The Role of CUEDC1 in Suppressing JAK1/STAT3 Signaling Pathway in Esophageal Cancer

The Role of CUEDC1 in Suppressing JAK1/STAT3 Signaling Pathway in Esophageal Cancer | 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 Article The Role of CUEDC1 in Suppressing JAK1/STAT3 Signaling Pathway in Esophageal Cancer Zhuo Li, Zhipeng Pan, Xuehan Su, Chunhong Li, Jian Zhang, Guohe Lin, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6618873/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract CUE domain containing protein 1 (CUEDC1) is implicated in tumor progression; however, its specific role in esophageal cancer (ESCA) remains unclear. In esophageal cancer, the expression of CUEDC1 is notably low, which correlates with reduced survival rates and adverse clinical outcomes. Overexpression of CUEDC1 results in decreased activity of the JAK1/STAT3 signaling pathway in cells, consequently diminishing their proliferation, migration, and invasion capabilities. This mechanism operates through the direct binding of CUEDC1 to STAT3, facilitating its ubiquitination and triggering the ubiquitin-proteasome degradation pathway, ultimately leading to a significant reduction in intracellular STAT3 levels. This study suggests that CUEDC1 can reduce intracellular STAT3 protein levels, thereby inhibiting JAK1/STAT3 signaling transduction and suppressing the progression of ESCA. This study aims to elucidate the regulatory mechanism of CUEDC1 on STAT3, which will enhance our understanding of the regulatory pathways involved in the treatment of esophageal cancer and potentially other tumors. Future breakthroughs and innovations may emerge from molecular research and development targeting this pathway. Biological sciences/Cancer/Cancer genetics Biological sciences/Cancer/Cancer therapy Biological sciences/Cancer Biological sciences/Genetics Health sciences/Molecular medicine CUEDC1 JAK1/STAT3 esophageal cancer ubiquitination proteasome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Esophageal cancer (ESCA) is a malignancy characterized by a poor prognosis, ranking 11th in incidence and 7th in mortality among all cancers. In 2022, it was estimated that there would be 511,000 new cases and 445,000 deaths 1 . Histologically, ESCA is classified into two main types: squamous cell carcinoma (SCC) and esophageal adenocarcinoma (EAC). The former constitutes approximately 85% of all cases, while the latter accounts for about 14% 2 . These two types exhibit relatively independent etiologies and geographical distributions. In regions such as North America and Western Europe, adenocarcinoma accounts for about two-thirds of cases, which is associated with obesity, gastroesophageal reflux disease, and Barrett's esophagus. Conversely, squamous cell carcinoma is the predominant subtype in East Asia and East Africa, closely linked to factors such as smoking, alcohol abuse, consumption of excessively salty or hot foods, and exposure to air pollution 3 . Moreover, the prognosis for esophageal cancer remains poor, posing a significant burden on global health 4 . The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is a critical cellular signaling mechanism that plays a vital role in various biological processes, including cell proliferation, differentiation, apoptosis, and immune response 5 . This pathway facilitates the growth and survival of cancer cells through the regulation of gene expression. For instance, in colorectal cancer, the activation of STAT3 and JAK1/2 is closely associated with the proliferation and invasion of tumor cells 6 . Furthermore, this pathway can promote tumor immune escape and drug resistance by modulating immune cells and inflammatory factors within the tumor microenvironment 7 , 8 . Targeting the JAK/STAT pathway offers a novel strategy for cancer treatment 9 . Future research should further investigate its specific mechanisms and potential clinical applications 10 . CUEDC1(CUE domain-containing protein 1) is a protein that contains the CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation) domain and is commonly expressed in cells 11 . Currently, there is limited research on CUEDC1. It has been reported that CUEDC1, as a functional target gene of estrogen receptor alpha (ERα), is associated with poor prognosis in breast cancer 12 . Additionally, there is a significant increase in CUEDC1 expression in metastatic cervical cancer compared to early tumors 13 . However, in contrast, overexpression of CUEDC1 in non-small cell lung cancer reduces the metastatic potential of cancer cells and inhibits the epithelial-mesenchymal transition (EMT) process 14 . In summary, the role of CUEDC1 appears to vary across different tumors, and the exact role of CUEDC1 in the metastasis and progression of ESCA remains unclear. Materials and methods Cell Culture and Lentivirus Infection The human ESCA cell line TE-1 and the human esophageal epithelial cell line BAR-T were obtained from Zishan Biology Co., Ltd in Wuhan, China. Cells were cultured in RPMI-1640 medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum(Biochannel, Nanjing, China) at 37°C and 5% CO2, with regular confirmation using short tandem repeat (STR) fingerprinting. The CUEDC1 overexpression sequence and gene knockout sequence for CUEDC1 were designed and synthesized by General Biosciences and packaged in lentivirus for transfection into TE-1 cells, yielding CUEDC1 overexpression (oe-CUEDC1) and silence (sh-CUEDC1) cell lines, respectively. The specific procedure involved evenly plating 1 x 10^6 TE-1 cells in a six-well plate and incubating overnight. The following day, the culture medium was discarded, and the cells were rinsed twice with phosphate-buffered saline (PBS). Subsequently, 2 ml of complete RPMI-1640 medium containing 1 x 10^6 viral particles and 8% polybrene was added. After 24 hours, the culture medium was replaced with fresh medium, and puromycin was added to select stable cell lines. The transfection efficiency was validated using RT-qPCR and Western blot analysis. The relevant sequences can be found in supplementary materials. Reverse transcription quantitative polymerase chain reaction Total RNA from TE-1 cells was extracted using an RNA rapid extraction kit (EnzyArtisan, Shanghai, China), followed by reverse transcription to synthesize cDNA, which was analyzed with a qPCR kit on appropriate instruments. GAPDH served as the internal control, and relative gene expression was normalized using the 2^-ΔΔCT method. This experiment was performed independently three times. Primer sequences can be found in Supplementary Table 1. Western blotting analysis To obtain proteins, cells were lysed using RIPA lysis buffer supplemented with protease and phosphatase inhibitors. The protein concentration was measured using a BCA protein detection kit (Abbkine, Wuhan, China) and subsequently diluted to 3 mg/ml. Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membrane (Millipore, USA.) The membrane was then blocked with a protein-free rapid blocking reagent at room temperature for 30 minutes and incubated overnight at 4 °C with the primary antibody. Following this, the membrane was incubated with an HRP-conjugated secondary antibody, and protein bands were detected using an ECL kit in the ECL Advance detection system. The grayscale of all bands was analyzed using ImageJ software. The experiment was independently repeated three times. Coimmunoprecipitation ( co-IP ) Co-IP was conducted using IgG and specific antibodies following the manufacturer's instructions. Briefly, the cell lysate was pre-incubated with antibodies on a rotating platform at 4 °C for 1 hour. Subsequently, protein A/G magnetic beads(TargetMol, USB) were added to the sample, and incubation continued overnight at 4 °C. The sample was then boiled in 1× SDS-PAGE loading buffer for 5 minutes, followed by five washes in immunoprecipitation detection lysis buffer to elute the complex. Cell Proliferation Assays Stable transfected TE-1 cells were inoculated into 96-well plates at a density of 4 × 10^4 cells per well and cultured for 24, 48, 72, and 96 hours. Following this, 10 µl of CCK-8 reagent was added to each well, and the cells were cultured for an additional 2 hours. Absorbance was measured at 450 nm, and the cell proliferation rate was calculated using the formula: Cell proliferation rate (%) = [(Absorbance at 48h/72h/96h of the negative control group) / (Absorbance at 24h of the negative control group)] × 100%. A growth curve was subsequently plotted, and the experiment was independently repeated three times. Cell cycle assay The EdU (Abbkine) incorporation assay serves as an effective method for detecting cell proliferation. Initially, inoculate 5 × 10³ cells into a 96-well plate. The following day, add 50 μM EdU and incubate at 37 °C for 2 hours. Subsequently, fix the cells in 4% formaldehyde for 30 minutes, followed by permeabilization in 0.5% Triton X-100 for 10 minutes. After rinsing the cells with PBS, incubate them for an additional 30 minutes. Nuclear DNA is then stained with DAPI (5 μg/ml) for 30 minutes. Images are observed under an inverted fluorescence microscope. For cell cycle analysis, resuspend the cells in 70% ethanol at -20 °C for 2 hours. Following centrifugation, resuspend the cell pellets in PI