Major-groove RNA triple helices are conserved tertiary structures formed when a third strand inserts into the major groove of a classic double helix either via Hoogsteen base pairing or reverse Hoogsteen base pairing. These structures are widely distributed in eukaryotic, prokaryotic, and viral RNAs. This paper systematically summarizes the structural classification, stability-influencing factors, and identification methods of RNA triple helices. A growing body of evidence indicates that RNA triple helices are extensively involved in diverse biological processes, including RNA stability regulation, translation regulation, riboswitch ligand recognition, transposition regulation, telomerase activity, and the assembly of the spliceosome catalytic core, as well as serving as scaffolds for molecular recruitment. Notably, dysregulation of RNA triple helices is closely associated with tumorigenesis, viral infections, and genetic diseases. Based on their structural and functional characteristics, multiple therapeutic strategies targeting RNA triple helices have been explored, such as small molecules and antisense oligonucleotides. Collectively, RNA triple helices represent a pivotal link between RNA structural biology and precision medicine, with promising potential as an important candidate target for future RNA structural drug development.
The modern era of precision oncology strives to identify novel and selective druggable targets that, on the one hand, achieve efficacious anti-tumor activity, and on the other hand inflict minimal adverse effects to healthy tissues. The realization that both the tumor microenvironment and the tumor immune microenvironment (TIME) modulate tumor progression and response to chemotherapeutics, has pushed tumor-associated immune cells to the forefront as bona fide druggable targets. Based on their unique overexpression pattern on the cell surface of specific human malignancies and suppressor immune cells, folate receptors FRα and FRβ constitute optimal facilitators of noninvasive cancer detection and localization, as well as efficacious selective delivery of potent therapeutic payloads. Along this vein, this review highlights recent advanced strategies utilizing FR-targeting for precise tumor and metastatic lesion localization for guided surgical precision resection, potent anti-cancer efficacy and reprogramming of the suppressive TIME. Notably, these activities were accomplished with minimal side effects to healthy tissues. AZD5335, a recent clinically tested FRα-targeted antibody-drug conjugate carrying a topoisomerase I inhibitor payload, exceeded its predecessors by demonstrating remarkable efficacy against low FRα-expressing tumors as well. Thus, FRs emerge as selective and promising targets for advanced precision oncology of various malignancies including those displaying chemoresistance.
Glioma is a highly aggressive brain tumor with poor prognosis and limited therapeutic options. Although temozolomide (TMZ) remains the standard chemotherapeutic agent for glioma, frequent recurrence and the development of therapeutic resistance continue to limit clinical benefit, highlighting the need for new molecular targets and treatment strategies. Here, we identify neuromedin U receptor 2 (NMUR2) as a driver of glioma progression and a potential therapeutic target. NMUR2 expression was markedly elevated in glioma tissues and positively associated with tumor grade. Functional analyses showed that NMUR2 promoted glioma cell proliferation and migration, whereas NMUR2 silencing attenuated these malignant phenotypes. Mechanistically, NMUR2 activated Gαq-dependent Ca²⁺ signaling, leading to STAT5 phosphorylation and subsequent transcriptional upregulation of the cell cycle-associated genes PIM1 and FOXM1. Drug-repurposing screening of 6,331 compounds identified NNC 05-2090 as a candidate NMUR2 antagonist. NNC 05-2090 blocked NMUR2-mediated Gαq/Ca²⁺/STAT5 signaling, which was associated with reduced PIM1 and FOXM1 expression, cell cycle arrest, and suppression of glioma growth in vitro and in vivo. In addition, combination treatment with TMZ produced synergistic anti-tumor effects in glioma models. Collectively, our findings define a previously unrecognized NMUR2/Gαq/STAT5/PIM1-FOXM1 signaling axis in glioma and support pharmacological inhibition of NMUR2 as a potential therapeutic strategy.
