GPR162 Drives Obesity-Associated Lung Adenocarcinoma Suppression via Fatty Acid Oxidation-Induced Cuproptosis and Metabolic-Immune Reprogramming

GPR162 Drives Obesity-Associated Lung Adenocarcinoma Suppression via Fatty Acid Oxidation-Induced Cuproptosis and Metabolic-Immune Reprogramming | 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 GPR162 Drives Obesity-Associated Lung Adenocarcinoma Suppression via Fatty Acid Oxidation-Induced Cuproptosis and Metabolic-Immune Reprogramming Yongguang Tao, Yao Long, Wei Wang, Can Cao, Molly S. C. Li, Lili Li, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8754789/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Obesity, a well-established risk factor for cancer development and progression, exerts its oncogenic effects primarily through metabolic reprogramming, particularly in lipid and amino acid metabolism. Despite its clinical significance, the molecular mechanisms linking obesity-induced metabolic alterations to lung adenocarcinoma progression remain elusive. In this study, we identify G protein-coupled receptor 162 (GPR162) as a pivotal metabolic-immune regulator in obesity-associated lung adenocarcinoma. Our findings reveal that GPR162 orchestrates tumor suppression through two distinct yet interconnected mechanisms: (1) GPR162 induces cell peroxidation by promoting the oxidation of medium-chain fatty acids such as decanoic acid and lauric acid. (2) GPR162 interacts with copper transporter SLC25A3 to form a functional complex in the mitochondria, driving copper influx and further promoting intracellular lipid peroxidation to induce cuproptosis, which directly kills tumor cells. The synergistic effect of fatty acid oxidation and cuproptosis not only reprogram the tumor metabolic microenvironment, but also induce the polarization of tumor-associated macrophages to M1 type, enhance CD8 + T cell infiltration and IFNγ secretion, and finally construct an immune microenvironment that inhibits tumor progression. This study provides a new target and theoretical basis for combined metabolic-immune treatment of obesity-related lung cancer. Biological sciences/Cell biology/Cell death Biological sciences/Cancer/Cancer microenvironment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Significance Statement This study identifies G protein-coupled receptor 162 (GPR162) as a critical protective factor in obesity-associated lung adenocarcinoma, uncovering its dual mechanisms of action. We demonstrate that GPR162 reprograms the tumor microenvironment by promoting medium-chain fatty acid oxidation and induces cuproptosis through its interaction with the mitochondrial copper transporter SLC25A3. Additionally, GPR162 enhances anti-tumor immunity by driving tumor-associated macrophages toward an M1-like phenotype. These findings not only advance our understanding of how obesity influences cancer metabolism and progression but also establish GPR162 as a promising therapeutic target for combating obesity-related lung adenocarcinoma. Introduction Lung cancer is one of the most common and deadly malignant tumors worldwide[ 1 ], with lung adenocarcinoma being the predominant pathological type[ 2 ] , [ 3 ]. It seriously threatens human life and health. Although smoking has been widely recognized as the primary risk factor for lung cancer, more and more studies have shown that in addition to traditional carcinogenic factors, systemic factors such as metabolism and inflammation also play an important role in the occurrence and development of lung cancer[ 4 – 6 ]. Obesity, as a chronic metabolic disease characterized by dysfunction of fat tissue and imbalance of energy metabolism, has a continuously rising prevalence worldwide and has become an important public health issue[ 7 – 9 ]. A large number of studies have shown that obesity can significantly promote the occurrence and progression of various malignant tumors by inducing chronic low-level inflammation, insulin resistance, imbalance in fat factor secretion, and remodeling of immune function[ 10 – 14 ]. In recent years, epidemiological investigations and basic research have further indicated that obesity is closely related to the occurrence, progression, and prognosis of lung cancer[ 15 , 16 ]. Another study has shown that obese patients with lung cancer have poor responses to immune checkpoint therapy[ 17 ]. However, the molecular mechanism by which obesity mediates the occurrence and development of lung cancer is still unclear, and the specific mechanisms of its regulation of tumor metabolic reprogramming and tumor immune microenvironment are also not clear. Therefore, in-depth analysis of the key molecular mechanisms by which obesity mediates the progression of lung cancer is expected to provide new theoretical basis and research directions for the precise intervention and targeted treatment of lung cancer. As the proportion of overweight and obese individuals worldwide continues to rise, obesity is widely regarded as a significant risk factor for various solid tumors and metabolic-related diseases. Moreover, most studies have indicated that the obese state is associated with a poor prognosis for cancer patients. A retrospective study of 794 cancer patients found that compared to normal patients, overweight or obese patients had a significantly lower 3-year overall survival rate (93.8% vs. 98.0%, P = 0.01), a significantly higher 3-year recurrence rate (10.5% vs. 5.8%, P = 0.02), and a significantly lower 3-year event-free survival rate (89.0% vs. 93.7%, P = 0.02) [ 18 ]. Another retrospective study based on 31,257 patients with advanced non-small cell lung cancer showed that in patients with BMI < 28, ICI treatment significantly reduced the risk of death (for example, when BMI was 24, HR = 0.81, 95% CI: 0.75–0.87); however, in patients with BMI ≥ 28, ICI treatment did not show significant survival benefits (for example, when BMI was 28, HR = 0.90, 95% CI: 0.81–1.00) [ 15 ]. These results suggest that persistent overweight or obesity may affect the survival outcome of patients by altering the biological characteristics of tumors or treatment responses. G protein-coupled receptors (GPCRs) are a broad class of membrane surface proteins that are also the largest family of drug targets[ 19 , 20 ]. GPCRs are involved in approximately 80% of cellular signaling processes and are important proteins in cell signaling[ 21 ]. Studies have shown that GPCRs are widely dysregulated in tumors, playing important roles in tumor proliferation, survival, angiogenesis, invasion, metastasis, resistance to treatment, and immune evasion[ 22 , 23 ]. Orphan receptors refer to receptors with unknown endogenous ligands, and about 200 of the approximately 800 human GPCR genes are orphan receptors[ 24 ]. Orphan receptors are involved in various physiological functions and play important roles, such as sensing, reproductive development, metabolism, and responsiveness, and may therefore be closely related to many diseases, such as central nervous system diseases, metabolic diseases, and cancer[ 25 – 28 ]. For orphan GPCRs, it is difficult to develop drugs due to a lack of understanding of endogenous ligands, and therefore, in-depth research into the functions and mechanisms of action of orphan receptors is of great importance for the development of new drugs. GPR162 belongs to the class of retinal A-type orphan GPCRs, and it is highly expressed in the brain and lung tissues. There are few studies on GPR162, suggesting that its mutation is associated with abnormal glucose levels in vivo and that changes in GPR162 are associated with reduced food intake in rats[ 29 – 31 ]. The TCGA database also shows that the activation of GPR162 is significantly enriched in EMT-related signaling pathways, and is closely related to metabolic-related pathways such as insulin-producing beta cells and glycolysis. There are already literature reports on the involvement of GPCRs in beta cell dysfunction, insulin resistance, and obesity-induced T2DM[ 32 , 33 ]. Therefore, it is crucial to further study the impact of GPR162 on the development of lung adenocarcinoma in obese mice and explore the relationship between GPR162 and the tumor microenvironment associated with obesity, which is important for the development of new drugs and the discovery of new strategies for cancer treatment. Several studies have indicated a close association between obesity and the oxidation of medium-chain fatty acids, as well as the potential of medium-chain fatty acids to mitigate and reverse metabolic syndrome induced by high-fat diets[ 34 , 35 ]. Furthermore, these medium-chain fatty acids and their ketone metabolites are activated by cell membrane receptors, leading to reduced fat deposition, improved insulin resistance, and regulation of glucose and lipid metabolism[ 36 , 37 ]. Given our identification of GPR162 as a protein located on the structural component of the mitochondrial membrane[ 38 ], it is pertinent to investigate whether mitochondrial GPR162 is influenced by lipid metabolites or impacts cellular lipid metabolism. Recent studies have shown that SLC25A3 is an important protein that transports copper into the mitochondrial matrix and participates in the respiratory chain on the inner mitochondrial membrane[ 39 , 40 ]. In 2022, it was first revealed that copper can abnormally bind to lipid-modified proteins and reduce iron-sulfur cluster proteins, causing significant damage to the mitochondrial respiratory chain and ultimately inducing a unique new type of cell death: cuproptosis[ 41 , 42 ]. The mitochondria, as the cell's energy factory, is a highly dynamic and stable organelle[ 43 ]. Imbalances in mitochondrial homeostasis are associated with virtually all human diseases[ 44 , 45 ]. Recent studies have revealed that a variety of GPCR members of membrane proteins can be found in non-plasma membrane sites such as endoplasmic reticulum, Golgi apparatus, mitochondria, nucleus, and even centriole to mediate signal initiation and transduction within the cell. As a result, we found the GPR162 and SLC25A3 interaction on the mitochondrial membrane, strongly suggests the biological significance of cuproptosis may participate in regulating cell death. Previous work has confirmed that GPR162 is closely related to the activation of tumor innate immunity, and GPCR can be an effective pro-inflammatory and anti-inflammatory regulator for key immune cells, serving as a bridge for the interaction between the nervous, endocrine, and immune systems[ 46 ]. There is literature to confirm that the macro- and microenvironment of obesity affect the biological changes of tumors, including mitosis and metabolism, promoting tumor growth[ 47 , 48 ]. We found that GPR162 is highly expressed in neutrophils, monocytes, and macrophages, and there is a significant correlation between GPR162 expression and macrophages. GPR162 can promote the polarization of macrophages towards M1, which is influenced by fatty acids, suggesting that GPR162 may play an important role in regulating fatty acid oxidation and immune cell function in the tumor microenvironment. Therefore, elucidating the molecular mechanisms by which GPR162 regulates fatty acid oxidation and understanding its function in immune cells, particularly macrophages, in the tumor microenvironment can help identify new therapeutic targets and improve the efficacy of immunotherapy, providing new insights for tumor immunotherapy strategies. Material and Methods Mice Mice were kept in pathogen-free conditions. When necessary, mice were acquired from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). Mice were fed an irradiated PicoLab Rodent Diet with a high-fat content of 60 kcal% (D12492), whereas the control diet had a fat content of 10 kcal% (D12450J). Mice classified as obese in this study were fed a high-fat diet for at least 9 weeks, beginning at age 6–7 weeks, and weighed at least 38 g (on average inside a cage). All mice utilized in the research were male. Animal research was conducted with the approval of Central South University's Xiangya School of Medicine's Institutional Animal Care and Use Committee, as well as in accordance with legislative and federal animal protection and care guidelines. Subcutaneous injections of GPR162-overexpressing cells and control cells (2 × 10 5 cells/mouse) were given to each mouse's axilla. Following that, tumor volume and mouse weight were recorded every three days until the mice were euthanized at 27 days. Tumors were weighed and lysed for flow cytometry analysis. Cell culture, viruses, stimulation, and transfection In this investigation, the following cell culture conditions were used: LLC(ATCC: CRL-1642), BMDM, RAW264.7(ATCC: SC-6003) cell lines were cultured in DMEM (Gibco, NY, USA) medium, A549 (ATCC: CCL-185) cell lines were cultured in 1:1 DME/F12 (HyClone, UT, USA) medium, PC9, H1299 (ATCC: CRL-5803) cell lines were cultured in RPMI1640 (Gibco) medium. Cells were cultured in a cell incubator at 37°C with 5% CO2 and the medium containing 10% (v/v) BCS. All cell lines were obtained from the cell bank of the Cancer Institute, Central South University. Supplementary Table 3 contains the sgRNA and shRNA sequences for GPR162 described in this study. The generated plasmid was introduced into cells and transfected using Lipofectamine Max. The colonies with stable expression were screened by puromycin (1 µg/ml). Western blot analysis and coimmunoprecipitation (Co-IP) assay Western blot analysis and coimmunoprecipitation (Co-IP) assay Following three 1×PBS washes, the harvested cells were lysed for one hour on ice in an IP lysis solution containing a protease inhibitor cocktail. The BCA method was used to determine the protein concentration, and the apparatus was set up. After being extracted from cell lysate using an SDS-polyacrylamide gel, the total proteins were transferred to a polyvinylidene fluoride membrane. Supplementary Table 4 lists primary antibodies used in Western blot analysis. Target protein antibodies were added to the magnetic bead-precleared proteins at 4°C, and the mixture was incubated for an entire night. Western blot analysis was used to detect the interaction between the proteins after they had been adsorbed by magnetic beads and denatured. Real-time quantitative polymerase chain reaction (RT-qPCR) TRIzol reagent (Takara, Kusatsu, Japan) was used to isolate the total RNA, and the kit (Takara, Kusatsu, Japan) was used to reverse transcribe the RNA into cDNA. Real-time PCR was performed on a Bio-rad CFX Connect real-time PCR instrument. β-actin served as the internal reference for gene expression. The primers used in this investigation are listed in Supplementary Table 1–2. Immunofluorescence microscopy A 24-well culture plate containing tiny glass discs was filled with logarithmic growth cells 24 hours before adherent growth cells were added. The culture plate was taken out when the degree of cell fusion was around 50%. Following three 1×PBS washes, 1 mL of methanol was added to each well, and the wells were fixed for 10 minutes at 20 degrees. Following two 5-minute PBS rinses, 1% (w/v) BSA in PBS was used to block the area for 30 minutes. three times for five minutes each, followed by an overnight incubation at 4°C with primary antibodies containing 1% (w/v) BSA. Depending on the characteristics of the primary antibody, either anti-rabbit IgG Alexa 594 fluorescent secondary antibody or anti-mouse IgM Alexa 488 fluorescent secondary antibody was chosen and incubated for one hour after washing with PBS. After DAPI labeling, they were put on slides and photographed using a Leica TCS SP8 confocal microscope. Living cells were stained with MitoTracker® Deep Red FM (Invitrogen, 644–665 nm, M22426) in a cell incubator set at 37°C with 5% CO 2 for 30 minutes. Separation of the cytoplasmic and mitochondria The Cell Mitochondria Isolation Kit (Beyotime, C3601) was utilized to isolate the proteins found in cellular mitochondria. Following three 1×PBS washes, the collected cells were lysed for fifteen minutes on ice in a mitochondrial separation reagent containing PMSF. After being moved to a glass homogenizer of the proper size, the cell suspension was homogenized for ten to thirty cycles. Centrifuging the cell suspension produced cellular mitochondria in the precipitate and cytoplasmic proteins in the supernatant. The BCA technique was used to determine the protein concentration. Flow cytometry The following antibodies were used. Biolegend: CD49b, CD4, CD8, CD45, CD206, CD3e, Ly-6G, F4/80, CD11b, CD86, CD11c, BB515, IFN-γ, GzmB and TNF. We used the eBioscience Fixable Viability Dye eFluor780 or DAPI to distinguish live from dying or dead cells. For intracellular staining, cells were treated with fixation and permeabilization reagents from eBioscience and labeled with appropriate antibodies before being analyzed. Data were analyzed using an LSRII, LSRFortessa, or FACSAria instrument (Becton Dickinson) and FlowJo software (FlowJo LLC). Cells were sorted on a BD FACSAria cell sorter. In vitro CD8 T cell differentiation CD8 + T cells were isolated from the spleen using the Pan T Cell Isolation Kit II (Miltenyi Biotec). A single-cell suspension from mouse spleen was prepared using the program m_spleen_01.01 on the gentleMACS™ Dissociator. T cells were isolated from this single-cell suspension using the Pan T Cell Isolation Kit II, an LS Column, and a MidiMACS™ Separator. Cells were fluorescently stained with the MC CD90.2 T Cell Cocktail, mouse as well as with CD3e-APC, and analyzed by flow cytometry using the MACSQuant® Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence. Statistics Except for the studies with mice, every study was carried out a minimum of three times. A mean SD, or SEM, is displayed for the data. The statistical analysis was performed