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PubMed2026

An SLC7A5-dependent nutrient-sensing circuit overcomes cisplatin tolerance via mTOR-autophagy signaling.

Cisplatin-based chemotherapy responses are highly heterogeneous across cancers, with the mechanisms governing drug sensitivity remaining incompletely understood. Using genome-wide CRISPR-Cas9 knockout screening, we systematically characterized regulators of cisplatin response and uncovered a counterintuitive finding: mTOR inhibition promotes cisplatin tolerance, contradicting the canonical view that PI3K-AKT-mTOR activation confers chemoresistance. Mechanistically, both mTOR suppression and cisplatin treatment converge to activate cytoprotective autophagy, which enhances cancer cell survival under therapeutic stress. The amino acid transporter SLC7A5 was identified and validated as a key integrator of the mTOR-autophagy axis that modulates cisplatin sensitivity. SLC7A5 expression positively correlates with cisplatin sensitivity across cancer cell lines, and its downregulation is associated with cisplatin resistance in multiple cancer types, supporting its potential as a mechanistically grounded predictive biomarker. Translationally, leucine supplementation sensitizes cancer cells to cisplatin in an SLC7A5-mTOR-autophagy-dependent manner. Collectively, our study defines a novel mTOR-autophagy adaptive loop governing cisplatin tolerance, positions SLC7A5 as a central regulatory node with both biomarker and therapeutic target value, and proposes leucine supplementation as a simple, translatable strategy to improve cisplatin efficacy in SLC7A5-expressing tumors.

Cisplatin↗
PubMed2026

Genome-wide CRISPRi screen in human iNeurons identifies novel negative mTOR regulator genes associated with focal cortical dysplasia.

Focal cortical dysplasia (FCD) is a common cause of focal epilepsy that typically results from brain mosaic mutations in the mTOR cell signaling pathway. To identify new potential FCD genes, we developed an in vitro CRISPRi screen in human neurons and used FACS enrichment based on the FCD biomarker, phosphorylated S6 ribosomal protein (pS6). Using whole-genome (110,000 gRNAs) and candidate (129 gRNAs) libraries, we discovered 6 new genes in which loss of function significantly increases pS6 levels: LRRC4, EIF3A, TSN, HIP1, PIK3R3, and URI1. Further analysis of the mTOR pathway showed that only two of the genes, PIK3R3 and HIP1, caused hyperphosphorylation throughout the AKT/mTOR/S6 signaling pathway. Importantly, potential pathogenic variants in these two genes have been reported in resected brain tissue from a single FCD patient each, supporting the predictive validity of our screen. Knocking down each of the 6 genes in iNeurons made mTOR signaling resistant to the loss of neurotrophic factor signaling, specifically GDNF; even without GDNF, pS6 levels remained comparable to GDNF-stimulated controls. Thus, we have identified negative regulators of neuronal mTOR signaling in the context of lost neurotrophic factor support. Our data expand the set of genes that are likely to regulate mTOR pathway signaling in neurons, provide biological confirmation for candidate genes identified in human tissue, and suggest additional targets for investigating somatic gene variants in resected FCD tissues. The identification of novel mTOR regulators using iNeurons also highlights the importance of genetic screening in disease-related cell types.

Brain mosaicism↗
PubMed2026

Crotonylome profiling identifies MLKL crotonylation in lupus nephritis associated with RAB1A-mTOR signalling and autophagy changes in tubular epithelial cells.

