USP1 activates the Wnt/β-catenin pathway by deubiquitinating KRT17, thereby facilitating thyroid cancer proliferation

USP1 activates the Wnt/β-catenin pathway by deubiquitinating KRT17, thereby facilitating thyroid cancer proliferation | 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 USP1 activates the Wnt/β-catenin pathway by deubiquitinating KRT17, thereby facilitating thyroid cancer proliferation Hong Zeng, Xuanrui Zhou, Xitong Geng, Hao Wan, Yongqi Ding, Minqin Zhou, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4092791/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Post-translational modification through ubiquitination is widely acknowledged for its pivotal regulatory role in tumor onset and progression. Ubiquitin ligases and deubiquitinases can modulate tumor advancement by impacting the expression of key proteins. Nonetheless, the precise contribution of ubiquitination and deubiquitinases in the onset of thyroid cancer (TC) remains to be comprehensively understood. Initially, Weighted Gene Co-expression Network Analysis (WGCNA) revealed the deubiquitinase USP1 as closely associated with TC and protein ubiquitination. Subsequently, our findings demonstrated the aberrant upregulation of USP1 expression in clinical TC samples. Moreover, interference with USP1 expression inhibited the proliferative ability of TC cells in vitro by colony formation and CCK8 assays. Notably, our findings have revealed that USP1 facilitates the proliferation of TC by modulating the Wnt/β-catenin pathway. Further, we identified KRT17 as a critical factor in the USP1-mediated Wnt/β-catenin pathway. Next, we validated that USP1 directly interacted with KRT17 and deubiquitinated it. Ultimately, we discovered that parthenolide exerted inhibitory effects on TC proliferation both in vivo and in vitro by modulating the USP1-KRT17-Wnt/β-catenin axis. To sum up, Our findings offer compelling evidence that underscores the pivotal role of USP1 in promoting TC proliferation. This is accomplished by stabilizing KRT17 expression through deubiquitination, which in turn activates the Wnt/β-catenin pathway. These results provide novel insights into potential therapeutic targets for TC treatment. USP1 KRT17 Wnt/β-catenin thyroid cancer cell proliferation deubiquitination Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1．Introduction The incidence of thyroid cancer (TC) renders it the most prevalent form of malignant neoplasm within the endocrine system. While the prognosis for primary TC is generally favorable [ 1 ], some late-stage TC patients may experience deterioration, leading to a more aggressive tumor [ 2 , 3 ]. Currently, the common treatment for TC remains surgical removal. However, due to the dysregulation of cell death and proliferation mechanisms in cancer, the issue of postoperative recurrence persists [ 4 ], resulting in unsatisfactory outcomes for TC prognosis. Revealing the molecular regulatory mechanisms of TC and identifying novel therapeutic targets is therefore imperative, which holds significant importance in seeking novel treatment strategies for TC. Ubiquitination and deubiquitination are two antagonistic biological processes executed by E1-E3 ubiquitin ligases and deubiquitinating enzymes (DUBs) [ 5 ]. Imbalance of these two processes often leads to overexpression of oncogenic proteins or inhibition of tumor suppressors, promoting tumor development [ 6 ]. In recent years, mounting evidence suggests a close association between DUB dysregulation and cancer progression [ 7 ]. USP1 (Ubiquitinyl Hydrolase 1) is a member of the ubiquitin-specific-processing (UBP) protease family and functions as a deubiquitinating enzyme (DUB) [ 8 ], possessing both His and Cys domains. This protein localizes in the cytoplasm and catalyzes the cleavage of ubiquitin moiety from both ubiquitin precursor and ubiquitinated protein substrates [ 9 ]. USP1 plays a critical role in cellular response to DNA damage and has been extensively studied in cancer. For instance, in hepatocellular carcinoma, USP1 stabilizes RPS16 protein through deubiquitination to drive the progression of liver cancer [ 10 ], while it facilitates the metastasis of gastric cancer by deubiquitinating and stabilizing ID2 [ 11 ]. However, there is currently no research on USP1 in TC. The Wnt/β-catenin pathway, a protein family critical in embryonic development, also maintains tissue homeostasis in adult organisms [ 12 ]. The canonical Wnt pathway predominantly governs cellular proliferation, whereas the non-canonical Wnt pathway modulates cell polarity and migration [ 13 ]. These two main pathways form an interconnected regulatory network. Dysregulation of the Wnt/β-catenin signaling often leads to the development of various tumors [ 14 ]. Prior investigations have shown that KDM1A facilitates the progression of TC and sustains stemness via modulation of the Wnt/β-catenin signaling pathway [ 15 ]. Another research revealed that lncRNA DOCK9-AS2 activates the Wnt/β-catenin pathway, promoting stemness, proliferation and progression of TC [ 16 ]. Given the complexity and significance of the dysregulated Wnt/β-catenin pathway in TC, further research into the specific molecular regulatory mechanisms is imperative. In our research, we utilized WGCNA to identify the hub gene USP1 associated with TC and ubiquitination. Subsequently, experimental validation revealed that USP1 regulates KRT17 to activate the Wnt/β-catenin signaling pathway, thereby impacting the proliferation of TC. Furthermore, we discovered that USP1 can stabilize KRT17 expression through its deubiquitination activity. Interestingly, this USP1-KRT17-Wnt/β-catenin axis can be targeted by the naturally derived compound parthenolide. This represents the first investigation into the ubiquitination molecular mechanism related to USP1 in TC. 2. Methods and Materials 2.1 Human specimens and cell culture We obtained human thyroid cancer (TC) specimens from a cohort of 50 patients underwent TC resection at the Second Affiliated Hospital of Nanchang University during March 2018 to June 2022. The acquisition of samples received appropriate informed consent from the patients, and all experimental protocols were approved by the research ethics committee of the Second Affiliated Hospital of Nanchang University. 2.2 Cell lines and cell culture The thyroid epithelial cell line Nthy was acquired from the European Collection of Animal Cell Cultures, while the human thyroid carcinoma cell lines (Bcpap, TPC-1 and 8505c) were procured from the Shanghai Institute of Cell Biology (Shanghai, China). The K1 cell line was obtained from the American Type Culture Collection. All cells were cultured in Dulbecco’s Modified Eagle’s media (DMEM) supplemented by 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO 2 . 2.3 Data Collection Our digital data was derived solely from The Cancer Genome Atlas (TCGA) ( https://cancergenome.nih.gov ) database and the Genotype-Tissue Expression (GTEx) ( https://www.gtexportal.org/ ) database. The TCGA-THCA dataset includes expression data from 571 samples, consisting of 59 normal samples and 512 tumor samples. For our research, we obtained GTEx-thyroid dataset which includes 276 samples from GTEx. 2.4 Weighted Gene Co-expression Network Analysis The WGCNA algorithm, which is widely employed for high-throughput gene coexpression profiling, was utilized in this study to identify gene coexpression networks associated with different diseases [ 17 ]. For constructing a scale-free co-expression network encompassing all genes, a soft threshold of 10 was applied. Furthermore, a dynamic tree cutting method was employed to identify modules within the network. To determine the modules significantly correlated with thyroid cancer and ubiquitination, the correlations between Eigengenes and THCA and GOBP_PROTEIN_POLYUBIQUITINATION, respectively, were analyzed. The gene connectivity within modules was assessed using the absolute value of Pearson correlation, allowing for the identification of hub genes exhibiting high within-module connectivity. 2.5 Protein-Protein Interaction, Molecular Docking and Gene set enrichment Analysis As previously described [ 18 ], we utilized the BioGRID ( https://thebiogrid.org/ ) interactome dataset to identify the interacting genes in TC patients. Subsequently, the construction of the protein-protein interaction (PPI) network was conducted using the Cytoscape software, followed by its visualization. The identification of protein nodes was accomplished using the Cytoscape plugin. The HDOCK ( http://hdock.phys.hust.edu.cn/ ), a commonly employed computational resource, employs a hybrid docking algorithm that combines template-based modeling and free docking for the automated utilization of binding data from the PDB [ 19 ]. To investigate the interaction between USP1 and KRT17, we employed HDOCK as a predictive tool to determine their docking mode. Subsequently, we utilized PyMOL software to visualize the obtained results. GSEA ( http://bioinfo.life.hust.edu.cn/GSEA ), a software package equipped with an extensive collection of 1,325 gene sets, offers a valuable tool for the interpretation of gene expression data [ 20 ]. The samples were classified into two groups based on the expression levels of THCA, and this approach was utilized to examine the enrichment of gene sets containing THCA. The analysis conducted 1,000 permutations and employed the h.All.V7.4 Symbols.gmt (Hallmarks) gene set database for comprehensive investigation. Significance was determined at P value < 0.05 and FDR < 0.05. 2.6 Quantitative real-time PCR (qrt-PCR) We used Trizol reagent (catalog number: 15596026, Invitrogen, USA) to extract total RNA. mRNA expression levels were assessed using SYBR Green assays with RT primers and SYBR Green from Takara Biotechnology (Catalog: DRR041A, TAKARA, Dalian, China). During the simultaneous process, we used human microtubule-associated protein as an internal control for amplification. The primer sequences used in this study are provided in Supplementary Table S1 . 2.7 Western blotting We prepared the total protein extract according to the method described earlier. We used RIPA buffer containing a mixture of protease inhibitors (manufactured by Shanghai Berry Times Company) to extract the protein. The antibodies employed in this study included anti-USP1 monoclonal antibody (1:1000 dilution; CST, 8033), anti-KRT17 monoclonal antibody (1:1000 dilution; CST, 12509), anti-β-catenin monoclonal antibody (1:1000 dilution; abcam, ab32572), anti-c-Myc monoclonal antibody (1:1000 dilution; abcam, ab32072), and anti-Tubulin monoclonal antibody (1:1000 dilution; Proteintech, 11224-1-AP). Following a 2-hour incubation with secondary antibodies at room temperature, the Quantity One software (Bio-Rad Laboratories Inc., Hercules CA, USA) was employed for quantitative analysis of protein band intensity. [ 21 ]. 2.8 Immunohistochemistry (IHC) The study involved the collection of TC samples and paired normal thyroid tissues, which underwent an antigen retrieval process. Heated the antigen retrieval solution (EDTA, pH 8.0) in a microwave for 40 minutes, then incubated with goat serum for 30 min. Subsequently, tissue slices were incubated with anti-USP1 monoclonal antibody (1:1000, CST, 8033) overnight at 4°C, followed by incubation with an HRP-conjugated secondary antibody (Boster) at room temperature for two hours. before immunostaining with the DAB Detection Kit (Maxim) for two minutes. Ultimately, the positive area proportion was semi-quantitatively evaluated by three pathologists blinded to the clinical parameters. 2.9 Plasmids and reagents In all diagrams, the shRNA structures of USP1 and KRT17 are provided in supplemental table S1 . These shrnas were integrated into the lentivirus pLKO vector obtained from a gene pharmaceutical company based in Shanghai. The overexpressed plasmid was sourced from another genetics company also located in Shanghai. Specifically, USP1, HIS-labeled KRT17, and HIS-labeled beta-catenin were inserted into the P-CMV vector. After 48 hour of transfection, the viral supernatant was collected and filtered as per the manufacturer's instructions for selecting lentiviral transduction cells using purinomycin. 2.10 Cell colony formation assay In colony formation experiments, the cells were seeded onto a 6-well plate. Once the colonies reached the desired size, they were fixed with 4% paraformaldehyde for 30 minutes and stained with 1.0% crystal violet for an additional 30 minutes until the clones became visible. Colonies comprising more than 50 cells were enumerated and subjected to analysis in order to investigate their correlation with the initial number of seed cells. 