with GraphPad Prism 9.0. The T-test was used to assess the significance of differences between two groups, and analysis of variance (ANOVA) was used to examine groups larger than two. The Parsons correlation coefficient was used for correlation analysis. Differences were considered statistically significant in the following cases: p < 0.05(*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Study approval The ethics committee at our hospital approved the project. The institutional Animal Care and Use Committee at Central South University gave its approval for the use of animal models in this study. The study was approved by the institutional review boards of all participating medical facilities. Each research subject signed a documented informed consent form before recruitment. Results GPR162 acts as a protective factor that inhibits the development of lung adenocarcinoma in obese mice. Based on the above research reports, we conducted a genome-wide association study (GWAS) to assess the correlations between obesity-related traits (BMI, body fat) and different types of lung cancer. The results showed that both BMI and body fat were significantly correlated with lung adenocarcinoma (Fig. 1 A), suggesting that obesity may be involved in the occurrence and development of lung adenocarcinoma. Furthermore, we divided the 514 lung adenocarcinoma samples from the TCGA database into four groups based on the patients' different weights: normal weight, overweight, obese, and extremely obese. The results showed that the expression of GPR162 was higher in the normal weight group than in the obese group (Fig. 1 B). Additionally, we analyzed the mRNA expression of 16,383 genes and classified them into two groups: high expression and low expression of GPR162. The gene set enrichment analysis (GSEA) revealed that in the tumor patients with high expression of GPR162, there was a significant enrichment in lipid metabolism-related pathways (Fig. 1 C). To further explore the relationship between obesity and lung adenocarcinoma, as well as the role played by GPR162 in this process, we established a diet-induced obesity (DIO) model using C57BL/6 mice fed a 60% high-fat diet (HFD) for 12 weeks (Fig. S1 A). After 12 weeks, peripheral blood of the mice was collected from the orbital vein, and then the levels of blood glucose (Fig. 1 H) and blood lipids (triglyceride, cholesterol, high-density lipoprotein, low-density lipoprotein) in peripheral blood of the mice were detected by biochemical experiments (Fig. S1 B-E). The results showed that the levels of blood glucose and blood lipid in the obese mice were significantly higher than those in the control group, which confirmed the successful construction of the obese mouse model. Next, we subcutaneously injected lung adenocarcinoma cells LLC into obese mice induced by high-fat diet to construct a subcutaneous tumor-bearing model. By monitoring the body weight, tumor growth curve and tumor weight of the mice, we found that the tumor growth rate of the obese mice was significantly accelerated, and the tumor volume was significantly larger than that of the control group (Fig. 1 D, Fig. S1 F-H). Notably, RT-qPCR results showed that GPR162 expression was significantly down-regulated in lung adenocarcinoma tissues of the obesity group compared with the control group (Fig. 1 E), suggesting its potential tumor-suppressive role in metabolic dysregulation contexts. To further confirm the effect of GPR162 on obesity-promoted lung adenocarcinoma progression. We subcutaneously transplanted mouse lung adenocarcinoma cells LLC stably overexpressing GPR162 into normal and diet-induced obese mice. The growth curve, tumor volume and tumor weight of subcutaneous tumors were dynamically monitored. We found that the tumor growth of obese mice was significantly accelerated compared with the normal diet group, while GPR162 overexpression could significantly inhibit the tumor growth rate, and the tumor volume and weight were significantly reduced compared with the control group (Fig. 1 F-G, Fig. S1 I-J). These results indicate that GPR162 overexpression can effectively inhibit obesity-promoted lung adenocarcinoma progression, suggesting that GPR162 may play an important tumor suppressor role in obesity-related lung adenocarcinoma. To mechanistically dissect this observation, we engrafted GPR162-overexpressing LLC cells into both lean and obese mice. we measured the glucose and lactate levels in the peripheral blood of normal and obese mice, and in the culture supernatant of lung adenocarcinoma cells in the control group and GPR162 overexpression group using an automatic biochemical analyzer system. The results showed that GPR162 overexpression significantly inhibited glucose consumption in PC9 and A549 lung adenocarcinoma cells (Fig. 1 I-J). This finding further supports GPR162 as a potential target for the treatment of obesity and other metabolic diseases. However, peripheral blood lactate level was not significantly altered in the obese mouse model, and lactate production in the culture medium of GPR162 overexpressed lung adenocarcinoma cells was not statistically different (Fig. 2 A-C). Since lactate is the end product of glycolysis, the amount of lactate produced was not statistically different, suggesting that the inhibitory effect of GPR162 on lung adenocarcinoma progression in obese mice may not be achieved by regulating the glycolytic pathway. Therefore, the specific metabolic reprogramming mechanisms and energy supply pathways of tumor cells in the obese microenvironment remain to be further studied. GPR162 promotes medium-chain fatty acid oxidation. To delineate the metabolic axis through which GPR162 modulates obesity-associated lung adenocarcinoma progression, we conducted RNA sequencing on tumors from diet-induced obese (DIO) mice with GPR162 overexpression. DESeq2-based pathway analysis revealed that 80% of differentially expressed genes (DEGs) mapped to lipid metabolism pathways, with pronounced enrichment in glycerophospholipid metabolism, sphingolipid cycling, and ether lipid biosynthesis, GSEA further corroborated these findings, showing strong associations with adipokine signaling and glycerolipid homeostasis (Fig. 2 D-E), positioning GPR162 as a central regulator of lipid metabolic rewiring in obese tumors. Non-targeted metabolomics of subcutaneous transplanted tumors identified pantothenate metabolism as the most significantly altered pathway in GPR162-overexpressing obese mice (Fig. 2 F). Given pantothenate's essential role as a CoA precursor in fatty acid metabolism, we focused on medium-chain fatty acid (MCFA) dynamics. Targeted lipidomics demonstrated 3.7-fold elevation of decanoic acid (C10:0) in GPR162-overexpressing tumors compared to obese controls, with parallel increases in octanoic and lauric acids (Fig. 2 G-H). Because there is a wide range of bidirectional regulatory relationships between molecules involved in the regulation of fatty acid metabolism and fatty acids, for example, the core regulatory molecules of fatty acid metabolism (such as PPARs, SREBP-1c, AMPK) are not only metabolic regulators, but also "sensors" of fatty acids and their derivatives. Therefore, to further test whether GPR162 is involved in fatty acid metabolism, we observed a significant up-regulation of GPR162 protein expression in lung adenocarcinoma cell culture media treated with pantothenic acid and fumaric acid, which are core cofactors of lipid metabolism (Fig. 2 I). In addition, treatment of lung adenocarcinoma cells with medium-chain fatty acids (capric acid, octanoic acid and lauric acid) similarly significantly increased GPR162 protein levels (Fig. 2 J). These results suggest that GPR162 may regulate medium-chain fatty acid metabolism. Subcellular fractionation confirmed predominant mitochondrial localization of GPR162 (Fig. S4 A-C). It is well known that mitochondria are the energy metabolism center of cells and are the site of energy metabolic processes such as oxidative phosphorylation, ATP synthesis, and fatty acid oxidation, suggesting that GPR162 is localized in mitochondria and participates in fatty acid energy metabolism. To determine the role of GPR162 in the regulation of mitochondrial energy metabolism, GPR162-overexpressing lung adenocarcinoma cells were treated with decanoic acid for 24 hours. Total RNA was extracted from the cells, and genes involved in fatty acid synthesis, fatty acid oxidation, and fatty acid breakdown were quantified by RT-qPCR. Fatty acid synthesis, oxidation and breakdown pathways were all activated in the overexpression group (Fig. 3 A-C). Moreover, GPR162 protein level was also significantly up-regulated after adding cell metabolism and function inhibitors in lung adenocarcinoma cells, further confirming that GPR162 is involved in fatty acid metabolism pathway (Fig. 3 D-E). To further explore the mechanism of GPR162 regulation on fatty acid metabolism, RNA-seq results of subcutaneous tumor tissues from normal and obese mice were cluster. The results showed that GPR162 overexpression in normal mice and GPR162 overexpression in obese mice significantly up-regulated the expression of Ppargc1a and Prl2c2 (Fig. 3 F). Since Prl2c2 is only expressed in mice, PPARGC1A mRNA levels were examined in LLC and PC9 cells overexpressing GPR162, and the results showed that GPR162 overexpression promoted PPARGC1A transcription (Fig. 3 G-H). Since PPARGC1A is a transcriptional coactivator of PPARγ and a major regulator of mitochondrial biogenesis, it can promote its transcriptional activity by binding to PPARγ, while PPARγ promotes fatty acid breakdown and energy production by activating the transcription of fatty acid oxidation related genes (such as CPT1, ACOX, etc.). Therefore, we isolated the cytoplasmic and nuclear proteins of LLC cells overexpressing GPR162, and the results showed that the expression level of PPARγ in the nucleus was significantly higher after GPR162 overexpression than in the control group (Fig. 3 I). ChIP results also showed that overexpression of GPR62 enhanced the binding between PPARγ and CPT1A in the promoter region (Fig. 3 J). These results suggest that GPR162 promotes medium-chain fatty acid oxidation by up-regulating PPARGC1A to drive PPARγ into the nucleus and enhance its binding with CPT1A at the promoter region. Co-culture experiments with primary adipocytes from obese mice revealed that GPR162 overexpression: (1) Upregulated fatty acid oxidation genes (Fig. 3 K), (2) Enhanced BODIPY-C12 uptake (Fig. 3 L-M). This metabolic plasticity occurred despite unchanged glucose-lactate coupling (Fig. 2 A-C), suggesting preferential utilization of lipid over glycolytic substrates. The bidirectional regulation between GPR162 and fatty acid metabolites (C8-C12) establishes a lipid-sensing paradigm where MCFAs both induce and are catabolized through GPR162-PPARGC1A signaling. GPR162 induces cuproptosis in lung adenocarcinoma cells by interacting with SLC25A3. As a mitochondrial membrane protein implicated in lipid metabolism, we sought to delineate GPR162's interactome through proteomic analysis of mitochondria-associated endoplasmic reticulum membranes (MAMs). Mass spectrometry identified 161 high-confidence interacting partners, including endoplasmic reticulum Ca²⁺-ATPase (SERCA), mitochondrial calcium transporters VDAC1/VDAC3, and copper carrier SLC25A3, Notably, SLC25A3 demonstrated the highest enrichment score (2.8-fold vs controls) among MAM components (Fig. 4 A-C). Co-immunoprecipitation validated this interaction (Fig. 4 D). while confocal microscopy confirmed mitochondrial co-localization of SLC25A3 with GPR162 (Fig. 4 E, Fig. S4 D), establishing a structural foundation for GPR162's metabolic regulatory role. Given SLC25A3's established function in mitochondrial copper transport and metalloenzyme maturation[ 40 ], we investigated GPR162's impact on copper homeostasis. Total RNA was extracted from PC9 cells overexpressing GPR162, and RT-qPCR was used to measure the transcriptional levels of copper related genes. The results showed that the transcriptional level of SLC25A3 was significantly upregulated after GPR162 overexpression, and the expression of another classical copper transporter ATP7B also showed the same trend. Moreover, other genes related to copper ions, such as SCO2,MTF1,DLD,PDHA1, PDHB,LIPT1, showed statistical differences after GPR162 overexpression (Fig. 4 F). Recent studies have shown that a variety of copper ionophore drugs, such as Elesclomol (ES), Disulfiram and NSC319726, can cause cell death. This copin-induced cell death is a new type of death, which is different from other programmed cell death (such as apoptosis, pyroptosis, necrosis and ferroptosis). Therefore, it was defined as cuproptosis. Thus, we hypothesized that GPR162 interacts with SLC25A3 in mitochondria to promote cuproptosis in lung adenocarcinoma cells. To test this hypothesis, we first examined the IC50 of ES-Cu-induced cuproptosis in different lung adenocarcinoma cells. The results showed that the IC50 of PC9 and H1299 cells was 130.8 nM and 88.9 nM, respectively, while the IC-50 of A549 cells was 226.3 nM. This also suggests that A549 cells are not sensitive to ES-Cu-induced cuproptosis (Fig. S4 G-I). Next, we examined the effect of GPR162 on ES-Cu-induced cuproptosis in PC9 cells, which were sensitive to cuproptosis, and in A549 cells, which were resistant to cuproptosis. The results showed that GPR162 promoted ES-Cu-induced cell death in PC9 cells, which was reversed by the copper chelator TTM. In contrast, GPR162 did not promote ES-Cu-induced cell death in elesclomole-resistant A549 cells (Fig. 4 G-H). Cuproptosis is mainly caused by copper ions directly binding to the lipoacylated proteins in the tricarboxylic acid cycle, which leads to the oligomerization and abnormal aggregation of lipoacylated proteins and the decrease of Fe-S cluster proteins[ 41 ]. Therefore, we also examined the oligomerization of the protein after GPR162 overexpression, which was shown to be more pronounced (Fig. 4 I). Western blot was used to detect the levels of ester acylation proteins DLAT, DBT, DLST, ferredoxin 1 (FDX1), and lipoacylation protein LIAS, and the results showed that overexpression of GPR162 significantly increased the protein levels of GPR162, which was opposite after knockdown of GPR162 (Fig. 4 J-L). Spatial coordination was confirmed by GPR162-DLAT mitochondrial co-localization (Fig. 5 A, Fig. S4 E-F) and direct GPR162-LIAS interaction (Fig. 5 B). The transcriptional levels of DLAT, DBT, DLST, FDX1, and LIAS were detected by RT-qPCR, and the results showed that the transcriptional levels of fatty acylation protein and Fe-S promoting protein related genes were significantly down-regulated after GPR162 overexpression, which further confirmed the above conclusion (Fig. 5 C). Previous studies have shown that GPR162 and SLC25A3 interact on the mitochondrial membrane and participate in the regulation of cuproptosis in lung adenocarcinoma cells. How GPR162 and SLC25A3 affect mitochondrial function is worthy of further exploration. Firstly, we examined the mitochondrial ROS levels in GPR162 overexpression cell lines after ES-Cu induction, and the results showed that the intracellular mitochondrial ROS levels were significantly upregulated in PC9 cells with GPR162 overexpression after ES-Cu induction (Fig. 5 D). In contrast, the addition of ES-Cu to GPR162 knockdown H1299 cells induced cell death, and mitochondrial ROS decreased (Fig. 5 E). We next examined mitochondrial membrane potential levels by flow cytometry using JC-1 as a probe, which showed that they were upregulated after GPR162 knockdown and decreased after GPR162 overexpression (Fig. 5 F-I). In addition, we further examined ATP levels in lung adenocarcinoma cells after GPR162 overexpression and knockdown using an ATP assay kit. Consistent with the membrane potential level, knockdown of GPR162 promoted ATP release and overexpression of GPR162 reduced ATP production (Fig. 5 J-M). Furthermore, GPR162 promoted mitochondrial ROS production, inhibited mitochondrial membrane potential and ATP level, and promoted cuproptosis of lung adenocarcinoma cells. Since mitochondrial MMP and ATP alterations are also important events in the process of apoptosis, to further test whether GPR162 affects apoptosis, we examined the apoptosis status by flow cytometry in lung adenocarcinoma cells overexpressing GPR162 after treatment with the apoptosis-inducing agent DBT for 24 hours. The results showed that overexpression of GPR162 did not affect the level of apoptosis (Fig. S5 A-D). It was further confirmed that GPR162 promoted apoptosis of lung adenocarcinoma cells by inhibiting mitochondrial membrane potential and ATP level, but did not affect apoptosis. In addition, we extracted subcutaneous inguinal adipose tissue from obese mice and cultured it in vitro, and then mouse lung adenocarcinoma cells LLC overexpressing GPR162 were co-cultured with primary adipocytes. Total RNA and total protein were extracted from tumor cells. GPR162 promoted the level of lipoacylated proteins, and the transcript levels of DLAT, DLST, FDX1, and LIAS were significantly decreased (Fig. 5 O-Q). These results suggest that GPR162 promotes cuproptosis in obesity-related lung adenocarcinoma cells. GPR162 drives the polarization of TAMs to M1 to enhance anti-tumor immunity. KEGG pathway analysis of GPR162-interacting proteins identified through mass spectrometry revealed significant enrichment in immune-related pathways, particularly in antigen processing/presentation and cytokine-cytokine receptor interaction (Fig. S6 A). Integrated analysis of subcutaneous xenograft RNA-seq data demonstrated GPR162 overexpression-associated enrichment in immunomodulatory pathways, including T cell receptor