LAMB3 Promotes Myofibrogenesis and Cytoskeletal Reorganization in Endometrial Stromal Cells via the RhoA/ROCK1/MYL9 Pathway.

LAMB3, a major extracellular matrix and basal membrane component, is involved in wound healing. We aimed to understand its role in Asherman's syndrome (AS), which is associated with infertility, by using bioinformatics analysis and cultured endometrial stromal cells (ESCs). MRNAs extracted from tissues obtained from control subjects and patients with severe intrauterine adhesion were sequenced and subjected to bioinformatics analysis and the RhoA/ROCK1/MYL9 pathway was implicated and this subsequently studied using cultured primary ESCs. The effects of overexpression and knockdown and activation and inhibition of LAMB3 on the mesenchymal to myofibroblastic phenotypic transformation of ECCs were assessed using PCR and western blot analysis. Phalloidin was used to localize the actin cytoskeletal proteins. Silencing of LAMB3 reversed the TGF-ß-induced ESC myofibroblast phenotype conversion, whereas overexpression of LAMB3 promoted this process. Activation and silencing of LAMB3 led to remodeling of the ESC cytoskeleton. Overexpression and silencing of LAMB3 caused activation and inhibition of ESCs, respectively. Y-27632 and LPA reversed the activation and inhibition of the RhoA/ROCK1/MYL9 pathway after overexpression and silencing, respectively. These results suggest that LAMB3 can regulate ESC fibrosis transformation and cytoskeleton remodeling via the RhoA/ROCK1/MYL9 pathway. This study provides a potential new target for gene therapy and drug intervention of AS.

RhoA Is a Crucial Regulator of Myoblast Fusion.

Satellite cells (SCs) are adult muscle stem cells that are mobilized when muscle homeostasis is perturbed. Here we show that RhoA in SCs is indispensable to have correct muscle regeneration and hypertrophy. In particular, the absence of RhoA in SCs prevents a correct SC fusion both to other RhoA-deleted SCs (regeneration context) and to growing control myofibers (hypertrophy context). We demonstrated that RhoA is dispensable for SCs proliferation and differentiation; however, RhoA-deleted SCs have an inefficient movement even if their cytoskeleton assembly is not altered. Proliferative myoblast and differentiated myotubes without RhoA display a decreased expression of Chordin, suggesting a crosstalk between these genes for myoblast fusion regulation. These findings demonstrate the importance of RhoA in SC fusion regulation and its requirement to achieve an efficient skeletal muscle homeostasis restoration.

RHOAL57V drives the development of diffuse gastric cancer through IGF1R-PAK1-YAP1 signaling.

Cancer-associated mutations in the guanosine triphosphatase (GTPase) RHOA are found at different locations from the mutational hotspots in the structurally and biochemically related RAS. Tyr42-to-Cys (Y42C) and Leu57-to-Val (L57V) substitutions are the two most prevalent RHOA mutations in diffuse gastric cancer (DGC). RHOAY42C exhibits a gain-of-function phenotype and is an oncogenic driver in DGC. Here, we determined how RHOAL57V promotes DGC growth. In mouse gastric organoids with deletion of Cdh1, which encodes the cell adhesion protein E-cadherin, the expression of RHOAL57V, but not of wild-type RHOA, induced an abnormal morphology similar to that of patient-derived DGC organoids. RHOAL57V also exhibited a gain-of-function phenotype and promoted F-actin stress fiber formation and cell migration. RHOAL57V retained interaction with effectors but exhibited impaired RHOA-intrinsic and GAP-catalyzed GTP hydrolysis, which favored formation of the active GTP-bound state. Introduction of missense mutations at KRAS residues analogous to Tyr42 and Leu57 in RHOA did not activate KRAS oncogenic potential, indicating distinct functional effects in otherwise highly related GTPases. Both RHOA mutants stimulated the transcriptional co-activator YAP1 through actin dynamics to promote DGC progression; however, RHOAL57V additionally did so by activating the kinases IGF1R and PAK1, distinct from the FAK-mediated mechanism induced by RHOAY42C. Our results reveal that RHOAL57V and RHOAY42C drive the development of DGC through distinct biochemical and signaling mechanisms.

