Search Results (277)

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive subtype of ALL characterized by unfavorable clinical outcomes. Despite significant progress in deciphering the genetic and epigenetic landscapes of T-ALL, the underlying molecular mechanisms, particularly in non-early T-cell precursor (non-ETP) T-ALL, remain incompletely understood. In this study, functional assays were performed using three well-characterized non-ETP T-ALL cell lines. In vivo therapeutic efficacy was evaluated using non-ETP T-ALL xenograft models. Transcriptomic profiling was performed by RNA sequencing (RNA-seq) followed by bioinformatic analysis. Publicly available clinical datasets from T-ALL patients were mined to analyze survival outcomes. We found that activation of CD40 ligand (CD40LG) or CD28 accelerates cell-cycle progression and enhances the migratory capacity of non-ETP T-ALL cells, with CD40LG uniquely upregulating CXCR4 to mediate bone marrow tropism. Further RNA-seq and functional validation identified Rho GTPase signaling, specifically RhoA/Rac1/Rac2, as a pivotal downstream effector of CD40LG/CD28, leading to therapeutic resistance to PI3K inhibition. Pharmacological blocking RhoA or Rac1 using small-molecule compounds not only induces remarkable cytotoxicity but also sensitizes resistant cells to PI3K inhibitors, both in vitro and in vivo. Clinically, elevated expression of CD40LG, CD28, RHOA, or RAC2 correlates with poor prognosis in non-ETP T-ALL patients. These findings uncover a novel CD40LG/CD28-Rho GTPase axis as a key driver of pathogenesis and a potential therapeutic vulnerability in non-ETP T-ALL, providing a new target for precision intervention and a promising strategy to overcome therapeutic resistance. Full article

Venous thromboembolism (VTE) is a major cause of morbidity and mortality, underscoring the need for new molecular markers to enable early detection and clarify underlying mechanisms. Iron metabolism is linked to oxidative stress, endothelial injury, and inflammation, all central to thrombosis, yet its transcriptomic contribution to VTE remains unclear. We analyzed gene expression profiles from GSE19151 and GSE48000 using differential expression and weighted gene co-expression network analysis (WGCNA), integrating results with an iron metabolism gene set. Three hub genes were identified, arachidonate 5-lipoxygenase (ALOX5), Rho GTPase activating protein 1 (ARHGAP1), and glucose-6-phosphate dehydrogenase (G6PD), all downregulated in VTE. Gene set enrichment indicated that ALOX5 is involved in endothelial regulation, lipid metabolism, and immune pathways. A three-gene signature showed high diagnostic accuracy (AUC = 0.924 in the discovery cohort; 0.705 in validation). Immune deconvolution revealed broad immune remodeling and associated ALOX5 with multiple immune cell subsets, especially M0 macrophages, and with regulators such as TGFB1 and IL6R. Western blot analysis further showed that ALOX5 protein expression was significantly increased in LPS-activated HUVECs, supporting its involvement in inflammatory endothelial injury. DrugBank screening identified 19 approved drugs targeting ALOX5, supporting its potential for mechanistic and clinical investigation. Full article

