Search

In recent years, increasing evidence shows that circular RNA (circRNA) disorder is closely related to tumorigenesis and cancer progression. However, the regulatory functions of most circRNAs in bladder cancer (BCa) remain unclear. This study was aimed at exploring the molecular regulatory mechanism of circRNAs in BCa. We obtained four datasets of circRNA, microRNA (miRNA), and messenger (mRNA) expression profiles from the Gene Expression Omnibus and The Cancer Genome Atlas microarray databases and identified 434, 367, and 4799/4841 differentially expressed circRNAs, miRNAs, and mRNAs, respectively. With these differentially expressed RNAs, we established a circRNA-miRNA-mRNA targeted interaction network. A total of 18, 24, and 51 central circRNAs, miRNAs, and mRNAs were identified, respectively. Among them, the top 10 mRNAs that had high connectivity with other circRNAs and miRNAs were regarded as hub genes. We detected the expression levels of these 10 mRNAs in 16 pairs of BCa tissues and adjacent normal tissues through quantitative real-time polymerase chain reaction. The differentially expressed mRNAs and central mRNAs were enriched in the processes and pathways that are associated with the growth, differentiation, proliferation, and apoptosis of tumor cells. The outstanding genes (CDCA4, GATA6, LATS2, RHOB, ZBTB4, and ZFPM2) also interacted with numerous drugs, indicating their potency as biomarkers and drug targets. The findings of this study provide a deep understanding of the circRNA-related competitive endogenous RNA regulatory mechanism in BCa pathogenesis.

The Ras homologous (Rho) protein family of GTPases (RhoA, RhoB and RhoC) are the members of the Ras superfamily and regulate cellular processes such as cell migration, proliferation, polarization, adhesion, gene transcription and cytoskeletal structure. Rho GTPases function as molecular switches that cycle between GTP-bound (active state) and GDP-bound (inactive state) forms. Leukaemia-associated RhoGEF (LARG) is a guanine nucleotide exchange factor (GEF) that activates RhoA subfamily GTPases by promoting the exchange of GDP for GTP. LARG is selective for RhoA subfamily GTPases and is an essential regulator of cell migration and invasion. Here, we describe the mechanisms by which LARG is regulated to facilitate the understanding of how LARG mediates functions like cell motility and to provide insight for better therapeutic targeting of these functions.

Background Evidence indicates that Gα12, Gα13, and its downstream effectors, RhoA and Rac1, regulate neuronal morphology affected by stress. This study was aimed at investigating whether repeated stress influences the expression of proteins related to the Gα12/13 intracellular signaling pathway in selected brain regions sensitive to the effects of stress. Furthermore, the therapeutic impact of β(1)adrenergic receptors (β1AR) blockade was assessed. Methods Restraint stress (RS) model in mice (2 h/14 days) was used to assess prolonged stress effects on the mRNA expression of Gα12, Gα13, RhoA, Rac1 in the prefrontal cortex (PFC), hippocampus (HIP) and amygdala (AMY). In a separate study, applying RS model in rats (3–4 h/1 day or 14 days), we evaluated stress effects on the expression of Gα12, Gα11, Gαq, RhoA, RhoB, RhoC, Rac1/2/3 in the HIP. Betaxolol (BET), a selective β1AR antagonist, was introduced (5 mg/kg/p.o./8–14 days) in the rat RS model to assess the role of β1AR in stress effects. RT-qPCR and Western Blot were used for mRNA and protein assessments, respectively. Results Chronic RS decreased mRNA expression of Gα12 and increased mRNA for Rac1 in the PFC of mice. In the mice AMY, decreased mRNA expression of Gα12, Gα13 and RhoA was observed. Fourteen days of RS exposure increased RhoA protein level in the rats’ HIP in the manner dependent on β1AR activity. Conclusions Together, these results suggest that repeated RS affects the expression of genes and proteins known to be engaged in neural plasticity, providing potential targets for further studies aimed at unraveling the molecular mechanisms of stress-related neuropsychiatric diseases.

An untargeted multi-omics study implicated the potential dysregulation of fatty acid, nucleotide, and energy metabolism in the brainstems of spontaneously hypertensive rats (SHRs). A further quantitative exploration of the alterations in the metabolic pathways is necessary for a deep understanding of the central nervous system in SHRs. Targeted metabolic profiling of 40 fatty acids (PeptideAtlas: PASS01671) and 32 metabolites of nucleotides and energy metabolism (PeptideAtlas: PASS01672) and parallel reaction monitoring analysis of 5 proteins (PeptideAtlas: PASS01673) were performed on the brainstems of SHRs (n = 8, 11 weeks old) and normotensive Wistar rats (n = 8, age-matched) using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem MS. The targeted profiling results of metabolites and proteins revealed decreased polyunsaturated fatty acid (PUFA) synthesis with a significant downregulation of cis-11,14-eicosadienoic acid, cis-13,16-docosadienoic acid, and docosatetraenoate and impaired PUFA oxidation with the accumulation of γ-linolenate induced by the significantly downregulated expression of 2,4-dienoyl-CoA reductase (p < 0.05). Dysregulated GTP and ATP metabolism was observed, with significantly decreased GDP and ADP (p < 0.05) correlated with reduced GTPases of guanine nucleotide-binding protein subunit beta-1 (GNB1), transforming protein RhoA (RHOA), and Rho-related GTP-binding protein RhoB (RHOB) in the brainstem of SHRs. In addition, protein-arginine deiminase type-2 was significantly reduced in the brainstems of SHRs (p < 0.05). The aberrant PUFA and energy metabolism might help to explain the alterations in the brainstem of SHRs. The findings on both metabolites and proteins could provide systemic insights into the pathology basis of altered PUFA and energy metabolism in hypertension, especially in the central nervous system.

Dietary intervention to prevent Helicobacter pylori (H. pylori)-associated gastric diseases seems to be ideal with no risk of bacterial resistance, safe long-term intervention, and correcting pathogenic mechanisms including rejuvenation of precancerous atrophic gastritis and anti-mutagenesis. A transcriptome as set of all RNAs transcribed by certain tissues or cells demonstrates gene functions and reveals the molecular mechanism of specific biological processes against diseases. Here, we have performed RNAseq and bioinformatic analysis to explain proof of concept that walnut intake can rescue from H. pylori infection and explore unidentified mode of actions of walnut polyphenol extract (WPE). As results, BIRC3, SLC25A4, f3 transcription, VEGFA, AZU1, HMOX1, RAB3A, RELBTNIP1, ETFB, INPP5J, PPME1, RHOB, TPI1, FOSL1, JUND.RELB, KLF2, MUC1, NDRG1, ALDOA, ENO1, PFKP, GPI, GDF15, and NRTN genes were newly discovered to be enriched with WPE, whereas CCR4, BLNK, CCR7, CXCR4, CDO1, KLSG1, SELE, RASGRP2, PIK3R3, TSPAN32, HOXC-AS3, HCG8, BTNL8, and CXCL3 genes as inhibitory targets by WPE in H. pylori infection. We identified additional genes what WPE afforded actions of avoiding H. pylori-driven onco-inflammation and rejuvenating precancerous atrophic gastritis. Conclusively, after applying RNAseq analysis in order to document walnut intake for precision medicine against H. pylori infection, significant transcriptomic profiling applicable for validation were drawn.

There are three RhoGDIs in mammalian cells, which were initially defined as negative regulators of Rho family small GTPases. However, it is now accepted that RhoGDIs not only maintain small GTPases in their inactive GDP-bound form but also act as chaperones for small GTPases, targeting them to specific intracellular membranes and protecting them from degradation. Studies to date with RhoGDIs have usually focused on the interactions between the "typical" or "classical" small GTPases, such as the Rho, Rac, and Cdc42 subfamily members, and either the widely expressed RhoGDI-1 or the hematopoietic-specific RhoGDI-2. Less is known about the third member of the family, RhoGDI-3 and its interacting partners. RhoGDI-3 has a unique N-terminal extension and is found to localize in both the cytoplasm and the Golgi. RhoGDI-3 has been shown to target RhoB and RhoG to endomembranes. In order to facilitate a more thorough understanding of RhoGDI function, we undertook a systematic study to determine all possible Rho family small GTPases that interact with the RhoGDIs. RhoGDI-1 and RhoGDI-2 were found to have relatively restricted activity, mainly binding members of the Rho and Rac subfamilies. RhoGDI-3 displayed wider specificity, interacting with the members of Rho, Rac, and Cdc42 subfamilies but also forming complexes with "atypical" small Rho GTPases such as Wrch2/RhoV, Rnd2, Miro2, and RhoH. Levels of RhoA, RhoB, RhoC, Rac1, RhoH, and Wrch2/RhoV bound to GTP were found to decrease following coexpression with RhoGDI-3, confirming its role as a negative regulator of these small Rho GTPases.

