DCAF13 promotes ovarian cancer progression by activating FRAS1-mediated FAK signaling pathway

DCAF13 promotes ovarian cancer progression by activating FRAS1-mediated FAK signaling pathway | 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 DCAF13 promotes ovarian cancer progression by activating FRAS1-mediated FAK signaling pathway Ze-Yi Tang, Xiaomin Wang, Chun-Wei Xu, Qing-Qing Sun, Yu-Xin Hua, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4539524/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Oct, 2024 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 5 You are reading this latest preprint version Abstract Cullin-RING ubiquitin ligase 4 (CRL4) is closely correlated with the incidence and progression of ovarian cancer. DDB1- and CUL4-associated factor 13 (DCAF13), a substrate-recognition protein in the CRL4 E3 ubiquitin ligase complex, is involved in the occurrence and development of ovarian cancer. However, its precise function and the underlying molecular mechanism in this disease remain unclear. In this study we confirmed that DCAF13 is highly expressed in human ovarian cancer and its expression is negatively correlated with the overall survival rate of patients with ovarian cancer. We then used CRISPR/Cas9 to knockout DCAF13 and found that its deletion significantly inhibited the proliferation, colony formation, and migration of human ovarian cancer cells. In addition, DCAF13 deficiency inhibited tumor proliferation in nude mice. Mechanistically, CRL4-DCAF13 was found to target Fraser extracellular matrix complex subunit 1 (FRAS1) for polyubiquitination and proteasomal degradation. FRAS1 was found to influence the proliferation and migration of ovarian cancer cell through induction of the focal adhesion kinase (FAK) signaling pathway. These findings collectively show that DCAF13 is an important oncogene which promotes tumorigenesis in ovarian cancer cells by mediating FRAS1/FAK signaling. Our findings pave the way to develop new potential targeted therapeutics for ovarian cancer treatment. ovarian cancer DCAF13 FRAS1 CRL4 E3 ubiquitin ligase FAK Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Ovarian cancer is a common malignant tumor of the female reproductive system and has a high mortality rate among gynecological cancers [1]. Ovarian cancer typically proceeds undetected and lacks specific clinical symptoms at the early stages [2]. Accordingly, more than 70% of patients with ovarian cancer are diagnosed in the late stages, which is associated with extensive metastasis and poor prognosis [3]. Currently, surgical resection combined with chemotherapy is considered the standard treatment for ovarian cancer. The 5-year survival rate for the patients with advanced disease is less than 40% [4]. With prolonged chemotherapy cycles and multiple relapses, the sensitivity of most ovarian cancers to chemotherapeutic drugs decreases gradually, ultimately leading to therapeutic recalcitrance [5]. Therefore, there is an urgent need to understand the pathogenesis of ovarian cancer to improve prognosis and reduce mortality. Cullin-RING ubiquitin ligase 4 (CRL4) is an important member of the E3 ubiquitin ligase family. The CRL4 E3 ubiquitin ligase consists of three components, the scaffold protein CUL4, RING finger protein RBX1 (also known as ROC1 or HRT1), and DNA damage binding protein 1 (DDB1) [6]. Numerous studies have shown that the CRL4 complex plays an important role in ovarian cancer [7-9]. DDB1- and CUL4-associated factor 13 (DCAF13), a substrate recognition protein for the CRL4 E3 ubiquitin ligase complex, is highly amplified in breast, liver, and lung cancer [10-14]. An early discovery by our group suggested that DCAF13 is involved in cell cycle regulation, apoptosis, tumor-related signaling pathways, and other processes in breast cancer, thus promoting breast cancer cell proliferation [11]. Moreover, DCAF13 overexpression in breast- and lung cancer is significantly associated with low survival rates, and therefore has the potential to be used as a tumor biomarker [11,14]. However, the function of CRL4 in ovarian cancer as well as the underlying molecular mechanism are currently elusive. Here we report that DCAF13 is a novel CRL4 adaptor that is prominently expressed in human ovarian cancer and is associated with poor prognosis. DCAF13 knockout inhibited the proliferation and migration of ovarian cancer cells in vitro and the growth of xenografted tumors in vivo . In addition, we demonstrated that DCAF13 regulated ovarian cancer cell proliferation and migration by affecting the ubiquitination of the Fraser extracellular matrix complex subunit 1 (FRAS1) and activating the focal adhesion kinase (FAK) signaling pathway. Our findings provide new biomarker options and give rise to new potential strategies for targeted molecular therapy for ovarian cancer. Results DCAF13 overexpression is positively correlated with histological grade and overall survival of ovarian cancer. To investigate the possible involvement of DCAF13 in ovarian cancer, we conducted H&E staining and immunohistochemical analyses on ovarian cancer tissue microarrays, aiming to determine the DCAF13 expression level. The tissue microarrays included 8 normal ovarian tissue samples and 152 ovarian cancer tissue samples from patients aged 20–75 years (mean, 48 years) (Fig. 1A). Based on the staining intensity and positivity rate, we divided the stained ovarian tissues into negative (−), positive (+), moderate (++) and strong positive (+++) classes (Fig. 1B). The positive rates of DCAF13 staining in these different ovarian cancer type tissues, such as serous adenocarcinoma, mucinous adenocarcinoma, endometrial carcinoma, and clear cell carcinoma were 98.9% (87/88), 97.4% (37/38), 100.0% (17/17), and 100.0% (3/3), respectively, while the strong positive rates were as high as 95.5% (84/88), 84.2% (32/38), 100.0% (17/17), and 100.0% (3/3), respectively (Table 1). However, there were no positive DCAF13 staining in normal ovarian tissue (0%; Table 1). We found that DCAF13 protein expression was higher in ovarian cancer tissues compared to normal ovarian tissues ( P < 0.05, Fig. 1C). To assess the correlation between DCAF13 protein expression and ovarian cancer progression, we analyzed the relationship between its overexpression and the clinicopathological features of ovarian cancers. The rate of DCAF13 positivity was significantly higher in grade III ovarian cancers than that in grade I/II ovarian cancers ( P = 0.008; Table 2 ) . Similarly, we found that the strong positive rate for DCAF13 protein expression was significantly higher in serous adenocarcinomas than in non-serous adenocarcinomas ( P = 0.001; Table 2). It was also higher in pathological stage II disease than in pathological stage I disease ( P < 0.001; Table 2). Based on the IHC scores, we found that DCAF13 protein expression was higher in serous and endometrioid adenomas compared to normal ovarian tissues (Fig. 1D). The subsequent analysis of tissue microarray data revealed that patients exhibiting elevated DCAF13 expression experienced a lower overall survival rate compared to those with lower DCAF13 expression ( P = 0.001, Fig. 1E). Human ovarian cancer tissues and adjacent tissues were also collected from patients at the hospital. Western blot analysis further showed that DCAF13 protein expression in ovarian cancer tissues was higher than in paracancerous tissues (Fig. 1F). In addition, western blot revealed that DCAF13 was highly expressed in the ovarian cancer cell lines A2780, OVCAR-3, and HO8910 (Fig. 1G). These results indicate that human ovarian cancer tissues have high expression levels of DCAF13 protein and that the DCAF13 expression intensity is related to the survival rate of patients and pathological type of the disease. DCAF13 mediates ovarian cancer cell proliferation, colony formation, and migration To further clarify the role of DCAF13 in ovarian cancer, we employed CRISPR/Cas9 technology to delete DCAF13 in the ovarian cancer cell lines OVCAR-3, A2780, and HO8910. We generated several DCAF13-partial-knockout cell lines and used immunoblotting to confirm the DCAF13 knockout efficacy (Fig. 2A and Supplementary Fig. S1A). Cell proliferation assay showed that partial DCAF13 knockout resulted in a consistent and significant decrease in cell proliferation (Fig. 2B). Colony formation assays demonstrated that the deletion of DCAF13 resulted in a decreased number of formed colonies (Fig. 2C). Similarly, the expression of p-Histone H3, Ki67 and p-AKT, i.e., established markers of cell proliferation, was significantly reduced in DCAF13-deficient cells based on both immunoblotting and immunofluorescence (Fig. 2A and 2D, Supplementary Fig. S1B and S1C). In addition, the scratch and transwell experiments revealed that the DCAF13 deletion significantly inhibited ovarian cancer cell migration (Fig. 2E and 2F). Corroboratively, qRT-PCR data showed that DCAF13 deletion decreased the expression of the cell migration markers Twist1 and N- cadherin in OVCAR-3 and A2780 cells (Fig. 2G). These findings support the crucial role that DCAF13 plays in promoting the proliferation and migration of ovarian cancer cells. DCAF13 deletion causes cell cycle arrests. We further investigated the mechanism by which DCAF13 deletion inhibits ovarian cancer cells. Flow cytometry demonstrated that DCAF13-deleted cells were halted in the G1 phase of their cell cycle (Fig. 3A). Cell cycle regulation is closely associated with DNA damage and senescence [15,16]. Western blotting further showed that expression of the cyclin-dependent kinase inhibitors P21 and P27 and the DNA damage marker p-H2AX were increased in DCAF13-deleted cells, indicating that DNA damage occurred in ovarian cancer cells as a consequence to the DCAF13 deletion (Fig. 3B and Supplementary Fig. S1D). Immunofluorescence results also showed that the expression of P21 and p-H2AX protein was increased in DCAF13-deleted cells (Fig. 3C), which was consistent with the western blot results (Fig. 3B). Furthermore, qRT-PCR results revealed that DCAF13 deletion boosted the expression of p53 and the p53 -downstream gene MDM2 , whereas the expression of CDK2 , cdc25A , and Abl was decreased, indicating that the DNA damage mechanism was activated at the G1/S checkpoint (Fig. 3D). These findings imply that DCAF13 deletion causes cell cycle arrest. DCAF13 deletion inhibits tumor growth in vivo . To determine the impact of DCAF13 on ovarian cancer cell proliferation in vivo , we subcutaneously transplanted equal numbers of WT or DCAF13-deleted ovarian cancer cells into the left and right flanks of nude mice respectively and measured the tumor volume during xenograft development. Tumor size and weight were lower in nude mice transplanted with DCAF13-deleted ovarian cancer cells compared to the wild-type group (Fig. 4A-C). qRT-PCR results revealed that expression of the cell cycle-related genes p27 , p53 , MDM2 , and CDK2 were altered in DCAF13-deficient tumor tissues (Fig. 4D), which was consistent with the in vitro results (Fig.3D). Immunohistochemistry results revealed that protein expression of the cell proliferation markers p-Histone H3 and Ki67 were decreased, whereas expression of the apoptosis marker cleaved caspase-3 was increased in DCAF13-deleted tumor tissue (Fig. 4E). Western blotting revealed that protein levels of p-Histone H3, p-AKT, and p-PI3K were decreased in the DCAF13-deleted group (Fig. 4F and Supplementary Fig. S1E). These results indicated that DCAF13 deletion inhibited tumor proliferation in vivo . CRL4 DCAF13 regulates cell proliferation by targeting FRAS1 for polyubiquitination and degradation To identify the specifically targeted substrate of DCAF13, we isolated the DCAF13-associated protein complex in HEK293T cells through tandem affinity purification followed by mass spectrometry analysis (Fig. 5A). Coomassie brilliant blue staining showed that proteins were pulled down with IgG and FLAG antibodies (Fig. 5B). The mass spectrometry results showed that DCAF13 potentially interacts with FRAS1 (Fig. 5C). FRAS1 is an extracellular matrix protein and plays a significant role in tumor invasion and migration[17-20]. In agreement with the mass spectrometry results, co-immunoprecipitation assays demonstrated that DCAF13 interacts directly with FRAS1(Fig. 5D). To identify the interaction domain of DCAF13 with FRAS1, we used two DCAF13 truncations, in which either the conserved SOF or WD domains of DCAF13 were deleted and found that both the DCAF13 SOF △ and WD △ truncations interacted with FRAS1, suggesting that both of these two domains are involved in the interaction (Supplementary Fig. S1F). Given that DCAF13 functions as a substrate receptor of CRL4 E3 ubiquitin ligase, we tried to analyze whether FRAS1 acts as a substrate of CRL4 E3 ubiquitin ligase. We examined the association between FRAS1 and DDB1, the linker protein of CRL4 E3 ubiquitin ligase, and found that FRAS1 also directly interacts with DDB1, and DDB1 overexpression strengthened the interaction between FRAS1 and DCAF13(Fig. 5E), suggesting that FRAS1 could form complexes with CRL4 E3 ligase. To further determine whether FRAS1 could be ubiquitinated by CRL4 DCAF13 E3 ligase, we found that levels of FRAS1 polyubiquitination significantly increased after DCAF13 or DDB1 overexpression (Fig. 5F), indicating that CRL4 DCAF13 E3 ligase targeted FRAS1 for polyubiquitination. Furthermore, we examined the degradation rates of FRAS1 by using the protein synthesis inhibitor cycloheximide (CHX). The FRAS1 protein was mostly degraded upon CHX treatment but was stabled in DCAF13-deficient cells (Fig. 5G and Supplementary Fig. S2A). Moreover, when DCAF13 was depleted by siRNAs, FRAS1 protein significantly increased (Fig.5 H). ROC1, a component of the CRL4 E3 ubiquitin ligase, was depleted with siRNA oligos, which similarly increased FRAS1 expression (Supplementary Fig. S2B). These results indicated that the CRL4 DCAF13 E3 ligase targets FRAS1 for ubiquitination and proteasomal degradation. To demonstrate that the observed decrease in DCAF13-deficient ovarian cancer cell