Rho Pathway Research Articles

The Hexosamine Biosynthetic Pathway alters the cytoskeleton to modulate cell proliferation and migration in metastatic prostate cancer.

Castration-resistant prostate cancer (CRPC) progresses despite androgen deprivation therapy, as cancer cells adapt to grow without testosterone, becoming more aggressive and prone to metastasis. CRPC biology complicates the development of effective therapies, posing challenges for patient care. Recent gene-expression and metabolomics studies highlight the Hexosamine Biosynthetic Pathway (HBP) as a critical player, with key components like GNPNAT1 and UAP1 being downregulated in metastatic CRPC. GNPNAT1 knockdown has been shown to increase cell proliferation and metastasis in CRPC cell lines, though the mechanisms remain unclear. To investigate the cellular basis of these CRPC phenotypes, we generated a CRISPR-Cas9 knockout model of GNPNAT1 in 22Rv1 CRPC cells, analyzing its impact on metabolomic, glycoproteomic, and transcriptomic profiles of cells. We hypothesize that HBP inhibition disrupts the cytoskeleton, altering mitotic progression and promoting uncontrolled growth. GNPNAT1 KO cells showed reduced levels of cytoskeletal filaments, such as actin and microtubules, leading to cell structure disorganization and chromosomal mis-segregation. GNPNAT1 inhibition also activated PI3K/AKT signaling, promoting proliferation, and impaired cell adhesion by mislocalizing EphB6, enhancing migration via the RhoA pathway and promoting epithelial-to-mesenchymal transition. These findings suggest that HBP plays a critical role in regulating CRPC cell behavior, and targeting this pathway could provide a novel therapeutic approach.

Calcium wave dynamics in the embryonic mouse gut mesenchyme: impact on smooth muscle differentiation

Intestinal smooth muscle differentiation is a complex physico-biological process involving several different pathways. Here, we investigate the properties of Ca2+ waves in the developing intestinal mesenchyme using GCamp6f expressing mouse embryos and investigate their relationship with smooth muscle differentiation. We find that Ca2+ waves are absent in the pre-differentiation mesenchyme and start propagating immediately following α-SMA expression. Ca2+ waves are abrogated by CaV1.2 and gap-junction blockers, but are independent of the Rho pathway. The myosine light-chain kinase inhibitor ML-7 strongly disorganized or abolished Ca2+ waves, showing that perturbation of the contractile machinery at the myosine level also affected the upstream Ca2+ handling chain. Inhibiting Ca2+ waves and contractility with CaV1.2 blockers did not perturb circular smooth muscle differentiation at early stages. At later stages, CaV1.2 blockers abolished intestinal elongation and differentiation of the longitudinal smooth muscle, leading instead to the emergence of KIT-expressing interstitial cells of Cajal at the gut periphery. CaV1.2 blockers also drove apoptosis of already differentiated, CaV1.2-expressing smooth muscle and enteric neural cells. We provide fundamental new data on Ca2+ waves in the developing murine gut and their relation to myogenesis in this organ.

Tumor-induced endothelial RhoA activation mediates tumor cell transendothelial migration and metastasis.

The endothelial barrier plays an active role in transendothelial tumor cell migration during metastasis, however, the endothelial regulatory elements of this step remain obscure. Here we show that endothelial RhoA activation is a determining factor during this process. Breast tumor cell-induced endothelial RhoA activation is the combined outcome of paracrine IL-8-dependent and cell-to-cell contact β 1 integrin-mediated mechanisms, with elements of this pathway correlating with clinical data. Endothelial-specific RhoA blockade or in vivo deficiency inhibited the transendothelial migration and metastatic potential of human breast tumor and three murine syngeneic tumor cell lines, similar to the pharmacological blockade of the downstream RhoA pathway. These findings highlight endothelial RhoA as a potent, universal target in the tumor microenvironment for anti-metastatic treatment of solid tumors.

G protein selectivity profile of GPR56/ADGRG1 and its effect on downstream effectors.

