyses were performed using Prism 5.0 software (GraphPad). The unpaired two-samples t-test was used to compare the mean of two independent groups, and one-way ANOVA was used to determine differences between the means of two or more independent groups. p values less than 0.05 were considered statistically significant. The data are presented as the mean ± the standard deviation (SD) of at least three independent experiments. The correlation between APE1 expression and lymph node metastasis or E-cadherin expression was tested by the chi-square test. Results High expression of APE1 is closely associated with EMT and lymph node metastasis in cervical cancer patients To investigate whether the high expression of APE1 in cervical cancer is related to EMT and metastasis, we performed gene set enrichment analysis (GSEA) using transcriptome data from four cervical cancer samples expressing high levels of APE1 and four cervical cancer samples expressing low levels of APE1 (Fig. 1 a and Fig. S2). The GSEA results showed that the APE1 expression level was closely associated with EMT in cervical cancer (Fig. 1 b). This association was further confirmed in an expanded 72 cervical cancer patient cohort where we directly compared APE1 to E-cadherin (loss of E-cadherin is a well-established hallmark of EMT) expression using IHC (Fig. 1 c). Our data showed that among the 30 cases with low APE1 expression, 26 cases (87%) showed high expression of E-cadherin, while among the 42 cases with high APE1 expression, 27 (64%) cases presented with high expression of E-cadherin (Fig. 1 d). This result suggests that high expression of APE1 is associated with EMT in cervical cancer patients. Because EMT stimulates cancer metastasis partially through the lymphatic system 27 , 28 , we next evaluated the associations between APE1 expression and lymph node metastasis. The results showed that 69% (29 cases) of cases with high APE1 expression (42 cases) had lymph node metastasis, while only 23% (7 cases) of cases with low APE1 expression (30 cases) had lymph node metastasis (Fig. 1 e and Table 1 ). Taken together, these findings indicate that APE1 may be involved in metastasis by stimulating EMT in cervical cancer patients. Table 1 Characteristics of patients with cervical cancer Variable Number of patients (%) p value APE1 high APE1 low Age(years) ≥ 50 <50 14 (33%) 28 (67%) 8 (27%) 22 (73%) 0.54 Histological subtypes Squamous carcinoma Adenocarcinoma Adenosquamous 34 (81%) 6 (14%) 2 (5%) 27 (90%) 2 (7%) 1 (3%) 0.56 Tumor grade Poorly differentiation Moderate differentiation High differentiation 28(67%) 10(24%) 4 (9%) 14 (48%) 8 (26%) 8 (26%) 0.11 Stage Stage I Stage II Stage III 23 (55%) 14 (33%) 5 (12%) 16 (53%) 11 (37%) 3 (10%) 0.94 Lymph node metastasis Positive Negative 29 (69%) 13 (31%) 7 (23%) 23 (77%) < 0.01 Ectopic expression of APE1 stimulates EMT and the invasion of cervical cancer cells To investigate whether APE1 directly stimulates EMT in cervical cancer cells, leading to metastasis, both HeLa and SiHa cells were transfected with APE1-expressing plasmid or APE1 siRNA (Fig. 2 a) and then subjected to detection of EMT-related protein expression and invasion assays. As shown in Fig. 2 b, western blot analysis showed that ectopic expression of APE1 inhibited the epithelial cell marker E-cadherin but upregulated the mesenchymal marker proteins vimentin and N-cadherin in both HeLa and SiHa cells. In contrast, silencing APE1 upregulated E-cadherin but downregulated vimentin and N-cadherin expression (Fig. 2 b). Consistent with this, IF also showed that silencing APE1 stimulated E-cadherin expression but inhibited vimentin expression in HeLa cells (Fig. 2 c). Of functional importance, cervical cancer cell invasion was significantly increased by ectopic expression of APE1 but suppressed by silencing of APE1 (Fig. 2 d). Taken together, these findings indicate that APE1 positively regulates cervical cancer cell EMT and invasion. APE1 promotes cervical cancer cell EMT and invasion via a redox-dependent mechanism APE1 exerts functions both in the DNA repair response and redox regulation of transcription factors 19 . To investigate which function is involved in EMT regulation and invasion, cervical cancer cells were treated with the APE1 redox inhibitor (APX3330) 29 or the APE1 DNA repair inhibitor (APE1 inhibitor III) 30 and then subjected to EMT marker protein detection and invasion analysis. Our data show that APX3330 treatment increased E-cadherin expression but suppressed N-cadherin and vimentin expression in both HeLa and SiHa cells (Fig. 3 a). In contrast, APE1 inhibitor III had no effect on EMT marker expression (Fig. 3 b), despite impairing APE1 DNA repair activity (Fig. S3). Consistent with the APX3330 treatment, overexpression of mutant APE1, C65S, which lack redox function 31 , increased E-cadherin expression and decreased N-cadherin and vimentin expression compared to overexpression of wild-type APE1 in HeLa cells (Fig. 3 c). In addition, cell invasion assays also showed inhibition when challenged with APX3330 but not APE1 inhibitor III (Fig. 3 d). Together, these findings suggest that APE1 stimulates cervical cancer cell EMT and invasion via a redox-dependent mechanism. APE1 inhibits E-cadherin by stimulating ZEB1 binding to the E-cadherin promoter ZEB1 is an EMT-activating factor 32 that induces EMT through inhibiting E-cadherin expression by directly binding to the E-cadherin promoter 33 . Given that we showed that APE1 inhibits EMT by inhibiting E-cadherin expression in cervical cancer cells (Fig. 2 b), we next investigated whether APE1 is involved in ZEB1-regulated inhibition of E-cadherin expression. The results showed that overexpression of ZEB1 inhibited E-cadherin expression and it was partially restored by silencing APE1 or APX3330 treatment in HeLa cells (Fig. 4 a), indicating that APE1 was involved in ZEB1-regulated E-cadherin expression. In addition, Co-IP of Flag-ZEB1 or HA-APE1 expressed in HeLa cells showed a direct interaction between APE1 and ZEB1 (Fig. 4 b). Importantly, EMSA results showed that APE1 silencing or APX330 treatment significantly reduced ZEB1 and E-cadherin promoter binding compared to controls (Fig. 4 c), suggesting that APE1 positively regulates the interaction between ZEB1 and the E-cadherin promoter in a redox-dependent manner. As another means of confirmation, we constructed a reporter assay that relies on interactions between ZEB1 and the E-cadherin promoter to suppress the expression of luciferase in HeLa cells. Our luciferase reporter assay showed that ectopic expression of ZEB1 inhibited luciferase activity but was restored by APE1 silencing (Fig. 4 d). Taken together, these findings indicate that APE1 inhibits E-cadherin expression by enhancing interactions between ZEB1 and the E-cadherin promoter by directly binding to ZEB1. Next, to map the interaction domain of ZEB1, truncated versions of ZEB1 (Fig. 5 a) were expressed in HeLa cells, and Co-IP experiments evaluating ZEB1-APE1 interactions were performed. Among the fragments, only the ZEB1 fragment encompassing 368–739 amino acids was able to pull down APE1 (Fig. 5 b). We noted that secondary structure prediction of that region of ZEB1 suggested that residues 573–621 had a high likelihood of forming an alpha helix (Fig. S4). We considered that the alpha helix might be important for binding to APE1 and designed mutations in residues 578–580 to disrupt the binding (Fig. 5 c and Fig. S5). Co-IP showed that the mutated version of ZEB1, ZEB1 MUT , was expressed but was unable to bind to APE1 (Fig. 5 d). Additionally, we tested ZEB1 MUT in the E-cadherin promoter-driven luciferase reporter assay. As with wild type, ZEB1 MUT overexpression inhibited E-cadherin promoter-driven luciferase expression (Fig. 5 e). However, this was not rescued by APE1 silencing (Fig. 5 e), supporting that the helix formed by ZEB1 residues 573–621 is important for the interaction between ZEB1 with APE1 and resulting functional activity at the E-cadherin promoter. Inhibition of APE1 redox function suppressed lymph node and distant metastasis of cervical cancer cells in vivo Our in vitro findings suggested a therapeutic opportunity for tumors overexpressing APE1 in the form of APE1 redox inhibitors such as APX3330. We therefore investigated whether APX3330 could suppress cervical cancer cell metastasis in vivo . An established model for studying lymph node metastases in cervical cancer was generated by the injection of GFP-expressing HeLa cells into the footpad of nude mice 26 . Treatment of such mice with APX3330 showed a dramatic reduction in the incidence of nodal metastases at the 3-week mark, with 75% of the mice in the control group developing lymph node metastasis versus only 25% of the mice in the APX3330 treatment group (Fig. 6 a). MRI and pathological examination showed that 50% of the mice in the control group developed hepatic metastasis, but no hepatic metastasis was found in the APX3330 treatment group (Fig. 6 b). These results were further confirmed in models of systemic metastases, including a model of liver, colon and mesenteric metastases (Fig. 6 c) and lung