Recent genetic studies have identified defects in the PIWI-interacting RNA (piRNA) pathway to cause nonobstructive azoospermia. However, limited knowledge of piRNA function in human spermatogenesis makes it difficult to quantify the number of cases affected. Central to the piRNA biogenesis are the transcription factor MYB proto-oncogene like 1 (MYBL1), which transcribes most piRNA clusters, and PIWI-like 1 (PIWIL1), which is critical for the processing of piRNAs. Here, we show that testicular biopsies with complete spermatogenesis (n = 55) and biopsies with spermatogenic arrest at the round spermatid stage (n = 32) consistently exhibit a robust expression of MYBL1 and PIWIL1 proteins in pachytene spermatocytes and until round spermatids. In contrast, MYBL1 and/or PIWIL1 expression was lost in 59% (19/32) and 42% (15/36) of the biopsies with homogeneous and heterogeneous arrest at the pachytene stage, respectively. Biopsies showing arrest at the earlier leptotene stage (n = 17) did not express MYBL1 or PIWIL1 because pachytene spermatocytes were absent. In biopsies without MYBL1 and PIWIL1 protein expression, RNA in situ hybridization revealed a concurrent decrease in PIWIL1, but not MYBL1, transcripts. Small RNA sequencing further demonstrated a significant and near-complete loss of pachytene piRNAs, but not pre-pachytene piRNAs, in biopsies without MYBL1 and PIWIL1 protein expression. Although we cannot establish direct causality, our data suggest that defective piRNA processing can be linked to arrest of spermatogenesis at the pachytene stage in half (34/68) of the cases. This establishes the piRNA pathway as a frequent and central determinant of human spermatogenic arrest with a distinctive molecular signature of impaired germ‑cell differentiation.
The insulin-like growth factor 2 mRNA-binding protein (IGF2BP) family is overexpressed in cancer and associated with poor prognosis. IGF2BP2 has been linked to single metabolic alterations by acting on its RNA targets. Here, we used a comprehensive approach to elucidate the effects of IGF2BP2 on primary and lipid metabolism. 13C-metabolic flux analysis (MFA) combined with RNA-Seq data revealed that IGF2BP2 affects mitochondrial fluxes by regulating the expression of several mitochondrial transporters, such as mitochondrial pyruvate carrier 1 (MPC1) and uncoupling protein 2 (UCP2). Methyl pyruvate reversed the gene expression patterns of UCP2 and CPT1A in HCT116 IGF2BP2 knockout (KO) cells by bypassing MPC1. Interestingly, an altered expression of the transporter UCP2 was also observed in a patient-derived tumor organoid (PDO), in which IGF2BP2 was knocked down. The altered glutamine metabolism seen in the 13C-MFA and the citrate label data derived from extracted mitochondria confirm a rerouting of glutamine almost exclusively into the mitochondria and a reduction of glycolytic carbon intake into the mitochondria. Due to changes in palmitate labeling patterns, lipid stainings were performed, suggesting lipid accumulation in KO cells. A lipidomic analysis revealed altered compositions across almost all lipid species. Further, lipogenic genes involved in fatty acid and cholesterol metabolism were differentially expressed. Most of the differentially expressed genes are potential direct targets of IGF2BP2 based on publicly available IGF2BP2 CLIP data. Overall, these results show the influence of IGF2BP2 on the central carbon metabolism of cancer cells, primarily through its effects on MPC1 and the resulting effects on UCP2. The complex interaction of IGF2BP2 with the metabolic network provides important insights into tumor metabolism, particularly relevant to tumor growth and resistance to therapy.