2.11 Cell counting kit-8 (CCK-8) assay The CCK-8 assay was conducted in accordance with the manufacturer's instructions using the CCK-8 kit. Transfected cells were incubated for 48 hours and then seeded into each well of a 96-well plate at a density of 5×10 3 cells per well. Following incubation for 24, 48, 72, 96, and 120 hours, each well was supplemented with 10 µL of CCK8 reagent and further incubated at a temperature of 37℃ for a duration of two hours. Absorbance at the wavelength of 450 nm was measured using an enzyme-labeled apparatus (ELX-800; Bio-Tek, Winooski, VT, USA). Moreover, the inhibitory effects of PTL were evaluated utilizing CCK-8 assays. Parthenolide (with a purity exceeding 98%) was procured from Absin Bioscience Inc (#20554-84-1), while Dimethyl sulfoxide (DMSO) was sourced from Sigma-Aldrich (#276855) [ 22 ]. Following a 24-hour incubation period, cells were exposed to PTL at different concentrations or DMSO for 24, 48, 72, 96, and 120 hours. The specific operation is as described above. 2.12 Luciferase reporter assay Cells were co-transfected with plasmids containing firefly luciferase reporters and USP1 plasmids in the TOP/FOP-Flash reporter assay. Following a 48-hour transfection period, cells were harvested and subjected to analysis using the Dual-Luciferase Reporter Assay System from Promega (Madison, WI, USA). Luciferase activity was assessed using the PerkinElmer EnSpire Multilabel Reader 2300 (PerkinElmer Inc., Waltham, MA, USA), with normalization to Renilla luciferase activity to ensure uniform transfection efficiency. 2.13 Co-immunoprecipitation experiment Cell lysates were subjected to overnight incubation at 4°C with 50 µl of protein G beads and 1 µg of the designated antibody. Subsequent centrifugation effectively separated the protein G beads from the solution. Following this, loading buffer was then introduced to the resulting mixture and subjected to thermal treatment at 100°C for a quarter. The immunoprecipitated proteins were then subjected to analysis via SDS-PAGE and immunoblotting. The intensity of the protein bands was quantitatively examined and assessed using the specialized software. 2.14 Mouse xenograft assay The animal experiments conducted in this study adhered to the ethical guidelines prescribed by the Animal Ethics Committee of the Second Affiliated Hospital of Nanchang University. Bcpap cells were suspended in a medium devoid of fetal bovine serum and subsequently subcutaneously injected into 6-week-old nude mice at a volume of 100 µl. Each experimental group, consisting of six mice, received daily intraperitoneal injections of either a vehicle solution (comprising 2% DMSO, 40% PEG400, and 2% Tween 80 in normal saline) or PTL (20 mg/kg) for a duration of 42 days. Regular assessments were made to monitor changes in body weight on a daily basis. Additionally, tumor measurements and dimensions were taken at intervals of 3 days employing a digital caliper. Meanwhlie, the corresponding tumor volumes were computed according to the dedicated formula [ 23 ]. 2.15 Statistical analysis The results are presented as mean ± SD and were evaluated through GraphPad Prism 5 (GraphPad Software, USA) based on data obtained from a minimum of three distinct experiments. Statistical analyses involved pairwise comparisons utilizing the Statistical comparisons among multiple groups were conducted using Student's t-test or one-way ANOVA. Furthermore, logistic regression models were utilized for both univariate and multivariate analyses. All P values were computed as two-tailed tests, with statistical significance defined at a threshold of < 0.05. 3. Results 3.1 USP1 was identified as the pivotal gene relevant to thyroid cancer and protein ubiquitination. In order to identify key genes in thyroid cancer (TC), we identified 10 874 differentially expressed genes (logFC > 1, p < 0.05) from the TCGA-THCA and GTEx-thyroid databases (Fig. 1 A). Following this, we conducted a weighted gene co-expression network analysis (WGCNA) with a soft threshold power of 10 to construct a scale-free network (Fig. 1 B). Analysis showed the identification of 5 gene co-expression modules through hierarchical clustering (Fig. 1 C). It is worth noting that the module trait correlation heatmap elucidated that the brown and blue modules were most strongly associated with THCA and GOBP_protein_polyubiquitination, respectively (Fig. 1 D). Furthermore, in consideration of the vital role of deubiquitinating enzymes (DUBs) in driving tumor development, we downloaded 126 genes encoding deubiquitinating enzymes from the integrated annotation of ubiquitin and ubiquitin-like conjugation databases (iUUCD). We then intersected these genes with the 5% hub genes of the brown and blue modules (Fig. 1 E), resulting in 4 genes: USP1, USP10, VCPIP1, and BRCC3 (Fig. 1 F). Among them, USP1 had the highest connectivity within the module (Fig. 1 G). In order to delve deeper into the functional aspects of USP1, we performed GO and KEGG analyses. The results indicated that USP1 was primarily abundant in pathways associated with monoubiquitinated protein deubiquitination, regulation of DNA repair, protein deubiquitination, cysteine-type deubiquitinase activity, deubiquitinase activity, ubiquitin-like peptidase activity and Fanconi anemia pathway (Fig. 1 H). In conclusion, USP1 is considered a key gene associated with THCA and protein ubiquitination. 3.2 High Expression of USP1 in Thyroid Cancer Tissues To assess USP1 expression in thyroid cancer (TC), we first employed TCGA and GTEx databases to compare USP1 expression patterns in normal and tumor tissues. Our investigation revealed a remarkable upregulation of USP1 in tumor tissues when compared to normal tissues (Fig. 2 A). To validate these results, we employed real-time quantitative PCR to investigate the expression of USP1 in 50 pairs of TC samples. Data exhibited a substantial elevation in the mRNA levels of USP1 in 31 out of the 50 pairs, providing further support for the heightened expression of USP1 in TC (Fig. 2 B). Furthermore, we utilized Western Blotting to evaluate the protein expression of USP1 in 50 TC specimens and their corresponding adjacent tissues. Our analysis demonstrated a marked elevation in the protein expression of USP1 in TC (Fig. 2 C), and representative results were shown in Fig. 2 D. Furthermore, we performed immunohistochemical staining on 50 TC samples and their corresponding normal tissues to assess the expression of USP1. The findings validated a marked augmentation in the protein expression of USP1 (Fig. 2 E). In conclusion, our findings provide compelling evidence of the heightened expression of USP1 in TC tissues. 3.3 USP1 facilitates in vitro proliferation Capacity of TC To further explore the cellular expression of USP1, we assessed the levels of USP1 mRNA and protein in various TC cell lines (Bcpap, K1, TPC-1, 8505c) along with a normal cell line (Nthy). Interestingly, both USP1 mRNA and protein expression exhibited substantial elevation in the TC cell lines relative to the Nthy (Figs. 3 A, B). In order to understand the influence of the irregular expression of USP1 on TC development, we utilized small hairpin RNA (shRNA) to suppress the expression of USP1 and evaluated the effectiveness of knockdown using qRT-PCR and Western Blotting analysis (Figs. 3 C, D). Subsequently, to assess the impact of transfection on the proliferation of TC cells, both the control group and the USP1 knockdown group were subjected to colony formation and CCK8 assays. The results obtained from the colony formation assay demonstrated that the suppression of USP1 resulted in a reduction in the colony-forming capacity of Bcpap cells (Figs. 3 E, F). Furthermore, the CCK8 assay demonstrated that the downregulation of USP1 markedly hindered cell proliferation (Fig. 3 G). In conclusion, these analyses collectively indicate that the knockdown of USP1 inhibits the in vitro proliferation of TC cells. 3.4 USP1 impacts the Proliferation Capacity of TC through the Wnt/β-Catenin Signaling Pathway We then investigated the precise mechanism underlying the regulation of TC cell proliferation by USP1. Previous investigations have highlighted the significant involvement of the Wnt/β-catenin signaling pathway in TC proliferation [ 24 ]. Therefore, we postulated that USP1 might modulate TC proliferation through this pathway. To substantiate our hypothesis, we initially performed gene set GSEA utilizing the TCGA database. Our findings uncovered a positive relevance between USP1 and the Wnt signaling pathway (Fig. 4 A). To further investigate the impact of USP1 on Wnt pathway activation, we knocked down USP1 in Bcpap cells and assessed the protein expression of β-catenin and c-MYC. Notably, the knockdown group exhibited downregulated expression of these three proteins (Fig. 4 B). Moreover, TOP-Flash luciferase analysis demonstrated that downregulation of USP1 hindered the activity of the Wnt/β-catenin pathway (Fig. 4 C). Following USP1 silencing, ectopic expression of β-catenin was performed, revealing a restoration of Wnt signaling pathway activity previously compromised by USP1 knockdown. (Fig. 4 D). These results affirm the pivotal role played by USP1 in regulating the Wnt/β-catenin pathway. Additionally, to probe whether USP1 influences cell proliferation by modulating the Wnt/β-catenin pathway, we conducted CCK8 experiments and demonstrated that overexpression of β-catenin can rescue the decreased proliferation ability of TC cells resulting from USP1 knockdown in vitro (Fig. 4 E). Consequently, our research provides compelling evidence demonstrating that USP1 exerts an impact on the proliferation capacity of TC via its regulation of the Wnt/β-catenin pathway. 3.5 KRT17 is significantly correlated with USP1 at the protein level in TC Delving into the mechanisms underlying USP1's impact on Wnt/β-catenin pathway in TC, our initial co-IP experiments revealed that USP1 didn’t directly bind to β-catenin (Figure S1 A). Moreover, at the mRNA level, a significant correlation was observed between USP1 and CTNNB1 (Figure S1 B). This prompted us to delve deeper into genes that can directly interact with USP1 while showing no correlation at the mRNA level. Initiating our exploration, we identified 45 genes that interact with USP1 using the BioGRID database (Fig. 5 A). Subsequently, leveraging the Timer online website, we pinpointed 10 genes that exhibited no correlation with USP1 at the mRNA level (Table 1 ). Further investigation into the varied gene expression patterns (Figure S1 C) highlighting that only KRT17 displayed significant upregulation in TC tissues (Fig. 5 B). To scrutinize the variance in KRT17 expression between normal and TC tissues, Western blotting assay was performed to assess the protein expression of KRT17 in 50 TC samples. Figure 5 C underscored representative results, a considerable elevation of KRT17 in TC tissues relative to the corresponding normal tissues. Subsequent exploration of the relationship between USP1 and KRT17 entailed an analysis of their expression patterns in human TC specimens, revealing a noteworthy correlation between the protein expressions of USP1 and KRT17 while lacking such association at the mRNA level (Figs. 5 D, E). Crucially, knocking down the mRNA expression of USP1 did not lead to a concomitant alteration in the mRNA expression of KRT17 (Fig. 5 F). Further investigations involved the examination of KRT17 and β-catenin expression in Bcpap cell lines subjected to shRNA and shUSP1 treatments, exposing a significant reduction in KRT17 and β-catenin protein expressions upon USP1 knockdown. In concordance, augmenting USP1 levels in 8505c cells resulted in increased protein expressions of KRT17 and β-catenin (Fig. 5 G). These findings collectively affirm a substantial correlation between USP1 and KRT17 at the protein level. Table 1 varX varY cor p SPANXN5 USP1 0.007205047 0.87118323 LGALS7 USP1 0.014510283 0.743986138 KRT17 USP1 0.02278842 0.607998338 CALML5 USP1 -0.025697358 0.562974535 SPANXN2 USP1 0.026369697 0.552801069 CALML3 USP1 -0.034385748 0.438871979 OPALIN USP1 -0.037065019 0.404024189 KRT4 USP1 0.058265099 0.189383914 DSG1 USP1 0.058466171 0.187858509 LRRC15 USP1 0.07565916 0.088157773 ALDH16A1 USP1 0.089278676 0.04408429 TAGLN2 USP1 0.099754177 0.024408355 CIRBP USP1 0.146782162 0.000895602 LYZ USP1 0.150934779 0.000634401 C4ORF49 USP1 0.170851241 0.000107231 UNC45A USP1 0.17177272 9.83E-05 DSC1 USP1 0.190166523 1.57E-05 PTGES2 USP1 -0.19568568 8.70E-06 ZCCHC10 USP1 0.234321325 8.88E-08 PKP1 USP1 0.257533248 3.73E-09 ANXA2P2 USP1 0.276272818 2.28E-10 JUP USP1 0.343463709 1.54E-15 SGOL1 USP1 0.34687435 7.75E-16 STAT2 USP1 0.357775415 8.14E-17 CCNF USP1 0.358611712 6.82E-17 UBE2A USP1 0.365510098 1.56E-17 RAD51AP1 USP1 0.388881895 8.00E-20 PHLPP1 USP1 0.451241419 6.69E-27 KPNA2 USP1 0.485518198 1.84E-31 FLG USP1 0.523481896 3.72E-37 MYH9 USP1 0.549004413 2.08E-41 WDR5 USP1 0.549834592 1.49E-41 HSPB1 USP1 -0.558109581 5.15E-43 WDR20 USP1 0.590712638 3.38E-49 PHLPP2 USP1 0.659138107 9.51E-65 CHIC1 USP1 0.685771294 5.72E-72 CSE1L USP1 0.720116537 1.64E-82 USP46 USP1 0.734198148 2.63E-87 WDR48 USP1 0.735631524 8.21E-88 VPS36 USP1 0.742990727 1.85E-90 USP4 USP1 0.774548728 6.34E-103 KPNA1 USP1 0.805446921 2.87E-117 VPS26A USP1 0.820348012 3.82E-125 SOCS6 USP1 0.82105832 1.54E-125 USP12 USP1 0.850009366 3.02E-143 3.6 KRT17 is pivotal in the USP1-regulated Wnt/β-catenin pathway. Building on the observed correlation between USP1 and KRT17, a thorough examination was carried out to ascertain the participation of KRT17 in the USP1-mediated Wnt/β-catenin signaling cascade. Utilizing Western blot experiments, KRT17 was upregulated in Bcpap cells upon USP1 downregulation, with concurrent monitoring of the protein expression levels of USP1, KRT17, and β-catenin. The results unveiled that the upregulation of KRT17 resulted in an augmented expression of β-catenin, effectively rescuing the diminished levels induced by USP1 knockdown (Fig. 6 A). Conversely, in 8505c cells, the downregulation of KRT17 resulted in a decrease in the expression of β-catenin, successfully reversing the elevated levels caused by USP1 upregulation (Fig. 6 B). Furthermore, CCK8 analysis demonstrated that the upregulation of KRT17 can restore the decreased proliferation capacity of TC cells resulting from USP1 knockdown (Fig. 6 C), whereas the downregulation of KRT17 inhibited the heightened proliferation capacity induced by USP1 overexpression (Fig. 6 D). Collectively, these findings suggest that USP1 regulates the Wnt/β-catenin pathway through its influence on KRT17 expression, consequently impacting the proliferation capacity of TC cells. 