Regulatory T cells (Tregs) maintain intestinal immune homeostasis, but their therapeutic potential is constrained by a fundamental paradox: the same plasticity that enables tissue repair renders FOXP3 vulnerable to degradation in chronic inflammation. Mechanistically, microbial metabolites (short-chain fatty acids, bile acids) and retinoic acid stabilize FOXP3 and induce RORγt⁺/GATA3⁺ Treg specialization. In contrast, inflammatory cytokines and succinate accumulation drive ER stress and post-translational FOXP3 degradation, leading to lineage instability in inflammatory bowel disease, colorectal cancer, and celiac disease. Current Tregs-based therapies-adoptive transfer, low-dose IL-2, CAR-Tregs, and microbiota consortia-have demonstrated safety profiles yet exhibit limited efficacy due to this inherent instability. Next-generation strategies therefore focus on actively stabilizing FOXP3 (e.g., gut-restricted HDAC inhibitors) and engineering exhaustion-resistant CAR-Tregs. Three questions remain for clinical translation: how to preserve Treg stability without compromising anti-tumor immunity; which biomarkers (succinate, TSDR methylation, FOXP3Δ2/FL ratio) predict response; and whether logic-gated CAR-Tregs can overcome exhaustion. Addressing these challenges will enable the development of precision Treg immunotherapy for intestinal diseases.
Preclinical research traditionally advances through hypothesis-driven experimentation that establishes mechanistic pathways to support translational development. While this approach has generated major biological insights, it may underemphasize alternative organizational patterns embedded within complex datasets, particularly in rare diseases where opportunities for experimental reiteration are limited. Recent advances in conversational artificial intelligence (AI) provide an opportunity to support structured analytical dialogue as a complementary approach for re-examining validated experimental observations. Here, we evaluated the feasibility and informative value of an investigator-led structured analytical dialogue to reinterpret a previously published preclinical study of Hutchinson-Gilford Progeria Syndrome (HGPS), a rare disorder characterized by accelerated cardiovascular aging. Investigators defined the analytical questions, established interpretative boundaries, and critically evaluated successive AI-generated outputs, while the AI platform functioned exclusively as an analytical support tool for exploring complementary conceptual organization of experimentally validated findings. The original study showed that delivery of the longevity-associated LAV-BPIFB4 gene preserved left ventricular diastolic function, reduced perivascular fibrosis, increased coronary arteriole density, and attenuated cellular senescence without modifying progerin accumulation. Structured analytical dialogue generated complementary hierarchical interpretations of these observations. By integrating graphical dispersion with individual-level numerical data, the investigator-led dialogue identified heterogeneous response trajectories and suggested that cardiovascular protection may be viewed as emerging from coordinated interactions between nuclear stress adaptation and vascular remodelling within a broader resilience framework. These interpretations are presented as hypothesis-generating conceptual extensions rather than new experimental findings. This study demonstrates the feasibility of structured investigator-led analytical dialogue as a complementary methodological approach for broadening interpretation of existing preclinical datasets while preserving the original experimental evidence. By making analytical reasoning more transparent and explicitly distinguishing validated observations from conceptual reinterpretation, this framework may assist prioritization of future mechanistic investigations, particularly in rare cardiovascular diseases where maximizing insight from existing datasets is especially important.
Tumor-associated macrophages (TAMs), a major glucose-consuming population within the tumor microenvironment (TME), utilize glycolysis to support tumor progression. Therefore, concentrating on the glycolysis of intratumoral TAMs appears to be a promising research direction for tumor therapy. In this study, we demonstrated that the key glycolytic component named glucose transporter protein 1 (GLUT1, also known as SLC2A1) is extensively expressed in TAMs and is frequently associated with tumor progression. To dissect the functions of GLUT1 in macrophages on influencing the TME, macrophage-specific Glut1 knockout mice were generated and tumor-bearing mouse models were established. It was found that macrophage-specific deletion of Glut1 retarded the development of tumors and reshaped the tumor immune microenvironment (TIME). Loss of Glut1 in macrophages limited the secretion of multiple inflammatory mediators, including IL-6, IL-10, VEGF, and CXCL2, by virtue of the GSK3β/β-catenin/CD36 signaling axis, thereby normalizing the tumor blood vessels. To pharmacologically manipulate the expression of Glut1 in macrophages, M2-PLGA@WZB117 (poly lactic-co-glycolic acid, PLGA) was constructed accordingly. Interestingly, M2-PLGA@WZB117 resulted in significant inhibition in tumor progression, accompanied by tumor vascular normalization and a concurrent increase in the infiltration of CD8+ T cells. Therefore, our study provides new insights into reprogramming TAMs to exert impacts on tumor endothelium and thus regulating the TME, thereby hindering the development of tumors.