Cisplatin resistance remains a major clinical challenge in intrahepatic cholangiocarcinoma (iCCA); however, its underlying mechanisms are poorly understood. Building on our previous study that identified Mucin 13 (MUC13) as an oncogenic driver, we aimed to investigate its contribution to cisplatin resistance. We uncovered a previously unrecognized positive feedback loop between MUC13 and histone H3K18 lactylation (H3K18la), which sustains metabolic reprogramming and drives cisplatin resistance. Mechanistically, MUC13 activates the β-catenin/c-Myc axis to upregulate the expression of glucose transporter GLUT1 and key glycolytic enzymes (HK2, PKM2, and LDHA), thereby enhancing lactate production. The resulting lactate level elevation increases H3K18la enrichment at the MUC13 promoter, transcriptionally upregulating MUC13 and reinforcing glycolytic reprogramming and resistance. Any disruption of this loop by LDHA inhibition, which reduces lactate-mediated H3K18 lactylation, alleviates cisplatin tolerance in vitro and in vivo models. Notably, patient-derived organoids with high MUC13 and H3K18la expression were effectively sensitized to cisplatin by the LDHA inhibitor GSK2837808A, highlighting the translational potential of targeting this feedback loop. Collectively, our findings identify a metabolic–epigenetic positive feedback circuit between H3K18la and MUC13 that contributes to cisplatin resistance in iCCA and highlights histone lactylation as a potentially targetable therapeutic vulnerability.
Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer (BC) with higher incidence rates in India. Ionizing radiation (IR) is a key component of TNBC treatment regimens. However, overall suboptimal response during the course of multiple repeat radiotherapy with eventual attainment of radioresistance remains a major challenge for this oncologic treatment modality. To overcome this limitation, it is important to understand molecular signatures that drive cells to transform into non-responsive populations against radiation therapy. Here, we demonstrate a constitutively high basal level of autophagy in TNBC cells, which might contribute to their relatively lower sensitivity to radiation-induced cell death. We also observed an elevated level of p62 in TNBC cells, which is regulated independently of autophagic flux. Concurrent upregulation of p62 and basal level of autophagy were found to be the driving forces in acquiring radioresistance in breast cancer cells. Ectopic expression of p62 enabled breast cancer cells to proliferate rapidly with enhanced migration potential. Reciprocally, posttranscriptional or pharmacological inhibition of p62 prevented the proliferation and migration potential of TNBC cells. Our high-throughput next-generation sequencing (NGS) data revealed VCAM-1 as a key mediator in p62-driven cell proliferation and acquired radioresistance. Likewise, impairment of autophagic flux, either by pharmacological inhibitors (CQ and bafA1) or by genetic deletion of ATG5, led to reversal of radioresistance. Collectively, our data highlight that an elevated basal level of autophagy with co-induction of p62 de novo protein synthesis confers radioresistance in TNBC.
CD19-directed CART therapy has improved outcomes in B-cell malignancies, but durable responses remain limited by antigen escape, in which loss of surface CD19 can drive antigen-negative relapse. Ibrutinib (IB) has been reported to enhance CART activity, yet whether IB directly modulates tumor-cell antigen presentation remains unclear. Here, we identify matriptase (MTP, encoded by ST14) as a tumor-cell-intrinsic regulator of CD19 stability and a mediator of IB-enhanced CART efficacy. In an HT xenograft model, combined IB and CD19-CART treatment prolonged survival, suppressed tumor growth, and increased apoptosis compared with either monotherapy. IB treatment induced coordinated upregulation of MTP and CD19 in vivo. Analysis of patient-derived DLBCL datasets and tissue microarrays further showed that high ST14 expression correlated with MYC-associated aggressive disease features, poor prognosis, and CD19 protein expression. Mechanistically, ST14 knockout in Ramos cells reduced total and surface CD19, accelerated CD19 degradation, and impaired membrane localization, whereas exogenous activated MTP restored CD19 abundance. Loss of ST14 also increased susceptibility to IB- and CART-induced CD19 downregulation, attenuated CART-mediated cytotoxicity, and abolished the synergistic benefit of IB in vitro. Phosphoproteomic and transcriptomic analyses revealed that MTP deficiency impaired CK2 activity and reduced PAX5 expression, linking MTP to a CK2-PAX5-CD19 regulatory axis. Finally, direct MTP inhibition with an anti-MTP monoclonal antibody enhanced IB-mediated tumor suppression in a dual Ramos/HT xenograft model without overt systemic toxicity, but reduced CD19 availability and slightly compromised CART cytotoxicity when combined with CART alone. Collectively, our findings reveal a context-dependent role for MTP in B-cell lymphoma, whereby MTP stabilizes CD19 to sustain CART recognition while also contributing to aggressive biology.