signaling and chemokine-mediated inflammation, suggesting its role in reshaping the obesity-associated tumor immune microenvironment (Fig. 6 A-C, S6B). GSEA further confirmed positive correlations between GPR162 expression and interferon-γ response, acute inflammatory response, and immune checkpoint activation (Fig. S6 C, S7A). These results suggest that GPR162 is significantly associated with tumor immunity, which provides us with new clues to study GPR162 mediating tumor metabolism and affecting lung cancer immunity. To continue to explore GPR162 influencing immune mediated tumor metabolic mechanism, we first by TIMER2.0 online website ( http://timer.cistrome.org/ ) analyzes the GPR162, expressed in all the immune cells in the The results showed that GPR162 was highly expressed in myeloid cells such as neutrophils, monocytes and macrophages (Fig. S7 C). Next, we predicted the association of GPR162 with various immune cells in lung adenocarcinoma and lung squamous cell carcinoma, and the results showed that GPR162 was positively correlated with macrophages (Fig. S6 D-E). Notably, single-cell RNA-seq clustering (GSE97168) showed exclusive GPR162 expression in tumor-associated macrophages (TAMs) within lung adenocarcinoma specimens (Fig. S7 D), suggesting myeloid-specific regulatory functions. The tumor tissues and spleens of normal and obese tumor-bearing mice were collected to detect the infiltration of immune cells by flow cytometry. The results showed that in the spleen, the infiltration of NK cells, NKT cells, CD8 + T cells, CD4 + T cells, macrophages and DC did not differ significantly between the control and GPR162 overexpression groups in normal and obese mice (Fig. S8 A-F). However, the infiltration of monocytes and neutrophils was significantly reduced in the overexpression group, and the difference was more obvious in the obese mice, which suggested that more inflammatory factors were released in the spleen tissue and activated the inflammatory response, However, in the tumor tissues, neutrophil infiltration was more obvious in the overexpression group of normal and obese mice, suggesting that more inflammatory factors were infiltrated in the tumor tissues (Fig. 6 F-G). In addition, the infiltration of NK cells, NKT cells, CD4 + T cells, monocytes and DC in the tumor tissues of normal and obese tumor-bearing mice was not significantly different between the control and GPR162 overexpression groups (Fig. S8 G-L), The infiltration of macrophages in GPR162 overexpression and obesity groups was higher than that in control group (Fig. 6 D-E). The infiltration of neutrophils in GPR162 overexpression and obesity groups was also higher than that in control group, and the infiltration of CD8 + T cells was significantly increased in GPR162 overexpression groups of normal and obese mice (Fig. 6 H). These results suggest that GPR162 is involved in the regulation of tumor immunity mainly through macrophage infiltration. Chemokines play a crucial role in regulating the infiltration of different immune cells into tumors; therefore, these molecules affect tumor immunity and the therapeutic outcome of patients. To determine whether GPR162 affects the induction of T cell function by macrophages, we used a transwell system. Jurkat T cells were co-cultured with GPR162-overexpressing lung adenocarcinoma PC9 cells or CD8 + T cells from primary spleen tissues of C57BL/6 mice with GPR162-overexpressing LLC cells for 48 hours. The mRNA levels of chemokines CCL2, GZMB, TNFα, and TGFβ were detected by RT-qPCR. The results showed that the co-culture of GPR162 overexpressing tumor cells with T cells did not activate T cells, suggesting that GPR162 promotes anti-tumor immunity through macrophages (Fig. 6 I-M, Fig. S10 B-F). To verify the above conclusions, we first co-cultured BMDM with LLC cells overexpressing GPR162 for 48 hours, and then extracted CD8 + T cells from the spleen of C57BL/6 mice were added to the upper chamber of the co-culture dish for further culture for 48 hours. CD8 + T cells in the upper chamber were collected, and the function of CD8 + T cells was detected by RT-qPCR. The results showed that overexpression of GPR162 significantly enhanced T cell function in this culture system (Fig. 6 N-R). Subsequently, BMDM were co-cultured with the supernatant of LLC cells overexpressing GPR162 for 48 hours, and then the extracted CD8 + T cells from the spleen of C57BL/6 mice were added to the upper chamber of the co-culture dish for further culture for 48 hours. CD8 + T cells in the upper chamber were collected and the function of CD8 + T cells was detected by RT-qPCR (Fig. S10 G-K). The results were consistent with that of co-culture with tumor cells. Co-culture of tumor cells and tumor cell supernatant with macrophages and then with CD8 + T cells could activate anti-tumor immunity. To verify the association between GPR162 and macrophages, BMDM from macrophages derived from mouse bone marrow were extracted (Fig. 7 A). In order to confirm the successful extraction of macrophages, we added LPS to induce macrophages to M1 polarization, IL4 and IL13 to induce macrophages to M2 polarization. We observed the morphology of M1 macrophages and M2 macrophages under light microscope, and found that there were significant differences in the morphology of M1 macrophages and M2 macrophages. M1 macrophages had a large irregular cell body, while M2 macrophages had a long spindle shape, smaller cell body and smoother cell surface than M1 macrophages (Fig. S9 A). Next, we detected polarization markers by RT-qPCR, and the results showed that the mRNA expression of pro-inflammatory factors CD86, IL12 and IL1β was significantly higher in M1 macrophages than in M0/M2 macrophages, while the expression of anti-inflammatory markers CD206, ARG1 and IL10 was significantly higher in M2 macrophages than in M0/M1 macrophages (Fig. S9 B-H), the polarization model was successfully constructed. Notably, in the co-culture system of GPR162 overexpressing LLC and BMDM, the expression of M1 markers was significantly up-regulated, while M2 markers showed no significant change (Fig. 7 B-D, Fig.S11A-C). These results suggest that GPR162 in lung adenocarcinoma cells can promote the polarization of macrophages to M1. To clarify the effect of GPR162 on the regulation of tumor metabolic reprogramming on immune cell polarization, we added capric acid and lauric acid to LLC-GPR162 over-expressing cells and macrophages co-culture system, respectively. Flow cytometry results showed that compared with the control group, the GPR162 overexpression group had a significantly lower M2 polarization level, a significantly lower proportion of CD206 + cells, and a significantly higher proportion of CD86 + cells in the basal state, while lauric acid treatment had no significant effect (Fig.S11I, K). After statistical analysis of the results, the addition of decanoic acid and lauric acid in the control group promoted the polarization of macrophages to M1 macrophages. In the overexpression group, GPR162 promoted the infiltration of cells to M1 macrophages, but the addition of decanoic acid in the overexpression group reversed the polarization of GPR162 to M1 macrophages, and lauric acid had no statistically significant difference (Fig. 7 E, Fig.S11J). These results suggest that decanoic acid may specifically antagonize its pro-M1 polarization effect by competitive inhibition of GPR162 mediated metabolic signaling pathway, suggesting that there is an interactive regulatory network between GPR162 and fatty acid metabolism. The mRNA levels of M1 markers IL6, NOS2, IL1β, TLR2 and TLR4, and M2 markers CD163, IL4 and IL13 were measured by RT-qPCR in lung adenocarcinoma cell lines treated with capric acid or lauric acid. Results Consistent with the above flow cytometry results, GPR162 overexpression significantly up-regulated the mRNA levels of M1 macrophage markers, while there was no significant difference in the mRNA levels of M2 macrophage markers. Moreover, treatment of cells with decanoic acid significantly reversed GPR162-promoted macrophage polarization (Fig. 7 F-H, Fig.S11D-H). These results suggest that decanoic acid affects tumor immunity by regulating GPR162-promoted macrophage polarization to M1. Based on previous studies, GPR162 mediates lipid metabolic reprogramming through the medium-chain fatty acid oxidation pathway (focusing on the regulation of decanoic acid mitochondrial β-oxidation), forms a functional complex with the mitochondrial copper transporter SLC25A3, and specifically induces cuproptosis in tumor cells by disrupting copper homeostasis. In this study, we further investigated the effect of this metabolic reprogramming on the tumor immune microenvironment. Primary mouse adipocytes and GPR162 overexpressed LLC lung adenocarcinoma cells were co-cultured in vitro for 48 hours. PPARγ agonist Troglitazone monotherapy group, cuproptosis inducer ES-Cu monotherapy group and combination treatment group were set up, respectively. The culture medium supernatant of the co-culture system was collected and co-cultured with BMDM, and the expression profile of macrophage polarization markers was detected by RT-qPCR. The results showed that: 1) GPR162 overexpression significantly promoted the polarization of BMDM to pro-inflammatory M1 phenotype, as indicated by significant up-regulation of CD80, CD86 and TLR4 mRNA levels; 2) In the single-agent intervention group, troglitazone and ES-Cu further increased the expression of M1 markers; 3) The combined treatment of the two drugs produced a synergistic effect, and the expression of CD80, CD86 and TLR4) reached the peak, while M2 markers (CD206 and ARG1) did not change significantly in all experimental groups (Fig. 7 I-M). This series of evidence suggests that GPR162 drives the polarization of tumor-associated macrophages to anti-tumor M1 phenotype through a dual regulatory mechanism, activation of medium-chain fatty acid oxidative metabolism pathway and induction of cuproptosis pathway, and this process can achieve synergistic effect by targeting PPARγ signaling and copper homeostasis. In conclusion, this study delineates a dual inhibitory mechanism through which GPR162 suppresses obesity-associated lung adenocarcinoma progression. First, GPR162 orchestrates lipid metabolic reprogramming by activating medium-chain fatty acid β-oxidation (specifically targeting decanoic acid catabolism in mitochondria). Second, it forms a functional complex with mitochondrial copper transporter SLC25A3 to selectively induce tumor-selective cuproptosis via copper homeostasis disruption. Crucially, these two pathways exhibit synergistic effects: 1) driving tumor-associated macrophage (TAM) polarization toward anti-tumor M1 phenotype, 2) enhancing CD8 + T cell infiltration and IFNγ secretion, and 3) collectively establishing an immune-hostile microenvironment that potently suppresses tumorigenesis (Fig. 8 A). These findings position GPR162 as a metabolic-immune nexus, these findings advocate combined targeting of lipid oxidation and copper death pathways for obesity-associated lung adenocarcinoma therapy. Discussion Human obesity has become a global health epidemic, with few safe and effective pharmacological therapies currently available. Previous research has revealed that GPCR agonist targets obesity and diabetes, and GPCR agonist G-1 decreases body weight, fat mass, and inflammation while increasing energy expenditure and improving glucose homeostasis in ovariectomized female mice[ 49 ]. A distinct class T GPCR subfamily TAS2Rs, expressed in extraoral tissues represent potential drug targets for addressing conditions such as obesity, asthma, diabetes, and metabolic diseases[ 50 ]. GPR3 is also a nonadrenergic activator of mouse and human thermogenic adipocytes, cold-induced lipolysis drives the expression of a constitutively active GPCR that regulates thermogenesis in mouse and human adipocytes independent of sympathetic or adrenergic inputs[ 51 ]. Nevertheless, there is a lack of studies examining the influence of GPCR on tumor growth in obese microenvironments, and whether GPR162 affects tumor growth in the obese environment needs to be investigated. Our study revealed that tumor growth was significantly higher in obese mice compared to the control group. Additionally, the expression of GPR162 was notably lower in the tumor tissue of obese mice than in the control group. This finding piqued our interest, suggesting that GPR162 may act as a protective factor against obesity-induced tumor growth. Given that GPR162 is a protein identified in the mitochondrial membrane structure, and that mitochondria play a crucial role in fatty acid oxidation (FAO) and metabolism, we are interested in investigating whether lipid metabolites regulate GPR162 within the mitochondria. In this study, we performed metabolomics sequencing of GPR162-overexpressing subcutaneous tumor tissues in obese mice. Through enrichment analysis, we found that medium-chain fatty acids played an important role in the regulation of GPR162. In addition, we found that decanoate synergistically upregulated the expression of several genes involved in fatty acid synthesis, oxidation, and catabolism. This suggests that GPR162 is both regulated by fatty acid metabolites and involved in the regulation of fatty acid metabolic processes. Our research demonstrates that GPR162 serves as a protective factor in obese settings by participating in the regulation of medium-chain fatty acid oxidation to impede the progression of lung adenocarcinoma. Medium-chain fatty acids (MCFAs) refer to saturated fatty acids with 6–12 carbon atoms, derived from medium-chain triglycerides (MCTs), which can be absorbed directly without digestion and transported to the liver via the portal vein for efficient metabolism and rapid energy supply[ 52 , 53 ]. Long-term high-fat diet intake plays a crucial role in the composition of the gut microbiome in animal models and human subjects and directly affects the production of MCFAs and host health[ 54 ]. Moreover, MCFAs can reduce and reverse metabolic syndrome caused by high-fat diets. Recently, MCTs have also been shown to have the ability to promote protein synthesis metabolism and inhibit catabolic metabolism[ 55 ]. More importantly, these medium-chain fatty acids and their ketone metabolic products are all triggered by cell membrane receptors, which can reduce fat deposition, improve insulin resistance, and regulate glucose and lipid metabolism[ 35 , 56 ]. There have been reports in the literature that the interaction between adipocytes and tumor fatty acid metabolism can promote the progression of cancer in obese individuals[ 37 , 57 , 58 ], therefore, a deeper understanding of GPR162's regulation of fatty acid metabolism could aid in the study of its impact on the progression of lung adenocarcinoma in obese mice. As one of the 53 transport proteins in the inner mitochondrial membrane, SLC25A3 can cause the accumulation of copper ions in the mitochondrial matrix and promote the maturation of the copper proenzymes cytochrome c oxidase and superoxide dismutase[ 59 ]. Several copper ion transporter drugs, such as Elesclomol, Disulfiram, and NSC319726, have been found to induce cell death through a mechanism known as cuproptosis[ 42 , 60 ]. This form of cell death is distinct from other programmed cell deaths (such as apoptosis, pyroptosis, necroptosis, and ferroptosis). Our study has revealed that GPR162 enhances cellular sensitivity to copper ions through its interaction with SLC25A3. As the intracellular copper ion concentration increases, the expression level of cysteinylated proteins decreases and the level of Fe-S cluster proteins falls, leading to mitochondrial protein toxicity stress and ultimately resulting in cell death due to copper overload. These findings suggest a close relationship between GPR162 and mitochondrial homeostasis. The induction of cuproptosis, a recently identified form of copper-dependent immunogenic cell death, is a promising approach for antitumor therapy[ 61 ]. In addition, a variety of recent studies have shown that cuproptosis can be induced in vitro to reprogram the tumor microenvironment and enhance the anti-tumor effect of immunotherapy[ 62 , 63 ]. By reviewing and analyzing GPR162 interacting proteins, we found that a large number of GPR162-binding proteins (32 in total) were enriched in immune pathways. RNA sequencing and immune cell infiltration analysis showed that macrophages were the most infiltrated immune cells. Then, bioinformatics analysis of single-cell databases of various types of tumors showed that GPR162 was mainly expressed in macrophages in most tumor tissues and was significantly associated with immune stress and immune response pathways. Therefore, we hypothesized that GPR162 may reprogram the tumor microenvironment by inducing cuproptosis in tumor cells in an obese environment. Tumor microenvironment(TME) has a central role in the development of tumors, a variety of immune cell infiltration characteristics of groups in TME has been shown to predict the prognosis of patients with some solid tumors[ 64 – 66 ]. Tumor-associated macrophages (TAMs) are the most abundant and important immune cells in the TME[ 67 , 68 ]. Based on molecular phenotype and functional characteristics, activated macrophages can be divided into two major classes: M1 macrophages (classical activation type) and M2 macrophages (alternative activation type). There are significant differences in gene expression patterns and regulation between the two. M1 macrophages mainly promote Th1 immune responses by phagocytosis of pathogens, presentation of antigens, production of IL-1β, TNFα, IL6, IL2, CCL2, and CCL3 cytokines, and killing tumor cells[ 69 ]. M2 macrophages lack cell-killing activity