Rho-ROCK Pathway Research Articles

PKCα-mediated phosphorylation of the diacylglycerol kinase ζ MARCKS domain switches cell migration modes by regulating interactions with Rac1 and RhoA

Cells can switch between Rac1 (lamellipodia-based) and RhoA (blebbing-based) migration modes, but the molecular mechanisms regulating this shift are not fully understood. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from their common inhibitor RhoGDI. DGKζ catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation; instead, DGKζ stimulates RhoA release via a kinase-independent scaffolding mechanism. The molecular determinants that mediate the selective targeting of DGKζ to Rac1 or RhoA signaling complexes are unknown. Here, we show that protein kinase Cα (PKCα)-mediated phosphorylation of the DGKζ MARCKS domain increased DGKζ association with RhoA and decreased its interaction with Rac1. The same modification also enhanced DGKζ interaction with the scaffold protein syntrophin. Expression of a phosphomimetic DGKζ mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGKζ expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the RhoA-ROCK pathway. These events were independent of DGKζ catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGKζ-null cells also required the PDZ-binding motif, suggesting that syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism whereby DGKζ phosphorylation by PKCα plays a role in the interconversion between Rac1 and RhoA signaling pathways that underlie different cellular migration modes.

Brain-derived neurotrophic factor enhances the therapeutic effect of oleuropein in the lipopolysaccharide-induced models of depression.

Major depressive disorder (MDD) is a heterogeneous disease, involving multiple mechanisms and factors, which commonly result in injury to the psychosocial function of the central nervous system, and even suicidality of patients. However, effective treatment for MDD is still lacking. Oleuropein is a newly discovered natural compound extracted from olive leaves, which has a strong antioxidative effect by reducing the production of reactive oxygen species (ROS). Oleuropein also reduces blood pressure in humans and experimental animals, and protects blood vessels. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family, which supports the function of the central nervous system. BDNF plays an important role in the development of the nervous system via the regulation of cellular differentiation, survival neurogenesis and synaptic plasticity; therefore, we hypothesized that overexpression of BDNF might contribute to the therapeutic effect of oleuropein. Here, we first demonstrated that oleuropein reverses depressive-like behaviour and restores the inflammatory response in a mouse lipopolysaccharide (LPS) model of MDD. We further established a cell model of BDNF overexpression and inhibition in SH-SY5Y cells, and found that the concentration of intercellular calcium was increased after treatment with oleuropein combined with BDNF overexpression, which may be mediated by the BDNF-TrkB-CaMKII signalling pathway. In addition, we observed that the expression of neurotrophic factors, including epidermal growth factor (EGF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4), was increased, which may be mediated by inhibition of the RhoA-ROCK signalling pathway.

Rock inhibition promotes NaV1.5 sodium channel-dependent SW620 colon cancer cell invasiveness

The acquisition of invasive capacities by carcinoma cells, i.e. their ability to migrate through and to remodel extracellular matrices, is a determinant process leading to their dissemination and to the development of metastases. these cancer cell properties have often been associated with an increased Rho-ROCK signalling, and ROCK inhibitors have been proposed for anticancer therapies. In this study we used the selective ROCK inhibitor, Y-27632, to address the participation of the Rho-ROCK signalling pathway in the invasive properties of SW620 human colon cancer cells. Contrarily to initial assumptions, Y-27632 induced the acquisition of a pro-migratory cell phenotype and increased cancer cell invasiveness in both 3- and 2-dimensions assays. This effect was also obtained using the other ROCK inhibitor Fasudil as well as with knocking down the expression of ROCK-1 or ROCK-2, but was prevented by the inhibition of NaV1.5 voltage-gated sodium channel activity. Indeed, ROCK inhibition enhanced the activity of the pro-invasive NaV1.5 channel through a pathway that was independent of gene expression regulation. In conclusions, our evidence identifies voltage-gated sodium channels as new targets of the ROCK signalling pathway, as well as responsible for possible deleterious effects of the use of ROCK inhibitors in the treatment of cancers.