Background/Objectives: Yes-associated protein 1 (YAP1) is a transcriptional cofactor that coordinates the complex interplay between cell proliferation, survival, differentiation, metabolism, biomechanics, and tissue regeneration. Previous studies have shown that YAP1 activity is reduced during aging, and replacing YAP1 function has been shown to rejuvenate old cells by mitigating senescence and its associated inflammation. Methods: As YAP1 is now confirmed to exert a profound regenerative influence on multiple organs, we wanted to gain more insight into the molecular signature of YAP1 expression relevant to brain cells. Since proteomics is a very powerful tool for discoveries, we generated SH-SY5Y cells stably expressing GFP-YAP1 and screened 8000 human proteins using multiplex arrays that utilize biotin-label-based antibody arrays. Results: We found YAP1 expression in astrocytes, microglia, neuronal and neuroblastoma cell lines, as well as human neurons. Importantly, YAP1 protein levels were significantly reduced selectively in the nuclear fractions of the brains of patients with Alzheimer’s disease (AD) relative to normal control (NC) subjects. The screen resulted in the identification of 283 differentially expressed proteins. In line with YAP1’s known role in the regulation of actin and cytoskeleton, we found a 2.53-fold upregulated level of Rho guanine nucleotide exchange factor 1 (ARHGEF1), a guanine nucleotide exchange factor (GEF) for the RhoA GTPase, which is crucial for dendritic spine regulation. A 6.19-fold upregulated level of NECAP endocytosis-associated 2 (NECAP2), the highest known increase for any protein in this screen, plays an essential role in clathrin-mediated endocytosis. Most importantly, another upregulated protein was Neudesin Neurotrophic Factor (NENF) (3.07-fold increase), also known as Neudesin, which primarily acts as a neurotrophic factor, and it promotes neuronal survival, enhances cell proliferation, and neurogenesis in neural progenitor cells. Neural Precursor Cell Expressed, Developmentally Down-Regulated 9(NEDD9) levels were also upregulated by 2.46-fold, and it affects neuronal cell number and synaptic connections through its role in neurite formation. However, it should be noted that these proteomic results are preliminary in nature as they are derived from single-sample data. The upregulated levels of ARHGEF1 and NEDD9 were confirmed by immunoblots. We also found a drastic reduction in the levels of p16INK4a, a marker of senescence. Conclusions: Thus, the anti-senescence effect of YAP1 may be mediated through p16INK4a, which in turn may be crucial for YAP1’s regenerative functions through NENF and NEDD9. Full article

Background: Merkel cell carcinoma (MCC) is a rare malignancy of the skin caused by Merkel cell polyomavirus (80% of cases) or by ultraviolet (UV) sun exposure (20% of cases). The isoprenoid biosynthesis pathway (IBP) is an essential metabolic pathway shown to be upregulated in tumorigenesis, causing aberrant activation of Ras and Rho GTPases and resulting in unregulated cellular proliferation and survival. Methods: Through the use of pharmacological inhibitors of the IBP, we assessed the role of the IBP and its downstream targets in viral-positive and viral-negative MCC cell lines. To identify the most critical IBP intermediates, cellular metabolic activity, cell death and cell cycle distribution were measured after IBP perturbation. Results: Across all cell lines, treatment with IBP inhibitors, especially fluvastatin, decreased metabolic activity; however, perturbation of different intermediates resulted in variable responses between viral-positive and viral-negative cell lines. Conclusions: Our findings demonstrate varying dependence on the IBP between viral-positive and viral-negative MCC and highlight the importance of cellular dynamics when determining a treatment regimen for patients with MCC. Full article

Toxin A and B from Clostridioides difficile are the main pathogenicity factors for clinical symptoms of C. difficile infections. Receptor-mediated endocytosis and endosomal escape are required for targeting substrate proteins of the Rho-GTPase family. We previously reported that Toxin B (TcdB) affects endo-lysosomal transport and autophagic flux of target cells. These effects are independent from pathogenic Rho inhibition. Here, we aimed at further characterization of this event by immunofluorescent characterization of the vesicular structures that are affected. We found large aggregates of damaged endolysosomal structures positive for EEA1, LAMP1, CHMP4B and TcdB, as well as an increase in perinuclear concentration of non-mature autophagosomes (amphisomes) positive for SQSTM, Rab7, and LC3B. We investigated whether Rab7, a regulator of late endosome transport, is causative for decreased lysosome function. Although TcdB induced an increase in active Rab7, as tested by an RILP pull-down assay, inhibition of Rab7 did not prevent TcdB-induced decrease in cathepsin D as a surrogate for lysosome dysfunction. It also indicates that the observed increase in Rab7 positive amphisomes is secondary to lysosomal dysfunction. By applying an autoproteolytic deficient mutant of TcdB we proved that the release of the glucosyltransferase domain is mandatory for triggering all of these effects. This suggests that after membrane perforation the toxin remnants leave an open leak in endolysosomes affecting ion homeostasis. Investigation of all large clostridial glucosyltransferases and other toxins revealed lysosomal dysfunction as a general effect of many but not of all toxins that integrate into the endosome membrane. Full article