Genomic and Reverse Translational Analysis Discloses a Role for Small GTPase RhoA Signaling in the Pathogenesis of Schizophrenia: Rho-Kinase as a Novel Drug Target.

Schizophrenia is one of the most serious psychiatric disorders and is characterized by reductions in both brain volume and spine density in the frontal cortex. RhoA belongs to the RAS homolog (Rho) family and plays critical roles in neuronal development and structural plasticity via Rho-kinase. RhoA activity is regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). Several variants in GAPs and GEFs associated with RhoA have been reported to be significantly associated with schizophrenia. Moreover, several mouse models carrying schizophrenia-associated gene variants involved in RhoA/Rho-kinase signaling have been developed. In this review, we summarize clinical evidence showing that variants in genes regulating RhoA activity are associated with schizophrenia. In the last half of the review, we discuss preclinical evidence indicating that RhoA/Rho-kinase is a potential therapeutic target of schizophrenia. In particular, Rho-kinase inhibitors exhibit anti-psychotic-like effects not only in Arhgap10 S490P/NHEJ mice, but also in pharmacologic models of schizophrenia (methamphetamine- and MK-801-treated mice). Accordingly, we propose that Rho-kinase inhibitors may have antipsychotic effects and reduce cognitive deficits in schizophrenia despite the presence or absence of genetic variants in small GTPase signaling pathways.

Novel cardiovascular protective effects of RhoA signaling and its therapeutic implications.

Ras homolog gene family member A (RhoA) belongs to the Rho GTPase superfamily, which was first studied in cancers as one of the essential regulators controlling cellular function. RhoA has long attracted attention as a key molecule involved in cell signaling and gene transcription, through which it affects cellular processes. A series of studies have demonstrated that RhoA plays crucial roles under both physiological states and pathological conditions in cardiovascular diseases. RhoA has been identified as an important regulator in cardiac remodeling by regulating actin stress fiber dynamics and cytoskeleton formation. However, its underlying mechanisms remain poorly understood, preventing definitive conclusions being drawn about its protective role in the cardiovascular system. In this review, we outline the characteristics of RhoA and its related signaling molecules, and present an overview of RhoA classical function and the corresponding cellular responses of RhoA under physiological and pathological conditions. Overall, we provide an update on the novel signaling under RhoA in the cardiovascular system and its potential clinical and therapeutic targets in cardiovascular medicine.

Src stimulates Abl-dependent phosphorylation of the guanine exchange factor Net1A to promote its cytosolic localization and cell motility.

The neuroepithelial cell transforming gene 1 (Net1) is a guanine nucleotide exchange factor for the small GTPase RhoA that promotes cancer cell motility and metastasis. Two isoforms of Net1 exist, Net1 and Net1A, both of which are sequestered in the nucleus in quiescent cells to prevent aberrant RhoA activation. Many cell motility stimuli drive cytosolic relocalization of Net1A, but mechanisms controlling this event are not fully understood. Here, we demonstrate that epithelial growth factor stimulates protein kinase Src- and Abl1-dependent phosphorylation of Net1A to promote its cytosolic localization. We show that Abl1 efficiently phosphorylates Net1A on Y373, and that phenylalanine substitution of Y373 prevents Net1A cytosolic localization. Furthermore, we found that Abl1-driven cytosolic localization of Net1A does not require S52, which is a phosphorylation site of a different kinase, c-Jun N-terminal kinase, that inhibits nuclear import of Net1A. However, we did find that MKK7-stimulated cytosolic localization of Net1A does require Y373. We also demonstrate that aspartate substitution at Y373 is sufficient to promote Net1A cytosolic accumulation, and expression of Net1A Y373D potentiates epithelial growth factor-stimulated RhoA activation, downstream myosin light chain 2 phosphorylation, and F-actin accumulation. Moreover, we show that expression of Net1A Y373D in breast cancer cells also significantly increases cell motility and Matrigel invasion. Finally, we show that Net1A is required for Abl1-stimulated cell motility, which is rescued by expression of Net1A Y373D, but not Net1A Y373F. Taken together, this work demonstrates a novel mechanism controlling Net1A subcellular localization to regulate RhoA-dependent cell motility and invasion.