3.7 USP1 exerts deubiquitination to stabilize KRT17 expression Having established the correlation between USP1 and KRT17, our study aimed to elucidate their potential regulatory mechanism. Prior research has highlighted USP1 as a deubiquitinase closely associated with cancer. Notably, a study unveiled the role of the E3 ligase TRIM21 in ubiquitinating and stabilizing KRT17 to trigger STAT3 activation in psoriasis [ 25 ]. Based on this background, we postulated that USP1 might deubiquitinate and stabilize KRT17. To test our hypothesis, we conducted co-immunoprecipitation experiments, unveiling a direct interaction between USP1 and KRT17 within Bcpap cells (Fig. 7 A). Molecular docking models and potential binding sites of these two proteins are depicted in Figs. 7 B-C. To investigate whether USP1 stabilizes KRT17 expression through the ubiquitin-proteasome pathway, we subjected 8305c cells transfected with USP1 shRNA or USP1 plasmid to treatments with or without 15 uM MG132. Our findings indicated that alterations in USP1 expression did not impact KRT17 protein levels compared to the control group (Fig. 7 D). Subsequently, we utilized 20 uM cycloheximide (CHX) as a translation inhibitor in Bcpap cells to assess KRT17 expression in the control and sh-USP1 groups at different time points. Strikingly, downregulation of USP1 significantly expedited the degradation of KRT17 (Figs. 7 F, G). Further experiments confirmed that upregulation of USP1 notably decreased the ubiquitination level of KRT17. Conversely, downregulation of USP1 exhibited contrasting effects (Fig. 7 H). Collectively, our data strongly supports the role of USP1 as a deubiquitinase for KRT17, thereby stabilizing its expression. 3.8 Parthenolide inhibits TC proliferation in vitro Previous studies have validated the potent inhibitory effect of parthenolide (PTL) on TC cell proliferation. To delve deeper into the underlying mechanism of PTL action in TC, we initially explored the association between USP1 expression levels and the half-maximal inhibitory concentration (IC50) of PTL based on GDCS database, revealing a significant positive correlation between them (Figure S2A). Further scrutiny indicated that lower USP1 expression corresponded to lower IC50 values of PTL, suggesting increased sensitivity of patients with diminished USP1 expression to PTL treatment (Figure S2B). Subsequently, we conducted molecular docking simulations of PTL with USP1, predicting their potential binding sites (Figs. 8 A, B). Next, CCK8 assays were conducted to assess the effects of different concentrations of PTL on cell proliferation. Our findings demonstrated the anticancer effects of PTL (Fig. 8 C). Furthermore, to elucidate the molecular mechanisms involved in PTL's action in TC, we conducted Western blot experiments which unveiled a downregulation in the protein expression levels of USP1, KRT17, β-catenin, and c-Myc following PTL treatment (Fig. 8 D). In summary, our study suggests that PTL might reduce the in vitro proliferative capacity of TC by inhibiting the expression of USP1, KRT17 and the activity of Wnt/β-catenin pathway. 3.9 Parthenolide Obstructs In Vivo Proliferation of TC To discern the anticancer properties of PTL in live organisms, we conducted a xenograft mouse model employing nude mice engrafted with Bcpap cells. PTL was intraperitoneally administered at a constant daily dose of 20 mg/kg. for a continuous span of 42 days (Fig. 8 E). The findings unequivocally demonstrated that PTL significantly impeded the growth of Bcpap cell xenograft tumors in terms of volume and weight (Figs. 8 F, G). Subsequently, we delved deeper into assessing whether PTL impedes the expression of USP1 and KRT17, while concurrently repressing the Wnt/β-catenin pathway related proteins influenced by them. Our Western blotting results underscored the downregulation of USP1, KRT17, β-catenin, and c-Myc protein expression after treatment with PTL (Fig. 8 H). To summarize, PTL has the potential to diminish the exuberant in vivo proliferation ability of TC by thwarting the expression of USP1-KRT17, consequently suppressing the Wnt/β-catenin pathway. 4. Discussion The imbalance in protein ubiquitination and deubiquitination has profound effects on tumor growth and proliferation [ 26 ]. Deubiquitinating enzymes (DUBs), as crucial regulatory factors in the deubiquitination process, play a pivotal role in cancer pathogenesis and represent promising targets for cancer diagnosis and treatment [ 7 ]. Considerable evidence supports a noteworthy association between aberrant expression of deubiquitinating enzymes and thyroid cancer (TC) manifestation, as observed in several research studies. In our research, weighted gene co-expression network analysis (WGCNA) was applied to recognize the core gene USP1, which was associated with TC and protein polyubiquitination. We were the first to report the mechanism by which USP1 regulates TC development. Initially, we observed significantly higher mRNA and protein expression of USP1 in TC compared to normal thyroid tissue. Furthermore, we elucidated that USP1 enhanced the proliferation capacity of TC cells in vitro and identified its oncogenic function in TC development. Moreover, we delved into the potential mechanisms through which USP1 regulates TC proliferation. Prior investigations have underscored the pivotal involvement of the Wnt/β-catenin pathway in the onset of TC, as elucidated in earlier studies [ 27 ].Additionally, c-Myc, a β-catenin-activated target molecule in Wnt signaling transduction, enhances tumor growth and metastasis in TC [ 28 ]. Another study suggested that USP8 positively regulates the progression of hepatocellular carcinoma through the Wnt/β-catenin pathway [ 29 ]. Building upon these findings, we propose that USP1 regulates TC proliferation by affecting the Wnt/β-catenin pathway. Firstly, through Gene Set Enrichment Analysis (GSEA), it was demonstrated that the Wnt signaling pathway functions as a downstream pathway of USP1, indicating a positive correlation between USP1 activity and the Wnt/β-catenin pathway. Secondly, knockdown of USP1 expression contributed to decreased expression of β-catenin and c-Myc. Consistently, overexpression of β-catenin significantly rescued the reduced activity of the Wnt/β-catenin pathway and the proliferation capacity induced by USP1 downregulation. These results unveiled the mechanism through which USP1 promotes TC proliferation by activating the Wnt/β-catenin pathway. USP1 (Ubiquitinyl Hydrolase 1), a member of the ubiquitin-specific processing (UBP) protease family, is known for its direct interaction with substrates to impact their ubiquitination degradation and modulate protein expression levels[ 8 , 30 ]. It was initially speculated that USP1 can directly interact with β-catenin and remove ubiquitin modifications from it. However, our findings reveal no direct interaction between USP1 and β-catenin. Meanwhile, USP1 and β-catenin show a significant correlation at the mRNA level. To identify the crucial mediator through which USP1 modulates the Wnt/β-catenin pathway and triggers TC proliferation, we conducted a PPI analysis utilizing the BioGRID platform, uncovering 45 proteins that physically interacted with USP1. Subsequently, we explored the relationship between these genes, USP1 expression, and their expression in TC. Intriguingly, only the mRNA expression of KRT17 appeared to be independent of USP1 and displayed upregulation in TC. Further examination of our clinical data unveiled a notable association between the protein expression of KRT17 and USP1, with mRNA expression showing no correlation. Given that deubiquitinating enzymes like USP1 regulate the expression of substrate proteins through post-translational modifications without affecting mRNA expression, the distinctive characteristic of KRT17 captured our attention. KRT17, a versatile cytoskeletal protein, participants in countless biological processes such as cell proliferation, growth and skin inflammation [ 31 ]. Notably, aberrant expression of KRT17 is observed in several diseases including cervical cancer [ 32 ], oral squamous cell carcinoma [ 33 ], and gastric cancer [ 34 ]. Recent evidence suggests that elevated KRT17 expression significantly activates the Wnt/β-catenin pathway, promoting proliferation and invasion in NSCLC, indicating a poor prognosis [ 35 ]. Consistent with these findings, our experimental results indicated that upregulating KRT17 can counteract the reduced TC proliferation resulting from USP1 knockdown-induced decrease in β-catenin expression, while downregulating KRT17 can inhibit the increased TC proliferation triggered by USP1 overexpression-induced elevation in β-catenin expression. Therefore, KRT17 emerged as a critical factor in mediating the proliferative capacity of TC regulated by USP1 through the Wnt/β-catenin pathway. To further investigate the role of USP1 in stabilizing KRT17 expression through deubiquitination, we conducted a series of experiments for validation. Co-immunoprecipitation experiments confirmed the direct binding of USP1 to KRT17. Additionally, we observed that downregulating USP1 decreased the half-life of KRT17, leading to an increased degradation rate. Furthermore, we discovered that downregulating USP1 significantly elevated the ubiquitination level of KRT17. These findings indicated that KRT17 can undergo ubiquitin-mediated proteolysis, and USP1 acts as the deubiquitinating enzyme for KRT17, playing a crucial role in its degradation process. In recent decades, there has been an increasing utilization of natural or herbal extracts, which have shown promising anti-cancer effects against various types of cancer [ 36 ]. Parthenolide (PTL), a sesquiterpene lactone found in the stems of Tanacetum balsamita, possesses potent anti-cancer and anti-inflammatory properties [ 37 ]. Previous studies have demonstrated that PTL inhibits ubiquitin-specific peptidase 7 (USP7), the Wnt signaling pathway, and the growth of colorectal cancer cells [ 23 ]. Moreover, Yuan et al. have suggested that PTL exhibits anti-cancer activity in TC, although the precise molecular mechanisms remain unclear [ 3 , 38 ]. Therefore, we hypothesized that PTL acts as an inhibitor of USP1, suppressing TC cell growth and proliferation by inhibiting KRT17 expression and modulating the Wnt/β-catenin pathway. In our research, we observed that PTL hindered the growth activity and proliferation ability of TC cells. Additionally, xenograft tumor experiments in nude mice demonstrated that PTL partially suppressed the growth and weight of heterotransplanted tumors. Since USP1 positively regulates the Wnt/β-catenin pathway through mediating deubiquitination of KRT17, further investigations revealed that PTL reduced the protein expression of USP1, KRT17, β-catenin, and c-Myc both in vitro and in vivo. Consequently, our research provides insights into the specific mechanism by which PTL inhibits TC proliferation. Specifically, it downregulates USP1 and KRT17 expression while modulating the Wnt/β-catenin pathway. 5. Conclusions To sum up, our investigation has illuminated a fresh aspect and regulatory mechanism involving USP1 in TC development. USP1 regulated KRT17 levels through its deubiquitinase activity, leading to the activation of the Wnt/β-catenin pathway and promoting TC cell proliferation. Notably, PTL demonstrated USP1 inhibitory effects and exhibited promise as a potential therapeutic option for TC. These findings strongly supported the significance of USP1 as a prospective target for TC treatment, underscoring its therapeutic potential (Figure S3). Declarations Acknowledgements We express our gratitude to TCGA, and GTEx for providing their resources at no cost. Author contributions The experiment was conducted by Hong Zeng, Xuanrui Zhou, and Xitong Geng. Hong Zeng and Hao Wan wrote and edited the manuscript. Data analysis was done by Yongqi Ding and Minqin Zhou. Formalized are Jingying Pan and Zichuan Yu. Da Huang amended the study and designed the experiment. Each author approved the manuscript's final draft and contributed significantly, directly, and intellectually to the project. Funding This study was supported by grants from Science and Technology Project of Jiangxi Provincial Health Commission (202210611) and Science and Technology project of traditional Chinese medicine in Jiangxi province (2023Z030) and Natural Science Foundation of Jiang xi Province Youth Science Foundation (20224BAB216055) and National Natural Science Foundation of China (82260472). Availability of data and materials Data supporting the conclusions of this research are shown in this article and the Supplemental Files. Ethics approval and consent to participate The studies involving human participants were reviewed and approved by the Ethics Committee of Second Affiliated Hospital of Nanchang University (Nanchang, China). