ID: 21355074
Title: Nelfinavir induces liposarcoma apoptosis through inhibition of regulated intramembrane proteolysis of SREBP-1 and ATF6.
Abstract: We previously reported that nelfinavir (NFV) induces G(1) cell-cycle block and apoptosis selectively in liposarcoma cell lines due to increased SREBP-1 (sterol regulatory element binding protein-1) expression in the absence of increased transcription. We postulate that NFV interferes with regulated intramembrane proteolysis of SREBP-1 and ATF6 (activating transcription factor 6). Time-lapse, confocal microscopic studies show that NFV inhibits the nuclear translocation of full-length SREBP-1-EGFP and ATF6-EGFP fusion proteins. siRNA-mediated knockdown of site-1 protease (S1P) and/or site-2 protease (S2P) leads to inhibition of SREBP-1 intracellular trafficking to the nucleus and reduces liposarcoma cell proliferation. Treatment of LiSa-2 liposarcoma cells with 3,4-dichloroisocoumarin, a serine protease inhibitor of S1P, did not affect SREBP-1 processing. In contrast, 1,10-phenanthroline, an S2P-specific inhibitor, reproduces the molecular and biological phenotypes observed in NFV-treated cells, which implicates S2P as a target of NFV. In vivo evaluation of NFV in a murine liposarcoma xenograft model leads to inhibition of tumor growth without significant toxicity. NFV-induced upregulation of SREBP-1 and ATF6 results from inhibition of S2P, which together with S1P mediates regulated intramembrane proteolysis from their precursor to their transcriptionally active forms. The resulting endoplasmic reticulum (ER) stress and concurrent inhibition of the unfolded protein response induce caspase-mediated apoptosis. These results provide new insight into the mechanism of NFV-mediated induction of ER stress and cell death in liposarcomas and are the first to report targeting S2P for cancer therapy.

ID: 26698881
Title: The Hepatitis C Virus-induced NLRP3 Inflammasome Activates the Sterol Regulatory Element-binding Protein (SREBP) and Regulates Lipid Metabolism.
Abstract: Hepatitis C virus (HCV) relies on host lipids and lipid droplets for replication and morphogenesis. The accumulation of lipid droplets in infected hepatocytes manifests as hepatosteatosis, a common pathology observed in chronic hepatitis C patients. One way by which HCV promotes the accumulation of intracellular lipids is through enhancing de novo lipogenesis by activating the sterol regulatory element-binding proteins (SREBPs). In general, activation of SREBPs occurs during cholesterol depletion. Interestingly, during HCV infection, the activation of SREBPs occurs under normal cholesterol levels, but the underlying mechanisms are still elusive. Our previous study has demonstrated the activation of the inflammasome complex in HCV-infected human hepatoma cells. In this study, we elucidate the potential link between chronic hepatitis C-associated inflammation and alteration of lipid homeostasis in infected cells. Our results reveal that the HCV-activated NLRP3 inflammasome is required for the up-regulation of lipogenic genes such as 3-hydroxy-3-methylglutaryl-coenzyme A synthase, fatty acid synthase, and stearoyl-CoA desaturase. Using pharmacological inhibitors and siRNA against the inflammasome components (NLRP3, apoptosis-associated speck-like protein containing a CARD, and caspase-1), we further show that the activation of the NLRP3 inflammasome plays a critical role in lipid droplet formation. NLRP3 inflammasome activation in HCV-infected cells enables caspase-1-mediated degradation of insulin-induced gene proteins. This subsequently leads to the transport of the SREBP cleavage-activating protein·SREBP complex from the endoplasmic reticulum to the Golgi, followed by proteolytic activation of SREBPs by S1P and S2P in the Golgi. Typically, inflammasome activation leads to viral clearance. Paradoxically, here we demonstrate how HCV exploits the NLRP3 inflammasome to activate SREBPs and host lipid metabolism, leading to liver disease pathogenesis associated with chronic HCV.

ID: 28645614
Title: Site-1 protease, a novel metabolic target for glioblastoma.
Abstract: Sterol regulatory element binding proteins (SREBPs) are transcriptional regulators of lipids which promote glioblastoma growth. Here, we investigate the effect of inhibiting expression of SREBP target genes in human glioblastoma cells. This was achieved by using PF-429242 to inhibit site-1 protease (S1P), an enzyme required for SREBP activation. Treatment with PF-429242 decreased glioblastoma cell viability, induced apoptosis and downregulated steroid, isoprenoid and unsaturated fatty acid biosynthetic pathways. Several pro-inflammatory genes were upregulated. Collectively, these results demonstrate the potential of S1P as a target for glioblastoma therapy.

ID: 29689241
Title: Pharmacologic inhibition of S1P attenuates ATF6 expression, causes ER stress and contributes to apoptotic cell death.
Abstract: Mammalian cells express unique transcription factors embedded in the endoplasmic reticulum (ER) membrane, such as the sterol regulatory element-binding proteins (SREBPs), that promote de novo lipogenesis. Upon their release from the ER, the SREBPs require proteolytic activation in the Golgi by site-1-protease (S1P). As such, inhibition of S1P, using compounds such as PF-429242 (PF), reduces cholesterol synthesis and may represent a new strategy for the management of dyslipidemia. In addition to the SREBPs, the unfolded protein response (UPR) transducer, known as the activating transcription factor 6 (ATF6), is another ER membrane-bound transcription factor that requires S1P-mediated activation. ATF6 regulates ER protein folding capacity by promoting the expression of ER chaperones such as the 78-kDa glucose-regulated protein (GRP78). ER-resident chaperones like GRP78 prevent and/or resolve ER polypeptide accumulation and subsequent ER stress-induced UPR activation by folding nascent polypeptides. Here we report that pharmacological inhibition of S1P reduced the expression of ATF6 and GRP78 and induced the activation of UPR transducers inositol-requiring enzyme-1α (IRE1α) and protein kinase RNA-like ER kinase (PERK). As a consequence, S1P inhibition also increased the susceptibility of cells to ER stress-induced cell death. Our findings suggest that S1P plays a crucial role in the regulation of ER folding capacity and also identifies a compensatory cross-talk between UPR transducers in order to maintain adequate ER chaperone expression and activity.

ID: 30046013
Title: Site-1 protease deficiency causes human skeletal dysplasia due to defective inter-organelle protein trafficking.
Abstract: Site-1 protease (S1P), encoded by MBTPS1, is a serine protease in the Golgi. S1P regulates lipogenesis, endoplasmic reticulum (ER) function, and lysosome biogenesis in mice and in cultured cells. However, how S1P differentially regulates these diverse functions in humans has been unclear. In addition, no human disease with S1P deficiency has been identified. Here, we report a pediatric patient with an amorphic and a severely hypomorphic mutation in MBTPS1. The unique combination of these mutations results in a frequency of functional MBTPS1 transcripts of approximately 1%, a finding that is associated with skeletal dysplasia and elevated blood lysosomal enzymes. We found that the residually expressed S1P is sufficient for lipid homeostasis but not for ER and lysosomal functions, especially in chondrocytes. The defective S1P function specifically impairs activation of the ER stress transducer BBF2H7, leading to ER retention of collagen in chondrocytes. S1P deficiency also causes abnormal secretion of lysosomal enzymes due to partial impairment of mannose-6-phosphate-dependent delivery to lysosomes. Collectively, these abnormalities lead to apoptosis of chondrocytes and lysosomal enzyme-mediated degradation of the bone matrix. Correction of an MBTPS1 variant or reduction of ER stress mitigated collagen-trafficking defects. These results define a new congenital human skeletal disorder and, more importantly, reveal that S1P is particularly required for skeletal development in humans. Our findings may also lead to new therapies for other genetic skeletal diseases, as ER dysfunction is common in these disorders.

ID: 30281916
Title: Alterations of EDEM1 functions enhance ATF6 pro-survival signaling.
Abstract: Activating transcription factor 6 alpha (referred to as ATF6 hereafter) is an endoplasmic reticulum (ER)-resident glycoprotein and one of the three sensors of the unfolded protein response (UPR). Upon ER stress, ATF6 is exported to the Golgi complex where it is cleaved by the S1P and S2P proteases thus releasing ATF6 cytosolic fragment and leading to the transcription of ATF6 target genes. In this study, we performed a phenotypic small-interfering RNA (siRNA) screening to better characterize the ER mechanisms involved in ATF6 activation upon ER stress. This revealed that silencing of ER-degradation-enhancing alpha-mannosidase-like protein-1 (EDEM1) increased the bioavailability of ER stress-induced ATF6 export to the Golgi complex through the stabilization of the natively unstable ATF6 protein. Moreover, we characterized a somatic variant of EDEM1 (N198I) found in hepatocellular carcinoma that alters ATF6 signaling and might provide a selective advantage to the transforming cells. Hence, our work confirms the natively unstable nature of ATF6 and links this property to potentially associated pro-oncogenic functions.

