A Novel Role of the ASS1-CCDC6 Complex in Orchestrating Mitochondrial Dynamics to Suppress Lung Adenocarcinoma

A Novel Role of the ASS1-CCDC6 Complex in Orchestrating Mitochondrial Dynamics to Suppress Lung Adenocarcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A Novel Role of the ASS1-CCDC6 Complex in Orchestrating Mitochondrial Dynamics to Suppress Lung Adenocarcinoma Yongguang Tao, Wei Wang, Yao Long, QiDong Cai, Zhenyu Zhao, Can Cao, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8712733/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract The persistent high burden of lung cancer in China highlights a critical demand for the identification of new therapeutic targets and intervention approaches. Our initial integrative analysis of metabolomic and transcriptomic data revealed a previously uncharacterized tumor-suppressive mechanism mediated by CCDC6 in lung adenocarcinoma. We discovered an interaction between CCDC6 and ASS1, concomitant with marked reductions in citrulline and aspartate within tumor tissues. compared to the adjacent normal tissues. Additionally, co-stimulation with citrulline and aspartate induces ASS1 localization in the mitochondria ASS1 localized in the mitochondria, but the underlying mechanism remains unclear. This study aimed to delineate the dual tumor-suppressive actions of ASS1: firstly, through the recruitment of the deubiquitinase OTUD7A to remove ubiquitin chains from MFN1/2 and OPA1, thereby stabilizing the mitochondrial fusion machinery and inducing hyperfused network formation; and secondly, via the CCDC6-ASS1 complex, which instigates mitochondrial reactive oxygen species accumulation, compromises ATP synthesis, and reduces mitochondrial membrane potential, consequently inducing a state of metabolic dormancy in tumor cells. This study elucidates the mechanism by which the ASS1-CCDC6 axis suppresses lung adenocarcinoma progression by remodeling mitochondrial dynamics and metabolic homeostasis,, thereby establishing a theoretical basis for mitochondria-targeted precision therapy. Biological sciences/Cancer Biological sciences/Cell biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Metabolic reprogramming has garnered significant attention in the context of tumor development, endowing cancer cells with the capacity to survive and proliferate even under adverse conditions such as nutrient deprivation 1 , 2 . The identification of the 'Warburg effect' has transformed our understanding of the metabolic networks within cancer cells; specifically, it reveals that a majority of tumor cells predominantly utilize aerobic glycolysis for energy production, thereby fulfilling their material and energetic demands necessary for unrestricted proliferation, invasion, and metastasis 3 . Furthermore, the dysregulation of anabolic and catabolic pathways involving fatty acids and amino acids—particularly glutamine, serine, and glycine—has been recognized as a crucial metabolic regulator that facilitates tumor cell proliferation 4 . Notably, there exists significant crosstalk between this dysregulated metabolic network and cellular signaling in tumor cells, which offers novel therapeutic strategies for addressing the malignant progression of lung adenocarcinoma through targeted modulation of tumor metabolism. CCDC6 is located in the q21.2 region of human chromosome 10 and is a tumor suppressor gene involved in cell apoptosis and DNA damage response 5 . Its encoded protein is widely expressed in various tissues, it is a proapoptotic protein substrate of the ataxia telangiectasia mutated gene (ATM) family and participates in ATM-mediated cellular responses 6 . Since CCDC6-RET is the second most common fusion gene after KIF5B-RET, the research on CCDC6 is more about its involvement in the progression of various diseases as a fusion gene 7 , 8 . However, the prognosis and biological function of CCDC6 in lung adenocarcinoma are still unclear, and the specific mechanism between CCDC6 and ASS1 remains to be further studied. ASS1, also known as ASS or CTLN1, encodes 412 amino acids and is the rate-limiting enzyme for arginine production 9 , 10 . Several studies have shown that ASS1 has a close and complex relationship with tumors, and its differential expression may lead to different tumor outcomes 11 , 12 . Studies have shown that the high expression or continuous activation of ASS1 can promote the proliferation and invasion of tumor cells in gastric cancer and colorectal cancer 13 , 14 . However, ASS1 inhibited cell proliferation and migration in HCC and breast cancer cells 11 , 15 . The mechanism of this dual action of ASS1 in different tumors is unknown. We focused on the mitochondrial localization of ASS1 in lung adenocarcinoma cells, which increased with the treatment of its substrates citrulline and aspartate, suggesting that ASS1 may have a urea cycle-dependent mitochondrial function in lung adenocarcinoma cells. Citrulline is a neutral α-amino acid formed by nitric oxide synthase in mitochondria 16 . It is a precursor of arginine and nitric oxide. It is a non-essential, non-protein amino acid (AA) 17 . It has been proven that the post-translational modification of arginine (known as argininization or deamination) plays an important role, it has a variety of effects such as anti-oxidation, immune regulation, cardiovascular protection, intestinal protection and neuroprotection, which is beneficial to human health 18 – 21 . In cells, citrulline is not one of the 20 amino acids encoded by DNA that make up proteins. Citrulline is mainly produced by ornithine and carbamoyl phosphate during the Urea Cycles and by nitric oxide synthase (NOS) as a by-product of arginine to NO 16 . Aspartic acid is an acidic amino acid, which is the precursor for the synthesis of a variety of amino acids (lysine, methionine, etc.) 22 . It has been reported that aspartic acid can play the role of a "rate-limiting device" in tumors, it is mainly involved in triggering the two most important metabolic pathways of cells 23 : Krebs Cycles and urea cycles. In the Krebs cycle, carbohydrates are broken down for energy, and aspartate helps deliver energy into mitochondria, as both citrulline and aspartic acid are precursors of arginine synthesis in the urea cycle, their cell biological functions are often abbreviated as ammonia detoxification of urea cycle, or indirectly as their downstream product arginine 24 . However, its direct biological effects on cells are rarely reported. Deubiquitinating enzymes are divided into several families according to their structural characteristics, among which the Ovarian Tumor Domain proteases (OTUs) family has attracted much attention due to their specificity in ubiquitin chain editing. OTU Deubiquitinase 7A (OTUD7A) is an important member of the deubiquitinase family. As a highly specific ubiquitin-editing enzyme, OTUD7A can precisely regulate the ubiquitination status of target proteins. It is involved in key biological processes such as cell signal transduction, immune response, apoptosis regulation and neurodevelopment 25 , 26 . The dynamic equilibrium of ubiquitination is maintained by ubiquitin ligase (E3) and deubiquitinating enzymes 27 , OTUD7A significantly affects the maturation and activity of proteasomes by regulating the biogenesis and assembly of proteasomes, thereby profoundly regulating the protein homeostasis of cells 25 . In our study, we found that citrulline in combination with aspartic acid drives ASS1 translocation from cytoplasm to mitochondria; ASS1 regulates lung adenocarcinoma progression through a dual mechanism: 1. ASS1 recruited deubiquitinating enzyme OTUD7A to antagonize the ubiquitination and degradation of MFN1/2 and OPA1, thereby stabilizing the expression of mitochondrial fusion protein and promoting the dynamic balance of mitochondrial network; The specific interaction between ASS1 I281M and R55L/N394Y mutation of CCDC6 triggers mitochondrial dysfunction, which is characterized by accumulation of MitoROS, inhibition of ATP synthesis and decrease of MMP. Finally, ASS1 induces tumor cells to enter metabolic dormancy and inhibits the malignant progression of lung adenocarcinoma. Material and Methods Cell culture, viruses, stimulation, and transfection In this study, we established the following cell culture conditions: 293T cell lines were maintained in DMEM (Gibco, NY, USA) medium, while A549 (ATCC: CCL-185) cell lines were grown in a 1:1 mixture of DME and F12 (HyClone, UT, USA) medium. The cell lines PC9, H1299 (ATCC: CRL-5803), H358, H23, and H1975 were cultured in RPMI1640 (Gibco) medium. All cells were incubated at 37°C with 5% CO 2 , supplemented with 10% (v/v) BCS. The cell lines were procured from the cell bank of the Cancer Institute at Central South University. Supplementary Table 1 lists the sgRNA and shRNA sequences for ASS1 and CCDC6 utilized in this research. The constructed plasmid was introduced into the cells and transfected using Lipofectamine Max. Stable expression colonies were selected using puromycin at a concentration of 1 µg/ml. Western blot analysis and coimmunoprecipitation (Co-IP) assay Western blot analysis and coimmunoprecipitation (Co-IP) assay Western blot analysis is a technique used to detect specific proteins in a sample. The process begins with preparing cell lysates to extract proteins, which are then separated using SDS-PAGE based on their size. After the proteins are transferred to a membrane, it is blocked to prevent non-specific binding. The membrane is incubated with a primary antibody that specifically recognizes the target protein, followed by a washing step to remove unbound antibodies. A secondary antibody, which is conjugated to an enzyme or dye, is then applied, and after another wash, a substrate is added to produce a signal. The resulting bands are visualized and quantified to assess protein expression levels. Coi mmunoprecipitation (Co-IP) is a method used to study interactions between proteins. The procedure starts with lysing cells to extract proteins, followed by centrifugation to clear the lysate. An antibody specific to the protein of interest is added and incubated to bind the target protein. Then, protein A or G beads are introduced to capture this antibody-protein complex. After several washes to remove non-specific interactions, the bound proteins are eluted from the beads. The eluted samples can be analyzed using Western blotting to confirm the presence of interacting proteins, providing insights into their associations within the cell. Supplementary Table 2 lists primary antibodies used in Western blot analysis. Mice Mice were kept in a pathogen-free environment, with some being sourced from Hunan SJA Laboratory Animal Co., LTD. (Changsha, China) as needed. They were fed an irradiated PicoLab rodent diet (10 kcal%, D12450J) and all subjects were female nude mice. The animal studies received approval from the Institutional Animal Care and Use Committee of Xiangya School of Medicine, Central South University, and were conducted in compliance with relevant legislation and federal animal care guidelines. Subcutaneous injections of 1 × 10 6 cells per mouse were administered, consisting of ASS1 and CCDC6 overexpression or knockdown cells, along with control cells, into the axilla of each mouse. Tumor volume and mouse weight were assessed every three days until the mice were sacrificed on day 27. Following euthanasia, tumors were weighed, photographed, and lysed for Western blot analysis. Real-time quantitative polymerase chain reaction (RT-qPCR) Real-time quantitative polymerase chain reaction (qPCR) is a technique used to amplify and quantify DNA sequences, typically aimed at measuring gene expression levels. The process begins by extracting RNA from cells or tissues, which is then reverse-transcribed into complementary DNA (cDNA). A reaction mix is prepared containing the cDNA, specific primers for the target gene, and a fluorescent dye or probe that emits a signal during DNA amplification. The mixture is placed in a real-time PCR machine that cycles through various temperatures to facilitate DNA synthesis while continuously monitoring fluorescence. The threshold cycle (Ct) value is recorded for each sample, indicating the amount of target DNA present; lower Ct values reflect higher quantities. Finally, data is analyzed by comparing Ct values to a housekeeping gene to determine relative gene expression across different samples or conditions, providing insights into biological processes. TRIzol reagent (Takara, Kusatsu, Japan) was used to isolate the total RNA, and the kit (Takara, Kusatsu, Japan) was used to reverse transcribe the RNA into cDNA. Real-time PCR was performed on a Bio-rad CFX Connect real-time PCR instrument. β-actin served as the internal reference for gene expression. The primers used in this investigation are listed in Supplementary Table 3. Immunofluorescence microscopy A 24-well culture plate containing small glass discs was prepared with logarithmically growing cells 24 hours prior to adding adherent cells. The culture plate was removed once the cells reached approximately 50% confluence. After washing three times with 1× PBS, 1 mL of methanol was added to each well to fix the cells for 10 minutes at room temperature. Following two 5-minute rinses with PBS, the wells were blocked for 30 minutes using 1% (w/v) BSA in PBS. The primary antibodies, also diluted in 1% BSA, were incubated overnight at 4°C. Depending on the primary antibody type, either anti-rabbit IgG Alexa 594 or anti-mouse IgM Alexa 488 fluorescent secondary antibodies were used and incubated for one hour after rinsing with PBS. After DAPI labeling, the samples were mounted onto slides and imaged using a Leica TCS SP8 confocal microscope. For live cell staining, MitoTracker® Deep Red FM (Invitrogen, 644–665 nm, M22426) was applied in a cell incubator set at 37°C with 5% CO 2 for 30 minutes. Separation of the cytoplasmic and mitochondria The Cell Mitochondria Isolation Kit (Beyotime, C3601) was employed to isolate mitochondrial proteins from the cells. After washing the collected cells three times with 1×PBS, they were lysed on ice for fifteen minutes using a mitochondrial separation reagent supplemented with PMSF. The lysate was then transferred into an appropriately sized glass homogenizer and homogenized for ten to thirty cycles. Subsequent centrifugation of the homogenate resulted in the pelleting of cellular mitochondria in the precipitate while cytoplasmic proteins remained in the supernatant. The protein concentration was subsequently determined using the BCA assay. Transmission electron microscopy (TEM) Following the collection of ASS1 overexpression and knockdown cells through trypsin digestion, the cells were washed twice with PBS. Subsequently, 1 mL of stationary solution was gently added along the walls of the centrifuge tube to avoid disturbing the cell pellet, and the tubes were then kept at 4°C overnight. Images were captured using a transmission electron microscope (Hitachi; HT7700) at the Department of Pathology, Xiangya Hospital. Mitochondrial ROS (MitoROS) Seed cells in a culture dish, and wait until the cells are fully adhered and in good condition, with a confluence of 70%-80%. Digest the cells with trypsin, centrifuge at 800 rpm for 5 minutes, and wash with PBS twice. Take 5 × 10 4 cells from each well and resuspend them in serum-free culture medium. Prepare the MitoSOX working solution (Invitrogen, M36008), incubate in a dark incubator for 