NINJ1 promotes lung adenocarcinoma progression via activation of the PI3K-AKT signaling axis and suppression of cell death pathways

NINJ1 promotes lung adenocarcinoma progression via activation of the PI3K-AKT signaling axis and suppression of cell death pathways | 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 NINJ1 promotes lung adenocarcinoma progression via activation of the PI3K-AKT signaling axis and suppression of cell death pathways Jixi Li, Xuehe Liu, Zihan Qi, Yajie Shen, Yanjie Zhang, Feiyan Xie, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6906860/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Lung adenocarcinoma (LUAD) remains a leading cause of cancer-related mortality, and current therapeutic options often fail to yield sustained responses. Ninjurin1 (NINJ1), a transmembrane protein implicated in plasma membrane rupture during lytic cell death, has recently emerged as a potential antitumor target, yet its role in LUAD pathogenesis remains poorly defined. Here, we comprehensively investigated the functional relevance and mechanistic regulation of NINJ1 in LUAD progression. We observed elevated NINJ1 expression in human LUAD tissues and cell lines. Genetic ablation of NINJ1 or pharmacological inhibition using NINJ1-derived short peptides significantly impaired tumor cell proliferation and migration, enhanced sensitivity to chemotherapeutic agents, and suppressed the growth of human iPSC-derived LUAD organoids and xenografted tumors in vivo . Mechanistically, NINJ1 was found to directly associate with PI3K, thereby activating the downstream AKT signaling pathway. Loss of NINJ1 led to widespread transcriptional dysregulation, affecting gene networks involved in cell cycle progression, epithelial-mesenchymal transition (EMT), and inflammatory responses. Functionally, NINJ1 deficiency induced G1 phase arrest, mitochondrial dysfunction, and increased susceptibility to cell death. These findings identify NINJ1 as a key molecular driver of LUAD malignancy and highlight its potential as a promising therapeutic target for lung cancer. Biological sciences/Cell biology/Cell death/Necroptosis Biological sciences/Cancer/Lung cancer/Non-small-cell lung cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Lung adenocarcinoma (LUAD) is the most prevalent subtype of non-small cell lung cancer and a major contributor to cancer-related mortality worldwide. Despite progress in surgical resection, chemotherapy, and targeted therapies, the overall five-year survival rate for LUAD remains low, primarily because of late-stage diagnosis and therapeutic resistance [ 1 ]. These clinical limitations highlight the urgent need to elucidate novel molecular mechanisms that drive LUAD progression and identify possible therapeutic targets to improve patient outcomes [ 2 , 3 ]. Ninjurin1 (NINJ1) is a conserved transmembrane protein that was initially identified as an adhesion molecule that is upregulated following nerve injury [ 4 , 5 ]. Recent studies have expanded its relevance to cancer biology, particularly through its role in regulating plasma membrane rupture (PMR) during various forms of programmed cell death (PCD), including apoptosis, pyroptosis, and necroptosis [ 4 , 6 ]. NINJ1 facilitates the release of intracellular components, such as lactate dehydrogenase (LDH) and damage-associated molecular patterns (DAMPs), which amplify inflammatory responses and may influence tumor progression [ 7 – 11 ]. Although NINJ1 does not directly initiate cell death, its function in mediating membrane rupture suggests its central role in modulating cell fate and immune activation [ 4 , 5 , 12 – 15 ]. Emerging evidence implicates NINJ1 in the regulation of tumorigenesis and inflammatory signaling [ 15 , 16 ]. Its overexpression has been associated with increased lung tumor burden in mice [ 17 – 19 ], whereas genetic deletion attenuates disease severity in models of pulmonary fibrosis and multiple sclerosis. Moreover, NINJ1 has been linked to ferroptosis [ 20 ], an iron-dependent form of cell death implicated in cancer invasion and metastasis. Despite these observations, the precise role of NINJ1 in LUAD remains unclear. In the present study, we comprehensively examined the functional significance of NINJ1 in LUAD. We demonstrated that NINJ1 deficiency altered key transcriptional programs associated with cell cycle progression, DNA replication, EMT, and inflammatory responses. Furthermore, both genetic ablation and pharmacological intervention using NINJ1-derived short peptides suppress tumor cell proliferation and migration, enhanced chemosensitivity, and impaired the growth of iPSC-derived organoids and tumors in vivo . These findings reveal that NINJ1 is a previously underappreciated regulator of LUAD pathogenesis and support its potential as a novel therapeutic target for lung cancer. Results NINJ1 is Highly Expressed in Human Lung Tumor Tissues To investigate the clinical relevance of NINJ1 in lung cancer, we analyzed its expression using the UALCAN cancer database, which integrates TCGA (The Cancer Genome Atlas (RRID:SCR_003193)) and CPTAC datasets (CPTAC (RRID:SCR_017135)) [21]. NINJ1 was significantly upregulated in lung adenocarcinoma (LUAD) at both the transcript and protein levels across disease stages (Fig. S1). Analysis of the CPTAC proteomic data confirmed higher NINJ1 protein levels in LUAD tissues than in normal tissues. Promoter methylation levels of NINJ1 are also reduced in tumor samples, suggesting transcriptional supression [14, 17, 22, 23]. To validate these findings, we performed immunohistochemical (IHC) analysis of 60 human lung cancer specimens. NINJ1 staining intensity was markedly elevated in LUAD, lung squamous cell carcinoma (LUSC), adenosquamous carcinoma (ASCC), and pulmonary sarcomatoid carcinoma (PSC) compared to adjacent normal tissues (Fig. 1A-B). These results support a role for NINJ1 in non-small cell lung cancer (NSCLC) progression. NINJ1 Deficiency Inhibits Proliferation and Migration of Lung Adenocarcinoma Cells Consistent with the patient data, we observed high NINJ1 expression in multiple lung cancer cell lines (Fig. S2A-B). To determine the functional role of NINJ1 in LUAD progression, we generated NINJ1 knockout A549 and H1299 cell lines by CRISPR-Cas9 and lentiviral transduction (Fig. S2C-F). Next, the proliferation and tumorigenicity of wild-type (WT) and NINJ1 knockout (KO) A549 and H1299 cells were detected. CCK-8 assays revealed that NINJ1 knockout significantly suppressed cell growth over 4 days compared to wild-type controls (Fig. 1C-D). Colony formation assays further showed a marked reduction in both colony number and size in NINJ1-deficient cells, indicating impaired tumorigenic potential (Fig. 1E-F). In addition, NINJ1-deficient A549 and H1299 cells exhibited a significant reduction in migratory capacity compared to their wild-type counterparts using wound healing assays (Fig. 1G-J). Together, these data suggest that NINJ1 promotes LUAD cell proliferation and migration, and its loss impairs tumor cell aggressiveness. NINJ1 Deficiency Enhances the Cytotoxic Effects of Chemotherapy in Lung Adenocarcinoma Cells and in the xenograft mouse model Cisplatin and its derivatives, including carboplatin and oxaliplatin, exert anticancer effects by disrupting DNA replication and repair, leading to impaired tumor cell proliferation [24]. Although effective across various cancer types, their clinical utility is limited by toxicity and severe side effects [25]. Given the critical role of NINJ1 in cell death, we explored whether targeting NINJ1 could enhance the therapeutic response to cisplatin and gefitinib in lung adenocarcinoma cells. Both cisplatin and gefitinib exhibited dose-dependent cytotoxicity in A549 and H1299 cells (Fig. 2A, 2C and 3E). Notably, NINJ1 knockout significantly increased sensitivity to these agents, as shown by decreased cell viability and enhanced membrane rupture (Fig. 2A-F). LDH release assays confirmed that drug-induced plasma membrane rupture was reduced in NINJ1-deficient cells, suggesting that NINJ1 contributed to chemotherapy-induced lytic cell death (Fig. 2D and 2F). Next, we assessed the role of NINJ1 in tumor growth in vivo using a xenograft model. BALB/c-nude mice (n = 28) were randomly divided into two groups (n = 14 per group) and injected subcutaneously with either WT or NINJ1-deficient A549 cells in both flanks (Fig. 2G). Tumor formation was observed on day 4 post injection, confirming successful engraftment. To evaluate the effect of chemotherapy in combination with NINJ1 depletion, the tumor-bearing mice were further subdivided into four groups (n = 7 per group). The two groups received intraperitoneal injections of cisplatin (5 mg/kg) starting on day 2 after the tumors were established. Over a 23-day observation period, we found that tumors derived from NINJ1-deficient A549 cells were significantly smaller than those derived from wild-type cells, indicating that NINJ1 loss suppresses LUAD tumor growth in vivo (Fig. 2H–L). Cisplatin treatment further inhibited tumor growth in wild-type A549 xenografts, whereas its effect on NINJ1-deficient tumors was less pronounced, suggesting that NINJ1 loss alone may confer strong antitumor activity. Importantly, body weight remained stable across all groups and no overt signs of systemic toxicity were observed (Fig. 2M), indicating that the combination treatment was well tolerated. These findings highlight the therapeutic potential of NINJ1 in lung adenocarcinoma. NINJ1-derived Short Peptides Inhibit Lung Tumor in the Xenograft Mouse Model To further inhibit NINJ1-mediated membrane rupture, we designed two NINJ1-derived short peptides (named Peptides 1 and 2) targeting the extracellular N-terminal domain based on the AlphaFold3-predicted full-length NINJ1 protein structure (Fig. 3A-B). These fluorophore (green) NBD-tagged peptides competitively bind to the NINJ1 N-terminus, preventing