GPR56, an adhesion G-protein coupled receptor (aGPCRs) with constitutive and ligand-promoted activity, is involved in many physiological and pathological processes. Whether the receptor's constitutive or ligand-promoted activation occur through the same molecular mechanism, and whether different activation modes lead to functional selectivity between G proteins is unknown. Here we show that GPR56 constitutively activates both G12 and G13. Unlike constitutive activation and activation with 3-a-acetoxydihydrodeoxygedunin (3αDOG), stimulation with an antibody, 10C7, directed against GPR56's extracellular domain (ECD) led to an activation that favors G13 over G12. An autoproteolytically deficient mutant, GPR56-T383A, was also activated by 10C7 indicating that the tethered agonist (TA) exposed through autocatalytic cleavage, is not required for this activation modality. In contrast, this proteolysis-resistant mutant could not be activated by 3αDOG indicating different modes of activation by the two ligands. We show that an N-terminal truncated GPR56 construct (GPR56-Δ1-385) is devoid of constitutive activity but was activated by 3αDOG. Similarly to 3αDOG, 10C7 promoted the recruitment of b-arrestin-2 but GPR56 internalization was β-arrestin independent. Despite the slow activation mode of 10C7 that favors G13 over G12, it efficiently activated the downstream Rho pathway in BT-20 breast cancer cells. These data show that different GPR56 ligands have different modes of activation yielding differential G protein selectivity but converging on the activation of the Rho pathway both in heterologous expressions system and in cancer cells endogenously expressing the receptor. 10C7 is therefore an interesting tool to study both the processes underlying GPR56 activity and its role in cancer cells.

G protein selectivity profile of GPR56/ADGRG1 and its effect on downstream effectors

GPR56, an adhesion G-protein coupled receptor (aGPCRs) with constitutive and ligand-promoted activity, is involved in many physiological and pathological processes. Whether the receptor’s constitutive or ligand-promoted activation occur through the same molecular mechanism, and whether different activation modes lead to functional selectivity between G proteins is unknown. Here we show that GPR56 constitutively activates both G12 and G13. Unlike constitutive activation and activation with 3-α-acetoxydihydrodeoxygedunin (3αDOG), stimulation with an antibody, 10C7, directed against GPR56’s extracellular domain (ECD) led to an activation that favors G13 over G12. An autoproteolytically deficient mutant, GPR56-T383A, was also activated by 10C7 indicating that the tethered agonist (TA) exposed through autocatalytic cleavage, is not required for this activation modality. In contrast, this proteolysis-resistant mutant could not be activated by 3αDOG indicating different modes of activation by the two ligands. We show that an N-terminal truncated GPR56 construct (GPR56-Δ1-385) is devoid of constitutive activity but was activated by 3αDOG. Similarly to 3αDOG, 10C7 promoted the recruitment of β-arrestin-2 but GPR56 internalization was β-arrestin independent. Despite the slow activation mode of 10C7 that favors G13 over G12, it efficiently activated the downstream Rho pathway in BT-20 breast cancer cells. These data show that different GPR56 ligands have different modes of activation yielding differential G protein selectivity but converging on the activation of the Rho pathway both in heterologous expressions system and in cancer cells endogenously expressing the receptor. 10C7 is therefore an interesting tool to study both the processes underlying GPR56 activity and its role in cancer cells.

I told you to stop: obscurin's role in epithelial cell migration.

The giant cytoskeletal protein obscurin contains multiple cell signaling domains that influence cell migration. Here, we follow each of these pathways, examine how these pathways modulate epithelial cell migration, and discuss the cross-talk between these pathways. Specifically, obscurin uses its PH domain to inhibit phosphoinositide-3-kinase (PI3K)-dependent migration and its RhoGEF domain to activate RhoA and slow cell migration. While obscurin's effect on the PI3K pathway agrees with the literature, obscurin's effect on the RhoA pathway runs counter to most other RhoA effectors, whose activation tends to lead to enhanced motility. Obscurin also phosphorylates cadherins, and this may also influence cell motility. When taken together, obscurin's ability to modulate three independent cell migration pathways is likely why obscurin knockout cells experience enhanced epithelial to mesenchymal transition, and why obscurin is a frequently mutated gene in several types of cancer.

