APOC3-Mediated Fatty Acid Metabolism Suppresses Lung Adenocarcinoma Progression by Inhibiting GNAI3/cAMP/PKA Pathway

APOC3-Mediated Fatty Acid Metabolism Suppresses Lung Adenocarcinoma Progression by Inhibiting GNAI3/cAMP/PKA Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article APOC3-Mediated Fatty Acid Metabolism Suppresses Lung Adenocarcinoma Progression by Inhibiting GNAI3/cAMP/PKA Pathway Liping Dai, Feifei Liang, Ying Chen, Yihao Liang, Bing-Hua Jiang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8140812/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Fatty acid metabolism is a key driver of tumor progression, yet its dysregulation in lung adenocarcinoma (LUAD) remains incompletely characterized. Here, we identify apolipoprotein C3 (APOC3)—previously linked to cardiovascular disease—as a novel suppressor of triglyceride (TG) hydrolysis and fatty acid oxidation, ultimately restraining LUAD growth and metastasis. Proteomic and tissue microarray analyses revealed that APOC3 expression was significantly downregulated in LUAD tissues compared with adjacent normal tissues, and low APOC3 levels correlated with poor prognosis in metastatic patients. Furthermore, plasma levels of APOC3 and TG showed a positive correlation in LUAD patients. Functionally, APOC3 overexpression suppressed TG hydrolysis, fatty acid oxidation, and the proliferation and metastasis of LUAD cells both in vitro and in vivo. Mechanistically, APOC3 attenuated the cAMP/PKA signaling pathway, leading to reduced expression of hormone-sensitive lipase (HSL), a key enzyme in TG hydrolysis, and PGC-1α, a master regulator of fatty acid oxidation. The inhibitory effects of APOC3 on TG hydrolysis and fatty acid oxidation were reversed by cAMP activators or knockdown of HSL or PGC-1α. Additionally, APOC3 was found to interact with GNAI3, a critical inhibitory regulator of the cAMP/PKA pathway. In summary, our study uncovers an APOC3-mediated pathway that constrains TG hydrolysis and fatty acid oxidation, the dysregulation of which contributes to LUAD progression, highlighting APOC3 as a potential therapeutic target in LUAD. Biological sciences/Cancer/Lung cancer/Non-small-cell lung cancer Biological sciences/Cell biology/Cell division/Cell growth Apolipoprotein C3 (APOC3) LUAD TG hydrolysis Fatty acid metabolism cAMP/PKA pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION According to recent global cancer statistics, lung cancer remains the most lethal type of cancer, ranking second only to breast cancer in terms of incidence [1]. In China, both the incidence and mortality rates of lung cancer are the highest among all cancers [2]. Non-small cell lung cancer (NSCLC) constitutes about 85% of all lung cancer cases, with lung adenocarcinoma (LUAD) representing 40% of all histological subtypes of NSCLC [3]. LUAD carries a substantial risk of metastasis, yet the precise molecular mechanisms underlying this process remain elusive [4]. Delving into the mechanisms governing LUAD initiation and progression, as well as identifying novel therapeutic targets, holds significant importance in enhancing patient survival rates. Lipid metabolism reprogramming is a crucial metabolic feature of tumor cells, influencing tumor initiation, progression, and immune modulation [5]. A recent study revealed a substantial enrichment of lipid droplets in LUAD cells, with fatty acid metabolism serving as the primary energy source for the malignant advancement of LUAD [6]. Moreover, heightened fatty acid synthesis in tumor cells leads to the activation of oxidative lipid degradation to produce ATP, meeting the demands for cell proliferation and survival during nutrient scarcity. Tumor cells modulate cell growth and metastasis signaling pathways through bioactive molecules generated by lipid metabolism [7]. Recent research indicates that increased lipid utilization promotes the growth and metastasis of various cancer cells, including lung cancer [8]. The aberrant activation of fatty acid synthesis and oxidative degradation metabolism significantly contributes to the metastasis, recurrence, and drug resistance observed in lung cancer [9, 10]. Inhibition of lipid metabolism and the Rap1 signaling pathway has been demonstrated to trigger apoptosis in LUAD cells [11]. Previous research has indicated that carnitine palmitoyl transferase 1A (CPT1A) enhances the metastatic potential of NSCLC through the regulation of fatty acid oxidation (FAO) [12]. Nonetheless, there is a scarcity of studies investigating the precise molecular pathways underlying lipid metabolism dysregulation in LUAD. Consequently, there is a pressing need for comprehensive investigations into the molecular underpinnings of lipid metabolism dysregulation in LUAD and the identification of novel therapeutic targets. The cAMP/PKA pathway is pivotal in regulating fatty acid oxidation, proliferation, and metastasis in malignant tumors [13, 14]. A crucial downstream effector of this pathway is the transcription factor CREB, which stimulates the expression of PGC-1α, a master regulator of mitochondrial biogenesis and fatty acid oxidation [15]. Activation of the cAMP/PKA pathway through intramuscular injection of lactate into the gastrocnemius muscle enhances the expression of key proteins involved in white adipose tissue lipolysis (AMPK, ATGL, HSL) and the mitochondrial marker PGC-1α [16]. Furthermore, this pathway upregulates the expression of hormone-sensitive triglyceride lipase (HSL), a critical enzyme in the second step of triglyceride hydrolysis, thereby modulating hydrolysis processes [17, 18]. Apolipoprotein C3 (APOC3), the predominant small molecule globulin in the C group, is essential for maintaining triglyceride homeostasis [19]. APOC3 exhibits dynamic interchange among different lipoproteins, its equilibrium state contingent upon an individual's metabolic status [20–22]. Numerous studies have highlighted the role of APOC3 in lipid metabolism and is a risk factor for cardiovascular diseases [23–25]. APOC3 expression is typically low in small cell lung cancer; however, its level rises in patients undergoing preoperative neoadjuvant chemotherapy [26]. APOC3 has been shown to boost the anti-tumor effects of CD8 + T cells by triggering macrophage inflammasome activation [27]. While APOC3 research has predominantly concentrated on cardiovascular diseases, particularly lacking in lung cancer investigations, specifically in LUAD. Our prior studies have shown that the expression level of APOC3 in LUAD patients is lower than that in benign pulmonary nodules (BPNs) patients [28]. As TG serves as a crucial energy source for the proliferation and spread of malignant tumor cells, further exploration into whether APOC3 modulates lipid metabolism to influence LUAD progression is necessary. In this study, we investigated the function and mechanism of APOC3 in repressing the progression of LUAD in vitro and in vivo. We demonstrated that APOC3 attenuates the cAMP/PKA pathway to inhibit TG hydrolysis and fatty acid oxidation, resulting in the suppression of LUAD. The identification of APOC3 as a key regulator fatty acid metabolism of LUAD indicated that APOC3 could be a potential biomarker and provided novel insights into LUAD therapy. MATERIALS AND METHODS Study population To investigate the expression of APOC3 in plasma samples, a total of 356 participants were consecutively recruited from a hospital in Henan Province (November 2017–January 2020), including 156 patients with lung adenocarcinoma (LUAD), 156 with benign pulmonary nodules (BPNs), and 44 normal controls (NCs). All pulmonary nodules were detected via thoracic low-dose computed tomography (LDCT) and pathologically confirmed as benign or malignant (with LC subtypes determined by biopsy). NCs were self-reported healthy individuals enrolled during routine medical examinations at the same hospital. Pre-treatment peripheral blood samples were collected from all patients prior to surgery or cancer-directed therapy. Detailed demographic and clinical characteristics are provided in Table S1 . Moreover, 336 blood samples were collected from LUAD patients with follow-up visits for prognostic evaluation. The detailed characteristics of participants and analysis of clinical factors are shown in Table S2 . This study was approved by the Ethics Committee of Zhengzhou University (2021-KY-1057-002). Written informed consent was obtained from all participants prior to enrollment. Blood sample collection and processing Peripheral venous blood (5 mL) was collected in EDTA-K2 anticoagulant tubes and transported to the laboratory within 2 h. Samples were centrifuged at 3000 rpm for 5 min to separate plasma, which was then aliquoted (500 µL per tube) into pre-labeled 1.5 mL Eppendorf tubes and stored at − 80°C until further analysis. Cell culture and establishment of stable cell lines The lung adenocarcinoma cell (A549) was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and lung adenocarcinoma cells (H1975 and PC-9) were derived from the ATCC Cell Bank of America. All three types of cells were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Solarbio, Beijing, China). All cell lines were incubated at 37℃ in a humidified atmosphere with 5% CO 2 . RNA extraction and quantitative Real-time PCR RNA extraction and quantitative Real-time PCR Total RNA from cells was isolated using TRIzol reagent and then reverse transcribed into cDNA using NovoScript Plus All-in-one 1st Strand cDNA Synthesis SuperMix (Novoprotein, Shanghai, China). qRT‒PCR was performed using NovoStart SYBR qPCR SuperMix Plus (Novoprotein, Shanghai, China) on a Light Cycler 96 system. The relative gene expression was calculated by the 2 −ΔΔCt method with GAPDH used for normalization. The primer sequences are shown in Supplementary Table S3 . Cell Proliferation Assays For the CCK-8 assay, 3 × 10 3 cells/well were seeded in a 96-well plate. Individually cultured tumor cells were used as controls. Ten microliters of CCK-8 (Meilunbio, Dalian, China) were added to each well at 0, 24, 48, and 72 h. The cells were incubated for 1 h at 37°C, and the absorbance values were measured at 450 nm. Each time point was assessed in replicates of at least three wells. For the colony formation assay, 6 × 10 2 cells/well were seeded in a 6-well plate and fixed for 1 h after 14 days. Crystal violet was added for staining overnight. Each well was subsequently washed three times and the number of colonies was counted via Image-Pro Plus 6.0 software. Migration assay and invasion assay For the cell migration, 5×10 4 cells were suspended in 200 µL of serum-free medium, placed in the top inserts of a 24-well Transwell plate (Corning, New York, USA), and then exposed to 10% serum culture medium in the lower chambers. The cells on the bottom surface of the membrane were fixed and stained with crystal violet after 24 h, and the cells that migrated were counted via Image-Pro Plus 6.0 software. The membrane for the invasion assay was covered with 40 µL of Matrigel (Corning, New York, USA) (diluted 1:8 with RPMI-1640) in advance. The tumor cells were seeded in the upper chambers; the lower chambers were filled with 600 µL including 10% serum culture medium. After 48 h of incubation, the cells that adhered to the lower filter surface were counted via Image-Pro Plus 6.0 software. Wound healing assay A monolayer of cells was scraped with 10-µL pipette tips when the cells had reached 90% confluence in a 6-well plate. Then, images were taken at appropriate time points (0 h/24 h). Image-Pro Plus 6.0 software was used to assess the relative wound width. Triglyceride measurement Intracellular lipid droplets were detected using Oil Red O staining and Nile Red fluorescence assays. Cells were suspended at a density of 5 × 10⁵ cells/mL and seeded in 6-well plates, followed by incubation at 37°C with 5% CO₂ for 24 h. Subsequent staining procedures were performed strictly according to the manufacturer's protocol. Mitochondrial activity assay The mitochondrial red fluorescent probe was used to detect mitochondrial activity. When the cells reach approximately 80% confluence with uniform distribution, carefully aspirate the original culture medium and replace it with the pre-warmed MitoTracker Red CMXRos working solution. Incubate the cells in a humidified 37°C, 5% CO₂ cell culture incubator for 25 min, protected from light. After incubation, gently remove the staining solution and wash the cells twice with warm PBS or fresh culture medium to remove excess probe. Finally, replenish with fresh pre-warmed culture medium and immediately observe the cells under a fluorescence microscope equipped with TRITC/Rhodamine filters. Capture images promptly while minimizing light exposure to prevent photobleaching. FFA, FAO and ATP content detection To measure the Intracellular FFA levels of LUAD cells, the FFA kit was purchased from Suzhou Keming Biotech Co. LTD (Cat. FFAD-1-W, Suzhou, China). The content is measured according to the manufacturer's protocol. Experiments were performed three times. The FAO activity was detected by a commercial reagent kit. The FAO enzyme-linked immunosorbent assay (ELISA) kit was purchased from Shanghai Meilian Biotech Co. LTD (Lot. ml060146, Shanghai, China), following the manufacturer’s instructions. Experiments were performed three times. In order to detect the intracellular ATP content, The ATP kit was purchased from Beijing Solarbio Biotech Co. LTD (Cat. BC0300, Beijing, China). The content is measured according to the manufacturer's protocol. Experiments were performed three times. Immunofluorescence To investigate protein co-localization, harvest well-grown cells and seed them evenly onto sterile coverslips at an appropriate density. After 6 h of adhesion, carefully retrieve the coverslips and fix the cells with 4% paraformaldehyde (PFA) at room temperature for 20 min. Subsequently, wash the samples three times with PBS, 5 min per wash. Permeabilize the cells with 0.2% Triton X-100 for 5 min at room temperature, followed by another three PBS washes. Block nonspecific binding sites by incubating the samples with 1% bovine serum albumin (BSA) for 1 h, then wash again with PBS three times. Incubate the samples overnight at 4°C with primary antibodies diluted in 1% BSA. The following day, remove unbound primary antibodies by washing three times with PBS, then incubate with fluorescently conjugated secondary antibodies (diluted in 1% BSA) for 1 h at room temperature protected from light. After three final PBS washes, mount the coverslips using an anti-fade mounting medium containing DAPI for nuclear counterstaining. Carefully invert the coverslips onto glass slides, seal the edges, and image using a fluorescence microscope equipped with appropriate filter sets. Western blot The whole cell lysates were extracted using RIPA Lysis buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (Solarbio, Beijing, China). The proteins were separated via 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% milk, the membrane was incubated with primary antibody overnight at 4°C. HRP-conjugated anti-mouse or anti-rabbit antibodies (1:5000, Proteintech, Wuhan, China) were used as secondary antibodies, and the antigen antibody reactions were visualized by ECL (HaiGene, Harbin, China). The primary antibodies that were used are listed in Table S4 . Tumor xenograft model A549 cells stably overexpressing APOC3 were resuspended in PBS at a density of 5×10⁷ cells/mL, and 100 µL of the cell suspension