Macrophage senescence is a pathological feature in aging or diseased kidneys. However, the role of senescent macrophages in kidney injury and aging has not been fully elucidated yet. We integrated the analysis of single-cell RNA sequencing datasets and the adoptively transfusion of pretreated bone marrow-derived macrophages to investigate the role of renal macrophage senescence in kidney injury. Here, we portrayed the senescence trajectory along multiple time points in infiltrating macrophages, and observed the persistent increase of macrophage-expressed purinergic receptor P2RX7 along the senescence trajectory in injured kidneys of septic mice. Importantly, our discovered small-molecule P2RX7 antagonist strikingly improved kidney function and pathological damage, as well as mitigated macrophage senescence in septic and aging mice. Mechanistically, P2RX7 antagonist could promote the wound healing, migration and proliferation capacity of senescent reparative macrophages, thus exerting anti-inflammatory effects and repairing kidney tissues. Together, our findings illustrate the crucial participation of senescent macrophages in septic kidney injury, and offer novel therapeutic strategy via intervening P2RX7 against immunosenescence-associated kidney injury and aging.
DNAJB4, a member of the DNAJ/HSP40 family, functions as a co-chaperone of HSP70, regulating protein homeostasis and cellular functions. However, the molecular mechanism underlying the biological effect of DNAJB4 on lipid metabolism remains unclear. We investigated the role of DNAJB4 and its molecular mechanism in hyperlipidemia and atheroprone apolipoprotein E-null (apoe-/- ) mice. Western blot analysis and immunohistochemistry were used to assess decreased DNAJB4 expression in apoe-/- mice. Moreover, the genetic deletion of DNAJB4 led to an increase in hepatic lipid accumulation and hyperlipidemia in apoe-/- mice, as evidenced by decreased expression of proteins related to cholesterol esterification and clearance, and an increased hepatic level of triglycerides, fatty acids, glycerol, free cholesterol, total cholesterol, and bile acid. Mechanistically, DNAJB4 deficiency impaired the protein stability of HSP70, reduced nuclear HSP70 association, and downregulated HSP70-induced LXRα transcription. The genetic deletion of DNAJB4 also promoted LXRα protein degradation and reduced LXRα autoregulation, thereby exacerbating the decrease in LXRα and LXRα-mediated gene expression. Furthermore, treatment with curcumin and andrographolide, the inducers of DNAJB4, did not reduce the atherosclerotic lesions at the aortic sinus in apoe-/-dnajb4-/- mice, suggesting that DNAJB4 is required for the atheroprotective effect of curcumin and andrographolide. Our findings indicate that DNAJB4 plays a crucial role in regulating the HSP70-LXRα axis in hyperlipidemia, hepatic lipid accumulation, and atherosclerosis. Here, we identify DNAJB4 as a critical HSP70 co-chaperone that stabilizes HSP70, promotes its nuclear association, and sustains LXRα autoregulation in hepatic lipid metabolism.