20 minutes, then centrifuge at 800 rpm for 5 minutes, discard the supernatant, resuspend in 200 µL of 1× PBS, and filter through a cell strainer. Finally, use flow cytometry to detect the mitochondrial ROS levels in the cells. Mitochondrial Membrane Potential (MMP) JC-1 is a widely used fluorescent probe for detecting mitochondrial membrane potential. When the membrane potential is high, JC-1 accumulates in the mitochondria and forms aggregates, resulting in red fluorescence; when the membrane potential is low, JC-1 exists as monomers, producing green fluorescence. Seed the cells in a culture dish and wait until the cells are fully adhered and in good condition, with a confluence of 70%-80%. Discard the culture medium and wash twice with PBS. Add 500 µL of staining working solution (Beyotime, C2006) and incubate in a dark incubator for 20 minutes. After incubation, discard the supernatant, wash twice with JC-1 staining buffer, and resuspend in 200 µL of staining buffer. Finally, use flow cytometry to detect the mitochondrial membrane potential. Adenosine 5'-triphosphate(ATP) Seed the cells in a 6-well plate and wait until the cells are fully adhered and in good condition, with a confluence of 70%-80%. Discard the culture medium and wash twice with PBS. Add 200 µL of ATP detection lysis buffer (Beyotime, S0027) to each well, and thoroughly lyse the cells on ice for 5 minutes. Transfer the cell suspension to a 1.5 mL centrifuge tube and centrifuge at 12,000 rcf for 5 minutes at 4°C, keeping the supernatant. In a 96-well plate, add 100 µL of ATP detection working solution and let it sit at room temperature for 5 minutes. Prepare ATP standard solutions (0, 0.01, 0.03, 1, 3, 10 µM concentration gradient) and add the cell supernatant and standard solutions to the ATP detection working solution. Measure the fluorescence using a microplate reader. After calculating the ATP concentration based on the standard curve, normalize it to the corresponding protein concentration. Annexin V/PI Collect 1 × 10^6 cells and wash them twice with pre-chilled PBS. Resuspend the cells in 500 µL of Apoptosis Positive Control Solution (MultiSciences, AP105) and incubate on ice for 30 minutes. Wash the cells with pre-chilled PBS and discard the supernatant. Resuspend the cells in 500 µL of Binding Buffer and add 1 × 10^6 untreated live cells. Complete the volume to 1.5 mL with Binding Buffer and divide into three tubes: one will be the blank control and two will be the single-staining tubes. Add 5 µL of Annexin V-APC or 10 µL of 7-AAD to the single-staining tubes and incubate at room temperature in the dark for 5 minutes. On the flow cytometer, adjust the voltage for the blank tube and set the fluorescence channels for the single-staining tubes. Use trypsin (without EDTA) to digest the cells, collect the cell suspension and supernatant, and wash twice with pre-chilled PBS. Take 1 × 10^5 cells, resuspend in 500 µL of Binding Buffer, and add 5 µL of Annexin V-APC and 10 µL of 7-AAD to each tube, mixing well. Incubate at room temperature in the dark for 5 minutes, then analyze by flow cytometry. Statistics All studies, except those involving mice, were conducted a minimum of three times, and the results are presented as mean ± SD or SEM. Statistical analyses were performed using GraphPad Prism 9.0. A T-test was employed to evaluate the significance of differences between two groups, while analysis of variance (ANOVA) was used for comparisons involving more than two groups. The Pearson correlation coefficient was applied for correlation analyses. Statistical significance was defined as p < 0.05, with levels of significance indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Study approval The project was approved by the ethics committee at our hospital, and the institutional Animal Care and Use Committee at Central South University granted approval for the use of animal models in this study. Additionally, the study received approval from the institutional review boards of all participating medical facilities. Prior to recruitment, each research subject provided written informed consent. Results Combined multi-omics analysis identified CCDC6 as a key regulatory target in lung adenocarcinoma. In order to explore new therapeutic targets and intervention strategies for lung adenocarcinoma, we collected 34 pairs (Table 4) of lung adenocarcinoma clinical tissue samples for combined untargeted metabolomics and transcriptomics analysis, and screened differentially expressed metabolites and genes by OPLSADA and limma (Linear Models for Microarray Data ) analysis (Fig. 1 a). The results of non-targeted metabolomics analysis showed that the overall levels of amino acids were significantly down-regulated in tumor tissues, among which citrulline and aspartic acid were most significantly reduced (Fig. 1 b), suggesting that citrulline may be involved in tumor metabolic reprogramming as energy substances. Based on the results of metabolomics data, the down-regulated differentially expressed metabolites in tumor tissues were screened and analyzed by the MetaboAnalyst platform (Fig. 1 c), and the KEGG pathway enrichment analysis of differentially expressed genes was further performed on the transcriptome results. ClusterProfiler package was used for functional annotation (Fig. S1 a), and the results showed that "Alanine, aspartate and glutamate metabolism" pathway was significantly down-regulated at both the metabolome and transcriptome levels. Further, gene set variation analysis (GSVA) was used to quantify pathway activity scores in tumor samples, and Spearman correlation analysis was performed with differential genes (Fig. S1 b). After correction for multiple testing, it was found that the expression level of CCDC6 was significantly positively correlated with the activity of the "Alanine, aspartate and glutamate metabolism" pathway (Fig. 1 d). To further verify the expression of CCDC6 in lung adenocarcinoma tissues, we collected 12 pairs of tumor tissues and paired adjacent tissues from lung adenocarcinoma patients. The results of Western blot showed that the protein expression level of CCDC6 was significantly decreased in lung adenocarcinoma tissues compared with adjacent normal tissues (Fig. 1 e-f). These results suggest that CCDC6 may be a key regulatory target in lung adenocarcinoma. In order to clarify its clinical significance and biological function in lung adenocarcinoma cells and its relationship with citrulline and aspartic acid, we first treated the cells with citrulline and aspartic acid, and detected the biological function of lung adenocarcinoma cells by CCK8 assay, colony formation assay, and transwell migration assay. The results showed that the combination of citrulline and aspartic acid promoted the proliferation, migration and colony formation ability of lung adenocarcinoma cells (Fig. S1 c-i). Next, we examined the protein levels of CCDC6 in different lung adenocarcinoma cell lines (Fig. S2 e). We selected A549 and H1299 cell lines with low expression of CCDC6 and stably overexpressed CCDC6 using lentiviral vector. shRNA technology was used to knock down CCDC6. As expected, the cell proliferation was much lower in CCDC6 overexpressed cells than that of control cells (Fig. 2 a-b). Furthermore, overexpression of CCDC6 dramatically decreased cell colony formation, migration, and invasion (Fig. 2 c-f). A xenograft model experiment was used to further investigate the influence of CCDC6 on tumor development in vivo. The injection of A549 cells overexpressing CCDC6 can drastically Inhibit tumor growth, volume, and weight when compared to the injection of control cells (Fig. 2 g-i). The expression of CCDC6 protein in subcutaneous tumor tissues of nude mice was detected by western-blot. The results further confirmed that CCDC6 inhibited lung adenocarcinoma cell progression in vitro and in vivo . Similarly, the physiological effects of CCDC6 knockdown on lung adenocarcinoma cells were examined, and the results showed that the above malignant phenotypes were significantly enhanced (Fig. 3 a-j). In general, these results indicate that the absence of CCDC6 significantly promotes the occurrence and development of tumors. ASS1 specifically binds to CCDC6 to inhibit the progression of lung adenocarcinoma. To further explore the mechanism by which CCDC6 regulates lung adenocarcinoma progression, we analyzed the proteins interacting with CCDC6 in H1975 cells by mass spectrometry (Fig. 4 a). The results showed that CCDC6 and ASS1 co-localized and interacted in the cytoplasm of lung adenocarcinoma cells (Fig. 4 b). Co-IP assay was used to verify the endogenous interaction between ASS1 and CCDC6 in H358 and PC9 cells (Fig. 4 c-f). Confocal laser scanning microscopy was used to confirm the co-localization of ASS1 and CCDC6 (Fig. 4 g-h). These findings suggest an interaction between CCDC6 and ASS1. Based on the clinical data and transcriptome data of 538 lung adenocarcinoma patients included in the TCGA database, the correlation between ASS1 expression level and clinical prognosis of patients was systematically evaluated. Kaplan-Meier survival analysis showed that compared with ASS1 low expression group, ASS1 high expression group showed better clinical outcomes, and OS and DFS were significantly improved (Log-rank test P < 0.05) (Fig. S3 a-b). This statistically significant difference suggests that the high expression status of ASS1 may serve as a potential molecular marker for good prognosis in patients with lung adenocarcinoma. After establishing the stable overexpression model of ASS1 in H23 and H1299 lung adenocarcinoma cell lines (Fig. S3 c-d), CCK8 cell proliferation assay and colony formation assay showed that compared with the control group, the overexpression of ASS1 significantly inhibited the proliferation (Fig. S3 e-f) and decreased the colony formation ability of lung adenocarcinoma cells (Fig. S3 g-h). Transwell migration and invasion assays showed that ASS1 overexpression significantly inhibited the migration and invasion ability of lung adenocarcinoma cells compared with the control group (Fig. S3 i-j). ASS1 stably overexpressing H1299 cell line was used to establish a subcutaneous xenograft tumor model in nude mice. The results showed that compared with the control group, the growth of transplanted tumors in the ASS1 overexpression group was significantly inhibited, and the final tumor volume and tumor weight were significantly reduced (Fig. S3 k-n). These in vivo data further confirmed the tumor suppressor function of ASS1 in lung adenocarcinoma progression, which was consistent with the in vitro results. Similarly, the physiological effects of ASS1 knockdown on lung adenocarcinoma cells were examined, and the results showed that the above malignant phenotypes were significantly enhanced (Fig. S4 a-j). In general, these results indicate that the absence of ASS1 significantly promotes the occurrence and development of tumors. In order to clarify the interaction mechanism between ASS1 and CCDC6, we screened the high-frequency mutation sites of ASS1 and CCDC6 ( https://www.cbioportal.org/ ), and constructed ASS1 (R86S, V103L, K165E, I281M, R404H) and CCDC6 (R55L, E160Q, D236N, N394Y) point mutation plasmids. Western blot analysis showed that none of these point mutations significantly affected the protein expression levels of ASS1 or CCDC6. However, the Co-IP assay showed that the binding ability of ASS1 mutants to CCDC6 was significantly reduced (Fig. 4 i). Similarly, when CCDC6 mutants (R55L, E160Q, D236N, N394Y) were transfected, although the protein expression of CCDC6 and ASS1 was not affected, R55L and N394Y mutations significantly weakened the interaction between CCDC6 and ASS1 (Fig. 4 j). To further explore the functional significance of these mutation sites, a cell model stably overexpressing ASS1 and its mutants was constructed in H1299 cell line. The results showed that overexpression of wild-type ASS1 significantly inhibited the proliferation ability, colony formation rate, migration and invasion ability of lung adenocarcinoma cells compared with the control group. However, these inhibitory effects were significantly reversed by ASS1 I281M mutation (Fig. 4 k-o). These results not only confirm the tumor suppressor function of ASS1, but more importantly reveal the decisive role of I281M as the key site of ASS1-CCDC6 interaction in maintaining the tumor suppressor activity of ASS1. This study provides an important experimental basis for further understanding the molecular mechanism of ASS1-CCDC6 in lung adenocarcinoma. The combination of citrulline and aspartic acid changes the metabolic microenvironment of ASS1 localization and reprogramming. Based on the classical metabolic pathway of urea cycle, citrulline is synthesized in mitochondria and transferred to the cytoplasm through specific transporters. Citrulline and aspartic acid act as a substrate to produce arginine succinate under the catalysis of ASS1 (Fig. 5 a). Metabolomics data showed that the expression of citrulline and aspartic acid in tumor tissues was significantly lower than that in adjacent tissues, suggesting that citrulline and aspartic acid may regulate the progression of lung adenocarcinoma by regulating the expression of ASS1 (Fig. 5 b-c). We therefore examined ASS1 expression in the cytoplasm and mitochondria after cells were treated with citrulline and aspartic acid (Fig. 5 d). Western blot showed that the expression of ASS1 protein in mitochondrial fraction was significantly up-regulated in citrullinate-aspartic acid combined treatment group compared with untreated control group (Fig. 5 e-f). Confocal analysis further confirmed the above findings, the co-localization signal of ASS1 with the mitochondria-specific probe MitoTracker Red was significantly enhanced after 48 hours of combined treatment with citrulline and aspartic acid (Fig. 5 g-j, Fig. S2 a-d). These results confirmed the role of citrullinate-aspartic acid metabolism axis in promoting ASS1 translocation to mitochondria from the perspective of subcellular spatial distribution. ASS1 recruits OTUD7A to stabilize mitochondrial fusion protein and promote mitochondrial fusion. Based on the previous study that found a large number of ASS1 translocated to mitochondria, we hypothesized that ASS1 may have mitochondrial related functions in addition to the key enzymes of urea cycle in lung adenocarcinoma. Considering that mitochondria are highly dynamic organelles that maintain normal morphology and function through continuous fusion and division processes, we further explored the effect of ASS1 on mitochondrial dynamics (Fig. 5 k). By observing ASS1 knockdown and overexpression in lung adenocarcinoma cell lines by transmission electron microscopy, we found that mitochondrial fission in ASS1 knockdown cells was significantly increased, and the average diameter of mitochondria was significantly shorter than that in the control group. In contrast, after overexpression of ASS1, mitochondria showed a pronounced elongation phenotype, with a significant increase in average length compared with the control group(Fig. 5 l-m). These results suggest that ASS1 may play a role in inhibiting lung adenocarcinoma progression by regulating mitochondrial dynamics and promoting mitochondrial fusion. Statistical results also showed that the number of mitochondria decreased and the average length increased after overexpression of ASS1, and conversely, the number of mitochondria increased