Background/Objectives: Epigenetic modifications, particularly DNA methylation, contribute to tumor progression and therapy resistance. Guadecitabine, a hypomethylating agent (HMA), has shown promising clinical activity when combined with carboplatin in preclinical models. We evaluated the combination of guadecitabine with carboplatin as a second-line treatment for extensive-stage small-cell lung cancer (SCLC; NCT03913455), one of the deadliest malignancies. Here, we report methylome changes in peripheral blood mononuclear cells (PBMCs) collected at baseline and during treatment from patients on the trial. Methods: PMBC DNA was analyzed using Infinium HumanMethylationEPIC v1.0 bead chips. Data were processed, and differentially methylated positions (DMPs) were identified and analyzed for pathway enrichment using bioinformatic approaches, and immune deconvolution analyses were conducted to investigate the impact on immune cell composition. Results: Direct comparison of PBMCs between cycle 2 day 5 (C2D5; post-treatment) vs. cycle 1 day 1 (C1D1; pre-treatment) revealed a greater number of hypomethylated DMPs (380 DMPs in C2D5 vs. C1D1 PBMCs; p < 0.05, |β| > 20%). Moreover, when first compared with normal PBMCs from cancer-free controls, the number of hypomethylated DMPs was even greater in C2D5 than in C1D1 (1771 vs. 237 DMPs, respectively; p < 0.05, |β| > 20%). Long interspersed nucleotide elements-1 (LINE-1) were significantly hypomethylated in PBMCs after HMA treatment (C2D5 vs. C1D1). Pathway analysis of hypomethylated DMPs revealed significant alterations in key signaling pathways, including NF-κB, Rho GTPase, and pulmonary fibrosis in C1D1 vs. C2D5. Normal PBMCs to C1D1 PBMCs revealed changes in IL-3 signaling, Fcγ receptor-mediated phagocytosis, and molecular mechanisms of cancer. Deconvolution analysis revealed a greater percentage of monocytes in C1D1 vs. normal PBMCs; after HMA treatment, percentages of monocytes and B cells decreased, while the eosinophil percentage increased in C1D1 vs. C2D5. Conclusions: HMA treatment has a global impact on PBMC methylomes in cancer patients. DNA methylation changes were associated with biological pathways related to PBMC function, and shifts in distinct immune cell populations were observed. Full article

Background: Glabridin, a natural compound derived from Glycyrrhiza glabra L., possesses skin-lightening effects. This study aims to further elucidate the depigmentation mechanism of glabridin by investigating its effects on melanogenesis and melanin transfer. Methods: We initially confirmed the anti-melanogenic effects of glabridin in MNT-1 human melanoma cells. Then, we investigated the mechanism of its anti-melanogenic effect by evaluating the protein expression of β-catenin and MITF via Western blot. To investigate melanin transfer, we compared glabridin’s efficacy with that of niacinamide, a recognized inhibitor of melanosome transfer and employed two complementary experimental models: (1) α-melanocyte-stimulating hormone (α-MSH)-stimulated MNT-1 cells to analyze dendrite formation, and (2) a UVB-irradiated co-culture system of MNT-1 cells and HaCaT keratinocytes to evaluate melanin transfer. Results: By measuring glabridin’s effects on melanin content, tyrosinase activity, and melanogenesis-related protein expression confirmed its inhibition of melanin synthesis. Further investigation demonstrated that glabridin suppresses melanogenesis by downregulating β-catenin and MITF, indicating inhibition of the Wnt/β-catenin pathway. Furthermore, in α-MSH-treated MNT-1 cells, both glabridin and niacinamide were found to suppress dendrite formation and elongation. In a UVB-exposed co-culture system, both glabridin and niacinamide inhibited melanin transfer to keratinocytes. Mechanistically, these effects were linked to the regulation of Rho GTPases (Rac1, RhoA, Cdc42) and suppression of F-actin reorganization. Conclusions: This study provides, for the first time, evidence that the skin-lightening effect of glabridin involves two complementary mechanisms: inhibition of melanogenesis through suppression of the Wnt/β-catenin pathway, and attenuation of both dendricity and melanin transfer via the influence of Rho family GTPases expression. Full article

