Search Results (711)

Hepatoblastoma (HB) is the most common malignant liver tumor in children, yet therapeutic options remain largely confined to conventional chemotherapy. To identify novel therapeutic targets, we performed gene set enrichment analysis on three publicly available HB datasets and found consistent activation of the proteasome pathway, with marked overexpression of the β5 proteolytic subunit encoded by PSMB5. High PSMB5 expression was associated with poor survival in adult hepatocellular carcinoma, highlighting the proteasome as a candidate for therapeutic vulnerability. Targeting the β5 proteolytic subunit with its selective inhibitor carfilzomib in HB patient-derived xenograft (PDX) models resulted in dose-dependent reductions in cell viability, proliferation, and clonogenic growth, accompanied by induction of apoptosis. Importantly, carfilzomib retained efficacy in three-dimensional PDX cultures, underscoring its activity in physiologically relevant tumor models. Bioinformatic analyses revealed that carfilzomib activates apoptosis and reactive oxygen species (ROS) signaling. Validation experiments in HB cells demonstrated increased ROS levels, with ROS induction correlating with drug sensitivity. Notably, pharmacological scavenging of ROS completely abrogated carfilzomib-induced cytotoxicity, establishing oxidative stress as a key mediator of therapeutic response. Together, these findings identify PSMB5 as a therapeutically actionable target in HB and support proteasome inhibition as a promising precision medicine strategy in HB. Full article

Proteasome inhibitors (PIs) are central to multiple myeloma (MM) therapy; however, resistance remains a major clinical challenge, particularly in relapsed/refractory disease. To identify functional mediators of carfilzomib (CFZ) resistance, we performed complementary gain-of-function CRISPR activation and pharmacological screening approaches. These unbiased strategies converged on the E3 ubiquitin ligase MDM2 as a modulator of PI response. MDM2 transactivation enhanced MM cell survival and accelerated recovery following CFZ exposure, supporting a causal role in proteotoxic stress tolerance. Pharmacologic inhibition of MDM2 with NVP-CGM097 synergized with CFZ across multiple PI-sensitive and PI-resistant MM cell lines, irrespective of TP53 status. Mechanistically, MDM2 inhibition induced p21 upregulation, cell-cycle arrest, and reduced c-MYC expression, accompanied by impaired activation of DNA damage response mediators. Genetic silencing of MDM2 phenocopied these effects and increased CFZ sensitivity. Importantly, the combination retained efficacy in MM–stromal co-culture models and in primary patient samples, including cases harboring del(17p), while sparing normal peripheral blood mononuclear cells. Collectively, these findings identify MDM2 as a functional driver of PI resistance and support combined MDM2 and proteasome inhibition as a rational therapeutic strategy in MM, including TP53-deficient contexts. Full article

Background: Esophageal squamous cell carcinoma (ESCC) remains a highly lethal malignancy with limited therapeutic options, in part due to frequent activation of nuclear factor erythroid 2-related factor 2 (NFE2L2 or NRF2). Gain-of-function mutations in NRF2 disrupt its negative regulation by Kelch-like ECH-associated protein 1 (KEAP1), resulting in sustained NRF2 signaling that promotes tumor growth and resistance to chemotherapy and radiation. We previously identified the FDA-approved drug pyrimethamine (PYR) as an NRF2 inhibitor and demonstrated that inhibition of dihydrofolate reductase (DHFR) represents the primary mechanism underlying its NRF2-suppressive activity, supporting its advancement into a Phase I window-of-opportunity clinical trial (NCT 05678348). Meanwhile, in NRF2^W24C-KYSE70 and NRF2^D77V-KYSE180 cells, PYR promoted NRF2^Mut ubiquitination and proteasomal degradation and shortened its half-life. This study aims to explore additional modes of action by which PYR inhibits NRF2. Methods: Cell cycle analysis was performed by flow cytometry. Cell proliferation, apoptosis and chemosensitivity were assessed by Live-Cell Analysis System, while radiosensitivity was evaluated using X-ray irradiation and the CellTiter-Glo assay. Molecular interactions between NRF2 and KEAP1 were examined through Co-IP and PLA, and the direct binding of PYR to KEAP1 was quantified using ITC and SPR. Molecular docking and dynamic simulations were employed to predict potential PYR-binding pockets within the Kelch domain. Results: Using genetically defined isogenic ESCC cell models, we show that activation of mutant NRF2 (NRF2^Mut) or wild-type NRF2 (NRF2^WT) produces distinct, context-dependent effects on squamous differentiation, proliferation, and therapeutic response. We further demonstrate that PYR restores sensitivity to chemotherapy and ionizing radiation in NRF2^Mut ESCC cells. Mechanistically, short-term PYR treatment promotes KEAP1-dependent proteasome-mediated degradation of NRF2^W24C. Biochemical and biophysical assays indicate that PYR enhances the interaction between KEAP1 and NRF2^W24C in a manner associated with KEAP1-dependent proteasomal degradation. Computational modeling further suggests that PYR may engage a pocket within the Kelch domain to facilitate the NRF2^W24C-KEAP1 interaction. Conclusions: These findings show that PYR functionally restores KEAP1-mediated NRF2 degradation of select NRF2^Mut through a glue-like effect and overcomes therapy resistance in ESCC. Although the proposed glue-like mechanism remains hypothetical, this work supports further investigation into the NRF2–KEAP1 interaction and may inform the development of KEAP1-targeted strategies for NRF2^Mut cancers, including ESCC. Full article