was subcutaneously injected into the right axillary region of mice. When the tumors reached approximately the size of a rice grain (typically 3–5 mm in diameter), tumor dimensions and mouse body weights were recorded every two days using digital calipers and an analytical balance, respectively. Approximately 40 days post-injection, mice were humanely euthanized via carbon dioxide asphyxiation. Tumor tissues were then carefully excised, photographed with a size reference, weighed using a precision balance, and processed for further analysis. Tumor growth curves were generated by plotting tumor volume (calculated as [length × width²]/2) against time, while body weight changes were monitored throughout the study period to assess potential toxicity. Construction of lung metastasis model Healthy, log-phase cells were harvested and resuspended in PBS at a concentration of 5×10⁶ cells/100 µL. Using a 29G insulin syringe, 100 µL of cell suspension was slowly injected into the tail vein (3 mm insertion depth) over 30 seconds, followed by immediate application of a sterile cotton ball with firm pressure for 2 min to ensure hemostasis. Mice were monitored for 30 min post-injection to confirm normal behavior and absence of adverse effects. After 28 days of standard housing, the mice were humanely euthanized by controlled CO₂ asphyxiation (20% chamber volume displacement per min), and complete lung tissues were excised for subsequent experimental analysis. RNA sequencing This study performed transcriptome sequencing analysis on three groups of A549 cell lines, including APOC3-overexpressing and normal APOC3-expressing groups, through collaboration with Zhengzhou Zhenhe Biotechnology Co., Ltd. The analytical pipeline consisted of the following steps: raw data processing→quality control→sequence alignment→gene expression quantification→differential expression analysis. Differentially expressed genes were identified based on the threshold criteria of |log2FC| > 1.5 with a significance level of P < 0.05. Subsequently, the identified differentially expressed genes were subjected to KEGG pathway enrichment analysis and Gene Ontology (GO) functional analysis to elucidate their potential biological roles and involved pathways. Immunohistochemical (IHC) Mouse tumor specimen sections were used for IHC. The sections were separately incubated with anti-APOC3 (1:400, Abcam, USA), anti-E-cadherin (1:500, CST, USA), anti-Vimentin (1:500, CST, USA) and anti-Ki67(1:500, Wuhan, China) antibodies. The scores were determined by combining the proportion of positively stained tumor cells and the intensity of staining. The proportion of positively stained tumor cells in a field was scored as follows: 0, no positive tumor cells; 1, < 10% positive tumor cells; 2, 10–35% positive tumor cells; 3, 35–75% positive tumor cells; and 4, ≥ 75% positive tumor cells. The staining index (SI) for each sample was obtained by multiplying the intensity and proportion values. An SI ≥ 7 was considered a high expression, and samples with an SI < 7 were considered to have low expression. HE staining Mouse lung tissues were fixed in 4% paraformaldehyde (PFA), dehydrated, and embedded in paraffin. Tissue sections (4–5µm thick) were prepared and stained with hematoxylin and eosin (H&E) for histological examination. Stained sections were digitally scanned, and quantitative analysis of the staining area was performed using Case Viewer software. Statistical analysis was conducted to evaluate differences in staining intensity and distribution. Co-immunoprecipitation (Co-IP), and mass spectrometry (MS) analysis Co-IP was performed to confirm protein-protein binding. Selleck Protein A/G Magnetic Bead System (#B23202, Houston, USA) and detailed protocol as previously described [29]. The antibodies used to incubate cell lysate was listed on Table S4 . The Co-IP complexes were analyzed by SDS-PAGE and IB (Immunoblot). And anti-mouse IgG (Cat: SE131, Solarbio) was used as the second antibody for Co-IP complexes detection and the Co-IP complexes. And the binding proteins of APOC3 were isolated by Co-IP and identified by mass spectrometry (MS) analysis applying Triple tof5600 and the results were processed by ProteinPilot (version 5.01). ELISA The concentrations of APOC3 in patients’ plasma were determined strictly following the manufacturer’s instructions. The ELISA kit used in the study was purchased from Cloud clone (Wuhan, China). The absorbance was measured at 450 nm. Statistical analysis The experimental results were quantified using ImageJ software, and statistical analysis was performed using GraphPad Prism 8.0. Each experiment was repeated at least three times. T-tests were used to analyze differences between two groups of data, while one-way ANOVA was employed to determine the statistical significance of data from three or more groups. Differences were considered statistically significant when P < 0.05, with significance levels indicated as * P < 0.05, ** P < 0.01, and *** P < 0.001. Results APOC3 is dramatically decreased in LUAD and positively correlated with the plasma TG levels We previously found that the plasma APOC3 levels were significantly lower in LUAD patients than those with benign pulmonary nodules or normal subjects[28]. To further investigate the APOC3 expression between LUAD and normal tissues, the proteomics data from 103 LUAD patients and paired normal adjacent tissues (NATs) were analyzed, we observed that APOC3 expression was significantly lower in LUAD tissues than that in NATs ( P < 0.001) (Fig. 1 A). Receiver Operating Characteristic (ROC) analysis showed that APOC3 could serve as a potential diagnostic indicator for LUAD, with an AUC (95% CI) of 0.862 (0.012–0.911) (Fig. 1 B). To further validate APOC3 expression levels in LUAD and paired normal adjacent tissues (NATs), we analyzed tissue microarrays comprising 92 LUAD samples and 88 matched NAT pairs. The results were consistent with our initial findings, confirming the differential expression pattern of APOC3 (Fig. 1 C, D). Moreover, low expression of APOC3 was strongly correlated with male ( P < 0.001, Mann-Whitney U test), EGFR negative patients ( P < 0.01, Mann-Whitney U test) and LUAD patients with positive lymph node metastasis ( P < 0.05, Mann-Whitney U test) (Figure S1A-I ).To further investigate the correlation between APOC3 and prognosis of LUAD patients, we collected plasma samples from 336 LUAD patients for prognostic analysis. Kaplan-Meier survival curves revealed a statistically significant association between elevated APOC3 expression and improved overall survival in the cohort of LUAD patients presenting with distant metastases (log-rank P = 0.023, HR = 2.24, 95% CI: 1.10–4.57) (Fig. 1 E). Further stratified analysis indicated that gender, age, stage, lymph node metastasis, nodule size, smoking history, and alcohol consumption were not significantly correlated with prognosis ( P > 0.05) (Figure S1J-X ). To identify clinical factors independently associated with APOC3 expression, univariate and multivariate regression analyses were performed, with the complete results detailed in Table S5 . Detection of APOC3 expression in plasma by ELISA method showed a positive correlation between APOC3 and TG levels, which were lower in LUAD patients than in benign lung nodules and normal controls (Fig. 1 F, G), suggesting that APOC3 may modulate lipid metabolism of LUAD. APOC3 inhibits the proliferation and metastasis of LUAD in vivo and in vitro To explore the role of APOC3 in LUAD progression, we examined its expression in normal lung epithelial cells (BASE-2B) and lung adenocarcinoma cell lines. Decreased expression of APOC3 was observed in the LUAD cell lines A549 and H1975 when compared with BEAS-2B. (Figure S2A ). We then established APOC3-overexpressing stable A549 and H1975 cells and APOC3- silenced stable PC-9 cells (Fig. 2 A-B, Figure S2B ). Colony-formation assays displayed a decreased clonogenic capacity of both A549 and H1975 cells (Fig. 2 C) and CCK8 assays confirmed the suppression of growth in both A549 and H1975 cells (Figure S2C-D ), but the converse result was observed in PC-9 cells (Figure S2E-F ). The results of wound healing experiments showed that overexpression of APOC3 significantly inhibited lateral migration ability in A549 and H1975 cells (Fig. 2 D) and silencing APOC3 promoted horizontal migration ability in PC-9 cells (Figure S2G ). And in the transwell assay, we prolonged the experimental time to allow more cell-movement and found overexpression APOC3 inhibited much more migration and invasion than APOC3-Vector in A549 and H1975 cells (Fig. 2 E and Figure S2H-J ). Moreover, APOC3 overexpression significantly induced E-cadherin, while inhibiting N-cadherin, vimentin levels(Fig. 2 F and Figure S2K ). To gain insights into the importance of APOC3 in repressing the progression of LUAD cells in vivo, we performed xenograft experiments using A549 cell lines. Overexpression of APOC3 significantly reduced tumor growth and volume (Fig. 2 G-H). Moreover, increased levels of E-cadherin and decreased levels of Ki67 and Vimentin were observed in APOC3-overexpression tumors (Fig. 2 I). Furthermore, we observed that fewer APOC3-overexpressd A549 cells metastasized to the lung and initiated secondary tumors (Figure S2L ). Taken together, these findings suggest a role for APOC3 in inhibiting proliferation and metastasis of LUAD cells both in vivo and in vitro. APOC3 inhibits fatty acid metabolism in lung adenocarcinoma Lipid metabolism reprogramming is crucial for LUAD progression. APOC3, an important apolipoprotein, is key to triglyceride homeostasis. To investigate whether APOC3 regulated tumor progression via fatty acid metabolism, the metascape analysis was conducted on genes negatively correlated with APOC3 expression in LUAD proteomic data (iProx Consortium, subproject ID: IPX0001804000). This analysis revealed significant enrichment of the lipid metabolism signaling pathway (Fig. 3 A), suggesting a potential mechanistic link between APOC3 and fatty acid metabolism in LUAD progression. The increased intracellular TG content and reduced mitochondrial activity were displayed in APOC3 overexpressed A549 and H1975 cells, while TG content decreased in APOC3-knockdown PC-9 cells (Fig. 3 B, C and S3A-E). We hypothesize that APOC3 inhibits triglyceride (TG) breakdown, thereby reducing free fatty acid (FFA) availability and limiting tumor cell energy derived from fatty acid oxidation. Consistent with this model, APOC3 overexpression in A549 and H1975 cells significantly decreased intracellular FFA levels (Fig. 3 D, E), suppressed fatty acid oxidase activity (Fig. 3 F, G) and downregulated key fatty acid oxidation markers (Fig. 3 H, I), and ultimately reduced cellular ATP production (Fig. 3 J, K), while, the FFA levels, fatty acid oxidase activity, key fatty acid oxidation markers and ATP levels increased in APOC3-knockdown PC-9 cells (Figure S3F-I ). In vivo experiments demonstrated that APOC3 overexpression increased triglyceride (TG) accumulation in mouse tumor tissues while reducing free fatty acid (FFA) content, fatty acid oxidase activity, expression of key fatty acid oxidation markers, and ATP levels (Fig. 3 L-P), which consistent with in vitro findings. Taken together, these findings demonstrate that APOC3 suppresses fatty acid metabolism in LUAD, both in vitro and in vivo. APOC3 inhibits fatty acid metabolism through the cAMP/PKA signaling pathway To investigate how APOC3 regulates TG breakdown and fatty acid oxidation, transcriptome sequencing was performed on A549 cells with normal and overexpressed APOC3. Screening identified 194 upregulated and 313 downregulated genes (|log2FC|>1.5, P < 0.05) (Fig. 4 A and Figure S4A ). KEGG analysis revealed significant enrichment of the cAMP pathway (Fig. 4 B). The decreased cAMP levels were validated in A549 and H1975 cells overexpressing APOC3 (Fig. 4 C and Figure S4B ), while APOC3 knockdown in PC-9 cells increased cAMP levels (Fig. 4 D). APOC3 overexpression significantly downregulated the cAMP downstream signaling such as the levels of p-PKA and p-CREB (Fig. 4 E, F and Figure S4C ), which was effectively reversed by the cAMP activator Forskolin. Concurrently, the expression of p-HSL, a rate-limiting enzyme in TG decomposition, and PGC-1α, a key enzyme in fatty acid oxidation, was also restored to higher levels upon Forskolin treatment (Fig. 4 G). In contrast, treatment of PC-9 cells with the cAMP inhibitor SQ22536 effectively inhibited the activation of the cAMP pathway and downregulated the expression of p-HSL and PGC-1α (Fig. 4 H). Forskolin also reversed the inhibitory effects of APOC3 on mitochondrial activity and fatty acid metabolism (Fig. 4 I, Figure S4D-F ). Moreover, the inhibitory effects of APOC3 overexpression on A549 cell migration and proliferation were reversed upon Forskolin treatment (Fig. 4 J-K). In mouse xenograft tumors, APOC3 also inhibits the cAMP/PKA pathway, leading to reduced expression levels of p-PKA and p-CREB, which is consistent with the in vitro results (Fig. 4 L). These results indicate that APOC3 suppresses the cAMP/PKA pathway both in vitro and in vivo. APOC3 inhibits TG decomposition through the cAMP-PKA-HSL axis and suppresses fatty acid metabolism through cAMP-PKA-CREB-PGC-1α Previous research has demonstrated that the expression of HSL and PGC-1α is regulated by the cAMP/PKA pathway [17, 30, 31]. To investigate the importance of HSL in APOC3-reduced TG breakdown, we detected the effects of HSL silencing on APOC3 knockdown PC-9 cells. The efficiency of HSL knockdown was confirmed by Western blot (Fig. 5 A). Moreover, the results indicated that HSL knockdown promoted TG accumulation (Fig. 5 B) and reduced FFA content (Fig. 5 C). To explore the necessity of PGC-1α for APOC3-inhibited mitochondrial activity and ATP production, we conducted dual knockdown of APOC3 and PGC-1α in PC-9 cells. Western blot showed significant inhibition of PGC-1α expression (Fig. 5 D). Mitochondrial activity assays demonstrated that PGC-1α knockdown markedly reduced mitochondrial activity, as well as ATP levels (Fig. 5 E-F). Collectively, these results indicate that APOC3 reduces energy utilization efficiency by inhibiting the expression of p-HSL in lipid droplets and diminishes fatty acid oxidation capacity by suppressing the expression of PGC-1α. APOC3 interacts with GNAI3 to inhibit the cAMP/PKA pathway in LUAD To investigate how APOC3 affects the cAMP/PKA pathway, therefore, the APOC3 protein was precipitated using an APOC3 antibody, and interacting proteins were identified via mass spectrometry. KEGG analysis showed that APOC3 immunoprecipitants enriched in the cAMP/PKA pathway (Fig. 6 A). We identified GNAI3, directly inhibits adenylate cyclase (AC) upon GTP binding and suppresses cAMP production and downstream PKA activation [32–34], that may be linked to APOC3 and the regulation of cAMP/PKA pathway (Fig. 6 B). We then transfected Flag-tagged APOC3 into A549 cells, immunoprecipitated the cell lysates with anti-Flag or anti-GNAI3 antibodies and immunoblotted the Co-IP complex. Our results indicated that APOC3 co-immunoprecipitated with GNAI3 but not with IgG in A549 cells (Fig. 6 C). In addition, we found a positive correlation between APOC3 and GNAI3 by using data from iProx Consortium with the subproject ID IPX0001804000 (Fig. 6 D). These data indicate that APOC3 may interact with GNAI3 to inhibit the cAMP/PKA pathway in LUAD. Discussion The high morbidity and