Natural products have been of much interest in research studies owing to their wide pharmacological applications, chemical diversity, low side effects, and multitarget activities. Examples of these compounds include matrine, sulforaphane, silibinin, curcumin, berberin, resveratrol, and quercetin. Some of the present anticancer drugs, such as taxol, vincristine, vinblastine, and doxorubicin are also derived from natural products. The anti-carcinogenic effects of these products are partly mediated through modulation of microRNA-21 (miR-21) expression. To date, numerous downstream targets of miR-21 have been recognized, which include phosphatase and tensin homolog (PTEN), ras homolog gene family member B (RHOB), phosphoinositide 3-kinase/protein kinase B (PI3K/Akt), programmed cell death 4 (PDCD4), signal transducer and activator of transcription (STAT)-3, and nuclear factor kappa B (NF-κB) pathways. These signaling pathways, their regulation by oncomiR-21 in cancer, and the modulating impact of natural products are the main focus of this review.

Background Tumor angiogenesis, an essential process for cancer proliferation and metastasis, has a critical role in prognostic of kidney renal clear cell carcinoma (KIRC), as well as a target in guiding treatment with antiangiogenic agents. However, tumor angiogenesis subtypes and potential epigenetic regulation mechanisms in KIRC patient remains poorly characterized. System evaluation of angiogenesis subtypes in KIRC patient might help to reveal the mechanisms of KIRC and develop more target treatments for patients. Method Ten independent tumor angiogenesis signatures were obtained from molecular signatures database (MSigDB) and gene set variation analysis was performed to calculate the angiogenesis score in silico using the Cancer Genome Atlas (TCGA) KIRC dataset. Tumor angiogenesis subtypes in 539 TCGA-KIRC patients were identified using consensus clustering analysis. The potential regulation mechanisms was studied using gene mutation, copy number variation, and differential methylation analysis (DMA). The master transcription factors (MTF) that cause the difference in tumor angiogenesis signals were completed by transcription factor enrichment analysis. Results The angiogenesis score of a prognosis related angiogenesis signature including 189 genes was significantly correlated with immune score, stroma score, hypoxia score, and vascular endothelial growth factor (VEGF) signal score in 539 TCGA KIRC patients. MMRN2, CLEC14A, ACVRL1, EFNB2, and TEK in candidate gene set showed highest correlation coefficient with angiogenesis score in TCGA-KIRC patients. In addition, all of them were associated with overall survival in both TCGA-KIRC and E-MTAB-1980 KIRC data. Clustering analysis based on 183 genes in angiogenesis signature identified two prognosis related angiogenesis subtypes in TCGA KIRC patients. Two clusters also showed different angiogenesis score, immune score, stroma score, hypoxia score, VEGF signal score, and microenvironment score. DMA identified 59,654 differential methylation sites between two clusters and part of these sites were correlated with tumor angiogenesis genes including CDH13, COL4A3, and RHOB. In addition, RFX2, SOX13, and THRA were identified as top three MTF in regulating angiogenesis signature in KIRC patients. Conclusion Our study indicate that evaluation the angiogenesis subtypes of KIRC based on angiogenesis signature with 183 genes and potential epigenetic mechanisms may help to develop more target treatments for KIRC patients.

Background The current study investigates the effect of transcription factor Prox1 on the proliferation, migration, and invasion ability of lung cancer. Methods Lung cancer cell lines (A549 and H446 cells) were transfected with Prox1NAD and siRNA, respectively. Thus, the A549 and H446 cells overexpressed Prox1 after transfection of Prox1NAD plasmids, and A549 and H446 cells have low expression of Prox1 after transfection with siRNA. Reverse transcriptase quantitative PCR and western blot analyses were used to detect Prox1 mRNA and protein expression in cells. Plate clone formation experiments and MTT experiments were used to detect cell proliferation. Western blot was used to detect the expression of Rho family-related proteins in cells. Results Compared to untransfected wild-type A549 and H446 that served as blank controls, the expression level of Prox1mRNA and protein in A549 and H446 cells overexpressing Prox1 after plasmid transfection was high, while the expression level of Prox1mRNA and protein in A549 and H446 cells with low expression of Prox1 after siRNA transfection was low. With the increase of Prox1 expression, the expression of RhoA and RhoC increased, while the expression of RhoB decreased. Conclusion The finding of this study may provide a new approach for the treatment of lung cancer using targeted gene therapy.

Background This study evaluated the effect of treatment with TKI-258 on apoptosis, involving Rho GTPases and their effectors in SCC-4 cells of oral squamous cell carcinoma. Methods Markers of cell death and apoptosis were analyzed in control and TKI-258 treated SCC-4 cells by flow cytometry. The involvement of Rho GTPases and effectors in the induction of apoptosis by TKI-258 was evaluated by quantification of cleaved PARP. Also gene expression analysis of those proteins was performed. Results The treatment with TKI-258 led to a significant increase in cell death (7-AAD) and apoptosis (Annexin V and cleaved PARP). When Rho GTPases were stimulated with LPA and inhibited with Toxin A Clostridium difficile, the percentage of apoptotic cells increased and decreased, respectively. A similar effect was found when the treatment was with TKI-258 combined with LPA and Toxin A. Treatment with TKI-258 significantly increased RhoA gene expression, while RhoB, RhoC, Rac1 and Cdc42 decreased significantly. ROCKs inhibitors (Y-27632 and HA-1077) reduced apoptosis compared with control. TKI-258 combined with Y-27632 or HA-1077 led to an increase in apoptosis compared with inhibitors only. Treatment with TKI-258 led to an increase in ROCK1 and ROCK2 gene expression, and a decrease in PAK1 and PAK2 gene expression. Conclusions TKI-258 stimulates apoptosis in SCC-4 cells of oral squamous cell carcinoma. Possibly, RhoA GTPase and their effectors ROCKs participate in the signalling pathway inhibited by TKI-258. Clinical relevance Therapies with multi-target inhibitors, such as TKI-258, may be promising alternatives for the clinical treatment of oral squamous cell carcinoma.

Alzheimer's disease (AD) poses a significant threat to human life and health. The intraneuronal accumulation of β-amyloid (Aβ) plaques in the brains of AD patients results in neuronal cell death, which is a key factor that triggers multiple changes in the pathogenesis of AD. The inhibition of Aβ-induced neuronal cell death may potentially help in the intervention and treatment of AD. Our previous study reported that tumor necrosis factor α-induced protein 1 (TNFAIP1) is induced by and promotes Aβ25-35-induced neurotoxicity in mouse neuronal cells, but the roles and regulatory mechanisms of TNFAIP1 are still largely unknown. In this study, our experimental results show that TNFAIP1 and p-TNFAIP1 (phosphorylation of TNFAIP1 at Ser280) are overexpressed in the neurons of the cortex and hippocampus in the brains of APP/PS1 mice, and the transcription factor NF-κB is involved in the Aβ-induced upregulation of TNFAIP1. Moreover, our results suggest that TNFAIP1 contributes to the Aβ-induced reactive oxygen species (ROS) production, decreased mitochondrial membrane potential (∆Ψm), and neuronal cell death in human SH-SY5Y cells. We further revealed that Aβ increases the binding of TNFAIP1 to RhoB, and knockdown of RhoB attenuates the TNFAIP1-induced apoptosis of human SH-SY5Y cells. These data suggest that TNFAIP1 is closely associated with AD pathogenesis, and overexpression of TNFAIP1 in the neurons of the brains of AD patients plays a role in apoptosis, at least in part, via RhoB signaling.

Since the discovery by Madaule and Axel in 1985 of the first Ras homologue (Rho) protein in Aplysia and its human orthologue RhoB, membership in the Rho GTPase family has grown to 20 proteins, with representatives in all eukaryotic species. These GTPases are molecular switches that cycle between active (GTP bound) and inactivate (GDP bound) states. The exchange of GDP for GTP on Rho GTPases is facilitated by guanine exchange factors (GEFs). Approximately 80 Rho GEFs have been identified to date, and only a few GEFs associate with microtubules. The guanine nucleotide exchange factor H1, GEF-H1, is a unique GEF that associates with microtubules and is regulated by the polymerization state of microtubule networks. This review summarizes the regulation and functions of GEF-H1 and discusses the roles of GEF-H1 in human diseases.