Rho GTPases are a group of guanosine triphosphate (GTP)-binding proteins with a relative molecular weight of about 20–30 kD, and 22 different Rho GTPases have been identified in mammalian cells, among which RhoA, Rac1 and Cdc42 are the most well-studied. Rho-associated coiled coil forming protein kinase (ROCK) is the most well-researched downstream effector of Rho GTPases. The Rho/ROCK signaling pathway widely participates in the reorganization of the cytoskeleton through cascade phosphorylation/dephosphorylation reactions and modulates cellular biological behaviors including cell adhesion, migration and phenotypic transformation. Abnormal activation of the Rho/ROCK signaling pathway is closely associated with the occurrence and progression of acute kidney injury, diabetic nephropathy, hypertension-related nephropathy and chronic allograft nephropathy, which contributes to podocyte injury, renal tubular epithelial-to-mesenchymal transition (EMT), mesangial cell proliferation and inflammatory infiltration in the kidney. This review focuses on the research progress and regulatory mechanisms of the Rho/ROCK signaling pathway in the above four major kidney diseases and discusses the therapeutic potential of targeting this pathway for kidney disease treatment, aiming to provide new insights for elucidating the pathogenesis of kidney diseases and developing novel therapeutic strategies. Full article

RhoB is an atypical Rho GTPase whose function is tightly linked to its subcellular localization and membrane trafficking, reflecting its unique post-translational modifications and association with endosomal membranes in addition to the plasma membrane. Despite its implication in membrane trafficking and cytoskeletal regulation, tools to directly monitor RhoB activity in space and time have been lacking. Here, we describe the development and validation of a single-chain, genetically encoded Förster resonance energy transfer (FRET) biosensor that enables direct visualization of RhoB activity in living cells while preserving its native membrane-targeting determinants. The biosensor exhibits a large dynamic range and resolves spatially heterogeneous RhoB activity during leading-edge protrusion–retraction cycles in migrating mouse embryonic fibroblasts. To demonstrate the utility of this tool, we performed multiplex live-cell imaging with a previously developed near-infrared FRET biosensor for the exocytic Rho GTPase TC10. Quantitative morphodynamic and cross-correlation analyses reveal coordinated yet antagonistic spatiotemporal patterns of RhoB and TC10 activities at the leading edge and show that perturbation of TC10 regulation reorganizes their spatial coupling. Together, this work introduces a robust biosensor for RhoB and establishes a multiplex imaging framework to study the coordination of trafficking and signaling during cell migration. Full article

Metastatic breast cancer (BC) remains a major clinical challenge, and identifying molecular mechanisms driving tumor cell migration and invasion is critical to develop effective therapeutic strategies. Clusterin (CLU), a secreted chaperone-like protein, is upregulated in BC and metastatic tissue; however, its functional contribution to tumor aggressiveness remains unclear. Here, we silenced CLU by siRNA in two BC cell lines with distinct aggressiveness and examined its impact on migration, invasion, and associated signaling pathways. Following CLU silencing, cell migration and invasion were assessed using transwell assays. Cytoskeletal organization was evaluated by F-actin staining, while downstream signaling pathways were analyzed by RT-PCR, Western blotting, and Rho GTPase pull-down. A comparative proteomic analysis was performed in CLU-expressing and CLU-silenced MDA-MB-231 cells. CLU knockdown significantly reduced migration and invasion in MDA-MB-231, concomitantly with loss of F-actin-rich membrane protrusions, reduced expression of MMP9, COL1A1, and COL4A1, and decreased activation of Akt, NF-κB, and RhoA. Proteomic profiling revealed extensive remodeling of pathways involved in cell adhesion, cytoskeletal dynamics, and extracellular matrix interactions. Differently, no or very mild effects were observed in CLU-silenced MCF-7 cells. These findings identify CLU as an upstream regulator of a pro-metastatic adhesion–cytoskeleton signaling in BC, selectively operative in EMT-engaged, basal-like cells, highlighting the importance of patient stratification for CLU-targeted therapeutic strategies. Full article