metastases (Fig. 6 d). All in vivo experiments showed that APX3330 treatment did not affect the body weight of the experimental animals (Fig. S6). Taken together, these data suggest that inhibition of APE1 redox function by APX3330 may be a therapeutic strategy for the treatment of cervical cancer metastasis. Discussion Lymph node and distant metastasis are major drivers of poor outcomes of cervical cancer 10 , 34 , 35 . Thus, a comprehensive understanding of the mechanisms underlying the development of metastasis is required to optimize treatment strategies and develop new therapeutic agents for cervical cancer. A previous study has shown that EMT is a primary process that involves increased cervical cancer metastasis with loss of EMT markers, such as E-cadherin, and a gain of mesenchymal markers, such as vimentin 10 . Here, we used clinical sample analysis to indicate that high expression of APE1 is closely associated with lymph node metastasis and low expression of E-cadherin in cervical cancer patients (Fig. 1 ), and in vitro functional experiments indicated that overexpression of APE1 significantly stimulates cervical cancer cell invasion, promotes the vimentin expression, and inhibits E-cadherin expression, while silencing of APE1 dramatically inhibits cervical cancer cell invasion (Fig. 2 ). Together, these findings suggest that aberrantly increased expression of APE1 in cervical cancer promotes metastasis by stimulating EMT, and APE1 is a target for the treatment of cervical cancer metastasis. Next, we elucidated the mechanism by which APE1 stimulates EMT in cervical cancer. Studies have shown that binding to ZEB1 and a dependence on ZEB1 to inhibit E-cadherin expression is a mechanism by which oncogenes promote EMT and metastasis in cancer. For example, telomerase reverse transcriptase (TERT) stimulates EMT in colorectal cancer through inhibition of E-cadherin expression by binding to ZEB1 36 ; histone H4K20-specific methyltransferase SET8 stimulates EMT in prostate cancer by inhibiting E-cadherin transcription through binding to ZEB1 37 . Here, we found that APE1 also inhibits E-cadherin expression through a similar mechanism in cervical cancer. Our data clearly showed that APE1 stimulates ZEB1 and E-cadherin promoter binding by directly binding to ZEB1, thereby inhibiting E-cadherin expression in cervical cancer cells (Figs. 4 and 5 ). To our knowledge, this is the first evidence that APE1 stimulates EMT via direct binding to ZEB1. Our findings may have therapeutic implications. One strategy might be to target ZEB1, given that ZEB1 is an essential driver of EMT activation and metastasis in cancer 38 . However, no specific ZEB1 inhibitors are available 38 . Here, we showed that targeting APE1 with the APE1 redox inhibitor APX3330 may be a feasible alternative. Our data showed that APX3330 inhibits ZEB1 and E-cadherin promoter binding, thereby restoring the E-cadherin expression inhibited by ZEB1 in cervical cancer cells (Fig. 4 ). Importantly, APX3330 treatment significantly inhibited cervical cancer cell EMT and invasion in vitro (Fig. 3 ) and cervical cancer lymph node and distant metastasis in vivo (Fig. 6 ). Our findings are supported by other research groups. Although the mechanisms are different, but the anti-EMT and anti-metastasis effects of APX3330 have been reported in various cancer types. For example, APX3330 treatment reverses the EMT phenotype in EGFR-mutated NSCLC via inhibition of TGF-β signaling 39 . Also, APX3330 treatment inhibits pancreatic cancer cell migration, but this was attributed to inhibition of STAT3 transcriptional activity 40 . Importantly, APX3330 is already in phase 1 clinical trials 41 , suggesting the clinical potential of targeting APE1 by APX3330 in cervical cancer treatment, at least for APE high expressing patients. Conclusion Overexpression of APE1 is a promising biomarker for the management of cervical cancer because of its role in promoting cervical cancer metastasis. These findings establish a mechanistic link between APE1 and inhibition of E-cadherin expression; specifically, APE1 enhances the ZEB1 interaction with the E-cadherin promoter by directly binding to ZEB1 (Fig. 7 ). These findings also raise the possibility of new therapeutic opportunities in the form of APE1 inhibition. Abbreviations APE1: apurinic/apyrimidinic endonuclease 1; ZEB1: zinc finger E-box binding homeobox 1; EMT: Epithelial-Mesenchymal Transition; EMSA: Electrophoretic Mobility Shift Assay. Declarations Ethics approval and consent to participate All specimens were obtained from Daping Hospital, with the approval of the Institutional Review Board. Consent for publication All authors have approved the manuscript and agree with submission to Journal of Experimental and Clinical Cancer Research. Availability of data and material All data generated of analyzed during this study are included in this published article and its supplementary information files. The datasets generated and used in this study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by Chongqing Natural Science Foundation (cstc2020jcyj-msxmX0119, to Q.L), Welch foundation (I-1829, to K.D.W.) and CPRIT (RP170373, to K.D.W.), the National Natural Science Foundation of China Young Scientists (81502241, to C.Y. Q). Authors’ contributions QL, ZWZ, WD, DW, KDW, and CXX conceived and designed the project; QL, ZWZ, WD, DZ, SNW, MSD, JMW, CYQ, CYM, and GS performed experiments. QL, ZWZ, and WD analyzed data; ZWZ, KDW, and CXX wrote the manuscript. Acknowledgements We thank all the people and patients who participated in this study. References Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2018. Ca-Cancer. J Clin 2018, 68(1): 7–30. https://doi.org/10.3322/caac.21442 . Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global Cancer Statistics. Ca-Cancer J Clin. 2011;61(2):69–90. https://doi.org/10.3322/caac.20107 . Gong Y, Wan JH, Zou W, Lian GY, Qin JL, Wang QM. MiR-29a inhibits invasion and metastasis of cervical cancer via modulating methylation of tumor suppressor SOCS1. Future oncology 2019. https://doi.org/10.2217/fon-2018-0497 . Lo HC, Zhang XH. EMT in Metastasis: Finding the Right Balance. Developmental cell. 2018;45(6):663–5. https://doi.org/10.1016/j.devcel.2018.05.033 . 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Transcription regulation of E-cadherin by zinc finger E-box binding homeobox proteins in solid tumors. BioMed research international 2014, 2014: 921564. https://doi.org/10.1155/2014/921564 . Wang WY, Cao YX, Zhou X, Wei B, Zhan L, Fu LT. HMGA2 gene silencing reduces epithelial-mesenchymal transition and lymph node metastasis in cervical cancer through inhibiting the ATR/Chk1 signaling pathway. American journal of translational research. 2018;10(10):3036–52. Li H, Wu X, Cheng X. Advances in diagnosis and treatment of metastatic cervical cancer. Journal of gynecologic oncology 2016, 27(4): e43. https://doi.org/10.3802/jgo.2016.27.e43 . Qin Y, Tang B, Hu CJ, Xiao YF, Xie R, Yong X, et al. An hTERT/ZEB1 complex directly regulates E-cadherin to promote epithelial-to-mesenchymal transition (EMT) in colorectal cancer. Oncotarget. 2016;7(1):351–61. https://doi.org/10.18632/oncotarget.5968 . Hou L, Li Q, Yu Y, Li M, Zhang D. SET8 induces epithelialmesenchymal transition and enhances prostate cancer cell metastasis by cooperating with ZEB1. Mol Med Rep. 2016;13(2):1681–8. https://doi.org/10.3892/mmr.2015.4733 . Zhang Y, Xu L, Li A, Han X. The roles of ZEB1 in tumorigenic progression and epigenetic modifications. Biomed Pharmacother. 2019;110:400–8. https://doi.org/10.1016/j.biopha.2018.11.112 . Yang X, Peng Y, Jiang X, Lu XF, Duan W, Zhang SH, et al. The regulatory role of APE1 in epithelial-to-mesenchymal transition and in determining EGFR-TKI responsiveness in non-small-cell lung cancer. Cancer Med-Us. 2018;7(9):4406–19. https://doi.org/10.1002/cam4.1717 . Cardoso AA, Jiang YL, Luo MH, Reed AM, Shahda S, He Y, et al. APE1/Ref-1 Regulates STAT3 Transcriptional Activity and APE1/Ref-1-STAT3 Dual-Targeting Effectively Inhibits Pancreatic Cancer Cell Survival. Plos One 2012, 7(10). https://doi.org/ARTN e4746210.1371/journal.pone.0047462.41 . Shah F, Logsdon D, Messmann RA, Fehrenbacher JC, Fishel ML, Kelley MR. Exploiting the Ref-1-APE1 node in cancer signaling and other diseases: from bench to clinic. NPJ Precis Oncol 2017, 1.https://doi.org/10.1038/s41698-017-0023-0 . Supplementary Files Supprevised.docx SuppFigsrevised.pdf RawdataforWB.pdf Cite Share Download PDF Status: Under Review Version 1 posted First submitted to journal 06 May, 2021 Editorial decision: Minor revision 17 Feb, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-152454","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":29162918,"identity":"77032a64-82b7-4c5a-b7b5-9a39924c1e52","order_by":0,"name":"qing li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYBACPmY2MC3Hz958wOCDgY0dQS1sUC3Gkj3HEgpnFKQlE9bCANGSuOFGjsFnng+HGBsIamFnS5Mu+GMDtOWA4WYbgwPMDOyHj24g4LBj0jN40oB+aUg2zjG4w8fAk5Z2A78W9jZpHonDIFuOAbU8Y2aQ4DEjQovBf6BfEtt/WxgcZmwgrAXoMJ6EA0AtyQzGDERqSbbmOZAMCmQGwx6DtGQ2Qn7h5z9meJvnjx3Q+/0fDH78sbHjZz98DK8WLPaSpnwUjIJRMApGATYAALwhQ6xylAlGAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-2652-6070","institution":"daping