functional oligomerization at the cell surface, as evidenced by confocal laser scanning microscopy (CLSM) (Fig. S3A) [26]. Immunofluorescence observations in HEK293T cells revealed that the peptides were localized to the plasma membrane and co-distributed with NINJ1-mCherry (Fig. 3C). Treatment with these peptides maintained low LDH release during drug exposure and enhanced chemotherapy-induced cytotoxicity in A549 and H1299 cells (Fig. 3D-G). To evaluate the therapeutic potential of the NINJ1-derived short peptides, we assessed their effect on lung adenocarcinoma cell viability under chemotherapeutic stress using CCK-8 assays. Treatment with either peptide 1 or peptide 2 significantly reduced the viability of A549 and H1299 cells, particularly in the presence of gefitinib (Fig. 3D and 3F). Concurrently, LDH release assays demonstrated that the peptides preserved the plasma membrane integrity (Fig. 3E and 3G), indicating a dual effect of inhibiting proliferation while preventing membrane rupture-associated cytotoxicity. Next, we established subcutaneous xenografts in nude mice by using A549 cells. Following tumor establishment, the mice were randomized into six treatment groups: PBS, Peptide 1, Peptide 2, Gefitinib, Peptide 1 + Gefitinib, and Peptide 2 + gefitinib (Fig. 3H). Monotherapy with either the NINJ1-derived peptide or gefitinib significantly suppressed tumor growth compared to the PBS control (Fig. 3I-J). Notably, tumors treated with peptides alone exhibited slower growth rates than those treated with gefitinib alone. Importantly, combination therapy with NINJ1-derived peptides and gefitinib produced a synergistic antitumor effect, resulting in markedly enhanced tumor inhibition compared with either treatment alone (Fig. 3K-L). These findings confirm that NINJ1-derived short peptides suppress lung adenocarcinoma cell proliferation both in vitro and in vivo , providing a promising adjunct strategy to improve the therapeutic efficacy in LUAD. NINJ1 Deficiency Promotes Programmed Cell Death in Lung Adenocarcinoma Cells The inhibition of tumor cell proliferation and tumorigenesis observed in NINJ1-deficient A549 cells may be partially attributed to increased programmed cell death and cell cycle arrest [27-29]. Apoptosis and other regulated death pathways are frequently suppressed in cancer, allowing for unrestrained cell growth and therapy resistance [30, 31]. Given NINJ1’s established role in mediating plasma membrane rupture (PMR) and inflammatory signaling via DAMP release, we hypothesized that its loss may alter cell death dynamics in LUAD. CCK-8 and LDH release assays revealed that NINJ1 deficiency significantly increased cytotoxicity and reduced proliferation of A549 and H1299 cells (Fig. 4A-B). Flow cytometry confirmed a modest increase in early and late apoptosis in the NINJ1-deficient A549 cells (Fig. 4B). Transmission electron microscopy (TEM) showed dramatic mitochondrial damage in NINJ1-deficient A549 cells, characterized by decreased mitochondrial length and cristae disruption, the effects of which were partially reversed by NINJ1 restoration (Fig. 4C-E). These findings suggest that NINJ1 may maintain mitochondrial integrity, contributing to its role in tumor cell survival. We further assessed the impact of NINJ1 on cell death, including apoptosis, pyroptosis, and necrosis. Apoptotic stimuli led to a greater loss of viability and increased Caspase-3 cleavage in NINJ1-deficient cells, whereas pyroptosis-induced membrane rupture was significantly impaired despite enhanced cell death (Fig. S4A-F). Notably, Western blot analysis revealed that cisplatin treatment induced GSDMD cleavage in wild-type A549 cells, but this effect was attenuated in NINJ1-deficient cells, suggesting the suppression of pyroptosis (Fig. 4F). In contrast, apoptosis was exacerbated, with increased cleaved Caspase-3 (p17 fragment) and reduced full-length Caspase-3 and upstream effectors CASP9 and Cytochrome C in NINJ1-deficient cells (Fig. 4G-J). These data indicate that NINJ1 loss reprograms the cell death landscape, enhancing apoptotic responses while impairing membrane rupture, thereby sensitizing LUAD cells to chemotherapeutic stress. Transcriptomic Profiling Reveals NINJ1 Regulates Cell Cycle, EMT, and PI3K/AKT Signaling in LUAD To elucidate the molecular functions of NINJ1, we performed a transcriptomic analysis in A549 lung adenocarcinoma cells following NINJ1 knockout. Compared with wild-type cells, NINJ1-deficient cells showed 598 upregulated and 1,088 downregulated genes. Gene Ontology (GO) and KEGG enrichment analyses identified pathways altered by NINJ1 loss, including extracellular matrix (ECM) organization; cellular response to interleukin-1; and valine, leucine, and isoleucine biosynthesis (Fig. 5A-C). These pathways are critical for tumor progression as they regulate structural integrity, inflammatory signaling, and amino acid metabolism, which are key processes in cancer cell growth and survival. RNA-seq results were validated by qPCR and western blotting. NINJ1 deletion downregulated the WNT5A-PI3K/AKT signaling pathway at both transcript and protein levels (Fig. 5D and K-L). Co-immunoprecipitation confirmed the interaction between NINJ1 and PI3K (Fig. 5E). Further analysis using TCGA data showed that NINJ1 expression was positively correlated with the PI3K regulatory subunit genes PIK3R1, PIK3R2, and PIK3R3 in LUAD patients (Fig. 5F-G). Additionally, using patient-derived organoids, we found that NINJ1-derived short peptides significantly inhibited organoid growth and viability, further supporting NINJ1’s role in LUAD tumor maintenance (Fig. 5H-J). Consistent with previous phenotypic findings, NINJ1 knockout induced G1 phase cell cycle arrest and downregulated Cyclin D1 expression while suppressing pro-proliferative genes (KRT19 and CES1) and upregulating tumor suppressors (ANGPTL4) (Fig. S3B). NINJ1 deficiency also inhibited EMT, as evidenced by increased E-cadherin and decreased N-Cadherin and Vimentin expression, thus providing a mechanistic basis for reduced cell migration (Fig. S3B). Collectively, these results indicate that NINJ1 modulates cell proliferation, apoptosis, EMT, and drug sensitivity by regulating key transcriptional networks and oncogenic signaling pathways (Fig. 5M). Discussion The intricate roles and mechanisms of NINJ1 in LUAD have attracted considerable interest, and our experimental findings provide a comprehensive view of its multifaceted functions in this malignancy [ 2 , 3 ]. Our study provides compelling evidence that NINJ1 functions as a critical regulator of lung adenocarcinoma (LUAD) progression. Elevated expression of NINJ1 was observed in LUAD tissues and cell lines, and was associated with aggressive tumor features and poor prognosis (Fig. 1 ). In addition, NINJ1-deficient cells exhibited reduced colony formation, migration, and tumor growth in xenograft models (Fig. 1 – 2 ). Moreover, treatment with NINJ1-derived short peptides suppressed proliferation and enhanced chemosensitivity, suggesting their therapeutic potential (Fig. 3 ). Mechanistically, NINJ1 regulates plasma membrane rupture (PMR) during programmed cell death. Its deficiency amplified apoptotic signaling while attenuating pyroptosis-associated membrane damage, highlighting its dual role in controlling tumor cell fate under stress (Fig. 4 ). Through transcriptomic and functional analyses, we demonstrated that NINJ1 deficiency alters key gene programs involved in cell cycle progression, EMT, and inflammatory signaling and identified that NINJ1 modulates the WNT5A-PI3K/AKT pathway (Fig. 5 ). Disruption of this signaling cascade in NINJ1-deficient cells likely contributed to the observed antitumor effects (Fig. 5 M). Both in vitro and in vivo studies demonstrated that loss of NINJ1 significantly impaired LUAD tumorigenicity, primarily through suppression of the WNT5A-PI3K/AKT signaling axis (Fig. 1 – 5 ). This finding contrasts with previous reports suggesting that NINJ1 functions as a metastasis suppressor by inhibiting the IL-6/STAT3 pathway and reducing ICAM-1 expression [ 22 ]. Other studies have proposed a pro-tumorigenic role for NINJ1, implicating it in the maintenance of cancer stem cells (CSCs), activation of canonical Wnt/β-catenin signaling, and regulation of inflammation and programmed cell death [ 17 ]. Additionally, NINJ1 has been shown to interact with tumor suppressor networks involving p53 [ 14 ], underscoring its context-dependent functions. Although previous studies have reported that glycine and a 12-residue peptide can disrupt NINJ1 oligomerization at the membrane [ 32 ], our data revealed that NINJ1-derived peptides specifically bind to NINJ1, inhibit its oligomerization during plasma membrane rupture (PMR), and sensitize LUAD cells to chemotherapeutic agents, resulting in a significantly reduced tumor burden in vivo (Fig. 3 ). Given its involvement in multiple oncogenic and immune signaling pathways, NINJ1 has emerged as a multifaceted regulator of LUAD. Future studies should investigate how the tumor microenvironment and inflammatory cues influence NINJ1 expression and activity, with the goal of developing NINJ1-targeted strategies for precise lung cancer therapy. Materials and methods Reagents and antibodies Reagents: cisplatin (HY-17394, MedChemExpress), gefitinib (HY-50895, MedChemExpress), human TNF-alpha (HY-P7058, MedChemExpress), nigericin sodium salt (HY-100381, MedChemExpress), Z-VAD-FMK (HY-16658B, MedChemExpress), lipopolysaccharides (HY-D1056, MedChemExpress), SM-164 (HY-15989, MedChemExpress). Antibodies: NINJ1 (Santa Cruz Biotechnology Cat# sc-136295, RRID: AB_10607827), PI3K (Proteintech Cat# 60225-1-Ig, RRID: AB_11042594), p-PI3K(Affinity Biosciences Cat# AF3241, RRID: AB_2834667), AKT(Proteintech Cat# 10176-2-AP, RRID: AB_2224574), p-AKT (ser473) (Proteintech Cat# 66444-1-Ig, RRID: AB_2782958), WNT5A (Proteintech Cat# 55184-1-AP, RRID: AB_2881285), CD44 (Bioss Cat# bsm-54767R, RRID: AB_3696666), Apaf-1(Huabio Cat# ET1607-12, RRID: AB_3069751), Caspase-3 (Huabio Cat# ET1608-64, RRID: AB_3069820), Caspase-8 (Huabio Cat# HA722181, RRID: AB_3675886), XIAP (Huabio Cat# HA722113, RRID: AB_3696667), Bax(Huabio Cat# ET1603-34, RRID: AB_3069679), Caspase-9 (Huabio Cat# R1308-12, RRID: AB_3073230), Cytochrome C (Huabio Cat# ET1610-60, RRID: AB_2940742), GSDME (Abcam Cat# ab215191, RRID: AB_2737000), GSDMD (Abcam Cat# ab210070, RRID: AB_2893325), GSDMD-CTD (Abcam Cat# ab215203, RRID: AB_2916166), FZD2 (Affinity Biosciences Cat# AF5282, RRID: AB_2837768). Anti-β-actin (Proteintech Cat# 66009-1-Ig, RRID: AB_2687938), Anti-Mouse IgG (H+L) (Proteintech Cat# SA00001-1, RRID: AB_2722565), and Anti-Rabbit IgG (H+L)(Proteintech Cat# SA00001-2, RRID: AB_2722564). Cell lines and culture The human non–small cell lung cancer (NSCLC) cell line A549 was obtained from the Cell Bank of the Chinese Academy of Sciences (RRID: CVCL_0023). H1299 and OE19 cells were generously gifted by Dr. Hua Gao (Tongji University, China) and Dr. Feng Shao (National Institute of Biological Sciences, China), respectively. HEK293T, HeLa, THP-1, HUVEC, MEF, HT-29, and HepG2 cells were maintained in our laboratory. A549, OE19, HEK293T, HeLa, MEF, HT-29, and HepG2 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM-HG; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 100 U/mL penicillin with 0.1 mg/mL streptomycin (P/S; C0222, Beyotime Biotechnology). H1299, THP-1, and HUVEC were maintained in RPMI-1640 medium (Gibco) supplemented with 10% FBS and P/S. All the cell lines were routinely tested and confirmed to be free of mycoplasma contamination. Primary mouse cell isolation and culture Primary peritoneal macrophages were isolated from 6–8-week-old C57BL/6 mice by flushing the peritoneal cavity with sterile PBS. Following red blood cell lysis, the cells were cultured in high-glucose DMEM (DMEM-HG) supplemented with 10% fetal bovine serum (FBS; Gibco) and 100 U/mL penicillin with 0.1 mg/mL streptomycin (P/S; Beyotime) for 24 h. The cell viability was consistently greater than 95%. To isolate primary alveolar cells, the tracheas of 6- to 8-week-old C57BL/6 mice were cannulated, and the lungs were lavaged with sterile PBS to collect alveolar cells. After red blood cell lysis, cells were cultured in DMEM-HG containing 10% FBS and P/S for 24 h. The average cell viability was found to be 97%. Mice Mice were raised in an animal facility at Fudan University under specific pathogen-free (SPF) conditions. All animal experiments were approved by the Institutional Animal Care and Use Committee of Fudan University. BALB/c nude (RRID: IMSR_RJ:BALB-C-NUDE)and C57BL/6 (RRID: MGI:7786639) mice were purchased from Cyagen Biosciences (Suzhou, China). A549 cells were xenografted into 5‐ to 7‐week‐old male and female BALB/c nude mice. The ratio of male-to-female mice was approximately 1:1. Immunofluorescence and confocal microscopy HeLa cells were seeded onto laser confocal dishes and pretreated with lipopolysaccharide (LPS) and TNF-α for 2 h. Cells were treated with various concentrations of pharmacological agents, including Nigericin, SM164, Z-VAD, and cycloheximide (CHX), for 4-6 h. Following treatment, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 10-15 min, and washed again. The cells were then incubated in immunostaining blocking buffer at room temperature for 1-3 h. After blocking, the cells were incubated with primary antibodies (Abcam, UK), washed three times with PBS, and then incubated with the appropriate secondary antibodies. Nuclei were counterstained with DAPI and the samples were mounted with an anti-fade mounting medium. Fluorescent images were acquired using a laser scanning confocal microscope (Zeiss LSM 880 with Airyscan Confocal Laser Scanning Microscope (RRID: SCR_020925)). Analysis of protein expression The cells were lysed in RIPA buffer (P0013B, Beyotime) supplemented with 1 mM PMSF (Phenylmethanesulfonyl fluoride; ST507, Beyotime). Protein concentrations were measured using an Enhanced BCA Protein Assay Kit (P0010, Beyotime). Analysis of mRNA expression Total RNA was extracted using a reagent kit (DP430, TIANGEN), and reverse transcription was performed using a HiScript III All-in-one RT SuperMix Perfect for qPCR kit (R333-01, Vazyme). The total amount of cDNA used for one reaction corresponded to approximately 100 ng of starting RNA. qPCR was performed using 2 × Taq Pro Universal SYBR qPCR Master Mix (Q712-02, Vazyme). All quantities were standardized to endogenous β-actin levels. The experiments were performed using an ABI Life Fluorescence Quantitative PCR instrument Q7. The primers used are listed in Table S1. Cell counting kit-8 assay A549, H1299, and HeLa cells (3 × 10 3 cells/well) were seeded in 96‐well plates and cultured for 24 h. Cell Counting Kit-8 solution was added at the indicated time points to each well and incubated for 2 h at 37°C (C0038; Beyotime Biotechnology). The absorbance was measured at 450 nm using a fluorescence microplate reader (Molecular Devices SpectraMax M5 Multimode Plate Reader (RRID: SCR_020300)). Lactate dehydrogenase (LDH) release assay A549, H1299, and HeLa cells were seeded in 96-well plates at a density of 3 × 10³ cells/well and cultured for 24 h. At the indicated time points, cytotoxicity was assessed using a lactate dehydrogenase (LDH) assay kit (C0016; Beyotime Biotechnology) according to the manufacturer’s instructions. Absorbance was measured at 490 and 600 nm using a fluorescence microplate reader (Molecular Devices SpectraMax M5 Multimode Plate Reader (RRID: SCR_020300)). Cell migration assay Cell migration was assessed using a wound healing assay. Cells were collected, counted, and resuspended at a density of 1 × 10 cells/mL. A total of 500 μL of cell suspension was seeded into each well of a 6-well culture plate, which had been marked with horizontal reference lines spaced 0.5 cm apart on the back. The cells were incubated overnight to allow monolayer formation and to achieve 100% confluence. Three parallel wells were used in each experiment. The following day, a linear scratch was made perpendicular to the marked lines using a sterile pipette tip. Detached cells were removed by washing the wells thrice with PBS, and the medium was replaced with serum-reduced culture medium (1% FBS). Cell migration into the wound area was monitored by capturing images of the same marked area at 0, 24, and 48 h, using an inverted microscope. The wound area was quantified using image analysis software, and the relative migration was calculated. All experiments were independently repeated thrice. Transmission electron microscopy (TEM) Mitochondrial morphology of lung adenocarcinoma cells was examined by transmission electron microscopy (TEM). Cells were fixed in 1% osmium tetroxide (OsO₄) in 0.1 M phosphate buffer (pH 7.4) for 2 hours at room temperature in the dark. After fixation, the samples were dehydrated using a graded ethanol series and embedded in epoxy resin. Ultrathin sections (60-80 nm) were cut using an ultramicrotome (Leica EM UC7 ultramicrotome (RRID: SCR_016694)) and mounted onto 150-mesh copper grids coated with a Formvar film. The sections were stained with 2% uranyl acetate, followed by 2.6% lead citrate, and imaged using a transmission electron microscope (Hitachi HT7700 Transmission Electron Microscope (RRID: SCR_020022)). Peptides The following peptides were synthesized by GL Biochem (Shanghai) Ltd.: Peptide 1 (PARWGWRHGPIN), Control Peptide 1 (PWAPRRHGNGWI), Peptide 2 (RWGWR), and Control Peptide 2 (WGRRW). PARWGWRHGPIN-NBD and RWGWR-NBD peptides were synthesized according to methods described in a previous study [26]. Unless otherwise specified, all peptides were used at a final concentration of 50 μM. Statistical analyses Data analyses and figure plotting were performed using GraphPad Prism software (version 9) or R statistical environment. The numbers of replicates and independent experiments are listed in the figure legends. Data are presented as the mean ± SD unless stated otherwise. A P-value of < 0.05 was considered to indicate statistical significance for all analyses. Statistical significance was determined using a two-tailed Student’s t-test, Mann-Whitney test, one-way analysis of variance (ANOVA), or Brown-Forsythe and Welch ANOVA tests. No statistical methods were used for the predetermined sample sizes, and the experiments were not randomized. During the experiments and outcome assessments, the investigators were not blinded to allocation. Declarations Declaration of Competing Interest The authors declare that they have no conflicts of interest. Funding Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (32161160323) and Shanghai Committee of Science and Technology (24490713600). Authorship Contributions J.L. conceived of and designed the study. X.L., Z.Q., Y.S., Y.Z., F.X., Y.X., Y.L., W.Y., Q.D., B.Z., and J.D. performed the experiments and analyzed the data. X.L. and J.L. analyzed the data and wrote the manuscript. All the authors discussed the results and commented on the manuscript. Ethics Statement All animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals with the approval of the Scientific Investigation Board of the School of Life Sciences, Fudan University (2020-JS-016). Data Availability The RNA-seq data generated in this study were deposited in the NCBI Sequence Read Archive (SRA). 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Ahn, B.J., et al., Ninjurin1 Deficiency Attenuates Susceptibility of Experimental Autoimmune Encephalomyelitis in Mice. Journal of Biological Chemistry, 2014. 289 (6): p. 3328-3338. Choi, S., et al., Ninjurin1 Plays a Crucial Role in Pulmonary Fibrosis by Promoting Interaction between Macrophages and Alveolar Epithelial Cells. Scientific Reports, 2018. 8 . Chen, S.Y., et al., NINJ1 regulates ferroptosis via xCT antiporter interaction and CoA modulation. Cell Death Dis, 2024. 