proliferation was due to DCAF13 dependent expression of FRAS1, we performed FRAS1 knockdown experiments by using RNA interference. The qRT-PCR results showed that FRAS1 mRNA was successfully suppressed in OVCAR-3 DCAF13 knockout cells(Fig. 5I). Cell counting assays and transwell assays showed that silencing FRAS1 partially rescued ovarian cancer cell proliferation and migration defect caused by DCAF13 deficiency(Fig. 5J-5K). To further explore the relationship between DCAF13 and FRAS1, we constructed a stable wild-type ovarian cancer cell line overexpressing DCAF13 using the Lenti-X VSVG lentivirus packaging system. According to the qRT-PCR results, FRAS1 expression was reduced in DCAF13-overexpressing cells (Fig. 5L and Supplementary Fig. S2C). We then overexpressed DCAF13 in DCAF13-deletion cells, which also decreased FRAS1 expression (Fig. 5M and Supplementary Fig. S2D). The proliferative capacity was also rescued in cells overexpressing DCAF13 (Fig. 5N), confirming that DCAF13 is crucial for cellular proliferation. These findings demonstrated that CRL4 DCAF13 regulates the proliferation and migration of ovarian cancer cells by targeting FRAS1 for polyubiquitination and degradation. CRL4 DCAF13 -mediated FRAS1/FAK signaling pathway is necessary for ovarian cancer cell proliferation Kiyozumi D et al study showed that FRAS1 contains RGD-motif capable for mediating binding to integrins[21]. Focal adhesion kinase (FAK) is the key tyrosine kinase in the integrin signaling pathway[22]. FAK is a cytoplasmic protein tyrosine kinase that is highly expressed and overactivated in many advanced solid tumors and is also associated with tumor growth and metastasis[23]. Thus, we hypothesized that FRAS1 may affect ovarian cancer cell proliferation and migration by regulating the FAK signaling pathway. We tested if FRAS1 affects the phosphorylation level of FAK, which indicate the activities of FAK signaling pathway, and found that overexpression of HA-FRAS1 significantly decreased p-FAK (Fig. 6A and Supplementary Fig. S2E), while FRAS1 was silenced, p-FAK significantly increased (Fig. 6C and Supplementary Fig. S2F), indicating that FRAS1 regulating the activity of the FAK signaling pathway. Cell proliferation assay showed that overexpression FRAS1 inhibited ovarian cancer cell proliferation (Fig. 6B).These results showed that FRAS1 negatively regulated FAK signaling pathway. The previous data showed that DCAF13 silence resulted in the increase of FRAS1 (Fig. 5H). We tested if DCAF13 affects the activity of FAK signaling pathway via FRAS1, and found that the level of p-FAK was significantly reduced in DCAF13-deficient ovarian cancer OVCAR-3 and A2780 cells (Fig. 6D, and Supplementary Fig. S2G-S2H). p-FAK expression was also reduced in DCAF13-deficient tumor tissue (Fig. 6E and Supplementary Fig. S2I). Moreover, the silence of FRAS1 in DCAF13 deficient ovarian cancer cell partially rescued the expression level of p-FAK, and p-AKT which are the downstream activity indicators of FAK signaling pathway (Fig. 6F and Supplementary Fig. S2J). These results demonstrated that DCAF13 regulates FAK signaling pathway via FRAS1. To examine if FAK signaling pathway regulates ovarian cancer cell proliferation and migration, we silenced the expression of FAK using RNA interference. The expression of FAK was successfully silenced (Fig. 6G and Supplementary Fig. S2K). The silence of FAK significantly inhibited ovarian cancer cell proliferation and migration (Fig. 6H and 6I). Moreover, we treated ovarian cancer cells with the FAK inhibitor defactinib (Def) at concentrations ranging from 0.001 to 10 μM, and found that with the treatment of Def, the level of p-FAK was significantly decreased, indicating that Def inhibited the activity of FAK signaling pathway (Fig. 6J and Supplementary Fig. S2L). Def also inhibited ovarian cancer cell proliferation and migration (Fig. 6K-6L). These results indicated that FAK signaling pathway regulates ovarian cancer cell proliferation and migration. In summary, in wild-type ovarian cancer cell, the CRL4 DCAF13 ubiquitin ligase complex target FRAS1 for polyubiquitination and degradation, resulting in activation of the FAK signaling pathway, thus promoting ovarian cancer cell proliferation and migration (Fig. 6M). Discussion Our study demonstrates that DCAF13 plays a critical role in ovarian cancer carcinogenesis. Previous studies have revealed that the increase of DCAF13 expression in breast cancer and hepatocellular carcinoma leads to poor prognosis [10-12]. In this research, we found that DCAF13 affects ovarian cancer, indicating that it might play a role in promoting pan-cancer development. Based on our findings, it appears that the deletion of DCAF13 suppresses both the proliferation and migration of ovarian cancer cells, which is consistent with previous findings in breast cancer [24]. Detailedly, DCAF13 deletion led to cell cycle arrest and enhanced expression of P21, P27, and p-H2AX. These results suggest that cell cycle arrest could be related to DNA damage, confirming the results of previous bioinformatic analyses [12,13]. In addition, our research revealed that DCAF13 not only promotes cancer cell proliferation but also contributes to cell migration and senescence. More importantly, we elucidated a novel molecular mechanism in which the CRL4 DCAF13 E3 ligase regulated the FAK signaling pathway by the ubiquitin-mediating degradation of FRAS1. MLN4924 is a selective inhibitor of CUL neddylation, a prerequisite for the activity of Cullin (CUL)-RING E3 ligase (CRL4 E3 ubiquitin ligase). MLN4924 exhibits potent toxicity and side effects [25]. Therefore, there is a need to identify superior target proteins to develop potential therapeutic drugs. DCAF13, a CRL4 E3 ubiquitin ligase substrate-binding protein, is more specific than CRL because of its targeting specificity, which theoretically would reduce side effects. DCAF13 might influence tumor cell proliferation via the PI3K–PTEN and P53 pathways [11]. However, our findings suggest that DCAF13 could also affect the FAK signaling pathway through FRAS1. Previous research has suggested that the FAK signaling pathway could be affected by ubiquitin ligases, potentially contributing to tumor occurrence and metastasis [26,27]. Our research revealed that DCAF13, the key protein in the CRL4 complex, might have an impact on the proliferation and migration of ovarian cancer cells by activating the FAK signaling pathway, further confirming this assumption. Website prediction analysis identified potential ubiquitin modification sites in FRAS1. Furthermore, our study revealed that the CRL4 complex affects the ubiquitination of FRAS1. Co-immunoprecipitation and ubiquitin co-immunoprecipitation assays revealed that DDB1 and DCAF13, the pivotal components of CRL4, modulate the ubiquitination of FRAS1. Moreover, FRAS1 affects the biological phenotype of cells via ubiquitin-mediated degradation. Our results, which involved the overexpression and knockdown of FRAS1, revealed its effect on the proliferation and migration of ovarian cells. Recent research indicated that mutations in FRAS1 might cause Fraser syndrome, a rare chromosomal disease with cryptic malformations and multiple organ hypoplasia [28]. FRAS1 has also been associated with various cancers, and its silencing suppresses the migration and invasion of non-small-cell lung cancer cells [29] and promotes liver metastasis in gastric cancer [30]. FRAS1 has also been identified as a promising diagnostic marker for endometrial carcinoma [31] and it is implicated in ovarian cancer resistance to carboplatin [18]. Moreover, FRAS1 expression in renal clear cell carcinoma tissues are significantly higher than those in normal tissues. Patients with reduced FRAS1 expression in tumors show an increased incidence of metastasis and a poor prognosis, highlighting it as a prospective target for treatment and a valuable prognostic biomarker for clear cell carcinoma [17]. Our results similarly showed that FRAS1 expression was increased after DCAF13 knockout, supporting these results. The effect of the FAK signaling pathway on both cell migration and proliferation has been consistently validated in numerous studies. In ovarian, breast, and gastric cancers, FAK is overactivated and promotes cell proliferation and migration [32-34]. The FAK protein is considered a potential target for anti-cancer drugs and is overexpressed in ovarian cancer [35-36]. We found that the FAK signaling pathway is suppressed after DCAF13 knockout in ovarian cancer cells. Moreover, the capacity of ovarian cancer cells to proliferate and migrate was reduced after DCAF13 knockout, which might also be connected to suppression of the FAK signaling pathway. Previous studies have shown that FRAS1 knockdown in lung cancer cells inhibits the FAK signaling pathway [29]. Our results differed from this, which could be due to tissue specificity or transcriptional differences. In addition, our results showed that the FAK protein was dephosphorylated, indicating that FRAS1 affects FAK phosphorylation. FAK activity increases after its phosphorylation, and this protein participates in multiple signaling pathways, such as PI3K/AKT and MAPK/ERK, to regulate cell growth and affect tumor occurrence and migration. Our results showed that FRAS1 affects ovarian cancer cell proliferation and migration through the FAK/PI3K/AKT signaling pathway, which is consistent with the aforementioned view. FAK has Y397 and Y925 phosphorylation sites, but only Y397, a common phosphorylation site, was detected in our experiment, whereas the other phosphorylation sites could be detected individually in follow-up experiments to explore how FRAS1 affects FAK dephosphorylation, highlighting the significant involvement of DCAF13 in the development of ovarian cancer. However, there are currently no available data on the effect of the DCAF13-FRAS1-FAK pathway on the proliferation of ovarian cancer cells. Thus, our findings indicate that DCAF13 is a promising target for ovarian cancer therapy. Materials And Methods References to supplementary tables and figures are indicated with prefix ‘S’. A comprehensive list of antibodies used in this study is presented in Table S1. Cell culture and stable cell line generation Human ovarian cancer cell lines A2780, C13, ES-2, HO8910, OVCAR-3, and SKOV3 were purchased the American Type Culture Collection (ATCC, Manassas, VA, USA). Human normal ovarian epithelial cell line IOSE was supplied by Heng-Yu Fan, Zhejiang University [37]. Cells were grown in DMEM (Gibco | Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco | Thermo Fisher Scientific) and 1% penicillin-streptomycin (Gibco | Thermo Fisher Scientific) at 37 ℃ in humidified atmosphere composed of 5% CO 2 and 95% air (standard culture conditions). DCAF13-deficient cells were established using CRISPR/Cas9 technology. The guide RNA sequences used for targeting human DCAF13 were: human DCAF13 - 1: 5’- AGCGGGACAGCAGTGAGCCC-3’; human DCAF13 - 2: 5’-GATGTGGATTACTCTCCCAC-3’. The construction of DCAF13-deficient cell lines was previously described [11]. Cell proliferation and colony formation assays A total of 1×10 5 cells were seeded per well in a 6-wells plate (Corning, NY, USA) (n = 3 per group). The cells were counted by hemocytometer at 24, 48, and 72 hours after seeding. Cell count was plotted as a function of time after seeding. The colony formation assay was conduct on soft agar. Six-wells plates were coated with 1.5 mL of 0.5% agar (Sigma-Aldrich, St. Louis, MO, USA) base layer. Subsequently, a suspension of 2 ×10 3 cells in 1.5 mL of 0.35% top agar was carefully added. To provide nutrients to the cells, 2 mL of cell culture medium was transferred onto the top layer twice a week. After a period of 3 weeks following plating, colonies were stained with 0.1% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) dissolved in PBS and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Scratch and Transwell assays A total of 5×10 5 cells were seeded in 6-wells plates in medium supplemented with 10% FBS. Once cells reached 90% confluence, the monolayer was scraped with a with 10-µL pipette tip across the center of each well and the cells were washed once with PBS. Next, 2 mL of fresh serum-free medium was added to each well to starve the cells. The plates were imaged immediately after scratching and washing (baseline) and at 24 hours using an inverted phase contrast microscope (CKX53, Olympus, Tokyo, Japan). The extent of cell migration was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and data are presented as percentage of cell-occupied area in the scratch channel at 24 hours versus baseline. Migration experiments were conducted in 24-wells plates using Transwell chambers equipped with 8-μm pore filters (Corning, NY, USA). Cells (1×10 4 ) were resuspended in 300 μL of FBS-free medium and transferred into the upper chamber. Next, 500 μL of medium containing 10% FBS was added to the lower chamber. After 24 hours, cells were fixed in methanol for 5 minutes. Stationary cells in the upper chamber were removed with cotton swabs. The cells that had migrated were stained with hematoxylin and quantified using ImageJ software (National Institutes of Health). Western blotting Protein from cells and tissues was extracted using RIPA lysis buffer (Beyotime Biotechnology, Haimen, China) and quantified with a bicinchoninic acid assay (BCA assay kit; Beyotime Biotechnology). 