CD8⁺ T-cell exhaustion is a distinct differentiation state driven by persistent antigen stimulation, and its establishment and maintenance are major barriers to effective cancer immunotherapy. Although the transcriptional and epigenetic landscapes of exhausted CD8⁺ T cells have been extensively characterized, it remains unclear how sustained external stimulation is integrated at the level of protein function to produce stable dysfunction and altered cell fate. Post-translational modifications constitute a key regulatory layer of protein function. They form a dynamic network that links persistent antigenic stimulation and tumor microenvironmental stress to cell fate, and therefore provide a critical entry point for understanding how exhaustion is initiated and maintained. In this review, we focus on how post-translational modifications convert persistent antigen stimulation and tumor microenvironmental stress into protein-level dysregulation and ultimately lock CD8⁺ T cells into an exhausted fate. We summarize how multiple classes of post-translational modifications drive exhaustion through effects on signal transduction, protein homeostasis, metabolic stress responses, and epigenetic reprogramming. We then discuss potential intervention strategies centered on critical regulatory nodes that may preserve the plasticity of precursor exhausted CD8⁺ T cells, restrain stabilization of the terminally exhausted state in CD8⁺ T cells, and optimize rational combination therapies. Finally, we outline the translational challenges and future directions of targeting post-translational modifications, and emphasize that identifying actionable modification nodes will be important for patient stratification and combination design in cancer immunotherapy.
The incidence of early-onset colorectal cancer (EO-CRC, <50 years) is rising worldwide, highlighting the need to better understand the underlying causes of this age-related divergence. We used paired patient-derived normal and tumor colorectal organoids to investigate the therapeutic window of chemotherapeutic agents and its modulation by calcitriol (the most active vitamin D metabolite). Dose-response analyses revealed marked interpatient variability, with SN38 being more potent than 5-fluorouracil (5-FU) and oxaliplatin. Normal organoids were more resistant than paired tumor counterparts to 5-FU and oxaliplatin in both EO-CRC and late-onset CRC (LO-CRC, >50 years), indicating selective tumor cytotoxicity. In contrast, the therapeutic window for SN38 was preserved in LO-CRC but not in EO-CRC organoids, revealing age-dependent differences in drug sensitivity. Moreover, calcitriol reduced the cytotoxicity of 5-FU and SN38 in normal organoids regardless of patient age, while in tumor organoids this protective effect was restricted to EO-CRC. As a consequence, calcitriol treatment selectively expanded the therapeutic window for 5-FU and SN38 in LO-CRC organoids. Mechanistically, these effects correlated with calcitriol-induced antiproliferative action and transcriptional regulation of drug metabolism-related pathways. Overall, our findings identify age-dependent differences in chemotherapy response and support the importance of maintaining adequate vitamin D status to reduce chemotherapy-associated toxicity.
Aberrant activation of macrophages and their amplification of inflammatory responses constitute the core pathological basis driving the progression of acute lung injury (ALI). Celastrol (CE), despite its potent anti-inflammatory activity, suffers from poor aqueous solubility and substantial systemic toxicity, which severely limit its clinical translation. Capitalizing on the metabolic signature of pro-inflammatory M1 macrophages, specifically their high expression of glucose transporter 1 (GLUT1), we designed a glucose-modified CE prodrug that self-assembled into carrier-free nanoparticles CG NPs. With markedly improved solubility and systemic stability, CG NPs rapidly and persistently accumulated in the inflammatory lungs of LPS-induced ALI mice facilitated by GLUT1-mediated targeting and uptake by M1 macrophages. Compared with free CE, CG NPs exhibited enhanced overall therapeutic efficacy while significantly reducing hepatorenal toxicity. Mechanistic studies revealed that by targeting Drp1, CG NPs disrupt Drp1-MiD51 interaction, thus inhibiting excessive mitochondrial fission and ROS accumulation, which blocks NF-κB-mediated inflammatory signaling and M1-driven cytokine release. Molecular docking suggested that glucose conjugation may confer CG with a superior ability to regulate mitochondrial homeostasis over CE, potentially driven by its unique U-shaped conformation that inserts into Drp1 and forms a denser hydrogen-bond network, which could contribute to enhanced binding affinity. In summary, this study proposes a nanoprodrug strategy that combines precise targeting with mitochondrial protection, offering a promising therapeutic avenue for inflammatory diseases such as ALI.