The infiltration of macrophages and the inflammatory response they mediate, which are initiated by endothelial dysfunction, are pivotal in both the initiation and progression of atherogenesis. Phospholipase C epsilon 1 (PLCE1) is significantly involved in cardiovascular diseases, in part due to its regulation of vascular inflammation. This study seeks to explore the specific function of PLCE1 in the development of atherosclerosis. Bioinformatic analysis of datasets from the GEO database demonstrated abundant PLCE1 expression in patients with restenosis or myocardial infarction. Consistently, PLCE1 was upregulated in atherosclerotic plaques of high-fat diet (HFD)-fed ApoE-/- mice as well as in endothelial cells stimulated with oxidized low-density lipoprotein (OX-LDL). Additionally, double immunofluorescence staining revealed a strong co-localization of PLCE1 with the endothelium. The deficiency of PLCE1 in ApoE-/- mice mitigated endothelial dysfunction, as evidenced by reduced expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and various chemokines, which subsequently inhibited pro-inflammatory M1 macrophage polarization. The observed effects corresponded with decreased formation of atherosclerotic plaques and lower plaque vulnerability. Consistent results were also obtained from in vitro studies. Furthermore, bone marrow transplantation experiments suggested that the improvement in atherosclerosis was primarily due to the deletion of PLCE1 in resident vascular endothelial cells. Mechanistically, PLCE1 mediated endothelial dysfunction, macrophage infiltration, and inflammation, in part through its direct interaction with CTNNB1. These findings indicate that PLCE1 is an important regulator of vascular endothelial dysfunction, leading to a classical macrophage polarization switch through its interaction with CTNNB1, which collectively accelerates the development of atherosclerosis. Our study suggests that PLCE1 is a promising therapeutic target for the management of atherogenesis.
Chronic respiratory diseases (CRDs) are a growing global health concern, characterized by persistent inflammation, airway remodeling, and progressive airflow limitation. Elevated levels of the pro-inflammatory cytokine TGF-β promote epithelial-to-mesenchymal transition (EMT), a central process in CRD pathogenesis. However, its impact on cellular energy metabolism, particularly glycolysis, in lung epithelial cells remains unclear. We investigated the role of glycolysis in TGF-β-induced EMT using A549 and BEAS-2B lung epithelial cells. Immunoblotting, RT-qPCR, migration assays, ChIP, Seahorse analysis, lactate assays, glucose uptake, and siRNA transfection were used to dissect the link between glycolysis and EMT. TGF-β-induced EMT was reflected by enhanced motility and changes in epithelial and mesenchymal markers, alongside Smad3 phosphorylation and recruitment to the Snail promoter. It also upregulated glycolytic enzymes and lactate production. Inhibition of glycolysis with 2-deoxy-D-glucose (2-DG) suppressed TGF-β-induced Smad3 phosphorylation and EMT. Smad3 siRNA decreased TGF-β-driven glycolytic enzyme expression, including 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase 3 (PFKFB3). Moreover, TGF-β-induced glycolytic lactate further promoted Smad3 phosphorylation, suggesting a positive feedback loop. In vivo, decreased E-cadherin and increased Snail expression correlated with elevated GLUT1 and PFKFB3 in the lung epithelium of house dust mite (HDM)-induced allergic asthma and bleomycin (BLM)-induced idiopathic pulmonary fibrosis (IPF) models. Our findings reveal a vicious TGF-β-lactate-TGF-β cycle in which TGF-β enhances glycolysis, elevates lactate, and amplifies Smad3 signaling to promote EMT. Targeting glycolytic reprogramming may provide novel therapeutic strategies for TGF-β-associated CRDs such as asthma and pulmonary fibrosis.