20 μg protein per well was separated using SDS-PAGE, transferred to PVDF membranes (Merck | Millipore, Burlington, MA, USA), and blocked using 5% powdered milk for 1 hour at RT. PVDF membranes were incubated overnight at 4 °C with primary antibodies against proteins of interest. Subsequently, the samples were incubated with a secondary antibody, specifically an anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology, Danvers, MA, USA). The resulting bands were then visualized using an enhanced chemiluminescence detection kit (Merck | Millipore). Data acquisition was carried out using an Imager 680 (Amersham | GE Healthcare, Chicago, IL, USA). Image analysis and quantification using ImageJ software (National Institutes of Health). Co-immunoprecipitation assay Cells were lysed in cell lysis buffer for Western and IP (Beyotime Biotechnology). Cell lysates were spiked with 50 μL of Protein A/G magnetic beads (MCE Magnetic, Mianyang, China) and washed 3 × with 400 μL binding/washing buffer (1 × PBS + 0.5% Tween-20). The corresponding primary antibody was incubated with magnetic beads at 4 ℃ for 4 hours, and subsequently the cell lysates were transferred into antibody-magnetic bead complex solution and incubated overnight at 4 ℃. The beads were thoroughly washed 5 × with a binding/washing buffer, followed by suspension in 1 × loading buffer, and heated at 95°C for 5 minutes. The resulting samples were then analyzed by Western blot. Protein Identification Analysis This experiment was performed using 293T cells. DCAF13 was overexpressed by transfecting the DCAF13-FLAG plasmid into 293T cells. When the cells grew to 80%, the cells were collected and lysed in cell lysis buffer for Western and IP (Beyotime Biotechnology). The cell lysates were supplemented with 50 μL of Protein A/G magnetic beads (MCE Magnetic) and subjected to three washes using 400 μL of binding/washing buffer (1 × PBS + 0.5% Tween-20). Sequently, the corresponding primary antibody was incubated with the magnetic beads 4 hours. Following this, the cell lysates were transferred into a solution containing the antibody-magnetic bead complex and incubated overnight at 4 ℃. The beads were thoroughly washed five times with a binding/washing buffer, followed by suspension in 1 × loading buffer, and heated at 95°C for 5 minutes. Control experiments using IgG antibodies. Proteins were separated using SDS-PAGE. The gel was stained with Coomassie Brilliant Blue for 30 minutes and destained by deionized water to remove background. The obtained samples were subjected to protein identification using LC-MS/MS by APT Biotechnology (Shanghai, China). Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from cultured cells or murine tumor tissues using TRIzol reagent (Invitrogen | Thermo Fisher Scientific) (n = 3 per group). Extracted RNA was converted to cDNA through reverse transcription using Prime Script RT Reagent Kit (Takara Bio, Shiga, Japan). Real-time PCR analysis was performed with TB Green Master Mix Kit (Takara Bio) on a Realplex 2 PCR System (Eppendorf, Hamburg, Germany). The mRNA levels of each gene were standardized to the expression levels of the housekeeping gene β-actin. Primer information is presented in Table S2. Plasmids and RNA interference Expression constructs coding for mouse Dcaf13 cDNA (Flag-DCAF13), Flag-DCAF13 SOF ∆ , Flag-DCAF13 WD ∆ , Flag-DDB1, and Myc-Ub plasmids were kindly provided by Dr. Heng-Yu Fan [38]. This protein is characterized by the presence of seven WD40 repeats at its N terminus and a SOF1 domain located at the C terminus. Human FRAS1 cDNA was cloned by Miaoling Biotechnology (Wuhan, China). FRAS1 is a remarkably conserved protein consisting of 1976 amino acid residues, with a molecular weight of 217 kDa. A total of 2×10 5 cells were seeded in six-well plates for 24h. Lipofectamine RNAiMAX reagent (Invitrogen) was used for siRNA transfection. After 48 hours of transfection (final siRNA concentration 80 nM), the cells were collected and analyzed by qRT-PCR or Western blot to assess interference efficiency. The siRNA sequences are listed in Table S3. Flow cytometry analysis Cells (1×10 6 ) were fixed with 70% ethanol for 24 hours. After centrifugation and a dual washing step with PBS, the cells were resuspended in 500 μL of PI + RNase staining buffer (BD Biosciences, Franklin Lakes, NJ, USA) and incubated at 37 ℃ in the dark for 30 minutes. Cells were assayed by flow cytometry (model flow cytometer; BD Biosciences). Data were analyzed using ModFit software (Verity Software House, Topsham, ME, USA). Mouse xenograft models All animal experiments were approved by the Jiaxing University’s institutional review board (registration no. JUMC2020-069). Specific pathogen free female BALB/c nude mice, aged 6-8 weeks, were obtained from Jiangsu Jicui Yaokang Biotechnology Co. (Nanjing, China). Animals were housed in a room with 12-hour light/dark cycles in individually ventilated cages with ad libitum access to sterilized food and water. The animals were treated in accordance with institutional guidelines and the National Institute of Health Guidelines for the Care and Use of Laboratory Animals (8 th edition). The mice were randomly assigned to three groups and anesthetized with ether. Subcutaneously, One group was injected with wild-type ovarian cancer cells in the right dorsal flank (n = 6/group), and the other two groups was injected with DCAF13-deleted cells (single bolus of 5 × 10 6 cells in PBS) in the right dorsal flank (n = 6/group). The tumor size of the mice was measured with a caliper every 2 - 3 days. The tumor volume was calculated using the formula: (width) 2 × height × 0.523 [39]. Nude mice were killed by cervical dislocation. A tumor diameter of > 15 mm constituted a human endpoint. Resected tumor tissue was fixed in 4% paraformaldehyde or stored at -80 ℃ until further use. Histochemistry and immunohistochemistry Paraffin-embedded human tissue samples from ovarian cancer tissue and paracancerous tissues were provided by the Affiliated Hospital of Jiaxing University (approval no. LS2020-148). Human ovarian cancer tissue microarrays were purchased from Shanghai Outdo Biotech Co. (Shanghai, China). Clinical information was provided by Shanghai Outdo Biotech Co. Method for analyzing experimental data generated by immunohistochemical experiments on tissue chips: 1. Staining intensity score: 0 points (negative), 1 points (1+), 2 points (2+), 3 points (3+). 2. Staining positive rate: 0 points (negative), 1 point (1-25%), 2 points (26%-50%), 3 points (51-75%), 4 points (76%-100%). 3. Grouping of high-low expression analysis: The product of "staining intensity score" and "staining positive rate score" was used as the total score for grouping, =6 was divided into high antibody expression group. Fixed mouse-derived tumor tissue was thawed, embedded in paraffin, sliced into 5-μm thick sections (Leica, Wetzlar, Germany), deparaffinized, and stained with hematoxylin and eosin (H&E). For immunohistochemistry (IHC), the deparaffinized sections underwent 10-minute incubation in 0.3% H 2 O 2 . Antigen retrieval was performed using 10 mM sodium citrate (pH = 6.0) for 15 minutes. Subsequently, the sections were incubated overnight at 4 °C with primary antibodies against Ki67 (1:400), p-histone H3 (1:200), and cleaved caspase-3 (1:200) (Table S1). Next, a biotinylated and peroxidase-conjugated secondary antibody was applied for 30 minutes (1:400, Cell Signaling Technology). The sections were counterstained utilizing a Vectastain ABC kit and a 3,3′-diaminobenzidine peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA). Immunofluorescence staining Cells (5×10 4 ) were seeded in 24-well plates, fixed with 4% paraformaldehyde for 30 minutes, and blocked using 5% bovine serum albumin for 1 hour. Subsequently, the cells were incubated with primary antibodies against Ki67 (1:400), p21 (1:800), and p-H2AX (1:400) (Table S1). Primary antibodies were removed with a single washing step, and cells were incubated with secondary antibodies labeled with Alexa488 or Alexa594 (Abcam, Cambridge, UK). Next, cells were counterstained with DAPI (Beijing Solarbio Science & Technology Co., Beijing, China). Digital images were captured with a confocal laser scanning microscope (FV3000, Olympus). Statistical analysis GraphPad Prism (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. Mean ± standard deviation represented the data and their differences. Samples with n < 8 were subjected to analysis using nonparametric tests. The normal distribution of data was assessed using ANOVA. The correlation between DCAF13 expression and clinicopathological characteristics was assessed using both the Chi-square test and Fisher's exact tests. Using the Kaplan-Meier method, survival rates after tumor removal were calculated, and differences in survival curves were evaluated using the Log-rank test. Additionally, a multivariate survival analysis using the Cox proportional hazard regression model was performed, integrating all relevant traits found in the univariate survival study. A P-value of < 0.05 was considered statistically significant. Declarations Acknowledgements We extend our gratitude to Yuan-Yuan Gao, Jing-Ya Zhong, Jing-Jian Dong, and Li-Li Shi for their invaluable technical support. We also acknowledge Dr. Kun-Liang Guan for providing the CRISPR/Cas9 plasmid and Dr. Heng-Yu Fan for the IOSE cells. This research was made possible through the generous support of the Key Laboratory of Medical Electronics and Digital Health of Zhejiang Province and Engineering Research Center of Intelligent Human Health Situation Awareness of Zhejiang Province, Jiaxing University, 314001, China. This work was supported by the Jiaxing talent pioneer innovation team, Jiaxing. Funding This work was supported by grants from National Natural Science Foundation of China (31871402) and The Natural Science Foundation of Zhejiang Province (LY21H160047, LGD21H160003, LQ23C070001, LY17H160060, Z20H160031, LGF20H160031, LGD22H030004). This work was supported by grants from Zhejiang Provincial Foreign Expert Grant (12.2018). This work was supported by Jiaxing Key Laboratory for Photonanomedicine and Experimental Therapeutics (12.2019). This work was supported by the Dutch Cancer Foundation (KWF, project 10666) and The Top-level Talent Project of Zhejiang Province. This work was supported by the Jiaxing talent pioneer innovation team (6.2021). Author contributions W-WP, Z-JZ, and S-QC were responsible for the overall conception and design of this experiment. W-WP and Z-JZ collated and summarized the experimental results and wrote the manuscript. Z-YT and X-MW participated in most of the experimental procedures in this study. J-YZ and Z-YW participated in primer design. Q-QS, Y-XH, and Q-YZ were involved in the management and sampling of experimental animals. H-YH was involved in the immunohistochemical experiments. X C, X Z, and A-JL participated in the collection of clinicopathological specimens. C-WX, S-BL, X-CZ, Y-JG, A-JL, and MH revised the final manuscript. All the authors participated in the analysis of the results and provided critical input. Conflict of interest The authors declare no competing interests. Ethics statement All clinical samples used in this study were approved by the Human Research Ethics Committee of the Affiliated Hospital of Jiaxing University (approval number:LS2021-KY-292). All patients provided written informed consent before enrollment. All archived samples were approved by the Institutional Review Board of Jiaxing University. All animal experiments involved in this study were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals following approval by the Laboratory Animal Ethics Committee of Jiaxing University (approval number: JUMC2021-151). Data availability No new datasets were generated during the current study. 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EMBO J 37(18):e98981. https://doi.org/10.15252/embj.201898981 Moroishi T, Hayashi T, Pan WW, Fujita Y, Holt MV, Qin J, et al (2016) The Hippo Pathway Kinases LATS1/2 Suppress Cancer Immunity. Cell 167(6):1525-1539. e17. https://doi.org/10.1016/j.cell.2016.11.005 Tables Table 1. Detailed pathological types and grades of ovarian cancer from the patient tissue microarray. Ovarian cancer type Case Positive n (%) P Strongly positive n (%) P (n) +++ ++ + − Normal ovarian tissue 8 0 (0) 0 (0) 0 (0) 8 (100.0%) 0 (0) < 0.0001 0 (0) < 0.0001 Serous adenocar-cinoma 88 71 (80.7) 13 (14.8%) 3 (3.4%) 1 (1.1%) 87 (98.9%) 84 (95.5%) Mucinous adenocar-cinoma 38 24 (63.2) 8 (21.1%) 5 (13.2%) 1 (2.6%) 37 (97.4%) 32 (84.2%) Endome-trioid adenoma 17 13 (76.5%) 4 (23.5%) 0 (0) 0 (0) 17 (100.0%) 17 (100.0%) Clear cell carcinoma 3 3 (100.0%) 0(0) 0 (0) 0 (0) 3 (100.0%) 3 (100.0%) Table 2. Correlation between DCAF13 expression and clinicopathological characteristics Variables DCAF13 expression Total χ 2 P Low High Age (years) 0.54 0.464 ≤ 50 38 27 65 > 51 36 33 69 null T stage 2.20 0.137 T1/T2 27 15 42 T3 47 46 93 TNM stage 2.20 0.137 Ι/II 27 15 42 III/IV 47 46 93 null N stage 2.05 0.152 N0 61 44 105 N1 13 17 30 null M stage 0.52 0.471 M0 64 50 114 M1 10 11 21 null Grade 7.08 0.008 I/II 24 11 35 III 31 44 75 Pathological subtype 11.8 0.001 Non-serous adenocarcinoma 45 19 64 Serous adenocarcinoma 29 42 71 Pathological subtype 19.3 0 I 52 24 76 II 13 34 47 Supplementary Files SupplementalFigure1.jpg SupplementalFigure2.jpg Supplementaldata.docx Cite Share Download PDF Status: Published Journal Publication published 05 Oct, 2024 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted Editorial decision: Major Revision 28 May, 2024 Reviewers agreed at journal 01 May, 2024 Reviewers invited by journal 01 May, 2024 Editor assigned by journal 23 Apr, 2024 First submitted to journal 20 Apr, 2024 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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1","display":"","copyAsset":false,"role":"figure","size":20740130,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression pattern of DCAF13 in ovarian cancer tissues and ovarian cancer cells.