hospital army medical university","correspondingAuthor":true,"prefix":"","firstName":"qing","middleName":"","lastName":"li","suffix":""},{"id":29162919,"identity":"5893e854-aaca-420d-9047-1f018f757adb","order_by":1,"name":"Zhi-Wei Zhou","email":"","orcid":"","institution":"Southwestern Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Zhi-Wei","middleName":"","lastName":"Zhou","suffix":""},{"id":29162920,"identity":"f935080c-7265-4899-83d5-a6c799c61d3e","order_by":2,"name":"Wei Duan","email":"","orcid":"","institution":"jingzhou central hospital","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Duan","suffix":""},{"id":29162921,"identity":"7675e1c1-46f1-4b7d-b671-4157043b7369","order_by":3,"name":"Cheng-Yuan Qian","email":"","orcid":"","institution":"daping hospital","correspondingAuthor":false,"prefix":"","firstName":"Cheng-Yuan","middleName":"","lastName":"Qian","suffix":""},{"id":29162922,"identity":"9ad09fc5-9f73-43de-b117-e9b7b79c971a","order_by":4,"name":"Shu-Nan Wang","email":"","orcid":"","institution":"daping hospital","correspondingAuthor":false,"prefix":"","firstName":"Shu-Nan","middleName":"","lastName":"Wang","suffix":""},{"id":29162923,"identity":"f98ca5df-5c58-474c-919e-ac9cd582e342","order_by":5,"name":"Meng-Sheng Deng","email":"","orcid":"","institution":"daping hospital","correspondingAuthor":false,"prefix":"","firstName":"Meng-Sheng","middleName":"","lastName":"Deng","suffix":""},{"id":29162924,"identity":"17a06a99-946a-4aa8-9f17-399676f3ff5a","order_by":6,"name":"Dan Zi","email":"","orcid":"","institution":"guizhou medical university","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Zi","suffix":""},{"id":29162925,"identity":"5c6168cd-8f2a-42f0-8d7a-3fdbe32b52a0","order_by":7,"name":"Jian-Min Wang","email":"","orcid":"","institution":"daping hospital","correspondingAuthor":false,"prefix":"","firstName":"Jian-Min","middleName":"","lastName":"Wang","suffix":""},{"id":29162926,"identity":"542275f5-11f0-4c36-ad96-41391fcd153b","order_by":8,"name":"Cheng-Yi Mao","email":"","orcid":"","institution":"daping hospital","correspondingAuthor":false,"prefix":"","firstName":"Cheng-Yi","middleName":"","lastName":"Mao","suffix":""},{"id":29162927,"identity":"4f1a5d99-474d-49ad-963c-b8047fc18e9b","order_by":9,"name":"Guanbin Song","email":"","orcid":"","institution":"chongqing university","correspondingAuthor":false,"prefix":"","firstName":"Guanbin","middleName":"","lastName":"Song","suffix":""},{"id":29162928,"identity":"f003c00e-d31b-40d9-990a-3ff3e599d691","order_by":10,"name":"Dong Wang","email":"","orcid":"","institution":"daping hospital","correspondingAuthor":false,"prefix":"","firstName":"Dong","middleName":"","lastName":"Wang","suffix":""},{"id":29162929,"identity":"7104f6f9-23b3-4f34-9a1d-2eb88d996fb7","order_by":11,"name":"Kenneth D. Westover","email":"","orcid":"","institution":"southwestern medical center","correspondingAuthor":false,"prefix":"","firstName":"Kenneth","middleName":"D.","lastName":"Westover","suffix":""},{"id":29162930,"identity":"bd94e9fc-7c58-4f69-a42d-c32d8380bb0b","order_by":12,"name":"Cheng-Xiong Xu","email":"","orcid":"","institution":"chongqing university","correspondingAuthor":false,"prefix":"","firstName":"Cheng-Xiong","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2021-01-21 13:16:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-152454/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-152454/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":9681613,"identity":"54e8e08a-df93-4124-85ef-ec6a8b1def8a","added_by":"auto","created_at":"2021-05-27 20:04:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":90797,"visible":true,"origin":"","legend":"APE1 expression associated with EMT and lymph node metastasis in cervical cancer patients. a Heatmap showing genes differentially expressed in cervical cancer with high expression (n=4) and low expression of APE1 (n=4). If the APE1 expression level in cancer tissues was higher than adjacent tissues, it was classified as the APE1 high expression group and if there was no difference, it was classified as the APE1 low expression group. b Gene set enrichment analysis of signaling pathways. c The expression levels of APE1 and E-cadherin were determined by immunohistochemistry (IHC) of 72 specimens of cervical cancer patients. The representative images are the standard scoring images used to evaluate the intensity of APE1 and E-cadherin staining. d The expression of APE1 and E-cadherin was negatively associated in cervical cancer patients. e A high expression level of APE1 was correlated with lymph node metastasis in cervical cancer patients. The difference was tested by the chi square test.","description":"","filename":"OnlineFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-152454/v1/cd327a64fb61ef23d89ddfda.png"},{"id":9681380,"identity":"f77da867-0a74-48ea-b2a4-c7ff482f766c","added_by":"auto","created_at":"2021-05-27 19:55:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":60715,"visible":true,"origin":"","legend":"APE1 stimulates EMT and invasion of cervical cancer cells. a APE1 expression levels detected by western blot. Indicated cells were transfected with the indicated plasmid or siRNA. Then, 72 hours after the transfection, the cells were subjected to western blot analysis. b Representative blots showing that APE1 positively regulates EMT in cervical cancer cells. Western blotting was performed 72 hours post-transfection. c Immunofluorescence (IF) showing that silencing APE1 stimulates E-cadherin expression and inhibits vimentin expression in HeLa cells. HeLa cells were transfected with the indicated siRNAs. After 72 hours of transfection, IF analysis was performed. d Cervical cancer cell invasion was stimulated or inhibited by the overexpression or knockdown of APE1, respectively. Vector, cells transfected with empty vector; APE1, cells transfected with APE1 expression plasmid; NC, cells transfected with nontargeting control siRNA; siAPE1, cells transfected with APE1 siRNA; Ctrl, cells not treated with anything. *, P\u003c0.05; **, P\u003c0.01; ***, P\u003c0.001.","description":"","filename":"OnlineFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-152454/v1/b22e9d6e89db7b39a4ed9d9f.png"},{"id":9681461,"identity":"defa511c-6cee-4b85-839c-3f1a2f8a8dac","added_by":"auto","created_at":"2021-05-27 19:58:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":46222,"visible":true,"origin":"","legend":"APE1 stimulates EMT and invasion through a redox-dependent mechanism in cervical cancer cells. a APX3330, an APE1 redox inhibitor, increased E-cadherin but inhibited N-cadherin and vimentin expression in cervical cancer cells. The indicated cells were treated with DMSO or 5 µM APEX3330 for 24 hours and then subjected to western blot analysis. b Treatment of cervical cancer cells with APE1 inhibitor III did not affect EMT marker protein expression in cervical cancer cells. Cells were treated with DMSO or 7.5 µM APE1 inhibitor III for 24 hours and then subjected to western blot analysis. c APE1 C65S, a mutant that ablates APE1 redox function, inhibited EMT compared to wild-type APE1. Western blotting was performed 72 hours post-transfection. d Cervical cancer cell invasion was inhibited by APX3330 but not by APE1 inhibitor III. *, P\u003c0.05; **, P\u003c0.01; ***, P\u003c0.001.","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-152454/v1/e03ab33841de010f162bd6cc.png"},{"id":9681574,"identity":"81a0459f-c518-4ea1-85d9-921b2f9d7208","added_by":"auto","created_at":"2021-05-27 20:01:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":41101,"visible":true,"origin":"","legend":"APE1 positively regulates ZEB1 and E-cadherin promoter binding in cervical cancer cells. a Silencing of APE1 or inhibition of APE1 redox function rescued ZEB1-mediated inhibition of E-cadherin expression in HeLa cells. Cells were transfected with indicated plasmid or nucleotides and Western blotting was performed after 72 hours of transfection. For APE1 inhibitor experiment, cells were treated with 5 µM APX3330 or DMSO for 24 hours after 48 hours of transfection. b Co-IP showing that APE1 binds to ZEB1 in HeLa cells. HeLa cells were transfected with the indicated plasmids. After 72 hours of transfection, cells were subjected to Co-IP. c EMSA showing that silencing APE1 or inhibition of the redox function of APE1 decreased ZEB1 and E-cadherin promoter binding in HeLa cells. HeLa cells were transfected with nontargeting siRNA or siRNA targeting APE1 for 72 hours and then subjected to total protein or nuclear protein extraction; HeLa cells were treated with DMSO or 5 µM APX3330 for 24 hours and then subjected to total protein or nuclear protein extraction. Western blots were conducted using total protein. d Silencing of APE1 rescued the ZEB1-mediated loss of luciferase activity in HeLa cells transfected with luciferase under the control of an E-cadherin promoter (HeLa-Luc). Western blot and luciferase assays were performed after 72 hours of transfection. Vector, cells transfected with empty vector; NC, cells transfected with