Rho GTPases Research Articles

Machine learning reveals CAT gene as a novel potential diagnostic and prognostic biomarker in non-small cell lung cancer

BackgroundNon-small cell lung cancer (NSCLC) represents one of the most prevalent forms of lung cancer, with a five-year survival rate of 21.7%. There is an urgent need to identify pertinent biomarkers to inform the diagnosis and prognosis of tumors, particularly those that can be applied to different age groups. Herein, we would apply machine learning methods to specifically analyze the issue of biomarker applicability across different age groups in NSCLC.MethodsStudies have shown a higher incidence of NSCLC in people over 40 years of age, and due to the limitations of data set, studies of individuals under 40 years of age were not included in this study. To simulate the human aging model as closely as possible, we gathered corresponding non-small cell lung cancer (NSCLC) samples from the UCSC Xena database based on patient age information. These samples were then categorized into three groups: 40–60, 60–80, and over 80 years old. Subsequently, we employed four machine learning methods—Random Forest, LASSO regression analysis, XGBoost, and GBM—to identify gene sets with significant diagnostic value for each age group. By taking the intersection of these sets, we identified the optimal gene and assessed its prognostic significance in NSCLC. Then, the diagnostic value of CAT gene was validated using global public databases, including the GSE32863, GSE43458, GSE68571, GSE10072, and GSE63459 datasets from the Americas, the GSE30219 and GSE102511 datasets from Europe, and the GSE31210 and GSE19804 datasets from Asia. Furthermore, immunohistochemical staining was performed in an independent cohort from a tissue microarray. Additionally, cell culture and RT-qPCR were employed for external validation.ResultsThrough the implementation of machine learning methods, we successfully identified the catalase (CAT) gene. Our analysis revealed that individuals with high expression of the CAT gene experienced improved survival rates. Additionally, these individuals exhibited elevated immune scores. We further discovered that the CAT gene synergizes with multiple components of neutrophils, including TLRs, FcRn, and the selective GEF of Rho-family GTPases. In addition, we identified a potential immune checkpoint, TNFSF15, which is applicable to the human aging model. Finally, we validated the CAT gene's diagnostic value using databases encompassing the Americas, Europe, and Asia regions. Through external RT-qPCR validation, we verified that CAT expression in BEAS-2B was higher than that of A549. In an independent human cohort, we also verified that CAT is lowly expressed in lung cancer tissues. In addition, higher CAT levels were associated with improved survival in the 40–60 and 60–80 age groups.ConclusionsIn our analysis of the NSCLC database, we pinpointed the CAT gene, which holds promise for potential diagnostic and prognostic applications in the context of human aging. Furthermore, it may offer insights into addressing age-related heterogeneity of NSCLC.

Model supports asymmetric regulation across the intercellular junction for collective cell polarization.

Symmetry breaking, which is ubiquitous in biological cells, functionally enables directed cell movement and organized embryogenesis. Prior to movement, cells break symmetry to form a well-defined cell front and rear in a process called polarization. In developing and regenerating tissues, collective cell movement requires the coordination of the polarity of the migration machineries of neighboring cells. Though several works shed light on the molecular basis of polarity, fewer studies have focused on the regulation across the cell-cell junction required for collective polarization, thus limiting our ability to connect tissue-level dynamics to subcellular interactions. Here, we investigated how polarity signals are communicated from one cell to its neighbor to ensure coordinated front-to-rear symmetry breaking with the same orientation across the group. In a theoretical setting, we systematically searched a variety of intercellular interactions and identified that co-alignment arrangement of the polarity axes in groups of two and four cells can only be achieved with strong asymmetric regulation of Rho GTPases or enhanced assembly of complementary F-actin structures across the junction. Our results held if we further assumed the presence of an external stimulus, intrinsic cell-to-cell variability, or larger groups. The results underline the potential of using quantitative models to probe the molecular interactions required for macroscopic biological phenomena. Lastly, we posit that asymmetric regulation is achieved through junction proteins and predict that in the absence of cytoplasmic tails of such linker proteins, the likeliness of doublet co-polarity is greatly diminished.