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Cha YJ and Koo JS. Next-generation sequencing in thyroid cancer. 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Positive Role of Chinese Herbal Medicine in Cancer Immune Regulation. Am J Chinese Med. 2020; 48(7):1577-1592.10.1142/S0192415X20500780 PMID:33202152 Sztiller-Sikorska M and Czyz M. Parthenolide as Cooperating Agent for Anti-Cancer Treatment of Various Malignancies. Pharmaceuticals-Base. 2020; 13(8).10.3390/ph13080194 PMID:32823992 Li X, Huang R, Li M, Zhu Z, Chen Z, Cui L, Luo H and Luo L. Parthenolide inhibits the growth of non-small cell lung cancer by targeting epidermal growth factor receptor. Cancer Cell Int. 2020; 20(1):561.10.1186/s12935-020-01658-1 PMID:33292235 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-4092791","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":281590185,"identity":"eeda1dfe-d082-405b-bd52-a038ed776f9a","order_by":0,"name":"Hong Zeng","email":"","orcid":"","institution":"Second Affiliated Hospital of Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Zeng","suffix":""},{"id":281590186,"identity":"1f8e98d7-94c3-46a8-b98e-cbc7239ea4cb","order_by":1,"name":"Xuanrui Zhou","email":"","orcid":"","institution":"Nanchang 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12:39:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4092791/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4092791/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53224497,"identity":"865c9b49-d477-4da3-a846-eb15143981a9","added_by":"auto","created_at":"2024-03-22 06:02:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":395386,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUSP1 was recognized as the pivotal gene relevant to THCA and proteinubiquitination. \u003c/strong\u003e(A) All genes expressed differently in TCGA-THCA, (B) Determination of soft threshold, (C) Tree diagram of five modules labelled with different colours, (D) Correlations between distinct modules, thyroid cancer, and GOBP_protein_polyubiquitination, (E) Venn diagram depicting the cross-correlation between brown and blue module feature genes and genes for deubiquitinating enzymes from iUUCD (\u003ca href=\"http://iuucd.biocuckoo.org/index.php\"\u003ehttp://iuucd.biocuckoo.org/index.php\u003c/a\u003e) (F) Four genes in the intersection of blue-5%hub genes with deubiquitinating enzymes, (G) Four intersecting genes and their connectivity rankings, (H) GO and KEGG analyses of USP1.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4092791/v1/3abb7ff167ff46a7bdf5864b.png"},{"id":53224500,"identity":"e7f35a37-be9e-42b7-a2fe-31a703d2172a","added_by":"auto","created_at":"2024-03-22 06:02:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2494729,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh Expression of USP1 in Thyroid Cancer Tissues. \u003c/strong\u003e(A) Bioinformatics methods to analyze the expression patterns of USP1 in normal and tumor tissues, (B) Real-time fluorescent quantitative PCR to investigate the expression of USP1 in 50 pairs of TC sample,(C, D) Utilized Western Blotting to assess the protein expression of USP1 in 50 TC specimens and their corresponding adjacent tissues, (E) Immunohistochemical staining on 100 TC samples and their corresponding normal tissues to evaluate the expression of USP1.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4092791/v1/45343e4b943b4740485f0af0.png"},{"id":53224941,"identity":"ec88d1c6-d546-428b-8160-1414fe218c21","added_by":"auto","created_at":"2024-03-22 06:10:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":797960,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUSP1 facilitates in vitro expansion of thyroid cancer cells. \u003c/strong\u003e(A) The levels of USP1 mRNA in various thyroid cell lines, (B) The levels of USP1 protein in various thyroid cell lines, (C, D)Utilized shRNA to suppress the expression of USP1 and evaluated the effectiveness of knockdown using qRT-PCR and Western Blotting analysis, (E) Colonies formed by Bcpap cells transfected with control shRNA or shRNA targeting USP1, (F) Quantification of the results of the colony formation assay. (G) CCK8 assay by Bcpap cells transfected.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4092791/v1/e13a742ce0574024baf8c6bf.png"},{"id":53224501,"identity":"d27703f9-a7cb-4ee6-af02-05bb9121264a","added_by":"auto","created_at":"2024-03-22 06:02:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":229527,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUSP1 Orchestrates the Proliferation Capacity of Thyroid Cancer via the Wnt/β-Catenin Signaling Pathway \u003c/strong\u003e(A) GSEA utilizing the TCGA database to determine the relationship between USP1 and the Wnt pathway. (B) Knocked down USP1 in Bcpap cells and assessed the protein expression levels of β-catenin and c-MYC. (C) TOP-Flash luciferase analysis demonstrated that downregulation of USP1 hindered the activity of the Wnt/β-catenin pathway. (D) Overexpressed β-catenin in the USP1 knockdown cells and observed that the upregulation of β-catenin restored the diminished activity of the Wnt pathway caused by USP1 knockdown. (E) Conducted CCK8 experiments and found that overexpression of β-catenin could rescue the decreased proliferation ability of thyroid cancer cells resulting from USP1 knockdown in vitro.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4092791/v1/3b7bea31925474dcc7a77435.png"},{"id":53224498,"identity":"5abd8b35-0f70-4869-b578-9bf93917357e","added_by":"auto","created_at":"2024-03-22 06:02:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":799462,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUSP1 upregulates the protein expression of KRT17 in thyroid cancer. \u003c/strong\u003e(A) The PPI network shows the associated genes of USP1, (B) Bioinformatics detection demonstrated that KRT17 expression was up-regulated in thyroid cancer tissues, (C) KRT17 protein levels in thyroid cancer tissues and paired non-tumor tissues by western blot, (D) Correlation of KRT17 and USP1 expression at the mRNA level, (E) Correlation of KRT17 and USP1 expression at the protein level, (F) Changes of KRT17 mRNA expression after knocking down USP1, (G) Effect of inhibiting or increasing the USP1 protein expression levels on the protein expression levels of KRT17 and β-catenin.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4092791/v1/f6c52e48a3ab5df52ebc1df6.png"},{"id":53224505,"identity":"7f8c8b1a-f02b-4855-80a6-a073e7e924c1","added_by":"auto","created_at":"2024-03-22 06:02:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":367762,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKRT17 is key for USP1-mediated thyroid carcinoma growth. \u003c/strong\u003e(A) (A)Western blot analysis of USP1 knockdown and KRT17 overexpression levels in Bcpap cells and their effects on β-catenin. (B) Western blot analysis of KRT17 knockdown and USP1 overexpression levels in 8505c cells and their effects on β-catenin; (C) CCK8 Assay showed that up-regulated KRT17 expression could significantly save β-catenin content in Bcpap-shUSP1 cells (*P\u0026lt;0.05; NS, not significant). (D) CCK8 Assay showed that upregulation of USP1 could significantly save β-catenin content in 8505c-shKRT17 cells (*P\u0026lt;0.05; NS, not significant).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4092791/v1/c5b8e43dedcfe8516ae3fed5.png"},{"id":53224504,"identity":"a8ed5a25-e7f4-4439-a140-4a6689aa7db9","added_by":"auto","created_at":"2024-03-22 06:02:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":713227,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUSP1 stabilizes KRT17 through deubiquitylation \u003c/strong\u003e(A) Co-IP between\u003c/p\u003e\n\u003cp\u003eendogenous KRT17 and USP1 in Bcpap and 8505c cells. USP1 was detected respectively in the immunoprecipitate when the anti-KRT17 antibodies was used as baits separately, (B) The predicted docking model between USP1 and KRT17, (C) The potential docking sites between USP1 and KRT17, (D) Bcpap cells transfected with USP1 shRNA were treated with MG132(15μM). Cells were collected at 6 h and immunoblotted with the antibodies indicated. (E) Bcpap cells were transfected with USP1 shRNA, and treated with 20μM cycloheximide (CHX). Cells were collected at different time points and immunoblotted with the antibodies indicated, (F) Quantitative results of relative KRT17 protein levels were analyzed (**p\u0026lt; 0.01, *p\u0026lt; 0.05), (G) the knockdown or exogenous expression of USP1 altered the ubiquitination of KTR17 in Bcpap cells. The cells in each group were treated with MG132 (15μM). The levels of ubiquitin-attached KTR17 were detected by Western blotting analysis with Ub antibody.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4092791/v1/336e894b423fa14254d5a5b1.png"},{"id":53224506,"identity":"69468b2b-c821-492c-8770-5f527a390338","added_by":"auto","created_at":"2024-03-22 06:02:36","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":789799,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe in vitro and in vivo efects of parthenolide in Bcpap cells. \u003c/strong\u003e(A) The predicted docking model between USP1 and parthenolide, (B) The potential docking sites between USP1 and parthenolide, (C) Bcpap cells were treated with different concentrations (0, 50, 100μM) of PTL, and cell viability was determined using the CCK8 assay, (D) Bcpap cells were pretreated with 50μM PTL, followed by western blotting assay to test the protein expression of USP1, KRT17, β-catenin and c-Myc, as well in control group, (E) Representative images of tumors of mice obtained at the end of the experiment, (F) The tumor size curve of mice subiected to parthenolide treatment up to day 42, (G) Tumor average weight of control group and the parthenolide-treated tumor up to harvest day, (H) Western blotting assay was used to test the protein expression of USP1, KRT17, β-catenin and c-Myc in parthenolide-treated group and control group.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4092791/v1/f4abde9e0a8fbb52946b1402.png"},{"id":53788649,"identity":"26e7b8e7-b6ac-4ff8-8afd-ec2b0177b68b","added_by":"auto","created_at":"2024-03-30 15:53:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3605260,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4092791/v1/aa093d22-eb8d-4434-8f00-114985800db2.pdf"},{"id":53224503,"identity":"4869b798-862a-4303-9c96-e48503a79321","added_by":"auto","created_at":"2024-03-22 06:02:36","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1326814,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4092791/v1/a7bcea9201e9cf15a808dfc8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"USP1 activates the Wnt/β-catenin pathway by deubiquitinating KRT17, thereby facilitating thyroid cancer proliferation","fulltext":[{"header":"1．Introduction","content":"\u003cp\u003eThe incidence of thyroid cancer (TC) renders it the most prevalent form of malignant neoplasm within the endocrine system. While the prognosis for primary TC is generally favorable [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], some late-stage TC patients may experience deterioration, leading to a more aggressive tumor [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Currently, the common treatment for TC remains surgical removal. However, due to the dysregulation of cell death and proliferation mechanisms in cancer, the issue of postoperative recurrence persists [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], resulting in unsatisfactory outcomes for TC prognosis. Revealing the molecular regulatory mechanisms of TC and identifying novel therapeutic targets is therefore imperative, which holds significant importance in seeking novel treatment strategies for TC.\u003c/p\u003e\u003cp\u003eUbiquitination and deubiquitination are two antagonistic biological processes executed by E1-E3 ubiquitin ligases and deubiquitinating enzymes (DUBs) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Imbalance of these two processes often leads to overexpression of oncogenic proteins or inhibition of tumor suppressors, promoting tumor development [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In recent years, mounting evidence suggests a close association between DUB dysregulation and cancer progression [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. USP1 (Ubiquitinyl Hydrolase 1) is a member of the ubiquitin-specific-processing (UBP) protease family and functions as a deubiquitinating enzyme (DUB) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], possessing both His and Cys domains. This protein localizes in the cytoplasm and catalyzes the cleavage of ubiquitin moiety from both ubiquitin precursor and ubiquitinated protein substrates [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. USP1 plays a critical role in cellular response to DNA damage and has been extensively studied in cancer. For instance, in hepatocellular carcinoma, USP1 stabilizes RPS16 protein through deubiquitination to drive the progression of liver cancer [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], while it facilitates the metastasis of gastric cancer by deubiquitinating and stabilizing ID2 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, there is currently no research on USP1 in TC.