Apoptosis, or programmed cell death, is a fundamental process essential for tissue homeostasis, development, and the elimination of damaged or potentially cancerous cells. Here, we identify the E3/E4 ubiquitin ligase UBE4B as a critical suppressor of apoptosis in p53-proficient tumor cells, functioning through a previously uncharacterized dual mechanism. Initially, an orthogonal ubiquitin transfer screening approach identified CCAR2 as a UBE4B substrate. We demonstrate that UBE4B interacts with and ubiquitinates CCAR2, promoting its proteasomal degradation. Furthermore, we found that UBE4B concurrently targets p53 itself for ubiquitin-dependent degradation. Functionally, UBE4B overexpression suppresses apoptosis, whereas rescue experiments indicate that restoring p53 expression reverses this suppression more effectively than restoring CCAR2, highlighting the dominance of the direct p53 degradation pathway. Mechanistically, UBE4B deficiency leads to CCAR2 accumulation, which inhibits SIRT1 activity, thereby enhancing p53 acetylation and stability; this effect is reversed upon CCAR2 co-depletion. Consistently, transcriptional profiling confirms that UBE4B downregulates key p53 target genes (e.g., BAX, PUMA) through this dual-pathway regulation. In summary, our study establishes that UBE4B acts as a key apoptosis suppressor by coordinately degrading both p53 and its positive regulator CCAR2, revealing a targetable vulnerability in p53-wild-type tumors. Full article

Bortezomib (BTZ), the first-generation proteasome inhibitor, has been approved for the treatment of relapsed, refractory, and newly diagnosed multiple myeloma. Despite its remarkable antitumor efficacy, BTZ treatment is severely limited by a high incidence of systemic adverse reactions, primarily due to its non-selective cytotoxicity toward rapidly dividing normal cells and its potent neurotoxic effects on peripheral neurons. Bortezomib-induced peripheral neurotoxicity (BIPN) manifests as neuropathic pain and sensory abnormalities, affecting up to 31% to 64% of patients and limiting BTZ’s clinical use. Currently, the underlying mechanisms of BIPN are poorly understood. To evaluate the effects of BTZ on the functions of peripheral nerves in mice, we administered an intraperitoneal injection treatment for four weeks. Results indicated that BIPN caused mechanical allodynia, gait abnormalities, and pathological changes in myelin and axons in mice. This study confirms that BTZ upregulates the expression of the activating transcription factor 3 (ATF3), which in turn mediates the increased expression of the copper transporter SLC31A1, causing dysregulation of intracellular copper ion homeostasis and subsequent copper accumulation, and ultimately inducing the development of peripheral neurotoxicity. Elevated intracellular copper concentration exerts a dual effect: it directly promotes the oligomerization of Dihydrolipoamide S-acetyltransferase (DLAT) and concurrently damages the iron–sulfur cluster protein ferredoxin 1 (FDX1), collectively triggering the onset of cuproptosis. Green tea has garnered attention for its rich content of catechins, with (−)-Epigallocatechin Gallate (EGCG) being the most abundant catechin present. This study uncovers the molecular mechanism by which EGCG inhibits BTZ-induced cuproptosis through targeted regulation of copper homeostasis. Analyses demonstrate that EGCG significantly downregulates the expression of the copper transporter SLC31A1, thereby effectively suppressing transmembrane influx of extracellular copper ions. This intervention markedly reduces intracellular copper overload, eliciting a dual regulatory effect: on one hand, the decreased copper concentration directly inhibits the oligomerization of DLAT; on the other hand, it effectively protects the iron–sulfur cluster protein FDX1 from damage. This study aims to systematically elucidate the molecular mechanisms underlying BIPN and to evaluate the therapeutic potential of EGCG in alleviating BIPN, offering a novel therapeutic strategy for the prevention and treatment of BIPN. Full article