solution and incubate at 37 °C for 30 minutes. The cell cycle is subsequently analyzed using flow cytometry (BD Biosciences, USA). Apoptosis analysis TE-1, oe-CUEDC1, and sh-CUEDC1 cells were seeded onto six-well plates under optimal conditions. Cells were harvested after treatment with EDTA-free trypsin before reaching 80% confluence. Following two washes with pre-cooled PBS, the cells were resuspended in 500 μL of binding buffer. Subsequently, 5 μL of Annexin V-AF647 staining solution and 5 μL of 7-AAD staining solution were added, mixed thoroughly, and incubated at room temperature for 15 minutes. Flow cytometry analysis was conducted using FlowJo V10 (BD Biosciences, USA) for data analysis, and the experiment was independently repeated three times. Wound Healing Assays TE-1, oe-CUEDC1, and sh-CUEDC1 cells were seeded into six-well plates and allowed to grow to a density of 90%. They were then treated with 20 μg/mL of mitomycin C for 2 hours. Subsequently, sterile 200 μL pipettes were employed to create scratches in the wells, and the displaced cells were removed by washing with phosphate-buffered saline (PBS). The width of the scratch was observed and recorded under a microscope, designating this time point as 0 hours. The same location was photographed at the 24-hour and 48-hour time points. The obtained images were processed using ImageJ to extract relevant data. This experiment was independently repeated three times. Transwell assays Cells exhibiting good growth status should be removed from the culture medium. Subsequently, a serum-free culture medium should be added, and the cells should be starved for 12 hours. The matrix gel, which has been melted overnight at 4°C on ice, should be diluted with serum-free culture medium to a concentration of 1 mg/ml. A pre-cooled pipette should be used to mix the solution until uniform. Next, 60 µL of this mixed solution should be vertically added to the Transwell chamber. The chamber should then be incubated at 37°C for 1-3 hours, after which the unbound matrix gel should be carefully removed. The starved cells should be resuspended in serum-free medium and inoculated into the Transwell chamber at a density of 20,000 cells per well, while 600 µL of culture medium containing 20% FBS should be placed in the lower chamber. The setup should be incubated for 24 hours, followed by fixation with 4% paraformaldehyde and staining with 0.1% crystal violet. Microscope images should be captured, and cell counts should be measured. Each experiment must be independently repeated three times. Transmission electron microscopy The cells were washed twice with phosphate-buffered saline (PBS) and subsequently digested using 0.25% trypsin. Following digestion, the cell suspension was centrifuged at 1000 rpm for 5 minutes. The resulting cell clusters were fixed in 2% glutaraldehyde at 4 °C, embedded, sliced, and stained with uranyl acetate and lead citrate. Subsequently, the samples were analyzed using transmission electron microscopy (TEM, HITACHI HT7800). Colony Formation Assay Transfected TE-1 cells were inoculated into a 6-well plate at a density of 1 × 10³ cells per well, with the culture medium replaced every 3 days for a duration of 3 weeks. Following this period, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Colonies containing more than 50 cells were randomly counted under a light microscope. Statistical Methods Data are presented as mean ± standard deviation (SD) and analyzed using a t-test in GraphPad Prism 8.0. A p-value of less than 0.05 was considered statistically significant. Results CUEDC1 is lowly expressed in ESCA and is associated with poor prognosis. The transcriptional expression of CUEDC1 across various cancers was investigated using the TIMER database (Fig. 1A). It was observed that CUEDC1 mRNA expression is downregulated in ESCA patients compared to normal esophageal tissue (Fig. 1B). To validate these findings, RT-qPCR was employed to assess CUEDC1 mRNA levels in ESCA cells and normal human esophageal epithelial cells (Fig. 1D). The results demonstrated that CUEDC1 mRNA expression in ESCA cells was significantly lower than that in normal human esophageal epithelial cells, corroborating the findings from the TIMER database. Furthermore, this disparity was also evident at the protein level (Fig. 1C). Subsequently, we examined the correlation between CUEDC1 expression levels and clinical pathological features, revealing that low CUEDC1 expression is associated with tumor staging and overall survival (Fig. 1E, F). This statistical analysis underscores that both transcriptional and protein levels of CUEDC1 are diminished in ESCA, suggesting that low CUEDC1 expression may be linked to the staging and prognosis of ESCA to a certain extent. Overexpression of CUEDC1 reduces TE-1 proliferation and increases apoptosis. To investigate the effect of CUEDC1 on the cytological behavior of ESCA cells, we employed lentiviral transfection technology to introduce the CUEDC1 overexpression plasmid into the cells, thereby establishing a stable CUEDC1 overexpressing TE-1 strain (oe-CUEDC1). Subsequently, quantitative reverse transcription PCR (RT-qPCR) and Western blotting were utilized to validate transfection efficiency at the transcriptional and protein levels (Fig. 2A, B). Compared to the untransfected group (control group) and the control group (oe-NC), the proliferation curve of oe-CUEDC1 exhibited a significant lag, with a notably lower number and proportion of proliferating cells, as well as a marked reduction in colony formation (Fig. 2C, D, E, F). Scratch and invasion assays further demonstrated a significant decrease in invasion and metastatic capabilities (Fig. 3B, C). Flow cytometry analysis and apoptosis fluorescence intensity indicated a substantial increase in the number and proportion of apoptotic cells (Fig. 3D). Additionally, transmission electron microscopy revealed a relative decrease in autophagosomes in the oe-CUEDC1 group (Fig. 3A). Corresponding RNA and protein analyses also indicated significant changes consistent with the aforementioned cellular behaviors, including elevated levels of P53 and P21, reduced levels of Snai1 and Vimentin, decreased expression of Bcl-2, and increased expression of Bax, among others (Fig. 3E, F, G; Fig. S1). In summary, the overexpression of CUEDC1 results in reduced proliferation, increased apoptosis, and diminished invasive capability of TE-1 cells. This suggests that CUEDC1 may exert an inhibitory effect on the progression of ESCA. CUEDC1 affects the proliferation and apoptosis of TE-1 cells by regulating the JAK1/STAT3 pathway. To further investigate the mechanism by which CUEDC1 affects ESCA cells, we conducted transcriptome sequencing on two cell lines: oe-CUEDC1 and oe-NC. The obtained data were processed using reference genome alignment (STAR), differential expression analysis, KEGG pathway enrichment analysis, and other methods to generate visual representations (Fig. 4A, B). The results indicated that differentially expressed genes were enriched in the JAK/STAT pathway, which was significantly downregulated upon overexpression of CUEDC1 (Fig. 4C). Subsequently, we focused on JAK1/STAT3 and its downstream pathway by analyzing the differentially expressed genes within this pathway and their biological significance. STAT3 is one of the most extensively studied proteins in the JAK/STAT pathway, and its constitutive activation is frequently observed in various tumors, including melanoma, pancreatic cancer, lung cancer, colorectal cancer, and ovarian cancer 15-20 . By querying the differences in STAT3 transcription levels between ESCA tissue and normal tissue on TIMER, we found that the STAT3 transcription level in ESCA tissue was significantly elevated. Subsequently, we employed Western blot analysis to detect key proteins in this pathway, specifically JAK1 and STAT3, along with their phosphorylated forms, p-JAK1 and p-STAT3. Based on the experimental results, we observed that when CUEDC1 was overexpressed, the levels of JAK1, STAT3, p-JAK1, and p-STAT3 were significantly reduced compared to both the control group and the oe-NC group (Fig. 4D; Fig. S2). Additionally, the results from the RT-qPCR experiments indicated that their mRNA levels were also diminished (Fig. 4E). CUEDC1 initiates the ubiquitination degradation pathway of STAT3 by directly binding to STAT3 . As a downstream protein of JAK1, STAT3 is activated by phosphorylated JAK1 (p-JAK1), leading to its phosphorylation and dimerization, which subsequently allows its translocation into the nucleus to regulate the transcription of target genes such as Bcl-2, c-Myc, and Cyclin D1, all of which are associated with cell proliferation and survival 21,22 . It is crucial to further investigate why both STAT3 and phosphorylated STAT3 (p-STAT3) levels significantly decrease upon CUEDC1 overexpression. CUEDC1 is intricately linked to the ubiquitination process through the CUE domain; this protein can recruit ubiquitin molecules to modify target proteins following its binding, thereby initiating the ubiquitin degradation pathway 23,24 . Overexpression of CUEDC1 results in a marked increase in the overall ubiquitination levels within cells (Fig. 5C, G). Following treatment with cycloheximide (CHX, TargetMol, USB), which inhibits protein synthesis, we observed a gradual decrease in STAT3 levels in the oe-NC group, while the STAT3 protein levels further declined in the oe-CUEDC1 group (Fig. 5E). This observation indicates that the reduction of STAT3 in response to CUEDC1 overexpression is attributable to enhanced protein degradation. Further experiments involving MG132 (TargetMol, USB), a known inhibitor of the ubiquitination degradation pathway, demonstrated its ability to reverse the reduction of STAT3 protein levels induced by CUEDC1 overexpression (Fig. 5D). In essence, CUEDC1 overexpression leads to increased degradation of STAT3 due to the activation of the ubiquitin degradation pathway. To ascertain whether CUEDC1 interacts with STAT3 directly or indirectly, we conducted molecular docking simulations, co-immunoprecipitation (co-IP), and immunofluorescence colocalization experiments. The results consistently indicated that CUEDC1 directly interacts with STAT3 (Fig. 5A, B, F). In conclusion, CUEDC1 can directly bind to STAT3, recruit ubiquitin molecules to ubiquitinate STAT3, and facilitate its transport to the proteasome for degradation. This process ultimately reduces the protein levels of STAT3 within cells, thereby attenuating the transmission of upstream signals and diminishing cell proliferation and survival (Fig. 7). Silencing CUEDC1 enhances the proliferation, metastasis, and survival of TE-1 cells . To further confirm the effect of CUEDC1 on ESCA cells, we constructed a CUEDC1-silenced line (sh-CUEDC1) by transfecting TE-1 cells with a lentivirus carrying sh-CUEDC1. When CUEDC1 is silenced, TE-1 cells exhibit increased proliferative activity, invasive ability, and resistance to apoptosis compared to the control group (Fig. 6A, B, C, D, F, G; Fig. S3). Correspondingly, these changes are also observed at the protein levels (Fig. 6E). DISCUSSION ESCA, recognized as a highly lethal disease, has seen some advancements in treatment in recent years; however, the prognosis and quality of life for patients remain poor 25 . The early symptoms of this malignancy are often subtle, and its rapid growth, high invasiveness, and propensity for recurrence frequently result in late-stage diagnoses, where most patients are limited to palliative care 26 , 27 . At this advanced stage, patients commonly experience eating disorders. Although postoperative esophageal reconstruction or endoscopic stent implantation may offer temporary relief, the duration and extent of this relief are often limited, ultimately leading to the patient's demise due to energy depletion 28 , 29 . Currently, targeted therapy represents a prominent area of research in cancer treatment owing to its superior efficacy and minimal side effects. However, the advancement of targeted therapy for ESCA has been notably sluggish. The dispersed nature of tumor driver genes and significant individual variability result in a lack of precise biomarkers for clinical treatment 30 . ESCA contributes significantly to the global cancer burden, with a high mortality rate each year, underscoring the necessity for dedicated research into targeted therapies for this malignancy 3 . Numerous studies have demonstrated that the JAK1/STAT3 pathway plays a crucial role in tumor progression. In the context of ESCA, this pathway can directly regulate gene expression, upregulating the levels of oncogenic proteins, thereby facilitating tumor progression. Our research aims to elucidate the regulatory mechanisms of the JAK1/STAT3 pathway in esophageal cells, with the hope of providing a new perspective for the advancement of molecular medicine. CUEDC1 contains a CUE domain, which can affect the degradation process of specific proteins by interacting with the ubiquitin system, thereby regulating protein levels in cells 23 , 31 . In this study, we found that CUEDC1 was downregulated in ESCA tissues, and low expression of CUEDC1 was associated with poor overall survival; however, we did not determine its association with the pathological type of this cancer. Additionally, we demonstrated for the first time that CUEDC1 upregulation inhibits ESCA cell proliferation and tumor progression. Conversely, downregulation of CUEDC1 significantly promoted the proliferation and survival of ESCA cell lines (anti-apoptosis), suggesting that low expression of CUEDC1 may be linked to drug resistance. Given the clinical and biological significance of CUEDC1, our study indicates that CUEDC1 may serve as a potential biomarker for the prognosis of ESCA and has potential therapeutic applications in the future. Mechanistically, we understand that ubiquitin molecules can bind to target proteins 32 , 33 . Both tumor suppression and tumor promotion pathways are regulated by ubiquitination 34 . Depending on the properties of the substrate, a single ubiquitin ligase can function as both an oncogene and a tumor suppressor 35 . This duality includes the ubiquitination-mediated degradation of key proteins, which affects their abundance, as well as the regulation of protein activity through ubiquitination modifications 36 – 38 . Consequently, the same ubiquitin ligase can exhibit varying effects on tumors, contingent upon the role its substrate plays in cancer progression 39 . Additionally, regulatory factors such as CUE domain-containing proteins can also assume completely opposing roles in different cancers due to these same underlying mechanisms 40 – 42 . We observed that following the upregulation of CUEDC1 expression, the intracellular ubiquitination level significantly increased, while the STAT3 protein level decreased. Subsequently, we monitored the dynamic changes of STAT3 protein through CHX and MG132 treatment, further suggesting that the decrease in STAT3 protein levels is achieved through ubiquitin-proteasome degradation. To explore the role of CUEDC1 in this process, we conducted molecular protein docking simulations, co-immunoprecipitation, and immunofluorescence co-localization experiments, revealing that CUEDC1 can directly bind to STAT3. Based on previous studies, we speculate that CUEDC1 recruits ubiquitin molecules to modify STAT3 after binding, leading to its degradation. However, we only predicted the relevant binding sites in molecular simulations and did not validate them through site-directed mutagenesis. Numerous types of ubiquitin E3 ligases are implicated in the degradation of STAT3 through ubiquitination. Among these, SLIM has been confirmed in previous studies, along with VHL, TRIM27, and Smurf1 43–46 . However, we have yet to determine which specific ligase plays a predominant role in this process. Research conducted by Paul S. Andrews indicates that Smurf1 and CUEDC1 can spatially associate; however, their functional interactions remain unreported 47 . It is plausible that Smurf1 and STAT3 may interact with each other through CUEDC1 as a mediator. This issue warrants clarification in future research. Conclusions Our results demonstrate that CUEDC1 is downregulated in ESCA, and its downregulation is closely related to the poor overall survival rate of ESCA patients. CUEDC1 can directly bind to STAT3 and initiate its ubiquitination and degradation, thereby inhibiting the role of the JAK/STAT3 signaling pathway in promoting cell proliferation, metastasis, and survival. Our data provide a new perspective on the regulation of STAT3 signaling in the pathogenesis of ESCA. CUEDC1 may serve as a potential target and prognostic factor for the treatment of this disease. Declarations Data availability Data is provided within the manuscript or supplementary information files. ACKNOWLEDGEMENTS We thank TCGA, KEGG, TIMER and GEPIA for providing data, and we are thankful to the Natural Science Fund of Heilongjiang Province grant (LH2023H097). Author contributions Z.L. and Z.P. conceptualized and designed the experiment. Z.L. and X.S. conducted experiments and collected data. C.L. and J.Z. analyzed the data and prepared charts. G.L. and H.L prepared pictures and formatted. Z.L. and Y.C. wrote and revised the manuscript. H.X. and C.Y. managed and reviewed the project and data. All authors contributed to this article and approved the final manuscript. Competing interests The authors report no competing interest. References Bray, F. et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. Cancer J. Clin. 74 , 229–263. 10.3322/caac.21834 (2024). Chen, W. et al. 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EMBO Rep. 9 , 536–542. 10.1038/embor.2008.93 (2008). Nakayama, K. I. & Nakayama, K. Ubiquitin ligases: cell-cycle control and cancer. Nat. Rev. Cancer . 6 , 369–381. 10.1038/nrc1881 (2006). Freedman, M. L. et al. ELF5 modulates the estrogen receptor cistrome in breast cancer. PLoS Genet. 16 10.1371/journal.pgen.1008531 (2020). Li, F. et al. CUEDC2 suppresses glioma tumorigenicity by inhibiting the activation of STAT3 and NF-κB signaling pathway. Int. J. Oncol. 51 , 115–127. 10.3892/ijo.2017.4009 (2017). Zhong, X. et al. CUE domain-containing protein 2 promotes the Warburg effect and tumorigenesis. EMBO Rep. 18 , 809–825. 10.15252/embr.201643617 (2017). Tanaka, T., Soriano, M. A. & Grusby, M. J. SLIM Is a Nuclear Ubiquitin E3 Ligase that Negatively Regulates STAT Signaling. Immunity 22 , 729–736. 