mortality rates of LUAD indicate a high degree of clinical need for the discovery of new effective therapeutic targets [35]. Our study reveals a previously unrecognized role of APOC3 in suppressing LUAD proliferation and metastasis by modulating fatty acid metabolism. The underlying mechanism involves two distinct pathways, on one pathway, APOC3 impedes triglyceride (TG) hydrolysis via the cAMP/PKA/HSL signaling axis, restricting energy substrate availability and reducing intracellular ATP levels. While on another pathway, APOC3 attenuates mitochondrial oxidative capacity and fatty acid β-oxidation through the cAMP/PKA/CREB/PGC-1α pathway, further compromising cellular bioenergetics. Collectively, these metabolic disruptions lead to significant inhibition of LUAD proliferation and metastatic potential, highlighting APOC3 as a promising therapeutic target for intervention (Fig. 7 ). Metabolic reprogramming of fatty acids has recently been recognized as a key driver of cancer progression [36]. LUAD cells acquire FAs via (i) microenvironment uptake, (ii) de novo lipogenesis, or (iii) TG hydrolysis[37]. serving as a key bioenergetic pathway for LUAD progression[6]. During starvation, FAs undergo mitochondrial oxidation for ATP production[38]. APOC3, a component of triglyceride-rich lipoproteins, is a hypertriglyceridemia risk factor, recent clinical trials have shown that inhibiting APOC3 is a promising approach for treating hypertriglyceridemia and preventing cardiovascular disease [36, 37]. Our study reveals reduced plasma APOC3 and TG levels in LUAD patients, showing positive correlation. Paradoxically, APOC3 overexpression in LUAD cells (A549/H1975) increases TG accumulation while suppressing proliferation and metastasis, contradicting TG's known pro-tumor role. This unexpected APOC3-mediated tumor suppression despite TG elevation requires mechanistic investigation. The cAMP/PKA/CREB axis promotes tumor malignancy by enhancing proliferation, invasion, metastasis, and drug resistance [14]. The cyclic AMP (cAMP) pathway, mediated by transmembrane (tmACs) and soluble (sAC) adenylate cyclases [39, 40], forms compartmentalized signaling complexes with GPCRs to regulate diverse cellular processes [41]. Through PKA-mediated CREB activation, this pathway controls critical functions including gene transcription, mitochondrial homeostasis, and cell proliferation [42], with dysregulation implicated in multiple cancer types [13]. Clinical evidence demonstrates its therapeutic relevance, as shown in colorectal cancer metastasis [43]. Recent studies have demonstrated that Lactate activates the cAMP/PKA pathway, upregulating lipolysis regulator HSL and mitochondrial biogenesis factor PGC-1α [16]. Our findings extend this paradigm to LUAD, where APOC3 overexpression suppresses cAMP levels and inhibits p-PKA/p-CREB activation, consequently attenuating tumor progression. Beyond canonical signaling, cAMP/PKA critically regulates energy metabolism through CREB-mediated PGC-1α expression (controlling mitochondrial biogenesis/FA oxidation) [44], and HSL phosphorylation (promoting TG hydrolysis). APOC3 inhibits both p-HSL and PGC-1α via cAMP/PKA suppression, with pathway activation reversing these effects and restoring tumorigenic potential. The cAMP/PKA pathway emerges as a central coordinator of LUAD progression through integrated control of oncogenic signaling and metabolic reprogramming. APOC3's tumor-suppressive effects via this axis highlight its potential as a therapeutic target, particularly given the reversible nature of its metabolic inhibition. While our study demonstrates that APOC3 suppresses LUAD progression by inhibiting the cAMP/PKA pathway and subsequent fatty acid metabolism, the precise molecular mechanism warrants further investigation. We identified a physical interaction between APOC3 and GNAI3, an inhibitory G protein of the cAMP pathway, along with their positive correlation in expression. However, whether APOC3 exerts its tumor-suppressive effects specifically through GNAI3-mediated cAMP signaling remains to be elucidated. This represents a limitation of our current work, as we cannot conclusively establish a causal relationship between the APOC3-GNAI3 interaction and the observed metabolic reprogramming in LUAD. Future studies employing GNAI3 knockdown or knockout models are needed to determine if GNAI3 is required for APOC3's inhibitory effects on cAMP signaling and tumor progression. Such investigations would provide more definitive evidence regarding the functional significance of this protein interaction in the context of LUAD pathogenesis and potentially identify novel therapeutic targets for metabolic intervention in LUAD. As expected, In vitro studies demonstrated APOC3 significantly modulates LUAD cell proliferation, migration and invasion. In vivo, APOC3 overexpression suppressed tumor growth and EMT progression, reducing tumor volume and pulmonary metastases. Mechanistically, APOC3 depletion enhanced HSL/PGC-1α-dependent fatty acid oxidation, while its overexpression inhibited lipolysis (via p-HSL downregulation) and fatty acid oxidation (through CPT1A suppression), ultimately reducing ATP production. RNA-seq implicated cAMP/PKA pathway involvement in these metabolic effects. Emerging evidence suggests APOC3 may enhance CD8 + T cell antitumor activity through macrophage inflammasome activation[27], indicating potential immune-mediated mechanisms contributing to its tumor-suppressive effects in LUAD. Moreover, our survival analysis of 336 lung adenocarcinoma patients revealed significantly prolonged overall survival in patients with higher APOC3 expression levels. This protective effect remained statistically significant when patients were stratified by metastatic status (both metastatic and non-metastatic subgroups). These clinical findings strongly corroborate our experimental data, providing compelling evidence for APOC3's metastasis-inhibitory function in LUAD. Overall, this study suggests for the first time that high expression of APOC3 reduced the fatty acid metabolism that inhibited LUAD progression. Our results shed new light on the role of APOC3 in the fatty acid metabolism of LUAD and provide a mechanistic basis for APOC3-mediated lipid metabolism induced progression in LUAD cells. Conclusions In the present study, we identify apolipoprotein C3 (APOC3) as a novel tumor suppressor in lung adenocarcinoma (LUAD) through regulation of fatty acid metabolism. Clinically, APOC3 is significantly downregulated in LUAD tissues and plasma, with its low expression correlating with metastatic progression and poor prognosis. Mechanistically, APOC3 interacts with GNAI3 to suppress the cAMP/PKA signaling pathway, leading to impaired triglyceride hydrolysis via reduced HSL phosphorylation and suppressed fatty acid oxidation through downregulation of PGC-1α. This metabolic rewiring results in accumulated lipid droplets, decreased ATP production, and ultimately inhibition of tumor growth and metastasis. Our findings establish the APOC3-GNAI3-cAMP/PKA axis as a crucial regulatory mechanism in LUAD, highlighting its therapeutic potential for metabolic intervention in lung cancer. Abbreviations APOC3 Apolipoprotein C3 CPT1A Carnitine Palmitoyl transferase 1A AC adenylate cyclase ATGL adipose triglyceride lipase MGL monoglyceride lipase FA fatty acid FAO Fatty acid oxidation FFA Free fatty acid HSL Hormone-sensitive triglyceride lipase LUAD Lung adenocarcinoma NSCLC Non-small cell lung cancer TG Triglyceride. Declarations Ethics approval and consent to participate Patient blood collection was followed the guidelines and principles of the Helsinki Declaration and received approval from the Ethics Committee of First Affiliated Hospital of Zhengzhou University (2021-KY-1057-002). Written informed consent was obtained from all study participants. All animal experiments were performed in accordance with the Basel Declaration and approved by the Animal Ethics Committee of Henan Institute of Medical and Pharmaceutical Sciences (2023-yyy-046). Competing Interests The authors have declared that no competing interest exists. Data availability The data used in the current study are available from the corresponding author upon reasonable request. Supporting Material Additional results, prognostic information of LUAD patients and extended analysis are provided as Supplementary Information. Acknowledgments This work was supported by the Leading Talents of Science and Technology Innovation in Henan Province (Grant Number 20420051008); the National Natural Science Foundation of China (No. 82203287); the project funded by China Postdoctoral Science Foundation (No. 2022M722874) and the Project of Basic Research Fund of Henan Institute of Medical and Pharmacological Sciences (No. 2025BP0203). The authors gratefully acknowledge Henan Key Laboratory of Pharmacology for Liver Diseases for providing experimental platform support. Author Contributions FFL, RLD and LPD designed experiments and prepared the manuscript. FFL and YC performed experiments and analyzed the data. YHL analyzed clinical samples. SYOY provided clinical sample. BHJ provided advice. All authors read and approved the final version of manuscript. References Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin. 2024; 74: 12-49. Wu Y, He S, Cao M, Teng Y, Li Q, Tan N, et al. Comparative analysis of cancer statistics in China and the United States in 2024. Chin Med J (Engl). 2024; 137: 3093-100. Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2022. CA Cancer J Clin. 2022; 72: 7-33. Fang H, Sun Q, Zhou J, Zhang H, Song Q, Zhang H, et al. m(6)A methylation reader IGF2BP2 activates endothelial cells to promote angiogenesis and metastasis of lung adenocarcinoma. Mol Cancer. 2023; 22: 99. Huang D, Tang E, Zhang T, Xu G. Characteristics of Fatty Acid Metabolism in Lung Adenocarcinoma to Guide Clinical Treatment. Front Immunol. 2022; 13: 916284. Han M, Bushong EA, Segawa M, Tiard A, Wong A, Brady MR, et al. Spatial mapping of mitochondrial networks and bioenergetics in lung cancer. Nature. 2023; 615: 712-9. Zhang L, Yao Y, Liu S. Targeting fatty acid metabolism for cancer therapy. Fundamental Research. 2024. Eltayeb K, La Monica S, Tiseo M, Alfieri R, Fumarola C. Reprogramming of Lipid Metabolism in Lung Cancer: An Overview with Focus on EGFR-Mutated Non-Small Cell Lung Cancer. Cells. 2022; 11. Vriens K, Christen S, Parik S, Broekaert D, Yoshinaga K, Talebi A, et al. Evidence for an alternative fatty acid desaturation pathway increasing cancer plasticity. Nature. 2019; 566: 403-6. Röhrig F, Schulze A. The multifaceted roles of fatty acid synthesis in cancer. Nat Rev Cancer. 2016; 16: 732-49. Zhang N, Liu Z, Lai X, Liu S, Wang Y. Silencing of CD147 inhibits cell proliferation, migration, invasion, lipid metabolism dysregulation and promotes apoptosis in lung adenocarcinoma via blocking the Rap1 signaling pathway. Respir Res. 2023; 24: 253. Ma L, Chen C, Zhao C, Li T, Ma L, Jiang J, et al. Targeting carnitine palmitoyl transferase 1A (CPT1A) induces ferroptosis and synergizes with immunotherapy in lung cancer. Signal Transduct Target Ther. 2024; 9: 64. Zhang H, Liu Y, Liu J, Chen J, Wang J, Hua H, et al. cAMP-PKA/EPAC signaling and cancer: the interplay in tumor microenvironment. J Hematol Oncol. 2024; 17: 5. Zhang H, Kong Q, Wang J, Jiang Y, Hua H. Complex roles of cAMP-PKA-CREB signaling in cancer. Exp Hematol Oncol. 2020; 9: 32. Ould Amer Y, Hebert-Chatelain E. Mitochondrial cAMP-PKA signaling: What do we really know? Biochim Biophys Acta Bioenerg. 2018; 1859: 868-77. Qu Y, Chen S, Zhou L, Chen M, Li L, Ni Y, et al. The different effects of intramuscularly-injected lactate on white and brown adipose tissue in vivo. Mol Biol Rep. 2022; 49: 8507-16. Abe T, Sato T, Murotomi K. Sudachitin and Nobiletin Stimulate Lipolysis via Activation of the cAMP/PKA/HSL Pathway in 3T3-L1 Adipocytes. Foods. 2023; 12. Ding L, Zhang F, Zhao MX, Ren XS, Chen Q, Li YH, et al. Reduced lipolysis response to adipose afferent reflex involved in impaired activation of adrenoceptor-cAMP-PKA-hormone sensitive lipase pathway in obesity. Sci Rep. 2016; 6: 34374. Yang LG, March ZM, Stephenson RA, Narayan PS. Apolipoprotein E in lipid metabolism and neurodegenerative disease. Trends Endocrinol Metab. 2023; 34: 430-45. Packard CJ, Pirillo A, Tsimikas S, Ference BA, Catapano AL. Exploring apolipoprotein C-III: pathophysiological and pharmacological relevance. Cardiovasc Res. 2024; 119: 2843-57. Taskinen MR, Borén J. Why Is Apolipoprotein CIII Emerging as a Novel Therapeutic Target to Reduce the Burden of Cardiovascular Disease? Curr Atheroscler Rep. 2016; 18: 59. Kohan AB. Apolipoprotein C-III: a potent modulator of hypertriglyceridemia and cardiovascular disease. Curr Opin Endocrinol Diabetes Obes. 2015; 22: 119-25. Tall AR. Increasing Lipolysis and Reducing Atherosclerosis. N Engl J Med. 2017; 377: 280-3. Klarin D, Damrauer SM, Cho K, Sun YV, Teslovich TM, Honerlaw J, et al. Genetics of blood lipids among ~300,000 multi-ethnic participants of the Million Veteran Program. Nat Genet. 2018; 50: 1514-23. Liu DJ, Peloso GM, Yu H, Butterworth AS, Wang X, Mahajan A, et al. Exome-wide association study of plasma lipids in >300,000 individuals. Nat Genet. 2017; 49: 1758-66. Shi J, Yang H, Duan X, Li L, Sun L, Li Q, et al. Apolipoproteins as Differentiating and Predictive Markers for Assessing Clinical Outcomes in Patients with Small Cell Lung Cancer. Yonsei Med J. 2016; 57: 549-56. Hu X, Ding S, Lu G, Lin Z, Liao L, Xiao W, et al. Apolipoprotein C-III itself stimulates the Syk/cPLA2-induced inflammasome activation of macrophage to boost anti-tumor activity of CD8(+) T cell. Cancer Immunol Immunother. 2023; 72: 4123-44. Zhang X, Ji L, Liu M, Li J, Sun H, Liang F, et al. Integrative Multianalytical Model Based on Novel Plasma Protein Biomarkers for Distinguishing Lung Adenocarcinoma and Benign Pulmonary Nodules. J Proteome Res. 2024; 23: 277-88. Jiang K, Yao G, Hu L, Yan Y, Liu J, Shi J, et al. MOB2 suppresses GBM cell migration and invasion via regulation of FAK/Akt and cAMP/PKA signaling. Cell Death Dis. 2020; 11: 230. Lei J, Wu L, Zhang N, Liu X, Zhang J, Kuang L, et al. Carcinoembryonic antigen potentiates non-small cell lung cancer progression via PKA-PGC-1ɑ axis. Mol Biomed. 2024; 5: 19. Yang M, Pan M, Huang D, Liu J, Guo Y, Liu Y, et al. Glucagon Promotes Gluconeogenesis through the GCGR/PKA/CREB/PGC-1α Pathway in Hepatocytes of the Japanese Flounder Paralichthys olivaceus. Cells. 2023; 12. Gilman AG. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987; 56: 615-49. Neves SR, Ram PT, Iyengar R. G protein pathways. Science. 2002; 296: 1636-9. O'Hayre M, Vázquez-Prado J, Kufareva I, Stawiski EW, Handel TM, Seshagiri S, et al. The emerging mutational landscape of G proteins and G-protein-coupled receptors in cancer. Nat Rev Cancer. 2013; 13: 412-24. Song L, Yu X, Wu Y, Zhang W, Zhang Y, Shao Y, et al. Integrin β8 Facilitates Macrophage Infiltration and Polarization by Regulating CCL5 to Promote LUAD Progression. Adv Sci (Weinh). 2025; 12: e2406865. Pham DV, Park PH. Adiponectin triggers breast cancer cell death via fatty acid metabolic reprogramming. J Exp Clin Cancer Res. 2022; 41: 9. Poudyal NR, Paul KS. Fatty acid uptake in Trypanosoma brucei: Host resources and possible mechanisms. Front Cell Infect Microbiol. 2022; 12: 949409. Rambold AS, Cohen S, Lippincott-Schwartz J. Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics. Dev Cell. 2015; 32: 678-92. Ostrom KF, LaVigne JE, Brust TF, Seifert R, Dessauer CW, Watts VJ, et al. Physiological roles of mammalian transmembrane adenylyl cyclase isoforms. Physiol Rev. 2022; 102: 815-57. Parker T, Wang KW, Manning D, Dart C. Soluble adenylyl cyclase links Ca(2+) entry to Ca(2+)/cAMP-response element binding protein (CREB) activation in vascular smooth muscle. Sci Rep. 2019; 9: 7317. Ferreira J, Levin LR, Buck J. Strategies to safely target widely expressed soluble adenylyl cyclase for contraception. Front Pharmacol. 2022; 13: 953903. Kilanowska A, Ziółkowska A, Stasiak P, Gibas-Dorna M. cAMP-Dependent Signaling and Ovarian Cancer. Cells. 2022; 11. Fujishita T, Kojima Y, Kajino-Sakamoto R, Mishiro-Sato E, Shimizu Y, Hosoda W, et al. The cAMP/PKA/CREB and TGFβ/SMAD4 Pathways Regulate Stemness and Metastatic Potential in Colorectal Cancer Cells. Cancer Res. 2022; 82: 4179-90. Halling JF, Pilegaard H. PGC-1α-mediated regulation of mitochondrial function and physiological implications. Appl Physiol Nutr Metab. 2020; 45: 927-36. Additional Declarations (Not answered) Supplementary Files Additionalfile1.docx Additional files OriginalqPCRdata.xls qPCR data OriginalWesternblotdata.pptx Full uncropped western blots Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8140812","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":559251307,"identity":"e9399cc1-5089-40aa-975c-be2b9be10e19","order_by":0,"name":"Liping Dai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYBACPgYGA4aECghHgigtbGAtZ0jWwthGkhbp5o0fHs6rszc4wHzwNg+DXR5hLTLHiiUSt7ExGxxgS7bmYUguJqxFIscAqIWHzeAAj5k0D8OBxAYitBj/SJwjwWNwgP8b0VrMJBIbDCSAtrARqUXmWJlFwrEEA8nDbMaWcwySCWvhl27efPNHTZ093/HmhzfeVNgR1oKIC2YQYUBQPQOx0TcKRsEoGAUjGgAAB74xrQqQ5DwAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-5531-1776","institution":"Zhengzhou University","correspondingAuthor":true,"prefix":"","firstName":"Liping","middleName":"","lastName":"Dai","suffix":""},{"id":559251308,"identity":"b87dd956-7c57-44fe-ba53-6a5cb7c632e4","order_by":1,"name":"Feifei Liang","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Feifei","middleName":"","lastName":"Liang","suffix":""},{"id":559251309,"identity":"75dda714-e813-424d-81d1-76d94adecf55","order_by":2,"name":"Ying Chen","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Chen","suffix":""},{"id":559251310,"identity":"8179cfb8-f2e4-40c5-886f-730a96ff0c8c","order_by":3,"name":"Yihao Liang","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yihao","middleName":"","lastName":"Liang","suffix":""},{"id":559251311,"identity":"e1a06988-bbee-4b8e-8ff8-2bd62f5b096b","order_by":4,"name":"Bing-Hua Jiang","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Bing-Hua","middleName":"","lastName":"Jiang","suffix":""},{"id":559251312,"identity":"31732395-c2a5-495a-8f6b-e78708127de4","order_by":5,"name":"Songyun Ouyang","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Songyun","middleName":"","lastName":"Ouyang","suffix":""}],"badges":[],"createdAt":"2025-11-18 04:30:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8140812/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8140812/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98749481,"identity":"9238af4e-8efd-4f70-9d5b-c84feea21603","added_by":"auto","created_at":"2025-12-22 09:01:53","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":157511,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/837203d7cedecf148fcc0efe.docx"},{"id":98779266,"identity":"7195630e-6356-41d7-906c-880051ec33b8","added_by":"auto","created_at":"2025-12-22 12:30:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4399595,"visible":true,"origin":"","legend":"","description":"","filename":"Figures.docx.docx","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/def6f51cf241b2032a2ceb4f.docx"},{"id":98749482,"identity":"31c385b5-39ad-4697-8628-af8c59ab5137","added_by":"auto","created_at":"2025-12-22 09:01:53","extension":"json","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7774,"visible":true,"origin":"","legend":"","description":"","filename":"CDDIS257379.json","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/8fdbbd96aab38754acb61d38.json"},{"id":98777296,"identity":"bd1cb88c-e117-40fb-88b5-febbf81240d5","added_by":"auto","created_at":"2025-12-22 12:26:30","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2613838,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/de325b4ae657e90674ff1c2a.docx"},{"id":98777458,"identity":"bc410d8b-879e-4c54-8c45-b02b2ad563df","added_by":"auto","created_at":"2025-12-22 12:27:27","extension":"pptx","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4788103,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalWesternblotdata.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/97d7b6994ca341ee6b16cd91.pptx"},{"id":98749491,"identity":"a8b78a89-3951-451a-a39c-d557a4b2ad4f","added_by":"auto","created_at":"2025-12-22 09:01:53","extension":"xls","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":150528,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalqPCRdata.xls","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/81e3a857d9a9b8ef3506d3fd.xls"},{"id":98749496,"identity":"b4894abf-a32c-4dbf-9424-81f5778bddb2","added_by":"auto","created_at":"2025-12-22 09:01:53","extension":"xml","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":94076,"visible":true,"origin":"","legend":"","description":"","filename":"CDDIS2573790enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/951846a1351128163c6df95d.xml"},{"id":98778138,"identity":"cdf15ef3-8a20-4213-b858-4a66a21da5c4","added_by":"auto","created_at":"2025-12-22 12:28:56","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6820792,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/d2f31a4b73153bec66b74f25.jpeg"},{"id":98749509,"identity":"348ce0cf-82ab-4383-ad05-d579c0b3f959","added_by":"auto","created_at":"2025-12-22 09:01:53","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6820792,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/185522db34b44b2182503555.jpeg"},{"id":98749507,"identity":"4c7a7c2c-4c29-4a56-b08e-4abc539976b4","added_by":"auto","created_at":"2025-12-22 09:01:53","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6820792,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/96de43deafcba7bce14032d0.jpeg"},{"id":98777753,"identity":"77635c96-630c-4655-95b7-defc90c328fd","added_by":"auto","created_at":"2025-12-22 12:28:25","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6820792,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/2db19abdc1a2b20b4687555a.jpeg"},{"id":98749511,"identity":"bd7bbeb6-6ece-459a-be38-5a4bf44c193b","added_by":"auto","created_at":"2025-12-22 09:01:53","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6820792,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/1d31f30980bf65b1b3ef51ca.jpeg"},{"id":98749501,"identity":"5da5b207-90c0-4967-a5e1-57c365ed8a83","added_by":"auto","created_at":"2025-12-22 09:01:53","extension":"jpeg","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6820792,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/029826041c89f48b6451e314.jpeg"},{"id":98749498,"identity":"5ea78cb5-6ca5-42a0-829a-d70f9aafe818","added_by":"auto","created_at":"2025-12-22 09:01:53","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":446188,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/16c5b6aa0ee10aefcd309710.png"},{"id":98779163,"identity":"b6acfcbe-82a0-49bc-886e-5ddb78fd8571","added_by":"auto","created_at":"2025-12-22 12:30:00","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":135472,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/01fd20944f5e51e887ddd6fe.png"},{"id":98779522,"identity":"870d4b49-0217-468b-af1c-be729554f4de","added_by":"auto","created_at":"2025-12-22 12:30:26","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":182517,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/90dd5cfc98e7276f2eaa69a6.png"},{"id":98749508,"identity":"df3aef3a-ae2c-4048-806b-f5e2ba4dc197","added_by":"auto","created_at":"2025-12-22 09:01:53","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126414,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/d0aba2541e29d6d97574f56e.png"},{"id":98778179,"identity":"bffb0d06-2179-4083-a483-8afb6c21f269","added_by":"auto","created_at":"2025-12-22 12:28:58","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":144143,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/9bc6de700ebe6e0c8961534b.png"},{"id":98749504,"identity":"538cbeb2-b8d2-4998-bc78-9b0e0e38c138","added_by":"auto","created_at":"2025-12-22 09:01:53","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":65238,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/304405a197ac81789b8cff31.png"},{"id":98749512,"identity":"be926fa2-a215-48ec-abb9-c7a82153199a","added_by":"auto","created_at":"2025-12-22 09:01:54","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":73280,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/3252d9b5a692639cb40986c3.png"},{"id":98777536,"identity":"12148e22-0def-4426-b15c-50c037385f49","added_by":"auto","created_at":"2025-12-22 12:27:57","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":47935,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/fb0522029345bf056b04305c.png"},{"id":98779577,"identity":"5bcc0e50-2c5c-4f43-91c2-52705c04e485","added_by":"auto","created_at":"2025-12-22 12:30:28","extension":"xml","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":90662,"visible":true,"origin":"","legend":"","description":"","filename":"CDDIS2573790structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/97fab51b2d876b4f5445ded2.xml"},{"id":98749514,"identity":"c693a587-dcc5-4e5d-a7ac-a511e50207ad","added_by":"auto","created_at":"2025-12-22 09:01:54","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":103290,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/c4c5b1a9f96a2b3fd38e8b55.html"},{"id":98749485,"identity":"f58550c2-5b0a-4acd-8f34-0b85d230390e","added_by":"auto","created_at":"2025-12-22 09:01:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":403368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAPOC3 is lowly expressed in LUAD and is positively correlated with TG levels in plasma. A \u003c/strong\u003eThe protein levels of APOC3 in 103 LUAD tissues and NATs. \u003cstrong\u003eB \u003c/strong\u003eROC analysis of APOC3 in LUAD. \u003cstrong\u003eC \u003c/strong\u003eTwo examples of human lung adenocarcinoma \u003ca href=\"https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/tissue-microarray\" title=\"Learn more about tissue microarrays from ScienceDirect's AI-generated Topic Pages\"\u003etissue microarrays\u003c/a\u003e, including tumor and adjacent tissues. \u003cstrong\u003eD \u003c/strong\u003eThe IHC score of APOC3 in 88 LUAD tissues and paired NATs (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). \u003cstrong\u003eE\u003c/strong\u003e The survival curve analysis of distant metastasis patients with high expression of APOC3. \u003cstrong\u003eF \u003c/strong\u003eCorrelation analysis of APOC3 and TG levels in blood samples of 146 patients with lung adenocarcinoma (r=0.182. \u003cem\u003eP\u003c/em\u003e=0.043). \u003cstrong\u003eG \u003c/strong\u003eThe concentration of TG in plasma of patients with 146 lung adenocarcinoma, 146 benign pulmonary nodules and 44 normal controls (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/ea77f5e646018993fce100f3.png"},{"id":98749487,"identity":"6e391a8b-3d34-47e0-a4ea-145264f13a5f","added_by":"auto","created_at":"2025-12-22 09:01:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":509462,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAPOC3 inhibits the proliferation, migration, invasion, and EMT of LUAD cells both in vivo and in vitro.\u003c/strong\u003e \u003cstrong\u003eA-B \u003c/strong\u003eWestern blot and qPCR were used to evaluate the overexpression efficiency of the APOC3-lentivirus infection in A549 and H1975 cells. \u003cstrong\u003eC-E\u003c/strong\u003e Colony formation (\u003cstrong\u003eC\u003c/strong\u003e), wound healing (\u003cstrong\u003eD\u003c/strong\u003e), transwell (\u003cstrong\u003eE\u003c/strong\u003e) assays were performed to detect the proliferation, migration and invasion ability of LUAD cells transfected with Lenti-APOC3 and Lenti-Vector. Scale bars, 50 μm. Data represent mean ± SD of three independent experiments, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, based on Student’s t-test.\u003cstrong\u003e F\u003c/strong\u003e Western blot analysis of the protein levels of E-cadherin, N-cadherin and Vimentin in A549 and H1975 cells transfected with APOC3 and Vector. \u003cstrong\u003eG-H\u003c/strong\u003e Photograph of subcutaneous tumors excised from Nod-scid mice treated with subcutaneous injection of A549 cells stably infected with Vector or APOC3 lentiviral particles (N = 4 per group). Tumors were observed and recorded by tumor volume. Data are shown as means ± SD. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. \u003cstrong\u003eI\u003c/strong\u003e Immunohistochemistry staining of APOC3, E-cadherin, Vimentin and Ki-67 in xenograft tissues. scale bars, 100 μm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/97dce301a767704fffa14f33.png"},{"id":98749483,"identity":"355527cf-2593-4c2f-a333-39fdfd22d1e7","added_by":"auto","created_at":"2025-12-22 09:01:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":396764,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAPOC3 inhibits fatty acid metabolism in lung adenocarcinoma. A \u003c/strong\u003eLipid metabolism signaling pathway was significantly enriched on genes negatively correlated with APOC3 expression in LUAD proteomic data (iProx Consortium, subproject ID: IPX0001804000). \u003cstrong\u003eB \u003c/strong\u003eRepresentative images of lipid droplets (LDs) in A549 and H1975 cells transfected with APOC3 and Vector as evaluated using APOC3 staining (green) and Nile red staining; cell nuclei were stained with 4',6-Diamidino-2-Phenylindole (blue). \u003cstrong\u003eC \u003c/strong\u003eThe mitochondrial metabolic activity in A549 and H1975 cells \u003cstrong\u003eD-E\u003c/strong\u003eConcentration of FFA and FAO activity in A549 and H1975 cells transfected with APOC3 and Vector. \u003cstrong\u003eF\u003c/strong\u003e Western blot analysis of CPT1A in APOC3-overexpressed stable A549 and H1975 cells. \u003cstrong\u003eG\u003c/strong\u003e Concentration of ATP in A549 and H1975 cells transfected with APOC3 and Vector. \u003cstrong\u003eH\u003c/strong\u003eConcentration of triglyceride (TG) of tumor tissues (left panel) and statistic (right panel). \u003cstrong\u003eI-J\u003c/strong\u003e FFA content and FAO activity in subcutaneous tumor tissue of mice. \u003cstrong\u003eK\u003c/strong\u003e Western blot analysis of CPT1A expression in tumor issues of mice. \u003cstrong\u003eL\u003c/strong\u003e The ATP levels in tumor tissue of mice. **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. Abbreviations: TG: triglyceride; FFA: free fatty acid; FAO: fatty acid oxidation\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/4353ffee707b70429cc868e8.png"},{"id":98749490,"identity":"268697d4-7b7f-4447-9f25-a2983c7ed8a9","added_by":"auto","created_at":"2025-12-22 09:01:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":501779,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAPOC3 inhibits fatty acid metabolism through the cAMP/PKA signaling pathway. A \u003c/strong\u003eSignificantly changing genes between vector and APOC3-overexpressed cells are shown in volcano plots with−log10(p.Value) on the Y axis and log2 fold change on the X axis. Red and Blue colors means upregulated and downregulated differentially expressed genes respectively (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and l FC l\u0026gt; 1.5 ). \u003cstrong\u003eB\u003c/strong\u003eKEGG showing the enriched pathways of downregulated genes. \u003cstrong\u003eC-D\u003c/strong\u003eConcentration of cAMP in A549 and H1975 cells. \u003cstrong\u003eE-F\u003c/strong\u003e Western blot analysis of PKA, p-PKA, CREB, and p-CREB expression in A549 cells and H1975 cells. \u003cstrong\u003eG\u003c/strong\u003eWestern blot analysis of PKA, p-PKA, CREB, p-CREB, HSL, p-HSL and PGC-1α expression when treated with the indicated concentrations of forskolin for 36 h in A549 cells. \u003cstrong\u003eH\u003c/strong\u003e Western blot analysis of PKA, p-PKA, CREB, p-CREB, HSL, p-HSL and PGC-1α expression when treated with the indicated concentrations of SQ22536 for 36 h in PC-9 cells. \u003cstrong\u003eI \u003c/strong\u003eUsing Mito-Tracker Red CMXRos to detect the effect of Forskolin on mitochondrial activity in A549 cells. Probes fluorescing red represent those that Mitochondria with biological activity. \u003cstrong\u003eJ-k\u003c/strong\u003eTranswell experiment showed the effect of Forskolin on ability of cell migration and invasion. \u003cstrong\u003eL\u003c/strong\u003e Detection of the expression levels of PKA, p-PKA, CREB, and p-CREB in tumor tissues via Western Blot analysis.