nontargeting control siRNA; Ctrl, cells not treated with anything. **, P\u003c0.01; ***, P\u003c0.001; ns, no significant difference.","description":"","filename":"OnlineFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-152454/v1/02df36f8082a690e0d129f51.png"},{"id":9681463,"identity":"15e78d5f-e5f9-4457-843f-615976a98a72","added_by":"auto","created_at":"2021-05-27 19:58:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":31156,"visible":true,"origin":"","legend":"ZEB1 amino acids 578-580 are essential for the interaction with APE1. a Schematic representation of ZEB1 protein fragments. b Co-IP showing that the middle fragment of ZEB1 binds to APE1 (F2). Co-IP was performed after 72 hours of transfection. c Secondary structure prediction of ZEB1-F2 suggesting the formation of an α helix. d Co-IP showing that mutation of ZEB1 578-580 disrupts the ZEB1-APE1 interaction. Co-IP was performed after 72 hours of transfection. e Silencing of APE1 is unable to rescue ZEB1 induced inhibition of luciferase activity in the setting of the ZEB1 578-580 mutation. Western blot and luciferase assays were performed after 72 hours of transfection. FL, full-length ZEB1; F1, ZEB1 fragment 1; F2, ZEB1 fragment 2; F3, ZEB1 fragment 3; Vector, cells transfected with empty vector; NC, cells transfected with nontargeting control siRNA; Ctrl, cells not treated with anything. Data are presented as the mean ± SD. ***, P\u003c0.001.","description":"","filename":"OnlineFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-152454/v1/db9bdee619be89b9f3b699f8.png"},{"id":9681468,"identity":"cb9ee16c-9125-412e-a693-d7c8058cf97b","added_by":"auto","created_at":"2021-05-27 19:58:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":646396,"visible":true,"origin":"","legend":"APX3330 suppressed cervical cancer metastasis in vivo. a APX3330 treatment reduced the incidence of lymph node metastasis in lymph node metastasis models. Lymph node metastatic models were generated with GFP-expressing HeLa cells (HeLa-GFP). Fluorescence image: Tumor metastasis was monitored by fluorescence imaging at day 28 after cell injection; Anatomic image: Arrow indicates metastatic tumors; LN H\u0026E: Hematoxylin and eosin (H\u0026E) staining of lymph nodes (LNs); Tumor cells: Green color indicates GFP-expressing HeLa cells in lymph nodes. blue, DAPI. Data were tested by the chi square test. b APX3330 treatment inhibited the incidence of liver metastasis in cervical cancer lymph node metastasis models. MRI image: Liver metastasis of tumor was detected by MRI at day 28 after cell injection; Anatomic image: The circle and arrow indicates metastatic tumor nodules on the surface of the liver; H\u0026E: H\u0026E staining of liver tissues; The circle and arrow indicates metastatic lesions in the liver. Tumor cells: Green indicates GFP-expressing HeLa cells in the liver. Blue, DAPI. Data were tested by the chi square test. c APX3330 treatment reduced cervical cancer abdominal cavity metastasis. Xenograft models were generated by intraperitoneal injection of HeLa-GFP cells into mice. Fluorescence image: Tumor metastasis was monitored by fluorescence imaging on day 21 after cell injection. Anatomic image: Arrow indicates metastatic tumors. H\u0026E: Hematoxylin and eosin (H\u0026E) staining shows metastatic lesions in the abdominal cavity. The circle and arrows indicate metastatic tumors. Data are presented as the mean ± SD and analyzed by unpaired t-tests. *, P\u003c0.05; **, P\u003c0.01; ***, P\u003c0.001. d APX3330 treatment inhibited cervical cancer cell lung metastasis. Lung metastatic models were generated by tail vein injection of HeLa-GFP cells into mice. Fluorescence image: Tumor metastasis was monitored by fluorescence imaging on day 28 after cell injection. Anatomic image: Arrow indicates metastatic tumor nodules on the lung surface. H\u0026E: Hematoxylin and eosin (H\u0026E) staining shows metastatic lesions in the lung. Arrows indicate the metastatic tumors. PBS, mice were treated with PBS by I.P. injection. **, P\u003c0.01; ***, P\u003c0.001.","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-152454/v1/1ba67f91331f4a82b513647b.png"},{"id":9681377,"identity":"ea589522-22fd-4c11-8814-c6c4d1376781","added_by":"auto","created_at":"2021-05-27 19:55:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":13544,"visible":true,"origin":"","legend":"A schematic regulatory mechanism showing that inhibition of APE1 results in EMT suppression.","description":"","filename":"OnlineFig7.png","url":"https://assets-eu.researchsquare.com/files/rs-152454/v1/d3c41ed092be5971b749ed6b.png"},{"id":13695716,"identity":"308577ce-32cc-449a-81fe-fa30473210c0","added_by":"auto","created_at":"2021-09-17 12:59:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1477614,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-152454/v1/2b666318-c339-4a6c-921b-52280a05893e.pdf"},{"id":9681571,"identity":"514b0697-e34b-4cf6-828f-e102c7261298","added_by":"auto","created_at":"2021-05-27 20:01:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16791,"visible":true,"origin":"","legend":"","description":"","filename":"Supprevised.docx","url":"https://assets-eu.researchsquare.com/files/rs-152454/v1/f81919e09484ba71702d243d.docx"},{"id":9681465,"identity":"4311cb11-4a5c-4d6b-ae76-43bcd0f9a628","added_by":"auto","created_at":"2021-05-27 19:58:54","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":370619,"visible":true,"origin":"","legend":"","description":"","filename":"SuppFigsrevised.pdf","url":"https://assets-eu.researchsquare.com/files/rs-152454/v1/11e94255a0a4251b14d156a1.pdf"},{"id":9681742,"identity":"ff952569-f4e6-41b4-af5c-79c9bbe639bd","added_by":"auto","created_at":"2021-05-27 20:07:54","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":802714,"visible":true,"origin":"","legend":"","description":"","filename":"RawdataforWB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-152454/v1/1f1072cb9063453175b9621e.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eInhibiting the Redox Function of APE1 Suppresses Cervical Cancer Metastasis via Disengagement of ZEB1 from E-cadherin in EMT\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eCervical cancer is the third most common cancer and the second leading cause of cancer-related death in women worldwide \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The major challenge in cervical cancer treatment is metastasis because most of the mortality associated with cervical cancer is caused by metastasis \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Unfortunately, no effective therapeutic strategies exist for preventing or inhibiting cervical cancer metastasis, in part because the mechanisms that underlie metastasis are incompletely understood.\u003c/p\u003e \u003cp\u003eGrowing evidence illustrates that epithelial-mesenchymal transition (EMT) plays a key role in tumor metastasis \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. EMT is a process in which epithelial tumor cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Notably, the clinical data show that EMT is closely related to a poor prognosis in cervical cancer patients \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. E-cadherin is a homotypic cell-to-cell adhesion molecule ubiquitously expressed on epithelial cells \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, E-cadherin is frequently downregulated in cervical cancer \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, and downregulation of E-cadherin is sufficient to induce EMT in tumor cells and promote tumor cell metastasis \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Importantly, clinical data show that low expression of E-cadherin is closely associated with metastasis and a poor prognosis in cervical cancer patients \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn cancer, one cause of downregulation of E-cadherin is abnormal overexpression of zinc finger E-box-binding homeobox 1 (ZEB1). ZEB1 is a transcriptional repressor that inhibits E-cadherin expression at the transcriptional level by binding to the E-cadherin promoter \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. According to Chen et al., ZEB1 is not expressed in normal cervical epithelial cells but is expressed in most invasive cervical carcinomas, and the ZEB1 expression level is strongly associated with lymph node metastasis in cervical cancer patients \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Consistent with this, ZEB1 silencing inhibits cervical cancer cell EMT and metastasis \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, suggesting that ZEB1 is a potential therapeutic target for EMT-induced cervical cancer metastasis treatment. Unfortunately, no specific ZEB1 inhibitors are available.