Extracellular vesicles (EVs) secreted by Fusarium oxysporum play an important role in the process of its infestation of the host, but the in vitro research system for EVs of F. oxysporum (Fo-EVs) has not yet been improved, and the mechanism of its action remains unclear. In this study, particle size distribution, particle concentration, number of particles per unit of protein, number of particles per unit of mycelial biomass, and concentration of contaminated proteins were used as indicators to evaluate the yield and purity of Fo-EVs. The optimal method for Fo-EV preparation and extraction was screened by comparing liquid culture, solid culture, and solid culture with enzymatic cell wall hydrolysis. The optimal system for Fo-EVs separation and purification was screened by a pairwise combination of three primary methods (Ultracentrifugation (UC), Ultrafiltration (UF), and Polyethylene glycol precipitation method (PEG)) and two secondary methods (Size-exclusion chromatography (SEC) and Aqueous two-phase system (ATPS)), respectively. The protein composition was identified via mass spectrometry technology, followed by GO annotation and GO enrichment analysis using whole-genome proteins as the background. Based on these steps, a Fo-EV protein library was constructed to reveal Fo-EV’s most active biological functions. The results showed that solid culture combined with the UC-SEC method could effectively enrich Fo-EVs with a typical cup-shaped membrane structure. The obtained Fo-EVs had an average particle size of 253.50 nm, a main peak value of 200.60 nm, a particle concentration of 2.04 × 10¹⁰ particles/mL, and a particle number per unit protein of 1.09 × 10⁸ particles/μg, which were significantly superior to those of other combined methods. Through proteomic analysis, 1931 proteins enriched in Fo-EVs were identified, among which 350 contained signal peptides and 375 had transmembrane domains. GO enrichment analysis revealed that these proteins were mainly involved in cell wall synthesis, vesicle transport, and pathogenicity-related metabolic pathways. Additionally, 9 potential fungal EV markers, including Hsp70, Rho GTPase family, and SNARE proteins, were screened. This study constructed an isolation system and a marker database for Fo-EVs, providing a methodological and theoretical basis for in-depth analysis of the biological functions of Fo-EVs. Full article

RhoGDI2 is a RhoGTPase regulator that has roles in cytoskeleton organization and cell survival, amongst others. It is differentially expressed in many cell types and tissues, including several human cancers, where its expression has been correlated with either good or bad prognosis. To identify the underlying mechanisms, we knocked down its expression in human cancer cell lines. We observed that repression of RhoGDI2 expression, but not that of the closely related RhoGDI1, significantly reduces their proliferation rate. In parallel, RhoGDI2 suppression induces supernumerary centrosomes and inhibits ciliogenesis. As RhoGDIs are regulators of GTPases, we checked whether key RhoGTPases are involved in these effects. We found that silencing RhoA partially rescued the induction of supernumerary centrosomes and ciliary defects observed upon RhoGDI2 silencing. It was previously shown that RhoGDI2 is strongly expressed in immune cells and that there are striking similarities between primary cilia and immune synapses. Based on this knowledge, we silenced RhoGDI2 in NK cells and could demonstrate that this strongly affects their immune synapse-related cancer cell-killing activity. Altogether, these data suggest novel roles for RhoGDI2 in centrosome functions in human cancer and immune synapses in immune cells, which provides an explanation for its reported dual role in cancer. Full article