Background/Objectives: MYC-driven tumors exhibit significant glutamine addiction, but the metabolic adaptation mechanisms enabling their survival under glutamine deprivation remain incompletely understood. Malic enzymes catalyze the oxidative decarboxylation of malate to pyruvate while generating NADPH, linking central carbon metabolism to redox homeostasis. This study investigates whether and how ME1 and ME2 mediate cell adaptation to glutamine starvation and explores their functional division in relation to p53 status. Methods: Using MYC-amplified, p53-mutant (G266E) SF188 glioblastoma cells, we performed siRNA-mediated knockdown, overexpression, and rescue experiments. Cell survival was assessed by trypan blue exclusion and Annexin V/PI staining. ROS levels and NADP⁺/NADPH ratios were measured by DCFH-DA fluorescence and enzymatic assays. Metabolite tracing was conducted using [U-¹³C₅] glutamine followed by LC-MS. Key findings were validated in additional cell lines including HCT116, U2OS and MDA-MB-231. Results: ME1 and ME2 promote SF188 cell survival under glutamine deprivation, an effect that depends on their catalytic activity but is independent of TCA cycle anaplerosis. ME1 maintains redox balance by generating NADPH, and antioxidant treatment rescues the survival defect caused by ME1 knockdown. In contrast, ME2 does not contribute to redox regulation but stabilizes mutant p53 (G266E) via proteasome inhibition. Both of these pro-survival functions are attenuated upon MYC knockdown, suggesting a dependency on MYC expression. Across all cell lines tested, ME1 and ME2 also promote survival through redox maintenance, although the isoform responsible for antioxidant function differs. Conclusions: ME1 and ME2 support metabolic adaptation to glutamine starvation through distinct, isoform-specific mechanisms that depend on MYC expression and p53 mutation status. These findings suggest malic enzymes as potential therapeutic targets in MYC-driven, p53-mutant tumors. Full article

Background: Idiopathic inflammatory myopathies (IIMs), characterized by muscle weakness and chronic inflammation, currently lack highly effective therapies. This study investigated the therapeutic potential and underlying mechanism of (5R)-5-hydroxytriptolide (LLDT-8), a triptolide derivative with reduced toxicity, using an experimental autoimmune myositis (EAM) mouse model and in vitro assays. Methods: Forty female BALB/c mice were randomly assigned to five groups: normal, vehicle, methylprednisolone (MP), LLDT-8 (0.0625 mg/kg), and LLDT-8 (0.125 mg/kg). EAM mice were treated with LLDT-8 (0.0625 or 0.125 mg/kg) or methylprednisolone as a positive control. Cellular experiments and molecular docking were performed to investigate potential mechanisms of LLDT-8. Results: LLDT-8 significantly attenuated clinicopathological features, including muscle weakness and pain sensitivity, while reducing serum levels of aspartate aminotransferase and lactate dehydrogenase. Histological analysis revealed that LLDT-8 reduced inflammatory cell infiltration and the presence of CD4⁺ and CD8⁺ T cells in muscle tissues. Mechanistically, LLDT-8 inhibited the expression of nucleotide-binding oligomerization domain receptor caspase recruitment domain 5 (NLRC5), a key transcriptional regulator of major histocompatibility complex-I (MHC-I). This suppression extended to downstream antigen presentation-related molecules, including the transporter associated with antigen processing and proteasome 20S subunit beta. Molecular docking further confirmed the high binding affinity of LLDT-8 to both NLRC5 and MHC-I. Conclusions: LLDT-8 alleviates inflammatory muscle injury by targeting the NLRC5/MHC-I signaling axis, suggesting it may be a promising therapeutic candidate for IIMs. Full article