Ghrelin may protect against vascular endothelial injury in Acute traumatic coagulopathy by mediating the RhoA/ROCK/MLC2 pathway.

Ghrelin exerts widespread effects in several diseases, but its role and mechanism in Acute Traumatic Coagulopathy (ATC) are largely unknown. The effect of ghrelin on cell proliferation was examined using three assays: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), Lactate Dehydrogenase (LDH), and flow cytometry. The barrier function of the endothelial cells was evaluated using the Trans-Endothelial Electrical Resistance (TEER) and the endothelial permeability assay. An ATC mouse model was established to evaluate the in vivo effects of ghrelin. The Ras homolog family member A (RhoA) overexpression plasmid or adenovirus was used to examine the molecular mechanism of ghrelin. Ghrelin enhanced Human Umbilical Vein Endothelial Cells (HUVEC) proliferation and endothelial cell barrier function and inhibited HUVEC permeability damage in vitro. Additionally, ghrelin decreased the activated Partial Thromboplastin Time (aPTT) and Prothrombin Time (PT) in mice blood samples in the ATC mouse model. Ghrelin also improved the pathological alterations in postcava. Mechanistically, ghrelin acts through the RhoA/ Rho-associated Coiled-coil Containing Kinases (ROCK)/ Myosin Light Chain 2 (MLC2) pathway. Furthermore, the protective effects of ghrelin, both in vitro and in vivo, were reversed by RhoA overexpression. Our findings demonstrate that ghrelin may reduce vascular endothelial cell damage and endothelial barrier dysfunction by blocking the RhoA pathway, suggesting that ghrelin may serve as a potential therapeutic target for ATC treatment.

Human organoids with an autologous tissue-resident immune compartment

The intimate relationship between the epithelium and immune system is crucial for maintaining tissue homeostasis, with perturbations therein linked to autoimmune disease and cancer1–3. Whereas stem cell-derived organoids are powerful models of epithelial function4, they lack tissue-resident immune cells that are essential for capturing organ-level processes. We describe human intestinal immuno-organoids (IIOs), formed through self-organization of epithelial organoids and autologous tissue-resident memory T (TRM) cells, a portion of which integrate within the epithelium and continuously survey the barrier. TRM cell migration and interaction with epithelial cells was orchestrated by TRM cell-enriched transcriptomic programs governing cell motility and adhesion. We combined IIOs and single-cell transcriptomics to investigate intestinal inflammation triggered by cancer-targeting biologics in patients. Inflammation was associated with the emergence of an activated population of CD8+ T cells that progressively acquired intraepithelial and cytotoxic features. The appearance of this effector population was preceded and potentiated by a T helper-1-like CD4+ population, which initially produced cytokines and subsequently became cytotoxic itself. As a system amenable to direct perturbation, IIOs allowed us to identify the Rho pathway as a new target for mitigation of immunotherapy-associated intestinal inflammation. Given that they recapitulate both the phenotypic outcomes and underlying interlineage immune interactions, IIOs can be used to study tissue-resident immune responses in the context of tumorigenesis and infectious and autoimmune diseases.

Serum Response Factor Expression in Excess Permits a Dual Contractile-Proliferative Phenotype of Airway Smooth Muscle.

The transcription factors (TFs) MyoCD (myocardin) and Elk-1 (ETS Like-1 protein) competitively bind to SRF (serum response factor) and control myogenic- and mitogenic-related gene expression in smooth muscle, respectively. Their functions are therefore mutually inhibitory, which results in a contractile-versus-proliferative phenotype dichotomy. Airway smooth muscle cell (ASMC) phenotype alterations occur in various inflammatory airway diseases, promoting pathological remodeling and contributing to airflow obstruction. We characterized MyoCD and Elk-1 interactions and their roles in phenotype determination in human ASMCs. MyoCD overexpression in ASMCs increased smooth muscle gene expression, force generation, and partially restored the loss of smooth muscle protein associated with prolonged culturing while inhibiting Elk-1 transcriptional activities and proliferation induced by EGF (epidermal growth factor). However, MyoCD overexpression failed to suppress these responses induced by FBS, as FBS also upregulated SRF expression to a degree that allowed unopposed function of both TFs. Inhibition of the RhoA pathway reversed said SRF changes, allowing inhibition of Elk-1 by MyoCD overexpression and suppressing FBS-mediated contractile protein gene upregulation. Our study confirmed that MyoCD in increased abundance can competitively inhibit Elk-1 function. However, SRF upregulation permits a dual contractile-proliferative ASMC phenotype that is anticipated to exacerbate pathological alterations, whereas therapies targeting SRF may inhibit pathological ASMC proliferation and contractile protein gene expression.