BACKGROUND: Hepatocellular carcinoma (HCC) is the leading cause of mortality worldwide. In recent years, the incidence of HCC induced by NAFLD is growing rapidly. OBJECTIVE: To screen for new pathogenic genes and related pathways both in NAFLD and HCC, and to explore the pathogenesis of progression from NAFLD to HCC. METHODS: Gene expression microarrays (GSE74656, GSE62232) were used for identifying differentially expressed genes (DEGs). Functional enrichment and pathway enrichment analyses indicated that these DEGs were related to cell cycle and extracellular exosome, which were closely related to NAFLD and HCC development. We then used the Search Tool for the Retrieval of Interacting Genes (STRING) to establish the protein-protein interaction (PPI) network and visualized them in Cytoscape. And the overall survival (OS) analysis and gene expression validation in TCGA of hub genes was performed. RESULTS: Seven hub genes, including CDK1, HSP90AA1, MAD2L1, PRKCD, ITGB3BP, CEP192, and RHOB were identified. Finally, we verified the expression level of ITGB3BP and CEP192 by quantitative real-time PCR in vitro. CONCLUSIONS: The present study implied possible DEGs, especially the new gene CEP192, in the progression of NAFLD developing to HCC. Further rigorous experiments are required to verify the above results.

LncRNA LOXL1-AS1 has been reported to be upregulated in several types of cancer and plays oncogenic roles. We analysed TCGA dataset and observed the downregulation of LOXL1-AS1 in cervical squamous cell carcinoma (CSCC). We therefore analysed the roles of LOXL1-AS1 in CSCC. We observed that LOXL1-AS1 was downregulated in CSCC. LOXL1-AS1 was predicted to interact with miR-21, while overexpression experiments showed that LOXL1-AS1 and miR-21 had no significant effects on the expression of each other. However, LOXL1-AS1 overexpression led to the upregulation of RHOB, a direct target of miR-21. Cell invasion and migration analysis showed decreased invasion and migration rates of CSCC cells after LOXL1-AS1 and RHOB overexpression. MiR-21 played an opposite role at reduced the effects of LOXL1-AS1 and RHOB overexpression. Therefore, LOXL1-AS1 may promote CSCC cell invasion and migration by sponging miR-21 to upregulate RHOB.

Introduction Emerging evidence has revealed the importance of long non-coding RNAs (lncRNAs) in carcinogenesis. The aim of this work was to investigate the roles of lncRNA growth arrest specific 5 (GAS5) in osteosarcoma (OS) progression. Methods Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to explore GAS5, microRNA-663a (miR-663a), and ras homolog family member B (RHOB) expression levels in OS tissues and cells. Moreover, cell counting kit-8 assay, wound-healing assay, and transwell invasion assay were conducted to investigate biological roles of GAS5 in OS progression. In addition, mechanisms underlying the functions of GAS5 in OS were investigated by bioinformatic analysis, luciferase activity reporter assay, and rescue experiments. Results The GAS5 expression level was significantly decreased in OS tissues and cells compared with normal tissues and cells, and could negatively regulate miR-663a expression. Moreover, we found RHOB expression can be negatively regulated by miR-663a. Overexpression of GAS5 and RHOB suppresses, while overexpression of miR-663a stimulates, OS cell proliferation, migration, and invasion in vitro. In summary, we revealed lncRNA GAS5 was a downregulated lncRNA in OS and impaired OS malignant behaviors. In addition, this regulation relied on miR-663a and its target gene, RHOB. Discussion To sum up, we showed lncRNA GAS5 regulates OS progression via regulating the miR-663a/RHOB axis.

N6-Methyladenosine (m6A) in messenger RNA (mRNA) regulates its stability, splicing, and translation efficiency. Here, we explored how the expression levels of small GTPase proteins are regulated by m6A modulators. We employed a high-throughput scheduled multiple-reaction monitoring (MRM)-based targeted proteomic approach to quantify systemically the changes in expression of small GTPase proteins in cells upon genetic ablation of METTL3 (the catalytic subunit of the major m6A methyltransferase complex), m6A demethylases (ALKBH5 and FTO), or m6A reader proteins (YTHDF1, YTHDF2, and YTHDF3). Depletions of METTL3 and ALKBH5 resulted in substantially diminished and augmented expression, respectively, of a subset of small GTPase proteins, including RHOB and RHOC. Our results also revealed that the stability of RHOB mRNA is significantly increased in cells depleted of METTL3, suggesting an m6A-elicited destabilization of this mRNA. Those small GTPases that are targeted by METTL3 and/or ALKBH5 also displayed higher discrepancies between protein and mRNA expression in paired primary/metastatic melanoma or colorectal cancer cells than those that are not. Together, this is the first comprehensive analysis of the alterations in small GTPase proteome regulated by epitranscriptomic modulators of m6A, and our study suggests the potential of an alternative therapeutic approach to target the currently "undruggable" small GTPases.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer with a high fatality rate in men and women worldwide. Recently, microRNAs (miRNAs) have been reported to be diagnostic biomarkers and therapeutic targets in NSCLC. MiR-223-3p was proved to act as a promoter in NSCLC progression. However, the regulatory mechanism of miR-223-3p in NSCLC remains little known. This study aimed to explore the regulatory mechanism between miR-223-3p and its target gene Ras homolog family member B (RHOB) in NSCLC. The mRNA level of miR-223-3p and RHOB was measured by quantitative reverse transcription PCR. Furthermore, cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometry was conducted to analyze cell apoptosis. Transwell assays and wound healing assay were employed to examine migration and invasion. The target relationship between miR-223-3p and RHOB was predicted by starBase online database and verified by dual-luciferase assay. The protein level of RHOB was tested by western blot. Our data suggested that miR-223-3p was upregulated in NSCLC tissues and cell lines and high level of miR-223-3p contributed to a poor survival in NSCLC patients. Knockdown of miR-223-3p exerted inhibitory effects on NSCLC cell viability, migration, and invasion and promotion effect on cell apoptosis. Furthermore, RHOB was directly targeted by miR-223-3p and constrained NSCLC progression. Moreover, knockdown of RHOB rescued the effect of anti-miR-223-3p on NSCLC progression. In vivo experiments indicated that miR-223-3p deletion suppressed tumor growth. MiR-223-3p could regulate the NSCLC cellular processes through targeting RHOB.

Breast cancer (BC), the most frequent cancer in women worldwide, is extremely heterogeneous. For effective and precise treatment and to cope with drug resistance in BC, we need to find more therapeutic molecular targets. In this study, we found that the Proteasome 26S Subunit, Non-ATPase 12 (PSMD12) was upregulated in BC samples, its expression was heterogeneous among different cell lines, and high levels of PSMD12 were related to poor prognosis of BC patients. Notably, the expression of PSMD12 increased in the nucleus. Cytological experiments revealed that PSMD12 knockdown inhibited cell growth and migration, and a genome-wide CRISPR-Cas9 knockout (GeCKO) screen also confirmed that PSMD12 is a crucial gene for the growth of BC cells. Flow cytometry showed that cell apoptosis increased in the PSMD12 knockdown, and RNA-seq indicated that the apoptosis pathway was activated, and the TXNIP, GADD45A, GADD45B, RHOB, and CDKN1A pro-apoptotic genes were highly expressed, a result that was validated by RT-qPCR and Western blot. Furthermore, restoration of PSMD12 expression decreased the expression of pro-apoptotic genes. A tumor-bearing mice assay demonstrated that BC growth was arrested by reduced PSMD12 levels in vivo. Taken together, PSMD12, a subunit of 19S regulator of 26S proteasome, was identified as a potential prognostic and therapeutic molecular target for BC, which provides a new insight for developing anticancer drugs that promote apoptosis based on the targeting of the 26S proteasome complex.

Zika virus (ZIKV) is a re-emerging flavivirus that has caused large-scale epidemics. Infection during pregnancy can lead to neurologic developmental abnormalities in children. There is no approved vaccine or therapy for ZIKV. To uncover cellular pathways required for ZIKV that can be therapeutically targeted, we transcriptionally upregulated all known human coding genes with an engineered CRISPR–Cas9 activation complex in human fibroblasts deficient in interferon (IFN) signaling. We identified Ras homolog family member V (RhoV) and WW domain-containing transcription regulator 1 (WWTR1) as proviral factors, and found them to play important roles during early ZIKV infection in A549 cells. We then focused on RhoV, a Rho GTPase with atypical terminal sequences and membrane association, and validated its proviral effects on ZIKV infection and virion production in SNB-19 cells. We found that RhoV promotes infection of some flaviviruses and acts at the step of viral entry. Furthermore, RhoV proviral effects depend on the complete GTPase cycle. By depleting Rho GTPases and related proteins, we identified RhoB and Pak1 as additional proviral factors. Taken together, these results highlight the positive role of RhoV in ZIKV infection and confirm CRISPR activation as a relevant method to identify novel host–pathogen interactions.