but can produce EGF, MMP, IL-10, TGFβ, and VEGF cytokines to promote Th1 immune responses, induce immune suppression, and mediate tumorigenesis and development, thereby playing a pro-tumor role[ 70 ]. Studies have shown that M1 and M2 macrophages can mutually transform due to changes in the tissue microenvironment. Targeting M2 macrophages and consuming them or converting them back to M1 macrophages in the TME will be a potential strategy for tumor immunotherapy by indirectly stimulating cytotoxic T cells or directly enhancing their phagocytic ability to eliminate tumor cells[ 71 , 72 ]. Therefore, deeply studying the key target molecules that regulate TAM function and targeting the polarization of TAMs to convert M2 macrophages to M1 macrophages, which are beneficial for tumor immunotherapy, has become a frontier and hotspot in current tumor research. Our study reveals that GPR162 functions as a critical tumor suppressor in obesity-associated lung adenocarcinoma through dual metabolic and immunologic mechanisms. We demonstrate that GPR162 simultaneously orchestrates tumor microenvironment reprogramming by enhancing medium-chain fatty acid oxidation and induces mitochondrial dysfunction through SLC25A3-mediated cuproptosis. Furthermore, GPR162 activation promotes anti-tumor immunity by driving M1-like macrophage polarization. These findings provide novel insights into the metabolic-immune axis in cancer progression and establish GPR162 as a promising therapeutic target for treating obesity-associated malignancies, offering new opportunities for developing targeted therapies that simultaneously address metabolic dysregulation and immune suppression in lung adenocarcinoma. Declarations Ethics approval and consent to participate The ethics committee of the Cancer Research Institute of Central South University has approved this study. Consent for publication Not applicable Availability of data and materials Not applicable Competing interests The authors declare that they have no competing interests. The authors declare no conflict of interest. This manuscript has been read and approved by all authors and is not under consideration for publication elsewhere. Funding Author contributions Conception and design: Y. Tao, S. Liu, D. Xiao Development of the methodology: Y. Long, Y. Tao, S. Liu Acquisition of the data: Y. Long, D. Xiao Analysis and interpretation of the data (e.g., statistical analysis, biostatistics, computational analysis): Y. Long, Y. Tao, S. Liu, D. Xiao Writing, review, and/or revision of the manuscript: Y. Long, Y. Tao Study supervision: Y. Tao Disclosure of Potential Conflicts of Interest The authors declare no potential conflicts of interest. 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Additional Declarations There is no conflict of interest Supplementary Files SupplementaryInformation.docx GPR162 Attenuates Obesity-Induced Lung Adenocarcinoma Progression via Dual Modulation of Fatty Acid Oxidation and Cuproptosis Fig.S2.pdf Figure S2 Fig.S7.pdf Figure S7 Fig.S5.pdf Figure S5 Fig.S1.pdf Figure S1 Fig.S8.pdf Figure S8 Fig.S4.pdf Figure S4 Fig.S3.pdf Figure S3 Fig.S9.pdf Figure S9 Fig.S6.pdf Figure S6 Cite Share Download PDF Status: Under Review Version 1 posted Review # 1 received at journal 14 Apr, 2026 Reviewer # 1 agreed at journal 26 Mar, 2026 Reviewers invited by journal 26 Mar, 2026 Submission checks completed at journal 02 Feb, 2026 First submitted to journal 02 Feb, 2026 Unknown event 01 Feb, 2026 Editor assigned by journal 01 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8754789","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":584652625,"identity":"bdefcfbb-27a6-4776-9fc2-c868dca3ab0b","order_by":0,"name":"Yongguang Tao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYBADOQjFRoRSHihtTLqWxAaitdizHz4m8XFHbXp//xkDhg9lhxn4ZzcQsIUnLU1y5pnjuTNu5Bgwzjh3mEHizgFCDssxk+ZtO5a7QYLHgJm37TCDgUQCAS38b8Ba0g34zxgw/yVKiwTYlpoEA4YcA2ZGorTceJZsObPtgOGMG2kFB3vOpfNI3CCghb0/+eCNj2118vz9hzc++FFmLcc/g4AWIGCRYGA4DGYdYEBEFF7A/IGBoY4YhaNgFIyCUTBSAQCORz7DHRIG5AAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-2354-5321","institution":"Central south university","correspondingAuthor":true,"prefix":"","firstName":"Yongguang","middleName":"","lastName":"Tao","suffix":""},{"id":584652626,"identity":"82aad376-9440-4686-b50d-009fba27b165","order_by":1,"name":"Yao Long","email":"","orcid":"","institution":"Cancer Research Institute; School of Basic Medicine, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Long","suffix":""},{"id":584652627,"identity":"3c145ca4-0648-4f66-99a1-3f34223f2cec","order_by":2,"name":"Wei Wang","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Wang","suffix":""},{"id":584652628,"identity":"fe9121c8-2bb3-4904-bd45-4098111c44bd","order_by":3,"name":"Can Cao","email":"","orcid":"","institution":"Central South University,","correspondingAuthor":false,"prefix":"","firstName":"Can","middleName":"","lastName":"Cao","suffix":""},{"id":584652629,"identity":"999ab225-9105-437c-9eee-786a9c0c8a6f","order_by":4,"name":"Molly S. C. Li","email":"","orcid":"","institution":"The Chinese University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Molly","middleName":"S. C.","lastName":"Li","suffix":""},{"id":584652630,"identity":"cd36baa4-2e9a-4326-b121-de750e816f6d","order_by":5,"name":"Lili Li","email":"","orcid":"","institution":"The Chinese University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"Li","suffix":""},{"id":584652631,"identity":"d48a4769-e6da-445f-948a-52e9cd9dc02f","order_by":6,"name":"Shuang Liu","email":"","orcid":"https://orcid.org/0000-0002-7206-7277","institution":"Department of Oncology, Institute of Medical Sciences, National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Shuang","middleName":"","lastName":"Liu","suffix":""},{"id":584652632,"identity":"e95286e3-3767-4dd9-8dba-473ce9d73a6a","order_by":7,"name":"Desheng Xiao","email":"","orcid":"https://orcid.org/0000-0003-2204-5042","institution":"Xiangya Hospital, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Desheng","middleName":"","lastName":"Xiao","suffix":""}],"badges":[],"createdAt":"2026-02-01 08:25:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8754789/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8754789/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105557185,"identity":"790d1f04-b6c7-4c64-b7d5-a42d41a88d08","added_by":"auto","created_at":"2026-03-27 11:12:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":186809,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGPR162 acts as a protective factor inhibits the development of lung adenocarcinoma in HFD-induced obese mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. GWAS analysis of the correlations between BMI and Fat and various types of lung cancer. B. The expression of GPR162 in lung adenocarcinoma patients of different weights. C. The gene set enrichment analysis (GSEA) was performed using R software package. D. To investigate the capacity of LLC cells with stable GPR162 overexpression to develop tumors (n = 10 mice per group), a tumor growth xenograft model was established. Tumor formation was tracked at the weight. E. qPCR analyses of GPR162 in tumor tissues. F-G. Tumor formation was tracked at the indicated times (F) and weight (G). H. Biochemical testing of normal and obese mice blood glucose. I-J. Biochemical testing glucose consumption in stable GPR162 overexpression PC9 (H) and A549 (I) cell culture medium.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8754789/v1/3d0a397f893a2013194bec1e.png"},{"id":105557252,"identity":"e052f619-8d40-4f56-aea2-d320c61ab672","added_by":"auto","created_at":"2026-03-27 11:12:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":310680,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGPR162 was significantly enriched in fatty acid metabolic pathways and correlated with medium-chain fatty acids.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Biochemical testing of normal and obese mice blood lactic acid level. B-C. Biochemical testing lactate production in stable GPR162 overexpression PC9 (B) and A549 (C) cell culture medium. D. GSEA of the whole transcriptome data in GPR162 overexpression cells were enriched in Adipocytokine signaling pathway, and Glycerolipid metabolism. E. KEGG pathway enrichment analysis of LLC cells with stable overexpression GPR162 xenograft tumor tissues. F-G Heatmap representation of untargeted metabolomics sequencing in LLC cells with stable overexpression GPR162 xenograft tumor tissues in HFD obese mice. H. Volcanic map was used to analyze untargeted metabolomics differences metabolites. I. Western-blot analysis of GPR162 protein levels in PC9 cells after treated with pantothenic acid and fumarate. J. Western-blot analysis of GPR162 and CD36 protein levels in H1299 and PC9 cells after being treated with decanoic acid, suet acid, and lauric acid.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8754789/v1/a0cd859d28b6c993fdce12b9.png"},{"id":105566889,"identity":"99306632-ac9b-4f62-936c-07a9b226e572","added_by":"auto","created_at":"2026-03-27 12:57:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":241007,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGPR162 promotes medium-chain fatty acid oxidation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Fatty acid metabolism pathway. B-C. qPCR analysis of fatty acid synthesis, fatty acid oxidation, and fatty acid decomposition-related genes in A549 (B) and PC9 (C) cells treated with decanoic acid after overexpressing GPR162. D-E. Western-blot analysis of GPR162 protein levels in H1299 (D) and PC9 (E) cells after being treated with etomoxir, FCCP, and troglitazone. F. Venn diagram showing overlapping numbers of co-upregulated genes among GPR162-overexpression vs obesity groups, GPR162-overexpression vs obesity control groups, and control vs overexpression groups. G-H. qPCR analysis of PPARGC1A mRNA levels in GPR162-overexpressing LLC (G) and PC9 (H) cells. I. Western-blot analysis of PPARγ protein levels in cytoplasmic and nuclear compartments. J. Chromatin Immunoprecipitation (ChIP) assay demonstrating PPARγ binding to the CPT1A promoter region in GPR162-overexpressing LLC cells. K. Primary mouse adipocytes were extracted and cultured in vitro, and the culture medium supernatant was collected and co-cultured with LLC cells stable overexpression GPR162 for 24h, qPCR analysis offatty acid synthesis, fatty acid oxidation, and fatty acid decomposition-related genes in LLC cells. L-M. Fatty acid uptake assay kit(Dojindo, LD06-1set) analysis of fatty acid uptake in PC9 and LLC cells stable overexpression GPR162 after co-culture.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8754789/v1/676c04f39272e6384bd48aeb.png"},{"id":105557153,"identity":"aa27ce0a-3485-4ad5-b801-3f91f4d84f93","added_by":"auto","created_at":"2026-03-27 11:12:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":457517,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGPR162 promotes cuproptosis in lung adenocarcinoma cells by interacting with SLC25A3.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Mass spectrometry analysis shows a protein interaction between GPR162 and SLC25A3. B-C. Enrichment analysis of mitochondrial membrane protein interacting with GPR162. D. IP identification of endogenous GPR162 and SLC25A3 interactions in PC9 cells. E. Confocal microscopy images of H1975 cells stained with Mito Tracker, anti-LSC25A3 antibodies, and DAPI. Scale bar, 10μm. F. qPCR analysis of SLC25A3 and cuproptosis-related genes in PC9 cells. G-H. The viability of PC9 (G) and A549 (H) cells overexpression GPR162 was assessed at the indicated times after treatment with CuCl\u003csub\u003e2\u003c/sub\u003e-elesclomol, TTM, TTM-CuCl\u003csub\u003e2\u003c/sub\u003e-elesclomol. I. Western-blot analysis of protein oligomerization in PC9 cells stable overexpression GPR162. J-K. Western-blot analysis of GPR162, SLC25A3, LIAS, and lipoylated proteins in PC9 (J) and A549 (K) cells stable overexpression GPR162. L. Western-blot analysis of GPR162, LIAS, and lipoylated proteins in H1299 (L) cells knockdown GPR162.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8754789/v1/d2b67d9b454bac7464cad7e8.png"},{"id":105557149,"identity":"7f072969-5fe3-4f3b-8d26-f6a65d7cf3ac","added_by":"auto","created_at":"2026-03-27 11:12:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":394848,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGPR162 promotes the generation of MitoROS and inhibits ATP.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. qPCR analysis of Fe-S cluster proteins and lipoylated proteins related genes in PC9 cells. B. IP identification of endogenous GPR162 and LIAS interactions in H1299 and H358 cells. C. Confocal microscopy images of H1975 cells stained with DLAT, anti-GPR162 antibodies, and DAPI. Scale bar, 10μm. D. MitoSox Red kit(Invitrogen, M36008) analysis of MitoROS in PC9 cells stable overexpression GPR162 after treatment with CuCl\u003csub\u003e2\u003c/sub\u003e-elesclomol. E. MitoSox Red kit analysis of MitoROS in H1299 cells knockdown GPR162 after treatment with CuCl\u003csub\u003e2\u003c/sub\u003e-elesclomol. F. Mitochondrial membrane potential assay kit(Beyotime, C2003S) analysis of JC-1 in H1299 cells knockout GPR162. G. Mitochondrial membrane potential assay kit analysis of JC-1 in PC9 cells overexpression GPR162. H. Mitochondrial ATP assay kit (S0027) analysis of ATP in H1299 cells knockout GPR162. I. Mitochondrial ATP assay kit analysis of ATP in PC9 cells overexpression GPR162. J. Primary mouse adipocytes were extracted and cultured in vitro, and the culture medium supernatant was collected and co-cultured with LLC cells stable overexpression GPR162 for 24h, qPCR analysis of GPR162 in LLC cells. K. Western-blot analysis of GPR162, SLC25A3, and lipoylated proteins in LLC cells stable overexpression GPR162 after co-culture. L. qPCR analysis of SLC25A3, DLAT, DLST, FDX1, and LIAS in LLC cells stable overexpression GPR162 after co-culture.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8754789/v1/dbe66adf7d943cc7e0547268.png"},{"id":105557152,"identity":"26ed6eaa-5547-42f4-8a57-4363d73cb87f","added_by":"auto","created_at":"2026-03-27 11:12:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":354889,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn the obese mice induced by high-fat diet, GPR162 can promote tumor-associated macrophage infiltration.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. GOMF pathway enrichment analysis of LLC cells with stable overexpression GPR162 xenograft tumor tissues. B-C. GSEA of the whole transcriptome data in GPR162 overexpression cells were enriched in Immune receptor activity (B), and Antigen processing and presentation (C). D. Flow cytometry gating strategy for TAMs. Flow cytometry was used to detect the effect of obesity and GPR162 overexpression on macrophage infiltration. E. Percentage CD45\u003csup\u003e+\u003c/sup\u003e within F4/80\u003csup\u003e+\u003c/sup\u003e and CD11b\u003csup\u003e+\u003c/sup\u003e gated live cells from HFD mice with representative histograms (n = 6 mice/treatment). F. Percentage CD45\u003csup\u003e+\u003c/sup\u003e within CD11b\u003csup\u003e+\u003c/sup\u003e and Ly6G\u003csup\u003e-\u003c/sup\u003e gated live cells from HFD mice with representative histograms (n = 6 mice/treatment).G. Percentage CD45\u003csup\u003e+\u003c/sup\u003e within CD11b\u003csup\u003e+\u003c/sup\u003e and Ly6G\u003csup\u003e+ \u003c/sup\u003egated live cells from HFD mice with representative histograms (n = 6 mice/treatment). H. Percentage CD45\u003csup\u003e+\u003c/sup\u003e within CD8\u003csup\u003e+\u003c/sup\u003e gated live cells from HFD-fed mice with representative histograms (n = 6 mice/treatment). I. Primitive CD8\u003csup\u003e+\u003c/sup\u003eT cells from mice spleen were cocultured with LLC cells in the lower chambers of a 5-μm Transwell plate. J-M. qPCR analysis of CCL2 (G), GZMB (H), TNFα (I), and TGFβ (J) in LLC cells overexpression GPR162 after co-culture with CD8\u003csup\u003e+\u003c/sup\u003eT cells. N. Mouse bone marrow-derived macrophages were co-cultured with LLC cells overexpression GPR162 and then cocultured with CD8\u003csup\u003e+\u003c/sup\u003eT cells in the upper chambers of a 5-μm Transwell plate. O-R. qPCR analysis of CCL2 (L), GZMB (M), TNFα (N), and TGFβ (O) in LLC cells overexpression GPR162 after co-culture with BMDM and CD8\u003csup\u003e+\u003c/sup\u003eT cells.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8754789/v1/c1797e2487d88f34f6e25ce3.png"},{"id":105557150,"identity":"ef41b7be-ca87-445b-8341-634944bbcc77","added_by":"auto","created_at":"2026-03-27 11:12:13","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":163356,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGPR162 acquires more pro-inflammatory characteristics through tumor-associated macrophages, promoting antigen presentation and T cell responses.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Tumor cells and macrophages were cultivating strategies. B-D. qPCR analysis of CD86 (B), TLR2 (C), TLR4 (D) in LLC cells stable overexpression GPR162 after co-culture with BMDM. E. Percentage CD45\u003csup\u003e+\u003c/sup\u003e within CD86\u003csup\u003e+\u003c/sup\u003e and CD206\u003csup\u003e-\u003c/sup\u003e gated live cells from LLC cells stable overexpression GPR162 after co-culture with BMDM and treatment with Decanoic acid and Lauric acid. F-H. qPCR analysis of IL6 (F), TLR2 (G), and TLR4 (H) in LLC cells stable overexpression GPR162 after co-culture with BMDM and treatment with Decanoic acid and Lauric acid. I-M. RT-qPCR analysis of mRNA levels for macrophage markers CD80 (I), CD86 (J), TLR4 (K), CD206 (L), and ARG1 (M) in the co-culture system following treatment with Troglitazone and ES-Cu.