Baicalin suppresses Th1 and Th17 responses and promotes Treg response to ameliorate sepsis-associated pancreatic injury via the RhoA-ROCK pathway

Recent studies have reported that the imbalance of T helper 1 cell (Th1), Th17 and regulatory T cell (Treg) have been confirmed to play a vital role in the development of sepsis and other inflammatory diseases. Baicalin (BA) has anti-inflammatory properties and improves survival in sepsis. We investigated whether baicalin could regulate Th1, Th17 and Treg responses to ameliorate sepsis-associated pancreatic injury through the ras homolog family member A (RhoA)-Rho kinase (ROCK) pathway. The sepsis model was established by using the cecal ligation and puncture (CLP) method. Fifty mice were randomly divided into five groups (n = 10): sham group, model group, low-dose group (BA-L, 100 mg/kg of baicalin), medium-dose group (BA-M, 200 mg/kg of baicalin) and highdose group (BA-H, 300 mg/kg of baicalin). The effects of baicalin on the pancreatic injury, on changes of Th1, Th17 and Treg cells in vivo and in vitro, on RhoA, ROCK1 and signal transducer and activator of transcription (STAT) signaling pathways, and on levels of interferon-γ (IFN-γ), interleukin-17 (IL-17) and IL-10 were examined. Treatment of the CLP mice with baicalin significantly reduced the extent, scope and severity of the pathological changes of sepsis-associated pancreatic injury. Baicalin evidently reduced Th1 and Th17 cells and increased Treg cells in peripheral blood, spleen, pancreatic tissue and significantly inhibited T-box protein expressed in T cells (T-bet), retinoic acid receptor-related orphan receptor γt (RORγt) and increased forkhead/winged helix transcription factor (Foxp3) expressions in the pancreatic tissue. Baicalin reduced the expressions of RhoA, ROCK1, phosphorylated STAT4 (p-STAT4), p-STAT3 and increased the expression of p-STAT5 in peripheral blood, spleen and pancreatic tissue. Baicalin reduced the expressions of IFN-γ and IL-17 and increased the IL-10 in serum and pancreatic tissue. Baicalin is capable of ameliorating sepsis-associated pancreatic injury and regulating Th1, Th17 and Treg responses in sepsis. The present study provided a potential adjunctive therapy for treating pancreatic injury in sepsis, and further study is needed to reveal its deeper mechanisms.

The Vasopressin Receptor 2 Mutant R137L Linked to the Nephrogenic Syndrome of Inappropriate Antidiuresis (NSIAD) Signals through an Alternative Pathway that Increases AQP2 Membrane Targeting Independently of S256 Phosphorylation.

NSIAD is a rare X-linked condition, caused by activating mutations in the AVPR2 gene coding for the vasopressin V2 receptor (V2R) associated with hyponatremia, despite undetectable plasma vasopressin levels. We have recently provided in vitro evidence that, compared to V2R-wt, expression of activating V2R mutations R137L, R137C and F229V cause a constitutive redistribution of the AQP2 water channel to the plasma membrane, higher basal water permeability and significantly higher basal levels of p256-AQP2 in the F229V mutant but not in R137L or R137C. In this study, V2R mutations were expressed in collecting duct principal cells and the associated signalling was dissected. V2R-R137L and R137C mutants had significantly higher basal pT269-AQP2 levels -independently of S256 and PKA-which were reduced to control by treatment with Rho kinase (ROCK) inhibitor. Interestingly, ROCK activity was found significantly higher in V2R-R137L along with activation of the Gα12/13–Rho–ROCK pathway. Of note, inhibition of ROCK reduced the basal elevated osmotic water permeability to control. To conclude, our data demonstrate for the first time that the gain-of-function mutation of the V2R, R137L causing NSIAD, signals through an alternative PKA-independent pathway that increases AQP2 membrane targeting through ROCK-induced phosphorylation at S/T269 independently of S256 of AQP2.