ID: 31827236
Title: SPHK1 deficiency protects mice from acetaminophen-induced ER stress and mitochondrial permeability transition.
Abstract: Acetaminophen (APAP) is the leading cause of drug-induced acute liver failure. Sphingosine-1-phosphate (S1P), whose formation is catalyzed by sphingosine kinase (SPHK)-1 or -2, is a bioactive lipid implicated in human health and disease. Here, we show that APAP-treated sphK1-deficient (sphK1-/-) mice exhibited markedly less liver damage and reduced inflammation compared with the wild-type mice. SPHK1 deficiency alleviated APAP-induced endoplasmic reticulum (ER) stress by affecting the phosphorylation of inositol-requiring enzyme 1α (IRE1α) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)-eukaryotic translation initiation factor 2α (eIF2α), levels of activating transcription factor 4 (ATF4), and activation of activating transcription factor 6 (ATF6). SPHK1 deficiency also inhibited mitochondrial permeability transition (MPT), as evidenced by the impaired phosphorylation of JNK, apoptosis signal-regulated kinase 1 (ASK1), and glycogen synthase kinase 3β (GSK3β). In addition, SPHK1 deficiency reduced the levels of histone deacetylase and promoted the acetylation of p65 and STAT1, thereby impairing the transcription of inflammatory genes. Supplementation with exogenous S1P significantly reversed the activation of the PERK-eIF2α-ATF4 pathway and ATF6 during ER stress as well as the activation of GSK3β, ASK1, and JNK during MPT. Both FTY720, a functional S1P receptor antagonist, and PF543, an SPHK1 inhibitor, significantly ameliorated APAP-induced liver injury and improved animal survival. Our study reveals a critical role for SPHK1 in mediating APAP-induced hepatotoxicity by promoting ER stress and MPT.

ID: 32393662
Title: Therapeutic Targeting of the Secreted Lysophospholipase D Autotaxin Suppresses Tuberous Sclerosis Complex-Associated Tumorigenesis.
Abstract: Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by multiorgan hamartomas, including renal angiomyolipomas and pulmonary lymphangioleiomyomatosis (LAM). TSC2 deficiency leads to hyperactivation of mTOR Complex 1 (mTORC1), a master regulator of cell growth and metabolism. Phospholipid metabolism is dysregulated upon TSC2 loss, causing enhanced production of lysophosphatidylcholine (LPC) species by TSC2-deficient tumor cells. LPC is the major substrate of the secreted lysophospholipase D autotaxin (ATX), which generates two bioactive lipids, lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). We report here that ATX expression is upregulated in human renal angiomyolipoma-derived TSC2-deficient cells compared with TSC2 add-back cells. Inhibition of ATX via the clinically developed compound GLPG1690 suppressed TSC2-loss associated oncogenicity in vitro and in vivo and induced apoptosis in TSC2-deficient cells. GLPG1690 suppressed AKT and ERK1/2 signaling and profoundly impacted the transcriptome of these cells while inducing minor gene expression changes in TSC2 add-back cells. RNA-sequencing studies revealed transcriptomic signatures of LPA and S1P, suggesting an LPA/S1P-mediated reprogramming of the TSC lipidome. In addition, supplementation of LPA or S1P rescued proliferation and viability, neutral lipid content, and AKT or ERK1/2 signaling in human TSC2-deficient cells treated with GLPG1690. Importantly, TSC-associated renal angiomyolipomas have higher expression of LPA receptor 1 and S1P receptor 3 compared with normal kidney. These studies increase our understanding of TSC2-deficient cell metabolism, leading to novel potential therapeutic opportunities for TSC and LAM. SIGNIFICANCE: This study identifies activation of the ATX-LPA/S1P pathway as a novel mode of metabolic dysregulation upon TSC2 loss, highlighting critical roles for ATX in TSC2-deficient cell fitness and in TSC tumorigenesis.

ID: 32497488
Title: Mutations in SREBF1, Encoding Sterol Regulatory Element Binding Transcription Factor 1, Cause Autosomal-Dominant IFAP Syndrome.
Abstract: IFAP syndrome is a rare genetic disorder characterized by ichthyosis follicularis, atrichia, and photophobia. Previous research found that mutations in MBTPS2, encoding site-2-protease (S2P), underlie X-linked IFAP syndrome. The present report describes the identification via whole-exome sequencing of three heterozygous mutations in SREBF1 in 11 unrelated, ethnically diverse individuals with autosomal-dominant IFAP syndrome. SREBF1 encodes sterol regulatory element-binding protein 1 (SREBP1), which promotes the transcription of lipogenes involved in the biosynthesis of fatty acids and cholesterols. This process requires cleavage of SREBP1 by site-1-protease (S1P) and S2P and subsequent translocation into the nucleus where it binds to sterol regulatory elements (SRE). The three detected SREBF1 mutations caused substitution or deletion of residues 527, 528, and 530, which are crucial for S1P cleavage. In vitro investigation of SREBP1 variants demonstrated impaired S1P cleavage, which prohibited nuclear translocation of the transcriptionally active form of SREBP1. As a result, SREBP1 variants exhibited significantly lower transcriptional activity compared to the wild-type, as demonstrated via luciferase reporter assay. RNA sequencing of the scalp skin from IFAP-affected individuals revealed a dramatic reduction in transcript levels of low-density lipoprotein receptor (LDLR) and of keratin genes known to be expressed in the outer root sheath of hair follicles. An increased rate of in situ keratinocyte apoptosis, which might contribute to skin hyperkeratosis and hypotrichosis, was also detected in scalp samples from affected individuals. Together with previous research, the present findings suggest that SREBP signaling plays an essential role in epidermal differentiation, skin barrier formation, hair growth, and eye function.

ID: 33469231
Title: Site-1 protease controls osteoclastogenesis by mediating LC3 transcription.
Abstract: Site-1 protease (S1P) is a Golgi-located protein that activates unique membrane-bound latent transcription factors, and it plays an indispensable role in endoplasmic reticulum stress, lipid metabolism, inflammatory response and lysosome function. A patient with S1P mutation exhibits severe skeletal dysplasia with kyphoscoliosis, dysmorphic facial features and pectus carinatum. However, whether S1P regulates bone remodeling by affecting osteoclastogenesis remains elusive. Here, we show that S1P is indeed a positive regulator of osteoclastogenesis. S1P ablation in mice led to significant osteosclerosis compared with wild-type littermates. Mechanistically, S1P showed upregulated during osteoclastogenesis and was identified as a direct target of miR-9-5p. S1P deletion in bone marrow monocytes (BMMs) inhibited ATF6 and SREBP2 maturation, which subsequently impeded CHOP/SREBP2-complex-induced LC3 expression and autophagy flux. Consistently, transfection of LC3 adenovirus evidently rescued osteoclastogenesis in S1P-deficient BMMs. We then identified the interaction regions between CHOP and SREBP2 by Co-immunoprecipitation (Co-IP) and molecular docking. Furthermore, S1P deletion or inhibitor efficaciously rescued ovariectomized (OVX)- and LPS-induced bone loss in vivo. Collectively, we showed that S1P regulates osteoclast differentiation in a LC3 dependent manner and so is a potential therapy target for osteoporosis.

ID: 36379916
Title: Genuine selective caspase-2 inhibition with new irreversible small peptidomimetics.
Abstract: Caspase-2 (Casp2) is a promising therapeutic target in several human diseases, including nonalcoholic steatohepatitis (NASH) and Alzheimer's disease (AD). However, the design of an active-site-directed inhibitor selective to individual caspase family members is challenging because caspases have extremely similar active sites. Here we present new peptidomimetics derived from the VDVAD pentapeptide structure, harboring non-natural modifications at the P2 position and an irreversible warhead. Enzyme kinetics show that these new compounds, such as LJ2 or its specific isomers LJ2a, and LJ3a, strongly and irreversibly inhibit Casp2 with genuine selectivity. In agreement with the established role of Casp2 in cellular stress responses, LJ2 inhibits cell death induced by microtubule destabilization or hydroxamic acid-based deacetylase inhibition. The most potent peptidomimetic, LJ2a, inhibits human Casp2 with a remarkably high inactivation rate (k3/Ki ~5,500,000 M-1 s-1), and the most selective inhibitor, LJ3a, has close to a 1000 times higher inactivation rate on Casp2 as compared to Casp3. Structural analysis of LJ3a shows that the spatial configuration of Cα at the P2 position determines inhibitor efficacy. In transfected human cell lines overexpressing site-1 protease (S1P), sterol regulatory element-binding protein 2 (SREBP2) and Casp2, LJ2a and LJ3a fully inhibit Casp2-mediated S1P cleavage and thus SREBP2 activation, suggesting a potential to prevent NASH development. Furthermore, in primary hippocampal neurons treated with β-amyloid oligomers, submicromolar concentrations of LJ2a and of LJ3a prevent synapse loss, indicating a potential for further investigations in AD treatment.