HUNK inhibits epithelial-mesenchymal transition of CRC via direct phosphorylation of GEF-H1 and activating RhoA/LIMK-1/CFL-1.

Epithelial-mesenchymal transition (EMT) is associated with the invasive and metastatic phenotypes in colorectal cancer (CRC). However, the mechanisms underlying EMT in CRC are not completely understood. In this study, we find that HUNK inhibits EMT and metastasis of CRC cells via its substrate GEF-H1 in a kinase-dependent manner. Mechanistically, HUNK directly phosphorylates GEF-H1 at serine 645 (S645) site, which activates RhoA and consequently leads to a cascade of phosphorylation of LIMK-1/CFL-1, thereby stabilizing F-actin and inhibiting EMT. Clinically, the levels of both HUNK expression and phosphorylation S645 of GEH-H1 are not only downregulated in CRC tissues with metastasis compared with that without metastasis, but also positively correlated among these tissues. Our findings highlight the importance of HUNK kinase direct phosphorylation of GEF-H1 in regulation of EMT and metastasis of CRC.

Septin11 promotes hepatocellular carcinoma cell motility by activating RhoA to regulate cytoskeleton and cell adhesion.

Septins as GTPases in the cytoskeleton, are linked to a broad spectrum of cellular functions, including cell migration and the progression of hepatocellular carcinoma (HCC). However, roles of SEPT11, the new member of septin, have been hardly understood in HCC. In the study, the clinical significance and biological function of SEPT11 in HCC was explored. SEPT11 was screened out by combining ATAC-seq with mRNA-seq. Role of SEPT11 in HCC was further investigated by using overexpression, shRNA and CRISPR/Cas9-mediated SEPT11-knockout cells or in vivo models. We found RNA-seq and ATAC-seq highlights LncRNA AY927503 (AY) induced SEPT11 transcription, resulting in Rho GTPase activation and cytoskeleton actin aggregation. The GTP-binding protein SEPT11 is thus considered, as a downstream factor of AY, highly expressed in various tumors, including HCC, and associated with poor prognosis of the patients. In vitro, SEPT11 overexpression promotes the migration and invasion of HCC cells, while SEPT11-knockout inhibits migration and invasion. In vivo, SEPT11-overexpressed HCC cells show high metastasis incidents but don't significantly affect proliferation. Meanwhile, we found SEPT11 targets RhoA, thereby regulating cytoskeleton rearrangement and abnormal cell adhesion through ROCK1/cofilin and FAK/paxillin signaling pathways, promoting invasion and migration of HCC. Further, we found SEPT11 facilitates the binding of GEF-H1 to RhoA, which enhances the activity of RhoA. Overall, our study confirmed function of SEPT11 in promoting metastasis in HCC, and preliminarily explored its related molecular mechanism. SEPT11 acts as an oncogene in HCC, also draws further interest regarding its clinical application as a potential therapeutic target.

Fluoride exposure disrupts the cytoskeletal arrangement and ATP synthesis of HT-22 cell by activating the RhoA/ROCK signaling pathway.