\u003c/p\u003e\u003cp\u003eThe Wnt/β-catenin pathway, a protein family critical in embryonic development, also maintains tissue homeostasis in adult organisms [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The canonical Wnt pathway predominantly governs cellular proliferation, whereas the non-canonical Wnt pathway modulates cell polarity and migration [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These two main pathways form an interconnected regulatory network. Dysregulation of the Wnt/β-catenin signaling often leads to the development of various tumors [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Prior investigations have shown that KDM1A facilitates the progression of TC and sustains stemness via modulation of the Wnt/β-catenin signaling pathway [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Another research revealed that lncRNA DOCK9-AS2 activates the Wnt/β-catenin pathway, promoting stemness, proliferation and progression of TC [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Given the complexity and significance of the dysregulated Wnt/β-catenin pathway in TC, further research into the specific molecular regulatory mechanisms is imperative.\u003c/p\u003e\u003cp\u003eIn our research, we utilized WGCNA to identify the hub gene USP1 associated with TC and ubiquitination. Subsequently, experimental validation revealed that USP1 regulates KRT17 to activate the Wnt/β-catenin signaling pathway, thereby impacting the proliferation of TC. Furthermore, we discovered that USP1 can stabilize KRT17 expression through its deubiquitination activity. Interestingly, this USP1-KRT17-Wnt/β-catenin axis can be targeted by the naturally derived compound parthenolide. This represents the first investigation into the ubiquitination molecular mechanism related to USP1 in TC.\u003c/p\u003e"},{"header":"2. Methods and Materials","content":"\u003cp\u003e\u003cb\u003e2.1 Human specimens and cell culture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe obtained human thyroid cancer (TC) specimens from a cohort of 50 patients underwent TC resection at the Second Affiliated Hospital of Nanchang University during March 2018 to June 2022. The acquisition of samples received appropriate informed consent from the patients, and all experimental protocols were approved by the research ethics committee of the Second Affiliated Hospital of Nanchang University.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.2 Cell lines and cell culture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe thyroid epithelial cell line Nthy was acquired from the European Collection of Animal Cell Cultures, while the human thyroid carcinoma cell lines (Bcpap, TPC-1 and 8505c) were procured from the Shanghai Institute of Cell Biology (Shanghai, China). The K1 cell line was obtained from the American Type Culture Collection. All cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s media (DMEM) supplemented by 10% fetal bovine serum (FBS) at 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.3 Data Collection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur digital data was derived solely from The Cancer Genome Atlas (TCGA) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cancergenome.nih.gov\u003c/span\u003e\u003cspan address=\"https://cancergenome.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database and the Genotype-Tissue Expression (GTEx) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.gtexportal.org/\u003c/span\u003e\u003cspan address=\"https://www.gtexportal.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database. The TCGA-THCA dataset includes expression data from 571 samples, consisting of 59 normal samples and 512 tumor samples. For our research, we obtained GTEx-thyroid dataset which includes 276 samples from GTEx.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.4 Weighted Gene Co-expression Network Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe WGCNA algorithm, which is widely employed for high-throughput gene coexpression profiling, was utilized in this study to identify gene coexpression networks associated with different diseases [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. For constructing a scale-free co-expression network encompassing all genes, a soft threshold of 10 was applied. Furthermore, a dynamic tree cutting method was employed to identify modules within the network. To determine the modules significantly correlated with thyroid cancer and ubiquitination, the correlations between Eigengenes and THCA and GOBP_PROTEIN_POLYUBIQUITINATION, respectively, were analyzed. The gene connectivity within modules was assessed using the absolute value of Pearson correlation, allowing for the identification of hub genes exhibiting high within-module connectivity.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.5 Protein-Protein Interaction, Molecular Docking and Gene set enrichment Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs previously described [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], we utilized the BioGRID (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://thebiogrid.org/\u003c/span\u003e\u003cspan address=\"https://thebiogrid.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) interactome dataset to identify the interacting genes in TC patients. Subsequently, the construction of the protein-protein interaction (PPI) network was conducted using the Cytoscape software, followed by its visualization. The identification of protein nodes was accomplished using the Cytoscape plugin.\u003c/p\u003e\u003cp\u003eThe HDOCK (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://hdock.phys.hust.edu.cn/\u003c/span\u003e\u003cspan address=\"http://hdock.phys.hust.edu.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), a commonly employed computational resource, employs a hybrid docking algorithm that combines template-based modeling and free docking for the automated utilization of binding data from the PDB [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. To investigate the interaction between USP1 and KRT17, we employed HDOCK as a predictive tool to determine their docking mode. Subsequently, we utilized PyMOL software to visualize the obtained results.\u003c/p\u003e\u003cp\u003eGSEA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinfo.life.hust.edu.cn/GSEA\u003c/span\u003e\u003cspan address=\"http://bioinfo.life.hust.edu.cn/GSEA\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), a software package equipped with an extensive collection of 1,325 gene sets, offers a valuable tool for the interpretation of gene expression data [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The samples were classified into two groups based on the expression levels of THCA, and this approach was utilized to examine the enrichment of gene sets containing THCA. The analysis conducted 1,000 permutations and employed the h.All.V7.4 Symbols.gmt (Hallmarks) gene set database for comprehensive investigation. Significance was determined at \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.6 Quantitative real-time PCR (qrt-PCR)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe used Trizol reagent (catalog number: 15596026, Invitrogen, USA) to extract total RNA. mRNA expression levels were assessed using SYBR Green assays with RT primers and SYBR Green from Takara Biotechnology (Catalog: DRR041A, TAKARA, Dalian, China). During the simultaneous process, we used human microtubule-associated protein as an internal control for amplification. The primer sequences used in this study are provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.7 Western blotting\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe prepared the total protein extract according to the method described earlier. We used RIPA buffer containing a mixture of protease inhibitors (manufactured by Shanghai Berry Times Company) to extract the protein. The antibodies employed in this study included anti-USP1 monoclonal antibody (1:1000 dilution; CST, 8033), anti-KRT17 monoclonal antibody (1:1000 dilution; CST, 12509), anti-β-catenin monoclonal antibody (1:1000 dilution; abcam, ab32572), anti-c-Myc monoclonal antibody (1:1000 dilution; abcam, ab32072), and anti-Tubulin monoclonal antibody (1:1000 dilution; Proteintech, 11224-1-AP). Following a 2-hour incubation with secondary antibodies at room temperature, the Quantity One software (Bio-Rad Laboratories Inc., Hercules CA, USA) was employed for quantitative analysis of protein band intensity. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.8 Immunohistochemistry (IHC)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe study involved the collection of TC samples and paired normal thyroid tissues, which underwent an antigen retrieval process. Heated the antigen retrieval solution (EDTA, pH 8.0) in a microwave for 40 minutes, then incubated with goat serum for 30 min. Subsequently, tissue slices were incubated with anti-USP1 monoclonal antibody (1:1000, CST, 8033) overnight at 4\u0026deg;C, followed by incubation with an HRP-conjugated secondary antibody (Boster) at room temperature for two hours. before immunostaining with the DAB Detection Kit (Maxim) for two minutes. Ultimately, the positive area proportion was semi-quantitatively evaluated by three pathologists blinded to the clinical parameters.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.9 Plasmids and reagents\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn all diagrams, the shRNA structures of USP1 and KRT17 are provided in supplemental table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. These shrnas were integrated into the lentivirus pLKO vector obtained from a gene pharmaceutical company based in Shanghai. The overexpressed plasmid was sourced from another genetics company also located in Shanghai. Specifically, USP1, HIS-labeled KRT17, and HIS-labeled beta-catenin were inserted into the P-CMV vector. After 48 hour of transfection, the viral supernatant was collected and filtered as per the manufacturer's instructions for selecting lentiviral transduction cells using purinomycin.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.10 Cell colony formation assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn colony formation experiments, the cells were seeded onto a 6-well plate. Once the colonies reached the desired size, they were fixed with 4% paraformaldehyde for 30 minutes and stained with 1.0% crystal violet for an additional 30 minutes until the clones became visible. Colonies comprising more than 50 cells were enumerated and subjected to analysis in order to investigate their correlation with the initial number of seed cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.11 Cell counting kit-8 (CCK-8) assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe CCK-8 assay was conducted in accordance with the manufacturer's instructions using the CCK-8 kit. Transfected cells were incubated for 48 hours and then seeded into each well of a 96-well plate at a density of 5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well. Following incubation for 24, 48, 72, 96, and 120 hours, each well was supplemented with 10 \u0026micro;L of CCK8 reagent and further incubated at a temperature of 37℃ for a duration of two hours. Absorbance at the wavelength of 450 nm was measured using an enzyme-labeled apparatus (ELX-800; Bio-Tek, Winooski, VT, USA). Moreover, the inhibitory effects of PTL were evaluated utilizing CCK-8 assays. Parthenolide (with a purity exceeding 98%) was procured from Absin Bioscience Inc (#20554-84-1), while Dimethyl sulfoxide (DMSO) was sourced from Sigma-Aldrich (#276855) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Following a 24-hour incubation period, cells were exposed to PTL at different concentrations or DMSO for 24, 48, 72, 96, and 120 hours. The specific operation is as described above.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.12 Luciferase reporter assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were co-transfected with plasmids containing firefly luciferase reporters and USP1 plasmids in the TOP/FOP-Flash reporter assay. Following a 48-hour transfection period, cells were harvested and subjected to analysis using the Dual-Luciferase Reporter Assay System from Promega (Madison, WI, USA). Luciferase activity was assessed using the PerkinElmer EnSpire Multilabel Reader 2300 (PerkinElmer Inc., Waltham, MA, USA), with normalization to Renilla luciferase activity to ensure uniform transfection efficiency.