ID: 37501400
Title: ATF6 aggravates apoptosis in early porcine embryonic development by regulating organelle homeostasis under high-temperature conditions.
Abstract: Activating transcription factor 6 (ATF6), one of the three sensor proteins in the endoplasmic reticulum (ER), is an important regulator of ER stress-induced apoptosis. ATF6 resides in the ER and, upon activation, is translocated to the Golgi apparatus, where it is cleaved by site-1 protease (S1P) to generate an amino-terminal cytoplasmic fragment. Although recent studies have made progress in elucidating the regulatory mechanisms of ATF6, its function during early porcine embryonic development under high-temperature (HT) stress remains unclear. In this study, zygotes were divided into four groups: control, HT, HT+ATF6 knockdown, and HT+PF (S1P inhibitor). Results showed that HT exposure induced ER stress, which increased ATF6 protein expression and led to a decrease in the blastocyst rate. Next, ATF6 expression was knocked down in HT embryos under microinjection of ATF6 double-stranded RNA (dsRNA). Results revealed that ATF6 knockdown (ATF6-KD) attenuated the increased expression of CHOP, an ER stress marker, and Ca 2+ release induced by HT. In addition, ATF6-KD alleviated homeostasis dysregulation among organelles caused by HT-induced ER stress, and further reduced Golgi apparatus and mitochondrial dysfunction in HT embryos. AIFM2 is an important downstream effector of ATF6. Results showed that ATF6-KD reduced the occurrence of AIFM2-mediated embryonic apoptosis at HT. Taken together, our findings suggest that ATF6 is a crucial mediator of apoptosis during early porcine embryonic development, resulting from HT-induced ER stress and disruption of organelle homeostasis. 激活转录因子 6 (activating transcription factor 6，ATF6) 是内质网中的三种传感器蛋白之一，是内质网应激诱导细胞凋亡的重要调节因子。 ATF6位于内质网中并在激活后转移到高尔基体，在那里它被 site-1-蛋白酶 (S1P) 切割以生成氨基末端胞质片段。 尽管最近的研究已在ATF6的调控机制方面取得了进展，但ATF6在高温(high temperature，HT) 情况下，对猪早期胚胎发育过程的影响还不清楚。在该研究中，胚胎被分为四组：对照组、HT组、HT+ATF6-KD组和 HT+PF（S1P抑制剂）组。结果表明，HT暴露诱导胚胎内质网应激，从而增加 ATF6 蛋白表达并导致囊胚率降低。由于显微注射ATF6 dsRNA，ATF6的表达水平在HT胚胎中被敲低，结果显示，ATF6-KD可以减弱由HT所导致的CHOP（内质网应激标记物）的表达增加以及Ca 2+的释放。 此外，ATF6-KD还减轻了HT诱导的ER应激所引起的细胞器稳态失衡，数据还显示ATF6-KD减少了HT胚胎中的高尔基体和线粒体功能障碍。AIFM2作为ATF6的重要下游蛋白，ATF6-KD减少了AIFM2介导的HT胚胎凋亡的发生。总之，这些结果表明 ATF6 是早期猪胚胎发育过程中，由 HT诱导的内质网应激和细胞器稳态失衡所引起细胞凋亡的重要介质。.

ID: 37865189
Title: Targeting YTHDF2 inhibits tumorigenesis of diffuse large B-cell lymphoma through ACER2-mediated ceramide catabolism.
Abstract: Epigenetic alterations play crucial roles in diffuse large B-cell lymphoma (DLBCL). Disturbances in lipid metabolism contribute to tumor progression. However, studies in epigenetics, especially its critical regulator YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), on lipid metabolism regulation in DLBCL are unidentified. Elucidate the prognostic value and biological functions of YTHDF2 in DLBCL and illuminate the underlying epigenetic regulation mechanism of lipid metabolism by YTHDF2 in DLBCL development. The expression and clinical value of YTHDF2 in DLBCL were performed in public databases and clinical specimens. The biological functions of YTHDF2 in DLBCL were determined in vivo and in vitro through overexpression and CRISPR/Cas9-mediated knockout of YTHDF2. RNA sequencing, lipidomics, methylated RNA immunoprecipitation sequencing, RNA immunoprecipitation-qPCR, luciferase activity assay, and RNA stability experiments were used to explore the potential mechanism by which YTHDF2 contributed to DLBCL progression. YTHDF2 was highly expressed in DLBCL, and related to poor prognosis. YTHDF2 overexpression exerted a tumor-promoting effect in DLBCL, and knockdown of YTHDF2 restricted DLBCL cell proliferation, arrested cell cycle in the G2/M phase, facilitated apoptosis, and enhanced drug sensitivity to ibrutinib and venetoclax. In addition, YTHDF2 knockout drastically suppressed tumor growth in xenograft DLBCL models. Furthermore, a regulatory role of YTHDF2 in ceramide metabolism was identified in DLBCL cells. Exogenous ceramide effectively inhibited the malignant phenotype of DLBCL cells in vitro. The binding of YTHDF2 to m6A sites on alkaline ceramidase 2 (ACER2) mRNA promoted its stability and expression. Enhanced ACER2 expression hydrolyzed ceramides, disrupting the balance between ceramide and sphingosine-1-phosphate (S1P), activating the ERK and PI3K/AKT pathways, and leading to DLBCL tumorigenesis. This study demonstrated that YTHDF2 contributed to the progression of DLBCL by regulating ACER2-mediated ceramide metabolism in an m6A-dependent manner, providing novel insights into targeted therapies.

ID: 41365058
Title: Characterising the effect of circulating sphingolipids on metastatic prostate cancer cells.
Abstract: Prostate cancer (PC) is a leading cause of cancer-related deaths in men, with advanced cases exhibiting resistance to androgen deprivation therapy. Dysregulated lipid metabolism has emerged as a hallmark of aggressive PC. This study investigates the biological underpinnings of a circulating three-lipid signature (3LS) previously validated as a prognostic biomarker in patients with metastatic castration-resistant prostate cancer (mCRPC). Using plasma samples from 16 mCRPC patients (8 positive and 8 negative for the 3LS), we assessed the impact of 3LS-positive plasma on three prostate cancer cell lines representative of disease progression. We investigated changes in cell viability and intracellular lipid metabolism associated with plasma from the two patient cohorts. Exposure to 3LS-positive plasma improved cell viability across AR-positive (LNCaP, C4-2B) and AR-negative (PC3) cell lines compared to exposure to 3LS-negative plasma. Lipidomic profiling revealed elevated sphingolipids and glycosphingolipids in 3LS-positive plasma-treated cells, accompanied by metabolic shifts characterised by increased monounsaturated and reduced polyunsaturated fatty acid levels. Inhibition of sphingosine kinase 1 abrogated the 3LS-positive plasma-treatment phenotype consistent with ceramide/sphingosine 1 phosphate (S1P) signalling being the mechanism of action. These findings suggest that the circulating 3LS is not just a prognostic biomarker, but an actionable signature reflecting changes in PC biology. The plasma lipid milieu identified by the 3LS drives a pro-survival phenotype by modifying lipid metabolism and upregulating ceramide/S1P signalling. The study provides the biological rationale to target ceramide-S1P signalling in patients, who are 3LS-positive. National Health and Medical Research Council of Australia Investigator grants (1196225; 2009965; 1197190); Cancer Institute New South Wales Translational Program Grant (TPG172146); Victorian Government Operational Infrastructure Support Program; Australian Government Research Training Program; University of Sydney merit award.