BACKGROUND: Fluoride, an environmental contaminant, is ubiquitously present in air, water, and soil. It usually enters the body through drinking water and may cause structural and functional disorders in the central nervous system in humans and animals. Fluoride exposure affects cytoskeleton and neural function, but the mechanism is not clear. METHODS: The specific neurotoxic mechanism of fluoride was explored in HT-22 cells. Cellular proliferation and toxicity detection were investigated by CCK-8, CCK-F, and cytotoxicity detection kits. The development morphology of HT-22 cells was observed under a light microscope. Cell membrane permeability and neurotransmitter content were determined using lactate dehydrogenase (LDH) and glutamate content determination kits, respectively. The ultrastructural changes were detected by transmission electron microscopy, and actin homeostasis was observed by laser confocal microscopy. ATP enzyme and ATP activity were determined using the ATP content kit and ultramicro-total ATP enzyme content kit, respectively. The expression levels of GLUT1 and 3 were assessed by Western Blot assays and qRT-PCR. RESULTS: Our results showed that fluoride reduced the proliferation and survival rates of HT-22 cells. Cytomorphology showed that dendritic spines became shorter, cellular bodies became rounder, and adhesion decreased gradually after fluoride exposure. LDH results showed that fluoride exposure increased the membrane permeability of HT-22 cells. Transmission electron microscopy results showed that fluoride caused cells to swell, microvilli content decreased, cellular membrane integrity was damaged, chromatin was sparse, mitochondria ridge gap became wide, and microfilament and microtubule density decreased. Western Blot and qRT-PCR analyses showed that RhoA/ROCK/LIMK/Cofilin signaling pathway was activated by fluoride. F-actin/G-actin fluorescence intensity ratio remarkably increased in 0.125 and 0.5 mM NaF, and the mRNA expression of MAP2 was significantly decreased. Further studies showed that GLUT3 significantly increased in all fluoride groups, while GLUT1 decreased (p < 0.05). ATP contents remarkably increased, and ATP enzyme activity substantially decreased after NaF treatment with the control. CONCLUSION: Fluoride activates the RhoA/ROCK/LIMK/Cofilin signaling pathway, impairs the ultrastructure, and depresses the connection of synapses in HT-22 cells. Moreover, fluoride exposure affects the expression of glucose transporters (GLUT1 and 3) and ATP synthesis. Sum up fluoride exposure disrupts actin homeostasis, ultimately affecting structure, and function in HT-22 cells. These findings support our previous hypothesis and provide a new perspective on the neurotoxic mechanism of fluorosis.

MiR-490-3p Sponged by lncRNA NEAT1 Can Attenuate Lung Adenocarcinoma Progression by Suppressing the RhoA/ROCK Signaling Pathway.

OBJECTIVE: Long noncoding RNAs (lncRNAs) are crucial regulators of lung adenocarcinoma (LUAD) progression. Herein, we explored the role of miR-490-3p and the underlying molecular mechanism involving key lncRNAs and pathways in LUAD. METHODS: Reverse transcription-quantitative PCR (RT-qPCR) was performed to detect the expression of lncRNA NEAT1 and miR-490-3p in LUAD cells and tissues. Western blotting was used to determine protein expression levels of the Ras homologous gene family member A/Rho-related protein kinase (RhoA/ROCK) signal pathway marker. Considering cell functions, Cell Counting Kit-8 (CCK-8), Transwell, and xenograft experiments were employed to evaluate LUAD cell proliferation, migration, and tumor growth, respectively. The relationship between miR-490-3p and lncRNA NEAT1 was analyzed using a luciferase reporter assay. RESULTS: Herein, we found that miR-490-3p expression was significantly low in LUAD cells and tissues. MiR-490-3p overexpression markedly suppressed tumor growth, the RhoA/ROCK signaling pathway, migration, and proliferation of LUAD cells. Moreover, lncRNA NEAT1, which is highly expressed in LUAD, was detected upstream of miR-490-3p. Upregulation of lncRNA NEAT1 exacerbated the behavior of LUAD cells and offset the suppressive influence of miR-490-3p-mediated upregulation on malignant LUAD cell behavior. CONCLUSION: MiR-490-3p sponging by lncRNA NEAT1 could hamper LUAD progression by inhibiting the RhoA/ROCK signaling pathway. These findings provide new insights for LUAD diagnosis and treatment.

GEF-H1/RhoA signaling pathway mediates pro-inflammatory effects of NF-κB on CD40L-induced pulmonary endothelial cells.