The Rho-guanine nucleotide exchange factor Solo decelerates collective cell migration by modulating the Rho-ROCK pathway and keratin networks.

Collective cell migration plays crucial roles in tissue remodeling, wound healing, and cancer cell invasion. However, its underlying mechanism remains unknown. Previously, we showed that the RhoA-targeting guanine nucleotide exchange factor Solo (ARHGEF40) is required for tensile force–induced RhoA activation and proper organization of keratin-8/keratin-18 (K8/K18) networks. Here, we demonstrate that Solo knockdown significantly increases the rate at which Madin-Darby canine kidney cells collectively migrate on collagen gels. However, it has no apparent effect on the migratory speed of solitary cultured cells. Therefore, Solo decelerates collective cell migration. Moreover, Solo localized to the anteroposterior regions of cell–cell contact sites in collectively migrating cells and was required for the local accumulation of K8/K18 filaments in the forward areas of the cells. Partial Rho-associated protein kinase (ROCK) inhibition or K18 or plakoglobin knockdown also increased collective cell migration velocity. These results suggest that Solo acts as a brake for collective cell migration by generating pullback force at cell–cell contact sites via the RhoA-ROCK pathway. It may also promote the formation of desmosomal cell–cell junctions related to K8/K18 filaments and plakoglobin.

Oxytocin induced epithelium-mesenchimal transition through Rho-ROCK pathway in ARPE-19 cells, a human retinal pigmental cell line

Previous reports suggest that oxytocin receptors (OXTRs) are expressed in the retinal pigment epithelium in primates. Oxytocinergic signaling activates the Rho-ROCK pathway, which reorganizes the actin cytoskeleton and alters other cellular biophysical characteristics. Such changes could be involved in the epithelial–mesenchymal transition and development of proliferative vitreous retinopathy. Here, we investigated whether oxytocin (OXT) binding to OXTRs in the retinal pigment epithelium can induce Rho-ROCK-mediated cellular activity. We performed four different assays of Rho-ROCK signaling in a human retinal pigment epithelium cell line (ARPE-19) such as induction of actin fibers, wound healing, cell growth, and collagen gel contraction. The assays were performed with or without OXT (100 nM) exposure, as well as with exposure to ripasudil, a specific ROCK inhibitor. The actin stress fiber formation, a phenotype mediated by activated Rho GTPase, was induced by OXT. OXT also accelerated wound closure 19 h after administration, increased cell growth 24 h afterwards, and induced stronger collagen gel contractions. All four cellular responses were inhibited with the addition of 50 μM ripasudil. Taken together, OXT-mediated activation of Rho-ROCK signal transduction could play a role in regulating epithelial–mesenchymal transition in the retinal pigment epithelium, and increase the possibility of subsequent proliferative vitreous retinopathy after vitrectomy.

RhoA-ROCK Signaling as a Therapeutic Target in Traumatic Brain Injury.

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. TBIs, which range in severity from mild to severe, occur when a traumatic event, such as a fall, a traffic accident, or a blow, causes the brain to move rapidly within the skull, resulting in damage. Long-term consequences of TBI can include motor and cognitive deficits and emotional disturbances that result in a reduced quality of life and work productivity. Recovery from TBI can be challenging due to a lack of effective treatment options for repairing TBI-induced neural damage and alleviating functional impairments. Central nervous system (CNS) injury and disease are known to induce the activation of the small GTPase RhoA and its downstream effector Rho kinase (ROCK). Activation of this signaling pathway promotes cell death and the retraction and loss of neural processes and synapses, which mediate information flow and storage in the brain. Thus, inhibiting RhoA-ROCK signaling has emerged as a promising approach for treating CNS disorders. In this review, we discuss targeting the RhoA-ROCK pathway as a therapeutic strategy for treating TBI and summarize the recent advances in the development of RhoA-ROCK inhibitors.

Rho-ROCK Signaling in Normal Physiology and as a Key Player in Shaping the Tumor Microenvironment.