OBJECTIVE: To investigate whether MLKL crotonylation is associated with tubular autophagy-lysosome pathway homeostasis in lupus nephritis (LN) and to explore its relationship with RAB1A-mechanistic target of rapamycin (mTOR) signalling. METHODS: Crotonylome proteomics was performed in peripheral blood mononuclear cells from patients with LN, patients with systemic lupus erythematosus without nephritis and healthy controls. Renal biopsy tissues were evaluated for tubulointerstitial fibrosis and autophagy-lysosome pathway-related markers. Mechanistic studies were conducted in lipopolysaccharide-stimulated HK-2 cells. Autophagic flux was assessed using bafilomycin A1. The dependency of mTOR/autophagy changes on RAB1A was tested by siRNA-mediated knockdown. RESULTS: MLKL was identified as a differentially crotonylated protein in LN, with increased crotonylation at K95 and K219. Kidney tissues from patients with LN showed increased fibronectin and collagen III deposition compared with controls, whereas no significant difference was observed between class IV and class V LN. LC3 signal did not differ significantly between groups, whereas LAMP1 expression and LC3-LAMP1 co-localisation were reduced in LN. In HK-2 cells, crotonylation-deficient MLKL mutants were associated with increased LC3-II and reduced p62, whereas K219Q showed the opposite pattern. Autophagic flux assays using bafilomycin A1 showed that K219R-expressing cells had higher LC3-II levels than WT cells both before and after lysosomal inhibition, with comparable BafA1-induced LC3-II accumulation, consistent with increased autophagosome formation rather than impaired lysosomal degradation. HDAC1 knockdown increased MLKL crotonylation and was accompanied by mTOR activation. MLKL crotonylation enhanced RAB1A guanriphosphat osphate (GTP) binding without altering total RAB1A abundance. RAB1A knockdown in MLKL WT-expressing cells attenuated mTOR phosphorylation and partly reversed the autophagy-suppressive marker profile. Sodium crotonate induced an autophagy-suppressive marker profile that was partly reversed by rapamycin. CONCLUSION: MLKL crotonylation is associated with activation of the RAB1A-mTOR axis and altered tubular autophagy-lysosome pathway homeostasis in LN. These findings suggest that tubular injury-related changes in LN may not be fully reflected by glomerulus-based classification alone.

Humans↗
PubMed2025

Dysregulation of mTOR signalling is a converging mechanism in lissencephaly.

Cerebral cortex development in humans is a highly complex and orchestrated process that is under tight genetic regulation. Rare mutations that alter gene expression or function can disrupt the structure of the cerebral cortex, resulting in a range of neurological conditions1. Lissencephaly ('smooth brain') spectrum disorders comprise a group of rare, genetically heterogeneous congenital brain malformations commonly associated with epilepsy and intellectual disability2. However, the molecular mechanisms underlying disease pathogenesis remain unknown. Here we establish hypoactivity of the mTOR pathway as a clinically relevant molecular mechanism in lissencephaly spectrum disorders. We characterized two types of cerebral organoid derived from individuals with genetically distinct lissencephalies with a recessive mutation in p53-induced death domain protein 1 (PIDD1) or a heterozygous chromosome 17p13.3 microdeletion leading to Miller-Dieker lissencephaly syndrome (MDLS). PIDD1-mutant organoids and MDLS organoids recapitulated the thickened cortex typical of human lissencephaly and demonstrated dysregulation of protein translation, metabolism and the mTOR pathway. A brain-selective activator of mTOR complex 1 prevented and reversed cellular and molecular defects in the lissencephaly organoids. Our findings show that a converging molecular mechanism contributes to two genetically distinct lissencephaly spectrum disorders.

Humans↗
PubMed2023

Activation of mTOR signaling in adult lung microvascular progenitor cells accelerates lung aging.

Reactivation and dysregulation of the mTOR signaling pathway are a hallmark of aging and chronic lung disease; however, the impact on microvascular progenitor cells (MVPCs), capillary angiostasis, and tissue homeostasis is unknown. While the existence of an adult lung vascular progenitor has long been hypothesized, these studies show that Abcg2 enriches for a population of angiogenic tissue-resident MVPCs present in both adult mouse and human lungs using functional, lineage, and transcriptomic analyses. These studies link human and mouse MVPC-specific mTORC1 activation to decreased stemness, angiogenic potential, and disruption of p53 and Wnt pathways, with consequent loss of alveolar-capillary structure and function. Following mTOR activation, these MVPCs adapt a unique transcriptome signature and emerge as a venous subpopulation in the angiodiverse microvascular endothelial subclusters. Thus, our findings support a significant role for mTOR in the maintenance of MVPC function and microvascular niche homeostasis as well as a cell-based mechanism driving loss of tissue structure underlying lung aging and the development of emphysema.