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/ef06f5ccd81f5255e4c401e8.png"},{"id":98749494,"identity":"2f0034f6-d125-4b39-8371-4114d4538dd6","added_by":"auto","created_at":"2025-12-22 09:01:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":229816,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAPOC3 inhibits TG decomposition through the cAMP-PKA-HSL axis and fatty acid metabolism through the cAMP-PKA-CREB-PGC1α axis. A\u003c/strong\u003e p-HSL expression in siHSL and siAPOC3-transfected PC-9 cells as examined by western blotting. \u003cstrong\u003eB \u003c/strong\u003eThe content of TG in siHSL and siAPOC3-transfected PC-9 cells as examined by Nile Red. Probes fluorescing red represent those that have been combined to lipid droplet. Scale bar, 100 μm. \u003cstrong\u003eC\u003c/strong\u003e The content of FFA in siHSL and siAPOC3-transfected PC-9 cells. \u003cstrong\u003eD\u003c/strong\u003e PGC-1α expression in siHSL and siAPOC3-transfected PC-9 cells as examined by western blotting. \u003cstrong\u003eE \u003c/strong\u003eUsing Mito-Tracker Red CMXRos to detect the mitochondrial activity in siPGC-1α and siAPOC3-transfected PC-9 cells. Probes fluorescing red represent those that Mitochondria with biological activity. \u003cstrong\u003eF \u003c/strong\u003eThe ATP levels of siPGC-1α and siAPOC3-transfected PC-9 cells. \u0026nbsp;**\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. Abbreviations: TG: triglyceride; FFA: free fatty acid\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/471b32516023672ae368ece4.png"},{"id":98777696,"identity":"041eaae9-f1ed-4bad-adfa-c279437c3e1d","added_by":"auto","created_at":"2025-12-22 12:28:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":199552,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAPOC3 interacts with GNAI3 to inhibit the cAMP/PKA pathway in LUAD.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003eKEGG pathway enrichment analysis of interacting proteins identified through immunoprecipitation-mass spectrometry (IP-MS). \u003cstrong\u003eB \u003c/strong\u003eThe peptide sequences of the GNAI3 protein as detected in the Co-IP complex by mass spectrometry. \u003cstrong\u003eC\u003c/strong\u003eThe correlation analysis between APOC3 and GNAI3. Data from iProx Consortium with the subproject ID IPX0001804000. \u003cstrong\u003eD\u003c/strong\u003e A549 cells were infected with Flag-APOC3 plasmids. Co-IP was performed and the IP samples were analyzed through Western blotting.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/8e156addaf9d50a5ceecc685.png"},{"id":98749497,"identity":"44ff783b-e3ed-4f90-9944-854824ef0fb2","added_by":"auto","created_at":"2025-12-22 09:01:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":218105,"visible":true,"origin":"","legend":"\u003cp\u003eA schematic model illustrating the role of APOC3 and its underlying mechanism in fatty acid metabolism and tumorigenesis.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/26ca03c9c35de7c1031f3aa8.png"},{"id":100146508,"identity":"51370c54-4426-4b74-a4b3-40373edc30e3","added_by":"auto","created_at":"2026-01-13 12:36:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3384447,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/ade6b7f4-3814-4e81-8d24-957c201b29b5.pdf"},{"id":98777280,"identity":"15513f3a-751a-43d5-a339-50bf13e07a2b","added_by":"auto","created_at":"2025-12-22 12:26:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2613838,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional files\u003c/p\u003e","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/e6f33fb07335f2ac4b90dc69.docx"},{"id":98749489,"identity":"6a7df59f-97a0-4b6b-b0ac-ae2c0e7b23f5","added_by":"auto","created_at":"2025-12-22 09:01:53","extension":"xls","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":150528,"visible":true,"origin":"","legend":"\u003cp\u003eqPCR data\u003c/p\u003e","description":"","filename":"OriginalqPCRdata.xls","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/12bf9c0ed0e1938a990fca62.xls"},{"id":98780181,"identity":"616eafa8-a343-4fb4-ab99-bf7039cf4636","added_by":"auto","created_at":"2025-12-22 12:31:08","extension":"pptx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4788103,"visible":true,"origin":"","legend":"Full uncropped western blots","description":"","filename":"OriginalWesternblotdata.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8140812/v1/07db33be32ce9feb3056eaa3.pptx"}],"financialInterests":"(Not answered)","formattedTitle":"APOC3-Mediated Fatty Acid Metabolism Suppresses Lung Adenocarcinoma Progression by Inhibiting GNAI3/cAMP/PKA Pathway","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAccording to recent global cancer statistics, lung cancer remains the most lethal type of cancer, ranking second only to breast cancer in terms of incidence [1]. In China, both the incidence and mortality rates of lung cancer are the highest among all cancers [2]. Non-small cell lung cancer (NSCLC) constitutes about 85% of all lung cancer cases, with lung adenocarcinoma (LUAD) representing 40% of all histological subtypes of NSCLC [3]. LUAD carries a substantial risk of metastasis, yet the precise molecular mechanisms underlying this process remain elusive [4]. Delving into the mechanisms governing LUAD initiation and progression, as well as identifying novel therapeutic targets, holds significant importance in enhancing patient survival rates.\u003c/p\u003e \u003cp\u003eLipid metabolism reprogramming is a crucial metabolic feature of tumor cells, influencing tumor initiation, progression, and immune modulation [5]. A recent study revealed a substantial enrichment of lipid droplets in LUAD cells, with fatty acid metabolism serving as the primary energy source for the malignant advancement of LUAD [6]. Moreover, heightened fatty acid synthesis in tumor cells leads to the activation of oxidative lipid degradation to produce ATP, meeting the demands for cell proliferation and survival during nutrient scarcity. Tumor cells modulate cell growth and metastasis signaling pathways through bioactive molecules generated by lipid metabolism [7]. Recent research indicates that increased lipid utilization promotes the growth and metastasis of various cancer cells, including lung cancer [8]. The aberrant activation of fatty acid synthesis and oxidative degradation metabolism significantly contributes to the metastasis, recurrence, and drug resistance observed in lung cancer [9, 10]. Inhibition of lipid metabolism and the Rap1 signaling pathway has been demonstrated to trigger apoptosis in LUAD cells [11]. Previous research has indicated that carnitine palmitoyl transferase 1A (CPT1A) enhances the metastatic potential of NSCLC through the regulation of fatty acid oxidation (FAO) [12]. Nonetheless, there is a scarcity of studies investigating the precise molecular pathways underlying lipid metabolism dysregulation in LUAD. Consequently, there is a pressing need for comprehensive investigations into the molecular underpinnings of lipid metabolism dysregulation in LUAD and the identification of novel therapeutic targets.\u003c/p\u003e \u003cp\u003eThe cAMP/PKA pathway is pivotal in regulating fatty acid oxidation, proliferation, and metastasis in malignant tumors [13, 14]. A crucial downstream effector of this pathway is the transcription factor CREB, which stimulates the expression of PGC-1α, a master regulator of mitochondrial biogenesis and fatty acid oxidation [15]. Activation of the cAMP/PKA pathway through intramuscular injection of lactate into the gastrocnemius muscle enhances the expression of key proteins involved in white adipose tissue lipolysis (AMPK, ATGL, HSL) and the mitochondrial marker PGC-1α [16]. Furthermore, this pathway upregulates the expression of hormone-sensitive triglyceride lipase (HSL), a critical enzyme in the second step of triglyceride hydrolysis, thereby modulating hydrolysis processes [17, 18].\u003c/p\u003e \u003cp\u003eApolipoprotein C3 (APOC3), the predominant small molecule globulin in the C group, is essential for maintaining triglyceride homeostasis [19]. APOC3 exhibits dynamic interchange among different lipoproteins, its equilibrium state contingent upon an individual's metabolic status [20\u0026ndash;22]. Numerous studies have highlighted the role of APOC3 in lipid metabolism and is a risk factor for cardiovascular diseases [23\u0026ndash;25]. APOC3 expression is typically low in small cell lung cancer; however, its level rises in patients undergoing preoperative neoadjuvant chemotherapy [26]. APOC3 has been shown to boost the anti-tumor effects of CD8\u003csup\u003e+\u003c/sup\u003e T cells by triggering macrophage inflammasome activation [27]. While APOC3 research has predominantly concentrated on cardiovascular diseases, particularly lacking in lung cancer investigations, specifically in LUAD. Our prior studies have shown that the expression level of APOC3 in LUAD patients is lower than that in benign pulmonary nodules (BPNs) patients [28]. As TG serves as a crucial energy source for the proliferation and spread of malignant tumor cells, further exploration into whether APOC3 modulates lipid metabolism to influence LUAD progression is necessary.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the function and mechanism of APOC3 in repressing the progression of LUAD in vitro and in vivo. We demonstrated that APOC3 attenuates the cAMP/PKA pathway to inhibit TG hydrolysis and fatty acid oxidation, resulting in the suppression of LUAD. The identification of APOC3 as a key regulator fatty acid metabolism of LUAD indicated that APOC3 could be a potential biomarker and provided novel insights into LUAD therapy.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy population\u003c/h2\u003e \u003cp\u003eTo investigate the expression of APOC3 in plasma samples, a total of 356 participants were consecutively recruited from a hospital in Henan Province (November 2017\u0026ndash;January 2020), including 156 patients with lung adenocarcinoma (LUAD), 156 with benign pulmonary nodules (BPNs), and 44 normal controls (NCs). All pulmonary nodules were detected via thoracic low-dose computed tomography (LDCT) and pathologically confirmed as benign or malignant (with LC subtypes determined by biopsy). NCs were self-reported healthy individuals enrolled during routine medical examinations at the same hospital. Pre-treatment peripheral blood samples were collected from all patients prior to surgery or cancer-directed therapy. Detailed demographic and clinical characteristics are provided in Table \u003cb\u003eS1\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eMoreover, 336 blood samples were collected from LUAD patients with follow-up visits for prognostic evaluation. The detailed characteristics of participants and analysis of clinical factors are shown in Table \u003cb\u003eS2\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThis study was approved by the Ethics Committee of Zhengzhou University (2021-KY-1057-002). Written informed consent was obtained from all participants prior to enrollment.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBlood sample collection and processing\u003c/h3\u003e\n\u003cp\u003ePeripheral venous blood (5 mL) was collected in EDTA-K2 anticoagulant tubes and transported to the laboratory within 2 h. Samples were centrifuged at 3000 rpm for 5 min to separate plasma, which was then aliquoted (500 \u0026micro;L per tube) into pre-labeled 1.5 mL Eppendorf tubes and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further analysis.\u003c/p\u003e\n\u003ch3\u003eCell culture and establishment of stable cell lines\u003c/h3\u003e\n\u003cp\u003eThe lung adenocarcinoma cell (A549) was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and lung adenocarcinoma cells (H1975 and PC-9) were derived from the ATCC Cell Bank of America. All three types of cells were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Solarbio, Beijing, China). All cell lines were incubated at 37℃ in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003ch3\u003eRNA extraction and quantitative Real-time PCR\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eRNA extraction and quantitative Real-time PCR\u003c/div\u003e \u003cp\u003eTotal RNA from cells was isolated using TRIzol reagent and then reverse transcribed into cDNA using NovoScript Plus All-in-one 1st Strand cDNA Synthesis SuperMix (Novoprotein, Shanghai, China). qRT‒PCR was performed using NovoStart SYBR qPCR SuperMix Plus (Novoprotein, Shanghai, China) on a Light Cycler 96 system. The relative gene expression was calculated by the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method with GAPDH used for normalization. The primer sequences are shown in Supplementary Table \u003cb\u003eS3\u003c/b\u003e.\u003c/p\u003e\n\u003ch3\u003eCell Proliferation Assays\u003c/h3\u003e\n\u003cp\u003eFor the CCK-8 assay, 3 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well were seeded in a 96-well plate. Individually cultured tumor cells were used as controls. Ten microliters of CCK-8 (Meilunbio, Dalian, China) were added to each well at 0, 24, 48, and 72 h. The cells were incubated for 1 h at 37\u0026deg;C, and the absorbance values were measured at 450 nm. Each time point was assessed in replicates of at least three wells.\u003c/p\u003e \u003cp\u003eFor the colony formation assay, 6 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e cells/well were seeded in a 6-well plate and fixed for 1 h after 14 days. Crystal violet was added for staining overnight. Each well was subsequently washed three times and the number of colonies was counted via Image-Pro Plus 6.0 software.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMigration assay and invasion assay\u003c/h2\u003e \u003cp\u003eFor the cell migration, 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells were suspended in 200 \u0026micro;L of serum-free medium, placed in the top inserts of a 24-well Transwell plate (Corning, New York, USA), and then exposed to 10% serum culture medium in the lower chambers. The cells on the bottom surface of the membrane were fixed and stained with crystal violet after 24 h, and the cells that migrated were counted via Image-Pro Plus 6.0 software.