The Rho-ROCK signaling network has a range of specialized functions of key biological importance, including control of essential developmental processes such as morphogenesis and physiological processes including homeostasis, immunity, and wound healing. Deregulation of Rho-ROCK signaling actively contributes to multiple pathological conditions, and plays a major role in cancer development and progression. This dynamic network is critical in modulating the intricate communication between tumor cells, surrounding diverse stromal cells and the matrix, shaping the ever-changing microenvironment of aggressive tumors. In this chapter, we overview the complex regulation of the Rho-ROCK signaling axis, its role in health and disease, and analyze progress made with key approaches targeting the Rho-ROCK pathway for therapeutic benefit. Finally, we conclude by outlining likely future trends and key questions in the field of Rho-ROCK research, in particular surrounding Rho-ROCK signaling within the tumor microenvironment.

An update on ripasudil for the treatment of glaucoma and ocular hypertension.

Ripasudil (K-115) is a novel Rho-associated protein kinase (ROCK) inhibitor. The Rho-ROCK pathway regulates key downstream effectors involved in many cellular functions, in particular in the actin cytoskeleton activity. The clinical effects of ripasudil expected on the eye include an intraocular pressure-lowering effect and a wound-healing activity on corneal endothelial cells, but many other functions are currently under investigation. To date, ripasudil has been approved in Japan (2014) for the treatment of glaucoma and ocular hypertension, and several clinical trials are currently investigating its role in the treatment of Fuchs' corneal dystrophy. In this review, we will discuss its pharmacokinetics, pharmacodynamics and clinical efficacy, focusing also on its safety and tolerability profile.

The CXCL12-CXCR4/CXCR7 axis as a mechanism of immune resistance in gastrointestinal malignancies.

Single agent checkpoint inhibitor therapy has not been effective for most gastrointestinal solid tumors, but combination therapy with drugs targeting additional immunosuppressive pathways is being attempted. One such pathway, the CXCL12-CXCR4/CXCR7 chemokine axis, has attracted attention due to its effects on tumor cell survival and metastasis as well as immune cell migration. CXCL12 is a small protein that functions in normal hematopoietic stem cell homing in addition to repair of damaged tissue. Binding of CXCL12 to CXCR4 leads to activation of G protein signaling kinases such as P13K/mTOR and MEK/ERK while binding to CXCR7 leads to β-arrestin mediated signaling. While some gastric and colorectal carcinoma cells have been shown to make CXCL12, the primary source in pancreatic cancer and peritoneal metastases is cancer-associated fibroblasts. Binding of CXCL12 to CXCR4 and CXCR7 on tumor cells leads to anti-apoptotic signaling through Bcl-2 and survivin upregulation, as well as promotion of the epithelial-to-mesechymal transition through the Rho-ROCK pathway and alterations in cell adhesion molecules. High levels of CXCL12 seen in the bone marrow, liver, and spleen could partially explain why these are popular sites of metastases for many tumors. CXCL12 is a chemoattractant for lymphocytes at lower levels, but becomes chemorepellant at higher levels; it is unclear exactly what gradient exists in the tumor microenvironment and how this influences tumor-infiltrating lymphocytes. AMD3100 (Plerixafor or Mozobil) is a small molecule CXCR4 antagonist and is the most frequently used drug targeting the CXCL12-CXCR4/CXCR7 axis in clinical trials for gastrointestinal solid tumors currently. Other small molecules and monoclonal antibodies against CXCR4 are being trialed. Further understanding of the CXCL12- CXCR4/CXCR7 chemokine axis in the tumor microenvironment will allow more effective targeting of this pathway in combination immunotherapy.