Recurrent RhoGAP gene fusion CLDN18-ARHGAP26 promotes RHOA activation and focal adhesion kinase and YAP-TEAD signalling in diffuse gastric cancer

ObjectiveGenomic studies of gastric cancer have identified highly recurrent genomic alterations impacting RHO signalling, especially in the diffuse gastric cancer (DGC) histological subtype. Among these alterations are interchromosomal translations leading...

Does actin polymerization contribute to closure of the avian ductus arteriosus (Gallus gallus)?

The ductus arteriosus (DA) is a cardiovascular embryonic shunt present in all developing land vertebrates. This oxygen-sensitive blood vessel plays the important role of connecting the pulmonary artery to the aorta during development and directs blood flow toward the fetal respiratory organ (placenta in mammals or chorioallantois in birds and reptiles). Permanent vessel closure upon birth or hatch is necessary to establish proper circulation in the neonate. Increased arterial PO2 at birth or hatch stimulates DA closure via smooth muscle contraction. Smooth muscle contraction, such as in the DA, is governed by myosin light chain kinase (MLCK) stimulating increased cross-bridge cycling, and through increases in actin polymerization. The Ras homologous protein family (Rho) of GTPases are involved in calcium sensitization and contraction of vascular smooth muscle but have mostly been studied for their role in cell migration and signaling. Rho GTPases activate the Rho activated kinase (ROCK) pathway, which works in conjunction with MLCK to generate smooth muscle contraction as well as activating LIM kinase which inhibits the actin depolymerizer cofilin. Rac and Cdc42 GTPase pathways increase contractile strength in smooth muscle cells by activating the nucleator Arp2/3, leading to increased actin polymerization. The role of these GTPase pathways during closure of the DA is unknown. We examined the role of the Rho, Rac, and Cdc42 GTPase pathways leading to actin polymerization for their role in the closure of the avian DA in late-term chicken ( Gallus gallus) embryos. We hypothesized that inhibiting the pathways that lead to actin polymerization will inhibit tension development in the DA. Using a Danish Myo Technology wire myograph, DA physiology was examined when exposed to 25% oxygen followed by activators and inhibitors of the Rho, Rac, and Cdc42 GTPase pathways. Activation of Rho A pathways with CN01 (0.2 units/ml) produced contraction of the DA. Blocking ROCK pathways with Y-27632 (10 mM) removed the O2-sensitivity of the vessel, while the vessel was still able to respond to phenylephrine (100 mM). This suggests the Rho pathway is involved in maintaining baseline tension and contractile response to O2. Exposure to inhibitors of Rac (10 uM EHT 1864) and Cdc42 (via ML141) GTPases blocked the ability of vessels to contract in response to 25% oxygen, even after 3 hours of constant O2. Similarly, inhibition of the Arp2/3 complex (100 uM CK666) reduced the ability of the vessel to respond to oxygen. These findings suggest that Rho A, Rac, and Cdc42 pathways play a role in active tension of the chicken DA during development by potentially increasing actin polymerization. This research was funded by Grant R15HL14887 from NIH-NHLBI. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CX3CL1 and its receptor CX3CR1 interact with RhoA signaling to induce paclitaxel resistance in gastric cancer