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8754789/v1/d68ce554d992f7ca7bc04749.png"},{"id":105566492,"identity":"0c8134b4-5d08-46ad-aec2-a015069aa1d4","added_by":"auto","created_at":"2026-03-27 12:56:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":258211,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of GPR162 attenuating obesity-induced lung adenocarcinoma progression through dual regulation of fatty acid oxidation and cuproptosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Schematic diagram of GPR162 promotes antitumor immunity by regulating fatty acid oxidation(FAO) and cuproptosis in obese mice with lung cancer: On the one hand, GPR162 induces cell peroxidation by promoting the oxidation of medium-chain fatty acids such as decanoic acid and lauric acid. On the other hand, GPR162 interacts with copper transporter SLC25A3 to form a functional complex in the mitochondria, driving copper influx and further promoting intracellular lipid peroxidation to induce copper death, which directly kills tumor cells. The synergistic effect of fatty acid oxidation and copper death not only reprogram the tumor metabolic microenvironment, but also induce the polarization of tumor-associated macrophages to M1 type, enhance CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration and IFNγ secretion, and finally construct an immune microenvironment that inhibits tumor progression.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8754789/v1/fc11bf32d7bfcefb98c4d305.png"},{"id":105570216,"identity":"7480c9d9-b40b-4eae-beb8-cc5468ad39c9","added_by":"auto","created_at":"2026-03-27 13:15:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3952952,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8754789/v1/dc9a0c49-aec6-4ac7-bdfb-255f3e73cc5a.pdf"},{"id":105567404,"identity":"5c8465a3-c95f-4e5b-8e62-06cd1dcf8fe8","added_by":"auto","created_at":"2026-03-27 12:59:20","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":37665,"visible":true,"origin":"","legend":"GPR162 Attenuates Obesity-Induced Lung Adenocarcinoma Progression via Dual Modulation of Fatty Acid Oxidation and Cuproptosis","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8754789/v1/4cd0125ddce2a05cc48ed416.docx"},{"id":105557189,"identity":"446ea6e2-e006-44e0-ad2e-4e268d54bf49","added_by":"auto","created_at":"2026-03-27 11:12:27","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":304475,"visible":true,"origin":"","legend":"Figure S2","description":"","filename":"Fig.S2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8754789/v1/d33e7fb2c0729867c454fc2f.pdf"},{"id":105567282,"identity":"c9e7e988-913a-4967-b647-89f8ebb67a79","added_by":"auto","created_at":"2026-03-27 12:58:46","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":155195,"visible":true,"origin":"","legend":"Figure S7","description":"","filename":"Fig.S7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8754789/v1/e973cb9ab24a19aa04182089.pdf"},{"id":105557148,"identity":"b29817bd-4b30-4182-bc59-7583ddb8616a","added_by":"auto","created_at":"2026-03-27 11:12:12","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":467308,"visible":true,"origin":"","legend":"Figure S5","description":"","filename":"Fig.S5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8754789/v1/26ecbe134360b97475104eb3.pdf"},{"id":105557187,"identity":"3033edd1-e6fd-4799-8325-c2f04e8f77e9","added_by":"auto","created_at":"2026-03-27 11:12:26","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":182382,"visible":true,"origin":"","legend":"Figure S1","description":"","filename":"Fig.S1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8754789/v1/633f48c86df5a5fc04782409.pdf"},{"id":105557254,"identity":"ad922484-da1d-416a-ae20-6eaab53abede","added_by":"auto","created_at":"2026-03-27 11:12:29","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":611014,"visible":true,"origin":"","legend":"Figure S8","description":"","filename":"Fig.S8.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8754789/v1/fc5d53001abd5a41aa8ab9ac.pdf"},{"id":105557184,"identity":"647b3ae9-78bb-41b1-8e5d-9a80e3037f33","added_by":"auto","created_at":"2026-03-27 11:12:25","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":329915,"visible":true,"origin":"","legend":"Figure S4","description":"","filename":"Fig.S4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8754789/v1/2d60a70028ef382a98e9f0f5.pdf"},{"id":105557234,"identity":"093ecf28-3e9d-443a-9ad4-67244218bdfc","added_by":"auto","created_at":"2026-03-27 11:12:28","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":513768,"visible":true,"origin":"","legend":"Figure S3","description":"","filename":"Fig.S3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8754789/v1/1795be985d51184864709089.pdf"},{"id":105557235,"identity":"c4956ebb-0178-4f77-be97-d74ea87ac2a4","added_by":"auto","created_at":"2026-03-27 11:12:28","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":344408,"visible":true,"origin":"","legend":"Figure S9","description":"","filename":"Fig.S9.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8754789/v1/43ff24282ec3ffad6ae1250a.pdf"},{"id":105557255,"identity":"7a4bfa7a-80a3-4f86-a494-1f2b8b6d586e","added_by":"auto","created_at":"2026-03-27 11:12:29","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":156387,"visible":true,"origin":"","legend":"Figure S6","description":"","filename":"Fig.S6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8754789/v1/5fdc4c9a46a05cb7ab6e34f5.pdf"}],"financialInterests":"There is no conflict of interest","formattedTitle":"GPR162 Drives Obesity-Associated Lung Adenocarcinoma Suppression via Fatty Acid Oxidation-Induced Cuproptosis and Metabolic-Immune Reprogramming","fulltext":[{"header":"Significance Statement","content":"\u003cp\u003eThis study identifies G protein-coupled receptor 162 (GPR162) as a critical protective factor in obesity-associated lung adenocarcinoma, uncovering its dual mechanisms of action. We demonstrate that GPR162 reprograms the tumor microenvironment by promoting medium-chain fatty acid oxidation and induces cuproptosis through its interaction with the mitochondrial copper transporter SLC25A3. Additionally, GPR162 enhances anti-tumor immunity by driving tumor-associated macrophages toward an M1-like phenotype. These findings not only advance our understanding of how obesity influences cancer metabolism and progression but also establish GPR162 as a promising therapeutic target for combating obesity-related lung adenocarcinoma.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eLung cancer is one of the most common and deadly malignant tumors worldwide[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], with lung adenocarcinoma being the predominant pathological type[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It seriously threatens human life and health. Although smoking has been widely recognized as the primary risk factor for lung cancer, more and more studies have shown that in addition to traditional carcinogenic factors, systemic factors such as metabolism and inflammation also play an important role in the occurrence and development of lung cancer[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Obesity, as a chronic metabolic disease characterized by dysfunction of fat tissue and imbalance of energy metabolism, has a continuously rising prevalence worldwide and has become an important public health issue[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A large number of studies have shown that obesity can significantly promote the occurrence and progression of various malignant tumors by inducing chronic low-level inflammation, insulin resistance, imbalance in fat factor secretion, and remodeling of immune function[\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In recent years, epidemiological investigations and basic research have further indicated that obesity is closely related to the occurrence, progression, and prognosis of lung cancer[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Another study has shown that obese patients with lung cancer have poor responses to immune checkpoint therapy[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, the molecular mechanism by which obesity mediates the occurrence and development of lung cancer is still unclear, and the specific mechanisms of its regulation of tumor metabolic reprogramming and tumor immune microenvironment are also not clear. Therefore, in-depth analysis of the key molecular mechanisms by which obesity mediates the progression of lung cancer is expected to provide new theoretical basis and research directions for the precise intervention and targeted treatment of lung cancer.\u003c/p\u003e \u003cp\u003eAs the proportion of overweight and obese individuals worldwide continues to rise, obesity is widely regarded as a significant risk factor for various solid tumors and metabolic-related diseases. Moreover, most studies have indicated that the obese state is associated with a poor prognosis for cancer patients. A retrospective study of 794 cancer patients found that compared to normal patients, overweight or obese patients had a significantly lower 3-year overall survival rate (93.8% vs. 98.0%, P\u0026thinsp;=\u0026thinsp;0.01), a significantly higher 3-year recurrence rate (10.5% vs. 5.8%, P\u0026thinsp;=\u0026thinsp;0.02), and a significantly lower 3-year event-free survival rate (89.0% vs. 93.7%, P\u0026thinsp;=\u0026thinsp;0.02) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Another retrospective study based on 31,257 patients with advanced non-small cell lung cancer showed that in patients with BMI\u0026thinsp;\u0026lt;\u0026thinsp;28, ICI treatment significantly reduced the risk of death (for example, when BMI was 24, HR\u0026thinsp;=\u0026thinsp;0.81, 95% CI: 0.75\u0026ndash;0.87); however, in patients with BMI\u0026thinsp;\u0026ge;\u0026thinsp;28, ICI treatment did not show significant survival benefits (for example, when BMI was 28, HR\u0026thinsp;=\u0026thinsp;0.90, 95% CI: 0.81\u0026ndash;1.00) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These results suggest that persistent overweight or obesity may affect the survival outcome of patients by altering the biological characteristics of tumors or treatment responses.\u003c/p\u003e \u003cp\u003eG protein-coupled receptors (GPCRs) are a broad class of membrane surface proteins that are also the largest family of drug targets[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. GPCRs are involved in approximately 80% of cellular signaling processes and are important proteins in cell signaling[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Studies have shown that GPCRs are widely dysregulated in tumors, playing important roles in tumor proliferation, survival, angiogenesis, invasion, metastasis, resistance to treatment, and immune evasion[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Orphan receptors refer to receptors with unknown endogenous ligands, and about 200 of the approximately 800 human GPCR genes are orphan receptors[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Orphan receptors are involved in various physiological functions and play important roles, such as sensing, reproductive development, metabolism, and responsiveness, and may therefore be closely related to many diseases, such as central nervous system diseases, metabolic diseases, and cancer[\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. For orphan GPCRs, it is difficult to develop drugs due to a lack of understanding of endogenous ligands, and therefore, in-depth research into the functions and mechanisms of action of orphan receptors is of great importance for the development of new drugs.\u003c/p\u003e \u003cp\u003eGPR162 belongs to the class of retinal A-type orphan GPCRs, and it is highly expressed in the brain and lung tissues. There are few studies on GPR162, suggesting that its mutation is associated with abnormal glucose levels in vivo and that changes in GPR162 are associated with reduced food intake in rats[\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The TCGA database also shows that the activation of GPR162 is significantly enriched in EMT-related signaling pathways, and is closely related to metabolic-related pathways such as insulin-producing beta cells and glycolysis. There are already literature reports on the involvement of GPCRs in beta cell dysfunction, insulin resistance, and obesity-induced T2DM[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Therefore, it is crucial to further study the impact of GPR162 on the development of lung adenocarcinoma in obese mice and explore the relationship between GPR162 and the tumor microenvironment associated with obesity, which is important for the development of new drugs and the discovery of new strategies for cancer treatment.\u003c/p\u003e \u003cp\u003eSeveral studies have indicated a close association between obesity and the oxidation of medium-chain fatty acids, as well as the potential of medium-chain fatty acids to mitigate and reverse metabolic syndrome induced by high-fat diets[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Furthermore, these medium-chain fatty acids and their ketone metabolites are activated by cell membrane receptors, leading to reduced fat deposition, improved insulin resistance, and regulation of glucose and lipid metabolism[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Given our identification of GPR162 as a protein located on the structural component of the mitochondrial membrane[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], it is pertinent to investigate whether mitochondrial GPR162 is influenced by lipid metabolites or impacts cellular lipid metabolism.\u003c/p\u003e \u003cp\u003eRecent studies have shown that SLC25A3 is an important protein that transports copper into the mitochondrial matrix and participates in the respiratory chain on the inner mitochondrial membrane[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In 2022, it was first revealed that copper can abnormally bind to lipid-modified proteins and reduce iron-sulfur cluster proteins, causing significant damage to the mitochondrial respiratory chain and ultimately inducing a unique new type of cell death: cuproptosis[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The mitochondria, as the cell's energy factory, is a highly dynamic and stable organelle[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Imbalances in mitochondrial homeostasis are associated with virtually all human diseases[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Recent studies have revealed that a variety of GPCR members of membrane proteins can be found in non-plasma membrane sites such as endoplasmic reticulum, Golgi apparatus, mitochondria, nucleus, and even centriole to mediate signal initiation and transduction within the cell. As a result, we found the GPR162 and SLC25A3 interaction on the mitochondrial membrane, strongly suggests the biological significance of cuproptosis may participate in regulating cell death.\u003c/p\u003e \u003cp\u003ePrevious work has confirmed that GPR162 is closely related to the activation of tumor innate immunity, and GPCR can be an effective pro-inflammatory and anti-inflammatory regulator for key immune cells, serving as a bridge for the interaction between the nervous, endocrine, and immune systems[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. There is literature to confirm that the macro- and microenvironment of obesity affect the biological changes of tumors, including mitosis and metabolism, promoting tumor growth[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. We found that GPR162 is highly expressed in neutrophils, monocytes, and macrophages, and there is a significant correlation between GPR162 expression and macrophages. GPR162 can promote the polarization of macrophages towards M1, which is influenced by fatty acids, suggesting that GPR162 may play an important role in regulating fatty acid oxidation and immune cell function in the tumor microenvironment. Therefore, elucidating the molecular mechanisms by which GPR162 regulates fatty acid oxidation and understanding its function in immune cells, particularly macrophages, in the tumor microenvironment can help identify new therapeutic targets and improve the efficacy of immunotherapy, providing new insights for tumor immunotherapy strategies.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eMice were kept in pathogen-free conditions. When necessary, mice were acquired from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). Mice were fed an irradiated PicoLab Rodent Diet with a high-fat content of 60 kcal% (D12492), whereas the control diet had a fat content of 10 kcal% (D12450J). Mice classified as obese in this study were fed a high-fat diet for at least 9 weeks, beginning at age 6\u0026ndash;7 weeks, and weighed at least 38 g (on average inside a cage). All mice utilized in the research were male. Animal research was conducted with the approval of Central South University's Xiangya School of Medicine's Institutional Animal Care and Use Committee, as well as in accordance with legislative and federal animal protection and care guidelines. Subcutaneous injections of GPR162-overexpressing cells and control cells (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mouse) were given to each mouse's axilla. Following that, tumor volume and mouse weight were recorded every three days until the mice were euthanized at 27 days. Tumors were weighed and lysed for flow cytometry analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture, viruses, stimulation, and transfection\u003c/h3\u003e\n\u003cp\u003eIn this investigation, the following cell culture conditions were used: LLC(ATCC: CRL-1642), BMDM, RAW264.7(ATCC: SC-6003) cell lines were cultured in DMEM (Gibco, NY, USA) medium, A549 (ATCC: CCL-185) cell lines were cultured in 1:1 DME/F12 (HyClone, UT, USA) medium, PC9, H1299 (ATCC: CRL-5803) cell lines were cultured in RPMI1640 (Gibco) medium. Cells were cultured in a cell incubator at 37\u0026deg;C with 5% CO2 and the medium containing 10% (v/v) BCS. All cell lines were obtained from the cell bank of the Cancer Institute, Central South University. Supplementary Table\u0026nbsp;3 contains the sgRNA and shRNA sequences for GPR162 described in this study. The generated plasmid was introduced into cells and transfected using Lipofectamine Max. The colonies with stable expression were screened by puromycin (1 \u0026micro;g/ml).