10.1016/j.immuni.2005.04.008 (2005). La Sala, G. et al. Selective inhibition of STAT3 signaling using monobodies targeting the coiled-coil and N-terminal domains. Nat. Commun. 11 10.1038/s41467-020-17920-z (2020). Zhang, H. X. et al. TRIM27 mediates STAT3 activation at retromer-positive structures to promote colitis and colitis-associated carcinogenesis. Nat. Commun. 9 10.1038/s41467-018-05796-z (2018). Hou, X. et al. Talin-1 inhibits Smurf1-mediated Stat3 degradation to modulate β-cell proliferation and mass in mice. Cell Death Dis. 14 10.1038/s41419-023-06235-8 (2023). Andrews, P. S. et al. Identification of Substrates of SMURF1 Ubiquitin Ligase Activity Utilizing Protein Microarrays. Assay Drug Dev. Technol. 8 , 471–487. 10.1089/adt.2009.0264 (2010). Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1.png Supplementary Figure 1. (A) By staining cells with DAPI and PI, and observing under a fluorescence microscope, it was found that oe-CUEDC1 had more bright red color, indicating more apoptotic cells. (B) Statistical chart of changes in mRNA levels in oe-CUEDC1 and oe-NC. SupplementaryFigure2.png Supplementary Figure 2. Statistical chart of changes in protein levels in oe-CUEDC1 and oe-NC. SupplementaryFigure3.png Supplementary Figure 3. (A) Wound healing test to migration effect and statistical analysis in sh-CUEDC1 and sh-NC. (B-F) Statistical chart of the differences between sh-CUEDC1 and sh-NC in functional experiments. Cite Share Download PDF Status: Published Journal Publication published 31 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 16 Sep, 2025 Reviews received at journal 04 Aug, 2025 Reviewers agreed at journal 22 Jul, 2025 Reviews received at journal 22 Jul, 2025 Reviewers agreed at journal 08 Jul, 2025 Reviewers agreed at journal 02 Jun, 2025 Reviewers invited by journal 28 May, 2025 Editor assigned by journal 28 May, 2025 Editor invited by journal 22 May, 2025 Submission checks completed at journal 22 May, 2025 First submitted to journal 08 May, 2025 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. 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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-6618873","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":463334425,"identity":"49727c65-c0ca-41ee-8c1e-7459e8fe3534","order_by":0,"name":"Zhuo Li","email":"","orcid":"","institution":"Department of Oncology, The Second Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhuo","middleName":"","lastName":"Li","suffix":""},{"id":463334426,"identity":"d5e522b1-19f6-4651-a292-490c3c282339","order_by":1,"name":"Zhipeng Pan","email":"","orcid":"","institution":"Department of Oncology, The Second Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhipeng","middleName":"","lastName":"Pan","suffix":""},{"id":463334427,"identity":"f8210db3-827f-4fed-b1ad-fc7312a62746","order_by":2,"name":"Xuehan Su","email":"","orcid":"","institution":"Department of Dermatology, The Second Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xuehan","middleName":"","lastName":"Su","suffix":""},{"id":463334428,"identity":"e53151b7-9d6d-452b-9158-ba8c046eefbe","order_by":3,"name":"Chunhong Li","email":"","orcid":"","institution":"Department of Breast Medical Oncology, Third Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chunhong","middleName":"","lastName":"Li","suffix":""},{"id":463334429,"identity":"3127f08b-6311-444a-8283-d5922f0dc052","order_by":4,"name":"Jian Zhang","email":"","orcid":"","institution":"Department of thoracic surgery, Harbin Medical University Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Zhang","suffix":""},{"id":463334430,"identity":"5dd57f5c-121b-47ff-949e-7d25c01d62a7","order_by":5,"name":"Guohe Lin","email":"","orcid":"","institution":"Department of Oncology, The Second Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guohe","middleName":"","lastName":"Lin","suffix":""},{"id":463334431,"identity":"c5d5480a-6dba-4bfa-8f5f-ecf42fbc77de","order_by":6,"name":"Haowu Li","email":"","orcid":"","institution":"Department of Oncology, The Second Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Haowu","middleName":"","lastName":"Li","suffix":""},{"id":463334432,"identity":"10d82e68-f86b-4ea4-a8b5-744d8c712657","order_by":7,"name":"Yue Cui","email":"","orcid":"","institution":"Department of Oncology, The Second Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Cui","suffix":""},{"id":463334433,"identity":"d78b53ea-47d0-45e2-bce3-a9830d9fbc76","order_by":8,"name":"Han Xuan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYBACgxvpBx9+/GMjx8befoBYLTnJxpINacZ8PGcSiNMiOSPBTIC34VDiPAkHA+K08EskpDFI7jiQ3ibBkMDwo2IbYS1s8g+PPSg8cye3TbrxAGPPmdtEaJFISDeQYHuW2yZzIIGZsY0ILUCHmUnwsB1OB+o1IE4LyPsSvG2HE4jXAg5kiTNphm3AQD5IlF/AUfmhwkZevr394IMfFURoQQEHSFQ/CkbBKBgFowAXAACFCECKXxuZqgAAAABJRU5ErkJggg==","orcid":"","institution":"Department of Oncology, The Second Affiliated Hospital of Anhui Medical University","correspondingAuthor":true,"prefix":"","firstName":"Han","middleName":"","lastName":"Xuan","suffix":""}],"badges":[],"createdAt":"2025-05-08 09:08:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6618873/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6618873/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-46681-w","type":"published","date":"2026-03-31T15:58:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83610077,"identity":"f91b5305-20c2-479f-be96-467fa036b2ad","added_by":"auto","created_at":"2025-05-29 12:05:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":516167,"visible":true,"origin":"","legend":"\u003cp\u003eCUEDC1 expression and clinical features in ESCA patients. (\u003cstrong\u003eA\u003c/strong\u003e) The mRNA expression of CUEDC1 in TIMER database. (\u003cstrong\u003eB\u003c/strong\u003e) The transcription level of CUEDC1 in TCGA database. (\u003cstrong\u003eC-D\u003c/strong\u003e) The CUEDC1 protein and mRNA levels in TE-1 and BAR-T. (\u003cstrong\u003eE\u003c/strong\u003e)The prognostic significance of CUEDC1 in esophageal cancer in the GEPIA database.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eF\u003c/strong\u003e) The correlation of ZCCHC4 transcriptional expression and cancer stages 1–4.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6618873/v1/ff9225d721bd484969c0bcd4.png"},{"id":83609619,"identity":"18097819-23bd-4e93-a212-551a76bc9445","added_by":"auto","created_at":"2025-05-29 11:57:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1001405,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of CUEDC1 can inhibit cell proliferation. (\u003cstrong\u003eA-B\u003c/strong\u003e) WB and RT-qPCR to analyze the expression of CUEDC1 in TE-1 cells transfected with oe-CUEDC1. (\u003cstrong\u003eC\u003c/strong\u003e) CCK-8 assay to analyze cell proliferation. (\u003cstrong\u003eD\u003c/strong\u003e) The colony formation test showed decreased colony number in gene overexpressing cells. (\u003cstrong\u003eE\u003c/strong\u003e) EdU staining to detect the cell proliferation of oe-CUEDC1 and oe-NC. (\u003cstrong\u003eF\u003c/strong\u003e) The levels of cell cycle were examined by flow cytometry assay. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6618873/v1/e121cd8df20cd44a7e21153c.png"},{"id":83610075,"identity":"7f9b9b1e-7ba9-43ae-b00a-0cc2275f99a2","added_by":"auto","created_at":"2025-05-29 12:05:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2777270,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of CUEDC1 can inhibit cell migration, invasion and promote apoptosis. (\u003cstrong\u003eA\u003c/strong\u003e) Transmission electron microscopy image of TE-1 cells (red arrows represent autophagosomes, blue arrows represent self-apoptotic bodies). (\u003cstrong\u003eB\u003c/strong\u003e) Wound healing test to migration effect. (\u003cstrong\u003eC\u003c/strong\u003e) Transwell assays to analyze invasion. (\u003cstrong\u003eD\u003c/strong\u003e) The levels of apoptosis were examined by flow cytometry assay. (\u003cstrong\u003eE-G\u003c/strong\u003e) Use WB to measure the abundance of proteins such as CUEDC1, c-MYC, Vimentin, Bax, etc. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6618873/v1/0dc7aa7ebb898d3e6fd83d7e.png"},{"id":83609626,"identity":"d01fc26c-bdb0-4806-b711-eff509105de5","added_by":"auto","created_at":"2025-05-29 11:57:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":792983,"visible":true,"origin":"","legend":"\u003cp\u003eCUEDC1 affects the cellular function of TE-1 by regulating the JAK1/STAT3 pathway. (\u003cstrong\u003eA\u003c/strong\u003e) Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation. (\u003cstrong\u003eB\u003c/strong\u003e) Heatmap of the top 25 upregulated and downregulated genes. (\u003cstrong\u003eC\u003c/strong\u003e) GSEA plot depicted enrichment of the JAK/STAT pathway in TE-1 cells with CUEDC1 overexpression. FDR, false discovery rate; NES, normalized enrichment score. (\u003cstrong\u003eD\u003c/strong\u003e) Western blot was applied to detect phosphorylated proteins in the JAK1/STAT3 signaling pathway. (\u003cstrong\u003eE\u003c/strong\u003e) RT-qPCR was applied to detect mRNA of proteins involved in the JAK1/STAT3 signaling pathway.