15 (10): p. 755. Chandrashekar, D.S., et al., UALCAN: An update to the integrated cancer data analysis platform. Neoplasia, 2022. 25 : p. 18-27. Jang, Y.S., et al., Ninjurin1 suppresses metastatic property of lung cancer cells through inhibition of interleukin 6 signaling pathway. Int J Cancer, 2016. 139 (2): p. 383-95. Cho, S.J., et al., Ninjurin1, a target of p53, regulates p53 expression and p53-dependent cell survival, senescence, and radiation-induced mortality. Proc Natl Acad Sci U S A, 2013. 110 (23): p. 9362-7. Romani, A.M.P., Cisplatin in cancer treatment. Biochem Pharmacol, 2022. 206 : p. 115323. Desoize, B. and C. Madoulet, Particular aspects of platinum compounds used at present in cancer treatment. Crit Rev Oncol Hematol, 2002. 42 (3): p. 317-25. Zhang, Q., et al., Unnatural Peptide Assemblies Rapidly Deplete Cholesterol and Potently Inhibit Cancer Cells. J Am Chem Soc, 2024. 146 (19): p. 12901-12906. Arbiser, J.L., M.Y. Bonner, and L.C. Gilbert, Targeting the duality of cancer. NPJ Precis Oncol, 2017. 1 . Helleday, T., S. Eshtad, and S. Nik-Zainal, Mechanisms underlying mutational signatures in human cancers. Nat Rev Genet, 2014. 15 (9): p. 585-98. O'Connor, M.J., Targeting the DNA Damage Response in Cancer. Mol Cell, 2015. 60 (4): p. 547-60. Ghobrial, I.M., T.E. Witzig, and A.A. Adjei, Targeting apoptosis pathways in cancer therapy. CA Cancer J Clin, 2005. 55 (3): p. 178-94. Tang, S.M., et al., Pharmacological basis and new insights of quercetin action in respect to its anti-cancer effects. Biomed Pharmacother, 2020. 121 : p. 109604. Cui, J., et al., Inhibiting NINJ1-dependent plasma membrane rupture protects against inflammasome-induced blood coagulation and inflammation. Elife, 2025. 12 . Additional Declarations (Not answered) Supplementary Files NINJ1SupplementaryMaterials.docx Supplementary Materials Originaluncroppegels.pptx Original uncroppe gels Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-6906860","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":486324305,"identity":"9aec334a-3eab-4e7d-904a-94e0e84b7642","order_by":0,"name":"Jixi Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYBACPmaGhAMMBjb8DAwJID4zYS1sEC1pkg3Ea4FQh0nRws7w8DBPwXkJg+PJzx4wVFgnNrCfPUDQYQdnGNyWMDjzzNyA4Ux6YgNPXgJBLQc+GNyuM7iRYCbB2HY4sUGCx4CwlgSDcxIGN9K/STD+I1bLB4MDQC05QFsaiNQC9EuyhOSZN2USCcfSjdt4cvBr4ec/k/yZ54+dBN/x9G0SH2qsZfvZz+DXwsDAk4Bgg5hsBNQDAfsBwmpGwSgYBaNgZAMA0+lBn8IOS+IAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-3463-3175","institution":"Fudan University","correspondingAuthor":true,"prefix":"","firstName":"Jixi","middleName":"","lastName":"Li","suffix":""},{"id":486324306,"identity":"93bbd5ef-dde0-4c4a-b925-ae71e6ff0ec1","order_by":1,"name":"Xuehe Liu","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Xuehe","middleName":"","lastName":"Liu","suffix":""},{"id":486324307,"identity":"2fa331d1-6aca-4124-9bc2-efe31753aae7","order_by":2,"name":"Zihan Qi","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Zihan","middleName":"","lastName":"Qi","suffix":""},{"id":486324308,"identity":"94e71218-6df1-4c37-bd97-f81b87f0548d","order_by":3,"name":"Yajie Shen","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yajie","middleName":"","lastName":"Shen","suffix":""},{"id":486324309,"identity":"579bd957-ba3f-45cc-9dc1-e983a93ac297","order_by":4,"name":"Yanjie Zhang","email":"","orcid":"","institution":"Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yanjie","middleName":"","lastName":"Zhang","suffix":""},{"id":486324310,"identity":"4a61955b-51b9-439b-b080-34a89e6d0c58","order_by":5,"name":"Feiyan Xie","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Feiyan","middleName":"","lastName":"Xie","suffix":""},{"id":486324311,"identity":"72e738bb-ca13-4240-bf1f-66f00b8ae951","order_by":6,"name":"Yuhong Xia","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yuhong","middleName":"","lastName":"Xia","suffix":""},{"id":486324312,"identity":"ccb933ee-02d2-4c0e-a245-62b4bf402fe6","order_by":7,"name":"Weichen Yu","email":"","orcid":"","institution":"Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Weichen","middleName":"","lastName":"Yu","suffix":""},{"id":486324313,"identity":"2981dbb4-c694-43c3-b706-bb42f95b7a08","order_by":8,"name":"Quanhui Dai","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Quanhui","middleName":"","lastName":"Dai","suffix":""},{"id":486324314,"identity":"ed3b2da7-7223-4855-8330-ba26e99a895f","order_by":9,"name":"Bing Zhao","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Bing","middleName":"","lastName":"Zhao","suffix":""},{"id":486324315,"identity":"e78c0f7a-6c95-4044-8509-f2ecd4cb63fb","order_by":10,"name":"Jin Ding","email":"","orcid":"","institution":"Center for Translational Medicine, Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Ding","suffix":""}],"badges":[],"createdAt":"2025-06-16 15:08:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6906860/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6906860/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87190654,"identity":"5a292f24-07f4-459e-9a3e-e54e6b37e1ee","added_by":"auto","created_at":"2025-07-21 11:12:24","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":958464,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNINJ1 is upregulated in lung adenocarcinoma and promotes tumor cell proliferation and migration \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Representative immunohistochemistry (IHC) images showing NINJ1 expression in lung adenocarcinoma (tumor) and adjacent normal (normal) tissues. Two tissue microarray (TMA) chips containing a total of 60 pairs of tumors and matched adjacent tissues were obtained from Shanghai Zhuoli Biotechnology Company Ltd. (\u003cstrong\u003eB\u003c/strong\u003e) Quantification of NINJ1 protein expression from panel \u003cstrong\u003eA\u003c/strong\u003e. Blue bars represent normal tissue and red bars represent tumor tissue. (\u003cstrong\u003eC–D\u003c/strong\u003e) CCK-8 assay showing reduced cell proliferation following NINJ1 knockout (NINJ1-KO) in A549 (\u003cstrong\u003eC\u003c/strong\u003e) and H1299 (\u003cstrong\u003eD\u003c/strong\u003e) cells. (\u003cstrong\u003eE–F\u003c/strong\u003e) Colony formation assay demonstrating decreased clonogenic capacity in NINJ1-KO A549 and H1299 cells. (\u003cstrong\u003eF\u003c/strong\u003e) Quantification of colony numbers from panel (\u003cstrong\u003eE\u003c/strong\u003e). (\u003cstrong\u003eG–J\u003c/strong\u003e) Wound healing assay assessing the migration of NINJ1-KO A549 (\u003cstrong\u003eG–H\u003c/strong\u003e) and H1299 (\u003cstrong\u003eI–J\u003c/strong\u003e) cells. Quantification is shown in (\u003cstrong\u003eH\u003c/strong\u003e) and (\u003cstrong\u003eJ\u003c/strong\u003e). NINJ1+ cell counts were analyzed using the ImageJ software. Scale bar: 50 μm. ns, not significant; *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6906860/v1/d35f0e3ddd64f56e4441784d.jpg"},{"id":87190941,"identity":"f2869b1f-9507-4396-a0e5-e2a0f339dc2a","added_by":"auto","created_at":"2025-07-21 11:20:24","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":579617,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNINJ1 deficiency enhances sensitivity to chemotherapy and suppresses lung adenocarcinoma tumor growth \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(\u003cstrong\u003eA, C, E\u003c/strong\u003e) CCK-8 assays showing the increased sensitivity of A549 and H1299 cells to cisplatin and gefitinib following NINJ1 knockout. (\u003cstrong\u003eB, D, F\u003c/strong\u003e) LDH release assays to assess plasma membrane integrity in NINJ1-deficient cells after drug treatment. (\u003cstrong\u003eG–M\u003c/strong\u003e) BALB/c-nude mice were subcutaneously injected with either wild-type (WT) or NINJ1-deficient A549 cells (N = 7 per group). (\u003cstrong\u003eG\u003c/strong\u003e) Schematic overview of the \u003cem\u003ein vivo\u003c/em\u003e experimental design. (\u003cstrong\u003eH\u003c/strong\u003e) Representative tumor images captured on day 23. (\u003cstrong\u003eI–L\u003c/strong\u003e) Quantification of tumor volume over time and at endpoint. (\u003cstrong\u003eM\u003c/strong\u003e) Average body weight of the mice in each group, indicating no significant systemic toxicity. ns, not significant; *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6906860/v1/4152bac54f8228335c0184bb.jpg"},{"id":87189418,"identity":"7553a6fb-7d80-40cb-a779-f82ff8be5f13","added_by":"auto","created_at":"2025-07-21 11:04:25","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1203101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNINJ1-derived peptides enhance the sensitivity of lung adenocarcinoma cells to chemotherapy drugs in a mouse model.