Magnesium Supplementation Modifies Arthritis Synovial and Splenic Transcriptomic Signatures Including Ferroptosis and Cell Senescence Biological Pathways

Background: Rheumatoid arthritis (RA) is a common systemic autoimmune inflammatory disease that can cause joint damage. We have recently reported that oral magnesium supplementation significantly reduces disease severity and joint damage in models of RA. Methods: In the present study, we analyzed the transcriptome of spleens and synovial tissues obtained from mice with KRN serum-induced arthritis (KSIA) consuming either a high Mg supplemented diet (Mg2800; n = 7) or a normal diet (Mg500; n = 7). Tissues were collected at the end of a 15-day KSIA experiment. RNA was extracted and used for sequencing and analyses. Results: There was an enrichment of differentially expressed genes (DEGs) belonging to Reactome and Gene Ontology (GO) pathways implicated in RA pathogenesis such as RHO GTPases, the RUNX1 pathway, oxidative stress-induced senescence, and the senescence-associated secretory phenotype. Actc1 and Nr4a3 were among the genes with the highest expression, while Krt79 and Ffar2 were among the genes with the lowest expression in synovial tissues of the Mg2800 group compared with the Mg500 group. Spleens had an enrichment for the metabolism of folate and pterines and the HSP90 chaperone cycle for the steroid hormone receptor. Conclusions: We describe the tissue transcriptomic consequences of arthritis-protecting Mg supplementation in KSIA mice. These results show that oral Mg supplementation may interfere with the response to oxidative stress and senescence and other processes known to participate in RA pathogenesis. We provide new evidence supporting the disease-suppressing effect of increased Mg intake in arthritis and its potential to become a new addition to the therapeutic options for RA and other autoimmune and inflammatory diseases.

Oscillatory dynamics of Rac1 activity in Dictyostelium discoideum amoebae.

Small GTPases of the Rho family play a central role in the regulation of cell motility by controlling the remodeling of the actin cytoskeleton. In the amoeboid cells of Dictyostelium discoideum, the active form of the Rho GTPase Rac1 regulates actin polymerases at the leading edge and actin filament bundling proteins at the posterior cortex of polarized cells. We monitored the spatiotemporal dynamics of Rac1 and its effector DGAP1 in vegetative amoebae using specific fluorescent probes. We observed that plasma membrane domains enriched in active Rac1 not only exhibited stable polarization, but also showed rotations and oscillations, whereas DGAP1 was depleted from these regions. To simulate the observed dynamics of the two proteins, we developed a mass-conserving reaction-diffusion model based on the circulation of Rac1 between the membrane and the cytoplasm coupled with its activation by GEFs, deactivation by GAPs and interaction with DGAP1. Our theoretical model accurately reproduced the experimentally observed dynamic patterns, including the predominant anti-correlation between active Rac1 and DGAP1. Significantly, the model predicted a new colocalization regime of these two proteins in polarized cells, which we confirmed experimentally. In summary, our results improve the understanding of Rac1 dynamics and reveal how the occurrence and transitions between different regimes depend on biochemical reaction rates, protein levels and cell size. This study not only expands our knowledge of the behavior of Rac1 GTPases in D. discoideum amoebae but also demonstrates how specific modes of interaction between Rac1 and its effector DGAP1 lead to their counterintuitively anti-correlated dynamics.

The food dye Tartrazine disrupts vascular formation both in zebrafish larvae and in human primary endothelial cells

Tartrazine (E102) is a controversial coloring agent whose potential impacts on human health are not fully understood. Our study reveals the vascular disrupting effects of tartrazine (TTZ) on developing zebrafish embryos in vivo and on human umbilical vein endothelial cells in vitro. The dye was shown to cause dose-dependent hemorrhages in zebrafish embryos. Analyzing transgenic zebrafish harboring fluorescent endothelial cells revealed that TTZ treatment disrupted cell organization into vessels in both the sub-intestinal vein and the brain area. Assays on human umbilical vein endothelial cells demonstrated that TTZ inhibited endothelial proliferation, tube formation, and migration in a dose-dependent manner. Taken together, our results indicate for the first time that TTZ can affect endothelial cell properties, possibly by disrupting Rho family GTPase pathways which control the cytoskeleton. Our finding provides a credible explanation for many reported human health impacts and offers prospective applications for biomedicine.