\u003c/p\u003e \u003cp\u003eThe membrane for the invasion assay was covered with 40 \u0026micro;L of Matrigel (Corning, New York, USA) (diluted 1:8 with RPMI-1640) in advance. The tumor cells were seeded in the upper chambers; the lower chambers were filled with 600 \u0026micro;L including 10% serum culture medium. After 48 h of incubation, the cells that adhered to the lower filter surface were counted via Image-Pro Plus 6.0 software.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWound healing assay\u003c/h3\u003e\n\u003cp\u003eA monolayer of cells was scraped with 10-\u0026micro;L pipette tips when the cells had reached 90% confluence in a 6-well plate. Then, images were taken at appropriate time points (0 h/24 h). Image-Pro Plus 6.0 software was used to assess the relative wound width.\u003c/p\u003e\n\u003ch3\u003eTriglyceride measurement\u003c/h3\u003e\n\u003cp\u003eIntracellular lipid droplets were detected using Oil Red O staining and Nile Red fluorescence assays. Cells were suspended at a density of 5 \u0026times; 10⁵ cells/mL and seeded in 6-well plates, followed by incubation at 37\u0026deg;C with 5% CO₂ for 24 h. Subsequent staining procedures were performed strictly according to the manufacturer's protocol.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial activity assay\u003c/h2\u003e \u003cp\u003eThe mitochondrial red fluorescent probe was used to detect mitochondrial activity. When the cells reach approximately 80% confluence with uniform distribution, carefully aspirate the original culture medium and replace it with the pre-warmed MitoTracker Red CMXRos working solution. Incubate the cells in a humidified 37\u0026deg;C, 5% CO₂ cell culture incubator for 25 min, protected from light. After incubation, gently remove the staining solution and wash the cells twice with warm PBS or fresh culture medium to remove excess probe. Finally, replenish with fresh pre-warmed culture medium and immediately observe the cells under a fluorescence microscope equipped with TRITC/Rhodamine filters. Capture images promptly while minimizing light exposure to prevent photobleaching.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFFA, FAO and ATP content detection\u003c/h2\u003e \u003cp\u003eTo measure the Intracellular FFA levels of LUAD cells, the FFA kit was purchased from Suzhou Keming Biotech Co. LTD (Cat. FFAD-1-W, Suzhou, China). The content is measured according to the manufacturer's protocol. Experiments were performed three times.\u003c/p\u003e \u003cp\u003eThe FAO activity was detected by a commercial reagent kit. The FAO enzyme-linked immunosorbent assay (ELISA) kit was purchased from Shanghai Meilian Biotech Co. LTD (Lot. ml060146, Shanghai, China), following the manufacturer\u0026rsquo;s instructions. Experiments were performed three times.\u003c/p\u003e \u003cp\u003eIn order to detect the intracellular ATP content, The ATP kit was purchased from Beijing Solarbio Biotech Co. LTD (Cat. BC0300, Beijing, China). The content is measured according to the manufacturer's protocol. Experiments were performed three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eTo investigate protein co-localization, harvest well-grown cells and seed them evenly onto sterile coverslips at an appropriate density. After 6 h of adhesion, carefully retrieve the coverslips and fix the cells with 4% paraformaldehyde (PFA) at room temperature for 20 min. Subsequently, wash the samples three times with PBS, 5 min per wash. Permeabilize the cells with 0.2% Triton X-100 for 5 min at room temperature, followed by another three PBS washes. Block nonspecific binding sites by incubating the samples with 1% bovine serum albumin (BSA) for 1 h, then wash again with PBS three times. Incubate the samples overnight at 4\u0026deg;C with primary antibodies diluted in 1% BSA. The following day, remove unbound primary antibodies by washing three times with PBS, then incubate with fluorescently conjugated secondary antibodies (diluted in 1% BSA) for 1 h at room temperature protected from light. After three final PBS washes, mount the coverslips using an anti-fade mounting medium containing DAPI for nuclear counterstaining. Carefully invert the coverslips onto glass slides, seal the edges, and image using a fluorescence microscope equipped with appropriate filter sets.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eThe whole cell lysates were extracted using RIPA Lysis buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (Solarbio, Beijing, China). The proteins were separated via 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% milk, the membrane was incubated with primary antibody overnight at 4\u0026deg;C. HRP-conjugated anti-mouse or anti-rabbit antibodies (1:5000, Proteintech, Wuhan, China) were used as secondary antibodies, and the antigen antibody reactions were visualized by ECL (HaiGene, Harbin, China). The primary antibodies that were used are listed in Table \u003cb\u003eS4\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTumor xenograft model\u003c/h2\u003e \u003cp\u003eA549 cells stably overexpressing APOC3 were resuspended in PBS at a density of 5\u0026times;10⁷ cells/mL, and 100 \u0026micro;L of the cell suspension was subcutaneously injected into the right axillary region of mice. When the tumors reached approximately the size of a rice grain (typically 3\u0026ndash;5 mm in diameter), tumor dimensions and mouse body weights were recorded every two days using digital calipers and an analytical balance, respectively. Approximately 40 days post-injection, mice were humanely euthanized via carbon dioxide asphyxiation. Tumor tissues were then carefully excised, photographed with a size reference, weighed using a precision balance, and processed for further analysis. Tumor growth curves were generated by plotting tumor volume (calculated as [length \u0026times; width\u0026sup2;]/2) against time, while body weight changes were monitored throughout the study period to assess potential toxicity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of lung metastasis model\u003c/h2\u003e \u003cp\u003eHealthy, log-phase cells were harvested and resuspended in PBS at a concentration of 5\u0026times;10⁶ cells/100 \u0026micro;L. Using a 29G insulin syringe, 100 \u0026micro;L of cell suspension was slowly injected into the tail vein (3 mm insertion depth) over 30 seconds, followed by immediate application of a sterile cotton ball with firm pressure for 2 min to ensure hemostasis. Mice were monitored for 30 min post-injection to confirm normal behavior and absence of adverse effects. After 28 days of standard housing, the mice were humanely euthanized by controlled CO₂ asphyxiation (20% chamber volume displacement per min), and complete lung tissues were excised for subsequent experimental analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing\u003c/h2\u003e \u003cp\u003eThis study performed transcriptome sequencing analysis on three groups of A549 cell lines, including APOC3-overexpressing and normal APOC3-expressing groups, through collaboration with Zhengzhou Zhenhe Biotechnology Co., Ltd. The analytical pipeline consisted of the following steps: raw data processing\u0026rarr;quality control\u0026rarr;sequence alignment\u0026rarr;gene expression quantification\u0026rarr;differential expression analysis. Differentially expressed genes were identified based on the threshold criteria of |log2FC| \u0026gt; 1.5 with a significance level of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Subsequently, the identified differentially expressed genes were subjected to KEGG pathway enrichment analysis and Gene Ontology (GO) functional analysis to elucidate their potential biological roles and involved pathways.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical (IHC)\u003c/h2\u003e \u003cp\u003eMouse tumor specimen sections were used for IHC. The sections were separately incubated with anti-APOC3 (1:400, Abcam, USA), anti-E-cadherin (1:500, CST, USA), anti-Vimentin (1:500, CST, USA) and anti-Ki67(1:500, Wuhan, China) antibodies. The scores were determined by combining the proportion of positively stained tumor cells and the intensity of staining. The proportion of positively stained tumor cells in a field was scored as follows: 0, no positive tumor cells; 1, \u0026lt;\u0026thinsp;10% positive tumor cells; 2, 10\u0026ndash;35% positive tumor cells; 3, 35\u0026ndash;75% positive tumor cells; and 4, \u0026ge;\u0026thinsp;75% positive tumor cells. The staining index (SI) for each sample was obtained by multiplying the intensity and proportion values. An SI\u0026thinsp;\u0026ge;\u0026thinsp;7 was considered a high expression, and samples with an SI\u0026thinsp;\u0026lt;\u0026thinsp;7 were considered to have low expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHE staining\u003c/h2\u003e \u003cp\u003eMouse lung tissues were fixed in 4% paraformaldehyde (PFA), dehydrated, and embedded in paraffin. Tissue sections (4\u0026ndash;5\u0026micro;m thick) were prepared and stained with hematoxylin and eosin (H\u0026amp;E) for histological examination. Stained sections were digitally scanned, and quantitative analysis of the staining area was performed using Case Viewer software. Statistical analysis was conducted to evaluate differences in staining intensity and distribution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation (Co-IP), and mass spectrometry (MS) analysis\u003c/h2\u003e \u003cp\u003eCo-IP was performed to confirm protein-protein binding. Selleck Protein A/G Magnetic Bead System (#B23202, Houston, USA) and detailed protocol as previously described [29]. The antibodies used to incubate cell lysate was listed on Table \u003cb\u003eS4\u003c/b\u003e. The Co-IP complexes were analyzed by SDS-PAGE and IB (Immunoblot). And anti-mouse IgG (Cat: SE131, Solarbio) was used as the second antibody for Co-IP complexes detection and the Co-IP complexes. And the binding proteins of APOC3 were isolated by Co-IP and identified by mass spectrometry (MS) analysis applying Triple tof5600 and the results were processed by ProteinPilot (version 5.01).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eThe concentrations of APOC3 in patients\u0026rsquo; plasma were determined strictly following the manufacturer\u0026rsquo;s instructions. The ELISA kit used in the study was purchased from Cloud clone (Wuhan, China). The absorbance was measured at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe experimental results were quantified using ImageJ software, and statistical analysis was performed using GraphPad Prism 8.0. Each experiment was repeated at least three times. T-tests were used to analyze differences between two groups of data, while one-way ANOVA was employed to determine the statistical significance of data from three or more groups. Differences were considered statistically significant when \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, with significance levels indicated as *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eAPOC3 is dramatically decreased in LUAD and positively correlated with the plasma TG levels\u003c/h2\u003e \u003cp\u003eWe previously found that the plasma APOC3 levels were significantly lower in LUAD patients than those with benign pulmonary nodules or normal subjects[28]. To further investigate the APOC3 expression between LUAD and normal tissues, the proteomics data from 103 LUAD patients and paired normal adjacent tissues (NATs) were analyzed, we observed that APOC3 expression was significantly lower in LUAD tissues than that in NATs (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Receiver Operating Characteristic (ROC) analysis showed that APOC3 could serve as a potential diagnostic indicator for LUAD, with an AUC (95% CI) of 0.862 (0.012\u0026ndash;0.911) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). To further validate APOC3 expression levels in LUAD and paired normal adjacent tissues (NATs), we analyzed tissue microarrays comprising 92 LUAD samples and 88 matched NAT pairs. The results were consistent with our initial findings, confirming the differential expression pattern of APOC3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). Moreover, low expression of APOC3 was strongly correlated with male (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, Mann-Whitney U test), EGFR negative patients (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Mann-Whitney U test) and LUAD patients with positive lymph node metastasis (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Mann-Whitney U test) (Figure \u003cb\u003eS1A-I\u003c/b\u003e).To further investigate the correlation between APOC3 and prognosis of LUAD patients, we collected plasma samples from 336 LUAD patients for prognostic analysis. Kaplan-Meier survival curves revealed a statistically significant association between elevated APOC3 expression and improved overall survival in the cohort of LUAD patients presenting with distant metastases (log-rank \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.023, HR\u0026thinsp;=\u0026thinsp;2.24, 95% CI: 1.10\u0026ndash;4.57) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Further stratified analysis indicated that gender, age, stage, lymph node metastasis, nodule size, smoking history, and alcohol consumption were not significantly correlated with prognosis (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Figure \u003cb\u003eS1J-X\u003c/b\u003e). To identify clinical factors independently associated with APOC3 expression, univariate and multivariate regression analyses were performed, with the complete results detailed in Table \u003cb\u003eS5\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eDetection of APOC3 expression in plasma by ELISA method showed a positive correlation between APOC3 and TG levels, which were lower in LUAD patients than in benign lung nodules and normal controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G), suggesting that APOC3 may modulate lipid metabolism of LUAD.