Mice↗
PubMed2025

NME2 modulates HCC progression through 4EBP1 phosphorylation and autophagy regulation independent of mTOR.

BACKGROUND: To investigate the role of nucleoside diphosphate kinase 2 (NME2) in HCC progression, assessing its therapeutic potential. METHODS: Utilizing transcriptome sequencing data from The Cancer Genome Atlas (TCGA) and immunohistochemical staining of tissue microarrays, we analyzed NME2 expression in HCC tumor tissues. The effects of NME2 on HCC cell proliferation and autophagy flux were assessed through knockdown and overexpression experiments. Additionally, the relationship between NME2 and 4EBP1 phosphorylation was explored through specific site mutation analysis. RESULTS: NME2 overexpression in HCC correlated with poor prognosis. NME2 knockdown significantly hindered HCC cell proliferation and induced autophagy flux. Notably, NME2 modulates 4EBP1 phosphorylation (Thr37/46) independently of mTOR, unveiling a novel axis in HCC pathogenesis. Additionally, NME2 modulates eukaryotic translation initiation factor 4F (eIF4F) complex formation and autophagy flux. CONCLUSIONS: NME2 plays a crucial role in HCC development by modulating 4EBP1 phosphorylation and autophagy through an mTOR-independent pathway. Our research underscores NME2's significance as a potential therapeutic target in HCC, meriting further exploration of its underlying mechanisms and clinical applicability.

Humans↗
PubMed2025

Core passive and facultative mTOR-mediated mechanisms coordinate mammalian protein synthesis and decay.

The maintenance of cellular homeostasis requires tight regulation of proteome concentration and composition. To achieve this, protein production and elimination must be robustly coordinated. However, the mechanistic basis of this coordination remains unclear. Here, we address this question using quantitative live-cell imaging, computational modeling, transcriptomics, and proteomics approaches. We found that protein decay rates systematically adapt to global alterations of protein synthesis rates. This adaptation is driven by a core passive mechanism supplemented by facultative changes in mechanistic/mammalian target of rapamycin (mTOR) signaling. Passive adaptation hinges on changes in the production rate of the machinery governing protein decay and allows for partial maintenance of the cellular proteome. Sustained changes in mTOR signaling provide an additional layer of adaptation unique to naive pluripotent stem cells, allowing for near-perfect maintenance of proteome composition. Our work unravels the mechanisms protecting the integrity of mammalian proteomes upon variations in protein synthesis rates. A record of this paper's transparent peer review process is included in the supplemental information.

TOR Serine-Threonine Kinases↗
PubMed2025

Lipidomic profiling of mouse brain and human neuron cultures reveals a role for Mboat7 in mTOR-dependent neuronal migration.

Mutations in lipid regulator genes are a frequent cause of autism spectrum disorder, including those regulating phosphatidylinositol (PI) and phosphoinositide 3-kinase signaling. MBOAT7 encodes a key acyltransferase in PI synthesis and is mutated in an autism-related condition with neurodevelopmental delay and epilepsy. Using liquid chromatography-tandem mass spectrometry, we analyzed the PI-associated glycerolipidome in mice and humans during neurodevelopment and found dynamic regulation at times corresponding to neural apoptosis in the brains of Mboat7 knockout mice. Mboat7 function was necessary for polyunsaturated lipid synthesis and cortical neural migration, and loss resulted in massive accumulation of the precursor lysophosphatidylinositol and hyperactive mTOR signaling. Inhibiting mTOR signaling rescued migration defects. Our findings demonstrate roles for lipid remodeling during neurodevelopment and implicate lipid regulation in neuronal migration, revealing potential paths to treatment for MBOAT7 deficiency.

Animals↗
PubMed2026

Activation of mTOR pathway by human cytomegalovirus promoting host ribosomal protein expression by coordinated transcriptional and translational controls.