LC–MS-based quantitation of proteomic changes induced by Norcantharidin in MTB-Treated macrophages

Tuberculosis drug resistance contributes to the spread of tuberculosis. Immunotherapy is an effective strategy for treating tuberculosis, with the regulation of macrophage-mediated anti-tuberculosis immunity being crucial. Norcantharidin (NCTD), a drug used in tumor immunotherapy, has significant immunomodulatory effects. Thus, NCTD may have an anti-tuberculosis role by regulating immunity. Understanding how NCTD affects the proteome of Mtb-infected macrophages can provide valuable insights into potential treatments. This study aimed to investigate the impact of NCTD (10 μg/mL) on the proteome of macrophages infected with Mtb H37Ra using liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis. A total of 69 differentially regulated proteins (DRPs) were identified, with 28 up-regulated and 41 down-regulated in the NCTD-treated group. Validation of six DRPs (CLTCL1, VAV1, SP1, TRIM24, MYO1G, and WDR70) by Western blot analysis confirmed the accuracy of the LC–MS/MS method used in this study. NCTD modulates various protein expressions involved in chromatin-modifying enzymes, RHO GTPases activating PAKs, Fc gamma R-mediated phagocytosis, T cell receptor signaling pathway, and antigen processing and presentation. Overall, the research provides new insights into the effects of NCTD on the proteome of Mtb-infected macrophages. The identified changes highlight potential targets for future therapeutic interventions aimed at enhancing host immunity against Mtb infection or developing new anti-TB drugs based on these findings.

Structural basis for the effects of Ser387 phosphorylation of MgcRacGAP on its GTPase-activating activities for CDC42 and RHOA

MgcRacGAP is a GTPase-activating protein (GAP) for the Rho family GTPases. During cytokinesis, MgcRacGAP localizes to the midbody, where it activates the GTPase activity of Rho family GTPases to facilitate cytokinesis. In the midbody, Aurora B phosphorylates Ser387 within the GAP domain of human MgcRacGAP, a modification that is suggested to influence GTPase preference. However, there are conflicting reports, with some studies indicating that Ser387 phosphorylation does not alter the GTPase preference of MgcRacGAP. This controversy highlights the need for a deeper understanding of the molecular interactions involved, which can be clarified through structural analyses. In the present study, we determined the crystal structures of the wild-type MgcRacGAP GAP domain complexed with CDC42•GDP•AlF4− and the S378D phosphomimetic mutant GAP domain fused with RHOA•GDP•AlF4−. Additionally, crystal structures of the GAP domains were determined for the S387D and S387A mutants. Our analysis revealed that neither GTPase binding nor S387D mutation affected the overall structure of the GAP domain. However, comparison of the CDC42•MgcRacGAP (wild-type) complex with the RHOA-MgcRacGAP(S378D) fusion protein structure indicated that the S387D mutation caused positional shifts in both CDC42 and RHOA relative to MgcRacGAP. These shifts reduced interactions with CDC42 more severely than those with RHOA. In fact, the S387D mutation decreased the GTPase-activating activity of MgcRacGAP toward CDC42, while its impact on RHOA was only moderate. This difference in the rate of activity reduction may play an important role in GTPase preference.