Anti-angiogenic therapy is a standard first-line treatment for advanced hepatocellular carcinoma (HCC), but drug resistance remains a major obstacle to its long-term efficacy. Our previous study found that apolipoprotein A2 (ApoA2) was highly expressed in anti-angiogenic drug-resistant HCC. However, the mechanism underlying ApoA2-mediated resistance to anti-angiogenic therapy remains unclear. Here, ApoA2 overexpression markedly promoted HCC cell proliferation, inhibited lenvatinib-induced apoptosis in vitro, and enhanced tumor progression as well as intrahepatic and lung metastasis in vivo. Mechanistically, conditioned medium from ApoA2-overexpressing cells facilitated HUVEC migration and tube formation. Tumors with ApoA2 overexpression showed significantly increased CD31 expression and microvessel density. ApoA2 upregulated HMGB1 expression and activated the IL-17A signaling pathway. Glycyrrhizic acid (Gly) and secukinumab (mab), specific inhibitors of HMGB1 and IL-17A, respectively, synergized with lenvatinib in vivo in subcutaneous xenograft models and reversed ApoA2-driven angiogenesis. Clinically, high ApoA2 expression was strongly correlated with elevated IL-17A expression and poor prognosis. Our findings provided a mechanistic explanation for the failure of anti-angiogenic therapy and suggested a potential therapeutic strategy to overcome ApoA2-mediated resistance in HCC.
Acetaminophen (APAP) overdose is the leading cause of drug-induced acute liver failure, yet early pathways linking hepatocellular stress to tissue integrity remain incompletely defined. Here, we demonstrate that canonical Hh signaling is rapidly activated in centrilobular hepatocytes upon APAP challenge, preceding significant inflammation and recovery. Using mice with hepatocyte-specific constitutive expression of Smoothened (SmoM2), we show that pre-activation of Hh signaling attenuates APAP-induced liver injury and immune cell infiltration. Functionally, this protection is independent of intracellular glutathione depletion or CYP2E1-mediated APAP metabolism. Instead, we identify a Vegfa-associated vascular/endothelial response, in which hepatocyte Smo activation increases vascular endothelial growth factor A (VEGFA) expression and is linked to improved vascular injury and barrier function, as well as reduced expression of adhesion molecules (ICAM-1 and E-selectin). Furthermore, sorafenib treatment and AAV8-mediated hepatic Vegfa knockdown attenuated the protective phenotype in SmoM2 mice. Collectively, our findings support a model in which hepatocyte Smo activation is associated with a Vegfa-dependent vascular/endothelial protective response, suggesting a potential vascular-focused direction for studying acute liver failure.
Toxic epidermal necrolysis-like (TEN-like) immune-related cutaneous adverse event (ircAE) induced by programmed death 1 (PD-1) inhibitors is life-threatening; however, its pathogenesis remains unclear. This study aimed to dynamically characterize molecular signatures and identify potential pathogenic mediator(s) of TEN-like ircAE. Initial candidate mediators were identified through comparative analysis of bulk RNA sequencing data from distinct blood samples, coupled with a protein-protein interaction network. Subsequently, the potential pathogenic mediator(s), along with their primary cellular sources and corresponding effector events, were determined using quantitative PCR, tissue staining, single-cell RNA sequencing, and flow cytometry. Finally, validation was performed via in vitro cell experiments and in vivo rat models. By multiple comparisons among the sequencing data of progressive phase (Advanced stage), post-treatment recovery phase (Recovered stage), and matched controls (Control group) and following in vitro validation at the nucleic acid and protein levels, ADAMTS2 was identified as a promising candidate for further investigation. Subsequently, we showed that lesional fibroblasts were the primary cellular sources of ADAMTS2. Cultured human keratinocytes (HaCaT) were treated with supernatants from ADAMTS2-overexpressing fibroblasts (HDF-a), and the results showed that secreted ADAMTS2 promoted keratinocyte apoptosis via indirect inhibition of the NF-κB pathway. Furthermore, intradermal injection of an ADAMTS2-expressing plasmid in rats induced epidermal necrolysis-like lesions and keratinocyte apoptosis. Our study suggests that ADAMTS2 may serve as a candidate pathogenic mediator in PD-1 inhibitor-induced TEN-like ircAE. We have laid the groundwork for future studies on the pathogenesis and intervention targets of this life-threatening AE. These findings require validation with larger samples.