and the average length decreased after knockdown of ASS1 (Fig. 5 n-q). To further explore the molecular mechanism of ASS1 in regulating mitochondrial dynamics, the effects of ASS1 knockdown and overexpression on the expression levels of mitochondrial fission and fusion related proteins were detected by Western blot. The results showed that ASS1 knockdown significantly inhibited the expression of mitochondrial fusion proteins (MFN1/2 and OPA1). In contrast, ASS1 overexpression significantly upregulated the expression levels of these fusion proteins (Fig. 5 r-s). It is worth noting that these protein expression changes were not accompanied by significant changes in their mRNA levels (Fig. 5 t-u). These results suggest that ASS1 may affect the mitochondrial fusion process by promoting the expression of mitochondrial fusion-related proteins through post-translational modification machinery rather than regulation at the transcriptional level. In order to explore the molecular mechanism of ASS1 regulating mitochondrial fusion proteins (MFN1/2, OPA1), based on the perspective of protein post-translational modification regulation, OTUD7A was identified as a key clue in the CCDC6 interaction proteome found by previous mass spectrometry analysis. It was speculated that ASS1 may recruit the deubiquitinating enzyme OTUD7A to stabilize mitochondrial fusion protein expression (Fig. 6 a). Firstly, the direct interaction between ASS1 and OTUD7A was confirmed by Co-IP using H358 and H1975 lung adenocarcinoma cell lines (Fig. 6 b-c). To further explore the molecular mechanism by which ASS1 regulated mitochondrial fusion proteins (MFN1/2 and OPA1), We treated the H23 cell line overexpressing ASS1 with CHX and MG132. The results showed that the degradation rate of MFN1/2 and OPA1 was slowed in the overexpression group after CHX treatment (Fig. 6 d). After MG132 treatment, the expression differences of MFN1/2 and OPA1 between the empty vector group and the overexpression group disappeared, and the two groups had the same intensity of protein accumulation (Fig. 6 e). Next, H358 ASS1 knockdown cells were treated with CHX and MG132. The results showed that after CHX treatment, the degradation rate of MFN1/2 and OPA1 was significantly increased after ASS1 knockdown (Fig. 6 f). The accumulation of MFN1/2 and OPA1 proteins in ASS1 knockdown group was more significant (Fig. 6 g). To further explore the molecular mechanism of ASS1 regulating mitochondrial fusion protein ubiquitination. After MG132 pretreatment in H23 and H1299 cell lines with stable overexpression of ASS1, the ubiquitination modification levels of MFN1/2 and OPA1 were decreased after overexpression of ASS1 (Fig. 6 h-k). The above results confirmed that ASS1 stabilized the expression of mitochondrial fusion proteins (MFN1/2 and OPA1) by recruiting OTUD7A, changing the mitochondrial morphology and promoting mitochondrial fusion. The CCDC6/ASS1 interaction axis disrupts mitochondrial homeostasis and promotes apoptosis of lung adenocarcinoma cells through mitochondrial pathway. Previous studies have confirmed that ASS1 affects mitochondrial dynamics by recruiting the deubiquitination enzyme OTUD7A. To further explore the effect of ASS1 on mitochondrial function, we used flow cytometry and fluorescent enzyme labeling to detect the changes of MitoROS, ATP production and MMP. The results showed that overexpression of ASS1 significantly increased MitoROS level in lung adenocarcinoma cells (Fig. 7 b-c), while ATP production was decreased (Fig. 7 h-i). In addition, JC-1 staining results showed that overexpression of ASS1 caused a significant decrease in MMP (Fig. 7 k-l). In ASS1 knockdown lung adenocarcinoma cells, MitoROS level was decreased (Fig. 7 a), accompanied by increased ATP production (Fig. 7 g) and significantly increased MMP (Fig. 7 j). However, treatment of cells with citrulline and aspartic acid alone did not affect intracellular mitochondrial ROS levels (Fig. 7 d-f). These results suggest that ASS1 may affect the balance of mitochondrial dynamics by increasing the level of mitochondrial oxidative stress, inhibiting energy metabolism and destroying the stability of mitochondrial membrane potential, and ultimately inhibit the growth and proliferation of lung adenocarcinoma cells. This finding provides a new experimental basis for elucidating the mechanism by which ASS1 acts as a tumor suppressor by regulating mitochondrial function. Based on previous studies that confirmed the protein-protein interaction between ASS1 and CCDC6 and that ASS1 can inhibit lung adenocarcinoma progression by regulating mitochondrial dynamics, we further explored the effect of CCDC6 on mitochondrial function. The results showed that ATP production and MMP were significantly decreased in lung adenocarcinoma cells overexpressing CCDC6. In contrast, in CCDC6 knockdown lung adenocarcinoma cells, ATP production was significantly increased, accompanied by a decrease in MMP (Fig. 7 m-r). These results suggest that CCDC6 may be involved in the regulation of lung adenocarcinoma progression by regulating mitochondrial energy metabolism and maintaining membrane potential stability, affecting mitochondrial dynamic balance. Combined with the interaction between ASS1 and CCDC6, we hypothesized that ASS1 may inhibit the malignant progression of lung adenocarcinoma by forming functional protein complexes with CCDC6 and disrupting mitochondrial homeostasis. In order to further explore the molecular mechanism of ASS1-CCDC6 complex inhibiting the development of lung adenocarcinoma, since previous studies have found that ASS1 and CCDC6 can affect mitochondrial homeostasis, combined with the close relationship between mitochondrial dynamics and apoptosis, a scientific hypothesis is proposed: CCDC6-ASS1 interaction may determine the fate of lung adenocarcinoma cells by regulating the apoptotic signaling pathway. Western blot analysis showed that in the CCDC6 overexpression group, the expression of pro-apoptotic protein Bax was significantly increased, while the expression of anti-apoptotic protein Bcl-xl was significantly decreased. In contrast, a trend of decreased Bax expression and increased Bcl-xl expression was observed in the CCDC6 knockdown group (Fig. S5 g-i). Consistent with the results of CCDC6 assay, ASS1 overexpression significantly up-regulated the expression level of pro-apoptotic protein Bax and inhibited the expression level of anti-apoptotic protein Bcl-xl. On the contrary, when ASS1 was knocked down, the expression level of Bax protein was significantly decreased, while the expression of Bcl-xl protein was significantly increased (Fig. S5 j-l). These findings suggest that the ASS1/CCDC6 protein complex may activate the mitochondrial pathway of apoptosis by regulating the expression balance of Bcl-2 family proteins. This study not only provides new experimental evidence for in-depth understanding of the biological function of ASS1/CCDC6 complex, but also provides an important theoretical basis for elucidating the molecular mechanism of ASS1/CCDC6 complex as a tumor suppressor. Next, we used flow cytometry combined with Annexin V/PI double staining to quantify the level of apoptosis by detecting the extroversion of phosphatidylserine (PS) on the cell membrane surface. The results showed that overexpression of CCDC6 significantly increased the proportion of Annexin V-positive cells in lung adenocarcinoma cells compared with the control group. Further analysis revealed that both the proportion of early apoptotic cells (Annexin V+/PI-) and late apoptotic cells (Annexin V+/PI+) showed a significant increase (Fig, S5 a-b). Similarly, quantitative analysis showed that ASS1 overexpression significantly increased the proportion of early apoptotic cells (Annexin V+/PI-) compared with the control group, while the proportion of late apoptotic cells (Annexin V+/PI+) did not show a statistically significant change (Fig. 5 c-d). This experimental phenomenon of specifically inducing early apoptosis suggests that ASS1 may participate in the regulatory process of inhibiting the malignant progression of lung adenocarcinoma by activating key signaling pathways in the initiation stage of apoptosis. In this study, we identified a key regulatory target of lung adenocarcinoma, CCDC6, and demonstrated that CCDC6 specifically binds to ASS1. Citrulline and aspartic acid were found to drive the metabolic reprogramming of lung adenocarcinoma by promoting the translocation of ASS1 in mitochondria. Finally, CCDC6 and ASS1 promote the apoptosis of lung adenocarcinoma cells and inhibit the progression of lung adenocarcinoma by recruiting the deubiquitinating enzyme OTUD7A to stabilize mitochondrial fusion protein and destroy mitochondrial homeostasis (Fig. 7 s). Discussion The dynamics of mitochondrial division and fusion are crucial for the formation and growth of tumors, and they also play a vital biological function in promoting the invasion and metastasis of tumor cells through energy metabolism and biosynthetic metabolism 28 , 29 . Mitochondria are extremely dynamic organelles that constantly fuse and fission to preserve their structure and functionality 30 . On the one hand, mitochondria exist in a dynamic equilibrium between fission and fusion under normal physiological conditions. The process of mitochondrial fission is essential for preserving cell growth and mitosis because it produces a suitable number of mitochondria, isolates irreparable damage within them, and allows for the mobility and redistribution of mitochondria. On the other hand, defective components within the mitochondria are replaced by a mixture of healthy and damaged mitochondria through a process known as mitochondrial fusion 31 . The cell is somewhat protected by this process, which encourages material interchange and energy conversion amongst the constituents. Recent studies have shown that the localization of ASS1 in cells can lead to its different functions 32 , ASS1 acetylation status in endothelial cells and its enzymatic activity in arginine biosynthesis regulate vascular homeostasis. Although a large number of previous studies have found that ASS1 downregulation is associated with poor prognosis, some groups still found that ASS1 expression in the nucleus is increased after DOX-induced DNA damage in colon cancer cells and fibroblasts, which maintains genomic stability and is dependent on P53 regulation 33 . Other studies have found that loss of ASS1 can increase the sensitivity of non-small cell lung cancer cells to Erastin in vitro and inhibit the growth of lung adenocarcinoma in vivo 34 . In this paper, the authors mainly focused on the sensitivity of ASS1 to erastin and interestingly confirmed that loss of ASS1 can promote ferroptosis in lung adenocarcinoma cells. It is interesting to note that the xenograft tumor experiments in the authors' study were performed in A549 cell line, which is P53 wild-type. In our study, we used H358 and H1299 cell lines, both of which are P53 null. This aroused our great interest, and it remains to be further investigated whether mutation or deletion of P53 regulates ASS1-induced tumor progression. In our study, we found a large translocation of ASS1 from the cytoplasm to mitochondria after treatment with Citr and Asp, which aroused our interest and concern because mitochondria are the key platform of a large number of cells signaling cascades, not only affecting and often coordinating metabolism, but also coordinating the timing of complex cell signaling and participating in the regulation of cell pluripotency. Such as division, differentiation, senescence, death, etc. Therefore, we wanted to further investigate the function of its translocation to mitochondria. In this connection, we observed the effect of ASS1 on mitochondrial morphology in lung adenocarcinoma cells by transmission electron microscopy, and the results showed that ASS1 can promote mitochondrial fusion, suggesting that ASS1 may have a new function of remodeling mitochondrial morphology. In addition, protein-protein interaction plays a crucial role in the study of cell signaling network, which is a prerequisite for affecting the physiological and pathological changes of cells. CCDC6 has been recognized as a fusion protein, and the CCDC6-RET fusion is also a key target for cancer therapy. However, the function of CCDC6 in lung adenocarcinoma cells and the mechanism by which CCDC6 affects tumor progression have not been reported. The interaction between CCDC6 and ASS1 can promote mitochondrial ROS production by promoting mitochondrial fusion, and ultimately promote cell apoptosis by reducing mitochondrial ATP and down-regulating mitochondrial membrane potential, and inhibit the progression of lung adenocarcinoma. In this study, the interaction domain between ASS1 and CCDC6 was characterized, and the function of its mutants in lung adenocarcinoma cells was analyzed. Although these point mutations did not significantly change the protein expression levels of ASS1 and CCDC6, co-immunoprecipitation assay showed that these point mutations significantly reduced the binding ability of ASS1 to CCDC6, which revealed the effect of the interaction between ASS1 and CCDC6 on their biological functions. Mutant plasmids transfected with CCDC6 (e.g. R55L, N394Y), although CCDC6 expression was not affected, significantly impaired its ability to interact with ASS1. This suggests that mutations in CCDC6 may play a role in the progression of lung adenocarcinoma by disrupting its binding to ASS1 and affecting cell signaling pathways 35 , 36 . As the core organelle of cellular energy metabolism, mitochondria homeostasis (the precise regulation of fusion and division) plays a decisive role in maintaining cellular homeostasis 37 . Recent studies have revealed that mitochondrial fission plays a key role in tumor invasion and metastasis by driving actin skeleton remodeling and pseudopodia formation, which provides new molecular targets for cancer therapy 38 , 39 . The present study is the first to reveal the mode of action of ASS1 in its non-classical function by regulating mitochondrial dynamics in lung adenocarcinoma. The expression level of ASS1 was significantly correlated with mitochondrial morphology: knockdown of ASS1 resulted in aggravated mitochondrial fragmentation, while overexpression of ASS1 induced mitochondrial network elongation. Further studies found that ASS1 affected mitochondrial homeostasis by regulating the stability of mitochondrial fusion proteins MFN1/2 and OPA1 rather than the transcriptional level. Notably, CHX tracking assay showed that ASS1 overexpression significantly delayed the degradation rate of fusion proteins, while the difference disappeared after treatment with proteasome inhibitor MG132, suggesting that ASS1 regulates the stability of these proteins through the ubiquitin-proteasome pathway. This finding echoes recent studies on mitochondrial quality control, such as that OPA1 stability is regulated by the E3 ubiquitin ligase MARCH5, whereas the present study suggests the existence of a molecular switch with reverse regulation 40 . Based on the previous mass spectrometry data, we targeted the deubiquitinating enzyme OTUD7A as the ASS1 target. Co-IP experiments confirmed a direct interaction between ASS1 and OTUD7A, which provided a mechanistic clue to explain the regulation of fusion protein stability. Further mechanistic studies showed that ASS1 could reduce the ubiquitination level of MFN1/2, which