Zaoui et al. provide the first mechanistic evidence supporting a regulatory mechanism for RhoB localization and stability by Arf6. These findings establish that Arf6 is essential for RhoB-specific subcellular targeting to endosomes and, thus, the regulation of membrane trafficking and cell motility following activation of the oncogenic receptor tyrosine kinase, Met., The ADP-ribosylation factor 6 (Arf6) is a small GTPase that regulates endocytic recycling processes in concert with various effectors. Arf6 controls cytoskeletal organization and membrane trafficking; however, the detailed mechanisms of regulation remain poorly understood. Here, we report that Arf6 forms a complex with RhoB. The interaction between RhoB and Arf6 is mediated by the GCI (glycine, cysteine, and isoleucine) residues (188–190) of RhoB. Specific targeting of Arf6 to plasma membrane or mitochondrial membranes promotes recruitment and colocalization of RhoB to these membrane microdomains. Arf6 depletion promotes the loss of RhoB from endosomal membranes and leads to RhoB degradation through an endolysosomal pathway. This results in defective actin and focal adhesion dynamics and increased 3D cell migration upon activation of the Met receptor tyrosine kinase. Our findings identify a novel regulatory mechanism for RhoB localization and stability by Arf6 and establish the strict requirement of Arf6 for RhoB-specific subcellular targeting to endosomes and biological functions.

As an isoform of Rho family GTPases, RhoB plays a pivotal role in cytoskeletal organization, cell proliferation, apoptosis and immune response. However, the regulatory mechanisms of RhoB expression in aquatic animals are still unknown. In the present study, we first construct Vibrio anguillarum infection model in S. maximus, including susceptible and resistant individuals. Then the temporal expression of RhoB was detected after V. anguillarum challenge using qRT-PCR and found that RhoB transcripts were significantly induced in the liver, gill and blood despite of differential expression levels and responsive time points. In addition, the mRNA levels of RhoB in resistant individuals were significantly higher than in susceptible ones. The length of 2083 bp sequences of RhoB promoter was cloned and characterized. Moreover, DNA methylation of the RhoB promoter was measured by bisulfite sequencing (BSP) and hypo-methylated was detected in the CpG islands. Three SNPs (−1590, −1575 and −1449) and two haplotypes in the promoter region of RhoB were identified to be associated with V. anguillarum resistance in turbot by association analysis in group 17-R and 17-S. Deletion analysis indicated that these SNPs could negatively mediate the activity of RhoB promoter. Site-directed mutagenesis and qRT-PCR of individuals with different genotypes demonstrated that −1575 T/A polymorphism affected promoter activity. Further study showed that this mutation altered the binding site of the transcription factor CREB. Co-transfection of SmCREB and RhoB promoter was performed in HEK293T cells which confirmed the −1575 allelic differences on transcriptional activity, with the susceptibility allele showing reduced activity. Taken together, our findings implicate that losing of binding of CREB to SmRhoB promoter due to −1575T/A polymorphisms enhances SmRhoB expression in resistant turbot, which provide insights into the effect of SmRhoB expression in response to V. anguillarum infection.

Introduction Osteosarcoma is the most common bone tumor with high metastasis and recurrence rate. MicroRNA-19a (miR-19a) has been reported to act as tumor oncogene in multiple cancers. The objective of the study was to explore the molecular mechanisms of miR-19a in osteosarcoma cell migration and invasion. Materials and methods Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting were employed to measure the levels of miR-19a and RhoB in osteosarcoma tissues and cell lines. Transwell assay was employed to analyze the tissues and cell lines' migratory and invasive abilities. Dual luciferase reporter assay was utilized to analyze the association between miR-19a and RhoB. Results MiR-19a was overexpressed in osteosarcoma tissues and cell lines. MiR-19a promoted osteosarcoma cell migration and invasion in vitro. RhoB was thus confirmed as a direct and functional target of miR-19a, and it could partially reverse the function of miR-19a. Knockdown miR-19a inhibited osteosarcoma cell epithelial-mesenchymal transition (EMT) and suppressed osteosarcoma xenograft growth. Conclusion MiR-19a enhanced cell migration, invasion and EMT through RhoB in osteosarcoma. The newly identified miR-19a/RhoB axis provides novel insight into the progression of osteosarcoma and offers a promising target for osteosarcoma therapy.

The aim of the present study was to identify genes under the effect of transforming growth factor-β (TGF-β1), high glucose (HG) and glucosamine (GlcN) in MES-13 mesangial cells and elucidate the molecular mechanisms of diabetic nephropathy (DN). The gene expression datasets GSE2557 and GSE2558 were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were independently screened using the GEO2R online tool. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Database for Annotation, Visualization, and Integrated Discovery. The protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes and Cytoscape software. The hub genes were identified by the NetworkAnalyzer plugin. Overlapping genes were subjected to molecular docking analysis using SystemsDock. A total of 202 upregulated and 158 downregulated DEGs from the HG-treated groups, 138 upregulated and 103 downregulated DEGs from the GlcN-treated groups, and 81 upregulated and 44 downregulated DEGs from the TGF-β1-treated groups were identified. The majority of the DEGs were independently enriched in 'nucleosome assembly', 'chromatin silencing' and 'xenobiotic glucuronidation'. In addition, KEGG pathways were significantly enriched in 'systemic lupus erythematosus', 'protein processing in endoplasmic reticulum' and 'aldarate metabolism pathway', and 'TNF signaling pathway' intersected in the TGF-β1-treated and HG-treated groups. In total, eight hub genes, Jun, prostaglandin-endoperoxide synthase 2 (Ptgs2), fibronectin 1 (Fn1), cyclin-dependent kinase (Cdk)2, Fos, heat shock protein family A (Hsp70) member 5 (Hspa5), Hsp90b1 and homo sapiens hypoxia upregulated 1 (Hyou1), and three overlapping genes, Ras homolog gene family, member B (RHOB), complement factor H (CFH) and Krüppel-like factor 15 (KLF15), were selected. Valsartan with RHOB, and fosinopril with CFH and KLF15 had preferential binding activity. In conclusion, Jun, Ptgs2, Fn1, Cdk2, Fos, Hspa5, Hsp90b1, Hyou1, RHOB, CFH and KLF15 may be potential therapeutic targets for mesangial cells associated with DN, which may provide insight into DN treatment strategies.

BackgroundIschemia–reperfusion injury (IRI) remains an inevitable and major challenge in renal transplantation. The current study aims to obtain deep insights into underlying mechanisms and seek prognostic genes as potential therapeutic targets for renal IRI (RIRI).MethodsAfter systematically screening the Gene Expression Omnibus (GEO) database, we collected gene expression profiles of over 1,000 specimens from 11 independent cohorts. Differentially expressed genes (DEGs) were identified by comparing allograft kidney biopsies taken before and after reperfusion in the discovery cohort and further validated in another two independent transplant cohorts. Then, graft survival analysis and immune cell analysis of DEGs were performed in another independent renal transplant cohort with long-term follow-ups to further screen out prognostic genes. Cell type and time course analyses were performed for investigating the expression pattern of prognostic genes in more dimensions utilizing a mouse RIRI model. Finally, two novel genes firstly identified in RIRI were verified in the mouse model and comprehensively analyzed to investigate potential mechanisms.ResultsTwenty DEGs upregulated in the process of RIRI throughout different donor types (living donors, cardiac and brain death donors) were successfully identified and validated. Among them, upregulation of 10 genes was associated with poor long-term allograft outcomes and exhibited strong correlations with prognostic immune cells, like macrophages. Furthermore, certain genes were found to be only differentially expressed in specific cell types and remained with high expression levels even months after RIRI in the mouse model, which processed the potential to serve as therapeutic targets. Importantly, two newly identified genes in RIRI, Btg2 and Rhob, were successfully confirmed in the mouse model and found to have strong connections with NF-κB signaling.ConclusionsWe successfully identified and validated 10 IRI-associated prognostic genes in renal transplantation across different donor types, and two novel genes with crucial roles in RIRI were recognized for the first time. Our findings offered promising potential therapeutic targets for RIRI in renal transplantation.