One of the key targets of the inflammatory response in acute lung injury (ALI) is the human pulmonary micro-vascular endothelial cells (HPMVECs). Owing to its role in the activation of endothelial cells (ECs), CD40L figures prominently in the pathogenesis of ALI. Increasing evidences have showed that CD40L mediates inflammatory effects on ECs, at least in part, by triggering NF-κB-dependent gene expression. However, the mechanisms of such signal transmission remain unknown. In this study, we found that CD40L stimulated the transactivation of NF-κB and expression of its downstream cytokines in a p38 MAPK-dependent mechanism in HPMVECs. In addition, CD40L-mediated inflammatory effects might be correlated with the activation of the IKK/IκB/NF-κB pathway and nuclear translocation of NF-κB, being accompanied by dynamic cytoskeletal changes. GEF-H1/RhoA signaling is best known for its role in regulating cytoskeletal rearrangements. An interesting finding was that CD40L induced the activation of p38 and IKK/IκB, and the subsequent transactivation of NF-κB via GEF-H1/RhoA signaling. The critical role of GEF-H1/RhoA in CD40L-induced inflammatory responses in the lung was further confirmed in GEF-H1 and RhoA knockout mouse models, both of which were established by adeno-associated virus (AAV)-mediated delivery of sgRNAs into mice with EC-specific Cas9 expression. These results taken together suggested that p38 and IKK/IκB-mediated signaling pathways, both of which lied downstream of GEF-H1/RhoA, may coordinately regulate the transactivation of NF-κB in CD40L-activated HPMVECs. These findings may help to determine key pharmacological targets of intervention for CD40L-activated inflammatory effects associated with ALI.

Serotonin/5-HT7 receptor provides an adaptive signal to enhance pigmentation response to environmental stressors through cAMP-PKA-MAPK, Rab27a/RhoA, and PI3K/AKT signaling pathways.

Serotonin (5-HT), a neurotransmitter, is essential for normal and pathological pigmentation processing, and its receptors may be therapeutical targets. The effect and behavior of the 5-HT7 receptor (5-HT7R) in melanogenesis in high vertebrates remain unknown. Herein, we examine the role and molecular mechanism of 5-HT7R in the pigmentation of human skin cells, human tissue, mice, and zebrafish models. Firstly, 5-HT7R protein expression decreased significantly in stress-induced depigmentation skin and vitiligo epidermis. Stressed mice received transdermal serotonin 5-HT7R selective agonists (LP-12, 0.01%) for 12 or 60 days. Mice might recover from persistent stress-induced depigmentation. The downregulation of tyrosinase (Tyr), microphthalmia-associated transcription factor (Mitf) expression, and 5-HT7R was consistently restored in stressed skin. High-throughput RNA sequencing showed that structural organization (dendrite growth and migration) and associated pathways were activated in the dorsal skin of LP-12-treated animals. 5-HT7R selective agonist, LP-12, had been demonstrated to enhance melanin production, dendrite growth, and chemotactic motility in B16F10 cells, normal human melanocytes (NHMCs), and zebrafish. Mechanistically, the melanogenic, dendritic, and migratory functions of 5-HT7R were dependent on the downstream signaling of cAMP-PKA-ERK1/2, JNK MAPK, RhoA/Rab27a, and PI3K/AKT pathway activation. Importantly, pharmacological inhibition and genetic siRNA of 5-HT7R by antagonist SB269970 partially/completely abolished these functional properties and the related activated pathways in both NHMCs and B16F10 cells. Consistently, htr7a/7b genetic knockdown in zebrafish could blockade melanogenic effects and abrogate 5-HT-induced melanin accumulation. Collectively, we have first identified that 5-HT7R regulates melanogenesis, which may be a targeted therapy for pigmentation disorders, especially those worsened by stress.

Follicular Helper T-Cell-derived Nodal Lymphomas: Study of Histomorphologic, Immunophenotypic, Clinical, and RHOA G17V Mutational Profile.