revealed a new mechanism of ASS1 involved in mitochondrial homeostasis by regulating the ubiquitination of MFN1/2. This study suggests that ASS1 may recruit OTUD7A to the mitochondrial microenvironment, and then deubiquitinize MFN1/2 and OPA to maintain their protein stability. This regulatory mechanism bears similarities to the known mode by which deubiquitinating enzymes regulate mitochondrial dynamics. For example, it has been shown that USP30 can regulate mitophagy by deubiquitylation of MFN2 41 ; Similarly, USP19 can specifically bind to FUNDC1 protein at the ER-mitochondrial contact site, promote Drp1 oligomerization and enhance its gtpase activity through deubiquitination, thereby driving mitochondrial division 42 . The innovation of this study is to reveal for the first time the new function of metabolic enzyme ASS1 involved in the deubiquitination regulatory network, breaking through the classical understanding of ASS1 and revealing that ASS1 stabilizes mitochondrial fusion proteins through OTUD7A-mediated deubiquitination mechanism, thereby regulating energy metabolism and survival homeostasis of tumor cells. However, this study still has limitations that need to be further explored. First, although the interaction between OTUD7A and ASS1 and its effect on fusion protein stability were confirmed, the deubiquitination activity of OTUD7A on MFN1/2 and OPA1 has not been directly verified. Secondly, the specific ubiquitination sites on these fusion proteins have not been clearly identified. In addition, the molecular mechanism by which ASS1-OTUD7A complex recognizes and binds fusion proteins remains to be elucidated. In order to solve these scientific problems, we plan to further explore them by deubiquitination experiments of recombinant proteins in vitro, identification of ubiquitination sites by mass spectrometry, and construction of specific site mutants in the future studies. Declarations Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant Nos. 82072594 to Y.T.; 82073097 and 82573321 to S.L.), the Natural Science Foundation of Hunan Province (Grant No. 2024JJ3048 to S.L.), the Hunan Provincial Science and Technology Innovation Plan Project (Grant No. 2020SK53424 to X.W.), and the Central South University Research Programme of Advanced Interdisciplinary (Grant No. 2023QYJC030 to X.W. and Y.T.). We thank the Department of Pathology, Xiangya Hospital, for providing clinical specimens and immunohistochemistry (IHC) tissue sections, and Dr. Bin Xie for technical assistance with IHC experiments. We also thank colleagues for their valuable discussions and technical assistance during the course of this study. Ethics approval and consent to participate The Ethics Committee of the Second Xiangya Hospital, Central South University approved this study. Consent for publication Not applicable Availability of data and materials Not applicable Competing interests The authors declare that they have no competing interests. The authors declare no conflict of interest. This manuscript has been read and approved by all authors and is not under consideration for publication elsewhere. Funding This work was supported by the National Natural Science Foundation of China [82072594 to Y.T. ; 82073097 to S.L.; 82573321 to S.L.]; the Natural Science Foundation of Hunan Province [2024JJ3048 to S.L.]; the Hunan Provincial Science and Technology Innovation Plan Project [2020SK53424 to X.W.] and the Central South University Research Programme of Advanced Interdisciplinary [2023QYJC030 to X.W. and Y.T.]. Disclosure of Potential Conflicts of Interest The authors declare no potential conflicts of interest. References Tufail, M., Jiang, C. H. & Li, N. Altered metabolism in cancer: insights into energy pathways and therapeutic targets. Mol Cancer 23, 203, doi: 10.1186/s12943-024-02119-3 (2024). DeBerardinis, R. J., Vousden, K. H. & Chandel, N. S. Cancer Metabolism: Aspirations for the Coming Decade. Cold Spring Harb Perspect Med 15, a041555, doi: 10.1101/cshperspect.a041555 (2025). Dey, P., Kimmelman, A. C. & DePinho, R. A. Metabolic Codependencies in the Tumor Microenvironment. 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Additional Declarations (Not answered) Supplementary Files unprocessedimages.pdf original SupFigS3.pdf Figure S3 SupFigS5.pdf Figure S5 SupFigS1.pdf Figure S1 SupFigS4.pdf Figure S4 SupFigS2.pdf Figure S2 Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: revise 09 Mar, 2026 Review # 2 received at journal 08 Mar, 2026 Review # 1 received at journal 26 Feb, 2026 Reviewer # 2 agreed at journal 22 Feb, 2026 Reviewer # 1 agreed at journal 13 Feb, 2026 Reviewers invited by journal 12 Feb, 2026 Submission checks completed at journal 28 Jan, 2026 First submitted to journal 27 Jan, 2026 Editor assigned by journal 27 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8712733","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":590103999,"identity":"88bacc3c-ff8c-4e62-bfc2-a97998ce2f35","order_by":0,"name":"Yongguang Tao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYBADOQjFRoRSHihtTLqWxAaitdizHz4m8XFHbXp//xkDhg9lhxn4ZzcQsIUnLU1y5pnjuTNu5Bgwzjh3mEHizgFCDssxk+ZtO5a7QYLHgJm37TCDgUQCAS38b8Ba0g34zxgw/yVKiwTYlpoEA4YcA2ZGorTceJZsObPtgOGMG2kFB3vOpfNI3CCghb0/+eCNj2118vz9hzc++FFmLcc/g4AWIGCRYGA4DGYdYEBEFF7A/IGBoY4YhaNgFIyCUTBSAQCORz7DHRIG5AAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-2354-5321","institution":"Central south university","correspondingAuthor":true,"prefix":"","firstName":"Yongguang","middleName":"","lastName":"Tao","suffix":""},{"id":590104000,"identity":"66aeaeb9-0e29-4e5e-a945-6d1559289c7e","order_by":1,"name":"Wei Wang","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Wang","suffix":""},{"id":590104001,"identity":"cd82ecad-842b-4679-ae8e-92ac80664919","order_by":2,"name":"Yao Long","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Long","suffix":""},{"id":590104002,"identity":"64acd13a-230e-454b-8823-71f6d5dc1753","order_by":3,"name":"QiDong Cai","email":"","orcid":"","institution":"The Second Xiangya Hospital of Central South University","correspondingAuthor":false,"prefix":"","firstName":"QiDong","middleName":"","lastName":"Cai","suffix":""},{"id":590104003,"identity":"d70edfb1-a750-4a71-a6d1-e3b73b733271","order_by":4,"name":"Zhenyu Zhao","email":"","orcid":"https://orcid.org/0000-0002-8697-1419","institution":"central south university","correspondingAuthor":false,"prefix":"","firstName":"Zhenyu","middleName":"","lastName":"Zhao","suffix":""},{"id":590104004,"identity":"cdce545c-9d3a-4384-b5ae-9012d157381d","order_by":5,"name":"Can Cao","email":"","orcid":"","institution":"Central South University,","correspondingAuthor":false,"prefix":"","firstName":"Can","middleName":"","lastName":"Cao","suffix":""},{"id":590104005,"identity":"2aef5558-de46-4f5e-aed3-bffc0d1e5568","order_by":6,"name":"Li Linghu","email":"","orcid":"","institution":"Cancer Research Institute; 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Pattern map of key regulatory targets in lung adenocarcinoma based on multi-omics integration analysis; b. Unbiased metabolomics showed that Citrulline and Aspartic acid were significantly down-regulated in tumor tissues. c. Differential metabolites were screened by metabolomics data, and KEGG pathway enrichment analysis was performed on the down-regulated differential metabolites in tumor tissues. d. The activity score of Alanine, aspartate and glutamate metabolism pathway was calculated by GSVA algorithm, and Spearman correlation analysis was performed with the differentially expressed genes. The expression level of CCDC6 was significantly positively correlated with the activity of alanine, aspartate and glutamate metabolism pathway. The correlation coefficient was 0.732. e. The protein level of CCDC6 was found to be lower in 12 paired lung cancer tissue samples compared to neighboring normal lung tissue samples from western-blot analysis. f. Quantitative results of panel e. (****p \u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8712733/v1/2b542e957930be761acd01f4.png"},{"id":102963327,"identity":"1a800e11-dfff-4a7b-bef4-c93c78b5cfc1","added_by":"auto","created_at":"2026-02-19 04:15:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":975406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of CCDC6 inhibits the proliferation, migration, and clonal formation of lung adenocarcinoma cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea. Schematics of biological function detection of H1299 and A549 cells. b. Overexpression of CCDC6 inhibits the proliferation of lung adenocarcinoma cells. c. Overexpression of CCDC6 inhibits the colony formation ability of lung adenocarcinoma cells. d-f. Overexpression of CCDC6 inhibits the migration and invasion of lung adenocarcinoma cells. g-j. To investigate the capacity of A549 cells with stable CCDC6 overexpression to develop tumors (n = 8 mice per group), a tumor growth xenograft model was established. Tumor formation was tracked at image (g), the indicated times (h), and weight (i). (***p \u0026lt; 0.001, ****p \u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8712733/v1/922c3cde07cc9ce437c68b34.png"},{"id":102843996,"identity":"f59ac8f6-8a35-4fa9-8f91-48afa1b4f9b5","added_by":"auto","created_at":"2026-02-17 12:45:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1023794,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCDC6 deficiency promotes lung adenocarcinoma cell proliferation, migration and colony formation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea. Schematics of biological function detection of H358 cells. b. Knockdown of CCDC6 promoted the proliferation of lung adenocarcinoma cells. c. Knockdown of CCDC6 promotes the colony formation ability of lung adenocarcinoma cells. d-f. Knockdown of CCDC6 promotes the migration and invasion of lung adenocarcinoma cells. g-i. To assess the capacity of H358 cells with stable GPR162 deletion to produce tumors (n = 7 mice per group), a tumor growth xenograft model was established. Tumor formation was tracked at the times indicated (g), pictures (h) and weight (i) are shown. j. CCDC6 expression levels in tumor tissue were determined by western blot analysis. (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8712733/v1/bcc14d245452b5b2fe9ccf32.png"},{"id":102843990,"identity":"0e62b912-3a07-44a9-83ca-caa88dc00bf3","added_by":"auto","created_at":"2026-02-17 12:45:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1194481,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eASS1 interacts with CCDC6.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea-b. The interaction protein ASS1 of CCDC6 was identified by mass spectrometry. c-f. Co-IP was used to detect the interaction between ASS1 and CCDC6 in H358 and PC9 cells. g-h. Confocal was analysis the co-localization of ASS1 and CCDC6 in H358 and PC9 cells. i. Co-IP and Western-blot results showed that ASS1 point mutation did not affect the protein level of ASS1 and CCDC6, but the binding of CCDC6 to ASS1 was reduced. j. Co-IP and Western-blot results showed that the point mutation of CCDC6 did not affect the protein level of ASS1 and CCDC6, but the binding of CCDC6 to ASS1 was reduced after R55L and N394Y mutations. k-l. The clone formation ability of H1299 cells was detected after transient transfection of ASS1 and mutations. m. The proliferation ability of H1299 cells was detected after transient transfection of ASS1 and mutations. n-o. The migration and invasion ability of H1299 cells was detected after transient transfection of ASS1 and mutations. (*p \u0026lt; 0.05, ****p \u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8712733/v1/a3210bf43d32a02807cba55e.png"},{"id":102964087,"identity":"49d600dd-829a-406b-8a11-317704bfc1fc","added_by":"auto","created_at":"2026-02-19 04:21:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":868780,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCitrulline and aspartic acid promote ASS1 translocation to mitochondria.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea. Relationship between citrulline, aspartic acid and ASS1 in urea cycle. b-c. Unbiased metabolomics showed that Citr and Asp were significantly down-regulated in tumor tissues. d. Citrulline and aspartic acid promote ASS1 translocation to mitochondria. e-f. After treatment with Citr and Asp for 48 hours in H358 and H1299 cells, the cytoplasmic and mitochondrial proteins were isolated, and Western-blot was analysis the expression of ASS1 in the cytoplasm and mitochondria. g-j. The colocalization of ASS1 and Mitotracker after treatment with citrulline and aspartic acid in PC9 and H358 cells was statistically analyzed. k. ASS1 promotes mitochondrial fusion. l. Transmission electron microscopy showed that mitochondrial diameter became longer and ridge fusion increased after ASS1 overexpression in lung adenocarcinoma cells. m. Transmission electron microscopy results showed that the mitochondrial diameter became thicker and the fragmentation increased after ASS1 knockdown in lung adenocarcinoma cells. n-o. Statistical analysis of the number of mitochondria in the electron microscopy results. p-q. Statistical analysis of mitochondrial length in electron microscopy results. r-s. Western blot was analysis the protein levels of mitochondrial fission and fusion related molecules after ASS1 knockdown or overexpression. t-u. RT-qPCR was used to analyze the mRNA levels of mitochondrial fission and fusion related molecules after ASS1 knockdown or overexpression. (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8712733/v1/d64062e8441f330f79a72537.png"},{"id":102963388,"identity":"0e870f5f-3db1-471d-85fa-f51cda3da32b","added_by":"auto","created_at":"2026-02-19 04:16:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":819939,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eASS1 inhibits the degradation of MFN1/2 and OPA1 through ubiquitin-proteasome pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea. Combined with OTUD7A, an interaction protein of CCDC6 identified by mass spectrometry, it is speculated that ASS1 may stabilize mitochondrial fusion protein expression by recruiting OTUD7A, a deubiquitinating enzyme.b-c. Co-IP was used to detect the binding of endogenous ASS1 and OTUD7A in H358 and H1975 lung adenocarcinoma cell lines. d. After stable overexpression of ASS1 in H23 cells and addition of CHX, the degradation rate of MFN1/2 and OPA1 was significantly slowed down. e. After H23 cells were treated with proteasome inhibitor MG132, the promoting effect of ASS1 on the expression of MFN1/2 and OPA1 proteins disappeared. f. The degradation rate of MFN1/2 and OPA1 was significantly increased in H358 cell line after ASS1 knockdown and CHX treatment. g. After treatment with the proteasome inhibitor MG132 in H358 cells, the loss of ASS1 resulted in the accumulation of MFN1/2 and OPA1 proteins. h-k. After stable overexpression of ASS1 in H23 and H1299 cell lines, MG132 was pretreated for 6 hours, and immunoprecipitation was performed using MFN1/2 and OPA1 antibodies. The expression levels of Ubiquitin, ASS1, MFN1/2 and OPA1 proteins were detected by Western blot. After stable overexpression of ASS1 in H23 and H1299, the ubiquitination levels of MFN1/2 and OPA1 decreased.