Background: Glioblastoma (GBM) is the most common and aggressive brain tumor in adults. Changes in the ubiquitination system in GBM cells can promote uncontrolled tumor growth and reduce the effectiveness of treatments. However, the exact targets and regulatory elements of the ubiquitin–proteasome system involved in GBM are still not well understood. Methods: All data were obtained by using in silico analysis, immunohistochemistry, Western blot, RT-qPCR, gene silencing and proliferation assay. Results: Computational and protein analyses show that aggressive gliomas have higher expression of the RING ligase RNF182, with significantly greater levels in glioblastoma (GBM) than in low-grade gliomas. Elevated RNF182 is strongly associated with GBM growth. Experiments using siRNA to inhibit RNF182 in the human glioblastoma cell line U87MG significantly reduced cell proliferation, suggesting that RNF182 promotes tumor growth and may be a potential therapeutic target. Conclusions: These findings create a connection between the ubiquitin–proteasome system and the unchecked growth observed in GBM, identifying RNF182 as a new marker associated with GBM proliferation and an additional target for GBM treatment. Full article

Background/Objectives: EBV is an oncogenic virus linked to NPC and GC, driving cisplatin resistance. Resveratrol has anticancer activity, but its targets and mechanisms against EBV-positive cancers remain unclear. Methods: We assessed resveratrol’s cytotoxicity in EBV-positive cells via functional assays, identified targets by chemical similarity search and molecular docking, and validated PTPN1 via in vitro experiments and nude mouse xenograft models. Results: Resveratrol inhibited EBV-positive cell viability in a time- and concentration- dependent manner, with IC50 values ranging from 35.85 to 145.7 μM across different cell lines at 24–72 h. Apoptosis rates increased by approximately 2- to 4-fold after 80 μM resveratrol treatment for 24 h. Resveratrol directly targeted PTPN1 (docking score = −4.89) and promoted its degradation via the proteasome pathway, as MG132 reversed this effect. Notably, resveratrol synergized with cisplatin (combination index < 1) to reverse cisplatin resistance in both in vitro and in vivo models. Furthermore, resveratrol induced EBV lytic reactivation through ROS production, as evidenced by the increased expression of BZLF1, BMRF1, and BALF2, which was attenuated by the ROS scavenger NAC. Conclusions: Our findings identify PTPN1 as a direct anticancer target of resveratrol in EBV-positive cancers. Resveratrol enhances the therapeutic efficacy of cisplatin via PTPN1 proteasomal degradation and induces EBV lytic reactivation through ROS accumulation. These findings provide a mechanistic basis for the development of novel combination therapies targeting EBV-associated malignancies. Full article

Intracerebral hemorrhage induces severe secondary brain injury characterized by excessive neuroinflammation and inefficient hematoma clearance, processes largely governed by microglial polarization and phagocytic activity. The immunoproteasome, an inducible proteasome isoform involved in immune regulation, has been implicated in inflammatory neurological disorders, but its role in microglial responses after ICH remains unclear. In this study, rat models of common hemorrhage, severe hemorrhage, and severe hemorrhage with hematoma aspiration were used to represent graded injury severity and post-evacuation recovery. Transcriptomic profiling at day 3 post-injury identified immunoproteasome-associated gene networks, while expression of the catalytic subunits LMP2 and LMP7, microglial polarization markers, and phagocytic receptors was analyzed by Western blotting and immunofluorescence. Severe hemorrhage markedly induced LMP2 and LMP7 expression, predominantly in Iba1⁺ microglia, accompanied by enhanced ER stress, NF-κB signaling, and M1-like polarization and reduced phagocytic marker expression. Hematoma aspiration attenuated immunoproteasome expression and restored M2-associated and phagocytic signatures. Consistently, pharmacological inhibition of immunoproteasomes in primary microglia enhanced erythrophagocytosis and promoted a reparative phenotype in vitro. These findings indicate that immunoproteasome activation links hemorrhagic severity to maladaptive microglial polarization and impaired hematoma clearance after ICH, and that reducing immunoproteasome expression may help rebalance inflammatory and phagocytic microglial functions. Full article