ID: 41465439
Title: Comparative Transcriptomic Analyses Reveal Potential Stp1 Regulatory Roles Independent of Sre1 in Phaffia rhodozyma.
Abstract: Sterol regulatory element-binding proteins (SREBPs) regulate lipid homeostasis in mammals via sequential activation by the site-1 (S1P) and site-2 (S2P) proteases. In the yeast Phaffia rhodozyma, homologs of SREBP (Sre1) and S2P (Stp1) were identified, with Sre1 cleaved by Stp1 and involved in the regulation of sterol and carotenoid biosynthesis. Additional regulatory roles of S2P have been described in other organisms, but such functions remain unexplored in P. rhodozyma, a question addressed in this study. Transcriptomic analyses of Δsre1, Δstp1, and Δsre1Δstp1 mutants were performed in both wild-type and Sre1-activated conditions. Potential genes regulated by Stp1 independently of Sre1 were identified, and their cellular roles were determined by KEGG mapping and Gene Ontology classification. As expected, most transcriptional changes in Δstp1 mutants reflected Sre1-mediated regulation. Notably, a subset of genes displayed differential expression independently of Sre1. These genes were linked to diverse aspects of cellular homeostasis, including metabolism, protein folding, ER stress response, and ribosome biogenesis. The transcriptomic analysis suggests that Stp1 regulates gene expression beyond the Sre1 transcription factor in P. rhodozyma, providing a framework for future studies to confirm and further explore these functions.

ID: 41639891
Title: Macrophage extracellular traps amplify retinal endothelial anoikis via the S1P-S1PR axis in diabetic retinopathy.
Abstract: OBJECTIVE: To investigate the pathogenic role of macrophage extracellular traps (METs) in proliferative diabetic retinopathy (PDR), focusing on endothelial dysfunction and inflammatory activation. METHODS: Transcriptomic data from PDR patients were first analyzed to identify METs-associated pathways. Anoikis-related subgroups were defined using the median gene set variation analysis (GSVA) enrichment score of the anoikis gene set, and enriched pathways were selected for subsequent validation. In vitro, diabetic retinopathy models were established using high-glucose and METs stimulation. Human retinal microvascular endothelial cells (HRMECs) were assessed for functional alterations and apoptosis. Rescue assays employed the AKT agonist SC79, the endocytosis inhibitor Dynasore, and DNase I. In vivo, streptozotocin (STZ)-induced diabetic mice received intravitreal METs with or without pharmacological interventions to evaluate vascular leakage, inflammatory responses, and neovascularization. RESULTS: Transcriptomic analysis revealed a strong association between METs-induced endothelial injury and activation of anoikis pathways, with sphingolipid signaling significantly enriched in the anoikis-high subgroup. In vitro, METs disrupted endothelial barrier integrity, induced ROS accumulation and mitochondrial damage, and activated anoikis and inflammatory signaling, accompanied by FAK dephosphorylation. These effects were partially reversed by SC79, DNase I, and sphingolipid-pathway inhibition. METs were internalized by HRMECs via endocytosis, triggering downstream signaling. In vivo, intravitreal METs induced vascular leakage, inflammatory cytokine elevation, and neovascularization, whereas inhibition of the SPHK1/S1P pathway (SKI-II) significantly mitigated these pathological changes. CONCLUSION: METs promote retinal vascular dysfunction by inducing endothelial anoikis and inflammatory activation through FAK/AKT and SPHK1/S1P/S1PR2/NF-κB signaling. Targeting METs-triggered lipid signaling may offer new therapeutic insights for diabetic retinopathy.

ID: 41735594
Title: Correction: Site-1 protease controls osteoclastogenesis by mediating LC3 transcription.
Abstract: 

ID: 41777869
Title: Plasma Sphingolipid Profiling Predicts Radiosensitivity in Hepatocellular Carcinoma.
Abstract: Radiotherapy constitutes a cornerstone in the management of hepatocellular carcinoma (HCC), but its efficacy is limited by radioresistance. Sphingolipids, a class of bioactive lipids, have been implicated in the metabolic reprogramming associated with treatment resistance. However, the potential of circulating sphingolipids as non-invasive biomarkers to predict radiosensitivity in HCC patients remains unexplored. This prospective study enrolled 61 HCC patients scheduled for radiotherapy (NCT06864221). Pre-treatment plasma samples were analyzed via LC-MS/MS to quantify 13 sphingolipid species. The primary endpoint was objective response rate (ORR) per mRECIST at 12 weeks. Predictive models were developed using multivariate logistic regression with forward selection and LASSO, evaluated by AUC with bootstrap validation, calibration, and decision curve analysis. Longitudinal analysis was performed in a sub-cohort (n=25) with paired pre- and post-radiotherapy plasma samples. The objective response rate was 54.1%. Univariable analysis identified a distinct sphingolipid signature in responders, characterized by significantly lower S1P and higher levels of CER(d18:1/20:0) and CER(d18:1/24:1). These candidate biomarkers, along with significant clinical variables, were entered into multivariate modeling. The optimal integrated model (Model 1), selected via forward selection, comprised S1P, CER(d18:1/20:0), and the clinical factors ALP and TBIL, and excelled at predicting response (bootstrap-corrected AUC=0.930). A second model based on ceramide/S1P balance (CER(d18:1/26:1)/S1P, Total CER(d18:1)/S1P, AFP) also performed robustly (bootstrap-corrected AUC=0.828). Both models showed clinical utility per decision curve analysis. Longitudinal analysis revealed a coordinated metabolic shift in responders, with reduced S1P and elevated CER(d18:1/26:0), supporting a radiation-induced "sphingolipid rheostat" shift toward apoptosis. This exploratory study provides the first clinical evidence that the baseline plasma sphingolipid profile is a potent, non-invasive predictor of HCC radiosensitivity, validating the "sphingolipid rheostat" theory. Our findings establish a framework for sphingolipid-guided precision radiotherapy and lay the necessary groundwork for future large-scale, multi-center validation trials, which hold significant potential to refine patient stratification and advance the development of novel metabolism-targeted interventions. Not all liver cancer patients benefit equally from radiotherapy, and doctors currently lack a good way to predict who will. Our study asked if a simple blood test could provide the answer by measuring specific fat molecules, called sphingolipids. We analyzed blood from 61 patients before their radiotherapy. We discovered that distinct patterns of these sphingolipids could accurately identify who would respond well to the treatment. We even built two prediction models that showed excellent accuracy. Interestingly, in patients who did respond well, we saw a helpful shift in these molecules after treatment: protective signals decreased while those that encourage cancer cell death increased. This means a straightforward blood test could one day help doctors personalize radiotherapy. By predicting a patient’s response in advance, we can better match them with the most effective therapies. This approach could spare those unlikely to benefit from unnecessary side effects, while ensuring those who will respond get the maximum benefit—ultimately leading to more precise and effective cancer care for everyone.

ID: 41857410
Title: S1P-S1PR1 signaling impairs CD8+ T cell metabolism and effector function in tumors.
Abstract: Sphingosine-1-phosphate receptor 1 (S1PR1) signaling has been linked to the regulation of immunosuppressive cell populations within the tumor microenvironment (TME); however, its role in shaping anti-tumor CD8⁺ T cell responses remains poorly defined. Herein, we demonstrate that intratumoral CD8⁺ T cells express S1PR1, with expression predominantly enriched in the terminally exhausted subset. Transcriptomic profiling, combined with pharmacological inhibition and genetic knockdown, reveals that S1PR1-S1P signaling activates the PERK (protein kinase R (PKR)-like endoplasmic reticulum kinase)-CHOP (C/EBP homologous protein) axis of the endoplasmic reticulum stress response. CHOP, in turn, upregulates transcription of Map3k13 and Map3k15, triggering downstream MAPK signaling and culminating in activation of p38MAPK. Activation of this pathway impairs CD8⁺ T cell metabolism and effector function while increasing apoptotic susceptibility. This ultimately limits the persistence and accumulation of functional CD8⁺ T cells within the TME, thereby compromising their responsiveness to anti-PD-1 therapy. Targeting the S1PR1-S1P axis or its downstream effectors offers a promising strategy to improve cancer immunotherapy outcomes.

ID: 42069319
Title: Lysosome-triggered nanotherapy packages osteoclast apoptotic bodies with pro-anabolic lipids to couple anti-resorption and bone formation.
Abstract: Osteoporosis remains challenging to treat because no approved therapy can simultaneously suppress bone resorption and stimulate bone formation. Here we report a lysosome-triggered nanotherapy that converts osteoclast death into a regenerative signal. We first synthesized docosahexaenoyl ceramide (DHA-ceramide) by replacing the native fatty acid of ceramide with docosahexaenoic acid and encapsulated it in aspartate-modified liposomes (Asp-Lip@Cer) to target osteoclasts. After cellular uptake, lysosomal acid ceramidase cleaves DHA-ceramide to release sphingosine (SPH) and DHA. Asp-Lip@Cer selectively disrupts the osteoclast lysosomal membrane and triggers apoptosis, and produces abundant osteoclast-derived apoptotic bodies (OC-ABs) enriched with SPH and DHA that are efficiently internalized by mesenchymal stem cells and endothelial progenitor cells. In recipient cells, DHA activates AKT signaling to promote osteogenesis, whereas SPH is converted to S1P to activate ERK signaling and enhance angiogenesis. In an ovariectomized mouse model of postmenopausal osteoporosis, systemic administration of Asp-Lip@Cer suppressed osteoclast activity, improved bone mineral density and trabecular architecture, increased osteopontin expression, and expanded CD31⁺/EMCN⁺ vasculature. This "one-stone-two-birds" strategy unites potent anti-resorptive activity with amplified pro-anabolic effects in a single platform, offering a promising therapeutic paradigm for osteoporosis and other disorders of impaired bone remodeling. ONE SENTENCE SUMMARY: Lysosome-triggered Asp-Lip@Cer nanotherapy turns osteoclast death into SPH/DHA-rich apoptotic bodies, simultaneously suppressing bone resorption and promoting bone formation.