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-8712733/v1/bb71420398c3c278d79de894.png"},{"id":102843992,"identity":"e841f4c5-c128-49de-a200-aebd328e1637","added_by":"auto","created_at":"2026-02-17 12:45:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":415242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe CCDC6/ASS1 interaction axis disrupts mitochondrial homeostasis and promotes apoptosis of lung adenocarcinoma cells through mitochondrial pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea-c. Flow cytometry was used to detect the level of MitoROS after knocking down or overexpressing ASS1 in lung adenocarcinoma cells. d-f. Flow cytometry was used to detect the level of MitoROS in lung adenocarcinoma cells treated with citrulline and aspartic acid. g-i. Fluorescence microplate reader was used to detect the mitochondrial ATP level after ASS1 knockdown and overexpression in lung adenocarcinoma cells. j-l. Flow cytometry was used to detect the changes of MMP after ASS1 knockdown and overexpression in lung adenocarcinoma cells. m-o. Fluorescence microplate reader was used to detect the mitochondrial ATP level after CCDC6 knockdown and overexpression in lung adenocarcinoma cells. p-r. Flow cytometry was used to detect the changes of MMP after CCDC6 knockdown and overexpression in lung adenocarcinoma cells. s. Citrulline and aspartic acid combine to drive the translocation of ASS1 from cytoplasm to mitochondria. ASS1 regulates lung adenocarcinoma progression through a dual mechanism: 1. ASS1 recruited deubiquitinating enzyme OTUD7A to antagonize the ubiquitination and degradation of MFN1/2 and OPA1, thereby stabilizing the expression of mitochondrial fusion protein and promoting the dynamic balance of mitochondrial network; The specific interaction between ASS1 I281M and R55L/N394Y mutation of CCDC6 triggers mitochondrial dysfunction, which is characterized by accumulation of MitoROS, inhibition of ATP synthesis and decrease of MMP. Finally, ASS1 induces tumor cells to enter metabolic dormancy and inhibits the malignant progression of lung adenocarcinoma. (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-8712733/v1/76c0a7fdb4de5a31fa5a4350.png"},{"id":103049456,"identity":"65ca9c27-cf5d-4803-8ef7-ffd30ab5e531","added_by":"auto","created_at":"2026-02-20 07:41:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7200435,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8712733/v1/d812b6e5-8c6b-4c89-8eec-a28d13321d81.pdf"},{"id":102843987,"identity":"4db9279c-16b1-4bd9-994b-ce72e22b3cff","added_by":"auto","created_at":"2026-02-17 12:45:46","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":535000,"visible":true,"origin":"","legend":"original","description":"","filename":"unprocessedimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8712733/v1/033cd3f030b8addacaf2bc42.pdf"},{"id":102963290,"identity":"fcea558c-9fc1-4b7b-9fc4-3d2d162b608d","added_by":"auto","created_at":"2026-02-19 04:15:14","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4483394,"visible":true,"origin":"","legend":"Figure S3","description":"","filename":"SupFigS3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8712733/v1/1ce0badb71b0cd4cd943dc26.pdf"},{"id":102843989,"identity":"e8d34b99-3ab4-4286-8388-b1b4a3de5d5b","added_by":"auto","created_at":"2026-02-17 12:45:46","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4556695,"visible":true,"origin":"","legend":"Figure S5","description":"","filename":"SupFigS5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8712733/v1/18e16173a6f18b9aa5c740cf.pdf"},{"id":102843997,"identity":"b5685347-fcda-46a8-a67e-da7dbb9a338b","added_by":"auto","created_at":"2026-02-17 12:45:46","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3734747,"visible":true,"origin":"","legend":"Figure S1","description":"","filename":"SupFigS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8712733/v1/9f7dda9a6d117fc20d238592.pdf"},{"id":102843994,"identity":"f0c5772e-896a-4813-bdf3-ee53bf9fea12","added_by":"auto","created_at":"2026-02-17 12:45:46","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":7945592,"visible":true,"origin":"","legend":"Figure S4","description":"","filename":"SupFigS4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8712733/v1/2a80def9eb400b9f9f70dd4d.pdf"},{"id":102843998,"identity":"31cd742a-ce4d-4727-a9af-1a688dded1f9","added_by":"auto","created_at":"2026-02-17 12:45:46","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":12869749,"visible":true,"origin":"","legend":"Figure S2","description":"","filename":"SupFigS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8712733/v1/61bfcdfe20d94396daa43458.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"A Novel Role of the ASS1-CCDC6 Complex in Orchestrating Mitochondrial Dynamics to Suppress Lung Adenocarcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMetabolic reprogramming has garnered significant attention in the context of tumor development, endowing cancer cells with the capacity to survive and proliferate even under adverse conditions such as nutrient deprivation\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The identification of the 'Warburg effect' has transformed our understanding of the metabolic networks within cancer cells; specifically, it reveals that a majority of tumor cells predominantly utilize aerobic glycolysis for energy production, thereby fulfilling their material and energetic demands necessary for unrestricted proliferation, invasion, and metastasis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Furthermore, the dysregulation of anabolic and catabolic pathways involving fatty acids and amino acids\u0026mdash;particularly glutamine, serine, and glycine\u0026mdash;has been recognized as a crucial metabolic regulator that facilitates tumor cell proliferation\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Notably, there exists significant crosstalk between this dysregulated metabolic network and cellular signaling in tumor cells, which offers novel therapeutic strategies for addressing the malignant progression of lung adenocarcinoma through targeted modulation of tumor metabolism.\u003c/p\u003e \u003cp\u003eCCDC6 is located in the q21.2 region of human chromosome 10 and is a tumor suppressor gene involved in cell apoptosis and DNA damage response\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Its encoded protein is widely expressed in various tissues, it is a proapoptotic protein substrate of the ataxia telangiectasia mutated gene (ATM) family and participates in ATM-mediated cellular responses\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Since CCDC6-RET is the second most common fusion gene after KIF5B-RET, the research on CCDC6 is more about its involvement in the progression of various diseases as a fusion gene\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. However, the prognosis and biological function of CCDC6 in lung adenocarcinoma are still unclear, and the specific mechanism between CCDC6 and ASS1 remains to be further studied.\u003c/p\u003e \u003cp\u003eASS1, also known as ASS or CTLN1, encodes 412 amino acids and is the rate-limiting enzyme for arginine production\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Several studies have shown that ASS1 has a close and complex relationship with tumors, and its differential expression may lead to different tumor outcomes\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Studies have shown that the high expression or continuous activation of ASS1 can promote the proliferation and invasion of tumor cells in gastric cancer and colorectal cancer\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, ASS1 inhibited cell proliferation and migration in HCC and breast cancer cells\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The mechanism of this dual action of ASS1 in different tumors is unknown. We focused on the mitochondrial localization of ASS1 in lung adenocarcinoma cells, which increased with the treatment of its substrates citrulline and aspartate, suggesting that ASS1 may have a urea cycle-dependent mitochondrial function in lung adenocarcinoma cells.\u003c/p\u003e \u003cp\u003eCitrulline is a neutral α-amino acid formed by nitric oxide synthase in mitochondria\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. It is a precursor of arginine and nitric oxide. It is a non-essential, non-protein amino acid (AA)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. It has been proven that the post-translational modification of arginine (known as argininization or deamination) plays an important role, it has a variety of effects such as anti-oxidation, immune regulation, cardiovascular protection, intestinal protection and neuroprotection, which is beneficial to human health\u003csup\u003e\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In cells, citrulline is not one of the 20 amino acids encoded by DNA that make up proteins. Citrulline is mainly produced by ornithine and carbamoyl phosphate during the Urea Cycles and by nitric oxide synthase (NOS) as a by-product of arginine to NO\u003csup\u003e16\u003c/sup\u003e. Aspartic acid is an acidic amino acid, which is the precursor for the synthesis of a variety of amino acids (lysine, methionine, etc.)\u003csup\u003e22\u003c/sup\u003e. It has been reported that aspartic acid can play the role of a \"rate-limiting device\" in tumors, it is mainly involved in triggering the two most important metabolic pathways of cells\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e: Krebs Cycles and urea cycles. In the Krebs cycle, carbohydrates are broken down for energy, and aspartate helps deliver energy into mitochondria, as both citrulline and aspartic acid are precursors of arginine synthesis in the urea cycle, their cell biological functions are often abbreviated as ammonia detoxification of urea cycle, or indirectly as their downstream product arginine\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. However, its direct biological effects on cells are rarely reported.\u003c/p\u003e \u003cp\u003eDeubiquitinating enzymes are divided into several families according to their structural characteristics, among which the Ovarian Tumor Domain proteases (OTUs) family has attracted much attention due to their specificity in ubiquitin chain editing. OTU Deubiquitinase 7A (OTUD7A) is an important member of the deubiquitinase family. As a highly specific ubiquitin-editing enzyme, OTUD7A can precisely regulate the ubiquitination status of target proteins. It is involved in key biological processes such as cell signal transduction, immune response, apoptosis regulation and neurodevelopment\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The dynamic equilibrium of ubiquitination is maintained by ubiquitin ligase (E3) and deubiquitinating enzymes\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, OTUD7A significantly affects the maturation and activity of proteasomes by regulating the biogenesis and assembly of proteasomes, thereby profoundly regulating the protein homeostasis of cells\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn our study, we found that citrulline in combination with aspartic acid drives ASS1 translocation from cytoplasm to mitochondria; ASS1 regulates lung adenocarcinoma progression through a dual mechanism: 1. ASS1 recruited deubiquitinating enzyme OTUD7A to antagonize the ubiquitination and degradation of MFN1/2 and OPA1, thereby stabilizing the expression of mitochondrial fusion protein and promoting the dynamic balance of mitochondrial network; The specific interaction between ASS1 I281M and R55L/N394Y mutation of CCDC6 triggers mitochondrial dysfunction, which is characterized by accumulation of MitoROS, inhibition of ATP synthesis and decrease of MMP. Finally, ASS1 induces tumor cells to enter metabolic dormancy and inhibits the malignant progression of lung adenocarcinoma.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture, viruses, stimulation, and transfection\u003c/h2\u003e \u003cp\u003eIn this study, we established the following cell culture conditions: 293T cell lines were maintained in DMEM (Gibco, NY, USA) medium, while A549 (ATCC: CCL-185) cell lines were grown in a 1:1 mixture of DME and F12 (HyClone, UT, USA) medium. The cell lines PC9, H1299 (ATCC: CRL-5803), H358, H23, and H1975 were cultured in RPMI1640 (Gibco) medium. All cells were incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e, supplemented with 10% (v/v) BCS. The cell lines were procured from the cell bank of the Cancer Institute at Central South University. Supplementary Table\u0026nbsp;1 lists the sgRNA and shRNA sequences for ASS1 and CCDC6 utilized in this research. The constructed plasmid was introduced into the cells and transfected using Lipofectamine Max. Stable expression colonies were selected using puromycin at a concentration of 1 \u0026micro;g/ml.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern blot analysis and coimmunoprecipitation (Co-IP) assay\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blot analysis and coimmunoprecipitation (Co-IP) assay\u003c/div\u003e \u003cp\u003eWestern blot analysis is a technique used to detect specific proteins in a sample. The process begins with preparing cell lysates to extract proteins, which are then separated using SDS-PAGE based on their size. After the proteins are transferred to a membrane, it is blocked to prevent non-specific binding. The membrane is incubated with a primary antibody that specifically recognizes the target protein, followed by a washing step to remove unbound antibodies. A secondary antibody, which is conjugated to an enzyme or dye, is then applied, and after another wash, a substrate is added to produce a signal. The resulting bands are visualized and quantified to assess protein expression levels.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCoi\u003c/strong\u003e \u003cp\u003emmunoprecipitation (Co-IP) is a method used to study interactions between proteins. The procedure starts with lysing cells to extract proteins, followed by centrifugation to clear the lysate. An antibody specific to the protein of interest is added and incubated to bind the target protein. Then, protein A or G beads are introduced to capture this antibody-protein complex. After several washes to remove non-specific interactions, the bound proteins are eluted from the beads. The eluted samples can be analyzed using Western blotting to confirm the presence of interacting proteins, providing insights into their associations within the cell. Supplementary Table\u0026nbsp;2 lists primary antibodies used in Western blot analysis.\u003c/p\u003e \u003c/p\u003e\n\u003ch3\u003eMice\u003c/h3\u003e\n\u003cp\u003eMice were kept in a pathogen-free environment, with some being sourced from Hunan SJA Laboratory Animal Co., LTD. (Changsha, China) as needed. They were fed an irradiated PicoLab rodent diet (10 kcal%, D12450J) and all subjects were female nude mice. The animal studies received approval from the Institutional Animal Care and Use Committee of Xiangya School of Medicine, Central South University, and were conducted in compliance with relevant legislation and federal animal care guidelines. Subcutaneous injections of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per mouse were administered, consisting of ASS1 and CCDC6 overexpression or knockdown cells, along with control cells, into the axilla of each mouse. Tumor volume and mouse weight were assessed every three days until the mice were sacrificed on day 27. Following euthanasia, tumors were weighed, photographed, and lysed for Western blot analysis.