Human cytomegalovirus (HCMV) profoundly reprograms host transcription and RNA metabolism, yet its impact on transcription start site (TSS) regulation of host genes remains poorly understood. Here, we employed NanoCap Analysis of Gene Expression sequencing (NanoCAGE-seq) to investigate HCMV-driven changes in alternative TSS usage across the host transcriptome. We identified widespread TSS switching, with ribosomal protein genes (RPGs) emerging as a highly enriched category. Alternative TSS usage produced isoforms with distinct 5'untranslated regions (UTRs), thereby altering cis-regulatory elements that shape translational efficiency. Integrative transcriptomic and proteomic analyses revealed a paradoxical accumulation of RPG proteins despite transcriptional downregulation during infection. Using 5' Rapid Amplification of cDNA Ends (5'RACE), we characterized four RPGs of RPL4, RPS11, RPS23, and RPS24 that generated 5'UTR variants through alternative TSS usage. Notably, isoforms containing a 5'terminal oligopyrimidine (5'TOP) motif were significantly enriched, correlating with mTOR activation induced by HCMV. Functional assays with bicistronic reporter constructs in HEK293 cells and infection models in human embryonic lung fibroblasts demonstrated that the RPL4 5'TOP isoform exhibited enhanced mTORC1-driven translation compared with non-5'TOP counterparts. Importantly, RPL4 upregulation facilitated viral protein synthesis and boosted production of infectious virions. Together, our findings reveal that dynamic TSS switching of RPGs provides a simple, yet effective, mechanism for fine-tuning mTORC1-responsive translation. By co-opting host transcriptional and translational programs, HCMV enhances ribosome function to optimize the cellular environment for productive viral replication.

Humans↗
PubMed2025

Panduratin A Induces Autophagy Through AMPK Activation Independent of mTOR Inhibition and Restricts Mycobacterium tuberculosis in Host Macrophages.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health burden, especially with the increasing prevalence of drug-resistant strains. There is an urgent need for new therapeutics that act via alternative mechanisms. Autophagy, a vital cell-autonomous defense process, allows macrophages to degrade intracellular pathogens such as Mtb and has gained attention as a potential target for host-directed therapy. In this study, we conducted a high-content imaging screen of herb-derived compounds to identify autophagy inducers in RAW264.7 macrophages. Panduratin A (NPA), a natural compound from Boesenbergia rotunda, was found to potently induce autophagy. NPA promoted autophagic vacuole formation in a dose-dependent fashion at low micromolar levels. Its autophagy-inducing effect was validated using RFP-GFP-LC3 dual fluorescence assays and immunoblotting in the presence of bafilomycin A1. Further mechanistic analysis revealed that NPA activates autophagy through AMPK activation, independent of mTOR inhibition. Importantly, NPA significantly promoted intracellular Mtb clearance and increased colocalization of Mtb with autophagosomes and lysosomes, in a manner dependent on Beclin-1. These findings highlight NPA as a potent enhancer of macrophage antimicrobial responses via autophagy, supporting its potential as a candidate for host-directed adjunctive therapy against TB.

Autophagy↗
PubMed2023

Dominant negative variants in KIF5B cause osteogenesis imperfecta via down regulation of mTOR signaling.

BACKGROUND: Kinesin motor proteins transport intracellular cargo, including mRNA, proteins, and organelles. Pathogenic variants in kinesin-related genes have been implicated in neurodevelopmental disorders and skeletal dysplasias. We identified de novo, heterozygous variants in KIF5B, encoding a kinesin-1 subunit, in four individuals with osteogenesis imperfecta. The variants cluster within the highly conserved kinesin motor domain and are predicted to interfere with nucleotide binding, although the mechanistic consequences on cell signaling and function are unknown. METHODS: To understand the in vivo genetic mechanism of KIF5B variants, we modeled the p.Thr87Ile variant that was found in two patients in the C. elegans ortholog, unc-116, at the corresponding position (Thr90Ile) by CRISPR/Cas9 editing and performed functional analysis. Next, we studied the cellular and molecular consequences of the recurrent p.Thr87Ile variant by microscopy, RNA and protein analysis in NIH3T3 cells, primary human fibroblasts and bone biopsy. RESULTS: C. elegans heterozygous for the unc-116 Thr90Ile variant displayed abnormal body length and motility phenotypes that were suppressed by additional copies of the wild type allele, consistent with a dominant negative mechanism. Time-lapse imaging of GFP-tagged mitochondria showed defective mitochondria transport in unc-116 Thr90Ile neurons providing strong evidence for disrupted kinesin motor function. Microscopy studies in human cells showed dilated endoplasmic reticulum, multiple intracellular vacuoles, and abnormal distribution of the Golgi complex, supporting an intracellular trafficking defect. RNA sequencing, proteomic analysis, and bone immunohistochemistry demonstrated down regulation of the mTOR signaling pathway that was partially rescued with leucine supplementation in patient cells. CONCLUSION: We report dominant negative variants in the KIF5B kinesin motor domain in individuals with osteogenesis imperfecta. This study expands the spectrum of kinesin-related disorders and identifies dysregulated signaling targets for KIF5B in skeletal development.