Pathological ocular neovascularization remains a leading cause of irreversible vision loss. This study investigates the role of Polo-like kinase 2 (PLK2) in coordinating the transition of endothelial cells into pathological phenotypes. Single-cell transcriptomic and clinical tissue analysis revealed that PLK2 is specifically enriched in the vascular component of human proliferative membranes and is markedly upregulated in human fibrovascular membrane tissue. PLK2 endothelial knockdown (eKD) inhibited vascular branching and tip cell filopodia formation in postnatal mice. Analysis of murine oxygen-induced retinopathy (OIR) single cell sequencing datasets identified PLK2 as a specific marker for actively sprouting tip cells and proliferative endothelial subpopulations during pathological neovascularization. PLK2 eKD or conditional knockout (eCKO) significantly attenuated pathological neovascularization in OIR. Additionally, PLK2 eKD inhibited laser-induced choroidal neovascularization (CNV) in adult mice. In vitro assays confirmed that PLK2 depletion via shRNA or CRISPR/Cas9 markedly inhibited endothelial cell proliferation, migration, and tube formation across various microvascular cells, whereas ectopic PLK2 overexpression promoted a robust pro-angiogenic phenotype. Mechanistically, PLK2 is a critical regulator of mitochondrial bioenergetics; genetic depletion led to a reduction of mitochondrial Complex I activity, decreased ATP levels, and impaired oxygen consumption, accompanied by mitochondrial membrane depolarization and the accumulation of reactive oxygen species (ROS). The anti-angiogenic effects and apoptotic induction caused by PLK2 deficiency were significantly rescued by glucose supplementation or antioxidant N-acetylcysteine (NAC) treatment. Thus, by maintaining mitochondrial bioenergetic efficiency and mitigating oxidative stress, PLK2 sustains the high-energy demands of pathological endothelial cell activation and sprouting. PLK2 represents a promising therapeutic strategy for pathological ocular angiogenesis.
Anthracyclines such as doxorubicin are highly effective chemotherapeutic agents, but their clinical use is limited by dose-dependent cardiotoxicity associated with mitochondrial dysfunction. Dysregulated mitochondrial dynamics, particularly excessive dynamin-related protein 1 (Drp1)-mediated fission, has been implicated in cardiac injury. We investigated whether DRP1i2, a novel small-molecule Drp1 inhibitor targeting a conserved domain, mitigates doxorubicin-induced cardiotoxicity while preserving anticancer efficacy. Cardioprotective effects were assessed in a murine model of chronic doxorubicin cardiotoxicity and in human induced pluripotent stem cell-derived cardiac microtissues, while anticancer activity was evaluated across multiple cancer cell lines in 2D and 3D systems. DRP1i2 preserved left ventricular systolic function in vivo, reduced interstitial fibrosis and cardiomyocyte atrophy, and attenuated myocardial proteomic remodelling. In human cardiac microtissues, DRP1i2 improved viability and restored contractile function despite persistent mitochondrial oxidative stress and metabolic dysfunction. In cancer models, DRP1i2 did not compromise doxorubicin efficacy across A549 lung cancer, OVCAR3 ovarian cancer, and MDA-MB-231 breast cancer cell lines, and exhibited modest anticancer activity in MG63 osteosarcoma cells. These findings demonstrate that DRP1i2 confers cardioprotection while maintaining anticancer efficacy via modulation of Drp1-dependent mitochondrial dynamics, supporting mitochondrial fission as a targetable pathway to mitigate doxorubicin-induced cardiotoxicity.