\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis and coimmunoprecipitation (Co-IP) assay\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blot analysis and coimmunoprecipitation (Co-IP) assay\u003c/div\u003e \u003cp\u003eFollowing three 1\u0026times;PBS washes, the harvested cells were lysed for one hour on ice in an IP lysis solution containing a protease inhibitor cocktail. The BCA method was used to determine the protein concentration, and the apparatus was set up. After being extracted from cell lysate using an SDS-polyacrylamide gel, the total proteins were transferred to a polyvinylidene fluoride membrane. Supplementary Table\u0026nbsp;4 lists primary antibodies used in Western blot analysis.\u003c/p\u003e \u003cp\u003eTarget protein antibodies were added to the magnetic bead-precleared proteins at 4\u0026deg;C, and the mixture was incubated for an entire night. Western blot analysis was used to detect the interaction between the proteins after they had been adsorbed by magnetic beads and denatured.\u003c/p\u003e\n\u003ch3\u003eReal-time quantitative polymerase chain reaction (RT-qPCR)\u003c/h3\u003e\n\u003cp\u003eTRIzol reagent (Takara, Kusatsu, Japan) was used to isolate the total RNA, and the kit (Takara, Kusatsu, Japan) was used to reverse transcribe the RNA into cDNA. Real-time PCR was performed on a Bio-rad CFX Connect real-time PCR instrument. β-actin served as the internal reference for gene expression. The primers used in this investigation are listed in Supplementary Table\u0026nbsp;1\u0026ndash;2.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence microscopy\u003c/h3\u003e\n\u003cp\u003eA 24-well culture plate containing tiny glass discs was filled with logarithmic growth cells 24 hours before adherent growth cells were added. The culture plate was taken out when the degree of cell fusion was around 50%. Following three 1\u0026times;PBS washes, 1 mL of methanol was added to each well, and the wells were fixed for 10 minutes at 20 degrees. Following two 5-minute PBS rinses, 1% (w/v) BSA in PBS was used to block the area for 30 minutes. three times for five minutes each, followed by an overnight incubation at 4\u0026deg;C with primary antibodies containing 1% (w/v) BSA. Depending on the characteristics of the primary antibody, either anti-rabbit IgG Alexa 594 fluorescent secondary antibody or anti-mouse IgM Alexa 488 fluorescent secondary antibody was chosen and incubated for one hour after washing with PBS. After DAPI labeling, they were put on slides and photographed using a Leica TCS SP8 confocal microscope. Living cells were stained with MitoTracker\u0026reg; Deep Red FM (Invitrogen, 644\u0026ndash;665 nm, M22426) in a cell incubator set at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 30 minutes.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSeparation of the cytoplasmic and mitochondria\u003c/h2\u003e \u003cp\u003eThe Cell Mitochondria Isolation Kit (Beyotime, C3601) was utilized to isolate the proteins found in cellular mitochondria. Following three 1\u0026times;PBS washes, the collected cells were lysed for fifteen minutes on ice in a mitochondrial separation reagent containing PMSF. After being moved to a glass homogenizer of the proper size, the cell suspension was homogenized for ten to thirty cycles. Centrifuging the cell suspension produced cellular mitochondria in the precipitate and cytoplasmic proteins in the supernatant. The BCA technique was used to determine the protein concentration.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFlow cytometry\u003c/h3\u003e\n\u003cp\u003eThe following antibodies were used. Biolegend: CD49b, CD4, CD8, CD45, CD206, CD3e, Ly-6G, F4/80, CD11b, CD86, CD11c, BB515, IFN-γ, GzmB and TNF. We used the eBioscience Fixable Viability Dye eFluor780 or DAPI to distinguish live from dying or dead cells. For intracellular staining, cells were treated with fixation and permeabilization reagents from eBioscience and labeled with appropriate antibodies before being analyzed. Data were analyzed using an LSRII, LSRFortessa, or FACSAria instrument (Becton Dickinson) and FlowJo software (FlowJo LLC). Cells were sorted on a BD FACSAria cell sorter.\u003c/p\u003e\n\u003ch3\u003eIn vitro CD8 T cell differentiation\u003c/h3\u003e\n\u003cp\u003eCD8\u0026thinsp;+\u0026thinsp;T cells were isolated from the spleen using the Pan T Cell Isolation Kit II (Miltenyi Biotec). A single-cell suspension from mouse spleen was prepared using the program m_spleen_01.01 on the gentleMACS\u0026trade; Dissociator. T cells were isolated from this single-cell suspension using the Pan T Cell Isolation Kit II, an LS Column, and a MidiMACS\u0026trade; Separator. Cells were fluorescently stained with the MC CD90.2 T Cell Cocktail, mouse as well as with CD3e-APC, and analyzed by flow cytometry using the MACSQuant\u0026reg; Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eExcept for the studies with mice, every study was carried out a minimum of three times. A mean SD, or SEM, is displayed for the data. The statistical analysis was performed with GraphPad Prism 9.0. The T-test was used to assess the significance of differences between two groups, and analysis of variance (ANOVA) was used to examine groups larger than two. The Parsons correlation coefficient was used for correlation analysis. Differences were considered statistically significant in the following cases: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05(*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStudy approval\u003c/h2\u003e \u003cp\u003e The ethics committee at our hospital approved the project. The institutional Animal Care and Use Committee at Central South University gave its approval for the use of animal models in this study. The study was approved by the institutional review boards of all participating medical facilities. Each research subject signed a documented informed consent form before recruitment.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eGPR162 acts as a protective factor that inhibits the development of lung adenocarcinoma in obese mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBased on the above research reports, we conducted a genome-wide association study (GWAS) to assess the correlations between obesity-related traits (BMI, body fat) and different types of lung cancer. The results showed that both BMI and body fat were significantly correlated with lung adenocarcinoma (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), suggesting that obesity may be involved in the occurrence and development of lung adenocarcinoma. Furthermore, we divided the 514 lung adenocarcinoma samples from the TCGA database into four groups based on the patients' different weights: normal weight, overweight, obese, and extremely obese. The results showed that the expression of GPR162 was higher in the normal weight group than in the obese group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Additionally, we analyzed the mRNA expression of 16,383 genes and classified them into two groups: high expression and low expression of GPR162. The gene set enrichment analysis (GSEA) revealed that in the tumor patients with high expression of GPR162, there was a significant enrichment in lipid metabolism-related pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further explore the relationship between obesity and lung adenocarcinoma, as well as the role played by GPR162 in this process, we established a diet-induced obesity (DIO) model using C57BL/6 mice fed a 60% high-fat diet (HFD) for 12 weeks (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). After 12 weeks, peripheral blood of the mice was collected from the orbital vein, and then the levels of blood glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH) and blood lipids (triglyceride, cholesterol, high-density lipoprotein, low-density lipoprotein) in peripheral blood of the mice were detected by biochemical experiments (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB-E). The results showed that the levels of blood glucose and blood lipid in the obese mice were significantly higher than those in the control group, which confirmed the successful construction of the obese mouse model.\u003c/p\u003e \u003cp\u003eNext, we subcutaneously injected lung adenocarcinoma cells LLC into obese mice induced by high-fat diet to construct a subcutaneous tumor-bearing model. By monitoring the body weight, tumor growth curve and tumor weight of the mice, we found that the tumor growth rate of the obese mice was significantly accelerated, and the tumor volume was significantly larger than that of the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF-H). Notably, RT-qPCR results showed that GPR162 expression was significantly down-regulated in lung adenocarcinoma tissues of the obesity group compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), suggesting its potential tumor-suppressive role in metabolic dysregulation contexts.\u003c/p\u003e \u003cp\u003eTo further confirm the effect of GPR162 on obesity-promoted lung adenocarcinoma progression. We subcutaneously transplanted mouse lung adenocarcinoma cells LLC stably overexpressing GPR162 into normal and diet-induced obese mice. The growth curve, tumor volume and tumor weight of subcutaneous tumors were dynamically monitored. We found that the tumor growth of obese mice was significantly accelerated compared with the normal diet group, while GPR162 overexpression could significantly inhibit the tumor growth rate, and the tumor volume and weight were significantly reduced compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-G, Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eI-J). These results indicate that GPR162 overexpression can effectively inhibit obesity-promoted lung adenocarcinoma progression, suggesting that GPR162 may play an important tumor suppressor role in obesity-related lung adenocarcinoma.\u003c/p\u003e \u003cp\u003eTo mechanistically dissect this observation, we engrafted GPR162-overexpressing LLC cells into both lean and obese mice. we measured the glucose and lactate levels in the peripheral blood of normal and obese mice, and in the culture supernatant of lung adenocarcinoma cells in the control group and GPR162 overexpression group using an automatic biochemical analyzer system. The results showed that GPR162 overexpression significantly inhibited glucose consumption in PC9 and A549 lung adenocarcinoma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-J). This finding further supports GPR162 as a potential target for the treatment of obesity and other metabolic diseases. However, peripheral blood lactate level was not significantly altered in the obese mouse model, and lactate production in the culture medium of GPR162 overexpressed lung adenocarcinoma cells was not statistically different (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). Since lactate is the end product of glycolysis, the amount of lactate produced was not statistically different, suggesting that the inhibitory effect of GPR162 on lung adenocarcinoma progression in obese mice may not be achieved by regulating the glycolytic pathway. Therefore, the specific metabolic reprogramming mechanisms and energy supply pathways of tumor cells in the obese microenvironment remain to be further studied.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGPR162 promotes medium-chain fatty acid oxidation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo delineate the metabolic axis through which GPR162 modulates obesity-associated lung adenocarcinoma progression, we conducted RNA sequencing on tumors from diet-induced obese (DIO) mice with GPR162 overexpression. DESeq2-based pathway analysis revealed that 80% of differentially expressed genes (DEGs) mapped to lipid metabolism pathways, with pronounced enrichment in glycerophospholipid metabolism, sphingolipid cycling, and ether lipid biosynthesis, GSEA further corroborated these findings, showing strong associations with adipokine signaling and glycerolipid homeostasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E), positioning GPR162 as a central regulator of lipid metabolic rewiring in obese tumors.\u003c/p\u003e \u003cp\u003eNon-targeted metabolomics of subcutaneous transplanted tumors identified pantothenate metabolism as the most significantly altered pathway in GPR162-overexpressing obese mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Given pantothenate's essential role as a CoA precursor in fatty acid metabolism, we focused on medium-chain fatty acid (MCFA) dynamics. Targeted lipidomics demonstrated 3.7-fold elevation of decanoic acid (C10:0) in GPR162-overexpressing tumors compared to obese controls, with parallel increases in octanoic and lauric acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-H). Because there is a wide range of bidirectional regulatory relationships between molecules involved in the regulation of fatty acid metabolism and fatty acids, for example, the core regulatory molecules of fatty acid metabolism (such as PPARs, SREBP-1c, AMPK) are not only metabolic regulators, but also \"sensors\" of fatty acids and their derivatives. Therefore, to further test whether GPR162 is involved in fatty acid metabolism, we observed a significant up-regulation of GPR162 protein expression in lung adenocarcinoma cell culture media treated with pantothenic acid and fumaric acid, which are core cofactors of lipid metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). In addition, treatment of lung adenocarcinoma cells with medium-chain fatty acids (capric acid, octanoic acid and lauric acid) similarly significantly increased GPR162 protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). These results suggest that GPR162 may regulate medium-chain fatty acid metabolism.\u003c/p\u003e \u003cp\u003eSubcellular fractionation confirmed predominant mitochondrial localization of GPR162 (Fig.\u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA-C). It is well known that mitochondria are the energy metabolism center of cells and are the site of energy metabolic processes such as oxidative phosphorylation, ATP synthesis, and fatty acid oxidation, suggesting that GPR162 is localized in mitochondria and participates in fatty acid energy metabolism. To determine the role of GPR162 in the regulation of mitochondrial energy metabolism, GPR162-overexpressing lung adenocarcinoma cells were treated with decanoic acid for 24 hours. Total RNA was extracted from the cells, and genes involved in fatty acid synthesis, fatty acid oxidation, and fatty acid breakdown were quantified by RT-qPCR. Fatty acid synthesis, oxidation and breakdown pathways were all activated in the overexpression group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). Moreover, GPR162 protein level was also significantly up-regulated after adding cell metabolism and function inhibitors in lung adenocarcinoma cells, further confirming that GPR162 is involved in fatty acid metabolism pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further explore the mechanism of GPR162 regulation on fatty acid metabolism, RNA-seq results of subcutaneous tumor tissues from normal and obese mice were cluster. The results showed that GPR162 overexpression in normal mice and GPR162 overexpression in obese mice significantly up-regulated the expression of Ppargc1a and Prl2c2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Since Prl2c2 is only expressed in mice, PPARGC1A mRNA levels were examined in LLC and PC9 cells overexpressing GPR162, and the results showed that GPR162 overexpression promoted PPARGC1A transcription (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H). Since PPARGC1A is a transcriptional coactivator of PPARγ and a major regulator of mitochondrial biogenesis, it can promote its transcriptional activity by binding to PPARγ, while PPARγ promotes fatty acid breakdown and energy production by activating the transcription of fatty acid oxidation related genes (such as CPT1, ACOX, etc.). Therefore, we isolated the cytoplasmic and nuclear proteins of LLC cells overexpressing GPR162, and the results showed that the expression level of PPARγ in the nucleus was significantly higher after GPR162 overexpression than in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). ChIP results also showed that overexpression of GPR62 enhanced the binding between PPARγ and CPT1A in the promoter region (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). These results suggest that GPR162 promotes medium-chain fatty acid oxidation by up-regulating PPARGC1A to drive PPARγ into the nucleus and enhance its binding with CPT1A at the promoter region.\u003c/p\u003e \u003cp\u003eCo-culture experiments with primary adipocytes from obese mice revealed that GPR162 overexpression: (1) Upregulated fatty acid oxidation genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK), (2) Enhanced BODIPY-C12 uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL-M). This metabolic plasticity occurred despite unchanged glucose-lactate coupling (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C), suggesting preferential utilization of lipid over glycolytic substrates. The bidirectional regulation between GPR162 and fatty acid metabolites (C8-C12) establishes a lipid-sensing paradigm where MCFAs both induce and are catabolized through GPR162-PPARGC1A signaling.