Animals↗
PubMed2026

Insights from changes in NDEV biomarkers of metabolism: effects of PPARγ and GLP1 receptor agonists on brain metabolism.

BACKGROUND: Insulin resistance (IR) is implicated in central nervous system disorders, including depression and Alzheimer's disease (AD). METHODS: We analyzed biological samples from two cohorts of clinical trial participants: (1) participants with unremitted depression after six months of treatment as usual who received pioglitazone (PPARγ agonist, N = 12) or placebo and (2) middle-aged participants at genetic risk for AD who received liraglutide (glucagon-like peptide 1 [GLP1] receptor agonist, N = 15) or placebo. These cohorts, which previously showed treatment-related improvements in peripheral IR, were used to assess the effects of pioglitazone and liraglutide on CNS insulin signaling using neuron-derived extracellular vesicles (NDEVs) as biomarkers. We utilized biological samples to measure biomarkers of IR in NDEVs. Eleven Akt-mTOR pathway proteins were measured before and after 12 weeks of treatment in both groups. RESULTS: Participants who received pioglitazone experienced broader changes, with significant increases in GSK3β (Ser9), mTOR (Ser2448), and RPS6 (Ser235/Ser236; all P ≤ .02) compared with placebo, and 77% of participants showed mTOR (Ser2448) response. Participants who received liraglutide demonstrated significantly increased NDEV-associated phosphorylated Akt (Ser473) and mTOR (Ser2448; P = .04 and P = .025, respectively) compared with placebo, with 40% and 30% of participants in the liraglutide group showing biomarker response in both Akt (Ser473) and mTOR (Ser2448), respectively. These effects appeared relatively independent from changes in fasting plasma insulin and glucose concentration at 120-minutes during the oral glucose tolerance test. DISCUSSION: Our findings demonstrate CNS-specific biomarker responses to both PPARγ agonists and GLP1 receptor agonists.

Humans↗
PubMed2026

Integrative proteomics reveals MSH6 to modulate PARP inhibitor sensitivity in BRCA1/2-proficient ovarian cancer.

Ovarian cancer remains a leading cause of gynecologic cancer-related deaths worldwide. Deficiencies in BRCA1/2 are well-established biomarkers that predict sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPis). However, emerging evidence indicates that a subset of BRCA-proficient tumors also responds to PARPi therapy, suggesting the presence of additional molecular mechanisms. We hypothesized that the composition of the PARP1 protein complex and PARylation-mediated signaling contribute to PARPi response in BRCA-proficient HGSOC. We assessed PARPi response across a panel of BRCA-proficient ovarian cancer cell lines and identified distinct sensitive and resistant groups. Chemical proteomics with rucaparib revealed different PARP1 complexes including higher enrichment of MSH6 in sensitive cells. Co-immunoprecipitation analyses further confirmed differential assembly of PARP1-MSH6-PARP2 complexes between sensitive and resistant models. To explore PARylation signaling, we performed ADP-ribosylation proteomics using clickable NAD⁺ analogs, revealing distinct PARylation profiles between sensitive and resistant cell lines. CHAF1A, a known MSH6 interactor and PARP1 substrate, showed more pronounced reduction in ADP-ribosylation in PARPi-sensitive cells. Targeting MSH6 using CRISPR or siRNA decreased PARPi sensitivity. In addition, mTOR signaling was reduced in sensitive, but increased in resistant cells, following rucaparib treatment. Notably, MSH6 knockdown led to increased CHAF1A expression regardless of rucaparib treatment. Importantly, knockdown of CHAF1A significantly impaired cell viability, especially in A2780 cells, and suppressed mTOR signaling, suggesting that CHAF1A acts downstream of MSH6 to regulate the mTOR axis. Furthermore, co-treatment with mTORC1 inhibitors enhanced the cellular effects of rucaparib in resistant cells, suggesting a therapeutic potential of targeting downstream mTOR effectors to overcome intrinsic resistance. In conclusion, this study identifies the PARP1-MSH6 interaction to modulate PARPi sensitivity via CHAF1A-mTOR signaling in BRCA-proficient ovarian cancer. By integrating chemical proteomics and ADP-ribosylation proteomics, we delineate the interplay between PARP1 complex composition and signaling dynamics, highlighting MSH6 as a critical modulator of PARPi response and potential biomarker to enhance therapeutic efficacy in BRCA-proficient HGSOC.