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.13 Co-immunoprecipitation experiment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCell lysates were subjected to overnight incubation at 4\u0026deg;C with 50 \u0026micro;l of protein G beads and 1 \u0026micro;g of the designated antibody. Subsequent centrifugation effectively separated the protein G beads from the solution. Following this, loading buffer was then introduced to the resulting mixture and subjected to thermal treatment at 100\u0026deg;C for a quarter. The immunoprecipitated proteins were then subjected to analysis via SDS-PAGE and immunoblotting. The intensity of the protein bands was quantitatively examined and assessed using the specialized software.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.14 Mouse xenograft assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe animal experiments conducted in this study adhered to the ethical guidelines prescribed by the Animal Ethics Committee of the Second Affiliated Hospital of Nanchang University. Bcpap cells were suspended in a medium devoid of fetal bovine serum and subsequently subcutaneously injected into 6-week-old nude mice at a volume of 100 \u0026micro;l. Each experimental group, consisting of six mice, received daily intraperitoneal injections of either a vehicle solution (comprising 2% DMSO, 40% PEG400, and 2% Tween 80 in normal saline) or PTL (20 mg/kg) for a duration of 42 days. Regular assessments were made to monitor changes in body weight on a daily basis. Additionally, tumor measurements and dimensions were taken at intervals of 3 days employing a digital caliper. Meanwhlie, the corresponding tumor volumes were computed according to the dedicated formula [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.15 Statistical analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and were evaluated through GraphPad Prism 5 (GraphPad Software, USA) based on data obtained from a minimum of three distinct experiments. Statistical analyses involved pairwise comparisons utilizing the Statistical comparisons among multiple groups were conducted using Student's t-test or one-way ANOVA. Furthermore, logistic regression models were utilized for both univariate and multivariate analyses. All P values were computed as two-tailed tests, with statistical significance defined at a threshold of \u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003e3.1 USP1 was identified as the pivotal gene relevant to thyroid cancer and protein ubiquitination.\u003c/h2\u003e \u003cp\u003eIn order to identify key genes in thyroid cancer (TC), we identified 10 874 differentially expressed genes (logFC\u0026thinsp;\u0026gt;\u0026thinsp;1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) from the TCGA-THCA and GTEx-thyroid databases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Following this, we conducted a weighted gene co-expression network analysis (WGCNA) with a soft threshold power of 10 to construct a scale-free network (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Analysis showed the identification of 5 gene co-expression modules through hierarchical clustering (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). It is worth noting that the module trait correlation heatmap elucidated that the brown and blue modules were most strongly associated with THCA and GOBP_protein_polyubiquitination, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Furthermore, in consideration of the vital role of deubiquitinating enzymes (DUBs) in driving tumor development, we downloaded 126 genes encoding deubiquitinating enzymes from the integrated annotation of ubiquitin and ubiquitin-like conjugation databases (iUUCD). We then intersected these genes with the 5% hub genes of the brown and blue modules (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), resulting in 4 genes: USP1, USP10, VCPIP1, and BRCC3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Among them, USP1 had the highest connectivity within the module (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). In order to delve deeper into the functional aspects of USP1, we performed GO and KEGG analyses. The results indicated that USP1 was primarily abundant in pathways associated with monoubiquitinated protein deubiquitination, regulation of DNA repair, protein deubiquitination, cysteine-type deubiquitinase activity, deubiquitinase activity, ubiquitin-like peptidase activity and Fanconi anemia pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). In conclusion, USP1 is considered a key gene associated with THCA and protein ubiquitination.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e3.2 High Expression of USP1 in Thyroid Cancer Tissues\u003c/h2\u003e \u003cp\u003eTo assess USP1 expression in thyroid cancer (TC), we first employed TCGA and GTEx databases to compare USP1 expression patterns in normal and tumor tissues. Our investigation revealed a remarkable upregulation of USP1 in tumor tissues when compared to normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To validate these results, we employed real-time quantitative PCR to investigate the expression of USP1 in 50 pairs of TC samples. Data exhibited a substantial elevation in the mRNA levels of USP1 in 31 out of the 50 pairs, providing further support for the heightened expression of USP1 in TC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Furthermore, we utilized Western Blotting to evaluate the protein expression of USP1 in 50 TC specimens and their corresponding adjacent tissues. Our analysis demonstrated a marked elevation in the protein expression of USP1 in TC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), and representative results were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD. Furthermore, we performed immunohistochemical staining on 50 TC samples and their corresponding normal tissues to assess the expression of USP1. The findings validated a marked augmentation in the protein expression of USP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In conclusion, our findings provide compelling evidence of the heightened expression of USP1 in TC tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.3 USP1 facilitates in vitro proliferation Capacity of TC\u003c/h2\u003e \u003cp\u003eTo further explore the cellular expression of USP1, we assessed the levels of USP1 mRNA and protein in various TC cell lines (Bcpap, K1, TPC-1, 8505c) along with a normal cell line (Nthy). Interestingly, both USP1 mRNA and protein expression exhibited substantial elevation in the TC cell lines relative to the Nthy (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). In order to understand the influence of the irregular expression of USP1 on TC development, we utilized small hairpin RNA (shRNA) to suppress the expression of USP1 and evaluated the effectiveness of knockdown using qRT-PCR and Western Blotting analysis (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). Subsequently, to assess the impact of transfection on the proliferation of TC cells, both the control group and the USP1 knockdown group were subjected to colony formation and CCK8 assays. The results obtained from the colony formation assay demonstrated that the suppression of USP1 resulted in a reduction in the colony-forming capacity of Bcpap cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F). Furthermore, the CCK8 assay demonstrated that the downregulation of USP1 markedly hindered cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). In conclusion, these analyses collectively indicate that the knockdown of USP1 inhibits the in vitro proliferation of TC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3.4 USP1 impacts the Proliferation Capacity of TC through the Wnt/β-Catenin Signaling Pathway\u003c/h2\u003e \u003cp\u003eWe then investigated the precise mechanism underlying the regulation of TC cell proliferation by USP1. Previous investigations have highlighted the significant involvement of the Wnt/β-catenin signaling pathway in TC proliferation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, we postulated that USP1 might modulate TC proliferation through this pathway. To substantiate our hypothesis, we initially performed gene set GSEA utilizing the TCGA database. Our findings uncovered a positive relevance between USP1 and the Wnt signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To further investigate the impact of USP1 on Wnt pathway activation, we knocked down USP1 in Bcpap cells and assessed the protein expression of β-catenin and c-MYC. Notably, the knockdown group exhibited downregulated expression of these three proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Moreover, TOP-Flash luciferase analysis demonstrated that downregulation of USP1 hindered the activity of the Wnt/β-catenin pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Following USP1 silencing, ectopic expression of β-catenin was performed, revealing a restoration of Wnt signaling pathway activity previously compromised by USP1 knockdown. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results affirm the pivotal role played by USP1 in regulating the Wnt/β-catenin pathway. Additionally, to probe whether USP1 influences cell proliferation by modulating the Wnt/β-catenin pathway, we conducted CCK8 experiments and demonstrated that overexpression of β-catenin can rescue the decreased proliferation ability of TC cells resulting from USP1 knockdown in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Consequently, our research provides compelling evidence demonstrating that USP1 exerts an impact on the proliferation capacity of TC via its regulation of the Wnt/β-catenin pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.5 KRT17 is significantly correlated with USP1 at the protein level in TC\u003c/h2\u003e \u003cp\u003eDelving into the mechanisms underlying USP1's impact on Wnt/β-catenin pathway in TC, our initial co-IP experiments revealed that USP1 didn\u0026rsquo;t directly bind to β-catenin (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Moreover, at the mRNA level, a significant correlation was observed between USP1 and CTNNB1 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). This prompted us to delve deeper into genes that can directly interact with USP1 while showing no correlation at the mRNA level. Initiating our exploration, we identified 45 genes that interact with USP1 using the BioGRID database (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Subsequently, leveraging the Timer online website, we pinpointed 10 genes that exhibited no correlation with USP1 at the mRNA level (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Further investigation into the varied gene expression patterns (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC) highlighting that only KRT17 displayed significant upregulation in TC tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). To scrutinize the variance in KRT17 expression between normal and TC tissues, Western blotting assay was performed to assess the protein expression of KRT17 in 50 TC samples. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC underscored representative results, a considerable elevation of KRT17 in TC tissues relative to the corresponding normal tissues. Subsequent exploration of the relationship between USP1 and KRT17 entailed an analysis of their expression patterns in human TC specimens, revealing a noteworthy correlation between the protein expressions of USP1 and KRT17 while lacking such association at the mRNA level (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E). Crucially, knocking down the mRNA expression of USP1 did not lead to a concomitant alteration in the mRNA expression of KRT17 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Further investigations involved the examination of KRT17 and β-catenin expression in Bcpap cell lines subjected to shRNA and shUSP1 treatments, exposing a significant reduction in KRT17 and β-catenin protein expressions upon USP1 knockdown. In concordance, augmenting USP1 levels in 8505c cells resulted in increased protein expressions of KRT17 and β-catenin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). These findings collectively affirm a substantial correlation between USP1 and KRT17 at the protein level.