\u003c/strong\u003e (\u003cstrong\u003eA–B\u003c/strong\u003e) Schematic illustration of NINJ1-derived short peptide sequences and their corresponding positions within the predicted NINJ1 protein structure. The 3D model is shown as a cartoon representation, with the targeted fragment (Pro26–Asn37) highlighted in stick format. Control peptides were designed with randomized sequences corresponding to the NINJ1 peptides. (\u003cstrong\u003eC\u003c/strong\u003e) Immunofluorescence colocalization of NINJ1-mCherry and NBD-labeled NINJ1-derived peptides in HEK293T cells. Red: mCherry; Green: NBD-labeled peptide; Blue: nuclei (DAPI); BF: bright field. Scale bar, 20 μm. (\u003cstrong\u003eD–G\u003c/strong\u003e) CCK-8 and LDH assays evaluating the impact of NINJ1-derived and control peptides on gefitinib sensitivity and plasma membrane integrity in A549 and H1299 cells. (\u003cstrong\u003eH–L\u003c/strong\u003e) BALB/c nude mice were subcutaneously injected with A549 cells and treated with PBS or NINJ1-derived short peptides (Peptide 1: PARWGWRHGPIN; Peptide 2: RWGWR) (N = 6 per group). (\u003cstrong\u003eH\u003c/strong\u003e) Schematic of the \u003cem\u003ein vivo\u003c/em\u003e experimental timeline. (\u003cstrong\u003eI, J\u003c/strong\u003e) Representative tumor images and tumor volumes on day 19. (\u003cstrong\u003eK\u003c/strong\u003e) Average body weights of mice in each group. (\u003cstrong\u003eL\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eTumor weights at the experimental endpoint. ns, not significant; *P \u0026lt; 0.05; ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6906860/v1/34aaf171f99260029e48bbeb.jpg"},{"id":87189416,"identity":"b2ebc06a-42cc-4a72-9dc4-46f1e4a774d4","added_by":"auto","created_at":"2025-07-21 11:04:25","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":913038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNINJ1 deficiency induces cell death and mitochondrial damage in A549 cells. \u003c/strong\u003e(\u003cstrong\u003eA–B\u003c/strong\u003e) Flow cytometric analysis of early and late apoptosis in wild-type (WT) and NINJ1-knockout (KO) A549 cells. GFP-positive live cells were gated using SSC-A/FSC-A and SSC-A/GFP (Alexa Fluor 488-A), while apoptotic cells were identified using Annexin V (PE-A) and Draq7 (Cy5-A) staining. (\u003cstrong\u003eC\u003c/strong\u003e) Transmission electron microscopy (TEM) images showing mitochondrial morphologies in WT, NINJ1-KO, and NINJ1-rescued A549 cells. Scale bar, 1 μm. (\u003cstrong\u003eD–E\u003c/strong\u003e) Quantification of damaged mitochondria and average mitochondrial length in WT, NINJ1-KO, and NINJ1-rescued A549 cells. (\u003cstrong\u003eF\u003c/strong\u003e) Western blot analysis of NINJ1, GSDMD, GSDME, and Caspase-3 expression in WT and NINJ1-KO A549 cells following cisplatin treatment. (\u003cstrong\u003eG–H\u003c/strong\u003e) Quantification of total Caspase-3 and cleaved Caspase-3 (p17) in panel (\u003cstrong\u003eF\u003c/strong\u003e), normalized to β-actin. (\u003cstrong\u003eI–J\u003c/strong\u003e) Western blotting and quantitative analysis of apoptotic pathway proteins in WT and NINJ1 KO A549 cells. ns, not significant; *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6906860/v1/4e7b94c2c2ed83237c124bc1.jpg"},{"id":87190657,"identity":"bb7f18a8-26a4-4ece-a56c-e8cba988ff23","added_by":"auto","created_at":"2025-07-21 11:12:25","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1076449,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNINJ1 is a critical modulator of PI3K/AKT signaling in LUAD. \u003c/strong\u003e(\u003cstrong\u003eA–C\u003c/strong\u003e) Transcriptomic profiling of WT and NINJ1-KO A549 cells using bulk RNA sequencing. (\u003cstrong\u003eA\u003c/strong\u003e) Volcano plot showing differentially expressed genes between WT and NINJ1-KO cells. (\u003cstrong\u003eB\u003c/strong\u003e) Heatmap of significantly altered gene expression between the two groups. (\u003cstrong\u003eC\u003c/strong\u003e) Heatmap of significantly altered gene expression between the two groups. (\u003cstrong\u003eD\u003c/strong\u003e) qPCR analysis of WNT5A, WNT5A-AS1, PIK3R1, AKT (including AKT1 and AKT2), CD44, and NINJ1 expression in WT and NINJ1-KO A549 cells. (\u003cstrong\u003eE\u003c/strong\u003e) Co-immunoprecipitation of NINJ1-mCherry with endogenous PI3K. (\u003cstrong\u003eF–G\u003c/strong\u003e) Correlation analysis between \u003cem\u003eNINJ1\u003c/em\u003eand PI3K p85 subunit genes (\u003cem\u003ePIK3R1\u003c/em\u003e, \u003cem\u003ePIK3R2\u003c/em\u003e, \u003cem\u003ePIK3R3\u003c/em\u003e) in LUAD based on TCGA data, using partial Spearman’s correlation. (\u003cstrong\u003eH–J\u003c/strong\u003e) Patient-derived LUAD tumor organoids treated with NINJ1-derived short peptides (200 μM) for nine days. (\u003cstrong\u003eH\u003c/strong\u003e) Representative images of organoids under different peptides treatment; scale bar: 250 μm. (\u003cstrong\u003eI–J\u003c/strong\u003e) Quantitative analysis of organoid size and viability. (\u003cstrong\u003eK–L\u003c/strong\u003e) Western blot analysis and quantification of PI3K/AKT signaling components (PI3K, AKT, and WNT5A) in WT and NINJ1-KO A549 cells. (\u003cstrong\u003eM\u003c/strong\u003e) Schematic model: NINJ1 interacts with PI3K to promote PI3K/AKT signal transduction and inhibit apoptotic complex formation. In contrast, NINJ1 deficiency or peptide-mediated inhibition destabilizes the PI3K/AKT pathway and induces mitochondrial damage. All experiments were performed in biological triplicates. Data represent the mean ± SD. ns, not significant; *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6906860/v1/ab3d4120a27dc9ea73486c32.jpg"},{"id":88644246,"identity":"229026a5-5247-4693-b476-71c48b6327e6","added_by":"auto","created_at":"2025-08-08 16:19:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5819498,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6906860/v1/454c979c-1080-43f8-9504-bb8d0dc020bc.pdf"},{"id":87189412,"identity":"fee3bac1-1049-473e-965b-da14f1755e8c","added_by":"auto","created_at":"2025-07-21 11:04:24","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1692473,"visible":true,"origin":"","legend":"Supplementary Materials","description":"","filename":"NINJ1SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6906860/v1/9c76c434a75baab5129afec9.docx"},{"id":87189438,"identity":"bf953292-8344-4953-8e9d-5d903e3aa151","added_by":"auto","created_at":"2025-07-21 11:04:27","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":196211091,"visible":true,"origin":"","legend":"Original uncroppe gels","description":"","filename":"Originaluncroppegels.pptx","url":"https://assets-eu.researchsquare.com/files/rs-6906860/v1/9e094d52136c4a6897fc1dcc.pptx"}],"financialInterests":"(Not answered)","formattedTitle":"NINJ1 promotes lung adenocarcinoma progression via activation of the PI3K-AKT signaling axis and suppression of cell death pathways","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLung adenocarcinoma (LUAD) is the most prevalent subtype of non-small cell lung cancer and a major contributor to cancer-related mortality worldwide. Despite progress in surgical resection, chemotherapy, and targeted therapies, the overall five-year survival rate for LUAD remains low, primarily because of late-stage diagnosis and therapeutic resistance [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. These clinical limitations highlight the urgent need to elucidate novel molecular mechanisms that drive LUAD progression and identify possible therapeutic targets to improve patient outcomes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNinjurin1 (NINJ1) is a conserved transmembrane protein that was initially identified as an adhesion molecule that is upregulated following nerve injury [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Recent studies have expanded its relevance to cancer biology, particularly through its role in regulating plasma membrane rupture (PMR) during various forms of programmed cell death (PCD), including apoptosis, pyroptosis, and necroptosis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. NINJ1 facilitates the release of intracellular components, such as lactate dehydrogenase (LDH) and damage-associated molecular patterns (DAMPs), which amplify inflammatory responses and may influence tumor progression [\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Although NINJ1 does not directly initiate cell death, its function in mediating membrane rupture suggests its central role in modulating cell fate and immune activation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEmerging evidence implicates NINJ1 in the regulation of tumorigenesis and inflammatory signaling [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Its overexpression has been associated with increased lung tumor burden in mice [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], whereas genetic deletion attenuates disease severity in models of pulmonary fibrosis and multiple sclerosis. Moreover, NINJ1 has been linked to ferroptosis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], an iron-dependent form of cell death implicated in cancer invasion and metastasis. Despite these observations, the precise role of NINJ1 in LUAD remains unclear.\u003c/p\u003e \u003cp\u003eIn the present study, we comprehensively examined the functional significance of NINJ1 in LUAD. We demonstrated that NINJ1 deficiency altered key transcriptional programs associated with cell cycle progression, DNA replication, EMT, and inflammatory responses. Furthermore, both genetic ablation and pharmacological intervention using NINJ1-derived short peptides suppress tumor cell proliferation and migration, enhanced chemosensitivity, and impaired the growth of iPSC-derived organoids and tumors \u003cem\u003ein vivo\u003c/em\u003e. These findings reveal that NINJ1 is a previously underappreciated regulator of LUAD pathogenesis and support its potential as a novel therapeutic target for lung cancer.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eNINJ1 is Highly Expressed in Human Lung Tumor Tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the clinical relevance of NINJ1 in lung cancer, we analyzed its expression using the UALCAN cancer database, which integrates TCGA (The Cancer Genome Atlas (RRID:SCR_003193)) and CPTAC datasets (CPTAC (RRID:SCR_017135)) [21]. NINJ1 was significantly upregulated in lung adenocarcinoma (LUAD) at both the transcript and protein levels across disease stages (Fig. S1). Analysis of the CPTAC proteomic data confirmed higher NINJ1 protein levels in LUAD tissues than in normal tissues. Promoter methylation levels of NINJ1 are also reduced in tumor samples, suggesting transcriptional supression [14, 17, 22, 23].\u003c/p\u003e\n\u003cp\u003eTo validate these findings, we performed immunohistochemical (IHC) analysis of 60 human lung cancer specimens. NINJ1 staining intensity was markedly elevated in LUAD, lung squamous cell carcinoma (LUSC), adenosquamous carcinoma (ASCC), and pulmonary sarcomatoid carcinoma (PSC) compared to adjacent normal tissues (Fig. 1A-B). These results support a role for NINJ1 in non-small cell lung cancer (NSCLC) progression. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eNINJ1 Deficiency Inhibits Proliferation and Migration of Lung Adenocarcinoma Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsistent with the patient data, we observed high NINJ1 expression in multiple lung cancer cell lines (Fig. S2A-B). To determine the functional role of NINJ1 in LUAD progression, we generated NINJ1 knockout A549 and H1299 cell lines by CRISPR-Cas9 and lentiviral transduction (Fig. S2C-F). Next, the proliferation and tumorigenicity of wild-type (WT) and NINJ1 knockout (KO) A549 and H1299 cells were detected. CCK-8 assays revealed that NINJ1 knockout significantly suppressed cell growth over 4 days compared to wild-type controls (Fig. 1C-D). Colony formation assays further showed a marked reduction in both colony number and size in NINJ1-deficient cells, indicating impaired tumorigenic potential (Fig. 1E-F). In addition, NINJ1-deficient A549 and H1299 cells exhibited a significant reduction in migratory capacity compared to their wild-type counterparts using wound healing assays (Fig. 1G-J). Together, these data suggest that NINJ1 promotes LUAD cell proliferation and migration, and its loss impairs tumor cell aggressiveness.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eNINJ1 Deficiency Enhances the Cytotoxic Effects of Chemotherapy in Lung Adenocarcinoma Cells and in the xenograft mouse model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCisplatin and its derivatives, including carboplatin and oxaliplatin, exert anticancer effects by disrupting DNA replication and repair, leading to impaired tumor cell proliferation [24]. Although effective across various cancer types, their clinical utility is limited by toxicity and severe side effects [25]. Given the critical role of NINJ1 in cell death, we explored whether targeting NINJ1 could enhance the therapeutic response to cisplatin and gefitinib in lung adenocarcinoma cells.\u003c/p\u003e\n\u003cp\u003eBoth cisplatin and gefitinib exhibited dose-dependent cytotoxicity in A549 and H1299 cells (Fig. 2A, 2C and 3E). Notably, NINJ1 knockout significantly increased sensitivity to these agents, as shown by decreased cell viability and enhanced membrane rupture (Fig. 2A-F). LDH release assays confirmed that drug-induced plasma membrane rupture was reduced in NINJ1-deficient cells, suggesting that NINJ1 contributed to chemotherapy-induced lytic cell death (Fig. 2D and 2F). Next, we assessed the role of NINJ1 in tumor growth \u003cem\u003ein vivo\u003c/em\u003e using a xenograft model. BALB/c-nude mice (n = 28) were randomly divided into two groups (n = 14 per group) and injected subcutaneously with either WT or NINJ1-deficient A549 cells in both flanks (Fig. 2G). Tumor formation was observed on day 4 post injection, confirming successful engraftment. \u003c/p\u003e\n\u003cp\u003eTo evaluate the effect of chemotherapy in combination with NINJ1 depletion, the tumor-bearing mice were further subdivided into four groups (n = 7 per group). The two groups received intraperitoneal injections of cisplatin (5 mg/kg) starting on day 2 after the tumors were established. Over a 23-day observation period, we found that tumors derived from NINJ1-deficient A549 cells were significantly smaller than those derived from wild-type cells, indicating that NINJ1 loss suppresses LUAD tumor growth in vivo (Fig. 2H\u0026ndash;L). Cisplatin treatment further inhibited tumor growth in wild-type A549 xenografts, whereas its effect on NINJ1-deficient tumors was less pronounced, suggesting that NINJ1 loss alone may confer strong antitumor activity. Importantly, body weight remained stable across all groups and no overt signs of systemic toxicity were observed (Fig. 2M), indicating that the combination treatment was well tolerated. These findings highlight the therapeutic potential of NINJ1 in lung adenocarcinoma.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eNINJ1-derived Short Peptides Inhibit Lung Tumor in the Xenograft Mouse Model \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further inhibit NINJ1-mediated membrane rupture, we designed two NINJ1-derived short peptides (named Peptides 1 and 2) targeting the extracellular N-terminal domain based on the AlphaFold3-predicted full-length NINJ1 protein structure (Fig. 3A-B). These fluorophore (green) NBD-tagged peptides competitively bind to the NINJ1 N-terminus, preventing functional oligomerization at the cell surface, as evidenced by confocal laser scanning microscopy (CLSM) (Fig. S3A) [26]. Immunofluorescence observations in HEK293T cells revealed that the peptides were localized to the plasma membrane and co-distributed with NINJ1-mCherry (Fig. 3C). Treatment with these peptides maintained low LDH release during drug exposure and enhanced chemotherapy-induced cytotoxicity in A549 and H1299 cells (Fig. 3D-G). \u003c/p\u003e\n\u003cp\u003eTo evaluate the therapeutic potential of the NINJ1-derived short peptides, we assessed their effect on lung adenocarcinoma cell viability under chemotherapeutic stress using CCK-8 assays. Treatment with either peptide 1 or peptide 2 significantly reduced the viability of A549 and H1299 cells, particularly in the presence of gefitinib (Fig. 3D and 3F). Concurrently, LDH release assays demonstrated that the peptides preserved the plasma membrane integrity (Fig. 3E and 3G), indicating a dual effect of inhibiting proliferation while preventing membrane rupture-associated cytotoxicity.\u003c/p\u003e\n\u003cp\u003eNext, we established subcutaneous xenografts in nude mice by using A549 cells. Following tumor establishment, the mice were randomized into six treatment groups: PBS, Peptide 1, Peptide 2, Gefitinib, Peptide 1 + Gefitinib, and Peptide 2 + gefitinib (Fig. 3H). Monotherapy with either the NINJ1-derived peptide or gefitinib significantly suppressed tumor growth compared to the PBS control (Fig. 3I-J). Notably, tumors treated with peptides alone exhibited slower growth rates than those treated with gefitinib alone.\u003c/p\u003e\n\u003cp\u003eImportantly, combination therapy with NINJ1-derived peptides and gefitinib produced a synergistic antitumor effect, resulting in markedly enhanced tumor inhibition compared with either treatment alone (Fig. 3K-L). These findings confirm that NINJ1-derived short peptides suppress lung adenocarcinoma cell proliferation both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, providing a promising adjunct strategy to improve the therapeutic efficacy in LUAD.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eNINJ1 Deficiency Promotes Programmed Cell Death in Lung Adenocarcinoma Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe inhibition of tumor cell proliferation and tumorigenesis observed in NINJ1-deficient A549 cells may be partially attributed to increased programmed cell death and cell cycle arrest [27-29]. Apoptosis and other regulated death pathways are frequently suppressed in cancer, allowing for unrestrained cell growth and therapy resistance [30, 31]. Given NINJ1\u0026rsquo;s established role in mediating plasma membrane rupture (PMR) and inflammatory signaling via DAMP release, we hypothesized that its loss may alter cell death dynamics in LUAD.\u003c/p\u003e\n\u003cp\u003eCCK-8 and LDH release assays revealed that NINJ1 deficiency significantly increased cytotoxicity and reduced proliferation of A549 and H1299 cells (Fig. 4A-B). Flow cytometry confirmed a modest increase in early and late apoptosis in the NINJ1-deficient A549 cells (Fig. 4B). Transmission electron microscopy (TEM) showed dramatic mitochondrial damage in NINJ1-deficient A549 cells, characterized by decreased mitochondrial length and cristae disruption, the effects of which were partially reversed by NINJ1 restoration (Fig. 4C-E). These findings suggest that NINJ1 may maintain mitochondrial integrity, contributing to its role in tumor cell survival.\u003c/p\u003e\n\u003cp\u003eWe further assessed the impact of NINJ1 on cell death, including apoptosis, pyroptosis, and necrosis. Apoptotic stimuli led to a greater loss of viability and increased Caspase-3 cleavage in NINJ1-deficient cells, whereas pyroptosis-induced membrane rupture was significantly impaired despite enhanced cell death (Fig. S4A-F). Notably, Western blot analysis revealed that cisplatin treatment induced GSDMD cleavage in wild-type A549 cells, but this effect was attenuated in NINJ1-deficient cells, suggesting the suppression of pyroptosis (Fig. 4F). In contrast, apoptosis was exacerbated, with increased cleaved Caspase-3 (p17 fragment) and reduced full-length Caspase-3 and upstream effectors CASP9 and Cytochrome C in NINJ1-deficient cells (Fig. 4G-J). These data indicate that NINJ1 loss reprograms the cell death landscape, enhancing apoptotic responses while impairing membrane rupture, thereby sensitizing LUAD cells to chemotherapeutic stress.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eTranscriptomic Profiling Reveals NINJ1 Regulates Cell Cycle, EMT, and PI3K/AKT Signaling in LUAD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the molecular functions of NINJ1, we performed a transcriptomic analysis in A549 lung adenocarcinoma cells following NINJ1 knockout. Compared with wild-type cells, NINJ1-deficient cells showed 598 upregulated and 1,088 downregulated genes. Gene Ontology (GO) and KEGG enrichment analyses identified pathways altered by NINJ1 loss, including extracellular matrix (ECM) organization; cellular response to interleukin-1; and valine, leucine, and isoleucine biosynthesis (Fig. 5A-C). These pathways are critical for tumor progression as they regulate structural integrity, inflammatory signaling, and amino acid metabolism, which are key processes in cancer cell growth and survival.