Simple Summary Glioblastoma multiforme (GBM) and metastatic triple-negative breast cancer (TNBC) with PTEN mutations are associated with brain tumor spreading and poor patient outcomes. GBM, and possibly TNBC, migrate on axons and blood vessels to disseminate in the brain; however, the mechanism is unresolved. There is a need for new therapeutic targets to blunt brain tumor spreading. Using 3D aligned printed microfibers mimicking brain structures proved that RHOB, in addition to ROCK and PTEN signaling, were essential for GBM and TNBC 3D cell migration. GBM and TNBC cell lines with PTEN loss of function and high RHOB expression exhibited amoeboid morphology with increased durotaxis, binding and migration speed on 3D microfibers, in contrast to the PTEN wildtype. Depending on the PTEN genotype, RHO-ROCK-PTEN inhibitors or PTEN rescue significantly regulated these properties. Regarding GBM and brain metastasizing TNBC, we conclude that RHOB inhibitors could play a novel role for improved therapy response and patient outcome. Abstract Background: Glioblastoma multiforme (GBM) and metastatic triple-negative breast cancer (TNBC) with PTEN mutations often lead to brain dissemination with poor patient outcome, thus new therapeutic targets are needed. To understand signaling, controlling the dynamics and mechanics of brain tumor cell migration, we implemented GBM and TNBC cell lines and designed 3D aligned microfibers and scaffolds mimicking brain structures. Methods: 3D microfibers and scaffolds were printed using melt electrowriting. GBM and TNBC cell lines with opposing PTEN genotypes were analyzed with RHO-ROCK-PTEN inhibitors and PTEN rescue using live-cell imaging. RNA-sequencing and qPCR of tumor cells in 3D with microfibers were performed, while scanning electron microscopy and confocal microscopy addressed cell morphology. Results: In contrast to the PTEN wildtype, GBM and TNBC cells with PTEN loss of function yielded enhanced durotaxis, topotaxis, adhesion, amoeboid migration on 3D microfibers and significant high RHOB expression. Functional studies concerning RHOB-ROCK-PTEN signaling confirmed the essential role for the above cellular processes. Conclusions: This study demonstrates a significant role of the PTEN genotype and RHOB expression for durotaxis, adhesion and migration dependent on 3D. GBM and TNBC cells with PTEN loss of function have an affinity for stiff brain structures promoting metastasis. 3D microfibers represent an important tool to model brain metastasizing tumor cells, where RHO-inhibitors could play an essential role for improved therapy.

Simple Summary The objective of the present study was to investigate the impact of sex in the development of long-term toxicities affecting quality of life in cancer survivors after systemic paclitaxel treatment and cardiac irradiation. Sex-specific differences may affect tumor biology, drug pharmacokinetics and dynamics, and response to local treatment such as radiation therapy (RT). However, sex is rarely taken into consideration when administering cancer therapies. Interestingly, female mice are protected from paclitaxel-induced neurotoxicity as well as from radiotherapy-induced cardiotoxicity, and deficiency in the small GTPase RhoB reversed the protection in females but not in males. In conclusion, our results are the first to identify sex- and organ-specific responses to systemic paclitaxel administration and localized RT. These results may have important implications for the management of cancer patients and implementation of personalized medicine in oncology. Abstract The impact of sex in the development of long-term toxicities affecting the quality of life of cancer survivors has not been investigated experimentally. To address this issue, a series of neurologic and cardiologic endpoints were used to investigate sex-based differences triggered by paclitaxel treatment and radiotherapy exposure. Male and female wild-type (WT) mice were treated with paclitaxel (150 and 300 mg/kg) administered weekly over 6 weeks or exposed to 19 Gy cardiac irradiation. Cohorts were analyzed for behavioral and neurobiologic endpoints to assess systemic toxicity of paclitaxel or cardiovascular endpoints to assess radiotherapy toxicity. Interestingly, female WT mice exhibited enhanced tolerance compared to male WT mice regardless of the treatment regimen. To provide insight into the possible sex-specific protective mechanisms, rhoB-deficient animals and elderly mice (22 months) were used with a focus on the possible contribution of sex hormones, including estrogen. In females, RhoB deficiency and advanced age had no impact on neurocognitive impairment induced by paclitaxel but enhanced cardiac sensitivity to radiotherapy. Conversely, rhoB-deficiency protected males from radiation toxicity. In sum, RhoB was identified as a molecular determinant driving estrogen-dependent cardioprotection in female mice, whereas neuroprotection was not sex hormone dependent. To our knowledge, this study revealed for the first time sex- and organ-specific responses to paclitaxel and radiotherapy.

Background Acute myeloid leukemia (AML) remains one of the most common hematological malignancies, posing a serious challenge to human health. HSPA8 is a chaperone protein that facilitates proper protein folding. It contributes to various activities of cell function and also is associated with various types of cancers. To date, the role of HSPA8 in AML is still undetermined. Methods In this study, public datasets available from the TCGA (Cancer Genome Atlas) and GEO (Gene Expression Omnibus) were mined to discover the association between the expression of HSPA8 and clinical phenotypes of CN-AML. A series of bioinformatics analysis methods, including functional annotation and miRNA-mRNA regulation network analysis, were employed to investigate the role of HSPA8 in CN-AML. Results HSPA8 was highly expressed in the AML patients compared to the healthy controls. The high HSPA8 expression had lower overall survival (OS) rate than those with low HSPA8 expression. High expression of HSPA8 was also an independent prognostic factor for overall survival (OS) of CN-AML patients by multivariate analysis. The differential expressed genes (DEGs) associated with HSPA8 high expression were identified, and they were enriched PI3k-Akt signaling, cAMP signaling, calcium signaling pathway. HSPA8 high expression was also positively associated with micro-RNAs (hsa-mir-1269a, hsa-mir-508-3p, hsa-mir-203a), the micro-RNAs targeted genes (VSTM4, RHOB, HOBX7) and key known oncogenes (KLF5, RAN, and IDH1), and negatively associated with tumor suppressors (KLF12, PRKG1, TRPS1, NOTCH1, RORA). Conclusions Our research revealed HSPA8 as a novel potential prognostic factor to predict the survival of CN-AML patients. Our data also revealed the possible carcinogenic mechanism and the complicated microRNA-mRNA network associated with the HSPA8 high expression in AML.

Capillary endothelial cells (ECs) maintain a semi-permeable barrier between the blood and tissue by forming inter-EC tight junctions (TJs), regulating selective transport of fluid and solutes. Overwhelming inflammation, as occurs in sepsis, disrupts these TJs, leading to leakage of fluid, proteins, and small molecules into the tissues. Mechanistically, disruption of capillary barrier function is mediated by small Rho-GTPases, such as RhoA, -B, and -C, which are activated by guanine nucleotide exchange factors (GEFs) and disrupted by GTPase-activating factors (GAPs). We previously reported that a mutation in a specific RhoB GAP (p190BRhoGAP) underlays a hereditary capillary leak syndrome. Tumor necrosis factor (TNF) treatment disrupts TJs in cultured human microvascular ECs, a model of capillary leak. This response requires new gene transcription and involves increased RhoB activation. However, the specific GEF that activates RhoB in capillary ECs remains unknown. Transcriptional profiling of cultured tight junction-forming human dermal microvascular endothelial cells (HDMECs) revealed that 17 GEFs were significantly induced by TNF. The function of each candidate GEF was assessed by short interfering RNA depletion and trans-endothelial electrical resistance screening. Knockown of ArhGEF10 reduced the TNF-induced loss of barrier which was phenocopied by RhoB or dual ArhGEF10/RhoB knockdown. ArhGEF10 knockdown also reduced the extent of TNF-induced RhoB activation and disruption at tight junctions. In a cell-free assay, immunoisolated ArhGEF10 selectively catalyzed nucleotide exchange to activate RhoB, but not RhoA or RhoC. We conclude ArhGEF10 is a TNF-induced RhoB-selective GEF that mediates TJ disruption and barrier loss in human capillary endothelial cells.

Canonical Wnt signaling plays an essential role in proper craniofacial morphogenesis, at least partially due to regulation of various aspects of cranial neural crest development. In an effort to gain insight into the etiology of craniofacial abnormalities resulting from Wnt signaling and/or cranial neural crest dysfunction, we sought to identify Wnt-responsive targets during chick cranial neural crest development. To this end, we leveraged overexpression of a canonical Wnt antagonist, Draxin, in conjunction with RNA-sequencing of cranial neural crest cells that have just activated their epithelial–mesenchymal transition (EMT) program. Through differential expression analysis, gene list functional annotation, hybridization chain reaction (HCR), and quantitative reverse transcription polymerase chain reaction (RT-qPCR), we validated a novel downstream target of canonical Wnt signaling in cranial neural crest –RHOB– and identified possible signaling pathway crosstalk underlying cranial neural crest migration. The results reveal novel putative targets of canonical Wnt signaling during cranial neural crest EMT and highlight important intersections across signaling pathways involved in craniofacial development.

Rho family GTPase RhoB is the critical signaling component controlling the inflammatory response elicited by pro-inflammatory cytokines. However, the underlying mechanisms of RhoB degradation in inflammatory response remain unclear. In this study, for the first time, we identified that TNFAIP1, an adaptor protein of Cullin3 E3 ubiquitin ligases, coordinated with Cullin3 to mediate RhoB degradation through ubiquitin proteasome system. In addition, we demonstrated that downregulation of TNFAIP1 induced the expression of pro-inflammatory cytokines IL-6 and IL-8 in TNFα-stimulated hepatocellular carcinoma cells through the activation of p38/JNK MAPK pathway via blocking RhoB degradation. Our findings revealed a novel mechanism of RhoB degradation and provided a potential strategy for anti-inflammatory intervention of tumors by targeting TNFAIP1-RhoB axis.