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e H\u0026amp;E staining and immunohistochemistry of DCAF13 protein expression in ovarian cancer tissue microarrays. \u003cstrong\u003eB\u003c/strong\u003e Immunohistochemistry for DCAF13 protein expression in normal ovarian tissue and ovarian cancer tissue in tissue microarray. The results were divided into negative (-), moderate (++) and strong positive (+++) staining pattern. \u003cstrong\u003eC\u003c/strong\u003eStatistical analysis of DCAF13 expression in normal ovarian tissue and ovarian cancer tissue of tissue microarray, *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. \u003cstrong\u003eD\u003c/strong\u003e Statistical analysis of DCAF13 expression in different pathological types of ovarian cancer tissue microarray, *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. \u003cstrong\u003eE\u003c/strong\u003e Kaplan-Meier analysis of cumulative survival rate of ovarian cancer tissue microarray, log-rank statistical test, \u003cem\u003eP\u003c/em\u003e= 0.001. \u003cstrong\u003eF\u003c/strong\u003e Western blot (upper panel) and quantification (lower panel) of DCAF13 protein expression in patient-derived ovarian cancer tissues and paracancerous tissues (NT represents paracancerous tissue, n= 2; CT refers to cancerous tissue, n= 5). \u003cstrong\u003eG\u003c/strong\u003e Western blot (upper panel) and quantification (lower panel) of DCAF13 expression in ovarian cancer cells (OVCAR-3, A2780, HO8910, SKOV3, ES-2, and C13), and immortalized mouse ovarian surface epithelium (IOSE). GAPDH was used as the loading control.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4539524/v1/a4996d4c3138fa80e47585b2.png"},{"id":59124024,"identity":"3c4b21fe-61a1-459e-9c6d-e5ccdaa9d475","added_by":"auto","created_at":"2024-06-26 15:20:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":20035406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDCAF13 deletion inhibits ovarian cancer cell proliferation, clone formation, and metastatic ability.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Detection of DCAF13 knockout efficiency in OVCAR-3, A2780 and HO8910 by immunoblotting. Two independent clones (22# and 15# in OVCAR-3; 18# and 9# in A2780; 6# and 18# in HO8910) are shown. \u003cstrong\u003eB\u003c/strong\u003e Growth curve of DCAF13 knockout ovarian cancer cells in OVCAR-3, A2780 and HO8910. This experiment was replicated three times. The error bars represent SD. Two-way ANOVA test was applied. ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003eC\u003c/strong\u003e Soft agar assay detection colony formation in DCAF13-deleted OVCAR-3, A2780 and HO8910 cells. Data are presented as mean ± SD. ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003eD\u003c/strong\u003e Immunofluorescence analysis of Ki67 (green) levels in WT and DCAF13-deficient OVCAR-3 and A2780 cells. Nuclei are counterstained with DAPI (blue). Scale bar, 20 μm. \u003cstrong\u003eE\u003c/strong\u003e Wound-healing assay for the migration ability of WT and DCAF13-deleted cells. Data are presented as mean ± SD for n = 3 per cell line. Student’s t-test was applied. **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. Scale bar, 50 μm. \u003cstrong\u003eF\u003c/strong\u003e Representative images (left panel) and quantification (right panel) of Transwell assay results showed that DCAF13 deletion inhibits ovarian cancer cell migration. This experiment was replicated three times. The error bars represent SD. **,\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, two-way ANOVA test. Scale bar, 50 μm. \u003cstrong\u003eG\u003c/strong\u003e qRT-PCR detection of cell migration-related genes in WT and DCAF13-deleted cells in OVCAR-3 and A2780. The error bars represent SD. Student’s t-test was applied. **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4539524/v1/1021cab8904d5ef5708c986b.png"},{"id":59124023,"identity":"a2104d10-dae0-4140-ac1e-405383876e5e","added_by":"auto","created_at":"2024-06-26 15:20:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8302172,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDCAF13 deletion affects the cell cycle and promotes cell senescence.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Flow cytometry was employed to analyze the changes in the cell cycle in DCAF13-deleted OVCAR-3 and A2780 cells. **,\u003cem\u003e P \u003c/em\u003e\u0026lt; 0.01 (nonparametric test). \u003cstrong\u003eB\u003c/strong\u003e Western blot analysis of the expression of P21, P27 and p-H2AX in WT and DCAF13-deleted OVCAR-3 and A2780 cells. \u003cstrong\u003eC\u003c/strong\u003e Representative images (left panel) and quantification (right panel) of immunofluorescence analysis of P21 (green) and p-H2AX (red) levels in WT and DCAF13-deleted OVCAR-3 and A2780 cells. Cells were counterstained with DAPI (blue). Scale bar, 20 μm. **,\u003cem\u003e P \u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 \u003cstrong\u003eD\u003c/strong\u003e qRT-PCR detection of cell cycle related genes in WT and DCAF13-deleted cells in OVCAR-3 and A2780. The error bars represent SD. Student’s t-test was applied. **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4539524/v1/d2f13ebc6f6e6ef211bd8e3a.png"},{"id":59124029,"identity":"63ecbf60-40dc-460a-98e8-994e277abd91","added_by":"auto","created_at":"2024-06-26 15:20:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":19546381,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDCAF13 deletion inhibits tumor growth \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Tumor size of HO8910 and OVCAR-3 wild-type and DCAF13 deletion ovarian cancer cells in nude mice. 5×10\u003csup\u003e6\u003c/sup\u003e cells were injected subcutaneously into flank of nude mice (n = 6). Some nude mice failed to form tumors. Scale bar, 10 mm. \u003cstrong\u003eB\u003c/strong\u003e Deletion of DCAF13 in HO8910 and OVCAR-3 cells inhibits tumor weight \u003cem\u003ein vivo\u003c/em\u003e. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, according to two-way ANOVA. \u003cstrong\u003eC\u003c/strong\u003e Tumor growth curve of nude mice. Tumor volume in each group was measured every 2-3 days. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, according to two-way ANOVA. \u003cstrong\u003eD\u003c/strong\u003e qRT-PCR detection of cell cycle-related factors in tumors. The error bars represent SD. Student’s t-test was applied. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003eE\u003c/strong\u003e Histochemistry and immunohistochemical analysis of p-Histone H3, Ki67 and Cleaved Caspase-3 in wild-type and DCAF13 deletion xenografts. Scale bar, 50 μm. Objective magnification 20x. \u003cstrong\u003eF\u003c/strong\u003e Western blot analysis of DCAF13, p-Histone H3, p-AKT, AKT, p-PI3K and PI3K expression in WT and DCAF13-deleted HO8910 and OVCAR-3 mouse tumor tissue. GAPDH was used as control.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4539524/v1/f57218f358e96720823f7e22.png"},{"id":59125370,"identity":"e5c2d407-08ce-4f1e-97fe-d238ba2c6a15","added_by":"auto","created_at":"2024-06-26 15:28:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":10721482,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFRAS1 is the key target of DCAF13 in ovarian cancer cells. A \u003c/strong\u003eSchematic representation of immunoprecipitation combined with liquid chromatography-mass spectrometry.\u003cstrong\u003e B \u003c/strong\u003eCoomassie brilliant blue staining of proteins pulled with IgG antibody and FLAG antibody. FLAG-DCAF13 plasmid was overexpressed in this experiment. \u003cstrong\u003eC\u003c/strong\u003e The base peak of protein sample by mass spectrometry. \u003cstrong\u003eD\u003c/strong\u003e Co-immunoprecipitation results showing FRAS1 interacts with DCAF13. 293T cells transfected with plasmids encoding the indicated proteins were lysed and subjected to IP with anti-FLAG or anti-HA beads. Input lysates were immunoblotted with antibodies against HA and FLAG. \u003cstrong\u003eE\u003c/strong\u003eCo-immunoprecipitation results showing FRAS1 interacts with DDB1 and DCAF13. The overexpression of DDB1 enhances the interaction between FRAS1 and DCAF13.\u003cstrong\u003eF\u003c/strong\u003e Co-immunoprecipitation experiments showing overexpression of DCAF13 and DDB1 increase FRAS1 polyubiquitination. \u003cstrong\u003eG\u003c/strong\u003e The Cycloheximide (CHX)-chasing experiment revealed the FRAS1 stability. OVCAR-3 WT and DCAF13-deleted cells were transfected with HA-FRAS1 plasmid. 10 μM CHX was used to inhibit protein synthesis in OVCAR-3. At the specified time points after CHX treatment, the cells were lysed for immunoblotting. \u003cstrong\u003eH\u003c/strong\u003e Western blot shows the expression of FRAS1 in OVCAR-3 cells transfected with control siRNA (siNC), siDCAF13 or siFRAS1(upper panel) and relative quantitative analysis (lower panel). **,\u003cem\u003e P \u003c/em\u003e\u0026lt; 0.01; ns, \u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05. \u003cstrong\u003eI\u003c/strong\u003e mRNA expression level of FRAS1 in WT or DCAF13 deficient OVCAR-3 cells transfected with control siRNA (siNC) or siFRAS1 ***,\u003cem\u003e P \u003c/em\u003e\u0026lt; 0.001. \u003cstrong\u003eJ\u003c/strong\u003e FRAS1 deletion rescues cell proliferation defects in DCAF13 deficient OVCAR-3 cells. ***,\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001.\u003cstrong\u003e K \u003c/strong\u003eFRAS1 deletion rescues cell migration ability in OVCAR-3 DCAF13 KO cells. Representative images from the Transwell assay (left panel) and quantification analysis (right panel) were shown. *,\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ***,\u003cem\u003e P \u003c/em\u003e\u0026lt; 0.001.\u003cstrong\u003e L\u003c/strong\u003e Western blot results of DCAF13 protein expression (left panel) and qRT-PCR results of FRAS1 mRNA expression in wild-type OVCAR-3 transfected with mouse DCAF13 cells. *,\u003cem\u003e P\u003c/em\u003e\u0026lt; 0.05. \u003cstrong\u003eM\u003c/strong\u003e Western blot results of DCAF13 protein expression (left panel) and qRT-PCR results of FRAS1 mRNA expression in WT, DCAF13 KO and DCAF13 KO transfected with encoding mouse DCAF13 cells. ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003eN\u003c/strong\u003eOverexpression of DCAF13 rescues the ability of cell proliferation. ***,\u003cem\u003e P\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4539524/v1/9a5956249cb03e9f1b93679e.png"},{"id":59125369,"identity":"279a40d3-5efb-4706-93cc-a14e3047b1d4","added_by":"auto","created_at":"2024-06-26 15:28:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":8567845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFRAS1 affects ovarian cancer cell proliferation and migration by mediating the FAK signaling pathway.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Western blot detection FAK, p-FAK, HA protein expression in cells. OVCAR-3 cells were transfected with HA-FRAS1 vector and or with control empty vector. GAPDH was used as control. \u003cstrong\u003eB\u003c/strong\u003e Cell proliferation assay of overexpressed HA-FRAS1 and control ovarian cancer cells. ***,\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003eC\u003c/strong\u003e Western blot results showing the FAK signaling pathway was activated after interfering with FRAS1. GAPDH was used as control. \u003cstrong\u003eD\u003c/strong\u003e Western blot analysis of FAK and p-FAK expression in WT and DCAF13-deleted OVCAR-3 cells. GAPDH was used as control.\u003cstrong\u003e E\u003c/strong\u003e Western blot analysis of FAK and p-FAK expression in WT and DCAF13-deleted OVCAR-3 mouse tumor tissue.\u003cstrong\u003e F\u003c/strong\u003e Western blot analysis of p-FAK, p-ERK1/2 and p-AKT expression in WT, DCAF13-deleted OVCAR-3 cells, DCAF13 and FRAS1 double knockdown cells .\u003cstrong\u003e G \u003c/strong\u003eWestern blot results for FAK siRNA interference efficiency. \u003cstrong\u003eH\u003c/strong\u003e FAK silencing inhibiting ovarian cancer cell proliferation. ***,\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003eI \u003c/strong\u003eFAK deletion inhibiting cell migration of ovarian cancer cells. Representative images from the Transwell assay (upper panel) and quantification analysis (lower panel) were shown. **,\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.01. \u003cstrong\u003eJ\u003c/strong\u003e OVCAR-3 cells were exposed to increasing doses of the FAK inhibitor Defactinib (Def) for 24 h. Western blot analysis of FAK and p-FAK. \u003cstrong\u003eK \u003c/strong\u003eOvarian cancer cell proliferation, after treatment with 1 μM Defactinib for 24 h. ***,\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001.\u003cstrong\u003e L\u003c/strong\u003e Defactinib inhibiting cell migration of ovarian cancer cells. Representative images from the Transwell assay (upper panel) and quantification analysis (lower panel) were shown. **,\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.01. \u0026nbsp;\u003cstrong\u003eM\u003c/strong\u003e A working model explaining how DCAF13 promotes ovarian cancer cell proliferation and migration. In wild-type ovarian cancer cells, DCAF13 ubiquitin degrades the FRAS1 substrate and activates the FAK signaling pathway, thus promoting the proliferation and migration of ovarian cancer cells. When DCAF13 is knocked out, FRAS1 accumulation leads to inhibition of the FAK signaling pathway and suppression of proliferation and migration of ovarian cancer cells.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4539524/v1/d5238f912f6b48228cb45489.png"},{"id":66098720,"identity":"31eaed93-5f61-4b20-b721-722530abcf41","added_by":"auto","created_at":"2024-10-07 16:23:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":108106370,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4539524/v1/e72d36dd-616f-46ee-8a1c-829ec7df98eb.pdf"},{"id":59124025,"identity":"e5f8b7a5-ddcd-4baa-9920-61eaf389885c","added_by":"auto","created_at":"2024-06-26 15:20:04","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5047280,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539524/v1/232e7f34c185c027edf28871.jpg"},{"id":59124028,"identity":"335babf2-8fa6-4640-a778-726023ef7bb6","added_by":"auto","created_at":"2024-06-26 15:20:05","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3285011,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4539524/v1/ab57352a02fb63afa7dd2bb7.jpg"},{"id":59125368,"identity":"b7002b43-87e1-4428-8f3d-56d8265144e9","added_by":"auto","created_at":"2024-06-26 15:28:04","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":32555,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaldata.docx","url":"https://assets-eu.researchsquare.com/files/rs-4539524/v1/99779c4c8a8e3b3591eeb829.docx"}],"financialInterests":"","formattedTitle":"DCAF13 promotes ovarian cancer progression by activating FRAS1-mediated FAK signaling pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOvarian cancer is a common malignant tumor of the female reproductive system and has a high mortality rate among gynecological cancers\u0026nbsp;[1]. Ovarian cancer typically proceeds undetected and lacks specific clinical symptoms at the early stages\u0026nbsp;[2]. Accordingly, more than 70% of\u0026nbsp;patients with ovarian cancer\u0026nbsp;are diagnosed in the late stages, which is associated with extensive metastasis and poor prognosis\u0026nbsp;[3]. Currently, surgical resection combined with chemotherapy is considered the standard treatment for ovarian cancer. The 5-year survival rate for the patients with advanced disease is less than 40%\u0026nbsp;[4]. With prolonged chemotherapy cycles and multiple relapses, the sensitivity of most ovarian cancers to chemotherapeutic drugs decreases gradually, ultimately leading to therapeutic recalcitrance\u0026nbsp;[5]. Therefore, there is an urgent need to understand the pathogenesis of ovarian cancer to improve prognosis and reduce mortality.