Humans↗
PubMed2026

Emerging Strategies Targeting the PI3K/AKT/mTOR Pathway in HR+/HER2- Advanced Breast Cancer.

Hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) breast cancer accounts for approximately 70% of breast cancer cases. Despite recent advances with cyclin-dependent kinase 4/6 inhibitors (CDK4/6i), resistance inevitably develops, often driven by activation of the phosphatidylinositol 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) pathway. Genetic alterations such as PIK3CA mutations (present in ~ 45% of HR+/HER2- tumors), AKT1 mutations, and PTEN loss contribute to endocrine resistance and poor outcomes. This review summarizes emerging strategies targeting this pathway to overcome resistance in advanced disease. Isoform-specific PI3K inhibitors, including alpelisib and inavolisib, have demonstrated clinically meaningful progression-free survival benefits in PIK3CA-mutated populations, with inavolisib showing improved tolerability and efficacy. In contrast, pan-PI3K inhibitors such as buparlisib have been constrained by toxicity. Targeting downstream signaling, AKT inhibitors have also shown benefit: capivasertib has demonstrated clinical efficacy leading to US Food and Drug Administration approval, while ipatasertib has yielded encouraging results, particularly in tumors harboring PIK3CA, AKT1, or PTEN alterations. Mammalian target of rapamycin inhibitors, notably everolimus, have shown efficacy irrespective of mutation status. The dual PI3K-mTOR inhibitor (gedatolisib) has also shown promising progression-free survival benefit in a PIK3CA wild-type population. Next-generation agents, including mutant-selective PI3Kα inhibitors and bi-steric mTOR complex 1 inhibitors, are under active investigation. Optimal sequencing of these agents alongside endocrine therapy and CDK4/6i options remain a critical question, as does integration of genomic testing to guide therapy. Future directions include rational combination strategies, improved biomarker-driven selection, and novel modalities such as proteolysis-targeting chimeras (PROTACs). Collectively, these advances aim to enhance durability of response, minimize toxicity, and improve survival in HR+/HER2- metastatic breast cancer.

Humans↗
PubMed2026

Crosstalk between the Wnt pathway and other signaling pathways.