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6618873/v1/768a5b09b6ae89b4d10e6e13.png"},{"id":83609628,"identity":"4398e15c-d1c4-4d7f-9066-e6208ca3830c","added_by":"auto","created_at":"2025-05-29 11:57:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1752288,"visible":true,"origin":"","legend":"\u003cp\u003eThe interaction between CUEDC1 and STAT3. (\u003cstrong\u003eA\u003c/strong\u003e) The protein-protein interaction diagram and the names of amino acid residues involved in hydrogen bonding interactions between proteins, where the blue part represents protein CUEDC1 and the khaki part represents protein STAT3. Hydrogen bonds are represented by yellow dashed lines. ARG187, ILE188, GLN192, PRO218, GLY219, GLN223, and ARG226 in protein CUEDC1 interact with GLN288, LYS290, LYS180, GLN205, GLN205, and GLN202 in protein STAT3 through hydrogen bonding. (\u003cstrong\u003eB\u003c/strong\u003e) Co-localization of CUEDC1 (green) and STAT3 (red) within the cell, with orange indicating the same location for both. (\u003cstrong\u003eC\u003c/strong\u003e)Relative content of intracellular K48 ubiquitin levels. (\u003cstrong\u003eD\u003c/strong\u003e) Changes of STAT3 after MG132 and CHX treatment in oe-CUEDC1. (\u003cstrong\u003eE\u003c/strong\u003e) CHX treatment further reduces STAT3 in oe-CUEDC1. (\u003cstrong\u003eF\u003c/strong\u003e) Co-IP for detecting the interaction between CUEDC and STAT3. (\u003cstrong\u003eG\u003c/strong\u003e) Co-IP for detecting the interaction between CUEDC and K48 ubiquitin.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6618873/v1/a552a75f8e47a5ccccb3fa5f.png"},{"id":83610766,"identity":"65a1875d-5661-471c-a421-8c445a1e1cbe","added_by":"auto","created_at":"2025-05-29 12:13:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2173462,"visible":true,"origin":"","legend":"\u003cp\u003eSilencing CUEDC1 expression in cells promoted cell proliferation, migration, invasion and inhibit apoptosis. (\u003cstrong\u003eA\u003c/strong\u003e) CCK-8 assay to analyze cell proliferation. (\u003cstrong\u003eB\u003c/strong\u003e) Transwell assays to analyze invasion. (\u003cstrong\u003eC\u003c/strong\u003e) The colony formation test to analyze cell proliferation. (\u003cstrong\u003eD\u003c/strong\u003e) EdU staining to detect the cell proliferation. (\u003cstrong\u003eE\u003c/strong\u003e) Use WB to measure the abundance of proteins. (\u003cstrong\u003eF-G\u003c/strong\u003e) The levels of apoptosis and cell cycle were examined by flow cytometry assay. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6618873/v1/95b5bf3332416138592a7d77.png"},{"id":83609629,"identity":"7bad2fbc-0267-4985-ab24-3fcb702e0670","added_by":"auto","created_at":"2025-05-29 11:57:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1512865,"visible":true,"origin":"","legend":"\u003cp\u003eCUEDC1 can directly bind to STAT3 and initiate its ubiquitination and degradation, thereby inhibiting the role of the JAK/STAT3 signaling pathway in promoting cell proliferation, metastasis, and survival.\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6618873/v1/d3d5516b4d8c67cd78adab76.png"},{"id":106343626,"identity":"548b6d7f-ca7c-4a9e-bbba-a2ec5d1ccfe1","added_by":"auto","created_at":"2026-04-07 16:07:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12326624,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6618873/v1/58ea0821-fdc6-469d-875d-a6f45f3d971c.pdf"},{"id":83609623,"identity":"c47c2df9-da3c-4486-a727-36dfeab7f785","added_by":"auto","created_at":"2025-05-29 11:57:44","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":643769,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 1. (\u003cstrong\u003eA\u003c/strong\u003e) By staining cells with DAPI and PI, and observing under a fluorescence microscope, it was found that oe-CUEDC1 had more bright red color, indicating more apoptotic cells. (\u003cstrong\u003eB\u003c/strong\u003e) Statistical chart of changes in mRNA levels in oe-CUEDC1 and oe-NC.\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6618873/v1/f2a84f5dfd97219a4ff5ae88.png"},{"id":83609620,"identity":"23d4ff2d-1887-4d0a-89b5-5235707303d3","added_by":"auto","created_at":"2025-05-29 11:57:44","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":181610,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 2. Statistical chart of changes in protein levels in oe-CUEDC1 and oe-NC.\u003c/p\u003e","description":"","filename":"SupplementaryFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6618873/v1/663bf91aee0e9dba008d5471.png"},{"id":83609627,"identity":"c47e0f5d-9c35-4632-8f26-4763a99c987f","added_by":"auto","created_at":"2025-05-29 11:57:44","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":987863,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 3. (\u003cstrong\u003eA\u003c/strong\u003e) Wound healing test to migration effect and statistical analysis in sh-CUEDC1 and sh-NC. (\u003cstrong\u003eB-F\u003c/strong\u003e) Statistical chart of the differences between sh-CUEDC1 and sh-NC in functional experiments.\u003c/p\u003e","description":"","filename":"SupplementaryFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6618873/v1/139c23d9dfd80b604c344545.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Role of CUEDC1 in Suppressing JAK1/STAT3 Signaling Pathway in Esophageal Cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEsophageal cancer (ESCA) is a malignancy characterized by a poor prognosis, ranking 11th in incidence and 7th in mortality among all cancers. In 2022, it was estimated that there would be 511,000 new cases and 445,000 deaths\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Histologically, ESCA is classified into two main types: squamous cell carcinoma (SCC) and esophageal adenocarcinoma (EAC). The former constitutes approximately 85% of all cases, while the latter accounts for about 14%\u003csup\u003e2\u003c/sup\u003e. These two types exhibit relatively independent etiologies and geographical distributions. In regions such as North America and Western Europe, adenocarcinoma accounts for about two-thirds of cases, which is associated with obesity, gastroesophageal reflux disease, and Barrett's esophagus. Conversely, squamous cell carcinoma is the predominant subtype in East Asia and East Africa, closely linked to factors such as smoking, alcohol abuse, consumption of excessively salty or hot foods, and exposure to air pollution\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Moreover, the prognosis for esophageal cancer remains poor, posing a significant burden on global health\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is a critical cellular signaling mechanism that plays a vital role in various biological processes, including cell proliferation, differentiation, apoptosis, and immune response\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. This pathway facilitates the growth and survival of cancer cells through the regulation of gene expression. For instance, in colorectal cancer, the activation of STAT3 and JAK1/2 is closely associated with the proliferation and invasion of tumor cells\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Furthermore, this pathway can promote tumor immune escape and drug resistance by modulating immune cells and inflammatory factors within the tumor microenvironment\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Targeting the JAK/STAT pathway offers a novel strategy for cancer treatment\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Future research should further investigate its specific mechanisms and potential clinical applications\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCUEDC1(CUE domain-containing protein 1) is a protein that contains the CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation) domain and is commonly expressed in cells\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Currently, there is limited research on CUEDC1. It has been reported that CUEDC1, as a functional target gene of estrogen receptor alpha (ERα), is associated with poor prognosis in breast cancer\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Additionally, there is a significant increase in CUEDC1 expression in metastatic cervical cancer compared to early tumors\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, in contrast, overexpression of CUEDC1 in non-small cell lung cancer reduces the metastatic potential of cancer cells and inhibits the epithelial-mesenchymal transition (EMT) process\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In summary, the role of CUEDC1 appears to vary across different tumors, and the exact role of CUEDC1 in the metastasis and progression of ESCA remains unclear.