Integrated analysis of disulfidptosis-related genes SLC7A11, SLC3A2, RPN1 and NCKAP1 across cancers

Disulfidptosis, a newly identified form of regulated cell death associated with disruption of disulfide bond formation in the endoplasmic reticulum, involves the dysregulation of disulfidptosis-related genes (DRGs) that may contribute to cancer development and progression. However, the molecular mechanisms and clinical implications of DRGs in different cancer types remain poorly characterized. Therefore, in this comprehensive study, we investigated the expression, prognostic value, and functional roles of four recently identified DRGs (SLC7A11, SLC3A2, RPN1, and NCKAP1) across various cancers. Especially, in clinical samples of glioblastoma, we found that RPN1 was significantly correlated with patient survival. Through mutation landscape analysis, we identified diverse missense mutations in these DRGs, with NCKAP1 exhibiting the highest mutation frequency (5.9% in skin cutaneous melanoma). Additionally, we observed positive correlations between these DRGs and tumor stemness (DNAss DNA stemness score and RNAss RNA stemness score) as well as RNA modifications, particularly m6A modification, in several cancer types. Furthermore, high expression of SLC7A11, RPN1, and NCKAP1 was positively associated with infiltration of T-helper type 2 (Th2) cells in various cancers, while high expression of SLC7A11, SLC3A2, and RPN1 correlated with tumor mutational burden (TMB) in 10, 4, and 8 tumor types, respectively. Utilizing a protein–protein interaction network, we identified the RHO GTPases Activate WASPs and WAVEs pathway as significantly enriched, suggesting the involvement of these DRGs in cancer-related signaling pathways. Collectively, our findings provide novel insights into the molecular mechanisms and clinical implications of DRGs in pan-cancer, highlighting their potential as biomarkers and therapeutic targets for cancer treatment.

The Rho effector ARHGAP18 coordinates a Hippo pathway feedback loop through YAP and Merlin to regulate the cytoskeleton and epithelial cell polarity.

The organization of the cell's cytoskeletal filaments is coordinated through a complex symphony of signaling cascades originating from internal and external cues. Two major actin regulatory pathways are signal transduction through Rho family GTPases and growth and proliferation signaling through the Hippo pathway. These two pathways act to define the actin cytoskeleton, controlling foundational cellular attributes such as morphology and polarity. In this study, we use human epithelial cells to investigate the interplay between the Hippo and Rho Family signaling pathways, which have predominantly been characterized as independent actin regulatory mechanisms. We identify that the RhoA effector, ARHGAP18, forms a complex with the Hippo pathway transcription factor YAP to address a long-standing enigma in the field. Using super resolution STORM microscopy, we characterize the changes in the actin cytoskeleton, on the single filament level, that arise from CRISPR/Cas9 knockout of ARHGAP18. We report that the loss of ARHGAP18 results in alterations of the cell that derive from both aberrant RhoA signaling and inappropriate nuclear localization of YAP. These findings indicate that the Hippo and Rho family GTPase signaling cascades are coordinated in their temporal and spatial control of the actin cytoskeleton.

RhoGDI in RBL-2H3 cells acts as a negative regulator of Rho GTPase signaling to inhibit granule exocytosis.

Mast cells are hematopoietic-derived immune cells that possess numerous cytoplasmic granules containing immune mediators such as cytokines and histamine. Antigen stimulation triggers mast cell granule exocytosis, releasing granule contents in a process known as degranulation. We have shown that Rho GTPase signaling is an essential component of granule exocytosis, however, the proteins that regulate Rho GTPases during this process are not well defined. Here we examined the role of Rho guanine-nucleotide dissociation inhibitors (RhoGDIs) in regulating Rho GTPase signaling using RBL-2H3 cells as a mast cell model. We found that RBL-2H3 cells express two RhoGDI isoforms which are primarily localized to the cytosol. Knockdown of RhoGDI1 and RhoGDI2 greatly reduced the levels of all Rho GTPases tested: RhoA, RhoG, Rac1, Rac2, and Cdc42. The reduction in Rho GTPase levels was accompanied by an increase in their membrane-localized fraction and an elevation in the levels of active Rho GTPases. All RhoGDI knockdown strains had altered resting cell morphology, although each strain was activation competent when stimulated. Live cell imaging revealed that the RhoGDI1/2 double knockdown (DKD) strain maintained its activated state for prolonged periods of time compared to the other strains. Only the RhoGDI1/2 DKD strain showed a significant increase in granule exocytosis. Conversely, RhoGDI overexpression in RBL-2H3 cells did not noticeably affect Rho GTPases or degranulation. Based on these results, RhoGDIs act as negative regulators of Rho GTPases during mast cell degranulation, and inhibit exocytosis by sequestering Rho GTPases in the cytosol.

BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

AbstractBardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of the BBSome, a cargo adaptor essential for export of transmembrane receptors from cilia. Although actin-dependent ectocytosis has been proposed to compensate defective cargo retrieval, its molecular basis remains unclear, especially in relation to BBS pathology. In this study, we investigated how actin polymerization and ectocytosis are regulated within the cilium. Our findings reveal that ciliary CDC42, a RHO-family GTPase triggers in situ actin polymerization, ciliary ectocytosis, and cilia shortening in BBSome-deficient cells. Activation of the Sonic Hedgehog pathway further enhances CDC42 activity specifically in BBSome-deficient cilia. Inhibition of CDC42 in BBSome-deficient cells decreases the frequency and duration of ciliary actin polymerization events, causing buildup of G protein coupled receptor 161 (GPR161) in bulges along the axoneme during Sonic Hedgehog signaling. Overall, our study identifies CDC42 as a key trigger of ciliary ectocytosis. Hyperactive ciliary CDC42 and ectocytosis and the resulting loss of ciliary material might contribute to BBS disease severity.

Molecular and genetic characterization of sex-linked orange coat color in the domestic cat.

The Sex-linked orange mutation in domestic cats causes variegated patches of reddish/yellow hair and is a defining signature of random X-inactivation in female tortoiseshell and calico cats. Unlike the situation for most coat color genes, there is no apparent homolog for Sex-linked orange in other mammals. We show that the Sex-linked orange is caused by a 5 kb deletion that leads to ectopic and melanocyte-specific expression of the Rho GTPase Activating Protein 36 ( Arhgap36 ) gene. Single cell RNA-seq studies from fetal cat skin reveal that red/yellow hair color is caused by reduced expression of melanogenic genes that are normally activated by the Melanocortin 1 receptor (Mc1r)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway, but the Mc1r gene and its ability to stimulate cAMP accumulation is intact. Instead, we show that increased expression of Arhgap36 in melanocytes leads to reduced levels of the PKA catalytic subunit (PKA C ); thus, Sex-linked orange is genetically and biochemically downstream of Mc1r . Our findings solve a comparative genomic conundrum, provide in vivo evidence for the ability of Arhgap36 to inhibit PKA, and reveal a molecular explanation for a charismatic color pattern with a rich genetic history.

Loss of Cdc42 causes abnormal optic cup morphogenesis and microphthalmia in mouse.

Congenital ocular malformations originate from defective morphogenesis during early eye development and cause 25% of childhood blindness. Formation of the eye is a multi-step, dynamic process; it involves evagination of the optic vesicle, followed by distal and ventral invagination, leading to the formation of a two-layered optic cup with a transient optic fissure. These tissue folding events require extensive changes in cell shape and tissue growth mediated by cytoskeleton mechanics and intercellular adhesion. We hypothesized that the Rho GTPase Cdc42 may be an essential, convergent effector downstream of key regulatory factors required for ocular morphogenesis. CDC42 controls actin remodeling, apicobasal polarity, and junction assembly. Here we identify a novel essential function for Cdc42 during eye morphogenesis in mouse; in Cdc42 mutant eyes expansion of the ventral optic cup is arrested, resulting in microphthalmia and a wide coloboma. Our analyses show that Cdc42 is required for expression of the polarity effector proteins PRKCZ and PARD6, intercellular junction protein tight junction protein 1, β-catenin, actin cytoskeleton F-actin, and contractile protein phospho myosin light chain 2. Expression of RPE fate determinants OTX2 and MITF, and formation of the RPE layer are severely affected in the temporal domain of the proximal optic cup. EdU incorporation is significantly downregulated. In addition, mitotic retinal progenitor cells mislocalize deeper, basal regions, likely contributing to decreased proliferation. We propose that morphogenesis of the ventral optic cup requires Cdc42 function for coordinated optic cup expansion and establishment of subretinal space, tissue tension, and differentiation of the ventral RPE layer.