The Wnt/β-catenin signaling pathway is a deeply conserved regulatory network that governs embryonic development, stem cell maintenance, and tissue homeostasis. Aberrant activation of the Wingless/Integrated protein (Wnt) signaling is a hallmark of numerous human diseases, most prominently in colorectal cancer, where it cooperates with additional oncogenic pathways to drive tumor initiation, progression, and therapeutic resistance (See Supplementary Table 1 for a list of the abbreviations used in this manuscript and their definitions.). Increasing evidence indicates that Wnt signaling does not function as an isolated linear cascade but rather as an integrative signaling hub that dynamically interfaces with major signaling pathways, including the RAS-RAF-MAPK and PI3K-AKT-mTOR pathways. Rat Sarcoma protein (RAS)- Rapidly Accelerated Fibrosarcoma protein (RAF)- Mitogen-Activated Protein Kinase (MAPK) and Phosphoinositide 3-Kinase (PI3K)- Ak strain transforming protein (AKT)- Mechanistic Target of Rapamycin (mTOR) pathways. These interactions occur at multiple molecular levels, encompassing shared kinases, transcriptional regulators, metabolic nodes, and cytoskeletal components, thereby coordinating proliferative, metabolic, and migratory programs. In this review, we synthesize current mechanistic and clinical insights into the crosstalk between Wnt signaling and the RAS-RAF-MAPK and PI3K-AKT-mTOR pathways, with particular emphasis on colorectal cancer. We discuss how these signaling networks converge to regulate β-catenin stability, transcriptional activity, cell adhesion, and metabolic reprogramming, thereby generating oncogenic phenotypes that cannot be explained by activation of individual pathways alone. To illustrate the evolutionary conservation and biological significance of these interactions, we integrate developmental paradigms from early Xenopus embryogenesis, where Wnt signaling governs zygotic genome activation, body axis formation, and the regulation of cell growth, protein stability, and biomass accumulation. Finally, we examine how an improved understanding of Wnt-centered signaling networks is informing emerging therapeutic strategies, including combinatorial pathway inhibition and nanoparticle-based drug delivery. Collectively, this review highlights Wnt signaling as a central integrator of developmental and oncogenic programs, providing a conceptual framework for understanding signaling network crosstalk and identifying new therapeutic opportunities in cancer.

Humans↗
PubMed2026

A CRISPR-Cas9 screen identifies LAPTM4A (lysosomal protein transmembrane 4 alpha) as a key host barrier against PRRSV infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) manipulates host intracellular processes, particularly macroautophagy/autophagy and lysosomal function, to facilitate its replication and spread. However, the precise host factors and molecular mechanisms by which PRRSV remodels the autophagy-lysosome axis remain poorly defined. Here, we performed a CRISPR-Cas9 knockout screen targeting 1,332 genes involved in protein degradation, metabolism, and vesicular trafficking, and identified LAPTM4A (lysosomal protein transmembrane 4 alpha) as a critical antiviral factor involved in the lysosomal pathway. A yeast two-hybrid screen identified LAPTM4A as an interactor of PRRSV GP5 (glycoprotein 5). Mechanistically, GP5 recruits the E3 ubiquitin ligase NEDD4 and the autophagy receptor SQSTM1/p62 to promote K63-linked polyubiquitination of LAPTM4A, leading to its autophagic degradation. This selective degradation activates the AMPK-ULK1-MAP1LC3/LC3 signaling cascade, initiating autophagy while facilitating MTOR-lysosome colocalization, thereby suppressing TFEB nuclear translocation and transcription of lysosome-related genes. The resulting incomplete autophagic flux enhances viral replication. Additionally, in terms of host defense, LAPTM4A maintains lysosomal homeostasis by restraining excessive autophagy through AMPK-ULK1-LC3 signaling and promoting TFEB-dependent lysosomal gene expression by impairing the binding of RPTOR/raptor to MTOR, thus providing broad antiviral protection against multiple RNA viruses. Collectively, our findings identify LAPTM4A as a central regulator of lysosome-autophagy homeostasis and reveal a viral strategy that dismantles this defense axis to facilitate infection.Abbreviations: ATG5: autophagy related 5; AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; Baf A1: bafilomycin A1; CHX: cycloheximide; Co-IP: co-immunoprecipitation; DMVT library: protein degradation, metabolism, and vesicular trafficking library; LAPTM4A: lysosomal protein transmembrane 4 alpha; MAGeCK: model-based analysis of genome-wide CRISPR-Cas9 knockout; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; NC: negative control; PAMs: porcine alveolar macrophages; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; PRRSV: porcine reproductive and respiratory syndrome virus; qRT-PCR: quantitative real-time PCR; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TCID50: 50% tissue culture infective dose; TFEB: transcription factor EB; Ub: ubiquitin; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type.

Animals↗
PubMed2026

EPS8 Differentially Regulates Antioxidant Defense and Mitochondrial Homeostatic Signaling in LNCaP and Enzalutamide-resistant LNCaP Cells.