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eAPOC3 inhibits the proliferation and metastasis of LUAD in vivo and in vitro\u003c/h2\u003e \u003cp\u003eTo explore the role of APOC3 in LUAD progression, we examined its expression in normal lung epithelial cells (BASE-2B) and lung adenocarcinoma cell lines. Decreased expression of APOC3 was observed in the LUAD cell lines A549 and H1975 when compared with BEAS-2B. (Figure \u003cb\u003eS2A\u003c/b\u003e). We then established APOC3-overexpressing stable A549 and H1975 cells and APOC3- silenced stable PC-9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B, Figure \u003cb\u003eS2B\u003c/b\u003e). Colony-formation assays displayed a decreased clonogenic capacity of both A549 and H1975 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) and CCK8 assays confirmed the suppression of growth in both A549 and H1975 cells (Figure \u003cb\u003eS2C-D\u003c/b\u003e), but the converse result was observed in PC-9 cells (Figure \u003cb\u003eS2E-F\u003c/b\u003e). The results of wound healing experiments showed that overexpression of APOC3 significantly inhibited lateral migration ability in A549 and H1975 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) and silencing APOC3 promoted horizontal migration ability in PC-9 cells (Figure \u003cb\u003eS2G\u003c/b\u003e). And in the transwell assay, we prolonged the experimental time to allow more cell-movement and found overexpression APOC3 inhibited much more migration and invasion than APOC3-Vector in A549 and H1975 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and Figure \u003cb\u003eS2H-J\u003c/b\u003e). Moreover, APOC3 overexpression significantly induced E-cadherin, while inhibiting N-cadherin, vimentin levels(Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and Figure \u003cb\u003eS2K\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTo gain insights into the importance of APOC3 in repressing the progression of LUAD cells in vivo, we performed xenograft experiments using A549 cell lines. Overexpression of APOC3 significantly reduced tumor growth and volume (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-H). Moreover, increased levels of E-cadherin and decreased levels of Ki67 and Vimentin were observed in APOC3-overexpression tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Furthermore, we observed that fewer APOC3-overexpressd A549 cells metastasized to the lung and initiated secondary tumors (Figure \u003cb\u003eS2L\u003c/b\u003e). Taken together, these findings suggest a role for APOC3 in inhibiting proliferation and metastasis of LUAD cells both in vivo and in vitro.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eAPOC3 inhibits fatty acid metabolism in lung adenocarcinoma\u003c/h2\u003e \u003cp\u003eLipid metabolism reprogramming is crucial for LUAD progression. APOC3, an important apolipoprotein, is key to triglyceride homeostasis. To investigate whether APOC3 regulated tumor progression via fatty acid metabolism, the metascape analysis was conducted on genes negatively correlated with APOC3 expression in LUAD proteomic data (iProx Consortium, subproject ID: IPX0001804000). This analysis revealed significant enrichment of the lipid metabolism signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), suggesting a potential mechanistic link between APOC3 and fatty acid metabolism in LUAD progression. The increased intracellular TG content and reduced mitochondrial activity were displayed in APOC3 overexpressed A549 and H1975 cells, while TG content decreased in APOC3-knockdown PC-9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C and S3A-E). We hypothesize that APOC3 inhibits triglyceride (TG) breakdown, thereby reducing free fatty acid (FFA) availability and limiting tumor cell energy derived from fatty acid oxidation. Consistent with this model, APOC3 overexpression in A549 and H1975 cells significantly decreased intracellular FFA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E), suppressed fatty acid oxidase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G) and downregulated key fatty acid oxidation markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, I), and ultimately reduced cellular ATP production (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ, K), while, the FFA levels, fatty acid oxidase activity, key fatty acid oxidation markers and ATP levels increased in APOC3-knockdown PC-9 cells (Figure \u003cb\u003eS3F-I\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eIn vivo experiments demonstrated that APOC3 overexpression increased triglyceride (TG) accumulation in mouse tumor tissues while reducing free fatty acid (FFA) content, fatty acid oxidase activity, expression of key fatty acid oxidation markers, and ATP levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eL-P), which consistent with in vitro findings. Taken together, these findings demonstrate that APOC3 suppresses fatty acid metabolism in LUAD, both in vitro and in vivo.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eAPOC3 inhibits fatty acid metabolism through the cAMP/PKA signaling pathway\u003c/h2\u003e \u003cp\u003eTo investigate how APOC3 regulates TG breakdown and fatty acid oxidation, transcriptome sequencing was performed on A549 cells with normal and overexpressed APOC3. Screening identified 194 upregulated and 313 downregulated genes (|log2FC|\u0026gt;1.5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eA \u003cb\u003eand\u003c/b\u003e Figure \u003cb\u003eS4A\u003c/b\u003e). KEGG analysis revealed significant enrichment of the cAMP pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The decreased cAMP levels were validated in A549 and H1975 cells overexpressing APOC3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eC \u003cb\u003eand\u003c/b\u003e Figure \u003cb\u003eS4B\u003c/b\u003e), while APOC3 knockdown in PC-9 cells increased cAMP levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). APOC3 overexpression significantly downregulated the cAMP downstream signaling such as the levels of p-PKA and p-CREB (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F \u003cb\u003eand\u003c/b\u003e Figure \u003cb\u003eS4C\u003c/b\u003e), which was effectively reversed by the cAMP activator Forskolin. Concurrently, the expression of p-HSL, a rate-limiting enzyme in TG decomposition, and PGC-1α, a key enzyme in fatty acid oxidation, was also restored to higher levels upon Forskolin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). In contrast, treatment of PC-9 cells with the cAMP inhibitor SQ22536 effectively inhibited the activation of the cAMP pathway and downregulated the expression of p-HSL and PGC-1α (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Forskolin also reversed the inhibitory effects of APOC3 on mitochondrial activity and fatty acid metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eI, Figure \u003cb\u003eS4D-F\u003c/b\u003e). Moreover, the inhibitory effects of APOC3 overexpression on A549 cell migration and proliferation were reversed upon Forskolin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ-K).\u003c/p\u003e \u003cp\u003eIn mouse xenograft tumors, APOC3 also inhibits the cAMP/PKA pathway, leading to reduced expression levels of p-PKA and p-CREB, which is consistent with the in vitro results (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eL). These results indicate that APOC3 suppresses the cAMP/PKA pathway both in vitro and in vivo.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eAPOC3 inhibits TG decomposition through the cAMP-PKA-HSL axis and suppresses fatty acid metabolism through cAMP-PKA-CREB-PGC-1α\u003c/h2\u003e \u003cp\u003ePrevious research has demonstrated that the expression of HSL and PGC-1α is regulated by the cAMP/PKA pathway [17, 30, 31]. To investigate the importance of HSL in APOC3-reduced TG breakdown, we detected the effects of HSL silencing on APOC3 knockdown PC-9 cells. The efficiency of HSL knockdown was confirmed by Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Moreover, the results indicated that HSL knockdown promoted TG accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and reduced FFA content (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). To explore the necessity of PGC-1α for APOC3-inhibited mitochondrial activity and ATP production, we conducted dual knockdown of APOC3 and PGC-1α in PC-9 cells. Western blot showed significant inhibition of PGC-1α expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Mitochondrial activity assays demonstrated that PGC-1α knockdown markedly reduced mitochondrial activity, as well as ATP levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F). Collectively, these results indicate that APOC3 reduces energy utilization efficiency by inhibiting the expression of p-HSL in lipid droplets and diminishes fatty acid oxidation capacity by suppressing the expression of PGC-1α.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eAPOC3 interacts with GNAI3 to inhibit the cAMP/PKA pathway in LUAD\u003c/h2\u003e \u003cp\u003eTo investigate how APOC3 affects the cAMP/PKA pathway, therefore, the APOC3 protein was precipitated using an APOC3 antibody, and interacting proteins were identified via mass spectrometry. KEGG analysis showed that APOC3 immunoprecipitants enriched in the cAMP/PKA pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). We identified GNAI3, directly inhibits adenylate cyclase (AC) upon GTP binding and suppresses cAMP production and downstream PKA activation [32\u0026ndash;34], that may be linked to APOC3 and the regulation of cAMP/PKA pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). We then transfected Flag-tagged APOC3 into A549 cells, immunoprecipitated the cell lysates with anti-Flag or anti-GNAI3 antibodies and immunoblotted the Co-IP complex. Our results indicated that APOC3 co-immunoprecipitated with GNAI3 but not with IgG in A549 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). In addition, we found a positive correlation between APOC3 and GNAI3 by using data from iProx Consortium with the subproject ID IPX0001804000 (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These data indicate that APOC3 may interact with GNAI3 to inhibit the cAMP/PKA pathway in LUAD.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe high morbidity and mortality rates of LUAD indicate a high degree of clinical need for the discovery of new effective therapeutic targets [35]. Our study reveals a previously unrecognized role of APOC3 in suppressing LUAD proliferation and metastasis by modulating fatty acid metabolism. The underlying mechanism involves two distinct pathways, on one pathway, APOC3 impedes triglyceride (TG) hydrolysis via the cAMP/PKA/HSL signaling axis, restricting energy substrate availability and reducing intracellular ATP levels. While on another pathway, APOC3 attenuates mitochondrial oxidative capacity and fatty acid β-oxidation through the cAMP/PKA/CREB/PGC-1α pathway, further compromising cellular bioenergetics. Collectively, these metabolic disruptions lead to significant inhibition of LUAD proliferation and metastatic potential, highlighting APOC3 as a promising therapeutic target for intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMetabolic reprogramming of fatty acids has recently been recognized as a key driver of cancer progression [36]. LUAD cells acquire FAs via (i) microenvironment uptake, (ii) de novo lipogenesis, or (iii) TG hydrolysis[37]. serving as a key bioenergetic pathway for LUAD progression[6]. During starvation, FAs undergo mitochondrial oxidation for ATP production[38]. APOC3, a component of triglyceride-rich lipoproteins, is a hypertriglyceridemia risk factor, recent clinical trials have shown that inhibiting APOC3 is a promising approach for treating hypertriglyceridemia and preventing cardiovascular disease [36, 37]. Our study reveals reduced plasma APOC3 and TG levels in LUAD patients, showing positive correlation. Paradoxically, APOC3 overexpression in LUAD cells (A549/H1975) increases TG accumulation while suppressing proliferation and metastasis, contradicting TG's known pro-tumor role. This unexpected APOC3-mediated tumor suppression despite TG elevation requires mechanistic investigation.\u003c/p\u003e \u003cp\u003eThe cAMP/PKA/CREB axis promotes tumor malignancy by enhancing proliferation, invasion, metastasis, and drug resistance [14]. The cyclic AMP (cAMP) pathway, mediated by transmembrane (tmACs) and soluble (sAC) adenylate cyclases [39, 40], forms compartmentalized signaling complexes with GPCRs to regulate diverse cellular processes [41]. Through PKA-mediated CREB activation, this pathway controls critical functions including gene transcription, mitochondrial homeostasis, and cell proliferation [42], with dysregulation implicated in multiple cancer types [13]. Clinical evidence demonstrates its therapeutic relevance, as shown in colorectal cancer metastasis [43]. Recent studies have demonstrated that Lactate activates the cAMP/PKA pathway, upregulating lipolysis regulator HSL and mitochondrial biogenesis factor PGC-1α [16]. Our findings extend this paradigm to LUAD, where APOC3 overexpression suppresses cAMP levels and inhibits p-PKA/p-CREB activation, consequently attenuating tumor progression. Beyond canonical signaling, cAMP/PKA critically regulates energy metabolism through CREB-mediated PGC-1α expression (controlling mitochondrial biogenesis/FA oxidation) [44], and HSL phosphorylation (promoting TG hydrolysis). APOC3 inhibits both p-HSL and PGC-1α via cAMP/PKA suppression, with pathway activation reversing these effects and restoring tumorigenic potential. The cAMP/PKA pathway emerges as a central coordinator of LUAD progression through integrated control of oncogenic signaling and metabolic reprogramming. APOC3's tumor-suppressive effects via this axis highlight its potential as a therapeutic target, particularly given the reversible nature of its metabolic inhibition.\u003c/p\u003e \u003cp\u003eWhile our study demonstrates that APOC3 suppresses LUAD progression by inhibiting the cAMP/PKA pathway and subsequent fatty acid metabolism, the precise molecular mechanism warrants further investigation. We identified a physical interaction between APOC3 and GNAI3, an inhibitory G protein of the cAMP pathway, along with their positive correlation in expression. However, whether APOC3 exerts its tumor-suppressive effects specifically through GNAI3-mediated cAMP signaling remains to be elucidated. This represents a limitation of our current work, as we cannot conclusively establish a causal relationship between the APOC3-GNAI3 interaction and the observed metabolic reprogramming in LUAD. Future studies employing GNAI3 knockdown or knockout models are needed to determine if GNAI3 is required for APOC3's inhibitory effects on cAMP signaling and tumor progression. Such investigations would provide more definitive evidence regarding the functional significance of this protein interaction in the context of LUAD pathogenesis and potentially identify novel therapeutic targets for metabolic intervention in LUAD.\u003c/p\u003e \u003cp\u003eAs expected, In vitro studies demonstrated APOC3 significantly modulates LUAD cell proliferation, migration and invasion. In vivo, APOC3 overexpression suppressed tumor growth and EMT progression, reducing tumor volume and pulmonary metastases. Mechanistically, APOC3 depletion enhanced HSL/PGC-1α-dependent fatty acid oxidation, while its overexpression inhibited lipolysis (via p-HSL downregulation) and fatty acid oxidation (through CPT1A suppression), ultimately reducing ATP production. RNA-seq implicated cAMP/PKA pathway involvement in these metabolic effects. Emerging evidence suggests APOC3 may enhance CD8\u0026thinsp;+\u0026thinsp;T cell antitumor activity through macrophage inflammasome activation[27], indicating potential immune-mediated mechanisms contributing to its tumor-suppressive effects in LUAD. Moreover, our survival analysis of 336 lung adenocarcinoma patients revealed significantly prolonged overall survival in patients with higher APOC3 expression levels. This protective effect remained statistically significant when patients were stratified by metastatic status (both metastatic and non-metastatic subgroups). These clinical findings strongly corroborate our experimental data, providing compelling evidence for APOC3's metastasis-inhibitory function in LUAD.\u003c/p\u003e \u003cp\u003eOverall, this study suggests for the first time that high expression of APOC3 reduced the fatty acid metabolism that inhibited LUAD progression. Our results shed new light on the role of APOC3 in the fatty acid metabolism of LUAD and provide a mechanistic basis for APOC3-mediated lipid metabolism induced progression in LUAD cells.