ARHGAP29 promotes keratinocyte proliferation and migration in vitro and is dispensable for in vivo wound healing.

RhoA GTPases play critical roles in actin cytoskeletal remodeling required for controlling a diverse range of cellular functions including cell proliferation, adhesion, migration and changes in cell shape, all required for cutaneous wound healing. RhoA cycles between an active GTP-bound and an inactive GDP-bound form, a process regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). ARHGAP29 is a GAP expressed in skin keratinocytes and is decreased in the absence of interferon regulator factor 6, a critical regulator of cell proliferation, migration, and wound healing. However, the role for ARHGAP29 in keratinocyte biology is unknown. We generated ARHGAP29 knockdown keratinocyte cell lines and show they displayed increased filamentous actin, phospho-myosin regulatory light chain, cell area and population doubling time. Furthermore, we found that ARHGAP29 knockdown keratinocytes displayed significant delays in scratch wound closure in both single and collective cell migration conditions; these delays were rescued by both adding back ARHGAP29 or adding a ROCK inhibitor to ARHGAP29 knockdown cells. In vivo, however, Arhgap29 heterozygotes or keratinocyte-specific knockouts showed on-time wound healing. These data demonstrate that ARHGAP29 is required for keratinocyte morphology, proliferation and migration in vitro but is dispensable during wound healing in vivo.

Abstract A020: Elevated extracellular glucose level in tumor microenvironment by hyperglycemia increases glycolysis rate, stiffness, contractility, and motility of breast cancer cells through the cAMP-RhoA-Rock and AMPK-YAP-RhoA axes to promote metastasis

Abstract Glucose is a preferred nutrient for most cells, including cancer cells. The tumor microenvironment is both hypoxic and hypoglycemic. Glucose levels in tumors are ten times lower than in normal tissues due to poor vascularization and high glucose consumption by cancer cells, which adapt to hypoglycemia. Obesity, often accompanied by hyperglycemia, hyperinsulinemia, and hypertriglyceridemia, is linked to increased breast cancer metastasis. High extracellular glucose levels have been shown to promote cancer cell motility, though the mechanisms remain unclear. Given cancer cells' reliance on aerobic glycolysis (the Warburg effect), we hypothesized that hyperglycemia in obesity promotes cancer metastasis by modulating glucose metabolism and related cell mechanics pathways. Using Triple-Negative Breast Cancer (TNBC) cells, we found that elevated extracellular glucose levels increase glycolysis, activating the RhoA GTPase signaling pathway. This results in greater cell stiffness and contractility, correlating with increased motility. Our unpublished data, which I will present, validate these findings in obese-TNBC mouse models and further elucidate the glycolysis-mediated activation of the RhoA-ROCK axis. We also identified a novel long-term effect of hyperglycemia in promoting metastasis via transcriptional regulation of the AMPK-YAP-RhoA axis. To confirm hyperglycemia's pro-metastatic effects, we developed two obese-TNBC mouse models using diet-induced obesity (DIO) in NSG and C57BL/6J mice with orthotopic implantation of human and mouse TNBC cells, respectively. RhoA GTPase activity was measured with a GTP-RhoA FRET sensor or pull-down assay. Cell stiffness was assessed using our innovative parallel microfiltration (PMF) assay or Atomic Force Microscopy (AFM). We disrupted glucose uptake by knocking out glucose transporter 3 (GLUT3) via CRISPR/Cas9 or using a GLUT3-selective inhibitor, G3iA. Cell motility was measured by scratch wound-healing and invasion assays. Bulk RNA-seq identified differentially expressed genes under high glucose conditions in human breast cancer cells. Our results show increased lung metastasis of breast cancer cells under hyperglycemic conditions in vivo. Besides the previously reported cAMP-RhoA-ROCK axis, we discovered the glycolysis-AMPK-YAP axis also regulates RhoA-ROCK activity transcriptionally. GLUT3 inhibition activated AMPK, reduced F-actin levels via the AMPK-VASP axis, and decreased non-muscle myosin activity through the AMPK-YAP-RhoA axis. These changes in cell mechanics correlated with reduced motility. Our study suggests that targeting glucose metabolism and cell mechanics could be a novel approach to inhibit cancer metastasis, particularly in obese patients. Citation Format: Tae-Hyung Kim, Aadil Q Bhat, Jun-Yong Choe, Donghoon Yoon. Elevated extracellular glucose level in tumor microenvironment by hyperglycemia increases glycolysis rate, stiffness, contractility, and motility of breast cancer cells through the cAMP-RhoA-Rock and AMPK-YAP-RhoA axes to promote metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A020.