Liver metastasis is a major factor contributing to the poor prognosis of pancreatic ductal adenocarcinoma (PDAC). The formation of pre-metastatic niche (PMN) initiates the process of liver metastasis. Exosomes (Exos) act as key mediators of crosstalk between the tumor microenvironment (TME) and the PMN to activate hepatic stellate cells (HSCs) and remodel the stiff extracellular matrix (ECM). In this study, we isolated Exos derived from PDAC cells cultured under acidic conditions and demonstrated that these Exos significantly activate HSCs and promote the remodeling of the stiff ECM, thereby promoting the stemness, migration, and invasion of PDAC cells. High expression of exosomal miR-1246 was screened by miRNA-sequencing, and Wiskott-Aldrich syndrome protein Family Member 3 (WASF3) was identified as the target of miR-1246. Mechanistically, exosomal miR-1246 activates HSCs to remodel the ECM by targeting WASF3 and stimulating the phosphatidylinositol 3-kinase-serine/threonine protein kinase (PI3K/Akt) pathway. Notably, RNA-binding protein immunoprecipitation (RIP) and miRNA pull-down assays were performed to identify that Human Antigen R (HuR) contributes to the enrichment of miR-1246 into Exos. Collectively, exosomal miR-1246 activates HSCs and remodels the stiff ECM to promote liver metastasis, and it may serve as a potential diagnostic and prognostic marker for PDAC liver metastasis.
Mitochondrial homeostasis has attracted increasing interest and is now recognized as playing a significant role in both kidney development and the progression of kidney disease. Among these, the latest approach, mitophagy, has been shown to be activated dynamically and reversibly under various physiological conditions, including reactive oxygen stress, nutrient deficiency, and cellular senescence, to maintain mitochondrial homeostasis and function. Moreover, findings indicate that mitophagy can also maintain mitochondrial quality through interactions and mutual regulation with mitochondrial dynamics. Crucially, a growing number of kidney diseases, such as acute kidney injury, diabetic kidney disease, and other chronic kidney diseases, are linked to abnormal levels of mitophagy. In this review, we comprehensively examined the vital role of mitophagy in kidney diseases, discussed the potential of mitophagy-targeted therapies, and described the detailed alterations in specific mitophagy-related proteins associated with kidney diseases.
Mitochondrial targeting represents a promising antitumor strategy by modulating cell differentiation, metabolic reprogramming, and immune responses. While chlorogenic acid (CGA) has demonstrated the ability to induce tumor cell differentiation and enhance antitumor immunity, the involvement of mitochondrial regulation in these effects remains unclear. This study investigated whether CGA mediates antitumor immune effects through mitochondrial regulation, thereby providing a theoretical framework for natural product-based, mitochondria-targeted therapies. Our findings reveal that CGA inhibits the translocation of mitochondrial transcription factor A (TFAM) into the mitochondria and promotes mtDNA leakage by disrupting the ATF5-mtHSP70 signaling axis. The cytosolic leakage of mtDNA activates the cGAS/STING pathway, triggering the activation of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), which ultimately facilitates antitumor immunity. In a mouse model, ATF5 knockout enhances cGAS/STING signaling and subsequent immune responses, leading to tumor growth inhibition. These results highlight a novel role of CGA in regulating mitochondrial-associated proteins, positioning it as a potential therapeutic strategy for cancer via the mtDNA-cGAS-STING pathway.