Drug-induced liver injury (DILI), particularly from isoniazid (INH), is a major clinical concern. Ferroptosis is implicated in DILI, yet direct GPX4 stabilizers with defined binding sites remain undiscovered. While Schisandra chinensis lignans exhibit hepatoprotective potential, their mechanistic interplay with ferroptosis remains unexplored. Here, we delineate a novel molecular axis by which Schisandrin A (SinA) and Schisandrin B (SinB) mitigate INH-induced hepatotoxicity through ferroptosis suppression. We demonstrate that SinA/SinB significantly attenuates hepatic injury markers (ALT/AST), iron overload, lipid peroxidation, and glutathione depletion in vitro and in vivo. Strikingly, GPX4 knockout abolished their protective effects, underscoring GPX4 as the pivotal target. ITC and SPR revealed high-affinity binding of SinA/SinB to GPX4, while molecular docking identified K31 and K90 as critical residues for GPX4 interaction. Mutagenesis studies confirmed that K31/K90 substitutions abolished SinA/SinB’s efficacy, highlighting a structure-dependent mechanism. Compound binding reduced the interaction between GPX4 and TRIM25, inhibited the ubiquitination of GPX4, as evidenced by Co-IP and MD. This stabilizes GPX4, suppresses lipid peroxidation/iron accumulation, and rescues INH-induced ferroptosis in vitro and in vivo. These findings establish a novel mechanism of GPX4 regulation and provide a structural blueprint for anti-ferroptotic drug design.
Gemcitabine(GEM)-based chemotherapy is considered the first-line regimen for pancreatic cancer(PC), but the poor responsiveness and chemoresistance greatly limit its application. Metabolic reprogramming, especially aerobic glycolysis, which is regulated by glycolytic enzymes, is closely associated with the malignant behavior of PC. Recently, triosephosphate isomerase 1(TPI1), as a key glycolytic enzyme involved in glycolysis, was reported to be increased in GEM-treated PC patients with poor outcome whereas the mechanism remains unclear. In this study, we identified vaccinia-related kinase 2 (VRK2), a serine/threonine protein kinase, which was upregulated in GEM-resistant PC cells and conferred GEM resistance by promoting TPI1-driven aerobic glycolysis. Further studies showed that phosphorylation and nuclear translocation of the transcriptional factor SP1 induced by VRK2 contributed to the interaction of SP1 with the promoter region of TPI1 and thus enhanced the transcriptional activity of TPI1. Taken together, our findings uncover a novel role for VRK2 in modulating glycolytic reprogramming and GEM-resistance, which implicates VRK2 as a potential therapeutic target in PC.
Neuropathic pain is associated with sensory neuron injury, and ferroptosis has been implicated in neuronal damage and death. However, the regulatory mechanism of ferroptosis in neuropathic pain remains unclear. This study aimed to investigate poly-(ADP-ribose) polymerase 1 (PARP1) in regulating sensory neuron ferroptosis and to assess its potential therapeutic role in neuropathic pain. A chronic constriction nerve injury (CCI) mouse model was established to examine how ferroptosis in sensory neurons contributes to CCI-induced neuropathic pain. We analyzed the expression of ferroptosis-related key gene (GPX4, FPN1, FSP1, TFR1, DMT1) and ferroptosis in dorsal root ganglion (DRG) neurons, along with PARP1 activity. Blocking PARP1 activity with PJ34 or olaparib, or conditionally knocking out neuronal PARP1, was performed to assess effects on GPX4 expression, ferroptosis, and neuropathic pain behavior. Nerve injury downregulated GPX4 expression and enhanced ferroptosis in DRG neurons, accompanied by increased PARP1 activity. Using sensory neuron-specific PARP1 conditional knockout mice, we demonstrated that neuronal PARP1 deletion upregulated GPX4 expression, reduced ferroptosis, and alleviated neuropathic pain-like behaviors. Blocking GPX4 activity induced pain sensitization in normal mice. Inhibition of ferroptosis or administration of PARP1 inhibitors significantly alleviated pain-like behaviors in CCI mice. Following nerve injury, PARP1 overactivation drives neuropathic pain by suppressing GPX4 and promoting ferroptosis in DRG neurons. Conditional knockout or pharmacological inhibition of PARP1 reversed these effects and alleviated pain. Thus, the PARP1-GPX4 axis represents a promising therapeutic target.