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e\u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003evarX\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003evarY\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ecor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSPANXN5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.007205047\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.87118323\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLGALS7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.014510283\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.743986138\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKRT17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02278842\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.607998338\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCALML5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.025697358\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.562974535\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSPANXN2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.026369697\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.552801069\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCALML3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.034385748\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.438871979\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOPALIN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.037065019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.404024189\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKRT4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.058265099\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.189383914\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDSG1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.058466171\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.187858509\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLRRC15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.07565916\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.088157773\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALDH16A1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.089278676\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.04408429\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTAGLN2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.099754177\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.024408355\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCIRBP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.146782162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.000895602\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLYZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.150934779\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.000634401\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC4ORF49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.170851241\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.000107231\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUNC45A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.17177272\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.83E-05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDSC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.190166523\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.57E-05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePTGES2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.19568568\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.70E-06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZCCHC10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.234321325\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.88E-08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePKP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.257533248\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.73E-09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eANXA2P2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.276272818\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.28E-10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJUP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.343463709\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.54E-15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSGOL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.34687435\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.75E-16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSTAT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.357775415\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.14E-17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCNF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.358611712\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.82E-17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBE2A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.365510098\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.56E-17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRAD51AP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.388881895\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.00E-20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePHLPP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.451241419\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.69E-27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKPNA2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.485518198\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.84E-31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFLG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.523481896\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.72E-37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMYH9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.549004413\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.08E-41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWDR5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.549834592\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.49E-41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHSPB1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.558109581\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.15E-43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWDR20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.590712638\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.38E-49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePHLPP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.659138107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.51E-65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCHIC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.685771294\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.72E-72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCSE1L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.720116537\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.64E-82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUSP46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.734198148\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.63E-87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWDR48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.735631524\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.21E-88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVPS36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.742990727\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.85E-90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUSP4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.774548728\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.34E-103\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKPNA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.805446921\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.87E-117\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVPS26A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.820348012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.82E-125\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSOCS6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.82105832\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.54E-125\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUSP12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.850009366\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.02E-143\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.6 KRT17 is pivotal in the USP1-regulated Wnt/β-catenin pathway.\u003c/h2\u003e \u003cp\u003eBuilding on the observed correlation between USP1 and KRT17, a thorough examination was carried out to ascertain the participation of KRT17 in the USP1-mediated Wnt/β-catenin signaling cascade. Utilizing Western blot experiments, KRT17 was upregulated in Bcpap cells upon USP1 downregulation, with concurrent monitoring of the protein expression levels of USP1, KRT17, and β-catenin. The results unveiled that the upregulation of KRT17 resulted in an augmented expression of β-catenin, effectively rescuing the diminished levels induced by USP1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Conversely, in 8505c cells, the downregulation of KRT17 resulted in a decrease in the expression of β-catenin, successfully reversing the elevated levels caused by USP1 upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Furthermore, CCK8 analysis demonstrated that the upregulation of KRT17 can restore the decreased proliferation capacity of TC cells resulting from USP1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), whereas the downregulation of KRT17 inhibited the heightened proliferation capacity induced by USP1 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Collectively, these findings suggest that USP1 regulates the Wnt/β-catenin pathway through its influence on KRT17 expression, consequently impacting the proliferation capacity of TC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.7 USP1 exerts deubiquitination to stabilize KRT17 expression\u003c/h2\u003e \u003cp\u003eHaving established the correlation between USP1 and KRT17, our study aimed to elucidate their potential regulatory mechanism. Prior research has highlighted USP1 as a deubiquitinase closely associated with cancer. Notably, a study unveiled the role of the E3 ligase TRIM21 in ubiquitinating and stabilizing KRT17 to trigger STAT3 activation in psoriasis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Based on this background, we postulated that USP1 might deubiquitinate and stabilize KRT17. To test our hypothesis, we conducted co-immunoprecipitation experiments, unveiling a direct interaction between USP1 and KRT17 within Bcpap cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Molecular docking models and potential binding sites of these two proteins are depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate whether USP1 stabilizes KRT17 expression through the ubiquitin-proteasome pathway, we subjected 8305c cells transfected with USP1 shRNA or USP1 plasmid to treatments with or without 15 uM MG132. Our findings indicated that alterations in USP1 expression did not impact KRT17 protein levels compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Subsequently, we utilized 20 uM cycloheximide (CHX) as a translation inhibitor in Bcpap cells to assess KRT17 expression in the control and sh-USP1 groups at different time points. Strikingly, downregulation of USP1 significantly expedited the degradation of KRT17 (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, G). Further experiments confirmed that upregulation of USP1 notably decreased the ubiquitination level of KRT17. Conversely, downregulation of USP1 exhibited contrasting effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). Collectively, our data strongly supports the role of USP1 as a deubiquitinase for KRT17, thereby stabilizing its expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Parthenolide inhibits TC proliferation in vitro\u003c/h2\u003e \u003cp\u003ePrevious studies have validated the potent inhibitory effect of parthenolide (PTL) on TC cell proliferation. To delve deeper into the underlying mechanism of PTL action in TC, we initially explored the association between USP1 expression levels and the half-maximal inhibitory concentration (IC50) of PTL based on GDCS database, revealing a significant positive correlation between them (Figure S2A). Further scrutiny indicated that lower USP1 expression corresponded to lower IC50 values of PTL, suggesting increased sensitivity of patients with diminished USP1 expression to PTL treatment (Figure S2B). Subsequently, we conducted molecular docking simulations of PTL with USP1, predicting their potential binding sites (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, B). Next, CCK8 assays were conducted to assess the effects of different concentrations of PTL on cell proliferation. Our findings demonstrated the anticancer effects of PTL (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Furthermore, to elucidate the molecular mechanisms involved in PTL's action in TC, we conducted Western blot experiments which unveiled a downregulation in the protein expression levels of USP1, KRT17, β-catenin, and c-Myc following PTL treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). In summary, our study suggests that PTL might reduce the in vitro proliferative capacity of TC by inhibiting the expression of USP1, KRT17 and the activity of Wnt/β-catenin pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Parthenolide Obstructs In Vivo Proliferation of TC\u003c/h2\u003e \u003cp\u003eTo discern the anticancer properties of PTL in live organisms, we conducted a xenograft mouse model employing nude mice engrafted with Bcpap cells. PTL was intraperitoneally administered at a constant daily dose of 20 mg/kg. for a continuous span of 42 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). The findings unequivocally demonstrated that PTL significantly impeded the growth of Bcpap cell xenograft tumors in terms of volume and weight (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF, G). Subsequently, we delved deeper into assessing whether PTL impedes the expression of USP1 and KRT17, while concurrently repressing the Wnt/β-catenin pathway related proteins influenced by them. Our Western blotting results underscored the downregulation of USP1, KRT17, β-catenin, and c-Myc protein expression after treatment with PTL (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH). To summarize, PTL has the potential to diminish the exuberant in vivo proliferation ability of TC by thwarting the expression of USP1-KRT17, consequently suppressing the Wnt/β-catenin pathway.