Arsenic exposure increases lung cancer risk, yet its molecular mechanisms remain unclear but are linked to peroxisome proliferator-activated receptor gamma (PPARγ). We investigated PPARγ-related molecules affected by sodium arsenite (NaAR) in non-small cell lung cancer (NSCLC) cells using immunochemical, gene knockdown, and immunoprecipitation approaches. PPARγ was critical for NSCLC growth, as high PPARγ-expressing A549 cells proliferated more than low-expressing H1299 cells after NaAR treatment. In A549 cells, NaAR upregulated polyubiquitinated PPARγ, activating cell cycle arrest and DNA damage response pathways. Rather than inducing significant caspase-dependent apoptosis, NaAR activated nuclear factor-kappa B and downregulated mixed lineage kinase domain-like (MLKL) via K63-linked polyubiquitinated receptor-interacting protein kinase 1, thereby inhibiting apoptosis and necroptosis. PPARγ knockdown or NAD⁺ supplementation induced PARP-1 hyperactivation and MLKL upregulation, leading to DNA damage and necroptosis. PARP-1 inhibition by 3-aminobenzamide induced apoptosis, indicating that PPARγ regulates apoptosis and necroptosis through PARP-1 activation. Proteasome inhibition increased polyubiquitinated PPARγ but not p53. Leptomycin B induced PPARγ degradation and p53 accumulation, promoting necroptosis and apoptosis, suggesting cytoplasmic p53 contributes to cell death. p62 interacted with PPARγ and p53, and its knockdown suppressed their NaAR-induced upregulation. In conclusion, NaAR-induced PPARγ promotes A549 cell survival by enhancing DNA repair and inhibiting apoptosis and necroptosis via cooperation with p53 and p62, highlighting PPARγ as a potential therapeutic target. Full article

Regulated cardiomyocyte death is a central contributor to myocardial infarction (MI)-associated injury. Mixed lineage kinase domain-like protein (MLKL), a key effector of necroptosis, has been implicated in cardiovascular disease; however, its role in MI remains incompletely defined. MLKL expression was evaluated in hypoxia-treated cardiomyocytes, infarcted murine hearts, and human cardiac tissue. MLKL function was investigated using siRNA-mediated knockdown in neonatal mouse cardiomyocytes and genetic deletion in mice subjected to left anterior descending (LAD) coronary artery ligation. Apoptosis- and pyroptosis-related signaling were assessed by immunoblotting and immunostaining. RIP3 expression and regulation were examined at both protein and mRNA levels, and the RIP3 inhibitor GSK’872 was used to assess pathway dependence. MLKL expression was increased in hypoxic cardiomyocytes, infarcted mouse hearts, and human failing cardiac tissue. Unexpectedly, MLKL deficiency was associated with aggravated myocardial injury, impaired cardiac function, and increased fibrosis following MI. Mechanistically, MLKL deficiency was associated with increased RIP3 protein abundance without a corresponding increase in RIP3 mRNA, consistent with post-transcriptional regulation. Further analyses indicated that MLKL deficiency reduced RIP3 ubiquitination and impaired proteasome-mediated degradation, resulting in RIP3 stabilization. Elevated RIP3 levels were accompanied by increased expression of apoptosis- and pyroptosis-related proteins, particularly at early time points after MI. Pharmacological inhibition of RIP3 with GSK’872 was associated with reduced apoptosis- and pyroptosis-related signaling and improved cardiac function. MLKL deficiency is associated with stabilization of RIP3 and enhanced activation of apoptosis- and pyroptosis-related signaling following MI, contributing to aggravated myocardial injury. These findings support a regulatory role for the MLKL–RIP3 axis in cardiomyocyte death and suggest that targeting RIP3 may represent a potential therapeutic strategy in myocardial infarction. Full article