\u003c/p\u003e\n\u003ch3\u003eReal-time quantitative polymerase chain reaction (RT-qPCR)\u003c/h3\u003e\n\u003cp\u003eReal-time quantitative polymerase chain reaction (qPCR) is a technique used to amplify and quantify DNA sequences, typically aimed at measuring gene expression levels. The process begins by extracting RNA from cells or tissues, which is then reverse-transcribed into complementary DNA (cDNA). A reaction mix is prepared containing the cDNA, specific primers for the target gene, and a fluorescent dye or probe that emits a signal during DNA amplification. The mixture is placed in a real-time PCR machine that cycles through various temperatures to facilitate DNA synthesis while continuously monitoring fluorescence. The threshold cycle (Ct) value is recorded for each sample, indicating the amount of target DNA present; lower Ct values reflect higher quantities. Finally, data is analyzed by comparing Ct values to a housekeeping gene to determine relative gene expression across different samples or conditions, providing insights into biological processes. TRIzol reagent (Takara, Kusatsu, Japan) was used to isolate the total RNA, and the kit (Takara, Kusatsu, Japan) was used to reverse transcribe the RNA into cDNA. Real-time PCR was performed on a Bio-rad CFX Connect real-time PCR instrument. β-actin served as the internal reference for gene expression. The primers used in this investigation are listed in Supplementary Table\u0026nbsp;3.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence microscopy\u003c/h3\u003e\n\u003cp\u003eA 24-well culture plate containing small glass discs was prepared with logarithmically growing cells 24 hours prior to adding adherent cells. The culture plate was removed once the cells reached approximately 50% confluence. After washing three times with 1\u0026times; PBS, 1 mL of methanol was added to each well to fix the cells for 10 minutes at room temperature. Following two 5-minute rinses with PBS, the wells were blocked for 30 minutes using 1% (w/v) BSA in PBS. The primary antibodies, also diluted in 1% BSA, were incubated overnight at 4\u0026deg;C. Depending on the primary antibody type, either anti-rabbit IgG Alexa 594 or anti-mouse IgM Alexa 488 fluorescent secondary antibodies were used and incubated for one hour after rinsing with PBS. After DAPI labeling, the samples were mounted onto slides and imaged using a Leica TCS SP8 confocal microscope. For live cell staining, MitoTracker\u0026reg; Deep Red FM (Invitrogen, 644\u0026ndash;665 nm, M22426) was applied in a cell incubator set at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 30 minutes.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSeparation of the cytoplasmic and mitochondria\u003c/h2\u003e \u003cp\u003eThe Cell Mitochondria Isolation Kit (Beyotime, C3601) was employed to isolate mitochondrial proteins from the cells. After washing the collected cells three times with 1\u0026times;PBS, they were lysed on ice for fifteen minutes using a mitochondrial separation reagent supplemented with PMSF. The lysate was then transferred into an appropriately sized glass homogenizer and homogenized for ten to thirty cycles. Subsequent centrifugation of the homogenate resulted in the pelleting of cellular mitochondria in the precipitate while cytoplasmic proteins remained in the supernatant. The protein concentration was subsequently determined using the BCA assay.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTransmission electron microscopy (TEM)\u003c/h3\u003e\n\u003cp\u003eFollowing the collection of ASS1 overexpression and knockdown cells through trypsin digestion, the cells were washed twice with PBS. Subsequently, 1 mL of stationary solution was gently added along the walls of the centrifuge tube to avoid disturbing the cell pellet, and the tubes were then kept at 4\u0026deg;C overnight. Images were captured using a transmission electron microscope (Hitachi; HT7700) at the Department of Pathology, Xiangya Hospital.\u003c/p\u003e\n\u003ch3\u003eMitochondrial ROS (MitoROS)\u003c/h3\u003e\n\u003cp\u003eSeed cells in a culture dish, and wait until the cells are fully adhered and in good condition, with a confluence of 70%-80%. Digest the cells with trypsin, centrifuge at 800 rpm for 5 minutes, and wash with PBS twice. Take 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells from each well and resuspend them in serum-free culture medium. Prepare the MitoSOX working solution (Invitrogen, M36008), incubate in a dark incubator for 20 minutes, then centrifuge at 800 rpm for 5 minutes, discard the supernatant, resuspend in 200 \u0026micro;L of 1\u0026times; PBS, and filter through a cell strainer. Finally, use flow cytometry to detect the mitochondrial ROS levels in the cells.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial Membrane Potential (MMP)\u003c/h2\u003e \u003cp\u003eJC-1 is a widely used fluorescent probe for detecting mitochondrial membrane potential. When the membrane potential is high, JC-1 accumulates in the mitochondria and forms aggregates, resulting in red fluorescence; when the membrane potential is low, JC-1 exists as monomers, producing green fluorescence.\u003c/p\u003e \u003cp\u003eSeed the cells in a culture dish and wait until the cells are fully adhered and in good condition, with a confluence of 70%-80%. Discard the culture medium and wash twice with PBS. Add 500 \u0026micro;L of staining working solution (Beyotime, C2006) and incubate in a dark incubator for 20 minutes. After incubation, discard the supernatant, wash twice with JC-1 staining buffer, and resuspend in 200 \u0026micro;L of staining buffer. Finally, use flow cytometry to detect the mitochondrial membrane potential.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAdenosine 5'-triphosphate(ATP)\u003c/h2\u003e \u003cp\u003eSeed the cells in a 6-well plate and wait until the cells are fully adhered and in good condition, with a confluence of 70%-80%. Discard the culture medium and wash twice with PBS. Add 200 \u0026micro;L of ATP detection lysis buffer (Beyotime, S0027) to each well, and thoroughly lyse the cells on ice for 5 minutes. Transfer the cell suspension to a 1.5 mL centrifuge tube and centrifuge at 12,000 rcf for 5 minutes at 4\u0026deg;C, keeping the supernatant. In a 96-well plate, add 100 \u0026micro;L of ATP detection working solution and let it sit at room temperature for 5 minutes. Prepare ATP standard solutions (0, 0.01, 0.03, 1, 3, 10 \u0026micro;M concentration gradient) and add the cell supernatant and standard solutions to the ATP detection working solution. Measure the fluorescence using a microplate reader. After calculating the ATP concentration based on the standard curve, normalize it to the corresponding protein concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnnexin V/PI\u003c/h2\u003e \u003cp\u003eCollect 1 \u0026times; 10^6 cells and wash them twice with pre-chilled PBS. Resuspend the cells in 500 \u0026micro;L of Apoptosis Positive Control Solution (MultiSciences, AP105) and incubate on ice for 30 minutes. Wash the cells with pre-chilled PBS and discard the supernatant. Resuspend the cells in 500 \u0026micro;L of Binding Buffer and add 1 \u0026times; 10^6 untreated live cells. Complete the volume to 1.5 mL with Binding Buffer and divide into three tubes: one will be the blank control and two will be the single-staining tubes. Add 5 \u0026micro;L of Annexin V-APC or 10 \u0026micro;L of 7-AAD to the single-staining tubes and incubate at room temperature in the dark for 5 minutes.\u003c/p\u003e \u003cp\u003eOn the flow cytometer, adjust the voltage for the blank tube and set the fluorescence channels for the single-staining tubes. Use trypsin (without EDTA) to digest the cells, collect the cell suspension and supernatant, and wash twice with pre-chilled PBS. Take 1 \u0026times; 10^5 cells, resuspend in 500 \u0026micro;L of Binding Buffer, and add 5 \u0026micro;L of Annexin V-APC and 10 \u0026micro;L of 7-AAD to each tube, mixing well. Incubate at room temperature in the dark for 5 minutes, then analyze by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eAll studies, except those involving mice, were conducted a minimum of three times, and the results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD or SEM. Statistical analyses were performed using GraphPad Prism 9.0. A T-test was employed to evaluate the significance of differences between two groups, while analysis of variance (ANOVA) was used for comparisons involving more than two groups. The Pearson correlation coefficient was applied for correlation analyses. Statistical significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, with levels of significance indicated as follows: *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStudy approval\u003c/h2\u003e \u003cp\u003e The project was approved by the ethics committee at our hospital, and the institutional Animal Care and Use Committee at Central South University granted approval for the use of animal models in this study. Additionally, the study received approval from the institutional review boards of all participating medical facilities. Prior to recruitment, each research subject provided written informed consent.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCombined multi-omics analysis identified CCDC6 as a key regulatory target in lung adenocarcinoma.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn order to explore new therapeutic targets and intervention strategies for lung adenocarcinoma, we collected 34 pairs (Table\u0026nbsp;4) of lung adenocarcinoma clinical tissue samples for combined untargeted metabolomics and transcriptomics analysis, and screened differentially expressed metabolites and genes by OPLSADA and limma (Linear Models for Microarray Data ) analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The results of non-targeted metabolomics analysis showed that the overall levels of amino acids were significantly down-regulated in tumor tissues, among which citrulline and aspartic acid were most significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), suggesting that citrulline may be involved in tumor metabolic reprogramming as energy substances.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the results of metabolomics data, the down-regulated differentially expressed metabolites in tumor tissues were screened and analyzed by the MetaboAnalyst platform (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), and the KEGG pathway enrichment analysis of differentially expressed genes was further performed on the transcriptome results. ClusterProfiler package was used for functional annotation (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea), and the results showed that \"Alanine, aspartate and glutamate metabolism\" pathway was significantly down-regulated at both the metabolome and transcriptome levels. Further, gene set variation analysis (GSVA) was used to quantify pathway activity scores in tumor samples, and Spearman correlation analysis was performed with differential genes (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb). After correction for multiple testing, it was found that the expression level of CCDC6 was significantly positively correlated with the activity of the \"Alanine, aspartate and glutamate metabolism\" pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). To further verify the expression of CCDC6 in lung adenocarcinoma tissues, we collected 12 pairs of tumor tissues and paired adjacent tissues from lung adenocarcinoma patients. The results of Western blot showed that the protein expression level of CCDC6 was significantly decreased in lung adenocarcinoma tissues compared with adjacent normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-f). These results suggest that CCDC6 may be a key regulatory target in lung adenocarcinoma.\u003c/p\u003e \u003cp\u003eIn order to clarify its clinical significance and biological function in lung adenocarcinoma cells and its relationship with citrulline and aspartic acid, we first treated the cells with citrulline and aspartic acid, and detected the biological function of lung adenocarcinoma cells by CCK8 assay, colony formation assay, and transwell migration assay. The results showed that the combination of citrulline and aspartic acid promoted the proliferation, migration and colony formation ability of lung adenocarcinoma cells (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec-i). Next, we examined the protein levels of CCDC6 in different lung adenocarcinoma cell lines (Fig.\u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ee). We selected A549 and H1299 cell lines with low expression of CCDC6 and stably overexpressed CCDC6 using lentiviral vector. shRNA technology was used to knock down CCDC6. As expected, the cell proliferation was much lower in CCDC6 overexpressed cells than that of control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-b). Furthermore, overexpression of CCDC6 dramatically decreased cell colony formation, migration, and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-f). A xenograft model experiment was used to further investigate the influence of CCDC6 on tumor development in vivo. The injection of A549 cells overexpressing CCDC6 can drastically Inhibit tumor growth, volume, and weight when compared to the injection of control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg-i). The expression of CCDC6 protein in subcutaneous tumor tissues of nude mice was detected by western-blot. The results further confirmed that CCDC6 inhibited lung adenocarcinoma cell progression \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilarly, the physiological effects of CCDC6 knockdown on lung adenocarcinoma cells were examined, and the results showed that the above malignant phenotypes were significantly enhanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-j). In general, these results indicate that the absence of CCDC6 significantly promotes the occurrence and development of tumors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eASS1 specifically binds to CCDC6 to inhibit the progression of lung adenocarcinoma.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further explore the mechanism by which CCDC6 regulates lung adenocarcinoma progression, we analyzed the proteins interacting with CCDC6 in H1975 cells by mass spectrometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The results showed that CCDC6 and ASS1 co-localized and interacted in the cytoplasm of lung adenocarcinoma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Co-IP assay was used to verify the endogenous interaction between ASS1 and CCDC6 in H358 and PC9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-f). Confocal laser scanning microscopy was used to confirm the co-localization of ASS1 and CCDC6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg-h). These findings suggest an interaction between CCDC6 and ASS1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the clinical data and transcriptome data of 538 lung adenocarcinoma patients included in the TCGA database, the correlation between ASS1 expression level and clinical prognosis of patients was systematically evaluated. Kaplan-Meier survival analysis showed that compared with ASS1 low expression group, ASS1 high expression group showed better clinical outcomes, and OS and DFS were significantly improved (Log-rank test P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ea-b). This statistically significant difference suggests that the high expression status of ASS1 may serve as a potential molecular marker for good prognosis in patients with lung adenocarcinoma.