ID: 42074265
Title: Structural Characterization, Toxicity Assessment and Molecular Modeling of Forced Degradation Products of Siponimod.
Abstract: Siponimod, a selective sphingosine 1-phosphate (S1P) receptor modulator, represents a next-generation therapeutic drug for active secondary progressive multiple sclerosis. This study conducted in-depth forced degradation studies of siponimod in solid state subjected to acidic, alkaline, oxidative, photolytic, and thermal conditions, in compliance with ICH guidelines Q1A (R2) and Q3A (R2). An HPLC method was developed to quantify siponimod and separate its degradation products (DPs). The DPs were characterized using LC-HRMS/MS and LC-MSn techniques. Moreover, the toxicological profiles of siponimod and its DPs were evaluated through the in silico tools ProTox 3.0 and ADMETlab 3.0, with molecular docking and dynamics simulations assessing their binding to the S1P1 receptor. Siponimod was stable to light but degraded under acidic, alkaline, oxidative, and thermal stress, producing five products: DP-1 (acidic), DP-2/3 (oxidative), DP-4 (hydrolytic), and DP-5 (thermal). The toxicity prediction suggested that neither siponimod nor its DPs exhibited carcinogenic or mutagenic potential, and the molecular modeling analysis revealed that DP-2 and DP-3 demonstrated favorable binding affinities, with stable dynamic profiles and thermodynamic properties that closely resembled those of siponimod. As far as we know, this is the first study on the structural elucidation of the DPs of siponimod by LC-HRMS/MS and LC-MSn.

ID: 42107070
Title: MEOX1 Coordinates Autocrine-Paracrine Programs via SPHK1/S1P to Promote Lymph Node Metastasis in Ovarian Cancer.
Abstract: Lymph node metastasis (LNM) is a pivotal determinant of poor prognosis in ovarian cancer (OC), yet how tumor-intrinsic programs remodel the microenvironment to enable spread remains unclear.Here, we identify the transcription factor mesenchymal homeobox 1 (MEOX1) as an upstream coordinator, whose overexpression associates with LNM, increased lymphatic density, and poor survival based on integrative analyses of public datasets and our 113-patient cohort. In an in vivo LNM model, MEOX1 overexpression enhances tumor burden, lymphatic vessel density, and LNM, whereas tumor-conditioned medium does not directly activate lymphatic endothelial cells (LECs), implicating stromal intermediates. Spatial transcriptomic and immunostaining analyses confirmed cancer-associated fibroblast (CAF)-LEC proximity and vascular endothelial growth factor-C (VEGF-C) localization within CAFs, supporting a CAF-dependent lymphangiogenic route. Mechanistically, MEOX1 binds the sphingosine kinase 1 (SPHK1) promoter to activate sphingosine-1-phosphate (S1P) synthesis, driving a dual autocrine-paracrine program: sphingosine-1-phosphate receptor 3 (S1PR3)-dependent signaling promotes tumor proliferation/migration, while S1P/S1PR1 reprograms fibroblasts into VEGF-C-secreting, alpha-smooth muscle actin (α-SMA)-positive CAFs that stimulate lymphangiogenesis and LNM; SPHK1 inhibition blunts these phenotypes, whereas S1P supplementation restores them. These findings provide novel insights into lymphatic metastasis and demonstrate that metastatic competence depends not only on intrinsic tumor aggressiveness but also on the acquired ability to construct a pro-dissemination niche.

ID: 42122966
Title: Higher Plasma Sphingosine-1-Phosphate Levels in Type 2 Diabetic Patients Have a Non-Linear Relationship with the Disease Prognostic Indices and Microvascular Complications: A Cross-Sectional Saudi Study.
Abstract: Background/Objectives: Sphingosine-1-phosphate (S1P) is implicated in glycemic control. However, its circulating levels and clinical significance in type 2 diabetes mellitus (T2DM) remain controversial. We assessed plasma S1P levels in T2DM patients, its associations with metabolic parameters and complications, and explored its biomarker potential and non-linear (U-/J-shaped) relationships. Methods: This cross-sectional study enrolled 140 patients with T2DM and 63 matching healthy controls. Plasma S1P was measured by competitive ELISA. Statistical analyses included comparisons, correlation, ROC analysis, multivariable logistic regression, and quadratic/spline regression for U-shaped relationships. Results: Plasma S1P was significantly elevated in T2DM patients [1256.7 (149.4-1510.0) ng/mL] compared to controls [1075.1 (202.0-1510.0) ng/mL; p < 0.001]. S1P correlated positively with age, disease duration, HbA1c, insulin resistance, TyG index, triglycerides, systolic blood pressure, and negatively with HDL-C. Patients with complications had higher S1P than those without (p = 0.001), with progressive increases from retinopathy to nephropathy to mixed complications. Insulin-treated patients exhibited the highest S1P levels (p < 0.001). ROC analysis showed moderate diagnostic accuracy (AUC = 0.724). S1P is an independent associated factor with complications (OR = 1.18 per 100 ng/mL, p = 0.003). Non-linear analysis revealed a U-shaped relationship with HDL-C (optimal S1P: 1100-1350 ng/mL) and a J-shaped relationship with complication risk (threshold ~1250 ng/mL). Conclusions: Plasma S1P is elevated in T2DM and correlates with disease severity, glycemic control, insulin resistance, and complications. S1P demonstrates moderate biomarker potential and exhibits non-linear U-/J-shaped relationships with metabolic parameters, suggesting an optimal therapeutic window of 1100-1280 ng/mL. These findings support S1P as a marker of cumulative disease burden and a potential therapeutic target.

ID: 42147971
Title: Decoding the S1P-S1PR axis in cancer: Mechanisms, pathways and therapeutic horizons (Review).
Abstract: Sphingosine-1-phosphate (S1P) and its five G protein-coupled receptors (S1PR1-S1PR5) regulate a broad range of processes that shape cancer progression, including proliferation, survival, angiogenesis, immune evasion and metastatic dissemination. Under physiological conditions, this signaling axis contributes to vascular integrity, immune cell trafficking and tissue homeostasis. In cancer, however, its output is not solely determined by ligand abundances. Rather, tumors reprogram the S1P-S1PR axis at a number of levels, coupling altered S1P production with receptor-specific changes in expression, localization and the signaling state to generate context-dependent malignant phenotypes. The present review provided a receptor-resolved synthesis of S1PR functions in cancer and examined the mechanisms that underlie pathway dysregulation, including transcriptional activation, epigenetic remodeling, microRNA loss, post-translational modifications and altered receptor trafficking and compartmentalization. It further discussed how metabolic amplification of S1P availability cooperates with receptor-level rewiring to sustain tumor progression, microenvironmental remodeling and therapeutic resistance. This framework positions the S1P-S1PR axis as a dynamically reprogrammed signaling network and highlights therapeutic strategies that concurrently target S1P production and receptor-mediated signaling as promising avenues for more-precise, biomarker-informed cancer treatment.