P2255Senescence associated secretory phenotype exacerbates overload pressure-cardiac hypertrophy

Abstract Background Advanced age is a significant risk factor for cardiovascular diseases such as hypertension and cardiac hypertrophy. The vascular system forms an essential component of cardiac tissue, to provide routes for circulation and transportation of nutrients and oxygen throughout the cardiac muscle. In addition to its function in vascular biology such as vasodilation and neovessel formation, endothelial cell (EC) also provides many secreted angiocrine factors that are crucially involved in maintaining tissue homeostasis. Ageing induces cellular senescence in various cells including EC. Senescent cells produce senescence-messaging secretomes that have deleterious effects on the tissue microenvironment, referred to as the senescence-associated secretory phenotype (SASP). Because of the crucial roles of EC in tissue homeostasis, EC senescence is presumed to play significant roles in age-related cardiac dysfunction, however, whether and the mechanism by which EC senescence affects age-related cardiac dysfunction remains to be elucidated. Purpose We aimed to investigate the role of senescent ECs in cardiac hypertrophy and heart function. Methods To investigate a contribution of senescent EC in age-related cardiac tissue dysfunction in vivo, we generated EC-specific progeroid mice that overexpress the dominant negative form of telomeric repeat-binding factor 2 (TRF2), which play a central role in the protection of chromosome ends, under the control of the vascular endothelial cadherin promoter (VEcad-TRF2DN-Tg). To induce pathological cardiac remodeling, Transverse Aortic Constriction (TAC) was performed in mice at the age of 10–12 weeks old. Cardiac function was assessed using fractional shortening percentage and ejection fraction measured with echocardiography every week until sacrifice day. Mice were sacrificed 4 weeks after TAC, heart tissue was collected for histological analysis, cardiac morphometry analysis, gene expression and protein expression analysis. In vitro, H9C2 rat cardiomyoblast cells were incubated with conditioned medium derived from control or senescent EC in the presence or absence of angiotensin II to induce cardiac hypertrophy. Results The serial echocardiographic analysis after TAC revealed the exacerbated LV dysfunction in VEcad-TRF2DN-Tg compared to that in wild-type mice. Morphometric and histological analysis 4 weeks after TAC showed increased heart weight and aggravated cardiac fibrosis in VEcad-TRF2DN-Tg mice. In vitro studies demonstrated that conditioned medium derived from senescent ECs enhanced cardiomyocyte hypertrophy in H9C2 cells. Of note, we found that treatment with Y2762, a Rho Kinase inhibitor, canceled the exacerbated cardiac hypertrophy caused by endothelial SASP. Conclusion These findings demonstrate for the first time that senescent ECs play causative roles in age-related cardiac disorders through the SASP, potentially by activating Rho-ROCK pathway in cardiomyocytes.

Protective effects of 18β-glycyrrhetinic acid on pulmonary arterial hypertension via regulation of Rho A/Rho kinsase pathway

PurposeExcessive proliferation, migration and anti-apoptosis of pulmonary artery smooth muscle cells (PASMCs) are the basis for the development of pulmonary vascular remodeling, and it is the driving force for pulmonary arterial hypertension (PAH). 18β-glycyrrhetinic acid (18β-GA) is the main active substance extracted from Chinese herbal medicine licorice, with outstanding anti-inflammatory, anti-oxidation and anti-proliferative effects. Our team found in previous studies that 18β-GA has protective effects on monocrotaline-induced PAH in rats. However, the anti-angiogenic effect of 18β-GA on PAH remains unclear. Therefore, in order to further investigate whether the beneficial effects of 18β-GA on PAH are related to its antiproliferative effect, we conducted experiments in vivo and in vitro. Methods and resultsIn vivo, 18β-GA relieved mean pulmonary arterial pressure, right ventricular systolic pressure, and right ventricular hypertrophy index, improving pulmonary remodeling. In vitro, 18β-GA significantly inhibited PDGF-BB-induced proliferation and DNA synthesis of HPASMCs, blocking the progression of G0/G1 to S phase of the cell cycle. Furthermore, after treatment with 18β-GA, the expression of Rho A, ROCK1, ROCK2 was decreased and ROCK activity was inhibited in HPASMC. In addition, 18β-GA also attenuated PDGF-induced changes in p27kip1, Bax and Bcl-2. ConclusionsIn summary, these results indicate that 18β-GA regulates the activity of RhoA-ROCK signaling pathway, inhibits the proliferation of HPASMCs, and has potential value in the treatment of PAH.