\u003c/p\u003e \u003cp\u003eApurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1) functions both as a redox regulator of transcription factor activation and as part of the DNA damage response \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Notably, recent studies showed that upregulated APE1 inhibits E-cadherin expression and stimulates EMT and metastasis in non-small-cell lung cancer \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In addition, Wei et al. reported that upregulated APE1 was closely associated with lymph node metastasis in gastric cancer \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. These findings suggest that APE1 may be involved in the regulation of E-cadherin-mediated EMT and tumor metastasis, but the mechanism is unclear. Aberrantly upregulated expression of APE1 has also been detected in cervical cancer \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, but the effects of APE1 on cervical cancer metastasis have not been studied.\u003c/p\u003e \u003cp\u003eIn this study, we found that high expression of APE1 was closely associated with EMT and lymph node metastasis in cervical cancer patients. Ectopic expression of APE1 inhibited E-cadherin expression and stimulated EMT and invasion in cervical cancer cells. In contrast, inhibition of APE1 redox function significantly suppressed lymph node and distant metastasis of cervical cancer cells \u003cem\u003ein vivo\u003c/em\u003e. Furthermore, we found that APE1 inhibited E-cadherin expression in a redox-dependent manner by enhancing the interaction between ZEB1 and the E-cadherin promoter by directly binding to ZEB1. In summary, our findings provide new insights into the underlying mechanism of metastasis in cervical cancer and provide a potential therapeutic target for metastatic cervical cancer therapy.\u003c/p\u003e "},{"header":"Materials And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and human specimens\u003c/h2\u003e \u003cp\u003eHeLa and SiHa cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco`s modified Eagle`s medium with 10% fetal bovine serum (HyClone, Logan, UT). Human samples were obtained from patients with cervical cancer by biopsy or surgery at Daping Hospital and Research Institute of Surgery. This study was approved by the Ethics Committee of Daping Hospital, Army Medical University. We obtained consent to publish from the participants (or legal parents or guardians for children) to report individual patient data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid constructs\u003c/h2\u003e \u003cp\u003eThe ZEB1 ORF was amplified by PCR from human cDNAs and inserted into the \u003cem\u003eBamH\u003c/em\u003eI and \u003cem\u003eHind\u003c/em\u003eIII sites of the pcDNA3.1-flag vector for expression in mammalian cells. The E-cadherin promoter region from \u0026minus;\u0026thinsp;670 to +\u0026thinsp;92 was amplified using primers with restriction enzyme sites \u003cem\u003eBgl\u003c/em\u003eII or \u003cem\u003eHind\u003c/em\u003eIII at each end and inserted into the upstream region of the firefly luciferase gene of the pGL3-Basic vector (Promega, Madison, WI, USA) \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. APE1 (NM_001641.4) and ZEB1 (NM_001323642.2) expression vectors were obtained from Shanghai GeneChem Co., Ltd. (Shanghai, China). All primer sequences used in this study are given in Table S1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC), immunofluorescence (IF), and western blot\u003c/h2\u003e \u003cp\u003eFor western blotting, cells were lysed in RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich, Saint Louis, MO, USA), and the protein concentration of the lysate was measured using a Bradford kit (Bio-Rad, Hercules, CA, USA). Equal amounts of proteins (30 \u0026micro;g) were separated by sodium dodecyl sulfate\u0026ndash;polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membrane was blocked for 1 hour in Tris-buffered saline with Tween 20 (TBST) containing 5% skim milk at room temperature (RT), and immunoblotting was performed by incubating the membranes overnight with their corresponding primary antibodies in 5% skim milk at 4\u0026deg;C. The membrane was washed with TBST and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at RT. After washing, the proteins were visualized with an enhanced chemiluminescence detection kit (Thermo Fisher Scientific) in accordance with the manufacturer\u0026rsquo;s recommendations.\u003c/p\u003e \u003cp\u003eFor IHC, the tumor tissues were fixed in 10% buffered formalin (Sigma-Aldrich), embedded in paraffin, and sectioned at 4 \u0026micro;M. The tissue sections were deparaffinized in xylene, rehydrated through an alcohol gradient, washed and incubated in 0.3% hydrogen peroxide (AppliChem, Darmstadt, Germany) for 15 min. After washing, the tissue sections were blocked with 5% bovine serum albumin in PBS for 1 hour. Then, primary antibodies were applied to the tissue sections overnight at 4\u0026deg;C. The following day, the tissue sections were washed and incubated with secondary HRP-conjugated antibodies for 1 hour at RT and counterstained with Mayer's hematoxylin (Dako, Carpinteria, CA, USA) for 10 seconds. Coverslips were mounted using Permount (Thermo Fisher Scientific). A panel of pathologists reviewed the IHC staining and scored it as follows: score 0, no staining positive tumor cells; score 1, staining positive tumor cells less than 10% of the total tumor cells; score 2, staining positive tumor cells more than 10% of the total tumor cells, but less than 50%; and score 3, staining positive tumor cells more than 50% of the total tumor cells. Scores of 0 and 1 were defined as low expression, and scores of 2 and 3 were defined as high expression.\u003c/p\u003e \u003cp\u003eFor IF, cells were grown on coverslips and transfected with the indicated oligonucleotides. After 72 hours of transfection, the cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.3% Triton X-100 for 10 min. The cells were washed with PBS and incubated with 3.5% bovine serum albumin (BSA) for 1 hour followed by incubation with the primary antibody in 3.5% BSA for 1 hour at RT. The cells were washed with PBS and incubated with FITC-conjugated secondary antibody for 1 hour at RT in the dark. Then, the cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 30 min. Antibodies against APE1, β-actin, E-cadherin, N-cadherin, vimentin, Flag, HA, and GAPDH and secondary antibodies were purchased from Abcam (Cambridge, MA, USA). The anti-ZEB1 antibody was obtained from Cell Signaling Technology (Danvers, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eInvasion assay\u003c/h2\u003e \u003cp\u003eCells were transfected with APE1 construct or siRNA (GeneChem Co., Shanghai, China) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer\u0026rsquo;s instructions. Sequences of the double-stranded siRNAs are antisense (5\u0026rsquo;-GUCUGGUACGACUGGAGUACC-3\u0026rsquo;, 5\u0026rsquo;-UACUCCAGUCGUACCAGACCU-3\u0026rsquo;) and nonsense (5\u0026rsquo;-CCAUGAGGUCAGCAUGGUCUG3\u0026rsquo;, 5\u0026rsquo;-GACCAUGCUGACCUCAUGGAA-3\u0026rsquo;) \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Nontargeting control siRNA was purchased from Qiagen (Hilden, Germany). After 48 hours of transfection, the cells were subjected to invasion assays. Briefly, 10,000 cells in medium without serum were seeded in the upper wells of invasion chambers (BD Biosciences, San Jose, CA, USA). The lower wells contained the same medium supplemented with 10% fetal bovine serum. After 24 hours, the cells that invaded to the other side of the chamber were fixed with 2.5% glutaraldehyde, stained with 0.1% crystal violet, and counted.\u003c/p\u003e \u003cp\u003eTo investigate the effects of the APE1 redox inhibitor APX3330 (Selleck, Houston, TX, USA) and the APE1 DNA repair inhibitor APE1 inhibitor III (Merck Millipore, Molsheim, France) on the invasion of cervical cancer cells, cells were pretreated with APE1 inhibitors for 24 hours. After 24 hours, the cells were subjected to invasion assays as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase reporter assay\u003c/h2\u003e \u003cp\u003ePlasmids and/or nucleotides were transfected into HeLa cells that were transfected with a firefly luciferase reporter construct containing the E-cadherin promoter. The Renilla luciferase plasmid was cotransfected as a transfection control (Promega). The cell extracts were processed 72 hours after transfection, and the luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer\u0026rsquo;s instructions. The luciferase activity was normalized to the activity of Renilla luciferase.