ID: 42182331
Title: Pyridoxine supplementation confers protection against SGPL1 R222Q variant sphingosine phosphate lyase insufficiency syndrome.
Abstract: Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is a rare condition causing nephrotic syndrome, neuropathy, and other manifestations. SPLIS is caused by mutations in SGPL1, which encodes sphingosine-1-phosphate lyase (SPL), a pyridoxal 5'-phosphate (PLP)-dependent enzyme needed to degrade the bioactive sphingolipid sphingosine-1-phosphate (S1P). Supplementation with the PLP precursor pyridoxine benefits some individuals with PLP-dependent enzymopathies. We sought to establish whether pyridoxine has therapeutic activity in SPLIS. Neurological improvement, plasma S1P normalization, and increased SPL activity in patient-derived fibroblasts were observed after pyridoxine supplementation in a patient with R222Q-variant SPLIS. Additionally, PLP dose-dependently augmented recombinant R222Q-variant SPL activity. To further explore pyridoxine's effects, gene editing was employed to create an R222Q-variant SPLIS mouse model. SPLR222Q mice fed pyridoxine-enriched chow lacked obvious phenotypes. However, SPL inactivation, S1P accumulation, wasting, anemia, proteinuria, and glomerulosclerosis developed in SPLR222Q but not WT mice fed chow with reduced pyridoxine. Ultrastructural analysis and super-resolution microscopy showed podocyte loss and foot process effacement. Transcriptional profiling revealed a pattern of cytokine upregulation and extracellular matrix remodeling. Inhibiting S1P production prevented nephrosis in SPLR222Q mice fed chow lacking pyridoxine. Our findings establish a novel SPLIS mouse model that recapitulates R222Q-variant SPLIS, demonstrates its responsiveness to pyridoxine, and implicates S1P in its pathophysiology.

ID: 42206708
Title: [Research progress of sphingosine-1-phosphate (S1P) in cancer].
Abstract: Sphingosine-1-Phosphate (S1P) is a bioactive phospholipid molecule that regulates multiple signaling pathways through its interaction with sphingosine-1-phosphate receptors (S1PRs). In cancer tissues, S1P is not only associated with the survival of cancer cells, but also related to the angiogenesis, inflammatory responses, and immune evasion in the tumor microenvironment (TME). Various inhibitors of the S1P kinase have shown significant anti-tumor effects in pre-clinical studies by directly inhibiting tumor growth and metastasis, and enhancing the therapeutic potential of both chemotherapy and immunotherapy. Nevertheless, the complex and pleiotropic nature of the S1P signaling pathway presents considerable challenges for the development of targeted therapeutic strategies. This paper reviews the biological functions and research progress of S1P and its related pathways in cancer, offering novel insights and potential directions for cancer therapy.

ID: 42256552
Title: Therapeutic potential of the sphingosine kinase 2 inhibitor opaganib.
Abstract: Opaganib is a proprietary, host-directed, potentially broadly potent, first-in-class oral sphingosine kinase 2 (SphK2) selective inhibitor developed by RedHill Biopharma Tel Aviv, Israel. It is currently the most widely used selective SphK2 inhibitor. It simultaneously inhibits three sphingolipid-metabolizing enzymes in human cells-SphK2, dihydroceramide desaturase (DES1), and glucosylceramide synthase (GCS)-leading to depletion of sphingosine 1-phosphate (S1P), accumulation of ceramides and dihydroceramides, and suppression of key pro-survival pathways including pERK, pAKT, and NF-κB. These events promote autophagy, apoptosis, and disruption of viral replication. A large body of evidence indicates that SphK plays an important role in health and disease. This study reviews the role and mechanisms of opaganib effects as anticancer, anti-inflammatory, and antiviral agent. The latest research directions for opaganib are described as gastrointestinal acute radiation syndrome (GI-ARS), chemical exposure indications, and COVID-19, Ebola, and other viruses, providing new therapeutic ideas and considerations for future research and clinical trials.

ID: 42343303
Title: Reduction of cancer cell quantity and viability in autologous blood salvaged from patients with liver cancer: efficacy of intraoperative cell salvage coupled with leukocyte depletion filtration.
Abstract: This in vitro study aims to evaluate the effects of intraoperative cell salvage (ICS) combined with leukocyte depletion filtration (LDF) on hepatocellular carcinoma (HCC) cell number and viability in salvaged autologous blood from patients undergoing liver cancer surgery. Twenty patients undergoing open radical resection for primary liver cancer with ICS were enrolled in the study.Blood samples of 20 ml each were procured at three distinct stages: from the surgical field(S1),post ICS treatment(S2),and post ICS treatment combined with leukocyte depletion filter(LDF) filtration(S3).Within these 20-ml blood samples,10 ml underwent cancer cell enrichment procedures, followed by identification and enumeration of cancer cells using immunofluorescence staining.The remaining 10 ml of blood samples were cultured for a duration of three weeks, with subsequent assessment of cell viability using immunofluorescence techniques subsequent to enrichment procedures. HCC cells were identified in samples obtained from S1(19/20),S2(18/20),and S3 (16/20) without a significant difference in the detection rate(P > 0.05).However, a significant reduction in HCC cells count was observed in samples from S2 and S3 when compared to S1(P < 0.05). Notably, no statistically significant differences were noted between HCC cells counts in samples from S2 and S3(P > 0.05). Following three weeks of culture, optical microscopy revealed the presence of liver cancer cell clusters exclusively in S1 samples, while such clusters were absent in samples from S2 and S3. Further examination under fluorescence microscopy indicated the presence of epithelial-mesenchymal hybrid-type HCC cells(S1: 400, S2: 14) and mesenchymal-type HCC cells(S1: 100, S2: 21) in both S1 and S2 samples, whereas no HCC cells were detected in S3 samples.Specifically, HCC cells in S1 samples manifested as liver cancer cell clusters, whereas such clusters were notably absent in samples from S2 and S3. Following treatment with ICS alone or in combination with LDF (ICS-LDF), both the number and viability of hepatocellular carcinoma (HCC) cells in salvaged autologous blood were markedly decreased, with no cell cluster formation, which lowered the risk of salvaging cancer cells to some extent. Nevertheless, LDF failed to completely remove HCC cells from all samples, and its filtration efficiency may be compromised when the HCC cell number exceeds a certain threshold.

ID: 42383100
Title: T Helper Cells and Cytokine Networks in the Immunopathogenesis of Multiple Sclerosis.
Abstract: Multiple sclerosis (MS) is a chronic autoimmune disorder of CNS with demyelination, neurodegeneration and compartmentalized inflammatory disorder. Excessive T-helper cell (CD4+) activation and unregulated cytokine signaling play a key role in its onset and progression. These changes impair communication between peripheral immune cells and CNS resident microglia, astrocytes and oligodendrocytes. This review provides an overview on the contribution of specific subsets of T-helper cells to MS pathology/immunity. Th1 cells release interferon-γ and lymphotoxin, that stimulate activation of myeloid cells/antigen presentation. Activated by IL-23, the Th17 cells produce IL-17A/F that lowers the blood-brain barrier (BBB) integrity, recruit neutrophils and monocytes, and enhance microglial killing. Activation of CD4+ T cells leads to activation of B cells via T follicular helper cells which couple these processes through the production of IL-21 and CXCR5. This leads to the development of tissue-like aggregates and intrathecal antibody production. T-cell plasticity adds to epitope spreading as well as chronic inflammation, IL-22, IL-9, IL-1β, IL-6, and TGF-β (these are additional mediators involved in the regulation of effector phenotypes). In MS, the regulation of dendritic cell co-stimulation and of glial activation often does not work. This is due to the lack of control of dendritic-cells co-stimulation and the lack of regulation of glial activation by regulatory pathways such as FOXP3+ regulatory T cells and Tr1 cells that secrete IL-10 and TGF-Beta. The review also explores the cytokine network biomarkers, CSF and serum signatures and single-cell immune states, as well as existing and new drugs. These include migration blockade, targeting of S1P-receptors, anti-CD20 therapy, targeting of Th17/GM-CSF and JAK-STAT pathways, low-dose IL-2, approaches of targeting antigens and engineered Tregs. Investigating the areas of stage and compartment-specific CD4+ T-cell circuits can help to advance targeted immunomodulation in progressive MS and neuro-repair.

ID: 42409594
Title: CerS2 Overexpression Enhances Palmitoyl-CoA-Induced Cytotoxicity and Alters Ceramide Species Composition in Breast Cancer Cells.
Abstract: Sphingolipid metabolism, particularly the balance between pro-apoptotic ceramide and pro-survival sphingosine-1-phosphate (S1P), has been associated with breast cancer progression. The aim of the present study was to examine whether ceramide synthase 2 (CerS2) overexpression is associated with changes in increased palmitoyl-CoA (PCA) based ceramide buildup and cell fate changes in MCF-7 breast cancer cells, a hormone receptor-positive model. CERS2 plasmid transfected cells were treated with PCA and/or the CerS inhibitor, fumonisin B1 (FB1). Cell viability, the proliferation marker proliferating cell nuclear antigen (PCNA), apoptosis (TUNEL and cleaved caspase-3), and sphingolipid profiles were evaluated. High-dose PCA (100 μM) significantly reduced MTT signal and PCNA expression and increased apoptotic markers, with stronger effects in CerS2-overexpressing cells. Across short- and longer-term exposures, the response pattern was concentration- and time-responsive but not uniformly monotonic. Lipidomic analysis revealed that PCA and CerS2 overexpression increased C16 ceramide and very-long-chain ceramides (C22-C24), respectively. FB1 decreased the level of ceramide, elevated S1P and partly counteracted PCA-induced cytotoxicity. FB1 pre-treatment which was applied sequentially restricted but did not eliminate apoptosis. These findings indicate that CerS2 overexpression reshapes the cellular response to substrate-driven sphingolipid stress by altering acyl-chain-specific ceramide composition and the balance between ceramide- and S1P-associated signaling. Rather than reflecting isolated pathway effects, the results support a context-dependent role for CerS2 in modulating apoptotic sensitivity in MCF-7 breast cancer cells.