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn the present study, we identify apolipoprotein C3 (APOC3) as a novel tumor suppressor in lung adenocarcinoma (LUAD) through regulation of fatty acid metabolism. Clinically, APOC3 is significantly downregulated in LUAD tissues and plasma, with its low expression correlating with metastatic progression and poor prognosis. Mechanistically, APOC3 interacts with GNAI3 to suppress the cAMP/PKA signaling pathway, leading to impaired triglyceride hydrolysis via reduced HSL phosphorylation and suppressed fatty acid oxidation through downregulation of PGC-1α. This metabolic rewiring results in accumulated lipid droplets, decreased ATP production, and ultimately inhibition of tumor growth and metastasis. Our findings establish the APOC3-GNAI3-cAMP/PKA axis as a crucial regulatory mechanism in LUAD, highlighting its therapeutic potential for metabolic intervention in lung cancer.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAPOC3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eApolipoprotein C3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCPT1A\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCarnitine Palmitoyl transferase 1A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eadenylate cyclase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eATGL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eadipose triglyceride lipase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMGL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emonoglyceride lipase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efatty acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFAO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFatty acid oxidation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFree fatty acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHSL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHormone-sensitive triglyceride lipase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLUAD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLung adenocarcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNSCLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNon-small cell lung cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTriglyceride.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatient blood collection was followed the guidelines and principles of the Helsinki Declaration and received approval from the Ethics Committee of First Affiliated Hospital of Zhengzhou University (2021-KY-1057-002). Written informed consent was obtained from all study participants.\u0026nbsp;All animal experiments were performed in accordance with the Basel Declaration and approved by the Animal Ethics Committee of Henan Institute of Medical and Pharmaceutical Sciences (2023-yyy-046).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no competing interest exists.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used in the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupporting Material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional results, prognostic information of LUAD patients and extended analysis are provided as Supplementary Information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Leading Talents of Science and Technology Innovation in Henan Province (Grant Number 20420051008); the National\u0026nbsp;Natural Science Foundation of China (No. 82203287); the project funded by China Postdoctoral Science Foundation (No. 2022M722874) and the Project of Basic Research Fund of Henan Institute of Medical and Pharmacological Sciences (No. 2025BP0203). The authors gratefully acknowledge Henan Key Laboratory of Pharmacology for Liver Diseases for providing experimental platform support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFFL, RLD and LPD designed experiments and prepared the manuscript. FFL and YC performed experiments and analyzed the data. YHL analyzed clinical samples. SYOY provided clinical sample. BHJ provided advice. All authors read and approved the final version of manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin. 2024; 74: 12-49.\u003c/li\u003e\n\u003cli\u003eWu Y, He S, Cao M, Teng Y, Li Q, Tan N, et al. Comparative analysis of cancer statistics in China and the United States in 2024. Chin Med J (Engl). 2024; 137: 3093-100.\u003c/li\u003e\n\u003cli\u003eSiegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2022. CA Cancer J Clin. 2022; 72: 7-33.\u003c/li\u003e\n\u003cli\u003eFang H, Sun Q, Zhou J, Zhang H, Song Q, Zhang H, et al. m(6)A methylation reader IGF2BP2 activates endothelial cells to promote angiogenesis and metastasis of lung adenocarcinoma. Mol Cancer. 2023; 22: 99.\u003c/li\u003e\n\u003cli\u003eHuang D, Tang E, Zhang T, Xu G. Characteristics of Fatty Acid Metabolism in Lung Adenocarcinoma to Guide Clinical Treatment. Front Immunol. 2022; 13: 916284.\u003c/li\u003e\n\u003cli\u003eHan M, Bushong EA, Segawa M, Tiard A, Wong A, Brady MR, et al. Spatial mapping of mitochondrial networks and bioenergetics in lung cancer. Nature. 2023; 615: 712-9.\u003c/li\u003e\n\u003cli\u003eZhang L, Yao Y, Liu S. Targeting fatty acid metabolism for cancer therapy. Fundamental Research. 2024.\u003c/li\u003e\n\u003cli\u003eEltayeb K, La Monica S, Tiseo M, Alfieri R, Fumarola C. Reprogramming of Lipid Metabolism in Lung Cancer: An Overview with Focus on EGFR-Mutated Non-Small Cell Lung Cancer. Cells. 2022; 11.\u003c/li\u003e\n\u003cli\u003eVriens K, Christen S, Parik S, Broekaert D, Yoshinaga K, Talebi A, et al. Evidence for an alternative fatty acid desaturation pathway increasing cancer plasticity. Nature. 2019; 566: 403-6.\u003c/li\u003e\n\u003cli\u003eR\u0026ouml;hrig F, Schulze A. The multifaceted roles of fatty acid synthesis in cancer. Nat Rev Cancer. 2016; 16: 732-49.\u003c/li\u003e\n\u003cli\u003eZhang N, Liu Z, Lai X, Liu S, Wang Y. Silencing of CD147 inhibits cell proliferation, migration, invasion, lipid metabolism dysregulation and promotes apoptosis in lung adenocarcinoma via blocking the Rap1 signaling pathway. Respir Res. 2023; 24: 253.\u003c/li\u003e\n\u003cli\u003eMa L, Chen C, Zhao C, Li T, Ma L, Jiang J, et al. Targeting carnitine palmitoyl transferase 1A (CPT1A) induces ferroptosis and synergizes with immunotherapy in lung cancer. Signal Transduct Target Ther. 2024; 9: 64.\u003c/li\u003e\n\u003cli\u003eZhang H, Liu Y, Liu J, Chen J, Wang J, Hua H, et al. cAMP-PKA/EPAC signaling and cancer: the interplay in tumor microenvironment. J Hematol Oncol. 2024; 17: 5.\u003c/li\u003e\n\u003cli\u003eZhang H, Kong Q, Wang J, Jiang Y, Hua H. Complex roles of cAMP-PKA-CREB signaling in cancer. Exp Hematol Oncol. 2020; 9: 32.\u003c/li\u003e\n\u003cli\u003eOuld Amer Y, Hebert-Chatelain E. Mitochondrial cAMP-PKA signaling: What do we really know? Biochim Biophys Acta Bioenerg. 2018; 1859: 868-77.\u003c/li\u003e\n\u003cli\u003eQu Y, Chen S, Zhou L, Chen M, Li L, Ni Y, et al. The different effects of intramuscularly-injected lactate on white and brown adipose tissue in vivo. Mol Biol Rep. 2022; 49: 8507-16.\u003c/li\u003e\n\u003cli\u003eAbe T, Sato T, Murotomi K. Sudachitin and Nobiletin Stimulate Lipolysis via Activation of the cAMP/PKA/HSL Pathway in 3T3-L1 Adipocytes. Foods. 2023; 12.\u003c/li\u003e\n\u003cli\u003eDing L, Zhang F, Zhao MX, Ren XS, Chen Q, Li YH, et al. Reduced lipolysis response to adipose afferent reflex involved in impaired activation of adrenoceptor-cAMP-PKA-hormone sensitive lipase pathway in obesity. Sci Rep. 2016; 6: 34374.\u003c/li\u003e\n\u003cli\u003eYang LG, March ZM, Stephenson RA, Narayan PS. Apolipoprotein E in lipid metabolism and neurodegenerative disease. Trends Endocrinol Metab. 2023; 34: 430-45.\u003c/li\u003e\n\u003cli\u003ePackard CJ, Pirillo A, Tsimikas S, Ference BA, Catapano AL. Exploring apolipoprotein C-III: pathophysiological and pharmacological relevance. Cardiovasc Res. 2024; 119: 2843-57.\u003c/li\u003e\n\u003cli\u003eTaskinen MR, Bor\u0026eacute;n J. Why Is Apolipoprotein CIII Emerging as a Novel Therapeutic Target to Reduce the Burden of Cardiovascular Disease? Curr Atheroscler Rep. 2016; 18: 59.\u003c/li\u003e\n\u003cli\u003eKohan AB. Apolipoprotein C-III: a potent modulator of hypertriglyceridemia and cardiovascular disease. Curr Opin Endocrinol Diabetes Obes. 2015; 22: 119-25.\u003c/li\u003e\n\u003cli\u003eTall AR. Increasing Lipolysis and Reducing Atherosclerosis. N Engl J Med. 2017; 377: 280-3.\u003c/li\u003e\n\u003cli\u003eKlarin D, Damrauer SM, Cho K, Sun YV, Teslovich TM, Honerlaw J, et al. Genetics of blood lipids among ~300,000 multi-ethnic participants of the Million Veteran Program. Nat Genet. 2018; 50: 1514-23.\u003c/li\u003e\n\u003cli\u003eLiu DJ, Peloso GM, Yu H, Butterworth AS, Wang X, Mahajan A, et al. Exome-wide association study of plasma lipids in \u0026gt;300,000 individuals. Nat Genet. 2017; 49: 1758-66.\u003c/li\u003e\n\u003cli\u003eShi J, Yang H, Duan X, Li L, Sun L, Li Q, et al. Apolipoproteins as Differentiating and Predictive Markers for Assessing Clinical Outcomes in Patients with Small Cell Lung Cancer. Yonsei Med J. 2016; 57: 549-56.\u003c/li\u003e\n\u003cli\u003eHu X, Ding S, Lu G, Lin Z, Liao L, Xiao W, et al. Apolipoprotein C-III itself stimulates the Syk/cPLA2-induced inflammasome activation of macrophage to boost anti-tumor activity of CD8(+) T cell. Cancer Immunol Immunother. 2023; 72: 4123-44.\u003c/li\u003e\n\u003cli\u003eZhang X, Ji L, Liu M, Li J, Sun H, Liang F, et al. Integrative Multianalytical Model Based on Novel Plasma Protein Biomarkers for Distinguishing Lung Adenocarcinoma and Benign Pulmonary Nodules. J Proteome Res. 2024; 23: 277-88.\u003c/li\u003e\n\u003cli\u003eJiang K, Yao G, Hu L, Yan Y, Liu J, Shi J, et al. MOB2 suppresses GBM cell migration and invasion via regulation of FAK/Akt and cAMP/PKA signaling. Cell Death Dis. 2020; 11: 230.\u003c/li\u003e\n\u003cli\u003eLei J, Wu L, Zhang N, Liu X, Zhang J, Kuang L, et al. Carcinoembryonic antigen potentiates non-small cell lung cancer progression via PKA-PGC-1ɑ axis. Mol Biomed. 2024; 5: 19.\u003c/li\u003e\n\u003cli\u003eYang M, Pan M, Huang D, Liu J, Guo Y, Liu Y, et al. Glucagon Promotes Gluconeogenesis through the GCGR/PKA/CREB/PGC-1\u0026alpha; Pathway in Hepatocytes of the Japanese Flounder Paralichthys olivaceus. Cells. 2023; 12.\u003c/li\u003e\n\u003cli\u003eGilman AG. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987; 56: 615-49.\u003c/li\u003e\n\u003cli\u003eNeves SR, Ram PT, Iyengar R. G protein pathways. Science. 2002; 296: 1636-9.\u003c/li\u003e\n\u003cli\u003eO\u0026apos;Hayre M, V\u0026aacute;zquez-Prado J, Kufareva I, Stawiski EW, Handel TM, Seshagiri S, et al. The emerging mutational landscape of G proteins and G-protein-coupled receptors in cancer. Nat Rev Cancer. 2013; 13: 412-24.\u003c/li\u003e\n\u003cli\u003eSong L, Yu X, Wu Y, Zhang W, Zhang Y, Shao Y, et al. Integrin \u0026beta;8 Facilitates Macrophage Infiltration and Polarization by Regulating CCL5 to Promote LUAD Progression. Adv Sci (Weinh). 2025; 12: e2406865.\u003c/li\u003e\n\u003cli\u003ePham DV, Park PH. Adiponectin triggers breast cancer cell death via fatty acid metabolic reprogramming. J Exp Clin Cancer Res. 2022; 41: 9.\u003c/li\u003e\n\u003cli\u003ePoudyal NR, Paul KS. Fatty acid uptake in Trypanosoma brucei: Host resources and possible mechanisms. Front Cell Infect Microbiol. 2022; 12: 949409.\u003c/li\u003e\n\u003cli\u003eRambold AS, Cohen S, Lippincott-Schwartz J. Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics. Dev Cell. 2015; 32: 678-92.\u003c/li\u003e\n\u003cli\u003eOstrom KF, LaVigne JE, Brust TF, Seifert R, Dessauer CW, Watts VJ, et al. Physiological roles of mammalian transmembrane adenylyl cyclase isoforms. Physiol Rev. 2022; 102: 815-57.\u003c/li\u003e\n\u003cli\u003eParker T, Wang KW, Manning D, Dart C. Soluble adenylyl cyclase links Ca(2+) entry to Ca(2+)/cAMP-response element binding protein (CREB) activation in vascular smooth muscle. Sci Rep. 2019; 9: 7317.\u003c/li\u003e\n\u003cli\u003eFerreira J, Levin LR, Buck J. Strategies to safely target widely expressed soluble adenylyl cyclase for contraception. Front Pharmacol. 2022; 13: 953903.\u003c/li\u003e\n\u003cli\u003eKilanowska A, Zi\u0026oacute;łkowska A, Stasiak P, Gibas-Dorna M. cAMP-Dependent Signaling and Ovarian Cancer. Cells. 2022; 11.\u003c/li\u003e\n\u003cli\u003eFujishita T, Kojima Y, Kajino-Sakamoto R, Mishiro-Sato E, Shimizu Y, Hosoda W, et al. The cAMP/PKA/CREB and TGF\u0026beta;/SMAD4 Pathways Regulate Stemness and Metastatic Potential in Colorectal Cancer Cells. Cancer Res. 2022; 82: 4179-90.\u003c/li\u003e\n\u003cli\u003eHalling JF, Pilegaard H. PGC-1\u0026alpha;-mediated regulation of mitochondrial function and physiological implications. Appl Physiol Nutr Metab. 2020; 45: 927-36.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Apolipoprotein C3 (APOC3), LUAD, TG hydrolysis, Fatty acid metabolism, cAMP/PKA pathway","lastPublishedDoi":"10.21203/rs.3.rs-8140812/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8140812/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFatty acid metabolism is a key driver of tumor progression, yet its dysregulation in lung adenocarcinoma (LUAD) remains incompletely characterized. Here, we identify apolipoprotein C3 (APOC3)\u0026mdash;previously linked to cardiovascular disease\u0026mdash;as a novel suppressor of triglyceride (TG) hydrolysis and fatty acid oxidation, ultimately restraining LUAD growth and metastasis. Proteomic and tissue microarray analyses revealed that APOC3 expression was significantly downregulated in LUAD tissues compared with adjacent normal tissues, and low APOC3 levels correlated with poor prognosis in metastatic patients. Furthermore, plasma levels of APOC3 and TG showed a positive correlation in LUAD patients. Functionally, APOC3 overexpression suppressed TG hydrolysis, fatty acid oxidation, and the proliferation and metastasis of LUAD cells both in vitro and in vivo. Mechanistically, APOC3 attenuated the cAMP/PKA signaling pathway, leading to reduced expression of hormone-sensitive lipase (HSL), a key enzyme in TG hydrolysis, and PGC-1α, a master regulator of fatty acid oxidation. The inhibitory effects of APOC3 on TG hydrolysis and fatty acid oxidation were reversed by cAMP activators or knockdown of HSL or PGC-1α. Additionally, APOC3 was found to interact with GNAI3, a critical inhibitory regulator of the cAMP/PKA pathway. In summary, our study uncovers an APOC3-mediated pathway that constrains TG hydrolysis and fatty acid oxidation, the dysregulation of which contributes to LUAD progression, highlighting APOC3 as a potential therapeutic target in LUAD.\u003c/p\u003e","manuscriptTitle":"APOC3-Mediated Fatty Acid Metabolism Suppresses Lung Adenocarcinoma Progression by Inhibiting GNAI3/cAMP/PKA Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-22 09:01:48","doi":"10.21203/rs.3.rs-8140812/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"daff915c-c0d9-4d47-91b6-f8ed769ace5e","owner":[],"postedDate":"December 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59538313,"name":"Biological sciences/Cancer/Lung cancer/Non-small-cell lung cancer"},{"id":59538314,"name":"Biological sciences/Cell biology/Cell division/Cell growth"}],"tags":[],"updatedAt":"2026-01-13T12:35:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-22 09:01:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8140812","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8140812","identity":"rs-8140812","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}