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation\u003c/h2\u003e \u003cp\u003eHeLa cells were transfected with the indicated plasmids. After 72 hours, the cells were lysed in lysis buffer [1% Nonidet P40, 0.1% SDS, 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 mM DTT and 0.5 mM EDTA containing protease inhibitors], and the protein concentration of the lysate was measured using a Bradford kit (Bio-Rad). Immunoprecipitation was performed using monoclonal antibodies as indicated. Immunocomplexes were collected on protein A/G-agarose beads (Merck Millipore), washed twice with lysis buffer, and then washed twice with wash buffer [10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.2 mM sodium orthovanadate, protease inhibitor cocktail]. The beads were dissolved in SDS loading buffer (Bio-Rad), denatured at 95\u0026deg;C for 10 min, and later subjected to western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSecondary structure prediction\u003c/h2\u003e \u003cp\u003eThe sequence of ZEB1 was retrieved from UniProt, and the corresponding secondary structure was generated using Jpred (Jnet version: 2.3.1) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.compbio.dundee.ac.uk/jpred/\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eElectrophoretic mobility shift assay (EMSA)\u003c/h2\u003e \u003cp\u003eEMSA was performed using the LightShift chemiluminescence EMSA kit (Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions as described previously \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Nuclear extracts were isolated from the indicated cells and incubated with 3\u0026rsquo;-biotin-labeled double-stranded oligonucleotide probes containing consensus sequences for ZEB1 binding sites (Fig. S1), then the samples were separated on a 5% polyacrylamide gel and transferred to a Zeta-Probe GT nylon membrane. The probes were detected by HRP-conjugated streptavidin. The probe sequences are shown in Table S1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiments\u003c/h2\u003e \u003cp\u003eAll xenograft models were generated using GFP-expressing HeLa cells (HeLa-GFP) in 6-week-old female BALB/c nude mice. For the lymph node metastatic model, HeLa-GFP cells (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/50 \u0026micro;l PBS per mouse) were directly injected into the footpad of the mice \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. For the abdominal cavity metastatic model, HeLa-GFP cells (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/200 \u0026micro;l PBS per mouse) were intraperitoneally (IP) injected into the mice. For the lung metastatic model, HeLa-GFP cells (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/100 \u0026micro;l PBS per mouse) were injected intravenously into the tail vein of the mice. One week after the cell injection, the mice were randomly divided into two groups. The control group mice were treated with PBS, and the treatment group mice were treated with APX3330 (12.5 mg/kg body weight) by IP injection once every two days. The mice were treated with APX3330 for 3 weeks for the lung and lymph node metastasis experiments, treated with APX3330 for 2 weeks in the abdominal cavity metastasis experiments, and their body weight was measured every 3 days. Tumor metastasis was monitored by an IVIS Spectrum \u003cem\u003eIn Vivo\u003c/em\u003e Imaging System (Perkin Elmer, Utah, USA) and MRI (7.0-T MRI, Bruker Biospec 70/20USR, Germany). All animal experiments complied with the Daping Hospital, Army Medical University Policy on the Care and Use of Laboratory Animals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using Prism 5.0 software (GraphPad). The unpaired two-samples t-test was used to compare the mean of two independent groups, and one-way ANOVA was used to determine differences between the means of two or more independent groups. p values less than 0.05 were considered statistically significant. The data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;the standard deviation (SD) of at least three independent experiments. The correlation between APE1 expression and lymph node metastasis or E-cadherin expression was tested by the chi-square test.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eHigh expression of APE1 is closely associated with EMT and lymph node metastasis in cervical cancer patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate whether the high expression of APE1 in cervical cancer is related to EMT and metastasis, we performed gene set enrichment analysis (GSEA) using transcriptome data from four cervical cancer samples expressing high levels of APE1 and four cervical cancer samples expressing low levels of APE1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea and Fig. S2). The GSEA results showed that the APE1 expression level was closely associated with EMT in cervical cancer (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). This association was further confirmed in an expanded 72 cervical cancer patient cohort where we directly compared APE1 to E-cadherin (loss of E-cadherin is a well-established hallmark of EMT) expression using IHC (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). Our data showed that among the 30 cases with low APE1 expression, 26 cases (87%) showed high expression of E-cadherin, while among the 42 cases with high APE1 expression, 27 (64%) cases presented with high expression of E-cadherin (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed). This result suggests that high expression of APE1 is associated with EMT in cervical cancer patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBecause EMT stimulates cancer metastasis partially through the lymphatic system \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, we next evaluated the associations between APE1 expression and lymph node metastasis. The results showed that 69% (29 cases) of cases with high APE1 expression (42 cases) had lymph node metastasis, while only 23% (7 cases) of cases with low APE1 expression (30 cases) had lymph node metastasis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Taken together, these findings indicate that APE1 may be involved in metastasis by stimulating EMT in cervical cancer patients.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eCharacteristics of patients with cervical cancer\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eVariable\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eNumber of patients (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eAPE1 high\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eAPE1 low\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAge(years)\u003c/p\u003e\n\u003cp\u003e\u0026ge;\u0026thinsp;50\u003c/p\u003e\n\u003cp\u003e\u0026lt;50\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14 (33%)\u003c/p\u003e\n\u003cp\u003e28 (67%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8 (27%)\u003c/p\u003e\n\u003cp\u003e22 (73%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.54\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHistological subtypes\u003c/p\u003e\n\u003cp\u003eSquamous carcinoma\u003c/p\u003e\n\u003cp\u003eAdenocarcinoma\u003c/p\u003e\n\u003cp\u003eAdenosquamous\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e34 (81%)\u003c/p\u003e\n\u003cp\u003e6 (14%)\u003c/p\u003e\n\u003cp\u003e2 (5%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e27 (90%)\u003c/p\u003e\n\u003cp\u003e2 (7%)\u003c/p\u003e\n\u003cp\u003e1 (3%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.56\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTumor grade\u003c/p\u003e\n\u003cp\u003ePoorly differentiation\u003c/p\u003e\n\u003cp\u003eModerate differentiation\u003c/p\u003e\n\u003cp\u003eHigh differentiation\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e28(67%)\u003c/p\u003e\n\u003cp\u003e10(24%)\u003c/p\u003e\n\u003cp\u003e4 (9%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14 (48%)\u003c/p\u003e\n\u003cp\u003e8 (26%)\u003c/p\u003e\n\u003cp\u003e8 (26%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.11\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eStage\u003c/p\u003e\n\u003cp\u003eStage I\u003c/p\u003e\n\u003cp\u003eStage II\u003c/p\u003e\n\u003cp\u003eStage III\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e23 (55%)\u003c/p\u003e\n\u003cp\u003e14 (33%)\u003c/p\u003e\n\u003cp\u003e5 (12%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16 (53%)\u003c/p\u003e\n\u003cp\u003e11 (37%)\u003c/p\u003e\n\u003cp\u003e3 (10%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.94\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLymph node metastasis\u003c/p\u003e\n\u003cp\u003ePositive\u003c/p\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e29 (69%)\u003c/p\u003e\n\u003cp\u003e13 (31%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7 (23%)\u003c/p\u003e\n\u003cp\u003e23 (77%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eEctopic expression of APE1 stimulates EMT and the invasion of cervical cancer cells\u003c/h2\u003e\n\u003cp\u003eTo investigate whether APE1 directly stimulates EMT in cervical cancer cells, leading to metastasis, both HeLa and SiHa cells were transfected with APE1-expressing plasmid or APE1 siRNA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea) and then subjected to detection of EMT-related protein expression and invasion assays. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb, western blot analysis showed that ectopic expression of APE1 inhibited the epithelial cell marker E-cadherin but upregulated the mesenchymal marker proteins vimentin and N-cadherin in both HeLa and SiHa cells. In contrast, silencing APE1 upregulated E-cadherin but downregulated vimentin and N-cadherin expression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). Consistent with this, IF also showed that silencing APE1 stimulated E-cadherin expression but inhibited vimentin expression in HeLa cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec). Of functional importance, cervical cancer cell invasion was significantly increased by ectopic expression of APE1 but suppressed by silencing of APE1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). Taken together, these findings indicate that APE1 positively regulates cervical cancer cell EMT and invasion.