ID: 42417903
Title: Engineering of siRNA molecules to silence SphK1 mRNA for therapeutic intervention in triple-negative breast cancer: An In Silico and In Vitro Analysis.
Abstract: Triple-negative breast cancer (TNBC) is an aggressive and therapeutically challenging subtype of breast cancer lacking estrogen, progesterone, and HER2 receptors. Sphingosine kinase 1 (SphK1), a key enzyme in the sphingosine-1-phosphate (S1P) signaling pathway, has been implicated in TNBC progression, metastasis, and chemoresistance. In this study, we employed a comprehensive computational and experimental approach to design and validate small interfering RNA (siRNA) molecules targeting SphK1 for therapeutic intervention. A panel of siRNAs was designed using multiple bioinformatic algorithms and evaluated through secondary structure prediction, off-target screening, and molecular docking against both SphK1 mRNA and the human Argonaute 2 (AGO2) protein. Molecular dynamics simulations confirmed the structural stability and functional compatibility of the top candidate, g6 siRNA, within the AGO2 binding pocket. Experimental validation using Lipofectamine-2000 mediated transfection in MDA-MB-231 TNBC cells demonstrated that g6 siRNA achieved approximately 75-80% reduction in SphK1 mRNA and protein expression, leading to marked inhibition of cell proliferation, migration, and colony formation, and a significant increase in apoptosis. These findings confirm the predictive reliability of our in-silico workflow and establish g6 siRNA as a potent candidate for targeted SphK1 silencing in TNBC. The study further highlights the translational potential of combining g6 siRNA with current therapeutic regimens, including PARP inhibitors, immune checkpoint inhibitors, or chemotherapeutic agents, to improve treatment efficacy and overcome drug resistance in TNBC.

ID: 42450239
Title: The Sphingolipid Balance and Endothelial Dysfunction in Lysosomal Storage Diseases: Shared Mechanisms in Gaucher, Niemann-Pick and Fabry Disease.
Abstract: Endothelial dysfunction underlies many cardiovascular and metabolic diseases. Lysosomal storage disorders, particularly sphingolipidoses, cause intracellular accumulation of specific sphingolipids due to inherited enzyme defects. This review focuses on Gaucher, Niemann-Pick (types A, B, A/B) and Fabry diseases, selected because they exhibit clinically significant cardiovascular manifestations and each accumulates a distinct sphingolipid-glucocerebroside, sphingomyelin, or globotriaosylceramide-allowing comparative analysis of how different metabolic defects converge on similar endothelial phenotypes. We summarize current knowledge on how substrate accumulation disrupts the ceramide/sphingosine-1-phosphate (S1P) rheostat, affecting NO synthase, vascular permeability, inflammation, angiogenesis, autophagy and cell death. Common and disease-specific changes in endothelial morphology and barrier function are discussed. Importantly, direct experimental evidence for endothelial involvement in Gaucher and Niemann-Pick diseases remains scarce; most mechanistic insights derive from non-endothelial cell models, highlighting a significant gap that underscores the need for targeted endothelial studies. Deficiencies of GBA1, SMPD1, and GLA each modulate S1P and ceramide production through distinct pathways, yet all three conditions share similar functional endothelial alterations driven by disrupted sphingolipid homeostasis. Understanding these common mechanisms opens new perspectives for diagnostic biomarkers and therapeutic strategies aimed at restoring sphingolipid balance in the endothelium, though further research is required to validate these findings in endothelial-specific contexts.

ID: 42454062
Title: Iron overload disrupts bone homeostasis via TfR1-dependent ferroptosis and cGAS/STING-driven pyroptosis in pyogenic spondylitis.
Abstract: Pyogenic spondylitis (PS) accompanies with diverse destruction, especially the subsequent bone destruction, which leads to spine instability and severe neurological disability. However, the mechanism underlying bone loss induced by infection has not been elucidated. In this study, we aimed to reveal a novel mechanism of bone destruction in PS. To certify the involvement of iron overload in PS-induced bone loss, vertebrae samples were collected and evaluated from patients with PS. Next Staphylococcus aureus (S. aureus, ATCC 25923) was used to induce bone infection in vivo and in vitro, and relevant markers were investigated. Then, experiments using siRNA targeting transferrin receptor-1 (TfR1), an iron chelator (DFO), and the TfR1 inhibitor Ferristatin II were conducted to investigate the role of TfR1-induced iron overload and ferroptosis in PS-induced bone destruction. Infected vertebral specimens from PS patients showed iron overload and increased TfR1 expression, which was also observed in S. aureus -infected MC3T3-E1 cells. Excessive iron leads to osteoblast ferroptosis and osteogenic activity via iron overload and oxidative stress injury, which was inhibited by TfR1 siRNA or DFO. Meanwhile, iron overload promoted mtDNA leakage and activated the cGAS/STING pathway, contributing to NLRP3-associated pyroptosis and impaired osteogenesis. In addition, S. aureus -induced iron overload in osteoclasts promoted osteoclastogenesis, which was also ameliorated by TfR1 siRNA or DFO. In vivo, Ferristatin II reduced iron deposition, suppressed TfR1 expression, and preserved trabecular architecture in PS rats. Our research indicates that S. aureus infection triggers iron overload in infected bone tissue via the promotion of TfR1 expression, finally contributing to osteoblast ferroptosis and bone destruction. Targeting TfR1-mediated iron influx and ferroptosis is a novel therapeutic strategy for the treatment of bone loss induced by PS.

ID: 42454501
Title: Exendin-4 improves high glucose-induced mitochondrial dysfunction of pancreatic β-cells via PKA/Drp1 signaling.
Abstract: The development of type 2 diabetes mellitus is closely associated with mitochondrial dysfunction of pancreatic β-cells, but the mechanisms by which glucagon-like peptide-1 receptor activation preserves mitochondrial homeostasis under glucotoxic conditions remain incompletely understood. Herein, we investigated whether Exendin-4 protects β-cells against chronic high glucose (HG)-induced mitochondrial injury by regulating the cAMP/PKA/Drp1 signaling pathway. INS-1 β-cells, pancreatic tissues from db/db mice, and isolated primary islets were used to assess oxidative stress, apoptosis, mitochondrial function and morphology, insulin secretion, and cAMP/PKA/Drp1 signaling. Prolonged HG exposure increased oxidative stress and apoptosis, impaired mitochondrial membrane potential, elevated mitochondrial ROS accumulation, reduced ATP content, and promoted mitochondrial fragmentation in INS-1 β-cells. These changes were accompanied by increased Drp1 expression, reduced cAMP levels and PKA activity, decreased inhibitory phosphorylation of Drp1 at Ser637, and increased Ser616 phosphorylation. Exendin-4 attenuated HG-induced oxidative stress and apoptosis, restored mitochondrial function, improved mitochondrial morphology, and partially restored Drp1 Ser637 phosphorylation, whereas it did not significantly affect HG-induced Ser616 phosphorylation. In db/db mice, Exendin-4 improved metabolic parameters and alleviated β-cell apoptosis, with partial recovery of Drp1 Ser637 phosphorylation in pancreatic islets. Furthermore, glucose-stimulated insulin secretion assays in isolated primary islets showed that Exendin-4 improved β-cell secretory function in islets isolated from db/db mice. Pharmacological inhibition of PKA with H89 attenuated Exendin-4-induced Drp1 Ser637 phosphorylation and mitochondrial protection. Collectively, these results suggest that Exendin-4 protects pancreatic β-cells against HG-induced mitochondrial dysfunction and β-cell injury by restoring PKA-associated inhibitory phosphorylation of Drp1 at Ser637 and improving mitochondrial dynamics.