\u003c/p\u003e\n\u003cp\u003eCullin-RING ubiquitin ligase 4 (CRL4) is an important member of the E3 ubiquitin ligase family. The CRL4 E3 ubiquitin ligase consists of three components, the scaffold protein CUL4, RING finger protein RBX1 (also known as ROC1 or HRT1), and\u0026nbsp;DNA damage binding protein 1 (DDB1)\u0026nbsp;[6]. Numerous studies have shown that the CRL4 complex plays an important role in ovarian cancer\u0026nbsp;[7-9]. DDB1- and CUL4-associated factor 13 (DCAF13), a substrate recognition protein\u0026nbsp;for the CRL4 E3 ubiquitin ligase complex,\u0026nbsp;is highly amplified in breast, liver, and lung cancer\u0026nbsp;[10-14]. An early discovery by our group suggested that DCAF13 is involved in\u0026nbsp;cell cycle regulation, apoptosis, tumor-related signaling pathways, and other processes in breast cancer, thus promoting breast cancer cell proliferation\u0026nbsp;[11]. Moreover, DCAF13 overexpression in breast- and lung cancer\u0026nbsp;is significantly associated with low survival rates, and therefore has the potential to be used as a tumor biomarker\u0026nbsp;[11,14]. However, the function of CRL4 in ovarian cancer as well as the underlying molecular mechanism are currently elusive.\u003c/p\u003e\n\u003cp\u003eHere we report\u0026nbsp;that DCAF13\u0026nbsp;is a novel CRL4 adaptor that is prominently expressed in human ovarian cancer and\u0026nbsp;is associated with poor prognosis. DCAF13 knockout inhibited the proliferation and migration of ovarian cancer cells \u003cem\u003ein vitro\u003c/em\u003e and the growth of xenografted tumors \u003cem\u003ein vivo\u003c/em\u003e. In addition, we demonstrated that DCAF13 regulated ovarian cancer cell proliferation and migration by affecting the ubiquitination of the Fraser extracellular matrix complex subunit 1 (FRAS1) and activating the focal adhesion kinase (FAK) signaling pathway. Our findings provide new biomarker options and give rise to new potential strategies for targeted molecular therapy for ovarian cancer.\u003c/p\u003e\n"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDCAF13 overexpression is positively correlated with histological grade and overall\u003c/strong\u003e \u003cstrong\u003esurvival of ovarian cancer.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the possible involvement of DCAF13 in ovarian cancer, we conducted H\u0026amp;E staining and immunohistochemical analyses on ovarian cancer tissue microarrays, aiming to determine the DCAF13 expression level.\u0026nbsp;The tissue microarrays included\u0026nbsp;8 normal ovarian tissue samples and 152 ovarian cancer tissue samples from patients aged 20\u0026ndash;75 years (mean, 48 years) (Fig. 1A).\u0026nbsp;Based on the staining intensity and positivity rate,\u0026nbsp;we divided\u0026nbsp;the stained ovarian tissues\u0026nbsp;into negative (\u0026minus;), positive (+), moderate (++) and strong positive (+++) classes (Fig. 1B).\u0026nbsp;The positive rates of DCAF13 staining in these different ovarian cancer type tissues, such as serous adenocarcinoma, mucinous adenocarcinoma, endometrial carcinoma, and clear cell carcinoma were 98.9% (87/88), 97.4% (37/38), 100.0% (17/17), and 100.0% (3/3), respectively, while the strong positive rates were as high as 95.5% (84/88), 84.2% (32/38), 100.0% (17/17), and 100.0% (3/3), respectively (Table 1). However, there were no positive DCAF13 staining in normal ovarian tissue (0%; Table 1).\u0026nbsp;We found that DCAF13 protein expression was higher in ovarian cancer tissues compared to normal ovarian tissues\u0026nbsp;(\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Fig. 1C).\u003c/p\u003e\n\u003cp\u003eTo assess the correlation between DCAF13 protein expression and ovarian cancer progression, we analyzed the relationship between its overexpression and the clinicopathological features of ovarian cancers. The rate of DCAF13 positivity was significantly higher in grade\u0026nbsp;III\u0026nbsp;ovarian cancers than that in grade\u0026nbsp;I/II\u0026nbsp;ovarian cancers (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e= 0.008; Table 2\u003cstrong\u003e)\u003c/strong\u003e. Similarly, we found that the strong positive rate for DCAF13 protein expression was significantly higher in\u0026nbsp;serous adenocarcinomas than in non-serous adenocarcinomas\u0026nbsp;(\u003cem\u003eP\u003c/em\u003e = 0.001; Table 2).\u0026nbsp;It was also higher in pathological stage\u0026nbsp;II\u0026nbsp;disease than\u0026nbsp;in pathological stage\u0026nbsp;I\u0026nbsp;disease (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001; Table 2).\u0026nbsp;Based on the IHC scores, we found that DCAF13 protein expression was higher in serous\u0026nbsp;and endometrioid adenomas compared to normal ovarian tissues\u0026nbsp;(Fig. 1D).\u0026nbsp;The subsequent analysis of tissue microarray data revealed that patients exhibiting elevated DCAF13 expression experienced a lower overall survival rate compared to those with lower DCAF13 expression (\u003cem\u003eP\u003c/em\u003e= 0.001, Fig. 1E).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHuman ovarian cancer\u0026nbsp;tissues and adjacent tissues were also collected from patients at the hospital. Western blot analysis further showed that DCAF13 protein expression in ovarian cancer tissues was higher than in\u0026nbsp;paracancerous tissues (Fig. 1F). In addition, western blot revealed that DCAF13 was highly expressed in\u0026nbsp;the ovarian cancer cell\u0026nbsp;lines A2780, OVCAR-3, and HO8910 (Fig. 1G).\u0026nbsp;These results indicate that human ovarian cancer tissues have high expression levels of DCAF13 protein and that the DCAF13 expression intensity is related to the survival rate of patients and pathological type of the disease.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDCAF13 mediates ovarian cancer cell proliferation, colony formation, and migration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further clarify the role of DCAF13 in ovarian cancer, we employed CRISPR/Cas9 technology to delete DCAF13 in the ovarian cancer cell lines OVCAR-3, A2780, and HO8910. We generated several DCAF13-partial-knockout cell lines and used immunoblotting to confirm the DCAF13 knockout efficacy (Fig. 2A and Supplementary Fig. S1A). Cell proliferation assay showed that partial DCAF13 knockout resulted in a consistent and significant decrease in cell proliferation (Fig. 2B). Colony formation assays demonstrated that the deletion of DCAF13 resulted in a decreased number of formed colonies (Fig. 2C). Similarly, the expression of p-Histone H3, Ki67 and p-AKT, i.e., established markers of cell proliferation, was significantly reduced in DCAF13-deficient cells based on both immunoblotting and immunofluorescence (Fig. 2A and 2D, Supplementary Fig. S1B and S1C). In addition, the scratch and transwell experiments revealed that the DCAF13 deletion significantly inhibited ovarian cancer cell migration (Fig. 2E and 2F). Corroboratively, qRT-PCR data showed that DCAF13 deletion decreased the expression of the cell migration markers \u003cem\u003eTwist1\u003c/em\u003e and \u003cem\u003eN-\u003c/em\u003e\u003cem\u003ecadherin\u003c/em\u003e in OVCAR-3 and A2780 cells (Fig. 2G). These findings support the crucial role that DCAF13 plays in promoting the proliferation and migration of ovarian cancer cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDCAF13 deletion causes cell cycle arrests.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe further investigated the mechanism by which DCAF13 deletion inhibits ovarian cancer cells. Flow cytometry demonstrated that DCAF13-deleted cells were halted in the G1 phase of their cell cycle (Fig. 3A). Cell cycle regulation is closely associated with DNA damage and senescence [15,16]. Western blotting further showed that expression of the cyclin-dependent kinase inhibitors P21 and P27 and the DNA damage marker p-H2AX were increased in DCAF13-deleted cells, indicating that DNA damage occurred in ovarian cancer cells as a consequence to the DCAF13 deletion (Fig. 3B and Supplementary Fig. S1D). Immunofluorescence results also showed that the expression of P21 and p-H2AX protein was increased in DCAF13-deleted cells (Fig. 3C), which was consistent with the western blot results (Fig. 3B). Furthermore, qRT-PCR results revealed that DCAF13 deletion boosted the expression of \u003cem\u003ep53\u003c/em\u003e and the \u003cem\u003ep53\u003c/em\u003e-downstream gene \u003cem\u003eMDM2\u003c/em\u003e, whereas the expression of \u003cem\u003eCDK2\u003c/em\u003e, \u003cem\u003ecdc25A\u003c/em\u003e, and \u003cem\u003eAbl\u003c/em\u003e was decreased, indicating that the DNA damage mechanism was activated at the G1/S checkpoint (Fig. 3D). These findings imply that DCAF13 deletion causes cell cycle arrest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDCAF13 deletion inhibits tumor growth \u003cem\u003ein vivo\u003c/em\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the impact of DCAF13 on ovarian cancer cell proliferation \u003cem\u003ein vivo\u003c/em\u003e, we subcutaneously transplanted equal numbers of WT or DCAF13-deleted ovarian cancer cells into the left and right flanks of nude mice respectively and measured the tumor volume during xenograft development. Tumor size and weight were lower in nude mice transplanted with DCAF13-deleted ovarian cancer cells compared to the wild-type group (Fig. 4A-C). qRT-PCR results revealed that expression of the cell cycle-related genes \u003cem\u003ep27\u003c/em\u003e, \u003cem\u003ep53\u003c/em\u003e, \u003cem\u003eMDM2\u003c/em\u003e, and \u003cem\u003eCDK2\u003c/em\u003e were altered in DCAF13-deficient tumor tissues (Fig. 4D), which was consistent with the \u003cem\u003ein vitro\u003c/em\u003e results (Fig.3D). Immunohistochemistry results revealed that protein expression of the cell proliferation markers p-Histone H3 and Ki67 were decreased, whereas expression of the apoptosis marker cleaved caspase-3 was increased in DCAF13-deleted tumor tissue (Fig. 4E). Western blotting revealed that protein levels of p-Histone H3, p-AKT, and p-PI3K were decreased in the DCAF13-deleted group (Fig. 4F and Supplementary Fig. S1E). These results indicated that DCAF13 deletion inhibited tumor proliferation \u003cem\u003ein vivo\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRL4\u003csup\u003eDCAF13\u003c/sup\u003e regulates cell proliferation by targeting FRAS1 for polyubiquitination and degradation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the specifically targeted substrate of DCAF13, we isolated the DCAF13-associated protein complex in HEK293T cells through tandem affinity purification followed by mass spectrometry analysis (Fig. 5A). Coomassie brilliant blue staining showed that proteins\u0026nbsp;were pulled down with IgG\u0026nbsp;and FLAG antibodies (Fig. 5B). The mass spectrometry results showed that DCAF13 potentially interacts with FRAS1 (Fig. 5C). FRAS1 is an extracellular matrix protein and plays a significant role in tumor invasion and migration[17-20]. In agreement with the mass spectrometry results,\u0026nbsp;co-immunoprecipitation assays demonstrated that DCAF13 interacts directly with FRAS1(Fig. 5D). To identify the interaction domain of DCAF13 with FRAS1, we used two DCAF13 truncations, in which either the conserved SOF or WD domains of DCAF13 were deleted and found that both the DCAF13 SOF\u003csup\u003e△\u003c/sup\u003e and WD\u003csup\u003e△\u003c/sup\u003e truncations interacted with FRAS1, suggesting that both of these two domains are involved in the interaction (Supplementary Fig. S1F). Given that DCAF13 functions as a substrate receptor of CRL4 E3 ubiquitin ligase, we tried to analyze whether FRAS1 acts as a substrate of CRL4 E3 ubiquitin ligase. We examined the association between FRAS1 and DDB1, the linker protein of CRL4 E3 ubiquitin ligase, and found that FRAS1 also directly interacts with DDB1, and DDB1 overexpression strengthened the interaction between FRAS1 and DCAF13(Fig. 5E), suggesting that FRAS1 could form complexes with CRL4 E3 ligase. To further determine whether FRAS1 could be ubiquitinated by CRL4\u003csup\u003eDCAF13\u003c/sup\u003e E3 ligase, we found that levels of FRAS1 polyubiquitination significantly increased after DCAF13 or DDB1 overexpression (Fig. 5F), indicating that CRL4\u003csup\u003eDCAF13\u003c/sup\u003e E3 ligase targeted FRAS1 for polyubiquitination. Furthermore, we examined the degradation rates of FRAS1 by using the protein synthesis inhibitor cycloheximide (CHX). The FRAS1 protein was mostly degraded upon CHX treatment but was stabled in DCAF13-deficient cells (Fig. 5G and Supplementary Fig. S2A). Moreover, when DCAF13 was depleted by siRNAs, FRAS1 protein significantly increased (Fig.5 H). ROC1, a component of the CRL4 E3 ubiquitin ligase, was depleted with siRNA oligos, which similarly increased FRAS1 expression (Supplementary Fig. S2B). These results indicated that the CRL4\u003csup\u003eDCAF13\u003c/sup\u003e E3 ligase targets FRAS1 for ubiquitination and proteasomal