Nephropathic cystinosis is a rare genetic lysosomal storage disorder caused by loss-of-function variants of the CTNS gene encoding for the lysosomal H+/cystine symporter cystinosin. The lack of cystinosin causes cystine accumulation in lysosomes and cell damage. Children with nephropathic cystinosis commonly exhibit skeletal abnormalities, including growth retardation, osteopenia and rickets. The only cystine-depleting drug available is cysteamine. However, several observations suggest that cysteamine, particularly at high doses, may have negative effects on the skeleton. In this study, we characterized the effects of cysteamine on bone cells and we identified Pepstatin A, a Cathepsin D-inhibitor, as a potential new therapeutic treatment to restore physiological bone remodeling in cystinosis. We observed a detrimental effect of cysteamine on bone cells inducing further impairment of osteoblast differentiation/activity and failure of cysteamine intraperitoneal injection to improve the somatic growth of pre-pubertal ctns-/- mice. Moreover, we demonstrated that Pepstatin A treatment increased bone formation in vitro acting on the Fibronectin/Sipa1/STAT3/JunB pathway, and rescued somatic growth and bone mass in pre-pubertal ctns-/- mice. Our results provide evidence that cysteamine negatively affects bone in cystinosis and that Cathepsin D inhibition could represent a new therapeutic approach for rescue of bone remodeling in cystinosis.
Hepatocellular carcinoma (HCC) represents a formidable oncological challenge characterized by complex molecular pathogenesis and limited therapeutic outcomes, particularly in the context of liver transplantation. As the sixth most commonly diagnosed cancer and the third leading cause of cancer-related mortality worldwide, HCC poses significant clinical challenges that demand innovative therapeutic approaches. Central to HCC development and progression is a pathogenic triad comprising nuclear factor-kappa B (NF-κB), hypoxia-inducible factor-1α (HIF-1α), and oxidative stress-three interconnected pathways that drive inflammation, angiogenesis, metabolic reprogramming, and cell survival. This comprehensive review examines the molecular mechanisms underlying this triad in HCC pathogenesis across different etiological contexts, including viral hepatitis and non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH). We critically analyse the unique clinical challenges posed by HCC in liver transplantation recipients, particularly the paradoxical requirement for immunosuppression alongside antitumor immunity, and constraints surrounding immunotherapy application. Furthermore, we present CIGB-552, a novel peptide therapeutic targeting COMMD1 (Copper Metabolism MURR1 Domain-containing protein 1), as a promising dual-function agent capable of simultaneously disrupting the pathogenic triad through NF-κB inhibition, HIF-1α suppression, and strategic modulation of oxidative stress via SOD1 regulation. The multimodal mechanism of CIGB-552 offers a theoretically rational therapeutic approach for HCC management in both pre-transplant and post-transplant settings. Clinical validation in the transplantation setting is required.
Dedifferentiated liposarcoma (DDLPS) is an aggressive mesenchymal malignancy coexisting with a low grade well-differentiated component. Pathways implicated in liposarcoma growth and dedifferentiation are promoted by heparan sulfate (HS) proteoglycans (HSPG) and their modifying enzymes including heparanase. HSPGs serve as co-receptors enhancing tyrosine kinase signaling and tumor aggressiveness. Targeting these interactions bears promise in attenuating liposarcoma growth. We employed an investigational HS mimetic, the non-anticoagulant heparin CX-01 (dociparstat), to assess its HS competition impact on deregulated adipogenic differentiation and growth of human DDLPS cell lines and patient-derived xenografts (PDXs). Remarkably, CX-01 reduced colony formation and invasive capacities of DDLPS cell lines, inducing cytoskeleton remodeling, lipid accumulation and reactivation of adipogenic program. Mechanistic studies into the anti-DDLPS activity of CX-01 unveiled Syndecan 1 (SDC1)/heparanase system and receptor tyrosine kinase-AKT signaling as targets of cell growth inhibition and induction of differentiation. CX-01 treatment of mice harboring DDLPS PDXs attenuated tumor growth, enhanced lipid content and consistently altered the transcriptome, modulating pathways associated with tumor dedifferentiation (adipogenesis and fatty acid metabolism) and tumor-microenvironment interaction (TGFβ signaling, inflammatory response). In two independent cohorts of DDLPS patients, genes downregulated in CX-01-treated PDXs (SDC1, TIMP1, FN1, COL5A1, and MMP14), were found preeminently expressed in the dedifferentiated, compared to the well-differentiated tumor component and normal fat. This suggests a role for these genes in disease progression. Collectively, this study demonstrates the remarkable potential of HS competition to simultaneously block multiple anti-adipogenic players representing metabolic vulnerabilities, and to promote a differentiated tumor phenotype markedly less aggressive.