\u003c/p\u003e \u003cp\u003eAfter establishing the stable overexpression model of ASS1 in H23 and H1299 lung adenocarcinoma cell lines (Fig.\u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ec-d), CCK8 cell proliferation assay and colony formation assay showed that compared with the control group, the overexpression of ASS1 significantly inhibited the proliferation (Fig.\u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ee-f) and decreased the colony formation ability of lung adenocarcinoma cells (Fig.\u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eg-h). Transwell migration and invasion assays showed that ASS1 overexpression significantly inhibited the migration and invasion ability of lung adenocarcinoma cells compared with the control group (Fig.\u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ei-j). ASS1 stably overexpressing H1299 cell line was used to establish a subcutaneous xenograft tumor model in nude mice. The results showed that compared with the control group, the growth of transplanted tumors in the ASS1 overexpression group was significantly inhibited, and the final tumor volume and tumor weight were significantly reduced (Fig.\u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ek-n). These in vivo data further confirmed the tumor suppressor function of ASS1 in lung adenocarcinoma progression, which was consistent with the in vitro results.\u003c/p\u003e \u003cp\u003eSimilarly, the physiological effects of ASS1 knockdown on lung adenocarcinoma cells were examined, and the results showed that the above malignant phenotypes were significantly enhanced (Fig.\u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003ea-j). In general, these results indicate that the absence of ASS1 significantly promotes the occurrence and development of tumors.\u003c/p\u003e \u003cp\u003eIn order to clarify the interaction mechanism between ASS1 and CCDC6, we screened the high-frequency mutation sites of ASS1 and CCDC6 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cbioportal.org/\u003c/span\u003e\u003cspan address=\"https://www.cbioportal.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and constructed ASS1 (R86S, V103L, K165E, I281M, R404H) and CCDC6 (R55L, E160Q, D236N, N394Y) point mutation plasmids. Western blot analysis showed that none of these point mutations significantly affected the protein expression levels of ASS1 or CCDC6. However, the Co-IP assay showed that the binding ability of ASS1 mutants to CCDC6 was significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). Similarly, when CCDC6 mutants (R55L, E160Q, D236N, N394Y) were transfected, although the protein expression of CCDC6 and ASS1 was not affected, R55L and N394Y mutations significantly weakened the interaction between CCDC6 and ASS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej).\u003c/p\u003e \u003cp\u003eTo further explore the functional significance of these mutation sites, a cell model stably overexpressing ASS1 and its mutants was constructed in H1299 cell line. The results showed that overexpression of wild-type ASS1 significantly inhibited the proliferation ability, colony formation rate, migration and invasion ability of lung adenocarcinoma cells compared with the control group. However, these inhibitory effects were significantly reversed by ASS1 I281M mutation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek-o). These results not only confirm the tumor suppressor function of ASS1, but more importantly reveal the decisive role of I281M as the key site of ASS1-CCDC6 interaction in maintaining the tumor suppressor activity of ASS1. This study provides an important experimental basis for further understanding the molecular mechanism of ASS1-CCDC6 in lung adenocarcinoma.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe combination of citrulline and aspartic acid changes the metabolic microenvironment of ASS1 localization and reprogramming.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBased on the classical metabolic pathway of urea cycle, citrulline is synthesized in mitochondria and transferred to the cytoplasm through specific transporters. Citrulline and aspartic acid act as a substrate to produce arginine succinate under the catalysis of ASS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Metabolomics data showed that the expression of citrulline and aspartic acid in tumor tissues was significantly lower than that in adjacent tissues, suggesting that citrulline and aspartic acid may regulate the progression of lung adenocarcinoma by regulating the expression of ASS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-c). We therefore examined ASS1 expression in the cytoplasm and mitochondria after cells were treated with citrulline and aspartic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Western blot showed that the expression of ASS1 protein in mitochondrial fraction was significantly up-regulated in citrullinate-aspartic acid combined treatment group compared with untreated control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee-f). Confocal analysis further confirmed the above findings, the co-localization signal of ASS1 with the mitochondria-specific probe MitoTracker Red was significantly enhanced after 48 hours of combined treatment with citrulline and aspartic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg-j, Fig.\u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ea-d). These results confirmed the role of citrullinate-aspartic acid metabolism axis in promoting ASS1 translocation to mitochondria from the perspective of subcellular spatial distribution.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eASS1 recruits OTUD7A to stabilize mitochondrial fusion protein and promote mitochondrial fusion.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBased on the previous study that found a large number of ASS1 translocated to mitochondria, we hypothesized that ASS1 may have mitochondrial related functions in addition to the key enzymes of urea cycle in lung adenocarcinoma. Considering that mitochondria are highly dynamic organelles that maintain normal morphology and function through continuous fusion and division processes, we further explored the effect of ASS1 on mitochondrial dynamics (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek). By observing ASS1 knockdown and overexpression in lung adenocarcinoma cell lines by transmission electron microscopy, we found that mitochondrial fission in ASS1 knockdown cells was significantly increased, and the average diameter of mitochondria was significantly shorter than that in the control group. In contrast, after overexpression of ASS1, mitochondria showed a pronounced elongation phenotype, with a significant increase in average length compared with the control group(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003el-m). These results suggest that ASS1 may play a role in inhibiting lung adenocarcinoma progression by regulating mitochondrial dynamics and promoting mitochondrial fusion. Statistical results also showed that the number of mitochondria decreased and the average length increased after overexpression of ASS1, and conversely, the number of mitochondria increased and the average length decreased after knockdown of ASS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003en-q).\u003c/p\u003e \u003cp\u003eTo further explore the molecular mechanism of ASS1 in regulating mitochondrial dynamics, the effects of ASS1 knockdown and overexpression on the expression levels of mitochondrial fission and fusion related proteins were detected by Western blot. The results showed that ASS1 knockdown significantly inhibited the expression of mitochondrial fusion proteins (MFN1/2 and OPA1). In contrast, ASS1 overexpression significantly upregulated the expression levels of these fusion proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003er-s). It is worth noting that these protein expression changes were not accompanied by significant changes in their mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003et-u). These results suggest that ASS1 may affect the mitochondrial fusion process by promoting the expression of mitochondrial fusion-related proteins through post-translational modification machinery rather than regulation at the transcriptional level.\u003c/p\u003e \u003cp\u003eIn order to explore the molecular mechanism of ASS1 regulating mitochondrial fusion proteins (MFN1/2, OPA1), based on the perspective of protein post-translational modification regulation, OTUD7A was identified as a key clue in the CCDC6 interaction proteome found by previous mass spectrometry analysis. It was speculated that ASS1 may recruit the deubiquitinating enzyme OTUD7A to stabilize mitochondrial fusion protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Firstly, the direct interaction between ASS1 and OTUD7A was confirmed by Co-IP using H358 and H1975 lung adenocarcinoma cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-c). To further explore the molecular mechanism by which ASS1 regulated mitochondrial fusion proteins (MFN1/2 and OPA1), We treated the H23 cell line overexpressing ASS1 with CHX and MG132. The results showed that the degradation rate of MFN1/2 and OPA1 was slowed in the overexpression group after CHX treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). After MG132 treatment, the expression differences of MFN1/2 and OPA1 between the empty vector group and the overexpression group disappeared, and the two groups had the same intensity of protein accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Next, H358 ASS1 knockdown cells were treated with CHX and MG132. The results showed that after CHX treatment, the degradation rate of MFN1/2 and OPA1 was significantly increased after ASS1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). The accumulation of MFN1/2 and OPA1 proteins in ASS1 knockdown group was more significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further explore the molecular mechanism of ASS1 regulating mitochondrial fusion protein ubiquitination. After MG132 pretreatment in H23 and H1299 cell lines with stable overexpression of ASS1, the ubiquitination modification levels of MFN1/2 and OPA1 were decreased after overexpression of ASS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh-k). The above results confirmed that ASS1 stabilized the expression of mitochondrial fusion proteins (MFN1/2 and OPA1) by recruiting OTUD7A, changing the mitochondrial morphology and promoting mitochondrial fusion.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe CCDC6/ASS1 interaction axis disrupts mitochondrial homeostasis and promotes apoptosis of lung adenocarcinoma cells through mitochondrial pathway.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have confirmed that ASS1 affects mitochondrial dynamics by recruiting the deubiquitination enzyme OTUD7A. To further explore the effect of ASS1 on mitochondrial function, we used flow cytometry and fluorescent enzyme labeling to detect the changes of MitoROS, ATP production and MMP. The results showed that overexpression of ASS1 significantly increased MitoROS level in lung adenocarcinoma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb-c), while ATP production was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh-i). In addition, JC-1 staining results showed that overexpression of ASS1 caused a significant decrease in MMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ek-l). In ASS1 knockdown lung adenocarcinoma cells, MitoROS level was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea), accompanied by increased ATP production (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg) and significantly increased MMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ej). However, treatment of cells with citrulline and aspartic acid alone did not affect intracellular mitochondrial ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed-f). These results suggest that ASS1 may affect the balance of mitochondrial dynamics by increasing the level of mitochondrial oxidative stress, inhibiting energy metabolism and destroying the stability of mitochondrial membrane potential, and ultimately inhibit the growth and proliferation of lung adenocarcinoma cells. This finding provides a new experimental basis for elucidating the mechanism by which ASS1 acts as a tumor suppressor by regulating mitochondrial function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on previous studies that confirmed the protein-protein interaction between ASS1 and CCDC6 and that ASS1 can inhibit lung adenocarcinoma progression by regulating mitochondrial dynamics, we further explored the effect of CCDC6 on mitochondrial function. The results showed that ATP production and MMP were significantly decreased in lung adenocarcinoma cells overexpressing CCDC6. In contrast, in CCDC6 knockdown lung adenocarcinoma cells, ATP production was significantly increased, accompanied by a decrease in MMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003em-r). These results suggest that CCDC6 may be involved in the regulation of lung adenocarcinoma progression by regulating mitochondrial energy metabolism and maintaining membrane potential stability, affecting mitochondrial dynamic balance. Combined with the interaction between ASS1 and CCDC6, we hypothesized that ASS1 may inhibit the malignant progression of lung adenocarcinoma by forming functional protein complexes with CCDC6 and disrupting mitochondrial homeostasis.\u003c/p\u003e \u003cp\u003eIn order to further explore the molecular mechanism of ASS1-CCDC6 complex inhibiting the development of lung adenocarcinoma, since previous studies have found that ASS1 and CCDC6 can affect mitochondrial homeostasis, combined with the close relationship between mitochondrial dynamics and apoptosis, a scientific hypothesis is proposed: CCDC6-ASS1 interaction may determine the fate of lung adenocarcinoma cells by regulating the apoptotic signaling pathway. Western blot analysis showed that in the CCDC6 overexpression group, the expression of pro-apoptotic protein Bax was significantly increased, while the expression of anti-apoptotic protein Bcl-xl was significantly decreased. In contrast, a trend of decreased Bax expression and increased Bcl-xl expression was observed in the CCDC6 knockdown group (Fig.\u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eg-i). Consistent with the results of CCDC6 assay, ASS1 overexpression significantly up-regulated the expression level of pro-apoptotic protein Bax and inhibited the expression level of anti-apoptotic protein Bcl-xl. On the contrary, when ASS1 was knocked down, the expression level of Bax protein was significantly decreased, while the expression of Bcl-xl protein was significantly increased (Fig.\u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003ej-l). These findings suggest that the ASS1/CCDC6 protein complex may activate the mitochondrial pathway of apoptosis by regulating the expression balance of Bcl-2 family proteins. This study not only provides new experimental evidence for in-depth understanding of the biological function of ASS1/CCDC6 complex, but also provides an important theoretical basis for elucidating the molecular mechanism of ASS1/CCDC6 complex as a tumor suppressor.