degradation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo demonstrate that the observed decrease in DCAF13-deficient ovarian cancer cell proliferation was due to DCAF13 dependent expression of \u003cem\u003eFRAS1,\u003c/em\u003e we performed FRAS1 knockdown experiments by using RNA interference. The qRT-PCR results showed that \u003cem\u003eFRAS1\u003c/em\u003e mRNA was successfully suppressed in OVCAR-3 \u003cem\u003eDCAF13\u003c/em\u003e knockout cells(Fig. 5I). Cell counting assays and transwell assays showed that silencing \u003cem\u003eFRAS1\u003c/em\u003e partially rescued ovarian cancer cell proliferation and migration defect caused by DCAF13 deficiency(Fig. 5J-5K). To further explore the relationship between DCAF13 and FRAS1, we constructed a stable wild-type ovarian cancer cell line overexpressing DCAF13 using the Lenti-X VSVG lentivirus packaging system. According to the qRT-PCR results, \u003cem\u003eFRAS1\u003c/em\u003e expression was reduced in DCAF13-overexpressing cells (Fig. 5L and Supplementary Fig. S2C). We then overexpressed DCAF13 in DCAF13-deletion cells, which also decreased FRAS1 expression (Fig. 5M and Supplementary Fig. S2D). The proliferative capacity was also rescued in cells overexpressing DCAF13 (Fig. 5N), confirming that DCAF13 is crucial for cellular proliferation. These findings demonstrated that CRL4\u003csup\u003eDCAF13\u003c/sup\u003e regulates the proliferation and migration of ovarian cancer cells by targeting FRAS1 for polyubiquitination and degradation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRL4\u003csup\u003eDCAF13\u003c/sup\u003e-mediated FRAS1/FAK signaling pathway is necessary for ovarian cancer cell proliferation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKiyozumi D\u0026nbsp;et al study showed\u0026nbsp;that FRAS1 contains\u0026nbsp;RGD-motif\u0026nbsp;capable for mediating binding to integrins[21]. Focal adhesion kinase (FAK) is the key tyrosine kinase in the integrin signaling pathway[22]. FAK is a cytoplasmic protein tyrosine kinase that is highly expressed and overactivated in many advanced solid tumors and is also associated with tumor growth and metastasis[23]. Thus, we hypothesized that FRAS1 may affect ovarian cancer cell proliferation and migration by regulating the FAK signaling pathway.\u003c/p\u003e\n\u003cp\u003eWe tested if FRAS1 affects the\u0026nbsp;phosphorylation level of\u0026nbsp;FAK, which indicate the activities of FAK signaling pathway, and found that\u0026nbsp;overexpression of HA-FRAS1 significantly decreased p-FAK\u0026nbsp;(Fig. 6A and\u0026nbsp;Supplementary Fig. S2E), while FRAS1 was silenced, p-FAK significantly increased\u0026nbsp;(Fig. 6C and\u0026nbsp;Supplementary Fig. S2F), indicating that FRAS1 regulating the activity of\u0026nbsp;the FAK signaling pathway. Cell proliferation assay showed that overexpression\u0026nbsp;FRAS1\u0026nbsp;inhibited ovarian cancer cell proliferation\u0026nbsp;(Fig. 6B).These results showed that FRAS1\u0026nbsp;negatively regulated FAK signaling pathway.\u003c/p\u003e\n\u003cp\u003eThe previous data showed that DCAF13 silence resulted in the increase of FRAS1 (Fig. 5H). We tested if DCAF13 affects the activity of FAK signaling pathway via FRAS1, and found that the level of p-FAK was significantly reduced in DCAF13-deficient ovarian cancer OVCAR-3 and A2780 cells (Fig. 6D, and\u0026nbsp;Supplementary Fig. S2G-S2H). p-FAK expression was also reduced in DCAF13-deficient tumor tissue (Fig. 6E and\u0026nbsp;Supplementary Fig. S2I). Moreover, the silence of FRAS1 in DCAF13 deficient ovarian cancer cell partially rescued the expression level of \u0026nbsp; p-FAK, and p-AKT which are the downstream activity indicators of FAK signaling pathway (Fig. 6F and\u0026nbsp;Supplementary Fig. S2J). These results demonstrated that DCAF13 regulates FAK signaling pathway via FRAS1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo examine if FAK signaling pathway regulates ovarian cancer cell proliferation and migration, we silenced the expression of FAK using RNA interference. The expression of FAK was successfully silenced (Fig. 6G and\u0026nbsp;Supplementary Fig. S2K). The silence of FAK significantly inhibited ovarian cancer cell proliferation and migration (Fig. 6H and 6I).\u0026nbsp;Moreover, we treated ovarian cancer cells with the FAK inhibitor defactinib (Def) at concentrations ranging from 0.001 to 10 \u0026mu;M, and found that with the treatment of Def, the level of p-FAK was significantly decreased, indicating that Def inhibited the activity of FAK signaling pathway (Fig. 6J and\u0026nbsp;Supplementary Fig. S2L). Def also inhibited ovarian cancer cell proliferation and migration (Fig. 6K-6L). These results indicated that FAK signaling pathway regulates ovarian cancer cell proliferation and migration.\u0026nbsp;In summary, in wild-type ovarian cancer cell, the CRL4\u003csup\u003eDCAF13\u003c/sup\u003e ubiquitin ligase complex target FRAS1 for polyubiquitination and degradation, resulting in activation of the FAK signaling pathway, thus promoting ovarian cancer cell proliferation and migration (Fig. 6M).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study demonstrates that DCAF13 plays a critical role in ovarian cancer carcinogenesis. Previous studies have revealed that the increase of DCAF13 expression in breast cancer and hepatocellular carcinoma leads to poor prognosis\u0026nbsp;[10-12]. In this research, we found that DCAF13 affects ovarian cancer, indicating that it might play a role in promoting pan-cancer development. Based on our findings, it appears that the deletion of DCAF13 suppresses both the proliferation and migration of ovarian cancer cells, which is consistent with previous findings in breast cancer [24]. Detailedly, DCAF13 deletion led to cell cycle arrest and enhanced expression of P21, P27, and p-H2AX. These results suggest that cell cycle arrest could be related to DNA damage, confirming the results of previous bioinformatic analyses\u0026nbsp;[12,13]. In addition, our research revealed that DCAF13 not only promotes cancer\u0026nbsp;cell proliferation but also contributes to cell migration and senescence. More importantly, we elucidated a novel molecular mechanism in which the CRL4\u003csup\u003eDCAF13\u0026nbsp;\u003c/sup\u003eE3 ligase\u0026nbsp;regulated\u0026nbsp;the FAK signaling pathway by\u0026nbsp;the ubiquitin-mediating degradation of FRAS1.\u003c/p\u003e\n\u003cp\u003eMLN4924 is a selective inhibitor of CUL neddylation, a prerequisite for the activity of Cullin (CUL)-RING E3 ligase (CRL4 E3 ubiquitin ligase). MLN4924 exhibits potent toxicity and side effects [25]. Therefore, there is a need to identify superior target proteins to develop potential therapeutic drugs. DCAF13, a CRL4 E3 ubiquitin ligase substrate-binding protein, is more specific than CRL because of its targeting specificity,\u0026nbsp;which theoretically would reduce side effects. DCAF13 might influence tumor cell proliferation via the PI3K\u0026ndash;PTEN and P53 pathways\u0026nbsp;[11]. However, our findings suggest that DCAF13 could also affect the FAK signaling pathway through FRAS1. Previous research has suggested that the FAK signaling pathway could be affected by ubiquitin ligases, potentially contributing to tumor occurrence and metastasis [26,27]. Our research revealed that DCAF13, the key protein in\u0026nbsp;the CRL4 complex, might have an impact on\u0026nbsp;the proliferation and migration of ovarian cancer cells by activating the FAK signaling pathway, further confirming this assumption.\u003c/p\u003e\n\u003cp\u003eWebsite prediction analysis identified potential ubiquitin modification sites in FRAS1. Furthermore, our study revealed that the CRL4 complex affects the ubiquitination of FRAS1. Co-immunoprecipitation and ubiquitin co-immunoprecipitation assays revealed that DDB1 and DCAF13, the pivotal components of CRL4, modulate the ubiquitination of FRAS1. Moreover, FRAS1 affects the biological phenotype of cells via ubiquitin-mediated degradation. Our results, which involved the overexpression and knockdown of FRAS1, revealed its effect on the proliferation and migration of ovarian cells. Recent research indicated that mutations in FRAS1 might cause Fraser syndrome, a rare chromosomal disease with cryptic malformations and multiple organ hypoplasia\u0026nbsp;[28]. FRAS1 has also been associated with various cancers, and its silencing suppresses\u0026nbsp;the migration and invasion of non-small-cell lung cancer cells [29] and promotes liver metastasis in gastric cancer [30]. FRAS1 has also been identified as a promising diagnostic marker\u0026nbsp;for endometrial carcinoma [31] and it\u0026nbsp;is implicated in ovarian cancer resistance to carboplatin\u0026nbsp;[18]. Moreover, FRAS1 expression in renal clear cell carcinoma\u0026nbsp;tissues are significantly higher than those in normal tissues. Patients with reduced FRAS1 expression in tumors show an increased incidence of metastasis and a poor prognosis, highlighting\u0026nbsp;it as a prospective target for treatment and a valuable prognostic biomarker for\u0026nbsp;clear cell carcinoma [17]. Our results similarly showed that FRAS1 expression was increased after\u0026nbsp;DCAF13 knockout,\u0026nbsp;supporting these results.\u003c/p\u003e\n\u003cp\u003eThe effect of the FAK signaling pathway on both cell migration and proliferation has been consistently validated in numerous studies. In ovarian, breast, and gastric cancers, FAK is overactivated and promotes cell proliferation and migration [32-34]. The FAK protein is considered a potential target for anti-cancer drugs and is overexpressed in ovarian cancer [35-36]. We found that the FAK signaling pathway is suppressed after DCAF13 knockout in ovarian cancer cells. Moreover, the capacity of ovarian cancer cells to proliferate and migrate was reduced after DCAF13 knockout, which might also be connected to suppression of the FAK signaling pathway. Previous studies have shown that FRAS1 knockdown in lung cancer cells inhibits the FAK signaling pathway [29]. Our results differed from this, which could be due to tissue specificity or transcriptional differences. In addition, our results showed that the FAK protein was dephosphorylated, indicating that FRAS1 affects FAK phosphorylation. FAK activity increases after its phosphorylation, and this protein participates in multiple signaling pathways, such as PI3K/AKT and MAPK/ERK, to regulate cell growth and affect tumor occurrence and migration. Our results showed that FRAS1 affects ovarian cancer cell proliferation and migration through the FAK/PI3K/AKT signaling pathway, which is consistent with the aforementioned view. FAK has Y397 and Y925 phosphorylation sites, but only Y397, a common phosphorylation site, was detected in our experiment, whereas the other phosphorylation sites could be detected individually in follow-up experiments to explore how FRAS1 affects FAK dephosphorylation, highlighting the significant involvement of DCAF13 in the development of ovarian cancer. However, there are currently no available data on the effect of the DCAF13-FRAS1-FAK pathway on the proliferation of ovarian cancer cells. Thus, our findings indicate that DCAF13 is a promising target for ovarian cancer therapy.\u0026nbsp;\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003eReferences to supplementary tables and figures are indicated with prefix \u0026lsquo;S\u0026rsquo;. A comprehensive list of antibodies used in this study is presented in Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture and stable cell line generation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman ovarian cancer cell lines A2780, C13, ES-2, HO8910, OVCAR-3, and SKOV3 were purchased the American Type Culture Collection (ATCC, Manassas, VA, USA). Human normal ovarian epithelial cell line IOSE was supplied by Heng-Yu Fan, Zhejiang University [37]. Cells were grown in DMEM (Gibco | Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco | Thermo Fisher Scientific) and 1% penicillin-streptomycin (Gibco | Thermo Fisher Scientific) at 37 ℃ in humidified atmosphere composed of 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% air (standard culture conditions).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDCAF13-deficient cells were established using CRISPR/Cas9 technology. The guide RNA sequences used for targeting human DCAF13 were:\u0026nbsp;human DCAF13 - 1: 5\u0026rsquo;- AGCGGGACAGCAGTGAGCCC-3\u0026rsquo;; human DCAF13 - 2: 5\u0026rsquo;-GATGTGGATTACTCTCCCAC-3\u0026rsquo;.\u0026nbsp;The construction of DCAF13-deficient cell lines was previously described\u0026nbsp;[11].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell proliferation and colony formation assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were seeded per well in a 6-wells plate (Corning, NY, USA) (n = 3 per group). The cells were counted by hemocytometer at 24, 48, and 72 hours after seeding. Cell count was plotted as a function of time after seeding.\u003c/p\u003e\n\u003cp\u003eThe colony formation assay was conduct on soft agar. Six-wells plates were coated with 1.5 mL of 0.5% agar (Sigma-Aldrich, St. Louis, MO, USA) base layer. Subsequently, a suspension of 2\u0026nbsp;\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells in 1.5 mL of 0.35% top agar was carefully added. To provide nutrients to the cells, 2 mL of cell culture medium was transferred onto the top layer\u0026nbsp;twice a week. After a period of 3 weeks following plating, colonies were stained with 0.1% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) dissolved in PBS and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScratch\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eand Transwell assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were seeded in 6-wells plates in medium supplemented with 10% FBS. Once cells reached 90% confluence, the monolayer was scraped with a with 10-\u0026micro;L pipette tip across the center of each well and the cells were washed once with PBS. Next, 2 mL of fresh serum-free medium was added to each well to starve the cells. The plates were imaged immediately after scratching and washing (baseline) and at 24 hours using an inverted phase contrast microscope (CKX53, Olympus, Tokyo, Japan). The extent of cell migration was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and data are presented as percentage of cell-occupied area in the scratch channel at 24 hours versus baseline.