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eCell Culture and Lentivirus Infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human ESCA cell line TE-1 and the human esophageal epithelial cell line BAR-T were obtained from Zishan Biology Co., Ltd in Wuhan, China. Cells were cultured in RPMI-1640 medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum(Biochannel, Nanjing, China) at 37°C and 5% CO2, with regular confirmation using short tandem repeat (STR) fingerprinting. The CUEDC1 overexpression sequence and gene knockout sequence for CUEDC1 were designed and synthesized by General Biosciences and packaged in lentivirus for transfection into TE-1 cells, yielding CUEDC1 overexpression (oe-CUEDC1) and silence (sh-CUEDC1) cell lines, respectively. The specific procedure involved evenly plating 1 x 10^6 TE-1 cells in a six-well plate and incubating overnight. The following day, the culture medium was discarded, and the cells were rinsed twice with phosphate-buffered saline (PBS). Subsequently, 2 ml of complete RPMI-1640 medium containing 1 x 10^6 viral particles and 8% polybrene was added. After 24 hours, the culture medium was replaced with fresh medium, and puromycin was added to select stable cell lines. The transfection efficiency was validated using RT-qPCR and Western blot analysis. The relevant sequences can be found in supplementary materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReverse transcription quantitative polymerase chain reaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA from TE-1 cells was extracted using an RNA rapid extraction kit (EnzyArtisan, Shanghai, China), followed by reverse transcription to synthesize cDNA, which was analyzed with a qPCR kit on appropriate instruments. GAPDH served as the internal control, and relative gene expression was normalized using the 2^-ΔΔCT method. This experiment was performed independently three times.\u003c/p\u003e\n\u003cp\u003ePrimer sequences can be found in Supplementary Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo obtain proteins, cells were lysed using RIPA lysis buffer supplemented with protease and phosphatase inhibitors. The protein concentration was measured using a BCA protein detection kit (Abbkine, Wuhan, China) and subsequently diluted to 3 mg/ml. Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membrane (Millipore, USA.) The membrane was then blocked with a protein-free rapid blocking reagent at room temperature for 30 minutes and incubated overnight at 4 °C with the primary antibody. Following this, the membrane was incubated with an HRP-conjugated secondary antibody, and protein bands were detected using an ECL kit in the ECL Advance detection system. The grayscale of all bands was analyzed using ImageJ software. The experiment was independently repeated three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCoimmunoprecipitation\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003eco-IP\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eCo-IP was conducted using IgG and specific antibodies following the manufacturer's instructions. Briefly, the cell lysate was pre-incubated with antibodies on a rotating platform at 4 °C for 1 hour. Subsequently, protein A/G magnetic beads(TargetMol, USB) were added to the sample, and incubation continued overnight at 4 °C. The sample was then boiled in 1× SDS-PAGE loading buffer for 5 minutes, followed by five washes in immunoprecipitation detection lysis buffer to elute the complex.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Proliferation Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStable transfected TE-1 cells were inoculated into 96-well plates at a density of 4 × 10^4 cells per well and cultured for 24, 48, 72, and 96 hours. Following this, 10 µl of CCK-8 reagent was added to each well, and the cells were cultured for an additional 2 hours. Absorbance was measured at 450 nm, and the cell proliferation rate was calculated using the formula: Cell proliferation rate (%) = [(Absorbance at 48h/72h/96h of the negative control group) / (Absorbance at 24h of the negative control group)] × 100%. A growth curve was subsequently plotted, and the experiment was independently repeated three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell cycle assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe EdU (Abbkine) incorporation assay serves as an effective method for detecting cell proliferation. Initially, inoculate 5 × 10³ cells into a 96-well plate. The following day, add 50 μM EdU and incubate at 37 °C for 2 hours. Subsequently, fix the cells in 4% formaldehyde for 30 minutes, followed by permeabilization in 0.5% Triton X-100 for 10 minutes. After rinsing the cells with PBS, incubate them for an additional 30 minutes. Nuclear DNA is then stained with DAPI (5 μg/ml) for 30 minutes. Images are observed under an inverted fluorescence microscope. For cell cycle analysis, resuspend the cells in 70% ethanol at -20 °C for 2 hours. Following centrifugation, resuspend the cell pellets in PI solution and incubate at 37 °C for 30 minutes. The cell cycle is subsequently analyzed using flow cytometry (BD Biosciences, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApoptosis analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTE-1, oe-CUEDC1, and sh-CUEDC1 cells were seeded onto six-well plates under optimal conditions. Cells were harvested after treatment with EDTA-free trypsin before reaching 80% confluence. Following two washes with pre-cooled PBS, the cells were resuspended in 500 μL of binding buffer. Subsequently, 5 μL of Annexin V-AF647 staining solution and 5 μL of 7-AAD staining solution were added, mixed thoroughly, and incubated at room temperature for 15 minutes. Flow cytometry analysis was conducted using FlowJo V10 (BD Biosciences, USA) for data analysis, and the experiment was independently repeated three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound Healing Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTE-1, oe-CUEDC1, and sh-CUEDC1 cells were seeded into six-well plates and allowed to grow to a density of 90%. They were then treated with 20 μg/mL of mitomycin C for 2 hours. Subsequently, sterile 200 μL pipettes were employed to create scratches in the wells, and the displaced cells were removed by washing with phosphate-buffered saline (PBS). The width of the scratch was observed and recorded under a microscope, designating this time point as 0 hours. The same location was photographed at the 24-hour and 48-hour time points. The obtained images were processed using ImageJ to extract relevant data. This experiment was independently repeated three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranswell assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells exhibiting good growth status should be removed from the culture medium. Subsequently, a serum-free culture medium should be added, and the cells should be starved for 12 hours. The matrix gel, which has been melted overnight at 4°C on ice, should be diluted with serum-free culture medium to a concentration of 1 mg/ml. A pre-cooled pipette should be used to mix the solution until uniform. Next, 60 µL of this mixed solution should be vertically added to the Transwell chamber. The chamber should then be incubated at 37°C for 1-3 hours, after which the unbound matrix gel should be carefully removed. The starved cells should be resuspended in serum-free medium and inoculated into the Transwell chamber at a density of 20,000 cells per well, while 600 µL of culture medium containing 20% FBS should be placed in the lower chamber. The setup should be incubated for 24 hours, followed by fixation with 4% paraformaldehyde and staining with 0.1% crystal violet. Microscope images should be captured, and cell counts should be measured. Each experiment must be independently repeated three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission electron microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cells were washed twice with phosphate-buffered saline (PBS) and subsequently digested using 0.25% trypsin. Following digestion, the cell suspension was centrifuged at 1000 rpm for 5 minutes. The resulting cell clusters were fixed in 2% glutaraldehyde at 4 °C, embedded, sliced, and stained with uranyl acetate and lead citrate. Subsequently, the samples were analyzed using transmission electron microscopy (TEM, HITACHI HT7800).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony Formation Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransfected TE-1 cells were inoculated into a 6-well plate at a density of 1 × 10³ cells per well, with the culture medium replaced every 3 days for a duration of 3 weeks. Following this period, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Colonies containing more than 50 cells were randomly counted under a light microscope.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± standard deviation (SD) and analyzed using a t-test in GraphPad Prism 8.0. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCUEDC1 is lowly expressed in ESCA and is associated with poor prognosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe transcriptional expression of CUEDC1 across various cancers was investigated using the TIMER database (Fig. 1A). It was observed that CUEDC1 mRNA expression is downregulated in ESCA patients compared to normal esophageal tissue (Fig. 1B). To validate these findings, RT-qPCR was employed to assess CUEDC1 mRNA levels in ESCA cells and normal human esophageal epithelial cells (Fig. 1D). The results demonstrated that CUEDC1 mRNA expression in ESCA cells was significantly lower than that in normal human esophageal epithelial cells, corroborating the findings from the TIMER database. Furthermore, this disparity was also evident at the protein level (Fig. 1C). Subsequently, we examined the correlation between CUEDC1 expression levels and clinical pathological features, revealing that low CUEDC1 expression is associated with tumor staging and overall survival (Fig. 1E, F). This statistical analysis underscores that both transcriptional and protein levels of CUEDC1 are diminished in ESCA, suggesting that low CUEDC1 expression may be linked to the staging and prognosis of ESCA to a certain extent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOverexpression of CUEDC1 reduces TE-1 proliferation and increases apoptosis.