C-X3-C motif chemokine ligand 1 (CX3CL1) is a transmembrane protein, and the membranal and soluble forms of CX3CL1 exhibit different functions, although both bind to the CX3CR1 chemokine receptor. The CX3CL1/CX3CR1 axis induces many cellular responses relevant to cancer, such as proliferation, migration, invasion, and apoptosis resistance. Here we attempt to elucidate whether CX3CL1/CX3CR1 is associated with paclitaxel (PTX) resistance in gastric cancer (GC). The Gene Expression Omnibus database was queried to screen for differentially expressed genes in GC cells caused by drug resistance, and CX3CL1 was selected as a candidate. CX3CL1 was overexpressed in PTX-resistant cells and tissues. CX3CL1 loss sensitized GC cells to PTX, promoted apoptosis and DNA damage, and inhibited cell proliferation, migration, and invasion. CX3CR1 reversed the ameliorative effect of CX3CL1 silencing on PTX sensitivity in GC cells. The promotion of PTX resistance by CX3CL1/CX3CR1 was inhibited by impairment of the small GTPase Ras homolog gene family member A (RhoA) pathway in vitro and in vivo. These findings indicate that the CX3CL1/CX3CR1 expedites PTX resistance through the RhoA signaling in GC cells.

Lipid-Binding Regions within PKC-Related Serine/Threonine Protein Kinase N1 (PKN1) Required for Its Regulation.

PKC-related serine/threonine protein kinase N1 (PKN1) is a protease/lipid-activated protein kinase that acts downstream of the RhoA and Rac1 pathways. PKN1 comprises unique regulatory, hinge region, and PKC homologous catalytic domains. The regulatory domain harbors two homologous regions, i.e., HR1 and C2-like. HR1 consists of three heptad repeats (HR1a, HR1b, and HR1c), with PKN1-(HR1a) hosting an amphipathic high-affinity cardiolipin-binding site for phospholipid interactions. Cardiolipin and C18:1 oleic acid are the most potent lipid activators of PKN1. PKN1-(C2) contains a pseudosubstrate sequence overlapping that of C20:4 arachidonic acid. However, the cardiolipin-binding site(s) within PKN1-(C2) and the respective binding properties remain unclear. Herein, we reveal (i) that the primary PKN1-(C2) sequence contains conserved amphipathic cardiolipin-binding motif(s); (ii) that trimeric PKN1-(C2) predominantly adopts a β-stranded conformation; (iii) that two distinct types of cardiolipin (or phosphatidic acid) binding occur, with the hydrophobic component playing a key role at higher salt levels; (iv) the multiplicity of C18 fatty acid binding to PKN1-(C2); and (v) the relevance of our lipid-binding parameters for PKN1-(C2) in terms of kinetic parameters previously determined for the full-length PKN1 enzyme. Thus, our discoveries create opportunities to design specific mammalian cell inhibitors that disrupt the localization of membrane-associated PKN1 signaling molecules.

Therapeutic potential of exosome derived from hepatocyte growth factor-overexpressing adipose mesenchymal stem cells in TGFβ1-stimulated hepatic stellate cells.

Cirrhosis is a familiar end-stage of multiple chronic liver diseases. The gene-modified mesenchymal stem cells (MSCs) have become one of the most promising schemes for the treatment of cirrhosis. MSCs exhibit their therapeutic role mainly by secreting hepatocyte growth factor (HGF). The aim of this research was to probe the anti-fibrosis role of exosomes secreted by HGF modified-mouse adipose MSCs (ADMSCs) on activated hepatic stellate cells (HSCs) and to preliminarily explore the possible mechanism. Firstly, mouse ADMSCs were isolated and identified. Quantitative real-time polymerase chain reaction verified the transfection efficiency of ADMSC transfected with HGF lentivirus. Exosomes derived from ADMSC transfecting negative control/HGF (ADMSCNC-Exo/ADMSCHGF-Exo) were extracted by density gradient centrifugation. HSCs were allocated to the control, TGF-β, TGF-β + ADMSC-Exo, TGF-β + ADMSCNC-Exo, and TGF-β + ADMSCHGF-Exo groups. Moreover, all mice were distributed to the control, CCl4 (40% CCl4 in olive oil), CCl4+ADMSC-Exo, CCl4+ADMSCNC-Exo, and CCl4+ADMSCHGF-Exo groups. Exosomes derived from ADMSCs with or without HGF transfection suppressed HSC activation, as evidenced by attenuating cell viability and cell cycle arrest at S phase but inducing apoptosis. Moreover, ADMSC-Exo, ADMSCNC-Exo, and ADMSCHGF-Exo effectively repressed the gene and protein levels of α-SMA, Col-I, Rho A, Cdc42, and Rac1 in TGF-β-treated HSCs, and ADMSCHGF-Exo had the best effect. ADMSCHGF-Exo had a stronger regulatory effect on serum liver index than ADMSCNC-Exo in CCl4-induced mice. In conclusion, ADMSCHGF-Exo alleviated liver fibrosis by weakening the Rho pathway, thus reducing collagen production.