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGPR162 induces cuproptosis in lung adenocarcinoma cells by interacting with SLC25A3.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs a mitochondrial membrane protein implicated in lipid metabolism, we sought to delineate GPR162's interactome through proteomic analysis of mitochondria-associated endoplasmic reticulum membranes (MAMs). Mass spectrometry identified 161 high-confidence interacting partners, including endoplasmic reticulum Ca\u0026sup2;⁺-ATPase (SERCA), mitochondrial calcium transporters VDAC1/VDAC3, and copper carrier SLC25A3, Notably, SLC25A3 demonstrated the highest enrichment score (2.8-fold vs controls) among MAM components (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). Co-immunoprecipitation validated this interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). while confocal microscopy confirmed mitochondrial co-localization of SLC25A3 with GPR162 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, Fig.\u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eD), establishing a structural foundation for GPR162's metabolic regulatory role.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven SLC25A3's established function in mitochondrial copper transport and metalloenzyme maturation[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], we investigated GPR162's impact on copper homeostasis. Total RNA was extracted from PC9 cells overexpressing GPR162, and RT-qPCR was used to measure the transcriptional levels of copper related genes. The results showed that the transcriptional level of SLC25A3 was significantly upregulated after GPR162 overexpression, and the expression of another classical copper transporter ATP7B also showed the same trend. Moreover, other genes related to copper ions, such as SCO2,MTF1,DLD,PDHA1, PDHB,LIPT1, showed statistical differences after GPR162 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eRecent studies have shown that a variety of copper ionophore drugs, such as Elesclomol (ES), Disulfiram and NSC319726, can cause cell death. This copin-induced cell death is a new type of death, which is different from other programmed cell death (such as apoptosis, pyroptosis, necrosis and ferroptosis). Therefore, it was defined as cuproptosis. Thus, we hypothesized that GPR162 interacts with SLC25A3 in mitochondria to promote cuproptosis in lung adenocarcinoma cells. To test this hypothesis, we first examined the IC50 of ES-Cu-induced cuproptosis in different lung adenocarcinoma cells. The results showed that the IC50 of PC9 and H1299 cells was 130.8 nM and 88.9 nM, respectively, while the IC-50 of A549 cells was 226.3 nM. This also suggests that A549 cells are not sensitive to ES-Cu-induced cuproptosis (Fig.\u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eG-I). Next, we examined the effect of GPR162 on ES-Cu-induced cuproptosis in PC9 cells, which were sensitive to cuproptosis, and in A549 cells, which were resistant to cuproptosis. The results showed that GPR162 promoted ES-Cu-induced cell death in PC9 cells, which was reversed by the copper chelator TTM. In contrast, GPR162 did not promote ES-Cu-induced cell death in elesclomole-resistant A549 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-H). Cuproptosis is mainly caused by copper ions directly binding to the lipoacylated proteins in the tricarboxylic acid cycle, which leads to the oligomerization and abnormal aggregation of lipoacylated proteins and the decrease of Fe-S cluster proteins[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Therefore, we also examined the oligomerization of the protein after GPR162 overexpression, which was shown to be more pronounced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Western blot was used to detect the levels of ester acylation proteins DLAT, DBT, DLST, ferredoxin 1 (FDX1), and lipoacylation protein LIAS, and the results showed that overexpression of GPR162 significantly increased the protein levels of GPR162, which was opposite after knockdown of GPR162 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ-L).\u003c/p\u003e \u003cp\u003eSpatial coordination was confirmed by GPR162-DLAT mitochondrial co-localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Fig.\u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eE-F) and direct GPR162-LIAS interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The transcriptional levels of DLAT, DBT, DLST, FDX1, and LIAS were detected by RT-qPCR, and the results showed that the transcriptional levels of fatty acylation protein and Fe-S promoting protein related genes were significantly down-regulated after GPR162 overexpression, which further confirmed the above conclusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have shown that GPR162 and SLC25A3 interact on the mitochondrial membrane and participate in the regulation of cuproptosis in lung adenocarcinoma cells. How GPR162 and SLC25A3 affect mitochondrial function is worthy of further exploration. Firstly, we examined the mitochondrial ROS levels in GPR162 overexpression cell lines after ES-Cu induction, and the results showed that the intracellular mitochondrial ROS levels were significantly upregulated in PC9 cells with GPR162 overexpression after ES-Cu induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). In contrast, the addition of ES-Cu to GPR162 knockdown H1299 cells induced cell death, and mitochondrial ROS decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). We next examined mitochondrial membrane potential levels by flow cytometry using JC-1 as a probe, which showed that they were upregulated after GPR162 knockdown and decreased after GPR162 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-I). In addition, we further examined ATP levels in lung adenocarcinoma cells after GPR162 overexpression and knockdown using an ATP assay kit. Consistent with the membrane potential level, knockdown of GPR162 promoted ATP release and overexpression of GPR162 reduced ATP production (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ-M). Furthermore, GPR162 promoted mitochondrial ROS production, inhibited mitochondrial membrane potential and ATP level, and promoted cuproptosis of lung adenocarcinoma cells. Since mitochondrial MMP and ATP alterations are also important events in the process of apoptosis, to further test whether GPR162 affects apoptosis, we examined the apoptosis status by flow cytometry in lung adenocarcinoma cells overexpressing GPR162 after treatment with the apoptosis-inducing agent DBT for 24 hours. The results showed that overexpression of GPR162 did not affect the level of apoptosis (Fig.\u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eA-D). It was further confirmed that GPR162 promoted apoptosis of lung adenocarcinoma cells by inhibiting mitochondrial membrane potential and ATP level, but did not affect apoptosis.\u003c/p\u003e \u003cp\u003eIn addition, we extracted subcutaneous inguinal adipose tissue from obese mice and cultured it in vitro, and then mouse lung adenocarcinoma cells LLC overexpressing GPR162 were co-cultured with primary adipocytes. Total RNA and total protein were extracted from tumor cells. GPR162 promoted the level of lipoacylated proteins, and the transcript levels of DLAT, DLST, FDX1, and LIAS were significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eO-Q). These results suggest that GPR162 promotes cuproptosis in obesity-related lung adenocarcinoma cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGPR162 drives the polarization of TAMs to M1 to enhance anti-tumor immunity.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eKEGG pathway analysis of GPR162-interacting proteins identified through mass spectrometry revealed significant enrichment in immune-related pathways, particularly in antigen processing/presentation and cytokine-cytokine receptor interaction (Fig.\u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eA). Integrated analysis of subcutaneous xenograft RNA-seq data demonstrated GPR162 overexpression-associated enrichment in immunomodulatory pathways, including T cell receptor signaling and chemokine-mediated inflammation, suggesting its role in reshaping the obesity-associated tumor immune microenvironment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C, S6B). GSEA further confirmed positive correlations between GPR162 expression and interferon-γ response, acute inflammatory response, and immune checkpoint activation (Fig.\u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eC, S7A). These results suggest that GPR162 is significantly associated with tumor immunity, which provides us with new clues to study GPR162 mediating tumor metabolism and affecting lung cancer immunity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo continue to explore GPR162 influencing immune mediated tumor metabolic mechanism, we first by TIMER2.0 online website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://timer.cistrome.org/\u003c/span\u003e\u003cspan address=\"http://timer.cistrome.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) analyzes the GPR162, expressed in all the immune cells in the The results showed that GPR162 was highly expressed in myeloid cells such as neutrophils, monocytes and macrophages (Fig.\u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eC). Next, we predicted the association of GPR162 with various immune cells in lung adenocarcinoma and lung squamous cell carcinoma, and the results showed that GPR162 was positively correlated with macrophages (Fig.\u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eD-E). Notably, single-cell RNA-seq clustering (GSE97168) showed exclusive GPR162 expression in tumor-associated macrophages (TAMs) within lung adenocarcinoma specimens (Fig. \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eD), suggesting myeloid-specific regulatory functions.\u003c/p\u003e \u003cp\u003eThe tumor tissues and spleens of normal and obese tumor-bearing mice were collected to detect the infiltration of immune cells by flow cytometry. The results showed that in the spleen, the infiltration of NK cells, NKT cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells, CD4\u003csup\u003e+\u003c/sup\u003e T cells, macrophages and DC did not differ significantly between the control and GPR162 overexpression groups in normal and obese mice (Fig.\u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003eA-F). However, the infiltration of monocytes and neutrophils was significantly reduced in the overexpression group, and the difference was more obvious in the obese mice, which suggested that more inflammatory factors were released in the spleen tissue and activated the inflammatory response, However, in the tumor tissues, neutrophil infiltration was more obvious in the overexpression group of normal and obese mice, suggesting that more inflammatory factors were infiltrated in the tumor tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-G). In addition, the infiltration of NK cells, NKT cells, CD4\u003csup\u003e+\u003c/sup\u003e T cells, monocytes and DC in the tumor tissues of normal and obese tumor-bearing mice was not significantly different between the control and GPR162 overexpression groups (Fig.\u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e G-L), The infiltration of macrophages in GPR162 overexpression and obesity groups was higher than that in control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-E). The infiltration of neutrophils in GPR162 overexpression and obesity groups was also higher than that in control group, and the infiltration of CD8\u003csup\u003e+\u003c/sup\u003e T cells was significantly increased in GPR162 overexpression groups of normal and obese mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). These results suggest that GPR162 is involved in the regulation of tumor immunity mainly through macrophage infiltration.\u003c/p\u003e \u003cp\u003eChemokines play a crucial role in regulating the infiltration of different immune cells into tumors; therefore, these molecules affect tumor immunity and the therapeutic outcome of patients. To determine whether GPR162 affects the induction of T cell function by macrophages, we used a transwell system. Jurkat T cells were co-cultured with GPR162-overexpressing lung adenocarcinoma PC9 cells or CD8\u003csup\u003e+\u003c/sup\u003e T cells from primary spleen tissues of C57BL/6 mice with GPR162-overexpressing LLC cells for 48 hours. The mRNA levels of chemokines CCL2, GZMB, TNFα, and TGFβ were detected by RT-qPCR. The results showed that the co-culture of GPR162 overexpressing tumor cells with T cells did not activate T cells, suggesting that GPR162 promotes anti-tumor immunity through macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI-M, Fig.\u003cspan refid=\"MOESM10\" class=\"InternalRef\"\u003eS10\u003c/span\u003eB-F). To verify the above conclusions, we first co-cultured BMDM with LLC cells overexpressing GPR162 for 48 hours, and then extracted CD8\u003csup\u003e+\u003c/sup\u003e T cells from the spleen of C57BL/6 mice were added to the upper chamber of the co-culture dish for further culture for 48 hours. CD8\u003csup\u003e+\u003c/sup\u003e T cells in the upper chamber were collected, and the function of CD8\u003csup\u003e+\u003c/sup\u003e T cells was detected by RT-qPCR. The results showed that overexpression of GPR162 significantly enhanced T cell function in this culture system (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eN-R). Subsequently, BMDM were co-cultured with the supernatant of LLC cells overexpressing GPR162 for 48 hours, and then the extracted CD8\u003csup\u003e+\u003c/sup\u003e T cells from the spleen of C57BL/6 mice were added to the upper chamber of the co-culture dish for further culture for 48 hours. CD8\u003csup\u003e+\u003c/sup\u003e T cells in the upper chamber were collected and the function of CD8\u003csup\u003e+\u003c/sup\u003e T cells was detected by RT-qPCR (Fig.\u003cspan refid=\"MOESM10\" class=\"InternalRef\"\u003eS10\u003c/span\u003eG-K). The results were consistent with that of co-culture with tumor cells. Co-culture of tumor cells and tumor cell supernatant with macrophages and then with CD8\u003csup\u003e+\u003c/sup\u003e T cells could activate anti-tumor immunity.\u003c/p\u003e \u003cp\u003eTo verify the association between GPR162 and macrophages, BMDM from macrophages derived from mouse bone marrow were extracted (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). In order to confirm the successful extraction of macrophages, we added LPS to induce macrophages to M1 polarization, IL4 and IL13 to induce macrophages to M2 polarization. We observed the morphology of M1 macrophages and M2 macrophages under light microscope, and found that there were significant differences in the morphology of M1 macrophages and M2 macrophages. M1 macrophages had a large irregular cell body, while M2 macrophages had a long spindle shape, smaller cell body and smoother cell surface than M1 macrophages (Fig.\u003cspan refid=\"MOESM9\" class=\"InternalRef\"\u003eS9\u003c/span\u003eA). Next, we detected polarization markers by RT-qPCR, and the results showed that the mRNA expression of pro-inflammatory factors CD86, IL12 and IL1β was significantly higher in M1 macrophages than in M0/M2 macrophages, while the expression of anti-inflammatory markers CD206, ARG1 and IL10 was significantly higher in M2 macrophages than in M0/M1 macrophages (Fig.\u003cspan refid=\"MOESM9\" class=\"InternalRef\"\u003eS9\u003c/span\u003eB-H), the polarization model was successfully constructed. Notably, in the co-culture system of GPR162 overexpressing LLC and BMDM, the expression of M1 markers was significantly up-regulated, while M2 markers showed no significant change (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-D, Fig.S11A-C). These results suggest that GPR162 in lung adenocarcinoma cells can promote the polarization of macrophages to M1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo clarify the effect of GPR162 on the regulation of tumor metabolic reprogramming on immune cell polarization, we added capric acid and lauric acid to LLC-GPR162 over-expressing cells and macrophages co-culture system, respectively. Flow cytometry results showed that compared with the control group, the GPR162 overexpression group had a significantly lower M2 polarization level, a significantly lower proportion of CD206\u003csup\u003e+\u003c/sup\u003e cells, and a significantly higher proportion of CD86\u003csup\u003e+\u003c/sup\u003e cells in the basal state, while lauric acid treatment had no significant effect (Fig.S11I, K). After statistical analysis of the results, the addition of decanoic acid and lauric acid in the control group promoted the polarization of macrophages to M1 macrophages. In the overexpression group, GPR162 promoted the infiltration of cells to M1 macrophages, but the addition of decanoic acid in the overexpression group reversed the polarization of GPR162 to M1 macrophages, and lauric acid had no statistically significant difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, Fig.S11J). These results suggest that decanoic acid may specifically antagonize its pro-M1 polarization effect by competitive inhibition of GPR162 mediated metabolic signaling pathway, suggesting that there is an interactive regulatory network between GPR162 and fatty acid metabolism.