An allosteric inhibitor of RhoGAP class-IX myosins suppresses the metastatic features of cancer cells

Aberrant Ras homologous (Rho) GTPase signalling is a major driver of cancer metastasis, and GTPase-activating proteins (GAPs), the negative regulators of RhoGTPases, are considered promising targets for suppressing metastasis, yet drug discovery efforts have remained elusive. Here, we report the identification and characterization of adhibin, a synthetic allosteric inhibitor of RhoGAP class-IX myosins that abrogates ATPase and motor function, suppressing RhoGTPase-mediated modes of cancer cell metastasis. In human and murine adenocarcinoma and melanoma cell models, including three-dimensional spheroid cultures, we reveal anti-migratory and anti-adhesive properties of adhibin that originate from local disturbances in RhoA/ROCK-regulated signalling, affecting actin-dynamics and actomyosin-based cell-contractility. Adhibin blocks membrane protrusion formation, disturbs remodelling of cell-matrix adhesions, affects contractile ring formation, and disrupts epithelial junction stability; processes severely impairing single/collective cell migration and cytokinesis. Combined with the non-toxic, non-pathological signatures of adhibin validated in organoids, mouse and Drosophila models, this mechanism of action provides the basis for developing anti-metastatic cancer therapies.

Autoinflammatory patients with Golgi-trapped CDC42 exhibit intracellular trafficking defects leading to STING hyperactivation and ER stress

Most autoinflammatory diseases are caused by mutations in innate immunity genes. Previously, four variants in the RHO GTPase CDC42 were discovered in patients affected by syndromes generally characterized by neonatal-onset of cytopenia and auto-inflammation, including hemophagocytic lymphohistiocytosis and rash in the most severe form (NOCARH syndrome). However, the mechanisms responsible for these phenotypes remain largely elusive. Here, we show that the recurrent p.R186C CDC42 variant, which is trapped in the Golgi apparatus, elicits a block in both anterograde and retrograde transports. Consequently, it favours STING accumulation in the Golgi in a COPI-dependent manner. This is also observed for the other Golgi-trapped p.*192 C*24 CDC42 variant, but not for the p.Y64C and p.C188Y variants that do not accumulate in the Golgi. We demonstrate that the two Golgi-trapped CDC42 variants are the only ones that exhibit overactivation of the STING pathway and the type I interferon response, and elicit endoplasmic reticulum stress. Consistent with these results, patients carrying Golgi-trapped CDC42 mutants present very high levels of circulating IFNα at the onset of their disease. In conclusion, we report further mechanistic insights on the impact of the Golgi-trapped CDC42 variants. This increase in STING activation provides a rationale for combination treatments for these severe cases.

RhoA Allosterically Activates Phospholipase Cε via its EF Hands.

Phospholipase Cε (PLCε) cleaves phosphatidylinositol lipids to increase intracellular Ca 2+ and activate protein kinase C (PKC) in response to stimulation of cell surface receptors. PLCε is activated via direct binding of small GTPases at the cytoplasmic leaflets of cellular membranes. In the cardiovascular system, the RhoA GTPase regulates PLCε to initiate a cardioprotective pathway, but the underlying molecular mechanism is not known. We present here the cryo-electron microscopy (cryo-EM) reconstruction of RhoA bound to PLCε. The G protein binds a unique insertion in the PLCε EF hands. Deletion of or mutations to this PLCε insertion decrease RhoA-dependent activation without impacting regulation by other G proteins. Together, our data support a model wherein RhoA binding to PLCε allosterically activates the lipase and increases its interactions with the membrane, resulting in maximum activity and cardiomyocyte survival.