\u003c/p\u003e \u003cp\u003eNext, we used flow cytometry combined with Annexin V/PI double staining to quantify the level of apoptosis by detecting the extroversion of phosphatidylserine (PS) on the cell membrane surface. The results showed that overexpression of CCDC6 significantly increased the proportion of Annexin V-positive cells in lung adenocarcinoma cells compared with the control group. Further analysis revealed that both the proportion of early apoptotic cells (Annexin V+/PI-) and late apoptotic cells (Annexin V+/PI+) showed a significant increase (Fig,\u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003ea-b). Similarly, quantitative analysis showed that ASS1 overexpression significantly increased the proportion of early apoptotic cells (Annexin V+/PI-) compared with the control group, while the proportion of late apoptotic cells (Annexin V+/PI+) did not show a statistically significant change (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-d). This experimental phenomenon of specifically inducing early apoptosis suggests that ASS1 may participate in the regulatory process of inhibiting the malignant progression of lung adenocarcinoma by activating key signaling pathways in the initiation stage of apoptosis.\u003c/p\u003e \u003cp\u003eIn this study, we identified a key regulatory target of lung adenocarcinoma, CCDC6, and demonstrated that CCDC6 specifically binds to ASS1. Citrulline and aspartic acid were found to drive the metabolic reprogramming of lung adenocarcinoma by promoting the translocation of ASS1 in mitochondria. Finally, CCDC6 and ASS1 promote the apoptosis of lung adenocarcinoma cells and inhibit the progression of lung adenocarcinoma by recruiting the deubiquitinating enzyme OTUD7A to stabilize mitochondrial fusion protein and destroy mitochondrial homeostasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003es).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe dynamics of mitochondrial division and fusion are crucial for the formation and growth of tumors, and they also play a vital biological function in promoting the invasion and metastasis of tumor cells through energy metabolism and biosynthetic metabolism\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Mitochondria are extremely dynamic organelles that constantly fuse and fission to preserve their structure and functionality\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. On the one hand, mitochondria exist in a dynamic equilibrium between fission and fusion under normal physiological conditions. The process of mitochondrial fission is essential for preserving cell growth and mitosis because it produces a suitable number of mitochondria, isolates irreparable damage within them, and allows for the mobility and redistribution of mitochondria. On the other hand, defective components within the mitochondria are replaced by a mixture of healthy and damaged mitochondria through a process known as mitochondrial fusion\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The cell is somewhat protected by this process, which encourages material interchange and energy conversion amongst the constituents.\u003c/p\u003e \u003cp\u003eRecent studies have shown that the localization of ASS1 in cells can lead to its different functions\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, ASS1 acetylation status in endothelial cells and its enzymatic activity in arginine biosynthesis regulate vascular homeostasis. Although a large number of previous studies have found that ASS1 downregulation is associated with poor prognosis, some groups still found that ASS1 expression in the nucleus is increased after DOX-induced DNA damage in colon cancer cells and fibroblasts, which maintains genomic stability and is dependent on P53 regulation\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Other studies have found that loss of ASS1 can increase the sensitivity of non-small cell lung cancer cells to Erastin in vitro and inhibit the growth of lung adenocarcinoma in vivo\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In this paper, the authors mainly focused on the sensitivity of ASS1 to erastin and interestingly confirmed that loss of ASS1 can promote ferroptosis in lung adenocarcinoma cells. It is interesting to note that the xenograft tumor experiments in the authors' study were performed in A549 cell line, which is P53 wild-type. In our study, we used H358 and H1299 cell lines, both of which are P53 null. This aroused our great interest, and it remains to be further investigated whether mutation or deletion of P53 regulates ASS1-induced tumor progression.\u003c/p\u003e \u003cp\u003eIn our study, we found a large translocation of ASS1 from the cytoplasm to mitochondria after treatment with Citr and Asp, which aroused our interest and concern because mitochondria are the key platform of a large number of cells signaling cascades, not only affecting and often coordinating metabolism, but also coordinating the timing of complex cell signaling and participating in the regulation of cell pluripotency. Such as division, differentiation, senescence, death, etc. Therefore, we wanted to further investigate the function of its translocation to mitochondria. In this connection, we observed the effect of ASS1 on mitochondrial morphology in lung adenocarcinoma cells by transmission electron microscopy, and the results showed that ASS1 can promote mitochondrial fusion, suggesting that ASS1 may have a new function of remodeling mitochondrial morphology.\u003c/p\u003e \u003cp\u003eIn addition, protein-protein interaction plays a crucial role in the study of cell signaling network, which is a prerequisite for affecting the physiological and pathological changes of cells. CCDC6 has been recognized as a fusion protein, and the CCDC6-RET fusion is also a key target for cancer therapy. However, the function of CCDC6 in lung adenocarcinoma cells and the mechanism by which CCDC6 affects tumor progression have not been reported. The interaction between CCDC6 and ASS1 can promote mitochondrial ROS production by promoting mitochondrial fusion, and ultimately promote cell apoptosis by reducing mitochondrial ATP and down-regulating mitochondrial membrane potential, and inhibit the progression of lung adenocarcinoma.\u003c/p\u003e \u003cp\u003eIn this study, the interaction domain between ASS1 and CCDC6 was characterized, and the function of its mutants in lung adenocarcinoma cells was analyzed. Although these point mutations did not significantly change the protein expression levels of ASS1 and CCDC6, co-immunoprecipitation assay showed that these point mutations significantly reduced the binding ability of ASS1 to CCDC6, which revealed the effect of the interaction between ASS1 and CCDC6 on their biological functions. Mutant plasmids transfected with CCDC6 (e.g. R55L, N394Y), although CCDC6 expression was not affected, significantly impaired its ability to interact with ASS1. This suggests that mutations in CCDC6 may play a role in the progression of lung adenocarcinoma by disrupting its binding to ASS1 and affecting cell signaling pathways \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs the core organelle of cellular energy metabolism, mitochondria homeostasis (the precise regulation of fusion and division) plays a decisive role in maintaining cellular homeostasis \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Recent studies have revealed that mitochondrial fission plays a key role in tumor invasion and metastasis by driving actin skeleton remodeling and pseudopodia formation, which provides new molecular targets for cancer therapy\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The present study is the first to reveal the mode of action of ASS1 in its non-classical function by regulating mitochondrial dynamics in lung adenocarcinoma. The expression level of ASS1 was significantly correlated with mitochondrial morphology: knockdown of ASS1 resulted in aggravated mitochondrial fragmentation, while overexpression of ASS1 induced mitochondrial network elongation. Further studies found that ASS1 affected mitochondrial homeostasis by regulating the stability of mitochondrial fusion proteins MFN1/2 and OPA1 rather than the transcriptional level. Notably, CHX tracking assay showed that ASS1 overexpression significantly delayed the degradation rate of fusion proteins, while the difference disappeared after treatment with proteasome inhibitor MG132, suggesting that ASS1 regulates the stability of these proteins through the ubiquitin-proteasome pathway. This finding echoes recent studies on mitochondrial quality control, such as that OPA1 stability is regulated by the E3 ubiquitin ligase MARCH5, whereas the present study suggests the existence of a molecular switch with reverse regulation \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBased on the previous mass spectrometry data, we targeted the deubiquitinating enzyme OTUD7A as the ASS1 target. Co-IP experiments confirmed a direct interaction between ASS1 and OTUD7A, which provided a mechanistic clue to explain the regulation of fusion protein stability. Further mechanistic studies showed that ASS1 could reduce the ubiquitination level of MFN1/2, which revealed a new mechanism of ASS1 involved in mitochondrial homeostasis by regulating the ubiquitination of MFN1/2. This study suggests that ASS1 may recruit OTUD7A to the mitochondrial microenvironment, and then deubiquitinize MFN1/2 and OPA to maintain their protein stability. This regulatory mechanism bears similarities to the known mode by which deubiquitinating enzymes regulate mitochondrial dynamics. For example, it has been shown that USP30 can regulate mitophagy by deubiquitylation of MFN2 \u003csup\u003e41\u003c/sup\u003e; Similarly, USP19 can specifically bind to FUNDC1 protein at the ER-mitochondrial contact site, promote Drp1 oligomerization and enhance its gtpase activity through deubiquitination, thereby driving mitochondrial division \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe innovation of this study is to reveal for the first time the new function of metabolic enzyme ASS1 involved in the deubiquitination regulatory network, breaking through the classical understanding of ASS1 and revealing that ASS1 stabilizes mitochondrial fusion proteins through OTUD7A-mediated deubiquitination mechanism, thereby regulating energy metabolism and survival homeostasis of tumor cells. However, this study still has limitations that need to be further explored. First, although the interaction between OTUD7A and ASS1 and its effect on fusion protein stability were confirmed, the deubiquitination activity of OTUD7A on MFN1/2 and OPA1 has not been directly verified. Secondly, the specific ubiquitination sites on these fusion proteins have not been clearly identified. In addition, the molecular mechanism by which ASS1-OTUD7A complex recognizes and binds fusion proteins remains to be elucidated. In order to solve these scientific problems, we plan to further explore them by deubiquitination experiments of recombinant proteins in vitro, identification of ubiquitination sites by mass spectrometry, and construction of specific site mutants in the future studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant Nos. 82072594 to Y.T.; 82073097 and 82573321 to S.L.), the Natural Science Foundation of Hunan Province (Grant No. 2024JJ3048 to S.L.), the Hunan Provincial Science and Technology Innovation Plan Project (Grant No. 2020SK53424 to X.W.), and the Central South University Research Programme of Advanced Interdisciplinary (Grant No. 2023QYJC030 to X.W. and Y.T.). We thank the Department of Pathology, Xiangya Hospital, for providing clinical specimens and immunohistochemistry (IHC) tissue sections, and Dr. Bin Xie for technical assistance with IHC experiments. We also thank colleagues for their valuable discussions and technical assistance during the course of this study.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe Ethics Committee of the Second Xiangya Hospital, Central South University approved this study.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests. The authors declare no conflict of interest. This manuscript has been read and approved by all authors and is not under consideration for publication elsewhere.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China [82072594 to Y.T. ; 82073097 to S.L.; 82573321 to S.L.]; the Natural Science Foundation of Hunan Province [2024JJ3048 to S.L.]; the Hunan Provincial Science and Technology Innovation Plan Project [2020SK53424 to X.W.] and the Central South University Research Programme of Advanced Interdisciplinary [2023QYJC030 to X.W. and Y.T.].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure of Potential Conflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTufail, M., Jiang, C. H. \u0026amp; Li, N. 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Our initial integrative analysis of metabolomic and transcriptomic data revealed a previously uncharacterized tumor-suppressive mechanism mediated by CCDC6 in lung adenocarcinoma. We discovered an interaction between CCDC6 and ASS1, concomitant with marked reductions in citrulline and aspartate within tumor tissues. compared to the adjacent normal tissues. Additionally, co-stimulation with citrulline and aspartate induces ASS1 localization in the mitochondria ASS1 localized in the mitochondria, but the underlying mechanism remains unclear. This study aimed to delineate the dual tumor-suppressive actions of ASS1: firstly, through the recruitment of the deubiquitinase OTUD7A to remove ubiquitin chains from MFN1/2 and OPA1, thereby stabilizing the mitochondrial fusion machinery and inducing hyperfused network formation; and secondly, via the CCDC6-ASS1 complex, which instigates mitochondrial reactive oxygen species accumulation, compromises ATP synthesis, and reduces mitochondrial membrane potential, consequently inducing a state of metabolic dormancy in tumor cells. This study elucidates the mechanism by which the ASS1-CCDC6 axis suppresses lung adenocarcinoma progression by remodeling mitochondrial dynamics and metabolic homeostasis,, thereby establishing a theoretical basis for mitochondria-targeted precision therapy.\u003c/p\u003e","manuscriptTitle":"A Novel Role of the ASS1-CCDC6 Complex in Orchestrating Mitochondrial Dynamics to Suppress Lung Adenocarcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-17 12:45:40","doi":"10.21203/rs.3.rs-8712733/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-03-09T09:56:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-08T15:22:07+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-26T06:25:36+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-23T00:23:00+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-13T09:41:22+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-02-12T08:14:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-28T11:45:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2026-01-27T16:26:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-27T16:26:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"674485a1-51d6-45e4-bfad-cee90205fbac","owner":[],"postedDate":"February 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":62789993,"name":"Biological sciences/Cancer"},{"id":62789994,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2026-03-09T10:03:55+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-17 12:45:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8712733","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8712733","identity":"rs-8712733","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}