Abstract 2659: IRE1á-dependent increase of Gá12, a gep oncogene, augments ER stress-induced catastrophic cell death

Abstract Hepatocellular carcinoma (HCC) belongs to the most deadly types of cancer since it has a poor prognosis due to its aggressive metastatic phenotype. Endoplasmic reticulum (ER) stress is closely associated with the incidences of a variety of liver diseases including liver injury, regeneration, fibrosis, cirrhosis and often HCC. G protein-coupled receptor (GPCR) signals lead to activation of their downstream pathways through G proteins. Previously, we reported that Gα12 overexpression enhances HCC malignancy, promoting cancer migration and microvascular invasion. However, the role of Gα12 in ER stress-associated pathophysiology in the liver was unclear. Given the link between ER stress and HCC malignancy, this study investigated the effect of ER stress on Gα12, the role of Gα12 in ER stress-mediated hepatocyte death, and the underlying pathway. In the analyses of RNA-sequencing data from mouse liver or primary mouse embryonic fibroblasts challenged with ER stress, Gα12 and C/EBP homologous protein (CHOP) levels correlated with each other. Increases in Gα12 level by unfolded protein response (UPR) under ER stress condition was verified using primary hepatocytes or hepatocyte-derived cell line, confirmative of the close association between Gα12 and UPR. Of note, enforced Gα12 expression under ER stress condition augmented IRE1α level in a RhoA/Rock-dependent pathway. Among the canonical pathways, IRE1α gene silencing distinctly inhibited Gα12 induction elicited by ER stress in hepatocytes. Consistently, overexpression of IRE1α enhanced Gα12 level upon tunicamycin treatment. Similar outcomes were obtained in cells or mice treated with acetaminophen, a representative liver toxicant inducing ER stress response. Overall, our results provide evidence that IRE1α-dependent increase of Gα12 potentiates ER stress response in the liver as mediated by the RhoA-ROCK pathway, enhancing catastrophic death of hepatocytes. Citation Format: Jihoon Tak, Tae Hyun Kim, Sang Geon Kim. IRE1á-dependent increase of Gá12, a gep oncogene, augments ER stress-induced catastrophic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2659.

Abstract 1029: Metastatic cancer cells activate endothelial RhoA-ROCK pathway for trans-endothelial migration

Abstract Metastasis is the process through which tumor cells disseminate from the primary tumor and colonize in distant parts of the body. Two of the steps in the metastatic process are the trans-migration of cancer cells through the endothelial lining of blood and lymphatic vessels, during entrance (intravasation) and exit (extravasation) from the vascular system. We and others have shown that the endothelial RhoA pathway plays important role in endothelial permeability. Translating these findings in the metastasis context we aim to explore the role of endothelial RhoA signaling pathway on cancer cell trans-endothelial migration and metastasis. In vitro, we have established a quantifiable, highly reproducible, transwell-based, two-cell co-culture model of trans-endothelial migration, where fluorescently-labeled cancer cells transmigrate through an endothelial monolayer. Pharmacological and molecular biology approaches were incorporated to dissect the role of endothelial RhoA signaling pathway. Primary and immortalized endothelial cells were used and cancer cells of both mouse and human origin were tested. In all cases, conditioned media from the cancer cells activated endothelial RhoA. Blockade of the RhoA pathway inhibited cancer cell trans-endothelial migration. In vivo, syngeneic and human cancer cell lines were tested in tail-vein and intra-cardiac models of experimental metastasis. We observed decreased lung metastatic nodules in endothelial-specific RhoA-deficient mice, compared to the littermate controls. Treatment with a clinically-relevant inhibitor of the RhoA pathway, Fasudil, also decreased the metastatic colonization of both human and cancer cells. The above findings demonstrate that the endothelial RhoA pathway has a pivotal role on cancer cell trans-endothelial migration. Citation Format: Md Sanaullah Sajib, Fatema Tuz Zahra, Jee Hyun Park, Paul Tullar, Sanjay K. Srivastava, Ulrich Bickel, Constantinos M Mikelis. Metastatic cancer cells activate endothelial RhoA-ROCK pathway for trans-endothelial migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1029.