\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eAPE1 promotes cervical cancer cell EMT and invasion via a redox-dependent mechanism\u003c/h2\u003e\n\u003cp\u003eAPE1 exerts functions both in the DNA repair response and redox regulation of transcription factors \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. To investigate which function is involved in EMT regulation and invasion, cervical cancer cells were treated with the APE1 redox inhibitor (APX3330) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e or the APE1 DNA repair inhibitor (APE1 inhibitor III) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and then subjected to EMT marker protein detection and invasion analysis. Our data show that APX3330 treatment increased E-cadherin expression but suppressed N-cadherin and vimentin expression in both HeLa and SiHa cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). In contrast, APE1 inhibitor III had no effect on EMT marker expression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb), despite impairing APE1 DNA repair activity (Fig. S3). Consistent with the APX3330 treatment, overexpression of mutant APE1, C65S, which lack redox function \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, increased E-cadherin expression and decreased N-cadherin and vimentin expression compared to overexpression of wild-type APE1 in HeLa cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec). In addition, cell invasion assays also showed inhibition when challenged with APX3330 but not APE1 inhibitor III (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed). Together, these findings suggest that APE1 stimulates cervical cancer cell EMT and invasion via a redox-dependent mechanism.\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eAPE1 inhibits E-cadherin by stimulating ZEB1 binding to the E-cadherin promoter\u003c/h2\u003e\n\u003cp\u003eZEB1 is an EMT-activating factor \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e that induces EMT through inhibiting E-cadherin expression by directly binding to the E-cadherin promoter \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Given that we showed that APE1 inhibits EMT by inhibiting E-cadherin expression in cervical cancer cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb), we next investigated whether APE1 is involved in ZEB1-regulated inhibition of E-cadherin expression. The results showed that overexpression of ZEB1 inhibited E-cadherin expression and it was partially restored by silencing APE1 or APX3330 treatment in HeLa cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea), indicating that APE1 was involved in ZEB1-regulated E-cadherin expression. In addition, Co-IP of Flag-ZEB1 or HA-APE1 expressed in HeLa cells showed a direct interaction between APE1 and ZEB1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). Importantly, EMSA results showed that APE1 silencing or APX330 treatment significantly reduced ZEB1 and E-cadherin promoter binding compared to controls (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec), suggesting that APE1 positively regulates the interaction between ZEB1 and the E-cadherin promoter in a redox-dependent manner. As another means of confirmation, we constructed a reporter assay that relies on interactions between ZEB1 and the E-cadherin promoter to suppress the expression of luciferase in HeLa cells. Our luciferase reporter assay showed that ectopic expression of ZEB1 inhibited luciferase activity but was restored by APE1 silencing (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed). Taken together, these findings indicate that APE1 inhibits E-cadherin expression by enhancing interactions between ZEB1 and the E-cadherin promoter by directly binding to ZEB1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, to map the interaction domain of ZEB1, truncated versions of ZEB1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea) were expressed in HeLa cells, and Co-IP experiments evaluating ZEB1-APE1 interactions were performed. Among the fragments, only the ZEB1 fragment encompassing 368\u0026ndash;739 amino acids was able to pull down APE1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb). We noted that secondary structure prediction of that region of ZEB1 suggested that residues 573\u0026ndash;621 had a high likelihood of forming an alpha helix (Fig. S4). We considered that the alpha helix might be important for binding to APE1 and designed mutations in residues 578\u0026ndash;580 to disrupt the binding (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec and Fig. S5). Co-IP showed that the mutated version of ZEB1, ZEB1\u003csup\u003eMUT\u003c/sup\u003e, was expressed but was unable to bind to APE1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed). Additionally, we tested ZEB1\u003csup\u003eMUT\u003c/sup\u003e in the E-cadherin promoter-driven luciferase reporter assay. As with wild type, ZEB1\u003csup\u003eMUT\u003c/sup\u003e overexpression inhibited E-cadherin promoter-driven luciferase expression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee). However, this was not rescued by APE1 silencing (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee), supporting that the helix formed by ZEB1 residues 573\u0026ndash;621 is important for the interaction between ZEB1 with APE1 and resulting functional activity at the E-cadherin promoter.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibition of APE1 redox function suppressed lymph node and distant metastasis of cervical cancer cells\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003ein vivo\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eOur \u003cem\u003ein vitro\u003c/em\u003e findings suggested a therapeutic opportunity for tumors overexpressing APE1 in the form of APE1 redox inhibitors such as APX3330. We therefore investigated whether APX3330 could suppress cervical cancer cell metastasis \u003cem\u003ein vivo\u003c/em\u003e. An established model for studying lymph node metastases in cervical cancer was generated by the injection of GFP-expressing HeLa cells into the footpad of nude mice \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Treatment of such mice with APX3330 showed a dramatic reduction in the incidence of nodal metastases at the 3-week mark, with 75% of the mice in the control group developing lymph node metastasis versus only 25% of the mice in the APX3330 treatment group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). MRI and pathological examination showed that 50% of the mice in the control group developed hepatic metastasis, but no hepatic metastasis was found in the APX3330 treatment group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb). These results were further confirmed in models of systemic metastases, including a model of liver, colon and mesenteric metastases (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec) and lung metastases (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed). All \u003cem\u003ein vivo\u003c/em\u003e experiments showed that APX3330 treatment did not affect the body weight of the experimental animals (Fig. S6). Taken together, these data suggest that inhibition of APE1 redox function by APX3330 may be a therapeutic strategy for the treatment of cervical cancer metastasis.\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":" \u003cp\u003eLymph node and distant metastasis are major drivers of poor outcomes of cervical cancer \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Thus, a comprehensive understanding of the mechanisms underlying the development of metastasis is required to optimize treatment strategies and develop new therapeutic agents for cervical cancer. A previous study has shown that EMT is a primary process that involves increased cervical cancer metastasis with loss of EMT markers, such as E-cadherin, and a gain of mesenchymal markers, such as vimentin \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Here, we used clinical sample analysis to indicate that high expression of APE1 is closely associated with lymph node metastasis and low expression of E-cadherin in cervical cancer patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and \u003cem\u003ein vitro\u003c/em\u003e functional experiments indicated that overexpression of APE1 significantly stimulates cervical cancer cell invasion, promotes the vimentin expression, and inhibits E-cadherin expression, while silencing of APE1 dramatically inhibits cervical cancer cell invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Together, these findings suggest that aberrantly increased expression of APE1 in cervical cancer promotes metastasis by stimulating EMT, and APE1 is a target for the treatment of cervical cancer metastasis.