The study was designed to review the demographic, clinical, and pathologic characteristics of follicular helper T cells (TFH)-derived nodal PTCL in India including angioimmunoblastic T-cell lymphoma (AITL), peripheral T-cell lymphoma (PTCL) with follicular helper T cell phenotype (P-TFH), and follicular T-cell lymphoma with additional immunohistochemistry (IHC) and RHOAG17V mutational analysis, as well as their impact on survival. This retrospective study included 88 cases of PTCL that were reclassified using IHC for TFH markers (PD1, ICOS, BCL6, and CD10) and dendritic-meshwork markers (CD21, CD23). Cases of TFH cell origin were evaluated for RHOAG17V mutation using Sanger sequencing and amplification-refractory mutation system-polymerase chain reaction (PCR) (validated using cloning and quantitative PCR) with detailed clinicopathologic correlation. Extensive re-evaluation with added IHC panel resulted in a total of 19 cases being reclassified, and the final subtypes were AITL (37 cases, 42%), PTCL-not otherwise specified (44, 50%), P-TFH (6, 7%), and follicular T-cell lymphoma (1, 1%). The presence of at least 2 TFH markers (>20% immunopositivity) determined the TFH origin. AITL patients tended to be male and showed increased presence of B-symptoms and hepatosplenomegaly. Histomorphology revealed that 92% of AITL cases had pattern 3 involvement. Sanger sequencing with conventional PCR did not yield any mutation, while RHOAG17V was detected by amplification-refractory mutation system-PCR in AITL (51%, P =0.027) and P-TFH (17%), which was validated with cloning followed by sequencing. Cases of RHOAG17V-mutant AITL had a worse Eastern Cooperative Oncology Group performance status initially but fared better in terms of overall outcome ( P =0.029). Although not specific for AITL, RHOAG17V mutation shows an association with diagnosis and requires sensitive methods for detection due to low-tumor burden. The mutant status of AITL could have prognostic implications and translational relevance.

RHOA Therapeutic Targeting in Hematological Cancers.

Primarily identified as an important regulator of cytoskeletal dynamics, the small GTPase Ras homolog gene family member A (RHOA) has been implicated in the transduction of signals regulating a broad range of cellular functions such as cell survival, migration, adhesion and proliferation. Deregulated activity of RHOA has been linked to the growth, progression and metastasis of various cancer types. Recent cancer genome-wide sequencing studies have unveiled both RHOA gain and loss-of-function mutations in primary leukemia/lymphoma, suggesting that this GTPase may exert tumor-promoting or tumor-suppressive functions depending on the cellular context. Based on these observations, RHOA signaling represents an attractive therapeutic target for the development of selective anticancer strategies. In this review, we will summarize the molecular mechanisms underlying RHOA GTPase functions in immune regulation and in the development of hematological neoplasms and will discuss the current strategies aimed at modulating RHOA functions in these diseases.

Activation of RhoA pathway participated in the changes of emotion, cognitive function and hippocampal synaptic plasticity in juvenile chronic stress rats.

Neuropsychiatric diseases are related to early life stress (ELS), patients often have abnormal learning, memory and emotion. But the regulatory mechanism is unclear. Hippocampal synaptic plasticity (HSP) changes are important mechanism. RhoA pathway is known to regulate HSP by modulating of dendritic spines (DS), whether it's involved in HSP changes in ELS hasn't been reported. So we investigated whether and how RhoA participates in HSP regulation in ELS. The ELS model was established by separation-rearing in juvenile. Results of IntelliCage detection etc. showed simple learning and memory wasn't affected, but spatial, punitive learning and memories reduced, the desire to explore novel things reduced, the anxiety-like emotion increased. We further found hippocampus was activated, the hippocampal neurons dendritic complexities reduced, the proportion of mature DS decreased. The full-length transcriptome sequencing techniques was used to screen for differentially expressed genes involved in regulating HSP changes, we found RhoA gene was up-regulated. We detected RhoA protein, RhoA phosphorylation and downstream molecules expression changes, results shown RhoA and p-RhoA, p-ROCK2 expression increased, p-LIMK, p-cofilin expression and F-actin/G-actin ratio decreased. Our study revealed HSP changes in ELS maybe regulate by activation RhoA through ROCK2/LIMK/cofilin pathway regulated F-actin/G-actin balance and DS plasticity, affecting emotion and cognition.

Eupafolin regulates non-small-cell lung cancer cell proliferation,migration, and invasion by suppressing MMP9 and RhoA via FAK/PI3K/AKT signaling pathway.