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe imbalance in protein ubiquitination and deubiquitination has profound effects on tumor growth and proliferation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Deubiquitinating enzymes (DUBs), as crucial regulatory factors in the deubiquitination process, play a pivotal role in cancer pathogenesis and represent promising targets for cancer diagnosis and treatment [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Considerable evidence supports a noteworthy association between aberrant expression of deubiquitinating enzymes and thyroid cancer (TC) manifestation, as observed in several research studies. In our research, weighted gene co-expression network analysis (WGCNA) was applied to recognize the core gene USP1, which was associated with TC and protein polyubiquitination. We were the first to report the mechanism by which USP1 regulates TC development. Initially, we observed significantly higher mRNA and protein expression of USP1 in TC compared to normal thyroid tissue. Furthermore, we elucidated that USP1 enhanced the proliferation capacity of TC cells in vitro and identified its oncogenic function in TC development.\u003c/p\u003e \u003cp\u003eMoreover, we delved into the potential mechanisms through which USP1 regulates TC proliferation. Prior investigations have underscored the pivotal involvement of the Wnt/β-catenin pathway in the onset of TC, as elucidated in earlier studies [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].Additionally, c-Myc, a β-catenin-activated target molecule in Wnt signaling transduction, enhances tumor growth and metastasis in TC [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Another study suggested that USP8 positively regulates the progression of hepatocellular carcinoma through the Wnt/β-catenin pathway [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Building upon these findings, we propose that USP1 regulates TC proliferation by affecting the Wnt/β-catenin pathway. Firstly, through Gene Set Enrichment Analysis (GSEA), it was demonstrated that the Wnt signaling pathway functions as a downstream pathway of USP1, indicating a positive correlation between USP1 activity and the Wnt/β-catenin pathway. Secondly, knockdown of USP1 expression contributed to decreased expression of β-catenin and c-Myc. Consistently, overexpression of β-catenin significantly rescued the reduced activity of the Wnt/β-catenin pathway and the proliferation capacity induced by USP1 downregulation. These results unveiled the mechanism through which USP1 promotes TC proliferation by activating the Wnt/β-catenin pathway.\u003c/p\u003e \u003cp\u003eUSP1 (Ubiquitinyl Hydrolase 1), a member of the ubiquitin-specific processing (UBP) protease family, is known for its direct interaction with substrates to impact their ubiquitination degradation and modulate protein expression levels[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. It was initially speculated that USP1 can directly interact with β-catenin and remove ubiquitin modifications from it. However, our findings reveal no direct interaction between USP1 and β-catenin. Meanwhile, USP1 and β-catenin show a significant correlation at the mRNA level. To identify the crucial mediator through which USP1 modulates the Wnt/β-catenin pathway and triggers TC proliferation, we conducted a PPI analysis utilizing the BioGRID platform, uncovering 45 proteins that physically interacted with USP1. Subsequently, we explored the relationship between these genes, USP1 expression, and their expression in TC. Intriguingly, only the mRNA expression of KRT17 appeared to be independent of USP1 and displayed upregulation in TC. Further examination of our clinical data unveiled a notable association between the protein expression of KRT17 and USP1, with mRNA expression showing no correlation. Given that deubiquitinating enzymes like USP1 regulate the expression of substrate proteins through post-translational modifications without affecting mRNA expression, the distinctive characteristic of KRT17 captured our attention. KRT17, a versatile cytoskeletal protein, participants in countless biological processes such as cell proliferation, growth and skin inflammation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Notably, aberrant expression of KRT17 is observed in several diseases including cervical cancer [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], oral squamous cell carcinoma [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and gastric cancer [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Recent evidence suggests that elevated KRT17 expression significantly activates the Wnt/β-catenin pathway, promoting proliferation and invasion in NSCLC, indicating a poor prognosis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Consistent with these findings, our experimental results indicated that upregulating KRT17 can counteract the reduced TC proliferation resulting from USP1 knockdown-induced decrease in β-catenin expression, while downregulating KRT17 can inhibit the increased TC proliferation triggered by USP1 overexpression-induced elevation in β-catenin expression. Therefore, KRT17 emerged as a critical factor in mediating the proliferative capacity of TC regulated by USP1 through the Wnt/β-catenin pathway.\u003c/p\u003e \u003cp\u003eTo further investigate the role of USP1 in stabilizing KRT17 expression through deubiquitination, we conducted a series of experiments for validation. Co-immunoprecipitation experiments confirmed the direct binding of USP1 to KRT17. Additionally, we observed that downregulating USP1 decreased the half-life of KRT17, leading to an increased degradation rate. Furthermore, we discovered that downregulating USP1 significantly elevated the ubiquitination level of KRT17. These findings indicated that KRT17 can undergo ubiquitin-mediated proteolysis, and USP1 acts as the deubiquitinating enzyme for KRT17, playing a crucial role in its degradation process.\u003c/p\u003e \u003cp\u003eIn recent decades, there has been an increasing utilization of natural or herbal extracts, which have shown promising anti-cancer effects against various types of cancer [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Parthenolide (PTL), a sesquiterpene lactone found in the stems of Tanacetum balsamita, possesses potent anti-cancer and anti-inflammatory properties [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Previous studies have demonstrated that PTL inhibits ubiquitin-specific peptidase 7 (USP7), the Wnt signaling pathway, and the growth of colorectal cancer cells [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Moreover, Yuan et al. have suggested that PTL exhibits anti-cancer activity in TC, although the precise molecular mechanisms remain unclear [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Therefore, we hypothesized that PTL acts as an inhibitor of USP1, suppressing TC cell growth and proliferation by inhibiting KRT17 expression and modulating the Wnt/β-catenin pathway. In our research, we observed that PTL hindered the growth activity and proliferation ability of TC cells. Additionally, xenograft tumor experiments in nude mice demonstrated that PTL partially suppressed the growth and weight of heterotransplanted tumors. Since USP1 positively regulates the Wnt/β-catenin pathway through mediating deubiquitination of KRT17, further investigations revealed that PTL reduced the protein expression of USP1, KRT17, β-catenin, and c-Myc both in vitro and in vivo. Consequently, our research provides insights into the specific mechanism by which PTL inhibits TC proliferation. Specifically, it downregulates USP1 and KRT17 expression while modulating the Wnt/β-catenin pathway.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eTo sum up, our investigation has illuminated a fresh aspect and regulatory mechanism involving USP1 in TC development. USP1 regulated KRT17 levels through its deubiquitinase activity, leading to the activation of the Wnt/β-catenin pathway and promoting TC cell proliferation. Notably, PTL demonstrated USP1 inhibitory effects and exhibited promise as a potential therapeutic option for TC. These findings strongly supported the significance of USP1 as a prospective target for TC treatment, underscoring its therapeutic potential (Figure S3).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe express our gratitude to TCGA, and GTEx for providing their resources at no cost.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiment was conducted by Hong Zeng, Xuanrui Zhou, and Xitong Geng. Hong Zeng and Hao Wan wrote and edited the manuscript. Data analysis was done by Yongqi Ding and Minqin Zhou. Formalized are Jingying Pan and Zichuan Yu. Da Huang amended the study\u0026nbsp;and designed the experiment. Each author approved the manuscript\u0026apos;s final draft and contributed significantly, directly, and intellectually to the project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from Science and Technology Project of Jiangxi Provincial Health Commission (202210611) and Science and Technology project of traditional Chinese medicine in Jiangxi province (2023Z030) and Natural Science Foundation of Jiang xi Province Youth Science Foundation (20224BAB216055) and National Natural Science Foundation of China (82260472).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the conclusions of this research are shown in this article and the Supplemental Files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe studies involving human participants were reviewed and approved by the Ethics Committee of Second Affiliated Hospital of Nanchang University (Nanchang, China).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCha YJ and Koo JS. 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Cancer Cell Int. 2020; 20(1):561.10.1186/s12935-020-01658-1 PMID:33292235\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"USP1, KRT17, Wnt/β-catenin, thyroid cancer, cell proliferation, deubiquitination","lastPublishedDoi":"10.21203/rs.3.rs-4092791/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4092791/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePost-translational modification through ubiquitination is widely acknowledged for its pivotal regulatory role in tumor onset and progression. Ubiquitin ligases and deubiquitinases can modulate tumor advancement by impacting the expression of key proteins. Nonetheless, the precise contribution of ubiquitination and deubiquitinases in the onset of thyroid cancer (TC) remains to be comprehensively understood. Initially, Weighted Gene Co-expression Network Analysis (WGCNA) revealed the deubiquitinase USP1 as closely associated with TC and protein ubiquitination. Subsequently, our findings demonstrated the aberrant upregulation of USP1 expression in clinical TC samples. Moreover, interference with USP1 expression inhibited the proliferative ability of TC cells in vitro by colony formation and CCK8 assays. Notably, our findings have revealed that USP1 facilitates the proliferation of TC by modulating the Wnt/β-catenin pathway. Further, we identified KRT17 as a critical factor in the USP1-mediated Wnt/β-catenin pathway. Next, we validated that USP1 directly interacted with KRT17 and deubiquitinated it. Ultimately, we discovered that parthenolide exerted inhibitory effects on TC proliferation both in vivo and in vitro by modulating the USP1-KRT17-Wnt/β-catenin axis. To sum up, Our findings offer compelling evidence that underscores the pivotal role of USP1 in promoting TC proliferation. This is accomplished by stabilizing KRT17 expression through deubiquitination, which in turn activates the Wnt/β-catenin pathway. These results provide novel insights into potential therapeutic targets for TC treatment.\u003c/p\u003e","manuscriptTitle":"USP1 activates the Wnt/β-catenin pathway by deubiquitinating KRT17, thereby facilitating thyroid cancer proliferation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-22 06:02:30","doi":"10.21203/rs.3.rs-4092791/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8506031a-9f90-4501-82be-ba1aa5919bc6","owner":[],"postedDate":"March 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-30T15:44:59+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-22 06:02:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4092791","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4092791","identity":"rs-4092791","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}