The molecular mechanisms underlying lung metastasis of colorectal cancer (CRC) remain largely elusive, and effective therapeutic agents are still lacking. In this study, we identify levistilide A (LeA) as a potential anti-metastatic agent against CRC lung metastasis. We demonstrate that heat shock protein 90α (HSP90α) is markedly upregulated in CRC and promotes lung metastasis by suppressing ferroptosis. Notably, combined treatment with LeA and the ferroptosis inducer RSL3 further alleviates lung metastatic burden in vivo. Mechanistically, we reveal that the E3 ubiquitin ligase ring finger protein 40 (RNF40) suppresses CRC cell proliferation by directly interacting with HSP90α, inducing ubiquitination at lysine 407 and promoting its proteasomal degradation. RNF40-mediated HSP90α downregulation leads to the accumulation of malondialdehyde (MDA) and reactive oxygen species (ROS), thereby inhibiting CRC cell growth. Collectively, our findings provide mechanistic insights into how LeA directly targets HSP90α or facilitates RNF40-HSP90α-mediated degradation of HSP90α to regulate ferroptosis in CRC.
CDC73 is a canonical tumor suppressor; however, its biological functions in lung adenocarcinoma (LUAD) and the extracellular vesicle (EV)-mediated mechanisms through which it exerts its effects remain unclear. In this study, we systematically evaluated the diagnostic value, biological functions, and EV-mediated regulatory roles of CDC73 in LUAD. First, quantitative proteomic analysis was performed on plasma-derived EVs from six patients with early-stage LUAD and three healthy donors, followed by further validation of EV-borne CDC73 expression in plasma-derived EV samples from eight LUAD patients and eight healthy donors. We found that the level of EV-borne CDC73 protein (EV-CDC73) was significantly reduced in LUAD, indicating its potential value as a diagnostic liquid-biopsy biomarker. To define its functional role, we established LUAD cell lines stably overexpressing CDC73 (A549^OE and H1299^OE) and performed rescue experiments in combination with PTEN knockdown using siRNA. The tumor-suppressive effects of CDC73 were systematically assessed through experiments examining cell proliferation, cell-cycle progression, apoptosis, migration, and related molecular mechanisms. Meanwhile, EVs were isolated by ultracentrifugation from the conditioned media of A549, H1299, and human bronchial epithelial (HBE) cells to analyze the effects of EVs with different CDC73 levels on recipient A549 and HBE cells, and xenograft models were used for in vivo validation. The results showed that CDC73 overexpression markedly inhibited LUAD cell proliferation and migration, induced G1-phase arrest, and promoted apoptosis. Mechanistically, CDC73 exerted its tumor-suppressive effects by upregulating PTEN and inhibiting AKT activation, whereas PTEN knockdown partially reversed these effects. In vivo experiments further confirmed that CDC73 overexpression suppressed tumor growth and migration. In addition, EVs derived from LUAD cells with high CDC73 expression showed a reduced ability to induce malignant transformation-related phenotypes in HBE cells and suppressed the PTEN/AKT signaling axis; conversely, EVs derived from HBE cells with high CDC73 expression inhibited the malignant progression of LUAD cells. Collectively, CDC73 suppresses the malignant progression of LUAD through the PTEN/AKT signaling axis, while EV-borne CDC73 not only has potential diagnostic value but also directly regulates malignant phenotypes in recipient cells, highlighting its translational potential in the diagnosis and treatment of LUAD.