The high mortality rate of cancer is strongly correlated with the development of distant metastases at secondary sites. Although Rho GTPases, such as RhoA, RhoB, RhoC, and RhoE, promote tumor metastasis, the main roles of Rho GTPases remain unidentified. It is also unclear whether rhosin, a Rho inhibitor, acts by suppressing metastasis by a downstream inhibition of Rho. In this study, we investigated this mechanism of metastasis in highly metastatic melanoma and breast cancer cells, and the mechanism of inhibition of metastasis by rhosin. We found that rhosin suppressed the RhoA and RhoC activation, the nuclear localization of YAP, but did not affect ERK1/2, Akt, or NF-&kappa, B activation in the highly metastatic cell lines B16BL6 and 4T1. High expression of YAP was associated with poor overall and recurrence-free survival in patients with breast cancer or melanoma. Treatment with rhosin inhibited lung metastasis in vivo. Moreover, rhosin inhibited tumor cell adhesion to the extracellular matrix via suppression of RHAMM expression, and inhibited SDF-1-induced cell migration and invasion by decreasing CXCR4 expression in B16BL6 and 4T1 cells. These results suggest that the inhibition of RhoA/C-YAP pathway by rhosin could be an extremely useful therapeutic approach in patients with melanoma and breast cancer.

Metastasis is the most prevalent cause of cancer-associated deaths amongst patients with cervical cancer. Epithelial–mesenchymal transition (EMT) is essential for carcinogenesis, and it confers metastatic properties to cancer cells. Gossypol is a natural polyphenolic compound with anti-inflammation, anti-oxidant, and anticancer activities. In this study, we investigated the antimetastatic and antitumour effects of gossypol on human cervical cancer cells (HeLa and SiHa cells). Gossypol exerted a strong inhibition effect on the migration and invasion of human cervical cancer cells. It reduced the focal adhesion kinase (FAK) pathway-mediated expression of matrix metalloproteinase-2 and urokinase-type plasminogen activator, subsequently inhibiting the invasion of SiHa cells. In addition, gossypol reversed EMT induced by transforming growth factor beta 1 (TGF-[Formula: see text]1) and up-regulated epithelial markers, such as E-cadherin but significantly suppressed Ras homolog family member (Rho)A, RhoB, and p-Samd3. The tail vein injection model showed that gossypol treatment via oral gavage reduced lung metastasis. Gossypol also decreased tumour growth in vivo in the nude mouse xenograft model. All these findings suggest that gossypol suppressed the invasion and migration of human cervical cancer cells by targeting the FAK signaling pathway and reversing TGF-[Formula: see text]1-induced EMT. Hence, gossypol warrants further attention for basic mechanistic studies and drug development.

Estrogen is a commonly used hormone in the adjuvant treatment of intrauterine adhesion (IUA), which can promote endometrial growth. Stem cell transplantation has also been reported to promote endometrial regeneration in IUA due to its potential differentiative capacity. Human Wharton's jelly mesenchymal stem cells (WJ‑MSCs) are isolated from the umbilical cord, possess strong self‑renewal and proliferative abilities, and are hypo‑immunogenic and non‑tumorigenic. Therefore, the present study aimed to investigate the therapeutic effects and underlying mechanism of WJ‑MSCs transplantation with estrogen treatment, separately or as a combined therapy, on IUA. The IUA model was established using the ethanol damage method. A total of 50 Sprague‑Dawley female rats were randomly divided into the control, IUA model, WJ‑MSCs treatment, estrogen treatment and WJ‑MSCs+ estrogen treatment groups (n=10/group). WJ‑MSCs were injected three times at 5‑day intervals. IUA rats in the estrogen group received 0.2 mg/kg estrogen through intragastric administration, once every 2 days for 8 weeks. Morphological changes were evaluated by hematoxylin‑eosin staining. Immunohistochemical evaluations of pan‑keratin, vimentin, transforming growth factor (TGF)‑β1, RhoA, RhoB, RhoC, Rho‑associated coiled‑coil‑containing protein kinase (ROCK)I, and ROCKII expression were performed in uterine tissue. After treatment, the uterine specimens were observed to have increased uterine thickness and gland numbers in all treatment groups compared with the IUA group; however, the degree of restoration in the independent WJ‑MSCs and estrogen treatment groups was better than in the combined treatment group. Immunohistochemical analysis demonstrated that pan‑keratin expression was increased, and RhoA, ROCKI and TGF‑β1 expression was significantly inhibited in the WJ‑MSCs and WJ‑MSCs + estrogen treatment groups compared with the IUA group; however, the expression levels of these proteins were similar among all treatment groups. No change in vimentin expression was detected in any treatment group. The expression levels of RhoB, RhoC and ROCKII were clearly not affected by WJ‑MSCs intervention alone. In conclusion, transplantation of WJ‑MSCs may repair endometrial damage in IUA rats via TGF‑β1‑mediated inhibition of RhoA/ROCKI signaling.

In addition to commonly associated environmental factors, genomic factors may cause cerebral palsy. We performed whole-exome sequencing of 250 parent-offspring trios, and observed enrichment of damaging de novo mutations in cerebral palsy cases. Eight genes had multiple damaging de novo mutations; of these, two (TUBA1A and CTNNB1) met genome-wide significance. We identified two novel monogenic etiologies, FBXO31 and RHOB, and showed that the RHOB mutation enhances active-state Rho effector binding while the FBXO31 mutation diminishes cyclin D levels. Candidate cerebral palsy risk genes overlapped with neurodevelopmental disorder genes. Network analyses identified enrichment of Rho GTPase, extracellular matrix, focal adhesion and cytoskeleton pathways. Cerebral palsy risk genes in enriched pathways were shown to regulate neuromotor function in a Drosophila reverse genetics screen. We estimate that 14% of cases could be attributed to an excess of damaging de novo or recessive variants. These findings provide evidence for genetically mediated dysregulation of early neuronal connectivity in cerebral palsy.

Background. Breast cancer (BC) is one of the most common malignant tumors in women around the world. Atorvastatin (ATO) was found to be associated with a decreased risk of recurrence and mortality in cancer. But the exact mechanism of its carcinostatic effects is unclear. The expression level of Ras homolog family member B (RhoB) in breast cancer cells was found to be upregulated after being treated with ATO. Thus, we conjecture that altered expression of RhoB induced by ATO may be decisive for the migration and progression of breast cancer. Methods. The effects of ATO on breast tumor cells in vivo and in vitro were detected by clone formation assay, CCK-8 assay, flow cytometry, wound healing, transwell assays, tumor xenograft model, and immunohistochemistry. Distribution of RhoB in different breast cancer tissues and its influence on prognosis were analyzed using the data from TCGA or GEO databases. The relationship between RhoB and PTEN/AKT pathway was detected by Western blotting and RT-qPCR. Results. ATO inhibits proliferation, invasion, EMT, and PTEN/AKT pathway and promotes apoptosis in breast tumor cells. In addition, ATO inhibits the volume and weight of breast tumor in tumor-bearing mice and upregulated RhoB in tumor tissues. The expression of RhoB in mRNA and protein level was upregulated in statin-treated breast cancer cells and downregulated in cancer tissues. Low expression of RhoB links with poor prognosis in patients with breast cancer (HR = 0.74[0.66–0.83], p =7e−8, log-rank test). Further research found that RhoB inhibits the proliferation, invasion, EMT, and PTEN/AKT signal pathway in breast tumor cells. Conclusions. The exact mechanism of ATO’s carcinostatic effects in breast cancer is related to downregulating PTEN/AKT pathway via promoting RhoB. Our study also demonstrates the potential applicability of RhoB as a therapeutic target for breast cancer.