\u003c/p\u003e\n\u003cp\u003eRNA-seq results were validated by qPCR and western blotting. NINJ1 deletion downregulated the WNT5A-PI3K/AKT signaling pathway at both transcript and protein levels (Fig. 5D and K-L). Co-immunoprecipitation confirmed the interaction between NINJ1 and PI3K (Fig. 5E). Further analysis using TCGA data showed that NINJ1 expression was positively correlated with the PI3K regulatory subunit genes PIK3R1, PIK3R2, and PIK3R3 in LUAD patients (Fig. 5F-G). Additionally, using patient-derived organoids, we found that NINJ1-derived short peptides significantly inhibited organoid growth and viability, further supporting NINJ1\u0026rsquo;s role in LUAD tumor maintenance (Fig. 5H-J).\u003c/p\u003e\n\u003cp\u003eConsistent with previous phenotypic findings, NINJ1 knockout induced G1 phase cell cycle arrest and downregulated Cyclin D1 expression while suppressing pro-proliferative genes (KRT19 and CES1) and upregulating tumor suppressors (ANGPTL4) (Fig. S3B). NINJ1 deficiency also inhibited EMT, as evidenced by increased E-cadherin and decreased N-Cadherin and Vimentin expression, thus providing a mechanistic basis for reduced cell migration (Fig. S3B). Collectively, these results indicate that NINJ1 modulates cell proliferation, apoptosis, EMT, and drug sensitivity by regulating key transcriptional networks and oncogenic signaling pathways (Fig. 5M).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe intricate roles and mechanisms of NINJ1 in LUAD have attracted considerable interest, and our experimental findings provide a comprehensive view of its multifaceted functions in this malignancy [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Our study provides compelling evidence that NINJ1 functions as a critical regulator of lung adenocarcinoma (LUAD) progression. Elevated expression of NINJ1 was observed in LUAD tissues and cell lines, and was associated with aggressive tumor features and poor prognosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In addition, NINJ1-deficient cells exhibited reduced colony formation, migration, and tumor growth in xenograft models (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Moreover, treatment with NINJ1-derived short peptides suppressed proliferation and enhanced chemosensitivity, suggesting their therapeutic potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Mechanistically, NINJ1 regulates plasma membrane rupture (PMR) during programmed cell death. Its deficiency amplified apoptotic signaling while attenuating pyroptosis-associated membrane damage, highlighting its dual role in controlling tumor cell fate under stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Through transcriptomic and functional analyses, we demonstrated that NINJ1 deficiency alters key gene programs involved in cell cycle progression, EMT, and inflammatory signaling and identified that NINJ1 modulates the WNT5A-PI3K/AKT pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Disruption of this signaling cascade in NINJ1-deficient cells likely contributed to the observed antitumor effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM).\u003c/p\u003e \u003cp\u003eBoth \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies demonstrated that loss of NINJ1 significantly impaired LUAD tumorigenicity, primarily through suppression of the WNT5A-PI3K/AKT signaling axis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This finding contrasts with previous reports suggesting that NINJ1 functions as a metastasis suppressor by inhibiting the IL-6/STAT3 pathway and reducing ICAM-1 expression [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Other studies have proposed a pro-tumorigenic role for NINJ1, implicating it in the maintenance of cancer stem cells (CSCs), activation of canonical Wnt/β-catenin signaling, and regulation of inflammation and programmed cell death [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Additionally, NINJ1 has been shown to interact with tumor suppressor networks involving p53 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], underscoring its context-dependent functions.\u003c/p\u003e \u003cp\u003eAlthough previous studies have reported that glycine and a 12-residue peptide can disrupt NINJ1 oligomerization at the membrane [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], our data revealed that NINJ1-derived peptides specifically bind to NINJ1, inhibit its oligomerization during plasma membrane rupture (PMR), and sensitize LUAD cells to chemotherapeutic agents, resulting in a significantly reduced tumor burden in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Given its involvement in multiple oncogenic and immune signaling pathways, NINJ1 has emerged as a multifaceted regulator of LUAD. Future studies should investigate how the tumor microenvironment and inflammatory cues influence NINJ1 expression and activity, with the goal of developing NINJ1-targeted strategies for precise lung cancer therapy.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eReagents and antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReagents: cisplatin (HY-17394, MedChemExpress), gefitinib (HY-50895, MedChemExpress), human TNF-alpha (HY-P7058, MedChemExpress), nigericin sodium salt (HY-100381, MedChemExpress), Z-VAD-FMK (HY-16658B, MedChemExpress), lipopolysaccharides (HY-D1056, MedChemExpress), SM-164 (HY-15989, MedChemExpress).\u003c/p\u003e\n\u003cp\u003eAntibodies: NINJ1 (Santa Cruz Biotechnology Cat# sc-136295, RRID: AB_10607827), PI3K (Proteintech Cat# 60225-1-Ig, RRID: AB_11042594), p-PI3K(Affinity Biosciences Cat# AF3241, RRID: AB_2834667), AKT(Proteintech Cat# 10176-2-AP, RRID: AB_2224574), p-AKT (ser473) (Proteintech Cat# 66444-1-Ig, RRID: AB_2782958), WNT5A (Proteintech Cat# 55184-1-AP, RRID: AB_2881285), CD44 (Bioss Cat# bsm-54767R, RRID: AB_3696666), Apaf-1(Huabio Cat# ET1607-12, RRID: AB_3069751), Caspase-3 (Huabio Cat# ET1608-64, RRID: AB_3069820), Caspase-8 (Huabio Cat# HA722181, RRID: AB_3675886), XIAP (Huabio Cat# HA722113, RRID: AB_3696667), Bax(Huabio Cat# ET1603-34, RRID: AB_3069679), Caspase-9 (Huabio Cat# R1308-12, RRID: AB_3073230), Cytochrome C (Huabio Cat# ET1610-60, RRID: AB_2940742), GSDME (Abcam Cat# ab215191, RRID: AB_2737000), GSDMD (Abcam Cat# ab210070, RRID: AB_2893325), GSDMD-CTD (Abcam Cat# ab215203, RRID: AB_2916166), FZD2 (Affinity Biosciences Cat# AF5282, RRID: AB_2837768). Anti-\u0026beta;-actin (Proteintech Cat# 66009-1-Ig, RRID: AB_2687938), Anti-Mouse IgG (H+L) (Proteintech Cat# SA00001-1, RRID: AB_2722565), and Anti-Rabbit IgG (H+L)(Proteintech Cat# SA00001-2, RRID: AB_2722564).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell lines and culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human non\u0026ndash;small cell lung cancer (NSCLC) cell line A549 was obtained from the Cell Bank of the Chinese Academy of Sciences (RRID: CVCL_0023). H1299 and OE19 cells were generously gifted by Dr. Hua Gao (Tongji University, China) and Dr. Feng Shao (National Institute of Biological Sciences, China), respectively. HEK293T, HeLa, THP-1, HUVEC, MEF, HT-29, and HepG2 cells were maintained in our laboratory.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA549, OE19, HEK293T, HeLa, MEF, HT-29, and HepG2 cells were cultured in high-glucose Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM-HG; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 100 U/mL penicillin with 0.1 mg/mL streptomycin (P/S; C0222, Beyotime Biotechnology). H1299, THP-1, and HUVEC were maintained in RPMI-1640 medium (Gibco) supplemented with 10% FBS and P/S. All the cell lines were routinely tested and confirmed to be free of mycoplasma contamination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrimary mouse cell isolation and culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary peritoneal macrophages were isolated from 6\u0026ndash;8-week-old C57BL/6 mice by flushing the peritoneal cavity with sterile PBS. Following red blood cell lysis, the cells were cultured in high-glucose DMEM (DMEM-HG) supplemented with 10% fetal bovine serum (FBS; Gibco) and 100 U/mL penicillin with 0.1 mg/mL streptomycin (P/S; Beyotime) for 24 h. The cell viability was consistently greater than 95%.\u003c/p\u003e\n\u003cp\u003eTo isolate primary alveolar cells, the tracheas of 6- to 8-week-old C57BL/6 mice were cannulated, and the lungs were lavaged with sterile PBS to collect alveolar cells. After red blood cell lysis, cells were cultured in DMEM-HG containing 10% FBS and P/S for 24 h. The average cell viability was found to be 97%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were raised in an animal facility at Fudan University under specific pathogen-free (SPF) conditions. All animal experiments were approved by the Institutional Animal Care and Use Committee of Fudan University. BALB/c nude (RRID: IMSR_RJ:BALB-C-NUDE)and C57BL/6 (RRID: MGI:7786639) mice were purchased from Cyagen Biosciences (Suzhou, China). A549 cells were xenografted into 5‐ to 7‐week‐old male and female BALB/c nude mice. The ratio of male-to-female mice was approximately 1:1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence and confocal microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeLa cells were seeded onto laser confocal dishes and pretreated with lipopolysaccharide (LPS) and TNF-\u0026alpha; for 2 h. Cells were treated with various concentrations of pharmacological agents, including Nigericin, SM164, Z-VAD, and cycloheximide (CHX), for 4-6 h. Following treatment, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 10-15 min, and washed again. The cells were then incubated in immunostaining blocking buffer at room temperature for 1-3 h. After blocking, the cells were incubated with primary antibodies (Abcam, UK), washed three times with PBS, and then incubated with the appropriate secondary antibodies. Nuclei were counterstained with DAPI and the samples were mounted with an anti-fade mounting medium. Fluorescent images were acquired using a laser scanning confocal microscope (Zeiss LSM 880 with Airyscan Confocal Laser Scanning Microscope (RRID: SCR_020925)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of protein expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cells were lysed in RIPA buffer (P0013B, Beyotime) supplemented with 1 mM PMSF (Phenylmethanesulfonyl fluoride; ST507, Beyotime). Protein concentrations were measured using an Enhanced BCA Protein Assay Kit (P0010, Beyotime).