Molecular subtype–guided therapy for breast cancer (BC) remains limited in a subset of patients by suboptimal efficacy, acquired resistance, and the presence of “undruggable” targets. Proteolysis-targeting chimeras (PROTACs) represent a targeted protein degradation (TPD) strategy that differs fundamentally from conventional occupancy-driven inhibition. By inducing ubiquitination of a protein of interest and subsequent proteasomal degradation, PROTACs can directly reduce pathogenic protein abundance and potentially abrogate non-catalytic or scaffolding functions, thereby enabling more durable pathway suppression in selected resistance contexts. This review comprehensively summarizes the mechanisms of action, key molecular design elements, and the developmental landscape of PROTACs, and maps target selection and research progress across BC molecular subtypes. In hormone receptor–positive/HER2-negative BC, clinical translation is most advanced for estrogen receptor alpha-directed PROTACs; Phase III evidence indicates biomarker-dependent efficacy, with clearer benefit signals in resistant subgroups such as estrogen receptor 1 mutations, suggesting that the net clinical benefit of TPD is more likely to be realized through precision stratification. In contrast, in solid-tumor settings, including human epidermal growth factor receptor 2 (HER2)-positive BC and triple-negative breast cancer, PROTAC translation is more frequently constrained by an “exposure–selectivity–therapeutic window” trade-off driven by physicochemical liabilities, insufficient tumor penetration, and broad target expression. Accordingly, engineering strategies—such as antibody/aptamer-mediated targeted delivery, stimulus-responsive prodrugs, nanocarriers, and local administration—are emerging as decisive approaches to enable safe and effective clinical implementation. Looking forward, further progress of PROTACs in BC will depend on expanding the spectrum of E3 ubiquitin ligases and recruitment modalities, establishing predictable and dynamically monitorable biomarker systems, optimizing rational combination/sequencing regimens with exposure- and schedule-guided dosing, and advancing scalable manufacturing and quality control capabilities, thereby translating mechanistic advantages of TPD into verifiable precision-therapy applications. Full article

Osteosarcoma (OS) is an aggressive bone malignancy with poor prognosis, characterized by high metastasis rates. Kinesin family member 18B (KIF18B), a key protein in cell division and mitosis, has emerged as a potential diagnostic and therapeutic target in various cancers, including OS. This study investigates the role of KIF18B in OS progression and its underlying mechanisms. We found that KIF18B expression is significantly upregulated in OS tissues and correlates with lymph node metastasis (N-stage) and clinical stage. Knockdown of KIF18B inhibited OS cell migration, invasion, proliferation, and tumorigenesis. Mechanistically, KIF18B promotes OS survival through the ubiquitin–proteasome system (UPS) by regulating Skp2 protein degradation. KIF18B knockdown accelerated Skp2 ubiquitination, leading to reduced Skp2 levels and inhibited OS cell viability and glycolytic metabolism. Overexpression of KIF18B enhanced OS cell viability and glycolysis in an Skp2-dependent manner. These findings suggest that the KIF18B-Skp2 axis plays a critical role in the metabolic reprogramming of OS cells and serves as a novel prognostic biomarker and therapeutic target in OS. Full article

Desulfovibrio spp. are sulfate-reducing bacteria (SRB) associated with conditions such as inflammatory bowel disease (IBD) that are linked to intestinal barrier dysfunction (leaky gut). Previously, we reported that Desulfovibrio vulgaris (DSV) caused increased intestinal permeability by upregulating nuclear transcription factor Snail. However, the signaling mechanisms underlying this effect remain unclear. Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that maintains intestinal barrier integrity and negatively regulates Snail and promotes its degradation by proteasomes. Rapamycin has been shown to protect the intestinal barrier and is also known to activate GSK-3. In this study, we investigated whether DSV disrupts intestinal barrier function through modulation of GSK-3 signaling and whether rapamycin could counteract these effects. Using a previously established DSV-induced paracellular permeability model using polarized Caco-2 monolayers, here, we showed that DSV induced inhibitory phosphorylation of GSK-3. Pretreatment of cells with rapamycin prevented DSV- induced phospho- inactivation of GSK-3, suppressed Snail expression and nuclear localization, and significantly reduced DSV-induced barrier permeability. Inhibition of proteasomal degradation with MG132 abolished the protective effects of rapamycin on barrier permeability, supporting a role for GSK-3–mediated proteasomal regulation of Snail. Together, these findings identify GSK-3 signaling as a novel mechanism underlying DSV-induced intestinal barrier dysfunction and highlight rapamycin as a potential therapeutic approach strategy to protect intestinal barrier integrity in response to DSV. Specifically, targeting the GSK-3/Snail pathway may represent a promising strategy to mitigate SRB-associated intestinal barrier disruption. Full article