ID: 42455831
Title: Sodium butyrate induces mitochondrial pathway apoptosis in liver cancer via ATF4/SLC7A11-mediated ferroptosis.
Abstract: Liver cancer, a prevalent and aggressive malignancy globally, is associated with high morbidity and mortality rates. Butyrate, a metabolite produced by intestinal microbiota, is capable of restricting cancer initiation and progression. However, the precise mechanisms underlying its effects on liver cancer remain poorly understood. This study utilized a CCK-8 cytotoxicity assay to demonstrate that sodium butyrate (NaB) suppresses liver cancer cell proliferation through ferroptosis and apoptosis. The involvement of ATF4/SLC7A11 signaling and mitochondrial dysfunction in NaB-induced ferroptosis and apoptosis was further investigated. Results revealed a decrease in ATF4 and SLC7A11 expression, an elevation in the levels of malondialdehyde (MDA) and reactive oxygen species (ROS), and a reduction in glutathione (GSH) in NaB-treated liver cancer cells. These ferroptosis-related alterations could be reversed by an ATF4 activator. Additionally, NaB-treated liver cancer cells presented a decrease in mitochondrial membrane potential (MMP), accumulation of mitochondrial ROS, and mitochondrial damage. These cellular changes disrupted the BAX/BCL-2 balance, leading to cytochrome C release, which subsequently activated caspase9 and caspase3, initiating mitochondrial pathway apoptosis. In vivo, NaB treatment resulted in increased iron content in liver cancer tissues, along with upregulated cytochrome C, activated caspase9, and caspase3 expression; these effects were counteracted by ferrostatin-1 (Fer-1). Collectively, this study elucidates that NaB induces mitochondrial damage via ferroptosis mediated by ATF4/SLC7A11, ultimately triggering mitochondrial pathway apoptosis in hepatoma cells. These findings may offer novel insights into therapeutic strategies for hepatoma.

ID: 42479117
Title: S1P, Generated by Sphingosine Kinase 1, Negatively Affects Corneal Wound Healing Process by Activating TGF-β/Smad Pathway.
Abstract: Abnormal healing of corneal injury can lead to corneal opacity, a leading cause of blindness. This process is extremely complex and involves precise interactions of multiple signaling pathways. A lack of understanding of these complex interactions provides a major challenge in developing therapeutic strategies. Both sphingosine-1-phosphate (S1P) and transforming growth factor beta (TGFβ) signaling have been associated with tissue fibrosis. The purpose of this study was to understand the interplay between S1P and TGFβ signaling in the process of corneal wound healing and fibrosis. Mice lacking the Sphingosine kinase 1 gene (Sphk1-/-) and their wildtype littermates were subjected to corneal injury by alkali burn. The progress of wound healing and the expression and activation of TGFβ signaling intermediates and profibrotic proteins were measured at different days postinjury (DPI). We observed that reduction of S1P signaling in Sphk1-/- mice induced the closure of corneal epithelial layer at a faster rate than its wildtype littermates following alkali burn. Reduced activation of profibrotic proteins Smad2 and 3, along with reduced expression of Smad4 and higher expression of antifibrotic protein Smad7, was also observed in the Sphk1-/- mice as compared to the wildtype littermates following corneal injury. Inhibition of S1P signaling by exogenous delivery of a small-molecule inhibitor of sphingosine kinase 1 (SphK1) could accelerate corneal wound healing in similarly injured wildtype (Sphk1+/+) mice. These results suggest that S1P signaling positively influences TGFβ signaling pathways that negatively affect corneal wound healing by delaying the process and inducing fibrotic development. Thus, inhibition of S1P signaling could be an effective therapeutic option to accelerate corneal wound healing and thereby prevent corneal fibrosis/scar formation.

ID: 42487268
Title: FZD7 Inhibitor SRI Attenuates High Glucose-Induced Retinal Pigment Epithelial Cell Injury Accompanied by Ferroptosis-Associated Changes via Suppression of the Wnt/β-Catenin Pathway.
Abstract: This study investigated the protective effects of the Frizzled-7 (FZD7) inhibitor SRI against high glucose-induced injury in human retinal pigment epithelial (ARPE-19) cells. It also explored the underlying mechanism, focusing on whether SRI attenuates ferroptosis and apoptosis by inhibiting the Wnt/β-catenin signalling pathway. ARPE-19 cells were exposed to high glucose (25 mM) and treated with a non-cytotoxic concentration of SRI (2 μmol/L), as determined by a CCK-8 assay. Wnt/β-catenin signalling was evaluated by measuring β-catenin expression and phosphorylation. The expression of GPX4, DHODH, and FSP1, together with intracellular Fe²⁺, ROS, and MDA levels, was assessed to evaluate ferroptosis and oxidative stress. Apoptosis was analysed by examining Bax and Bcl-2 expression, whereas mitochondrial ultrastructure was evaluated using quantitative transmission electron microscopy. Under hyperglycaemic conditions, SRI suppressed Wnt/β-catenin signalling by reducing β-catenin expression and promoting its phosphorylation. SRI activated a GPX4-independent ferroptosis defence pathway, evidenced by increased DHODH and FSP1 expression without affecting GPX4 levels. Furthermore, SRI reduced intracellular Fe²⁺, ROS and MDA accumulation, downregulated Bax, upregulated Bcl-2 and improved mitochondrial morphology with partial restoration of cristae integrity. Collectively, these findings demonstrate that SRI alleviates high glucose-induced oxidative stress, ferroptosis, apoptosis and mitochondrial damage. SRI protects ARPE-19 cells from high glucose-induced injury by reducing ferroptosis-associated oxidative stress, apoptosis and mitochondrial dysfunction. These protective effects are mediated, at least in part, through inhibition of Wnt/β-catenin signalling and activation of a DHODH/FSP1-dependent but GPX4-independent ferroptosis defence pathway. These findings suggest that FZD7 represents a promising therapeutic target for diabetic retinopathy.

ID: 42490423
Title: Distinct roles of COPI proteins attenuated in cell senescence.
Abstract: In senescent cells, functional alterations in organelles like mitochondria and lysosomes are well characterized, but senescence-associated changes in Golgi function are not. An RNA interference screen revealed that silencing subunits of the coatomer protein I (COPI) complex, critical for intracellular transport involving the Golgi, reduced extracellular vesicle uptake. In proliferating WI-38 fibroblasts, silencing COPI constituents COPA, COPB1, COPB2, or COPD induced ATF4 production and disrupted autophagy, apoptosis, and cytokine signaling, all hallmarks of impaired Golgi-to-endoplasmic reticulum transport, while silencing COPI constituents COPG1, COPE, or COPZ1 altered the production of extracellular matrix proteins. Furthermore, individual COPI proteins associated with Golgi and endosomal proteins, supporting roles in vesicular trafficking. In senescent WI-38 fibroblasts, silencing COPI proteins did not elicit these phenotypes. Our findings underscore the distinct, multifunctional actions of individual COPI proteins and their key roles in intracellular homeostatic transport networks that become attenuated during senescence.

ID: 42496794
Title: N6-Methyladenosine-Driven Nuclear Export of circKCNN2 Impairs GRP75 Function to Promote Neuronal Apoptosis.
Abstract: Emerging evidence implicates circular RNAs (circRNAs) in Alzheimer's disease (AD) pathogenesis. Notably, circKCNN2 correlates with clinical dementia severity in AD patients; however, its biological functions and regulatory mechanisms in neuronal systems remain poorly understood. Here, we elucidated the molecular mechanism by which circKCNN2 regulated neuronal apoptosis and delineated the underlying post-transcriptional regulatory axis. Using SH-SY5Y cells as an in vitro neuronal model, we manipulated circKCNN2 expression through overexpression and siRNA-mediated knockdown. Apoptosis was assessed by flow cytometry and TUNEL assays. Methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), MS2-based RNA pull-down coupled with mass spectrometry, fluorescence in situ hybridization, and nuclear/cytoplasmic fractionation were employed to characterize the underlying mechanism. circKCNN2 overexpression significantly induced apoptosis, whereas its knockdown attenuated apoptosis. circKCNN2 underwent N6-methyladenosine (m6A) modification. Mechanistically, METTL3-catalyzed m6A modification enabled YTHDC2 binding and facilitated the nuclear export of circKCNN2. Both METTL3 depletion and YTHDC2 overexpression caused nuclear retention and attenuated apoptosis. Furthermore, MS2-based RNA pull-down coupled with mass spectrometry identified GRP75 as a cytoplasmic interactor of circKCNN2. Functionally, GRP75 overexpression rescued circKCNN2-induced apoptosis. Our findings revealed a novel "modification-localization-function" cascade wherein the METTL3-YTHDC2-m6A axis dictates circKCNN2 nuclear-cytoplasmic trafficking and the subsequent inhibition of GRP75, thereby driving neuronal apoptosis. This mechanism established circKCNN2 as a pathogenic driver in AD and highlighted the m6A regulatory machinery as a potential target for further investigation.