Intrahepatic cholangiocarcinoma (iCCA) is the second most prevalent liver cancer with a high mortality and recurrence rate, and remains a poorly understood disease. The tumor margin, as the transition zone between normal tissue and tumor, was not appreciated before. We performed mass cytometry (cytometry by time of flight, CyTOF) on 30 samples from iCCA tumor, paratumor and margin tissue. We found that the number of CD4+ central memory T cells (CD4+ Tcm) increased in the margin zone. Single cell RNA-seq (scRNA-seq) further discovered that CD4+ Tcm cells enriched in the margin zone of iCCA exhibited upregulated Annexin A1 (ANXA1) expression. Fibroblasts recruited CD4+ ANXA1+ Tcm cells via Chemokine (C-C motif) ligand 19 (CCL19)-Chemokine (C-C motif) receptor 7 (CCR7) signaling pathway. The molecular characteristics of CD4+ ANXA1+ Tcm in iCCA were characterized, and the interactions between these T cells and other cells were determined. Patients with enriched CD4+ ANXA1+ Tcm cells in the tumor margin exhibited a worse prognosis. Specifically, CD4+ ANXA1+ Tcm cells could recruit macrophages to the tumor margin and regulate the macrophage polarization via the ANXA1-Formyl Peptide Receptor 1 (FPR1) signaling axis. CD4+ ANXA1+ Tcm cell-activated macrophages could enhance the invasion and proliferation of tumor cells by exerting different cytokines. In conclusion, our study systematically characterized the features and distribution of CD4+ ANXA1+ Tcm cells in the margin zone of iCCA. We investigated the potential mechanisms by which CD4+ ANXA1+ Tcm cells affected tumor progression, and provided novel understanding of the function of these CD4+ Tcm cells in iCCA.
Angiomotin-like protein 2 (AMOTL2) regulates cell polarity and cytoskeletal regulation, but its role in gastric cancer (GC) remains undefined. Here, we show that AMOTL2 is frequently downregulated in GC tissues and cell lines, and its low expression is associated with deeper tumor invasion, lymph node metastasis, and shorter overall survival. Gain- and loss-of-function assays demonstrated that AMOTL2 suppresses GC cell proliferation, migration, and invasion. AMOTL2 overexpression also impaired endothelial and lymphatic-endothelial tube formation and reduced the expression of angiogenic and lymphangiogenic factors. In the subcutaneous, orthotopic, and popliteal lymph node metastasis models, AMOTL2 inhibited tumor growth and lymphatic dissemination. Mechanistically, AMOTL2 suppressed TGF-β/Smad signaling, as evidenced by reduced BMP2 and TGF-β1 expression, decreased Smad1/5/9 and Smad2/3 phosphorylation, and impaired Smads nuclear accumulation. Notably, pharmacological Smad1/5/9 activation by SB4 partially reversed the suppressive phenotypes of AMOTL2 in vitro and in vivo. Co-immunoprecipitation, GST pull-down, and ubiquitination assays identified SMURF1 as an AMOTL2-interacting E3 ubiquitin ligase that binds through its WW domains and promotes AMOTL2 ubiquitination and proteasomal degradation. AMOTL2 restoration counteracted SMURF1-driven malignant phenotypes. Collectively, these findings define AMOTL2 as a potential suppressor of GC progression that is destabilized by SMURF1, suggesting that the SMURF1-AMOTL2-Smad signaling axis may contribute to GC progression and lymphatic dissemination, representing a candidate therapeutic target that warrants further validation.