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of mRNA expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using a reagent kit (DP430, TIANGEN), and reverse transcription was performed using a HiScript III All-in-one RT SuperMix Perfect for qPCR kit (R333-01, Vazyme). The total amount of cDNA used for one reaction corresponded to approximately 100\u0026thinsp;ng of starting RNA. qPCR was performed using 2\u0026thinsp;\u0026times;\u0026thinsp;Taq Pro Universal SYBR qPCR Master Mix (Q712-02, Vazyme). All quantities were standardized to endogenous \u0026beta;-actin levels. The experiments were performed using an ABI Life Fluorescence Quantitative PCR instrument Q7. The primers used are listed in Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell counting kit-8 assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA549, H1299, and HeLa cells (3\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e3\u003c/sup\u003e cells/well) were seeded in 96‐well plates and cultured for 24\u0026thinsp; h. Cell Counting Kit-8 solution was added at the indicated time points to each well and incubated for 2\u0026thinsp;h at 37\u0026deg;C (C0038; Beyotime Biotechnology). The absorbance was measured at 450\u0026thinsp;nm using a fluorescence microplate reader (Molecular Devices SpectraMax M5 Multimode Plate Reader (RRID: SCR_020300)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLactate dehydrogenase (LDH) release assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA549, H1299, and HeLa cells were seeded in 96-well plates at a density of 3 \u0026times; 10\u0026sup3; cells/well and cultured for 24 h. At the indicated time points, cytotoxicity was assessed using a lactate dehydrogenase (LDH) assay kit (C0016; Beyotime Biotechnology) according to the manufacturer\u0026rsquo;s instructions. Absorbance was measured at 490 and 600 nm using a fluorescence microplate reader (Molecular Devices SpectraMax M5 Multimode Plate Reader (RRID: SCR_020300)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell migration assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell migration was assessed using a wound healing assay. Cells were collected, counted, and resuspended at a density of 1 \u0026times; 10 cells/mL. A total of 500 \u0026mu;L of cell suspension was seeded into each well of a 6-well culture plate, which had been marked with horizontal reference lines spaced 0.5 cm apart on the back. The cells were incubated overnight to allow monolayer formation and to achieve 100% confluence. Three parallel wells were used in each experiment. The following day, a linear scratch was made perpendicular to the marked lines using a sterile pipette tip. Detached cells were removed by washing the wells thrice with PBS, and the medium was replaced with serum-reduced culture medium (1% FBS). Cell migration into the wound area was monitored by capturing images of the same marked area at 0, 24, and 48 h, using an inverted microscope. The wound area was quantified using image analysis software, and the relative migration was calculated. All experiments were independently repeated thrice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission electron microscopy (TEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMitochondrial morphology of lung adenocarcinoma cells was examined by transmission electron microscopy (TEM). Cells were fixed in 1% osmium tetroxide (OsO₄) in 0.1 M phosphate buffer (pH 7.4) for 2 hours at room temperature in the dark. After fixation, the samples were dehydrated using a graded ethanol series and embedded in epoxy resin. Ultrathin sections (60-80 nm) were cut using an ultramicrotome (Leica EM UC7 ultramicrotome (RRID: SCR_016694)) and mounted onto 150-mesh copper grids coated with a Formvar film. The sections were stained with 2% uranyl acetate, followed by 2.6% lead citrate, and imaged using a transmission electron microscope (Hitachi HT7700 Transmission Electron Microscope (RRID: SCR_020022)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeptides\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe following peptides were synthesized by GL Biochem (Shanghai) Ltd.: Peptide 1 (PARWGWRHGPIN), Control Peptide 1 (PWAPRRHGNGWI), Peptide 2 (RWGWR), and Control Peptide 2 (WGRRW). PARWGWRHGPIN-NBD and RWGWR-NBD peptides were synthesized according to methods described in a previous study [26]. Unless otherwise specified, all peptides were used at a final concentration of 50 \u0026mu;M.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData analyses and figure plotting were performed using GraphPad Prism software (version 9) or R statistical environment. The numbers of replicates and independent experiments are listed in the figure legends. Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD unless stated otherwise. A P-value of \u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance for all analyses. Statistical significance was determined using a two-tailed Student\u0026rsquo;s t-test, Mann-Whitney test, one-way analysis of variance (ANOVA), or Brown-Forsythe and Welch ANOVA tests. No statistical methods were used for the predetermined sample sizes, and the experiments were not randomized. During the experiments and outcome assessments, the investigators were not blinded to allocation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Acknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (32161160323) and Shanghai Committee of Science and Technology (24490713600).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.L. conceived of and designed the study. X.L., Z.Q., Y.S., Y.Z., F.X., Y.X., Y.L., W.Y., Q.D., B.Z., and J.D. performed the experiments and analyzed the data. X.L. and J.L. analyzed the data and wrote the manuscript. All the authors discussed the results and commented on the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals with the approval of the Scientific Investigation Board of the School of Life Sciences, Fudan University (2020-JS-016).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe RNA-seq data generated in this study were deposited in the NCBI Sequence Read Archive (SRA). Raw data, including fastq files, were uploaded under the accession numbers SRR31420437, SRR31420436, SRR31420435, SRR31420440, SRR31420439, and SRR31420438.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWhisstock, J.C. and R.H.P. 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Adjei, \u003cem\u003eTargeting apoptosis pathways in cancer therapy.\u003c/em\u003e CA Cancer J Clin, 2005. \u003cstrong\u003e55\u003c/strong\u003e(3): p. 178-94.\u003c/li\u003e\n\u003cli\u003eTang, S.M., et al., \u003cem\u003ePharmacological basis and new insights of quercetin action in respect to its anti-cancer effects.\u003c/em\u003e Biomed Pharmacother, 2020. \u003cstrong\u003e121\u003c/strong\u003e: p. 109604.\u003c/li\u003e\n\u003cli\u003eCui, J., et al., \u003cem\u003eInhibiting NINJ1-dependent plasma membrane rupture protects against inflammasome-induced blood coagulation and inflammation.\u003c/em\u003e Elife, 2025. \u003cstrong\u003e12\u003c/strong\u003e.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6906860/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6906860/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLung adenocarcinoma (LUAD) remains a leading cause of cancer-related mortality, and current therapeutic options often fail to yield sustained responses. Ninjurin1 (NINJ1), a transmembrane protein implicated in plasma membrane rupture during lytic cell death, has recently emerged as a potential antitumor target, yet its role in LUAD pathogenesis remains poorly defined. Here, we comprehensively investigated the functional relevance and mechanistic regulation of NINJ1 in LUAD progression. We observed elevated NINJ1 expression in human LUAD tissues and cell lines. Genetic ablation of NINJ1 or pharmacological inhibition using NINJ1-derived short peptides significantly impaired tumor cell proliferation and migration, enhanced sensitivity to chemotherapeutic agents, and suppressed the growth of human iPSC-derived LUAD organoids and xenografted tumors \u003cem\u003ein vivo\u003c/em\u003e. Mechanistically, NINJ1 was found to directly associate with PI3K, thereby activating the downstream AKT signaling pathway. Loss of NINJ1 led to widespread transcriptional dysregulation, affecting gene networks involved in cell cycle progression, epithelial-mesenchymal transition (EMT), and inflammatory responses. Functionally, NINJ1 deficiency induced G1 phase arrest, mitochondrial dysfunction, and increased susceptibility to cell death. These findings identify NINJ1 as a key molecular driver of LUAD malignancy and highlight its potential as a promising therapeutic target for lung cancer.\u003c/p\u003e","manuscriptTitle":"NINJ1 promotes lung adenocarcinoma progression via activation of the PI3K-AKT signaling axis and suppression of cell death pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-21 11:04:20","doi":"10.21203/rs.3.rs-6906860/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d0665597-62e8-4503-9769-2f6fc15a01d0","owner":[],"postedDate":"July 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51627890,"name":"Biological sciences/Cell biology/Cell death/Necroptosis"},{"id":51627891,"name":"Biological sciences/Cancer/Lung cancer/Non-small-cell lung cancer"}],"tags":[],"updatedAt":"2025-08-08T16:11:46+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-21 11:04:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6906860","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6906860","identity":"rs-6906860","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}