\u003c/p\u003e\n\u003cp\u003eMigration experiments were conducted in 24-wells plates using Transwell chambers equipped with 8-\u0026mu;m pore filters (Corning, NY, USA). Cells (1\u0026times;10\u003csup\u003e4\u003c/sup\u003e) were resuspended in 300 \u0026mu;L of FBS-free medium and transferred into the upper chamber. Next, 500 \u0026mu;L of medium containing 10% FBS was added to the lower chamber. After 24 hours, cells were fixed in methanol for 5 minutes. Stationary cells in the upper chamber were removed with cotton swabs. The cells that had migrated were stained with hematoxylin and quantified using ImageJ software (National Institutes of Health).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein from cells and tissues was extracted using RIPA lysis buffer (Beyotime Biotechnology, Haimen, China) and quantified with a bicinchoninic acid assay (BCA assay kit; Beyotime Biotechnology). 20 \u0026mu;g protein per well was separated using SDS-PAGE, transferred to PVDF membranes (Merck | Millipore, Burlington, MA, USA), and blocked using 5% powdered milk for 1 hour at RT. PVDF membranes were incubated overnight at 4 \u0026deg;C with primary antibodies against proteins of interest. Subsequently, the samples were incubated with a secondary antibody, specifically an anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology, Danvers, MA, USA). The resulting bands were then visualized using an enhanced chemiluminescence detection kit (Merck | Millipore). Data acquisition was carried out using an Imager 680 (Amersham | GE Healthcare, Chicago, IL, USA). Image analysis and quantification using ImageJ software (National Institutes of Health).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-immunoprecipitation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were lysed in cell lysis buffer for Western and IP (Beyotime Biotechnology). Cell lysates were spiked with 50 \u0026mu;L of Protein A/G magnetic beads\u0026nbsp;(MCE Magnetic, Mianyang, China) and washed 3 \u0026times; with 400 \u0026mu;L binding/washing buffer (1 \u0026times; PBS + 0.5% Tween-20). The corresponding primary antibody was incubated with magnetic beads at 4 ℃ for 4 hours, and subsequently the cell lysates were transferred into antibody-magnetic bead complex solution and incubated overnight at 4 ℃. The beads were thoroughly washed 5 \u0026times; with a binding/washing buffer, followed by suspension in 1 \u0026times; loading buffer, and heated at 95\u0026deg;C for 5 minutes. The resulting samples were then analyzed by Western blot.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein Identification Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis experiment was performed using 293T cells. DCAF13 was overexpressed by transfecting the DCAF13-FLAG plasmid into 293T cells. When the cells grew to 80%, the cells were collected and lysed in cell lysis buffer for Western and IP (Beyotime Biotechnology). The cell lysates were supplemented with 50 \u0026mu;L of Protein A/G magnetic beads (MCE Magnetic) and subjected to three washes using 400 \u0026mu;L of binding/washing buffer (1 \u0026times; PBS + 0.5% Tween-20). Sequently, the corresponding primary antibody was incubated with the magnetic beads 4 hours. Following this, the cell lysates were transferred into a solution containing the antibody-magnetic bead complex and incubated overnight at 4 ℃. The beads were thoroughly washed five times with a binding/washing buffer, followed by suspension in 1 \u0026times; loading buffer, and heated at 95\u0026deg;C for 5 minutes. Control experiments using IgG antibodies. Proteins were separated using SDS-PAGE. The gel was stained with Coomassie Brilliant Blue for 30 minutes and destained by deionized water to remove background.\u0026nbsp;The obtained samples were subjected to protein identification using LC-MS/MS by APT Biotechnology (Shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from cultured cells or murine tumor tissues using TRIzol reagent (Invitrogen | Thermo Fisher Scientific) (n = 3 per group). Extracted RNA was converted to cDNA through reverse transcription using Prime Script RT Reagent Kit (Takara Bio, Shiga, Japan). Real-time PCR analysis was performed with TB Green Master Mix Kit (Takara Bio) on a Realplex\u003csup\u003e2\u003c/sup\u003e PCR System (Eppendorf, Hamburg, Germany). The mRNA levels of each gene were standardized to the expression levels of the housekeeping gene \u0026beta;-actin. Primer information is presented in\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eTable S2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmids and RNA interference\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression constructs coding for mouse Dcaf13 cDNA (Flag-DCAF13), Flag-DCAF13 SOF\u003csup\u003e∆\u003c/sup\u003e, Flag-DCAF13 WD\u003csup\u003e∆\u003c/sup\u003e, Flag-DDB1, and Myc-Ub plasmids were kindly provided by Dr. Heng-Yu Fan [38]. This protein is characterized by the presence of seven WD40 repeats at its N terminus and a SOF1 domain located at the C terminus.\u0026nbsp;Human FRAS1 cDNA was cloned by Miaoling Biotechnology (Wuhan, China). FRAS1 is a remarkably conserved protein consisting of 1976 amino acid residues, with a molecular weight of 217 kDa.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA total of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were seeded in six-well plates for 24h. Lipofectamine RNAiMAX reagent (Invitrogen) was used for siRNA transfection. After 48 hours of transfection (final siRNA concentration 80 nM), the cells were collected and analyzed by qRT-PCR or Western blot to assess interference efficiency. The siRNA sequences are listed in Table S3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e) were fixed with 70% ethanol for 24 hours. After centrifugation and a dual washing step with PBS, the cells were resuspended in 500 \u0026mu;L of PI + RNase staining buffer (BD Biosciences, Franklin Lakes, NJ, USA) and incubated at 37 ℃ in the dark for 30 minutes. Cells were assayed by flow cytometry (model flow cytometer; BD Biosciences). Data were analyzed using ModFit software (Verity Software House, Topsham, ME, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMouse xenograft models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Jiaxing University\u0026rsquo;s institutional review board (registration no. JUMC2020-069).\u0026nbsp;Specific pathogen free female BALB/c nude mice, aged 6-8 weeks, were obtained from Jiangsu Jicui Yaokang Biotechnology Co. (Nanjing, China). Animals were housed in a room with 12-hour light/dark cycles in individually ventilated cages with ad libitum access to sterilized food and water. The animals were treated in accordance with institutional guidelines and the \u003cem\u003eNational Institute of Health Guidelines for the Care and Use of Laboratory Animals\u0026nbsp;\u003c/em\u003e(8\u003csup\u003eth\u003c/sup\u003e edition).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe mice were randomly assigned to three groups and anesthetized with ether. Subcutaneously, One group was injected with wild-type ovarian cancer cells in the right dorsal flank (n = 6/group), and the other two groups was injected with \u0026nbsp;DCAF13-deleted cells (single bolus of 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells in PBS) in the right dorsal flank (n = 6/group). The tumor size of the mice was measured with a caliper every 2 - 3 days. The tumor volume was calculated using the formula: (width)\u003csup\u003e2\u003c/sup\u003e \u0026times; height \u0026times; 0.523 [39]. Nude mice were killed by cervical dislocation. A tumor diameter of \u0026gt; 15 mm constituted a human endpoint. Resected tumor tissue was fixed in 4% paraformaldehyde or stored at -80 ℃ until further use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistochemistry and immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParaffin-embedded human tissue samples from ovarian cancer tissue and paracancerous tissues were provided by the Affiliated Hospital of Jiaxing University (approval no. LS2020-148).\u0026nbsp;Human ovarian cancer tissue microarrays were purchased from Shanghai Outdo Biotech Co. (Shanghai, China). Clinical information was provided by Shanghai Outdo Biotech Co.\u003c/p\u003e\n\u003cp\u003eMethod for analyzing experimental data generated by immunohistochemical experiments on tissue chips: 1. Staining intensity score: 0 points (negative), 1 points (1+), 2 points (2+), 3 points (3+). 2. Staining positive rate: 0 points (negative), 1 point (1-25%), 2 points (26%-50%), 3 points (51-75%), 4 points (76%-100%). 3. Grouping of high-low expression analysis: The product of \u0026quot;staining intensity score\u0026quot; and \u0026quot;staining positive rate score\u0026quot; was used as the total score for grouping, \u0026lt;6 was divided into low antibody expression group, \u0026gt;=6 was divided into high antibody expression group.\u003c/p\u003e\n\u003cp\u003eFixed mouse-derived tumor tissue was thawed, embedded in paraffin, sliced into 5-\u0026mu;m thick sections (Leica, Wetzlar, Germany), deparaffinized, and stained with hematoxylin and eosin (H\u0026amp;E). For immunohistochemistry (IHC), the deparaffinized sections underwent 10-minute incubation in 0.3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Antigen retrieval was performed using 10 mM sodium citrate (pH = 6.0) for 15 minutes. Subsequently, the sections were incubated overnight at 4 \u0026deg;C with primary antibodies against Ki67 (1:400), p-histone H3 (1:200), and cleaved caspase-3 (1:200) (Table S1). Next, a biotinylated and peroxidase-conjugated secondary antibody was applied for 30 minutes (1:400, Cell Signaling Technology). The sections were counterstained utilizing a Vectastain ABC kit and a 3,3\u0026prime;-diaminobenzidine peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells (5\u0026times;10\u003csup\u003e4\u003c/sup\u003e) were seeded in 24-well plates, fixed with 4% paraformaldehyde for 30 minutes, and blocked using 5% bovine serum albumin for 1 hour. Subsequently, the cells were incubated with primary antibodies against Ki67 (1:400), p21 (1:800), and p-H2AX (1:400)\u0026nbsp;(Table S1).\u0026nbsp;Primary antibodies were removed with a single washing step, and cells were incubated with secondary antibodies labeled with Alexa488 or Alexa594 (Abcam, Cambridge, UK). Next, cells were counterstained with DAPI (Beijing Solarbio Science \u0026amp; Technology Co., Beijing, China). Digital images were captured with a confocal laser scanning microscope (FV3000, Olympus).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGraphPad Prism (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. Mean\u0026nbsp;\u0026plusmn;\u0026nbsp;standard deviation represented the data and their differences. Samples with n \u0026lt; 8 were subjected to analysis using nonparametric tests. The normal distribution of data was assessed using ANOVA. The correlation between DCAF13 expression and clinicopathological characteristics was assessed using both the Chi-square test and Fisher\u0026apos;s exact tests. Using the Kaplan-Meier method, survival rates after tumor removal were calculated, and differences in survival curves were evaluated using the Log-rank test. Additionally, a multivariate survival analysis using the Cox proportional hazard regression model was performed, integrating all relevant traits found in the univariate survival study. A P-value of \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our gratitude to Yuan-Yuan Gao, Jing-Ya Zhong, Jing-Jian Dong, and Li-Li Shi for their invaluable technical support. We also acknowledge Dr. Kun-Liang Guan for providing the CRISPR/Cas9 plasmid and Dr. Heng-Yu Fan for the IOSE cells. This research was made possible through the generous support of the Key Laboratory of Medical Electronics and Digital Health of Zhejiang Province and Engineering Research Center of Intelligent Human Health Situation Awareness of Zhejiang Province, Jiaxing University, 314001, China. This work was supported by the Jiaxing talent pioneer innovation team, Jiaxing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from\u0026nbsp;National Natural Science Foundation of China (31871402) and The Natural Science Foundation of Zhejiang Province (LY21H160047, LGD21H160003, LQ23C070001, LY17H160060, Z20H160031, LGF20H160031, LGD22H030004).\u0026nbsp;This work was supported by grants from Zhejiang Provincial Foreign Expert Grant (12.2018). This work was supported by Jiaxing Key Laboratory for Photonanomedicine and Experimental Therapeutics (12.2019). This work was supported by the Dutch Cancer Foundation (KWF, project 10666) and The Top-level Talent Project of Zhejiang Province. This work was supported by the Jiaxing talent pioneer innovation team (6.2021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eW-WP, Z-JZ, and S-QC were responsible for the overall conception and design of this experiment. W-WP and Z-JZ collated and summarized the experimental results and wrote the manuscript. Z-YT and X-MW participated in most of the experimental procedures in this study. J-YZ and Z-YW participated in primer design. Q-QS,\u0026nbsp;Y-XH,\u0026nbsp;and Q-YZ were involved in the management and sampling of experimental animals. H-YH was involved in the immunohistochemical experiments. X C, X Z, and A-JL participated in the collection of clinicopathological specimens. C-WX, S-BL, X-CZ, Y-JG, A-JL,\u0026nbsp;and MH revised the final manuscript. All the authors participated\u0026nbsp;in the analysis of the results and provided critical input.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll clinical samples used in this study were approved by the Human Research Ethics Committee of the Affiliated Hospital of Jiaxing University (approval number:LS2021-KY-292).