FRET verification of crucial interaction sites in RhoA regulation mediated by RhoGDI

The small GTPase Rho family are the major factors in mediating actin cytoskeleton dynamics. Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) serve as important negative regulators by complexing with inactive Rho into the cytoplasm. However, how these two molecules interact still needs experimental verification. Based on fluorescence resonance energy transfer (FRET) measurements, we would demonstrate crucial sites in RhoGDI and RhoA for this regulatory role. Cotransfection of RhoGDI markedly reduced RhoA or Cdc42 activity in airway smooth muscle (ASM) cells, while D185R-RhoGDI mutant reversed this decrease, indicating that RhoGDI Asp185 residue is essential for the molecular interaction. R68D-RhoA (mutation in the switch II region) resulted in a deficiency in RhoGDI regulation, while TV37/38NG-RhoA (in the switch I region) displayed low RhoA activity. Hence, the Arg68 site in RhoA is indispensable for regulation by RhoGDI, and Thr37Val38 site is important for maintaining RhoA activity. Additionally, microtubule but not actin cytoskeleton showed inhibitory role in RhoA activity, while the dissolution of either cytoskeleton did not change the regulatory role of RhoGDI. In checking the downstream effect, reduction of RhoA activity induced by PDGF stimulation or RhoGDI decreased cellular stress fibers. In this study, FRET visualization was applied to have experimentally demonstrated the interaction sites and crucial role of RhoGDI in regulating RhoA activity.Graphical

Abstract 4141496: Methylation changes in monocytes of type 2 diabetes patients with cardiovascular disease are associated with apoptosis and inflammation pathways.

Background: Monocytes have been implicated in the initiation and progression of cardiovascular (CV) complications of type 2 diabetes (T2D). Aim: We investigated diabetes-induced methylation changes and enriched pathways in circulating monocytes of T2D patients. We compared T2D vs. non-T2D participants and analyzed T2D patients according to diabetes control. Further, we assessed methylation changes in a subset of patients with poor diabetes control and high CV risk following a 6-month treatment intensification. Methods: We recruited consecutively 200 participants, 160 with T2D and 40 non-diabetes controls. Circulating monocytes were sorted using the FACSAria2 ™ cell sorter. Samples were sequenced using enhanced reduced representation bisulfite sequencing. Methylation data was processed with Lumi and limma R packages to obtain batch and covariate corrected differential expression values for the indicated comparisons. Ingenuity Pathway Analysis (Qiagen) was used to identify enriched pathways for each tested condition. Differentially methylated genes used for pathway analysis were those with a p-value < 0.05 and an absolute logFC value ≥ 0.5. Results: There were 1122 differentially methylated genes when comparing T2D to non-T2D patients. Among the top five enriched canonical pathways, apoptosis signaling was predicted to be activated, while the RHO GTPase cycle and LPS-stimulated MAPK signaling pathways were predicted to be inhibited. Within patients with diabetes, there were 1670 differentially methylated genes comparing those with poor control (HbA1c ≥ 7%) vs. good control (HbA1c<7%). Among the top five enriched pathways, synthesis of DNA, DNA replication signaling, and cell cycle checkpoints were activated while inhibition of ARE-mediated mRNA degradation pathway was inhibited. In patients who responded to treatment intensification (Δ HbA1c = -1.5%, p= 0.02), 1377 differentially methylated genes and differentially inhibited pathways such as non-homologous end-joining and cellular response to heat stress were identified. In non-responders, we identified 1582 differentially methylated genes and activated pathways, including inhibition of mRNA degradation pathways and p14/p19 ARF signaling. Conclusion: Treatment intensification in T2D patients with CVD and poor control showed the presence of differentially methylated genes affecting pathways regulating cell proliferation and inflammation and are known to be associated with diabetic CVD complications.