Background/Objectives: Rho small GTPases (RSG), which regulates metastasis, constitute eight subfamilies—“classical” Rho, Rac, cdc42, and “atypical” Rif, Rnd, Wrch, RhoH, and RhoBTB. Their downstream signaling requires switching between GTP-bound active and GDP-bound inactive forms. Classical RSGs, but not atypical RSGs, require regulation by guanine nucleotide exchange factors (GEF), GTPase-activating proteins (GAP) and guanine nucleotide dissociation inhibitors (GDI) to achieve this switch. The objective of this review is to summarize the roles of RSGs in metastatic prostate cancer (mPCa) and their interaction with the androgen receptor (AR), which regulates this disease. Methods: We summarize the literature that describes the role of RSGs in mPCa, and their interaction with the AR. Results: Classical RSGs mostly promote metastasis (except RhoB), whereas atypical RSGs, with exceptions, mostly prevent it. Their role, however, is context-dependent—e.g., RhoB is tumor-suppressive in AR-null PCa but oncogenic in AR-positive tumors. The AR modulates RSG expression transcriptionally, but also affects their function through modulation of GEFs, GAPs, and GDIs. In turn, RSGs also regulate AR transcriptional activity. Interestingly, RSGs and the AR have non-genomic interactions via membrane-localized AR (mAR) not affected by AR inhibitors. Conclusions: Drugs that target RSGs are needed along with AR inhibitors to prevent mPCa progression. Full article

The small GTPase RhoA and its downstream effector Rho-kinase (ROCK) have emerged as pivotal regulators of vascular smooth muscle cell (VSMC) contraction, endothelial function, and vascular remodeling. Activation of the RhoA/ROCK pathway enhances calcium (Ca²⁺) sensitivity by inhibiting myosin light chain phosphatase (MLCP), thereby promoting sustained vascular tone independent of intracellular Ca²⁺ levels. In endothelial cells (ECs), RhoA/ROCK signaling contributes to nitric oxide (NO) dysregulation, oxidative stress, cytoskeletal reorganization, and inflammatory activation. Cumulative evidence implicates this pathway in the development and progression of hypertension and other cardiovascular diseases, where maladaptive vascular remodeling, VSMC proliferation, and endothelial dysfunction drive increased vascular resistance. Translational studies have identified ROCK inhibitors and indirect modulators such as statins as promising therapeutic strategies. This review integrates recent mechanistic insights into RhoA/ROCK regulation of vascular function with clinical and translational perspectives on targeting this pathway in hypertension. Full article

Background/Objectives: Targeting Hsp90β with isoform-selective inhibitors offers a promising therapeutic strategy with reduced toxicity compared to pan-Hsp90 inhibition. However, mechanisms of resistance to Hsp90β-selective inhibition remain poorly defined. This study aimed to identify molecular determinants of Hsp90β dependency and pharmacologic resistance across cancer types. Methods: We integrated gene dependency, transcriptomic, proteomic, metabolomic, and drug sensitivity data from the Cancer Cell Line Encyclopedia with in vitro validation using the Hsp90β-selective inhibitor, NDNB-25. Comparative and correlation analyses were performed to identify resistance-associated pathways, followed by network and combination drug testing to validate functional interactions. Results: Resistant cell lines exhibited extensive rewiring of Rho GTPase signaling, cytoskeletal remodeling, and metabolic adaptation, including mitochondrial dysfunction and redox imbalance. Integrated analyses linked these phenotypes to aryl hydrocarbon receptor (AHR) activation and compensatory Hsp90α expression. Experimental validation confirmed increased kynurenine levels, a known endogenous AHR ligand, in NDNB-25–acquired resistant cells. Gene–drug network integration revealed collateral sensitivity to carboplatin, which synergized with Hsp90β inhibition in resistant models. Conclusions: This study defines the molecular features and adaptive programs underlying resistance to Hsp90β-selective inhibition and identifies therapeutic vulnerabilities that can be exploited to overcome it. The findings establish a systems-level framework for predicting Hsp90β inhibitor response and support rational combination strategies, including carboplatin co-treatment, for future preclinical development. Full article