Overexpression of HER2 in breast cancer is correlated with poor prognosis. HER2-targeted drugs, such as trastuzumab and lapatinib, have been successful to treat HER2-positive breast cancers, however, the acquisition of the drug resistance of the cells is recognized. Here we suggest the novel molecular targets to cure HER2-positive breast cancers. The oncogenic roles of Rac1, a Rho family small GTPase, in a variety of cancers have been demonstrated. For example, the elevated expression or hyperactivation of Rac1 is frequently observed in human cancers, correlating with their aggressiveness and poor prognosis. In breast cancers, upregulation of Rac1 GEF (GTP exchange factor) and downregulation of Rac1 GAP (GTPase activating protein) have been reported. Moreover, activation of Rac1 contributed to trastuzumab resistance, which poses a serious problem during chemotherapy. In the present study, we first investigated in detail in which subtypes of breast cancers mRNA expression of Rac1 is correlated with their poor prognosis. Using the METABRIC database, we found that high mRNA expression of Rac1 significantly correlated with the poor prognosis of HER2-positive breast cancer (p=0.0012, High: n=49, Low: n=171). On the other hands, other three types (basal, claudin-low, or luminal-B type) did not show significant correlation between the expression levels of Rac1 mRNA and their prognosis (p=0.15, High: n=97, Low: n=102; p=0.052, High: n=110, Low: n=89; p=0.17, High: n=70, Low: n=391; respectively). In luminal-A type breast cancer, low mRNA expression of Rac1 significantly correlated with poor prognosis (p=0.0046, High: n=492, Low: n=187). We next investigated the molecular mechanism underlying Rac1 activation in HER2-positive breast cancer cells, SKBR-3 cells. We found that Cullin-3 (CUL3, a subunit of a RING ubiquitin E3 ligase complex) and its adaptor protein KCTD10 are essential for Rac1 activation. Mechanistically, CUL3/KCTD10 ubiquitinate RhoB, a Rho family small GTPase that suppresses the activation of Rac1, leading to the degradation RhoB. We also found that HER2 signaling is essential for the activation of Rac1. Conclusions: This study reveals that the novel molecular axis CUL3/KCTD10/RhoB regulates the Rac1 activation in HER2-positive breast cancer cells. The interference of CUL3/KCTD10 complex formation may be a new strategy to the treatment of HER2- and Rac1-positive breast cancers. Citation Format: Murakami A, Maekawa M, Kusakabe E, Yamasawa H, Aoki R, Komatsu S, Taguchi K, Nishiyama K, Yamashita M, Sugimori W, Kawai K, Nakayama J, Araki N, Semba K, Taguchi T, Kamei Y, Takada Y, Higashiyama S. Novel mechanisms of Rac1 activation by the Cullin-3/KCTD10 ubiquitin E3 complex in HER2-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-05-02.

The Ras homologous protein (Rho) GTPase subfamily, including RhoA, RhoB, and RhoC are small molecules (~21 kDa) that act as molecular switches in a wide range of signaling pathways to orchestrate biological processes associated with both physiological and tumorigenic cellular states. The Rho GTPases are crucial regulators of actin cytoskeleton rearrangements and FA dynamics and are required for effective cell migration and invasion, as well as cell cycle progression and apoptosis. The Rho GTPases activity is regulated by conformational switching between GTP-bound (active) and GDP-bound (inactive) states. This GTP/GDP cycling is tightly controlled by the guanine nucleotide exchange factors (GEFs), which function as activators by catalyzing the exchange of GDP for GTP and by the GTPase-activating proteins (GAPs), which enable hydrolysis of GTP leading to the Rho GTPase inactivation. Here, we describe a detailed protocol to perform a RhoB G-LISA activation assay to detect the level of GTP-loaded RhoB in vitro. This is the first colorimetric assay designed to specifically measure RhoB activation. This method was developed by adapting the RhoA G-LISA Activation Assay Kit (Cytoskeleton, Inc.) and allow the precise measurement of RhoB activity in less than 3 hours. This rapid methodology can be broadly used to assess the level of GTP-loaded RhoB in any kind of cellular models, to appreciate either the role RhoB activation in physiological processes, diseases, oncogenic transformation or for drug discovery in high throughput screens.

There is a known reciprocation between the chronic exertion of force on tissue and both increased tissue density (e.g., bone) and hypertrophy (e.g., heart). This can also be seen in cervical tissue where the excessive gravitational forces associated with multiple fetal pregnancies promote preterm births. While there is a well-known regulation of cervical remodeling (CR) by sex steroid hormones and growth factors, the role of mechanical force is less appreciated. Using proteome-wide technology, we previously provided evidence for the presence of and alteration in mechano-related signaling molecules in the mouse cervix during pregnancy. Here, we profile the expression of select cytoskeletal factors (filamin-A, gelsolin, vimentin, actinin-1, caveolin-1, transgelin, keratin-8, profilin-1) and their associated signaling molecules [focal adhesion kinase (FAK) and the Rho GTPases CDC42, RHOA, and RHOB] in cervices of pregnant mice by real-time PCR and confocal immunofluorescence microscopy. Messenger RNA and protein levels increased for each of these 12 factors, except for 3 (keratin-8, profilin-1, RHOA) that decreased during the course of pregnancy and this corresponded with an increase in gravitational force exerted by the fetus on the cervix. We therefore conclude that size or weight of the growing fetus likely plays a key role in CR through mechanotransduction processes.

To interfere with cell function, many scientists rely on methods that target DNA or RNA due to the ease with which they can be applied. Proteins are usually the final executors of function but are targeted only indirectly by these methods. Recent advances in targeted degradation of proteins based on proteolysis-targeting chimaeras (PROTACs), ubiquibodies, deGradFP (degrade Green Fluorescent Protein) and other approaches have demonstrated the potential of interfering directly at the protein level for research and therapy. Proteins can be targeted directly and very specifically by antibodies, but using antibodies inside cells has so far been considered to be challenging. However, it is possible to deliver antibodies or other proteins into the cytosol using standard laboratory equipment. Physical methods such as electroporation have been demonstrated to be efficient and validated thoroughly over time. The expression of intracellular antibodies (intrabodies) inside cells is another way to interfere with intracellular targets at the protein level. Methodological strategies to target the inside of cells with antibodies, including delivered antibodies and expressed antibodies, as well as applications in the research areas of neurobiology, viral infections and oncology, are reviewed here. Antibodies have already been used to interfere with a wide range of intracellular targets. Disease-related targets included proteins associated with neurodegenerative diseases such as Parkinson’s disease (α-synuclein), Alzheimer’s disease (amyloid-β) or Huntington’s disease (mutant huntingtin [mHtt]). The applications of intrabodies in the context of viral infections include targeting proteins associated with HIV (e.g. HIV1-TAT, Rev, Vif, gp41, gp120, gp160) and different oncoviruses such as human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV) and Epstein-Barr virus, and they have been used to interfere with various targets related to different processes in cancer, including oncogenic pathways, proliferation, cell cycle, apoptosis, metastasis, angiogenesis or neo-antigens (e.g. p53, human epidermal growth factor receptor-2 [HER2], signal transducer and activator of transcription 3 [STAT3], RAS-related RHO-GTPase B (RHOB), cortactin, vascular endothelial growth factor receptor 2 [VEGFR2], Ras, Bcr-Abl). Interfering at the protein level allows questions to be addressed that may remain unanswered using alternative methods. This review addresses why direct targeting of proteins allows unique insights, what is currently feasible in vitro, and how this relates to potential therapeutic applications.

One of the main research areas in biology from the mid‐1980s through the 1990s was the elucidation of signaling pathways governing cell responses. These studies brought, among other molecules, the small GTPase Rho to the epicenter. Rho signaling research has since expanded to all areas of biology and medicine. Here, we describe how Rho emerged as a key molecule governing cell morphogenesis and movement, how it was linked to actin reorganization, and how the study of Rho signaling has expanded from cultured cells to whole biological systems. We then give an overview of the current research status of Rho signaling in development, brain, cardiovascular system, immunity and cancer, and discuss the future directions of Rho signaling research, with emphasis on one Rho effector, ROCK*. *The Rho GTPase family. Rho family GTPases have now expanded to contain 20 members. Amino acid sequences of 20 Rho GTPases found in human were aligned and the phylogenetic tree was generated by ClustalW2 software (EMBL‐EBI) based on NJ algorithm. The subfamilies of the Rho GTPases are highlighted by the circle and labeled on the right side. Rho cited in this review refers to the original members of Rho subfamily, RhoA, RhoB and RhoC, that are C3 substrates, and, unless specified, not to other members of the Rho subfamily such as Rac, Cdc42, and Rnd.

The RhoA GTPase controls endothelial cell migration, adhesion, and barrier formation but the role of RhoB is unclear. Kovačević et al. now discover that RhoB is ubiquitinated by the CUL3–Rbx1–KCTD10 complex and that this is a prerequisite for lysosomal degradation of RhoB and the maintenance of endothelial barrier integrity., RhoGTPases control endothelial cell (EC) migration, adhesion, and barrier formation. Whereas the relevance of RhoA for endothelial barrier function is widely accepted, the role of the RhoA homologue RhoB is poorly defined. RhoB and RhoA are 85% identical, but RhoB’s subcellular localization and half-life are uniquely different. Here, we studied the role of ubiquitination for the function and stability of RhoB in primary human ECs. We show that the K63 polyubiquitination at lysine 162 and 181 of RhoB targets the protein to lysosomes. Moreover, we identified the RING E3 ligase complex Cullin-3–Rbx1–KCTD10 as key modulator of endothelial barrier integrity via its regulation of the ubiquitination, localization, and activity of RhoB. In conclusion, our data show that ubiquitination controls the subcellular localization and lysosomal degradation of RhoB and thereby regulates the stability of the endothelial barrier through control of RhoB-mediated EC contraction.