A54 SHP2 FUNCTION IN NORMAL AND APC-MUTANT INTESTINAL EPITHELIAL CELLS

Abstract Background Somatic SHP2 gain-of-function mutations were found in some colorectal tumours. However, its function in colorectal epithelium remains to be determined. SHP2 is a tyrosine phosphatase necessary for RAS/MAPK pathway activation by most, if not all, tyrosine kinase receptors, as well as by GPCRs and cytokine receptors. SHP2 can also regulate the PI3K, Jak/Stat, RhoA and Hippo pathways in different cell settings. As a result, SHP2 can control cellular processes including growth, migration and differentiation. Previously, we observed that SHP2 levels are enhanced in human colorectal tumours, notably in late adenomas. Likewise, SHP2 silencing inhibited ERK activation and tumour properties of established human CRC cell lines. Hence, these findings support a model in which SHP2 activation promotes tumorigenesis in the intestinal epithelium. But how? Aims To analyze the exact cellular and molecular mechanisms underlying SHP2 pro-oncogenic function in intestinal epithelial cells. Methods The impact of SHP2 pharmaco-inhibition (RMC-4550 and/or SHP099) on intracellular signaling, cell cycle, proliferation and survival was evaluated in three complementary models: 1) HIEC-6 cells, normal human fetal intestinal crypt-like cells; 2) organoids derived from normal mouse small intestine or ApcMin/+ tumors; 3) Caco-2/15 cells, colorectal cancer cells with APC mutation. Results 1- Pharmaco-inhibition of SHP2 blocks proliferation of HIEC-6 cells (loss of pRb phosphorylation, decreased EdU staining) and induces cell enlargement and darkening of the outside of perinuclear area. This phenotype was reminiscent of cellular senescence, and we indeed observed increased senescence-associated β-galactosidase activity (SA-β-Gal) and INK4A gene expression. 2- As expected, SHP2 inhibition in normal mouse organoids resulted in a reduction in the number of protrusions and EdU positive cells. RNA sequencing was performed, and gene set enriched revealed significant down regulation of several Ras signaling-dependant genes including cell cycle regulated genes (E2F targets, G2/M checkpoint) and Myc targets 48h after SHP2 inhibition when compared to DMSO treated organoids. Genes associated with PI3K signaling were also diminished in SHP099 treated organoids but no significant modulation of genes associated with Wnt/b-catenin signaling was observed. 3- Notably, SHP2 pharmaco-inhibition in Caco-2/15 cells as well as APC-mutant organoids rapidly induced cell death associated with increased expression of stem cell markers. Conclusions These results suggest that SHP2 is required for proliferation of normal IEC and protects against senescence. Notably, its inhibition restrains the proliferative and tumorigenic potential conferred by APC inactivation. Overall, our data suggest that SHP2 could represent a potential target to counter colorectal tumour initiation. Funding Agencies CIHRFRQS

Sfrp1 inhibits lung fibroblast invasion during transition to injury-induced myofibroblasts.