Non-small-cell lung cancer (NSCLC) predominates lung cancer with a striking percentage of 85%. Eupafolin is documented to possess anti-tumor efficacy, which prompts efforts to uncover its impacts on the pathology of diseases including cancers. Focal adhesion kinase (FAK)-mediated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) has been found to be associated with several carcinomas. Nevertheless, how eupafolin exerts its effects in NSCLC and whether FAK/PI3K/AKT is related to the corresponding mechanism remain unclear. Thus, the relevant experiments were carried out with NSCLC cells treated with eupafolin and/or LY294002 at first. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, wound healing, and transwell assays were used to assess cell viability, proliferation, migration, and invasion, respectively. Western blot assay was performed to measure the relative protein expressions of phosphorylated (p)-FAK/FAK, p-PI3K/ PI3K, p-AKT/AKT, matrix metalloproteinase 9 (MMP9), and ras homolog gene family member A (RhoA), and to determine transfection efficiency. From experimental results, it was found that eupafolin inhibited the viability, proliferation, migration, and invasion of NSCLC cells, and inactivated the FAK/PI3K/AKT pathway by downregulating the ratios of p-FAK/FAK, p-PI3K/PI3K, and p-AKT/AKTand the expressions of MMP9 and RhoA. On the contrary, overexpressed FAK upregulated the expressions of FAK, MMP9, and RhoA and the ratios of p-PI3K/ PI3K and p-AKT/AKT, and promoted cell proliferation, migration, and invasion. LY294002, conversely, could partly reverse the effects of FAK on the aforementioned aspects of NSCLC cells. Collectively, it was verified in our study that eupafolin regulates the proliferation, migration, and invasion of NSCLC cells by downregulating MMP9 and RhoA expressions via the FAK/PI3K/AKT axis, which may provide a promising avenue for cancer therapy.

RhoA G17E/Vav1 Signaling Induces Cancer Invasion via Matrix Metalloproteinase-9 in Gastric Cancer.

BACKGROUND: RAS homolog family member A (RhoA), a member of the Rho family of small GTPases, and Vav1, a guanine nucleotide exchange factor for Rho family GTPases, have been reported to activate pathways related to the actin cytoskeleton and regulation of cell shape, attachment, and motility. The interaction between these molecules in lymphoma is involved in malignant signaling, but its function in epithelial malignancy is unknown. Here, we investigated the malignant signal of mutant RhoA in gastric cancer and demonstrated the potential of RhoA G17E/Vav1 as a therapeutic target for diffuse gastric cancer. METHODS: The RhoA mutants R5W, G17E, and Y42C were retrovirally transduced into the gastric cancer cell line MKN74. The stably transduced cells were used for morphology, proliferation, and migration/invasion assays in vitro. MKN74 cells stably transduced with ectopic wild-type RhoA and mutant RhoA (G17E) were used in a peritoneal xenograft assay. RESULTS: The RhoA mutations G17E and Y42C induced morphological changes in MKN74. G17E induced Vav1 expression at the mRNA and protein levels and promoted the migration and invasion of MKN74. An RNA interference assay of Vav1 revealed that RhoA G17E enhanced cancer cell invasion via Vav1. Furthermore, immunoprecipitation revealed that Vav1 and RhoA G17E specifically bind and function together through matrix metalloproteinase -9. In a peritoneal xenograft model of nude mice, RhoA G17E promoted peritoneal dissemination, whereas Vav1 knockdown suppressed it. CONCLUSION: Overall, our findings indicate that RhoA G17E is associated with Vav1 and promoted cancer invasion via matrix metalloproteinase -9 in gastric cancer cells. Thus, RhoA G17E/Vav1 signaling in diffuse gastric cancer may be a useful therapeutic target.

RhoA regulation in space and time.