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the effect of CUEDC1 on the cytological behavior of ESCA cells, we employed lentiviral transfection technology to introduce the CUEDC1 overexpression plasmid into the cells, thereby establishing a stable CUEDC1 overexpressing TE-1 strain (oe-CUEDC1). Subsequently, quantitative reverse transcription PCR (RT-qPCR) and Western blotting were utilized to validate transfection efficiency at the transcriptional and protein levels (Fig. 2A, B). Compared to the untransfected group (control group) and the control group (oe-NC), the proliferation curve of oe-CUEDC1 exhibited a significant lag, with a notably lower number and proportion of proliferating cells, as well as a marked reduction in colony formation (Fig. 2C, D, E, F). Scratch and invasion assays further demonstrated a significant decrease in invasion and metastatic capabilities (Fig. 3B, C). Flow cytometry analysis and apoptosis fluorescence intensity indicated a substantial increase in the number and proportion of apoptotic cells (Fig. 3D). Additionally, transmission electron microscopy revealed a relative decrease in autophagosomes in the oe-CUEDC1 group (Fig. 3A). Corresponding RNA and protein analyses also indicated significant changes consistent with the aforementioned cellular behaviors, including elevated levels of P53 and P21, reduced levels of Snai1 and Vimentin, decreased expression of Bcl-2, and increased expression of Bax, among others (Fig. 3E, F, G;\u0026nbsp;Fig. S1).\u003c/p\u003e\n\u003cp\u003eIn summary, the overexpression of CUEDC1 results in reduced proliferation, increased apoptosis, and diminished invasive capability of TE-1 cells. This suggests that CUEDC1 may exert an inhibitory effect on the progression of ESCA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCUEDC1 affects the proliferation and apoptosis of TE-1 cells by regulating the JAK1/STAT3 pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the mechanism by which CUEDC1 affects ESCA cells, we conducted transcriptome sequencing on two cell lines: oe-CUEDC1 and oe-NC. The obtained data were processed using reference genome alignment (STAR), differential expression analysis, KEGG pathway enrichment analysis, and other methods to generate visual representations (Fig. 4A, B). The results indicated that differentially expressed genes were enriched in the JAK/STAT pathway, which was significantly downregulated upon overexpression of CUEDC1 (Fig. 4C). Subsequently, we focused on JAK1/STAT3 and its downstream pathway by analyzing the differentially expressed genes within this pathway and their biological significance.\u003c/p\u003e\n\u003cp\u003eSTAT3 is one of the most extensively studied proteins in the JAK/STAT pathway, and its constitutive activation is frequently observed in various tumors, including melanoma, pancreatic cancer, lung cancer, colorectal cancer, and ovarian cancer\u003csup\u003e15-20\u003c/sup\u003e. By querying the differences in STAT3 transcription levels between ESCA tissue and normal tissue on TIMER, we found that the STAT3 transcription level in ESCA tissue was significantly elevated. Subsequently, we employed Western blot analysis to detect key proteins in this pathway, specifically JAK1 and STAT3, along with their phosphorylated forms, p-JAK1 and p-STAT3. Based on the experimental results, we observed that when CUEDC1 was overexpressed, the levels of JAK1, STAT3, p-JAK1, and p-STAT3 were significantly reduced compared to both the control group and the oe-NC group (Fig. 4D; Fig. S2). Additionally, the results from the RT-qPCR experiments indicated that their mRNA levels were also diminished (Fig. 4E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCUEDC1 initiates the ubiquitination degradation pathway of STAT3 by directly binding to STAT3\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eAs a downstream protein of JAK1, STAT3 is activated by phosphorylated JAK1 (p-JAK1), leading to its phosphorylation and dimerization, which subsequently allows its translocation into the nucleus to regulate the transcription of target genes such as Bcl-2, c-Myc, and Cyclin D1, all of which are associated with cell proliferation and survival\u003csup\u003e21,22\u003c/sup\u003e. It is crucial to further investigate why both STAT3 and phosphorylated STAT3 (p-STAT3) levels significantly decrease upon CUEDC1 overexpression. CUEDC1 is intricately linked to the ubiquitination process through the CUE domain; this protein can recruit ubiquitin molecules to modify target proteins following its binding, thereby initiating the ubiquitin degradation pathway\u003csup\u003e23,24\u003c/sup\u003e. Overexpression of CUEDC1 results in a marked increase in the overall ubiquitination levels within cells (Fig. 5C, G). Following treatment with cycloheximide (CHX, TargetMol, USB), which inhibits protein synthesis, we observed a gradual decrease in STAT3 levels in the oe-NC group, while the STAT3 protein levels further declined in the oe-CUEDC1 group (Fig. 5E). This observation indicates that the reduction of STAT3 in response to CUEDC1 overexpression is attributable to enhanced protein degradation. Further experiments involving MG132 (TargetMol, USB), a known inhibitor of the ubiquitination degradation pathway, demonstrated its ability to reverse the reduction of STAT3 protein levels induced by CUEDC1 overexpression (Fig. 5D). In essence, CUEDC1 overexpression leads to increased degradation of STAT3 due to the activation of the ubiquitin degradation pathway. To ascertain whether CUEDC1 interacts with STAT3 directly or indirectly, we conducted molecular docking simulations, co-immunoprecipitation (co-IP), and immunofluorescence colocalization experiments. The results consistently indicated that CUEDC1 directly interacts with STAT3 (Fig. 5A, B, F). In conclusion, CUEDC1 can directly bind to STAT3, recruit ubiquitin molecules to ubiquitinate STAT3, and facilitate its transport to the proteasome for degradation. This process ultimately reduces the protein levels of STAT3 within cells, thereby attenuating the transmission of upstream signals and diminishing cell proliferation and survival (Fig. 7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSilencing CUEDC1 enhances the proliferation, metastasis, and survival of TE-1 cells\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further confirm the effect of CUEDC1 on ESCA cells, we constructed a CUEDC1-silenced line (sh-CUEDC1) by transfecting TE-1 cells with a lentivirus carrying sh-CUEDC1. When CUEDC1 is silenced, TE-1 cells exhibit increased proliferative activity, invasive ability, and resistance to apoptosis compared to the control group (Fig. 6A, B, C, D, F, G; Fig. S3). Correspondingly, these changes are also observed at the protein levels (Fig. 6E).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eESCA, recognized as a highly lethal disease, has seen some advancements in treatment in recent years; however, the prognosis and quality of life for patients remain poor\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The early symptoms of this malignancy are often subtle, and its rapid growth, high invasiveness, and propensity for recurrence frequently result in late-stage diagnoses, where most patients are limited to palliative care\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. At this advanced stage, patients commonly experience eating disorders. Although postoperative esophageal reconstruction or endoscopic stent implantation may offer temporary relief, the duration and extent of this relief are often limited, ultimately leading to the patient's demise due to energy depletion\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Currently, targeted therapy represents a prominent area of research in cancer treatment owing to its superior efficacy and minimal side effects. However, the advancement of targeted therapy for ESCA has been notably sluggish. The dispersed nature of tumor driver genes and significant individual variability result in a lack of precise biomarkers for clinical treatment\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. ESCA contributes significantly to the global cancer burden, with a high mortality rate each year, underscoring the necessity for dedicated research into targeted therapies for this malignancy\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNumerous studies have demonstrated that the JAK1/STAT3 pathway plays a crucial role in tumor progression. In the context of ESCA, this pathway can directly regulate gene expression, upregulating the levels of oncogenic proteins, thereby facilitating tumor progression. Our research aims to elucidate the regulatory mechanisms of the JAK1/STAT3 pathway in esophageal cells, with the hope of providing a new perspective for the advancement of molecular medicine.