BACKGROUND/AIM: Epidermal growth factor receptor pathway substrate 8 (EPS8) is an adaptor protein implicated in tumor progression and therapeutic resistance; however, its role in mitochondrial homeostatic signaling and antioxidant regulation remains unclear. This study examined the effects of EPS8 modulation in lymph node carcinoma of the prostate (LNCaP) and enzalutamide-resistant LNCaP (LNCaP-Enz) cells. MATERIALS AND METHODS: LNCaP-Enz cells were generated by long-term exposure to enzalutamide and maintained in 5 μM enzalutamide. EPS8 expression was modulated by plasmid-mediated overexpression or shRNA-mediated knockdown. Superoxide dismutase (SOD) activity and cellular adenosine triphosphate (ATP) levels were measured using colorimetric assays. Mitochondrial membrane potential (ΔΨm) was evaluated using JC-1 fluorescence, and mitochondrial staining patterns were qualitatively examined using MitoTracker Green staining. Protein expression associated with antioxidant defense, mitochondrial dynamics, mitochondrial stress response, mitochondrial biogenesis, and AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling was analyzed by western blotting. RESULTS: EPS8 overexpression increased SOD activity and the expression of SOD1 and SOD2, whereas EPS8 knockdown reduced these antioxidant parameters. Conversely, EPS8 silencing increased cellular ATP levels and enhanced JC-1 red fluorescence patterns. EPS8 silencing increased MFN1 and OPA1 expression and reduced DRP1 expression, consistent with a fusion-associated mitochondrial profile. EPS8 silencing also increased SIRT1, PGC-1α, NRF1, TFAM, p-AMPK/AMPK, and p-mTOR/mTOR, but reduced HSP60, LONP1, ATF5, and CEBPβ expression. CONCLUSION: EPS8 differentially regulates SOD-associated antioxidant capacity and mitochondrial homeostatic signaling in LNCaP-based cell models. Further studies are required to determine whether EPS8 modulation affects enzalutamide responsiveness.

Humans↗
PubMed2024

DGKH-mediated phosphatidic acid oncometabolism as a driver of self-renewal and therapy resistance in HCC.

BACKGROUND AND AIMS: HCC is characterized by metabolic pathway aberrations, which enable cancer cells to meet their energy demands and accelerate malignant progression. Identifying novel metabolic players governing therapy resistance and self-renewal in HCC is crucial, as these properties are likely responsible for tumor recurrence. APPROACH AND RESULTS: Clinical traits and RNA-seq of patients with HCC in The Cancer Genome Atlas were used for weighted gene coexpression network analysis, where 1 module was significantly correlated with advanced pathological stage and stem cell population maintenance. Further analysis of this module by integrating data obtained from HCC patient nonresponders to tyrosine kinase inhibitors identified 361 commonly deregulated genes. Intriguingly, these genes are significantly enriched in the intracellular signal transduction pathway, with diacylglycerol kinase eta (DGKH) ranked as the most enriched gene in poorly differentiated HCC tumors. Clinically, DGKH was elevated in tumor tissues compared to nontumor tissues. Patients with higher DGKH expression exhibited a more undifferentiated state and were less responsive to tyrosine kinase inhibitors. Functional assays using DGKH-manipulated HCC cell lines demonstrated that DGKH augmented aggressive features, including cancer stemness, therapy resistance, and metastasis. Upstream of DGKH , we discovered that the E1A-associated protein p300 (EP300) binds to DGKH's promoter region, thereby increasing its transcriptomic expression. Mechanistically, DGKH promotes mTOR signaling by producing phosphatidic acid. In an immunocompetent mouse model, cotreatment with sorafenib and liver-directed AAV8-mediated Dgkh depletion significantly reduced tumor burden, self-renewal, phosphatidic acid production, and mTOR signaling. CONCLUSIONS: Our research demonstrated that DGKH is a crucial oncometabolic regulator of cancer stemness and therapy resistance, suggesting that inhibiting DGKH may lead to more effective HCC treatment.

Humans↗