RhoGTPases are well known for being controllers of cell cytoskeleton and share common features in the way they act and are controlled. These include their switch from GDP to GTP states, their regulations by different guanine exchange factors (GEFs), GTPase-activating proteins and guanosine dissociation inhibitors (GDIs), and their similar structure of active sites/membrane anchors. These very similar features often lead to the common consideration that the differences in their biological effects mainly arise from the different types of regulators and specific effectors associated with each GTPase. Focusing on data obtained through biosensors, live cell microscopy and recent optogenetic approaches, we highlight in this review that the regulation of RhoA appears to depart from Cdc42 and Rac1 modes of regulation through its enhanced lability at the plasma membrane. RhoA presents a high dynamic turnover at the membrane that is regulated not only by GDIs but also by GEFs, effectors and a possible soluble conformational state. This peculiarity of RhoA regulation may be important for the specificities of its functions, such as the existence of activity waves or its putative dual role in the initiation of protrusions and contractions.

Overexpressions of RHOA, CSNK1A1, DVL2, FZD8, and LRP5 genes enhance gastric cancer development in the presence of Helicobacter pylori.

BACKGROUND AND STUDY AIMS: Intestinal metaplasia (IM), and Helicobacter pylori (HP) infection can be shown as risk factors in the development of gastric cancer (GC). WNT signaling pathway plays a critical role in carcinogenesis. However, the literature studies are limited on the significance of this pathway for the transition from IM to GC. PATIENTS AND METHODS: We aimed to investigate the importance of the genes of WNT signaling pathways diagnostic and prognostic markers in the presence and absence of HP in conversion from IM to GC. 104 patients, (GC group n = 35, IM group n = 45, control group n = 25) were included in this case-control study. Expression of genes in WNT signalling were searched in study groups with qRT-PCR array and qRT-PCR method. Data were analysed using PCR array data analysis software. RESULTS: Statistically significant overexpression of RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes was detected in the GC and IM groups compared to the control group (p < 0.05). Statistically significant overexpression of RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes was observed in patients with metastatic GC compared to patients with GC without metastasis (p < 0.05). It was found that the RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes were statistically significantly over-expressed in diffuse GC patients compared to non-diffuse GC patients (p < 0.05). Statistically significant overexpression of RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes was detected in HP positive IM patients compared to HP negative IM patients (p < 0.05). CONCLUSION: Overexpression of RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes in IM may suggest that these genes are important markers in the development of IM and inflammation with HP. In addition, these genes are linked to tumor burden in the GC group. Consequently, we can conclude that these genes are poor prognosis biomarkers for GC and have the potential to be used as markers for future treatment monitoring.

Knockdown of RhoA Expression Reverts Enzalutamide Resistance via the p38 MAPK Pathway in Castration-resistant Prostate Cancer.

BACKGROUND: Enzalutamide has been approved clinically for the treatment of castrationresistant prostate cancer (CRPC) but is limited by the emergence of resistance. RhoA has been shown to play a vital role in carcinogenesis, invasion, and metastasis. However, the role of RhoA in enzalutamide-resistant prostate cancer (PCa) remains unclear. OBJECTIVES: This study investigated the role of RhoA and the associated mechanisms of RhoA depletion in enzalutamide resistance in CRPC. METHODS: Western blotting, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and colony formation assays were used to assess protein expression, survival, and proliferation of PCa cells, respectively. Xenograft experiments and hematoxylin and eosin (H&E) staining were used to detect further effects of RhoA on enzalutamide resistance in vivo. RESULTS: In the present study, the expression of RhoA, ROCK2, p38, p-p38, and AR was upregulated in enzalutamide-resistant PCa cells treated with enzalutamide, and silencing of RhoA or ROCK2 attenuated enzalutamide-resistant cell proliferation and colony formation. Furthermore, the deletion of RhoA dramatically increased the efficacy of enzalutamide in inhibiting 22RV1-derived xenograft tumor growth. Additionally, there was no significant change in ROCK1 expression in C4-2R cells treated with or without enzalutamide. Mechanistically, the knockdown of RhoA expression reverted the resistance to enzalutamide via RhoA/ROCK2/p38 rather than RhoA/ROCK1/p38. CONCLUSION: Our results suggested that RhoA is a promising therapeutic target. As the inhibition of RhoA reverted enzalutamide resistance, it may increase its effectiveness in CRPC.