Fibroblast-to-myofibroblast conversion is a major driver of tissue remodelling in organ fibrosis. Distinct lineages of fibroblasts support homeostatic tissue niche functions, yet their specific activation states and phenotypic trajectories during injury and repair have remained unclear. We combined spatial transcriptomics, multiplexed immunostainings, longitudinal single-cell RNA-sequencing and genetic lineage tracing to study fibroblast fates during mouse lung regeneration. Our findings were validated in idiopathic pulmonary fibrosis patient tissues in situ as well as in cell differentiation and invasion assays using patient lung fibroblasts. Cell differentiation and invasion assays established a function of SFRP1 in regulating human lung fibroblast invasion in response to transforming growth factor (TGF)β1. We discovered a transitional fibroblast state characterised by high Sfrp1 expression, derived from both Tcf21-Cre lineage positive and negative cells. Sfrp1 + cells appeared early after injury in peribronchiolar, adventitial and alveolar locations and preceded the emergence of myofibroblasts. We identified lineage-specific paracrine signals and inferred converging transcriptional trajectories towards Sfrp1 + transitional fibroblasts and Cthrc1 + myofibroblasts. TGFβ1 downregulated SFRP1 in noninvasive transitional cells and induced their switch to an invasive CTHRC1+ myofibroblast identity. Finally, using loss-of-function studies we showed that SFRP1 modulates TGFβ1-induced fibroblast invasion and RHOA pathway activity. Our study reveals the convergence of spatially and transcriptionally distinct fibroblast lineages into transcriptionally uniform myofibroblasts and identifies SFRP1 as a modulator of TGFβ1-driven fibroblast phenotypes in fibrogenesis. These findings are relevant in the context of therapeutic interventions that aim at limiting or reversing fibroblast foci formation.

Transcriptome analysis of adenomyosis eutopic endometrium reveals molecular mechanisms involved in adenomyosis-related implantation failure and pregnancy disorders

BackgroundWomen with adenomyosis are characterized by having defective decidualization, impaired endometrial receptivity and/or embryo-maternal communication, and implantation failure. However, the molecular mechanisms underlying adenomyosis-related infertility remain unknown, mainly because of the restricted accessibility and the difficult preservation of endometrial tissue in vitro. We have recently shown that adenomyosis patient-derived endometrial organoids, maintain disease-specific features while differentiated into mid-secretory and gestational endometrial phase, overcoming these research barriers and providing a robust platform to study adenomyosis pathogenesis and the associated molecular dysregulation related to implantation and pregnancy disorders. For this reason, we aim to characterize the dysregulated mechanisms in the mid-secretory and gestational endometrium of patients with adenomyosis by RNA-sequencing.MethodsEndometrial organoids were derived from endometrial biopsies collected in the proliferative phase of women with adenomyosis (ADENO) or healthy oocyte donors (CONTROL) (n = 15/group) and differentiated into mid-secretory (-SECorg) and gestational (-GESTorg) phases in vitro. Following RNA-sequencing, the significantly differentially expressed genes (DEGs) (FDR < 0.05) were identified and selected for subsequent functional enrichment analysis and QIAGEN Ingenuity Pathway Analysis (IPA). Statistical differences in gene expression were evaluated with the Student’s t-test or Wilcoxon test.ResultsWe identified 1,430 DEGs in ADENO-SECorg and 1,999 DEGs in ADENO-GESTorg. In ADENO-SECorg, upregulated genes included OLFM1, FXYD5, and RUNX2, which are involved in impaired endometrial receptivity and implantation failure, while downregulated genes included RRM2, SOSTDC1, and CHAC2 implicated in recurrent implantation failure. In ADENO-GESTorg, upregulated CXCL14 and CYP24A1 and downregulated PGR were related to pregnancy loss. IPA predicted a significant inhibition of ID1 signaling, histamine degradation, and activation of HMGB1 and Senescence pathways, which are related to implantation failure. Alternatively, IPA predicted an inhibition of D-myo-inositol biosynthesis and VEGF signaling, and upregulation of Rho pathway, which are related to pregnancy loss and preeclampsia.ConclusionsIdentifying dysregulated molecular mechanisms in mid-secretory and gestational endometrium of adenomyosis women contributes to the understanding of adenomyosis-related implantation failure and/or pregnancy disorders revealing potential therapeutic targets. Following experimental validation of our transcriptomic and in silico findings, our differentiated adenomyosis patient-derived organoids have the potential to provide a reliable platform for drug discovery, development, and personalized drug screening for affected patients.