Endothelial RhoA Regulates Breast Cancer Metastasis

Metastasis is the cause of more than 90% of cancer‐related deaths. An important step in metastatic process is the migration of the disseminating cancer cells through the endothelial monolayer during entrance or exit from the vasculature. Activation of endothelial RhoA‐ROCK pathway has been implicated in the regulation of vascular permeability in various pathophysiological conditions. In the present study, we have investigated the role of this pathway in trans‐endothelial migration and metastasis of breast cancer cells. RhoA activation was identified by RhoA pull‐down experiments of murine and human breast cancer cell supernatant on primary endothelial cells. Cancer cell trans‐endothelial migration efficiency was explored through an in vitro transwell‐based two‐cell co‐culture model. RhoA signaling pathway was blocked by both pharmacological inhibition and knockdown experiments. In vivo metastatic potential was identified by experimental metastasis models of murine and human breast cancer cell lines using endothelial‐specific RhoA‐deficient mice or clinically relevant ROCK inhibitors. We identified that the diverse metastatic potential of a panel of human breast cancer cell lines correlated with the RhoA activation efficiency of their supernatant on primary endothelial cells. Secretome analysis revealed that IL‐8 (over)expression was relevant to breast cancer cell aggressiveness and its functionality was tested with gain‐and loss‐of‐function experiments. In vivo, experimental metastasis experiments with the syngeneic breast cancer cell line E0771 showed that endothelial RhoA deficiency decreased the metastatic potential. Similar inhibition was obtained with treatment with the clinically‐relevant ROCK inhibitor, Fasudil, in experimental metastasis experiments of murine and human cell lines using immune‐competent and immune‐deficient mice respectively. Collectively, our in vitro and in vivo findings demonstrate that the endothelial RhoA‐ROCK pathway affects breast cancer trans‐endothelial migration and metastasis and propose endothelial RhoA inhibition as a novel therapy for breast cancer metastases.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SHROOM2 inhibits tumor metastasis through RhoA\u2013ROCK pathway-dependent and -independent mechanisms in nasopharyngeal carcinoma

SHROOM2 is a key mediator of RhoA–ROCK pathway that regulates cell motility and actin cytoskeleton organization. However, the functions of SHROOM2 beyond RhoA/ROCK signaling remain poorly understood. Here, we report that SHROOM2 not only participates in RhoA–ROCK-induced stress fiber formation and focal adhesion, but also had an unanticipated role in suppressing epithelial-to-mesenchymal transition (EMT) and tumor metastasis. Depletion of SHROOM2 in nasopharyngeal carcinoma (NPC) cells enhances mesenchymal characteristics and reduces epithelial markers, concomitant with increased motility, enabling the development of invasion and tumor metastasis, which are largely ROCK-independent, as ROCK inhibitor Y-27632 did not cause EMT phenotype; furthermore, combination of ROCK inhibition and SHROOM2 depletion resulted in the most robust increases in cell migration and invasion, indicating that SHROOM2 and ROCK work synergistically rather than epistatic. Analysis of clinical samples suggested that SHROOM2 is downregulated in NPC and the expression of SHROOM2 in metastatic NPC was even lower than in the primary tumors. Our findings uncover a non-canonical role of SHROOM2 as a potent antagonist for EMT and NPC metastasis.