The presence or absence of lymph node metastasis (LNM) determines the staging and treatment strategy for cervical cancer. However, preoperative diagnosis of LNM remains insufficiently accurate and the underlying mechanisms are not fully understood. This study aimed to identify proteomic biomarkers associated with LNM and investigate the role of AKR1C2 in promoting LNM. Data-independent acquisition proteomic analysis indicated that LNM was significantly associated with extracellular matrix organization. A model with AKR1C2, ARHGAP26 and ATP6AP2 demonstrated optimal sensitivity of 95.8% and specificity of 72.7% (AUC, 0.890; 95% CI, 0.824–0.955; p < 0.001), which was confirmed in the validation cohort. Functional assays showed that AKR1C2 promotes migration, invasion and LNM in cervical cancer cells. RNA sequencing further supported its involvement in extracellular matrix reorganization. In vitro and rescue assays showed that SPINT2 mediates the effect of AKR1C2 on migration and invasion. GEPIA database analysis also revealed that SPINT2 is associated with worse prognosis. Furthermore, we found that AKR1C2 directly interacts with H4C13 in the nucleus, regulating SPINT2 expression and consequently influencing migration and invasion ability of cervical cancer cells. In conclusion, a proteomic model based on AKR1C2, ARHGAP26 and ATP6AP2 accurately predicts LNM in cervical cancer and could assist in clinical decision-making between radical surgery and concurrent chemoradiotherapy. AKR1C2-H4C13 interaction regulates SPINT2 expression, ultimately changing extracellular matrix and promoting LNM in cervical cancer.
The majority of patients with colorectal cancer (CRC) fail to respond to anti-PD-1/PD-L1 immunotherapy, highlighting an urgent need for alternative immunotherapeutic targets. The immunosuppressive ligand CD155 potently dampens natural killer (NK) cell cytotoxicity via inhibitory receptors, but how CRC cells sustain high CD155 surface levels remains elusive. Here, we identify aberrant CD155 N-glycosylation as a critical driver of CRC immune evasion. We find that the glycosyltransferase STT3A contributes to N-glycosylation of CD155. This modification is essential for maintaining CD155 protein stability, thereby sustaining its surface expression to suppress NK cell-mediated antitumor immunity. Consistent with a role for N-glycosylation in maintaining CD155 expression, pharmacological inhibition of oligosaccharyltransferase activity with NGI-1 promotes CD155 turnover, effectively abrogating its immunosuppressive function and enhancing NK cell-mediated tumor clearance in vivo. Collectively, these findings implicate the STT3A-CD155 glycosylation axis as a therapeutic vulnerability, providing a mechanistic rationale for reprogramming the immunosuppressive CRC microenvironment.
Hepatocellular carcinoma (HCC) exhibits a high propensity for metastasis and recurrence, leading to poor prognosis. Intrahepatic metastasis is more common than extrahepatic spread. Metastases arise through diverse mechanisms, including the involvement of cancer stem cells (CSCs) and the immune-modulatory role of the liver tissue microenvironment. As hepatic resident macrophages, Kupffer cells play a critical role in immune surveillance by phagocytosing disseminated HCC cells. However, the molecules regulating HCC cell stemness properties, Kupffer cell phagocytosis, and further HCC intrahepatic metastasis remain poorly understood. In the present study, we found that the long noncoding RNA LINC02709 was upregulated in HCC and correlated with stemness properties, intrahepatic metastasis, and poor prognosis. LINC02709 not only enhanced stemness properties of HCC cells, but also suppressed the phagocytosis of HCC cells by Kupffer cells, leading to increased liver metastasis. LINC02709 upregulated CD24 and CD47, which mediated the effects of LINC02709 on HCC cell stemness properties and Kupffer cell phagocytosis. LINC02709 was identified as an m6A-modified transcript. m6A-modified LINC02709 bound and recruited the m6A reader FXR1 and the DNA demethylase TET1 to the CD24 and CD47 promoters, inducing DNA demethylation and transcriptional activation of CD24 and CD47. The effects of LINC02709 on CD24/CD47, stemness properties, phagocytosis, and liver metastasis were all dependent on m6A modification. Our findings demonstrate that m6A-modified LINC02709 is a novel and promising biomarker for HCC recurrence and survival, enhancing HCC cell stemness properties, suppressing Kupffer cell phagocytosis, and driving HCC intrahepatic metastasis by epigenetically activating CD24 and CD47. Targeting m6A-modified LINC02709 represents a promising therapeutic strategy for HCC.