Repurposing of multiple sclerosis (MS) and graft versus host disease (GVHD) therapeutics to inhibit chronic rejection of transplanted organs

Chronic rejection of transplanted organs is an incurable event in transplantation. Macrophages infiltrating allograft induce vessel occlusion and tissue fibrosis, which over the months-years post-transplantation cause organ failure. Patients require re-transplantation, which is extremely problematic because of the permanent lack of organ donors. Our studies showed that chronic rejection depends on the function of macrophages and the RhoA/ROCK pathway. Genetic (RhoA knockout) or pharmacologic (ROCK inhibitors) interference with the RhoA pathway inhibits macrophage functions and prevents chronic rejection in the rodent transplantation model. Most commercially available RhoA pathway inhibitors are not approved for clinical use. However, we found two compounds: fingolimod (Gilenya, FTY720) and belumosudil (Rezurock), which are clinically approved for relapsing multiple sclerosis (MS) and chronic graft versus host disease (cGVHD), respectively, which also inhibit the RhoA pathway. We tested these two drugs in the rodent transplantation model, and both inhibited chronic rejection. Thus, we proposed to repurpose these drugs for organ transplantation. A clinical trial on the effect of fingolimod in kidney transplant patients is ongoing, and the belumosudil trial is pending.

Anoctamin-1 is induced by TGF-β and contributes to lung myofibroblast differentiation.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-β (TGF-β) is one of the most established drivers of fibrotic processes. TGF-β promotes the transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-β robustly upregulates the expression of the calcium-activated chloride channel anoctamin-1 (ANO1) in human lung fibroblasts (HLFs) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle α-actin (SMA)-positive myofibroblasts. TGF-β-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1, and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate profibrotic TGF-β signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-β treatment of HLFs results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing "with-no-lysine (K)" kinase (WNK1). WNK1 siRNA significantly attenuates TGF-β-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that 1) ANO1 is a TGF-β-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-β; and 2) ANO1 mediates TGF-β-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein but independent of WNK1 kinase activity.NEW & NOTEWORTHY This study describes a novel mechanism of differentiation of human lung fibroblasts (HLFs) to myofibroblasts: the key process in the pathogenesis of pulmonary fibrosis. Transforming growth factor-β (TGF-β) drives the expression of calcium-activated chloride channel anoctmin-1 (ANO1) leading to an increase in intracellular levels of chloride. The latter recruits chloride-sensitive with-no-lysine (K) kinase (WNK1) to activate profibrotic RhoA and AKT signaling pathways, possibly through activation of mammalian target of rapamycin complex-2 (mTORC2), altogether promoting myofibroblast differentiation.

TMIC-76. GLIOBLASTOMA VESSEL CO-OPTION OCCURS AS A RESISTANCE MECHANISM TO CHEMORADIATION VIA INDUCTION OF A NOVEL CELL STATE

Abstract Glioblastoma (GB) is one of the deadliest types of human cancer. Despite a very aggressive treatment regime-including resection, chemo-radiation, its recurrence rate is more than 90%. Recurrence is mostly caused by highly resistant and invasive cells that spread from tumor bulk and are not removed by resection. To develop an effective therapeutic approach, we need to better understand underlying molecular and cellular mechanism driving therapy resistance and invasion in GB. To dynamically track the changes post-therapy and chemoradiation-resistant cells, we employed multiple bulk and single cell transcriptomics, phosphoproteome, in vitro and in vivo real time imaging, organotypic cultures, functional analysis, digital pathology and spatial transcriptomics on patient material and preclinical models of GB. We demonstrated that the chemoradiation and brain vasculature induce a transition to invasive functional cell state, which we rename as VC-Resist. Better cell survival, G2M arrest, senescence/stemness pathway induction, makes this GB state much more resistant. Notably, these GB cells are highly vessel co-opting allowing homing to perivascular niche, which in turn increases their transition to this cell state. Molecularly, the transition to VC-Resist cell state takes place through FGF-FGFR1 signaling that leads to activation of DNA damage repair, YAP and Rho pathways. These findings demonstrate that the perivascular niche and GB cell plasticity jointly generates a vicious loop that leads to resistance and brain infiltration during GB recurrence.