\u003c/p\u003e \u003cp\u003eThe mRNA levels of M1 markers IL6, NOS2, IL1β, TLR2 and TLR4, and M2 markers CD163, IL4 and IL13 were measured by RT-qPCR in lung adenocarcinoma cell lines treated with capric acid or lauric acid. Results Consistent with the above flow cytometry results, GPR162 overexpression significantly up-regulated the mRNA levels of M1 macrophage markers, while there was no significant difference in the mRNA levels of M2 macrophage markers. Moreover, treatment of cells with decanoic acid significantly reversed GPR162-promoted macrophage polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF-H, Fig.S11D-H). These results suggest that decanoic acid affects tumor immunity by regulating GPR162-promoted macrophage polarization to M1.\u003c/p\u003e \u003cp\u003eBased on previous studies, GPR162 mediates lipid metabolic reprogramming through the medium-chain fatty acid oxidation pathway (focusing on the regulation of decanoic acid mitochondrial β-oxidation), forms a functional complex with the mitochondrial copper transporter SLC25A3, and specifically induces cuproptosis in tumor cells by disrupting copper homeostasis. In this study, we further investigated the effect of this metabolic reprogramming on the tumor immune microenvironment. Primary mouse adipocytes and GPR162 overexpressed LLC lung adenocarcinoma cells were co-cultured in vitro for 48 hours. PPARγ agonist Troglitazone monotherapy group, cuproptosis inducer ES-Cu monotherapy group and combination treatment group were set up, respectively. The culture medium supernatant of the co-culture system was collected and co-cultured with BMDM, and the expression profile of macrophage polarization markers was detected by RT-qPCR. The results showed that: 1) GPR162 overexpression significantly promoted the polarization of BMDM to pro-inflammatory M1 phenotype, as indicated by significant up-regulation of CD80, CD86 and TLR4 mRNA levels; 2) In the single-agent intervention group, troglitazone and ES-Cu further increased the expression of M1 markers; 3) The combined treatment of the two drugs produced a synergistic effect, and the expression of CD80, CD86 and TLR4) reached the peak, while M2 markers (CD206 and ARG1) did not change significantly in all experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI-M). This series of evidence suggests that GPR162 drives the polarization of tumor-associated macrophages to anti-tumor M1 phenotype through a dual regulatory mechanism, activation of medium-chain fatty acid oxidative metabolism pathway and induction of cuproptosis pathway, and this process can achieve synergistic effect by targeting PPARγ signaling and copper homeostasis.\u003c/p\u003e \u003cp\u003eIn conclusion, this study delineates a dual inhibitory mechanism through which GPR162 suppresses obesity-associated lung adenocarcinoma progression. First, GPR162 orchestrates lipid metabolic reprogramming by activating medium-chain fatty acid β-oxidation (specifically targeting decanoic acid catabolism in mitochondria). Second, it forms a functional complex with mitochondrial copper transporter SLC25A3 to selectively induce tumor-selective cuproptosis via copper homeostasis disruption. Crucially, these two pathways exhibit synergistic effects: 1) driving tumor-associated macrophage (TAM) polarization toward anti-tumor M1 phenotype, 2) enhancing CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration and IFNγ secretion, and 3) collectively establishing an immune-hostile microenvironment that potently suppresses tumorigenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). These findings position GPR162 as a metabolic-immune nexus, these findings advocate combined targeting of lipid oxidation and copper death pathways for obesity-associated lung adenocarcinoma therapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHuman obesity has become a global health epidemic, with few safe and effective pharmacological therapies currently available. Previous research has revealed that GPCR agonist targets obesity and diabetes, and GPCR agonist G-1 decreases body weight, fat mass, and inflammation while increasing energy expenditure and improving glucose homeostasis in ovariectomized female mice[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. A distinct class T GPCR subfamily TAS2Rs, expressed in extraoral tissues represent potential drug targets for addressing conditions such as obesity, asthma, diabetes, and metabolic diseases[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. GPR3 is also a nonadrenergic activator of mouse and human thermogenic adipocytes, cold-induced lipolysis drives the expression of a constitutively active GPCR that regulates thermogenesis in mouse and human adipocytes independent of sympathetic or adrenergic inputs[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Nevertheless, there is a lack of studies examining the influence of GPCR on tumor growth in obese microenvironments, and whether GPR162 affects tumor growth in the obese environment needs to be investigated. Our study revealed that tumor growth was significantly higher in obese mice compared to the control group. Additionally, the expression of GPR162 was notably lower in the tumor tissue of obese mice than in the control group. This finding piqued our interest, suggesting that GPR162 may act as a protective factor against obesity-induced tumor growth.\u003c/p\u003e \u003cp\u003eGiven that GPR162 is a protein identified in the mitochondrial membrane structure, and that mitochondria play a crucial role in fatty acid oxidation (FAO) and metabolism, we are interested in investigating whether lipid metabolites regulate GPR162 within the mitochondria. In this study, we performed metabolomics sequencing of GPR162-overexpressing subcutaneous tumor tissues in obese mice. Through enrichment analysis, we found that medium-chain fatty acids played an important role in the regulation of GPR162. In addition, we found that decanoate synergistically upregulated the expression of several genes involved in fatty acid synthesis, oxidation, and catabolism. This suggests that GPR162 is both regulated by fatty acid metabolites and involved in the regulation of fatty acid metabolic processes. Our research demonstrates that GPR162 serves as a protective factor in obese settings by participating in the regulation of medium-chain fatty acid oxidation to impede the progression of lung adenocarcinoma.\u003c/p\u003e \u003cp\u003eMedium-chain fatty acids (MCFAs) refer to saturated fatty acids with 6\u0026ndash;12 carbon atoms, derived from medium-chain triglycerides (MCTs), which can be absorbed directly without digestion and transported to the liver via the portal vein for efficient metabolism and rapid energy supply[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Long-term high-fat diet intake plays a crucial role in the composition of the gut microbiome in animal models and human subjects and directly affects the production of MCFAs and host health[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Moreover, MCFAs can reduce and reverse metabolic syndrome caused by high-fat diets. Recently, MCTs have also been shown to have the ability to promote protein synthesis metabolism and inhibit catabolic metabolism[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. More importantly, these medium-chain fatty acids and their ketone metabolic products are all triggered by cell membrane receptors, which can reduce fat deposition, improve insulin resistance, and regulate glucose and lipid metabolism[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. There have been reports in the literature that the interaction between adipocytes and tumor fatty acid metabolism can promote the progression of cancer in obese individuals[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], therefore, a deeper understanding of GPR162's regulation of fatty acid metabolism could aid in the study of its impact on the progression of lung adenocarcinoma in obese mice.\u003c/p\u003e \u003cp\u003eAs one of the 53 transport proteins in the inner mitochondrial membrane, SLC25A3 can cause the accumulation of copper ions in the mitochondrial matrix and promote the maturation of the copper proenzymes cytochrome c oxidase and superoxide dismutase[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Several copper ion transporter drugs, such as Elesclomol, Disulfiram, and NSC319726, have been found to induce cell death through a mechanism known as cuproptosis[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. This form of cell death is distinct from other programmed cell deaths (such as apoptosis, pyroptosis, necroptosis, and ferroptosis). Our study has revealed that GPR162 enhances cellular sensitivity to copper ions through its interaction with SLC25A3. As the intracellular copper ion concentration increases, the expression level of cysteinylated proteins decreases and the level of Fe-S cluster proteins falls, leading to mitochondrial protein toxicity stress and ultimately resulting in cell death due to copper overload. These findings suggest a close relationship between GPR162 and mitochondrial homeostasis.\u003c/p\u003e \u003cp\u003eThe induction of cuproptosis, a recently identified form of copper-dependent immunogenic cell death, is a promising approach for antitumor therapy[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. In addition, a variety of recent studies have shown that cuproptosis can be induced in vitro to reprogram the tumor microenvironment and enhance the anti-tumor effect of immunotherapy[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. By reviewing and analyzing GPR162 interacting proteins, we found that a large number of GPR162-binding proteins (32 in total) were enriched in immune pathways. RNA sequencing and immune cell infiltration analysis showed that macrophages were the most infiltrated immune cells. Then, bioinformatics analysis of single-cell databases of various types of tumors showed that GPR162 was mainly expressed in macrophages in most tumor tissues and was significantly associated with immune stress and immune response pathways. Therefore, we hypothesized that GPR162 may reprogram the tumor microenvironment by inducing cuproptosis in tumor cells in an obese environment.\u003c/p\u003e \u003cp\u003eTumor microenvironment(TME) has a central role in the development of tumors, a variety of immune cell infiltration characteristics of groups in TME has been shown to predict the prognosis of patients with some solid tumors[\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Tumor-associated macrophages (TAMs) are the most abundant and important immune cells in the TME[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Based on molecular phenotype and functional characteristics, activated macrophages can be divided into two major classes: M1 macrophages (classical activation type) and M2 macrophages (alternative activation type). There are significant differences in gene expression patterns and regulation between the two. M1 macrophages mainly promote Th1 immune responses by phagocytosis of pathogens, presentation of antigens, production of IL-1β, TNFα, IL6, IL2, CCL2, and CCL3 cytokines, and killing tumor cells[\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. M2 macrophages lack cell-killing activity but can produce EGF, MMP, IL-10, TGFβ, and VEGF cytokines to promote Th1 immune responses, induce immune suppression, and mediate tumorigenesis and development, thereby playing a pro-tumor role[\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Studies have shown that M1 and M2 macrophages can mutually transform due to changes in the tissue microenvironment. Targeting M2 macrophages and consuming them or converting them back to M1 macrophages in the TME will be a potential strategy for tumor immunotherapy by indirectly stimulating cytotoxic T cells or directly enhancing their phagocytic ability to eliminate tumor cells[\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Therefore, deeply studying the key target molecules that regulate TAM function and targeting the polarization of TAMs to convert M2 macrophages to M1 macrophages, which are beneficial for tumor immunotherapy, has become a frontier and hotspot in current tumor research.\u003c/p\u003e \u003cp\u003eOur study reveals that GPR162 functions as a critical tumor suppressor in obesity-associated lung adenocarcinoma through dual metabolic and immunologic mechanisms. We demonstrate that GPR162 simultaneously orchestrates tumor microenvironment reprogramming by enhancing medium-chain fatty acid oxidation and induces mitochondrial dysfunction through SLC25A3-mediated cuproptosis. Furthermore, GPR162 activation promotes anti-tumor immunity by driving M1-like macrophage polarization. These findings provide novel insights into the metabolic-immune axis in cancer progression and establish GPR162 as a promising therapeutic target for treating obesity-associated malignancies, offering new opportunities for developing targeted therapies that simultaneously address metabolic dysregulation and immune suppression in lung adenocarcinoma.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe ethics committee of the Cancer Research Institute of Central South University has approved this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests. The authors declare no conflict of interest. This manuscript has been read and approved by all authors and is not under consideration for publication elsewhere.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthor contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eConception and design: Y. Tao, S. Liu, D. Xiao\u003c/p\u003e\n\u003cp\u003eDevelopment of the methodology:\u0026nbsp;Y. Long, Y. Tao, S. Liu\u003c/p\u003e\n\u003cp\u003eAcquisition of the data: Y. Long, D. Xiao\u003c/p\u003e\n\u003cp\u003eAnalysis and interpretation of the data (e.g., statistical analysis, biostatistics, computational analysis):\u0026nbsp;Y. Long, Y. Tao, S. Liu, D. Xiao\u003c/p\u003e\n\u003cp\u003eWriting, review, and/or revision of the manuscript:\u0026nbsp;Y. Long, Y. Tao\u003c/p\u003e\n\u003cp\u003eStudy supervision: Y. Tao\u003c/p\u003e\n\u003cp\u003eDisclosure of Potential Conflicts of Interest\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, Jemal A: Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. \u003cem\u003eCA Cancer J Clin\u003c/em\u003e 2024, 74(3):229\u0026ndash;263.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQiu H, Cao S, Xu R: Cancer incidence, mortality, and burden in China: a time-trend analysis and comparison with the United States and United Kingdom based on the global epidemiological data released in 2020. \u003cem\u003eCancer Commun (Lond)\u003c/em\u003e 2021, 41(10):1037\u0026ndash;1048.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiao D, Zhao J, Han Y, Zhou J, Li X, Zhang T, Li W, 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11(3):1016\u0026ndash;1030.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8754789/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8754789/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eObesity, a well-established risk factor for cancer development and progression, exerts its oncogenic effects primarily through metabolic reprogramming, particularly in lipid and amino acid metabolism. Despite its clinical significance, the molecular mechanisms linking obesity-induced metabolic alterations to lung adenocarcinoma progression remain elusive. In this study, we identify G protein-coupled receptor 162 (GPR162) as a pivotal metabolic-immune regulator in obesity-associated lung adenocarcinoma. Our findings reveal that GPR162 orchestrates tumor suppression through two distinct yet interconnected mechanisms: (1) GPR162 induces cell peroxidation by promoting the oxidation of medium-chain fatty acids such as decanoic acid and lauric acid. (2) GPR162 interacts with copper transporter SLC25A3 to form a functional complex in the mitochondria, driving copper influx and further promoting intracellular lipid peroxidation to induce cuproptosis, which directly kills tumor cells. The synergistic effect of fatty acid oxidation and cuproptosis not only reprogram the tumor metabolic microenvironment, but also induce the polarization of tumor-associated macrophages to M1 type, enhance CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration and IFNγ secretion, and finally construct an immune microenvironment that inhibits tumor progression. This study provides a new target and theoretical basis for combined metabolic-immune treatment of obesity-related lung cancer.\u003c/p\u003e","manuscriptTitle":"GPR162 Drives Obesity-Associated Lung Adenocarcinoma Suppression via Fatty Acid Oxidation-Induced Cuproptosis and Metabolic-Immune Reprogramming","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-27 11:11:22","doi":"10.21203/rs.3.rs-8754789/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-04-14T05:27:03+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-03-26T09:43:26+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-03-26T07:37:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-02T23:58:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2026-02-02T10:35:05+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2026-02-02T01:51:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-01T08:23:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"674485a1-51d6-45e4-bfad-cee90205fbac","owner":[],"postedDate":"March 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":62195458,"name":"Biological sciences/Cell biology/Cell death"},{"id":62195459,"name":"Biological sciences/Cancer/Cancer microenvironment"}],"tags":[],"updatedAt":"2026-03-27T11:11:23+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-27 11:11:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8754789","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8754789","identity":"rs-8754789","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}