Genome-wide Screen for Culture Adaptation and Tumorigenicity-Related Genes in Human Pluripotent Stem Cells

SummaryHuman pluripotent stem cells (hPSCs) acquire genetic changes during their propagation in culture that can affect their use in research and future therapies. To identify the key genes involved in selective advantage during culture adaptation and tumorigenicity of hPSCs, we generated a genome-wide screening system for genes and pathways that provide a growth advantage either in vitro or in vivo. We found that hyperactivation of the RAS pathway confers resistance to selection with the hPSC-specific drug PluriSIn-1. We also identified that inactivation of the RHO-ROCK pathway gives growth advantage during culture adaptation. Last, we demonstrated the importance of the PI3K-AKT and HIPPO pathways for the teratoma formation process. Our screen revealed key genes and pathways relevant to the tumorigenicity and survival of hPSCs and should thus assist in understanding and confronting their tumorigenic potential.

Statin treatment before stroke reduces pro-inflammatory cytokine levels after stroke

ABSTRACTObjective: In this clinical case-control study, we investigated statin treatment in stroke patients on a range of inflammatory effectors in peripheral blood. We focus on RhoA GTPase and its downstream effectors as a future inflammatory target in stroke treatment.Methods: Data from 10 patients already on statins at stroke onset (Pre-S group) was compared with data from both 29 patients starting statin treatment right after stroke onset (Post-S group) and with 8 healthy controls. In T-cells isolated from stroke patients, we analyzed the activity of the main cytoskeletal regulator RhoA GTPase and its downstream effectors: rho-associated protein kinase (ROCK), myosin phosphatase targeting protein subunit 1 (pMYPT1), myosin light chain kinase (pMLC) and cofilin. In the blood samples, we further determined levels of 12 key plasma cytokines as well as C-reactive protein (CRP) and kallikrein.Results: Compared to healthy controls, the Post-S group achieved significantly higher RhoA and ROCK activities, while the Pre-S did not differ from controls. Levels of pMYPT1, pMLC and cofilin did not differ from controls in the Pre-S and Post-S groups. At day 90 after stroke, interferon γ and IL-18 were significantly increased in the Post-S group compared to the Pre-S group. We found a positive correlation between CRP and NIHSS, whereas kallikrein levels showed no correlation with NIHSS at any of the days.Conclusion: Stroke induces changes in the RhoA-ROCK pathway in T-cells. CRP and NIHSS score correlated positively in the study. Statins may have an anti-inflammatory effect as statin treatment before stroke reduces post-stroke pro-inflammatory levels. RhoA GTPase and its downstream effectors are possibly the key to improve statin treatment in stroke.

Total flavones of Rhododendron simsii Planch flower protect isolated rat heart from ischaemia-reperfusion injury and its mechanism of UTR-RhoA-ROCK pathway inhibition.

Total flavones of Rhododendron simsii Planch flower (TFR) are an effective part extracted from the flower. The present study was designed to investigate the protective effect of TFR in isolated rat heart following global ischaemia-reperfusion and the possible underlying mechanisms. Langendorff perfusion apparatus was used to perfuse isolated rat heart which was subjected to global ischaemia-reperfusion. The hemodynamic parameters were continuously monitored. Coronary flow as well as lactate dehydrogenase (LDH), creatine phosphokinase-MB (CK-MB) and cardiac troponin I (cTnI) in coronary effluents was measured. RhoA activity and urotensin receptor (UTR) and Rho-related coiled-coil-forming protein kinase (ROCK) protein expressions in rat myocardium were examined, respectively. Cardiac dysfunction was indicated by the alterations of hemodynamic parameters and the reduced coronary flow. Total flavones of Rhododendron simsii Planch flower significantly improved ischaemia-reperfusion-induced cardiac dysfunction and leakages of LDH, CK-MB and cTnI, and inhibited myocardial ischaemia-reperfusion-increased RhoA activity and UTR, ROCK1 and ROCK2 protein expressions. The improvement of TFR in the cardiac dysfunction and the leakage of LDH, CK-MB and cTnI were markedly attenuated under the UTR blockade and ROCK inhibition. TFR-inhibited RhoA activity was decreased under the UTR blockade. Total flavones of Rhododendron simsii Planch flower had a protective effect on ischaemia-reperfusion injury in isolated rat heart, which may be attributed to the blocking of UTR and subsequent inhibition of the RhoA-ROCK pathway.