The gene transactivation function of nuclear EGFR (nEGFR) has been studied by investigating the genomic co-occupancies of nEGFR and RNA Polymerase II (RNAPII). However, due to RNAPII pausing, the co-recruitment of RNAPII and nEGFR does not necessarily represent productive transactivation. In this study, we integrated gatekeepers of productive transcriptional elongation such as Integrator and Super Elongation Complex (SEC) to interrogate the function of nEGFR. By analyzing publicly available ChIP-seq and RNA-seq data, we aims to 1) explore the function of nEGFR, 2) unravel nEGFR target genes, and 3) discuss potential mechanisms of nEGFR chromatin recruitment. EGF treatment in HeLa cells instigated chromatin recruitment of nEGFR, ERK, RNAPII, Integrator, and SEC in a cluster of 61 EGF-responsive genes. The function of nEGFR was identified as gene-activating rather than gene-repressing. Within the cluster of EGF-responsive genes, nEGFR targeted eleven Immediate Early Genes (IEGs) — JUN, EGR1, JUNB, IER2, KLF2, FOS, FOSL1, RHOB, CCNL1, DUSP2, and DUSP5, which up-regulated >2-fold after EGF stimulation. The promoter of these target genes commonly harbors AT-rich minimal consensus sequences for nEGFR binding. In addition, TCGA data analysis demonstrated positive correlations between EGFR and JUN/FOSL1/RHOB expressions, as well as clinical correlations in specific cancer types. To our knowledge, this is the first study to compare the genome-wide distribution of nEGFR versus Integrator and SEC, providing novel insight into supporting the gene-activating function of nEGFR. We revealed a panel of eleven nEGFR target genes, which concurrently recruited nEGFR, RNAPII, Integrator, and SEC for productive transcriptional elongation.

Macrophages are innate immune cells that constantly patrol an organism to fulfill protective and homeostatic roles. Previous studies have shown that Rho GTPase activity is required for macrophage mobility, yet the roles of upstream regulatory proteins controlling Rho GTPase function in these cells are not well defined. Previously we have shown that the RhoA GEF Net1 is required for human breast cancer cell motility and extracellular matrix invasion. To assess the role of Net1 in macrophage motility, we isolated bone marrow macrophage (BMM) precursors from wild type and Net1 knockout mice. Loss of Net1 did not affect the ability of BMM precursors to differentiate into mature macrophages in vitro, as measured by CD68 and F4/80 staining. However, Net1 deletion significantly reduced RhoA activation, F-actin accumulation, adhesion, and motility in these cells. Nevertheless, similar to RhoA/RhoB double knockout macrophages, Net1 deletion did not impair macrophage recruitment to the peritoneum in a mouse model of sterile inflammation. These data demonstrate that Net1 is an important regulator of RhoA signaling and motility in mouse macrophages in vitro, but that its function may be dispensable for macrophage recruitment to inflammatory sites in vivo.

It was reported that statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase that are used to prevent hypercholesterolemia, have antitumor activity in several cancers. In this study, we investigated the cell viability of statins in Cisplatin-resistant HCP4 and PCDP5 cells compared with their parent Hela and PC3 cells, respectively, and found that HCP4 and PCDP5 cells were 37-fold and 18-fold more resistant to Cisplatin but 13-fold and 7-fold more sensitive to Lovastatin by cell proliferation assay. Lovastatin induced the apoptosis of HCP4 cells more rapidly and to greater extent than in Hela cells as assessed by flow cytometry and western blotting analyses. The MVA pathway was not involved in this acquired Cisplatin resistance. To elucidate the mechanism underlying the reduced viability to Lovastatin, we performed cDNA microarray analysis and identified 65 and 54 genes that were induced more than 2-fold by Lovastatin in HCP4 and PCDP5 cells, respectively. Of these, only three genes, KLF2, KLF6, and RHOB, were commonly induced between HCP4 and PCDP5 cells. These mRNAs were strongly induced by Lovastatin with transcriptional regulation in HCP4 cells. Consistent with transcription, the protein expression of RHOB also was induced by Lovastatin. The induction of these genes was associated with cell cycle arrest and apoptosis. Combination treatment with Cisplatin and Lovastatin resulted in an agonistic effect in Hela and PC3 cells and an antagonistic effect in HCP4 and PCDP5 cells. These results suggest that statins might have the potential to overcome Cisplatin resistance as single-agent therapy.

Active remodeling of the actin cytoskeleton in endothelial cells is necessary for allowing leukocytes to cross the barrier during the process of transendothelial migration (TEM). Involvement of RhoGTPases to regulate actin organization is inevitable, and we recently reported on the local function of RhoA in limiting vascular leakage during leukocyte TEM. As a follow-up we investigated here the possible involvement of two other closely-related GTPases; RhoB and RhoC, in regulating leukocyte TEM and vascular barrier maintenance. Physiological flow experiments showed no substantial involvement of either endothelial RhoB or RhoC in neutrophil adhesion and transmigration efficiency. Besides neutrophil TEM, we did not observe a role for endothelial RhoB or RhoC in limiting vascular leakage in both inflammatory conditions and during TEM. In conclusion, endothelial RhoB and RhoC are both dispensable for regulating leukocyte diapedesis and for maintaining vascular barrier function under inflammatory conditions and during leukocyte diapedesis.

Many microRNAs participate in the development, differentiation and function preservation of the embryonic and adult inner ear, but many details still need to be elucidated regarding the numerous microRNAs in the inner ear. Based on previous investigations on the microRNA profile in the inner ear, we confirmed that several microRNAs are expressed in the inner ear, and we detected the spatial expression of these microRNAs in the neonatal mouse inner ear. Then we focused on miR-194 for its specific expression with a dynamic spatiotemporal pattern during inner ear development. Overexpression of miR-194 in cultured spiral ganglion cells significantly affected the dendrites of differentiated neurons, with more branching and obviously dispersed nerve fibres. Furthermore, the cytoskeleton of cultured cells was markedly affected, as disordered actin filaments resulting from miR-194 overexpression and enhanced filaments resulting from miR-194 knockdown were observed. Together with the bioinformatic methods, the RT-qPCR and western blot results showed that RhoB is a candidate target of miR-194 in the morphogenesis of spiral ganglion neurons. Additionally, the double luciferase reporter system was used to identify RhoB as a novel target of miR-194. Finally, the inhibition of RhoB activation by Clostridium difficile toxin B disturbed the organization of the actin filament, similar to the effects of miR-194 overexpression. In summary, we investigated microRNA expression in the mouse inner ear, and demonstrated that miR-194 is dynamically expressed during inner ear development; importantly, we found that miR-194 affects neuron morphogenesis positively through Rho B-mediated F-actin rearrangement.

RhoGTPases are known regulators of intracellular actin dynamics that are important for maintaining endothelial barrier function. RhoA is most extensively studied as a key regulator of endothelial barrier function, however the function of the 2 highly homologous family-members (> 88%) RhoB and RhoC in endothelial barrier function is still poorly understood. This study aimed to determine whether RhoA, RhoB and RhoC have overlapping or distinct roles in barrier function and permeability in resting and activated endothelium. By using primary endothelial cells in combination with siRNA transfection to establish individual, double or triple knockdown of the RhoA/B/C RhoGTPases, we found that RhoB, but not RhoA or RhoC, is in resting endothelium a negative regulator of permeability. Loss of RhoB accounted for an accumulation of VE-cadherin at cell-cell contacts. Thrombin-induced loss of endothelial integrity is mediated primarily by RhoA and RhoB. Combined loss of RhoA/B showed decreased phosphorylation of Myosin Light Chain and increased expression of VE-cadherin at cell-cell contacts after thrombin stimulation. RhoC contributes to the Rac1-dependent restoration of endothelial barrier function. In summary, this study shows that these highly homologous RhoGTPases differentially control the dynamics of endothelial barrier function.

Toll-like receptors (TLRs) are the primary sensors detecting conserved molecular patterns on microorganisms, thus acting as important components of innate immunity against invading pathogens. Many positive and negative regulators of TLR-triggered signaling have been identified. The Rho GTPase RhoB plays a key role in cell migration, division and polarity; however, the function and regulatory mechanisms of RhoB in TLR ligand-triggered innate immune responses remain to be investigated. Here, we report that the expression of RhoB is induced by TLR agonists (lipopolysaccharide (LPS), CpG, poly(I:C)) in macrophages. Knockdown of RhoB expression markedly decreased TLR ligand-induced activation of mitogen activated protein kinases and nuclear factor-κB (NF-κB), and the production of tumor necrosis factor α (TNFα), interleukin (IL)-6 and IL-1β in macrophages stimulated with TLR ligands. Furthermore, we demonstrated that RhoB interacts with major histocompatibility complex class II (MHCII) α chain, but not β chain, in endosomes of macrophages. Knockdown of MHCII expression greatly reduced the interaction of RhoB with Btk, and attenuated the induction of NF-κB and interferon β activity by RhoB upon LPS stimulation. These findings suggest that RhoB is a positive physiological regulator of TLRs signaling via binding to MHCII in macrophages, and therefore RhoB may be a potential therapeutic target in inflammatory diseases.