\u0026nbsp;All patients provided written informed consent before enrollment. All archived samples were approved by the Institutional Review Board of Jiaxing University.\u0026nbsp;All animal experiments involved in this study were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals following approval by the Laboratory Animal Ethics Committee of Jiaxing University (approval number: JUMC2021-151).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo new datasets were generated during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al (2021) Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. 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Oncogene 30(32):3522-3536. https://doi.org/10.1038/onc.2011.70\u003c/li\u003e\n\u003cli\u003eZhang YL, Zhao LW, Zhang J, Le R, Ji SY, Chen C, et al (2022) DCAF13 promotes pluripotency by negatively regulating SUV39H1 stability during early embryonic development. EMBO J 37(18):e98981. https://doi.org/10.15252/embj.201898981\u003c/li\u003e\n\u003cli\u003eMoroishi T, Hayashi T, Pan WW, Fujita Y, Holt MV, Qin J, et al (2016) The Hippo Pathway Kinases LATS1/2 Suppress Cancer Immunity. Cell 167(6):1525-1539. e17. https://doi.org/10.1016/j.cell.2016.11.005\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eDetailed pathological types and grades of ovarian cancer from the patient tissue microarray.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"654\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.145038167938932%\" rowspan=\"2\"\u003e\n \u003cp\u003eOvarian cancer type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.412213740458015%\"\u003e\n \u003cp\u003eCase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.51145038167939%\" colspan=\"4\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.297709923664122%\" rowspan=\"2\"\u003e\n \u003cp\u003ePositive n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.702290076335878%\" rowspan=\"2\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.229007633587786%\" rowspan=\"2\"\u003e\n \u003cp\u003eStrongly positive n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.702290076335878%\" rowspan=\"2\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.844036697247706%\"\u003e\n \u003cp\u003e(n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.547400611620795%\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.629969418960243%\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.629969418960243%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.34862385321101%\"\u003e\n \u003cp\u003e\u0026minus;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.145038167938932%\"\u003e\n \u003cp\u003eNormal\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eovarian\u0026nbsp;\u003c/p\u003e\n \u003cp\u003etissue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.412213740458015%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.755725190839694%\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.297709923664122%\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.297709923664122%\"\u003e\n \u003cp\u003e0\u0026nbsp;(0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003cp\u003e(100.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.297709923664122%\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.702290076335878%\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.229007633587786%\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.702290076335878%\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.145038167938932%\"\u003e\n \u003cp\u003eSerous adenocar-cinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.412213740458015%\"\u003e\n \u003cp\u003e88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.755725190839694%\"\u003e\n \u003cp\u003e71\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;(80.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.297709923664122%\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003cp\u003e(14.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.297709923664122%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003cp\u003e(3.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003cp\u003e(1.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.297709923664122%\"\u003e\n \u003cp\u003e87\u003c/p\u003e\n \u003cp\u003e(98.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.702290076335878%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.229007633587786%\"\u003e\n \u003cp\u003e84\u003c/p\u003e\n \u003cp\u003e(95.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.702290076335878%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.145038167938932%\"\u003e\n \u003cp\u003eMucinous adenocar-cinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.412213740458015%\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.755725190839694%\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;(63.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.297709923664122%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003cp\u003e(21.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.297709923664122%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003cp\u003e(13.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003cp\u003e(2.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.297709923664122%\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003cp\u003e(97.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.702290076335878%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.229007633587786%\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003cp\u003e(84.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.702290076335878%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.145038167938932%\"\u003e\n \u003cp\u003eEndome-trioid adenoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.412213740458015%\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.755725190839694%\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003cp\u003e(76.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.297709923664122%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003cp\u003e(23.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.297709923664122%\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.297709923664122%\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003cp\u003e(100.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.702290076335878%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.229007633587786%\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003cp\u003e(100.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.702290076335878%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.145038167938932%\"\u003e\n \u003cp\u003eClear cell carcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.412213740458015%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.755725190839694%\"\u003e\n \u003cp\u003e3\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(100.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.297709923664122%\"\u003e\n \u003cp\u003e0(0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.297709923664122%\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.297709923664122%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003cp\u003e(100.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.702290076335878%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.229007633587786%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003cp\u003e(100.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.702290076335878%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Correlation between DCAF13 expression and clinicopathological characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"546\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\" rowspan=\"2\"\u003e\n \u003cp\u003eVariables\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.007326007326007%\" colspan=\"2\"\u003e\n \u003cp\u003eDCAF13\u0026nbsp;expression\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\" rowspan=\"2\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" rowspan=\"2\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.51012145748988%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.05263157894737%\"\u003e\n \u003cp\u003eLow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.43724696356275%\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e0.464\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\"\u003e\n \u003cp\u003e\u0026le; 50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\"\u003e\n \u003cp\u003e\u0026gt; 51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n \u003cp\u003e69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\"\u003e\n \u003cp\u003enull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003eT stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e2.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e0.137\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\"\u003e\n \u003cp\u003eT1/T2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n 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width=\"13.91941391941392%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\"\u003e\n \u003cp\u003eIII/IV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n \u003cp\u003e93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\"\u003e\n \u003cp\u003enull\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003eN stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e2.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e0.152\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\"\u003e\n \u003cp\u003eN0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n 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width=\"8.608058608058608%\"\u003e\n \u003cp\u003e114\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\"\u003e\n \u003cp\u003eM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n 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width=\"15.567765567765568%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e7.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\"\u003e\n \u003cp\u003eI/II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\"\u003e\n \u003cp\u003eIII\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003ePathological subtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e11.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\"\u003e\n \u003cp\u003eNon-serous adenocarcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\"\u003e\n \u003cp\u003eSerous adenocarcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n \u003cp\u003e71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003ePathological subtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e19.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n \u003cp\u003e76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.23076923076923%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.567765567765568%\"\u003e\n \u003cp\u003eII\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.523809523809524%\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.483516483516482%\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.91941391941392%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ovarian cancer, DCAF13, FRAS1, CRL4 E3 ubiquitin ligase, FAK ","lastPublishedDoi":"10.21203/rs.3.rs-4539524/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4539524/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Cullin-RING ubiquitin ligase 4 (CRL4) is closely correlated with the incidence and progression of ovarian cancer. DDB1- and CUL4-associated factor 13 (DCAF13), a substrate-recognition protein in the CRL4 E3 ubiquitin ligase complex, is involved in the occurrence and development of ovarian cancer. However, its precise function and the underlying molecular mechanism in this disease remain unclear. In this study we confirmed that DCAF13 is highly expressed in human ovarian cancer and its expression is negatively correlated with the overall survival rate of patients with ovarian cancer. We then used CRISPR/Cas9 to knockout DCAF13 and found that its deletion significantly inhibited the proliferation, colony formation, and migration of human ovarian cancer cells. In addition, DCAF13 deficiency inhibited tumor proliferation in nude mice. Mechanistically, CRL4-DCAF13 was found to target Fraser extracellular matrix complex subunit 1 (FRAS1) for polyubiquitination and proteasomal degradation. FRAS1 was found to influence the proliferation and migration of ovarian cancer cell through induction of the focal adhesion kinase (FAK) signaling pathway. These findings collectively show that DCAF13 is an important oncogene which promotes tumorigenesis in ovarian cancer cells by mediating FRAS1/FAK signaling. Our findings pave the way to develop new potential targeted therapeutics for ovarian cancer treatment.","manuscriptTitle":"DCAF13 promotes ovarian cancer progression by activating FRAS1-mediated FAK signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-26 15:19:59","doi":"10.21203/rs.3.rs-4539524/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2024-05-28T11:03:20+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-05-01T13:57:48+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-01T13:49:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-23T04:20:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2024-04-21T03:46:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9f33ad35-6204-4894-a120-bacd62c0f028","owner":[],"postedDate":"June 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-07T16:09:08+00:00","versionOfRecord":{"articleIdentity":"rs-4539524","link":"https://doi.org/10.1007/s00018-024-05446-2","journal":{"identity":"cellular-and-molecular-life-sciences","isVorOnly":false,"title":"Cellular and Molecular Life Sciences"},"publishedOn":"2024-10-05 15:58:19","publishedOnDateReadable":"October 5th, 2024"},"versionCreatedAt":"2024-06-26 15:19:59","video":"","vorDoi":"10.1007/s00018-024-05446-2","vorDoiUrl":"https://doi.org/10.1007/s00018-024-05446-2","workflowStages":[]},"version":"v1","identity":"rs-4539524","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4539524","identity":"rs-4539524","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}