\u003c/p\u003e \u003cp\u003eNext, we elucidated the mechanism by which APE1 stimulates EMT in cervical cancer. Studies have shown that binding to ZEB1 and a dependence on ZEB1 to inhibit E-cadherin expression is a mechanism by which oncogenes promote EMT and metastasis in cancer. For example, telomerase reverse transcriptase (TERT) stimulates EMT in colorectal cancer through inhibition of E-cadherin expression by binding to ZEB1 \u003csup\u003e36\u003c/sup\u003e; histone H4K20-specific methyltransferase SET8 stimulates EMT in prostate cancer by inhibiting E-cadherin transcription through binding to ZEB1 \u003csup\u003e37\u003c/sup\u003e. Here, we found that APE1 also inhibits E-cadherin expression through a similar mechanism in cervical cancer. Our data clearly showed that APE1 stimulates ZEB1 and E-cadherin promoter binding by directly binding to ZEB1, thereby inhibiting E-cadherin expression in cervical cancer cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). To our knowledge, this is the first evidence that APE1 stimulates EMT via direct binding to ZEB1.\u003c/p\u003e \u003cp\u003eOur findings may have therapeutic implications. One strategy might be to target ZEB1, given that ZEB1 is an essential driver of EMT activation and metastasis in cancer \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. However, no specific ZEB1 inhibitors are available \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Here, we showed that targeting APE1 with the APE1 redox inhibitor APX3330 may be a feasible alternative. Our data showed that APX3330 inhibits ZEB1 and E-cadherin promoter binding, thereby restoring the E-cadherin expression inhibited by ZEB1 in cervical cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Importantly, APX3330 treatment significantly inhibited cervical cancer cell EMT and invasion \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and cervical cancer lymph node and distant metastasis \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Our findings are supported by other research groups. Although the mechanisms are different, but the anti-EMT and anti-metastasis effects of APX3330 have been reported in various cancer types. For example, APX3330 treatment reverses the EMT phenotype in EGFR-mutated NSCLC via inhibition of TGF-β signaling \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Also, APX3330 treatment inhibits pancreatic cancer cell migration, but this was attributed to inhibition of STAT3 transcriptional activity \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Importantly, APX3330 is already in phase 1 clinical trials \u003csup\u003e41\u003c/sup\u003e, suggesting the clinical potential of targeting APE1 by APX3330 in cervical cancer treatment, at least for APE high expressing patients.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eOverexpression of APE1 is a promising biomarker for the management of cervical cancer because of its role in promoting cervical cancer metastasis. These findings establish a mechanistic link between APE1 and inhibition of E-cadherin expression; specifically, APE1 enhances the ZEB1 interaction with the E-cadherin promoter by directly binding to ZEB1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These findings also raise the possibility of new therapeutic opportunities in the form of APE1 inhibition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eAPE1: apurinic/apyrimidinic endonuclease 1; ZEB1: zinc finger E-box binding homeobox 1; EMT: Epithelial-Mesenchymal Transition; EMSA: Electrophoretic Mobility Shift Assay.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll specimens were obtained from Daping Hospital, with the approval of the Institutional Review Board.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have approved the manuscript and agree with submission to Journal of Experimental and Clinical Cancer Research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated of analyzed during this study are included in this published article and its supplementary information files. The datasets generated and used in this study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Chongqing Natural Science Foundation (cstc2020jcyj-msxmX0119, to Q.L), Welch foundation (I-1829, to K.D.W.) and CPRIT (RP170373, to K.D.W.), the National Natural Science Foundation of China Young Scientists (81502241, to C.Y. Q).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQL, ZWZ, WD, DW, KDW, and CXX conceived and designed the project; QL, ZWZ, WD, DZ, SNW, MSD, JMW, CYQ, CYM, and GS performed experiments. QL, ZWZ, and WD analyzed data; ZWZ, KDW, and CXX wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all the people and patients who participated in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Miller KD, Jemal A. Cancer Statistics, 2018. Ca-Cancer. J Clin 2018, 68(1): 7\u0026ndash;30.\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3322/caac.21442\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global Cancer Statistics. 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NPJ Precis Oncol 2017, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e1.https://doi.org/10.1038/s41698-017-0023-0\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-experimental-and-clinical-cancer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jecc","sideBox":"Learn more about [Journal of Experimental \u0026 Clinical Cancer Research](http://jeccr.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jecc/default.aspx","title":"Journal of Experimental \u0026 Clinical Cancer Research","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"APE1, EMT, E-cadherin, ZEB1, cervical cancer metastasis","lastPublishedDoi":"10.21203/rs.3.rs-152454/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-152454/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eMetastasis is a major challenge in cervical cancer treatment. Previous studies have shown that the dual functional protein apurinic/apyrimidinic endonuclease 1 (APE1) promotes tumor metastasis and is overexpressed in cervical cancer. However, the biological role and mechanism of APE1 in cervical cancer metastasis have rarely been studied.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eWe used\u003cstrong\u003e \u003c/strong\u003egene set enrichment analysis (GSEA) to determine the APE1-related signaling pathways in cervical cancer. To investigate the role and mechanism of APE1 in cervical cancer metastasis, invasion, immunohistochemistry, immunofluorescence, western blotting, secondary structure prediction, coimmunoprecipitation, luciferase reporter, and electrophoretic mobility shift assays were performed. The inhibitory effects of the APE1 redox function inhibitor APX3330 on cervical cancer metastasis were evaluated using animal models.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eClinical data showed that high expression of APE1 was associated with lymph node metastasis in cervical cancer patients. GSEA results showed that APE1 was associated with epithelial to mesenchymal transition (EMT) in cervical cancer. Ectopic expression of APE1 promoted EMT and invasion of cervical cancer cells, whereas inhibition of APE1 suppressed EMT and invasion of cervical cancer cells in a redox function-dependent manner. Notably, APE1 redox function inhibitor APX3330 treatment dramatically suppressed cervical cancer cell lymph node and distant metastasis \u003cem\u003ein vivo\u003c/em\u003e. Furthermore, we found that APE1 enhanced the interaction between ZEB1 and the E-cadherin promoter by binding to ZEB1, thereby suppressing the expression of E-cadherin, a negative regulator of EMT.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eOur findings help to elucidate the role played by APE1 in cervical cancer metastasis and targeting APE1 redox function may be a novel strategy for inhibiting cervical cancer metastasis.\u003c/p\u003e","manuscriptTitle":"Inhibiting the Redox Function of APE1 Suppresses Cervical Cancer Metastasis via Disengagement of ZEB1 from E-cadherin in EMT","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-05-27 19:55:52","doi":"10.21203/rs.3.rs-152454/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"submitted","content":"Journal of Experimental \u0026 Clinical Cancer Research","date":"2021-05-06T05:49:26+00:00","index":"","fulltext":""},{"type":"decision","content":"Minor revision","date":"2021-02-18T02:42:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-experimental-and-clinical-cancer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jecc","sideBox":"Learn more about [Journal of Experimental \u0026 Clinical Cancer Research](http://jeccr.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jecc/default.aspx","title":"Journal of Experimental \u0026 Clinical Cancer Research","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d2c4753f-7d7f-40b4-be9f-40dbf3cc3dc8","owner":[],"postedDate":"May 27th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":4576930,"name":"Cancer Biology"},{"id":4576931,"name":"Oncology"}],"tags":[],"updatedAt":"2021-06-04T22:23:06+00:00","versionOfRecord":[],"versionCreatedAt":"2021-05-27 19:55:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-152454","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-152454","identity":"rs-152454","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}