\u003c/p\u003e \u003cp\u003eCUEDC1 contains a CUE domain, which can affect the degradation process of specific proteins by interacting with the ubiquitin system, thereby regulating protein levels in cells\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In this study, we found that CUEDC1 was downregulated in ESCA tissues, and low expression of CUEDC1 was associated with poor overall survival; however, we did not determine its association with the pathological type of this cancer. Additionally, we demonstrated for the first time that CUEDC1 upregulation inhibits ESCA cell proliferation and tumor progression. Conversely, downregulation of CUEDC1 significantly promoted the proliferation and survival of ESCA cell lines (anti-apoptosis), suggesting that low expression of CUEDC1 may be linked to drug resistance. Given the clinical and biological significance of CUEDC1, our study indicates that CUEDC1 may serve as a potential biomarker for the prognosis of ESCA and has potential therapeutic applications in the future.\u003c/p\u003e \u003cp\u003eMechanistically, we understand that ubiquitin molecules can bind to target proteins\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Both tumor suppression and tumor promotion pathways are regulated by ubiquitination\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Depending on the properties of the substrate, a single ubiquitin ligase can function as both an oncogene and a tumor suppressor\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. This duality includes the ubiquitination-mediated degradation of key proteins, which affects their abundance, as well as the regulation of protein activity through ubiquitination modifications\u003csup\u003e\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Consequently, the same ubiquitin ligase can exhibit varying effects on tumors, contingent upon the role its substrate plays in cancer progression\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Additionally, regulatory factors such as CUE domain-containing proteins can also assume completely opposing roles in different cancers due to these same underlying mechanisms\u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe observed that following the upregulation of CUEDC1 expression, the intracellular ubiquitination level significantly increased, while the STAT3 protein level decreased. Subsequently, we monitored the dynamic changes of STAT3 protein through CHX and MG132 treatment, further suggesting that the decrease in STAT3 protein levels is achieved through ubiquitin-proteasome degradation. To explore the role of CUEDC1 in this process, we conducted molecular protein docking simulations, co-immunoprecipitation, and immunofluorescence co-localization experiments, revealing that CUEDC1 can directly bind to STAT3. Based on previous studies, we speculate that CUEDC1 recruits ubiquitin molecules to modify STAT3 after binding, leading to its degradation.\u003c/p\u003e \u003cp\u003eHowever, we only predicted the relevant binding sites in molecular simulations and did not validate them through site-directed mutagenesis. Numerous types of ubiquitin E3 ligases are implicated in the degradation of STAT3 through ubiquitination. Among these, SLIM has been confirmed in previous studies, along with VHL, TRIM27, and Smurf1\u003csup\u003e43\u0026ndash;46\u003c/sup\u003e. However, we have yet to determine which specific ligase plays a predominant role in this process. Research conducted by Paul S. Andrews indicates that Smurf1 and CUEDC1 can spatially associate; however, their functional interactions remain unreported\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. It is plausible that Smurf1 and STAT3 may interact with each other through CUEDC1 as a mediator. This issue warrants clarification in future research.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur results demonstrate that CUEDC1 is downregulated in ESCA, and its downregulation is closely related to the poor overall survival rate of ESCA patients. CUEDC1 can directly bind to STAT3 and initiate its ubiquitination and degradation, thereby inhibiting the role of the JAK/STAT3 signaling pathway in promoting cell proliferation, metastasis, and survival. Our data provide a new perspective on the regulation of STAT3 signaling in the pathogenesis of ESCA. CUEDC1 may serve as a potential target and prognostic factor for the treatment of this disease.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank TCGA, KEGG, TIMER and GEPIA for providing data, and we are thankful to the Natural Science Fund of Heilongjiang Province grant (LH2023H097).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.L. and Z.P. conceptualized and designed the experiment. Z.L. and X.S. conducted experiments and collected data. C.L. and J.Z. analyzed the data and prepared charts. G.L. and H.L prepared pictures and formatted. Z.L. and Y.C. wrote and revised the manuscript. H.X. and C.Y. managed and reviewed the project and data. All authors contributed to this article and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no competing interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray, F. et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. \u003cem\u003eCancer J. Clin.\u003c/em\u003e \u003cb\u003e74\u003c/b\u003e, 229\u0026ndash;263. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3322/caac.21834\u003c/span\u003e\u003cspan address=\"10.3322/caac.21834\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, W. et al. 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Technol.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 471\u0026ndash;487. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1089/adt.2009.0264\u003c/span\u003e\u003cspan address=\"10.1089/adt.2009.0264\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2010).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"CUEDC1, JAK1/STAT3, esophageal cancer, ubiquitination, proteasome","lastPublishedDoi":"10.21203/rs.3.rs-6618873/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6618873/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCUE domain containing protein 1 (CUEDC1) is implicated in tumor progression; however, its specific role in esophageal cancer (ESCA) remains unclear. In esophageal cancer, the expression of CUEDC1 is notably low, which correlates with reduced survival rates and adverse clinical outcomes. Overexpression of CUEDC1 results in decreased activity of the JAK1/STAT3 signaling pathway in cells, consequently diminishing their proliferation, migration, and invasion capabilities. This mechanism operates through the direct binding of CUEDC1 to STAT3, facilitating its ubiquitination and triggering the ubiquitin-proteasome degradation pathway, ultimately leading to a significant reduction in intracellular STAT3 levels. This study suggests that CUEDC1 can reduce intracellular STAT3 protein levels, thereby inhibiting JAK1/STAT3 signaling transduction and suppressing the progression of ESCA. This study aims to elucidate the regulatory mechanism of CUEDC1 on STAT3, which will enhance our understanding of the regulatory pathways involved in the treatment of esophageal cancer and potentially other tumors. Future breakthroughs and innovations may emerge from molecular research and development targeting this pathway.\u003c/p\u003e","manuscriptTitle":"The Role of CUEDC1 in Suppressing JAK1/STAT3 Signaling Pathway in Esophageal Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-29 11:57:39","doi":"10.21203/rs.3.rs-6618873/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-16T05:18:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-04T09:43:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"41643469056876124111725047294400359290","date":"2025-07-22T16:03:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-22T14:29:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"277841798289043316108380290151773576055","date":"2025-07-08T14:30:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"82120895301610448267771387988191840295","date":"2025-06-02T08:14:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-28T05:26:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-28T05:23:17+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-22T15:38:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-22T04:38:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-08T09:05:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cc209b16-e749-465a-ada8-ffe413753d0b","owner":[],"postedDate":"May 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":49198454,"name":"Biological sciences/Cancer/Cancer genetics"},{"id":49198455,"name":"Biological sciences/Cancer/Cancer therapy"},{"id":49198456,"name":"Biological sciences/Cancer"},{"id":49198457,"name":"Biological sciences/Genetics"},{"id":49198458,"name":"Health sciences/Molecular medicine"}],"tags":[],"updatedAt":"2026-04-07T16:04:57+00:00","versionOfRecord":{"articleIdentity":"rs-6618873","link":"https://doi.org/10.1038/s41598-026-46681-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-03-31 15:58:46","publishedOnDateReadable":"March 31st, 2026"},"versionCreatedAt":"2025-05-29 11:57:39","video":"","vorDoi":"10.1038/s41598-026-46681-w","vorDoiUrl":"https://doi.org/10.1038/s41598-026-46681-w","workflowStages":[]},"version":"v1","identity":"rs-6618873","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6618873","identity":"rs-6618873","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}