Discovery of a dual Ras and ARF6 inhibitor from a GPCR endocytosis screen.

Internalization and intracellular trafficking of G protein-coupled receptors (GPCRs) play pivotal roles in cell responsiveness. Dysregulation in receptor trafficking can lead to aberrant signaling and cell behavior. Here, using an endosomal BRET-based assay in a high-throughput screen with the prototypical GPCR angiotensin II type 1 receptor (AT1R), we sought to identify receptor trafficking inhibitors from a library of ~115,000 small molecules. We identified a novel dual Ras and ARF6 inhibitor, which we named Rasarfin, that blocks agonist-mediated internalization of AT1R and other GPCRs. Rasarfin also potently inhibits agonist-induced ERK1/2 signaling by GPCRs, and MAPK and Akt signaling by EGFR, as well as prevents cancer cell proliferation. In silico modeling and in vitro studies reveal a unique binding modality of Rasarfin within the SOS-binding domain of Ras. Our findings unveil a class of dual small G protein inhibitors for receptor trafficking and signaling, useful for the inhibition of oncogenic cellular responses.

ADP Ribosylation Factor 6 Promotes Contraction and Proliferation, Suppresses Apoptosis and Is Specifically Inhibited by NAV2729 in Prostate Stromal Cells.

The presumed ADP ribosylation factor (ARF) 6 inhibitor NAV2729 inhibits human prostate smooth muscle contraction and proliferation of stromal cells, which are driving factors of voiding symptoms in benign prostatic hyperplasia (BPH). However, its specificity and a confirmed role of ARF6 for smooth muscle contraction are still pending. Here, we generated monoclonal ARF6 knockouts in human prostate stromal cells (WPMY-1), and characterized phenotypes of contractility, growth-related functions, and susceptibility to NAV2729 in knockout and control clones. ARF6 knockout was verified by Western blot. Knockout clones showed impaired contraction and actin organization, reduced proliferation and viability, and increased apoptosis and cell death. In ARF6-expressing control clones, NAV2729 (5 µM) strongly inhibited contraction (67% inhibition across all three control clones), actin organization (72%), proliferation (97%), and viability (up to 82%), and increased apoptosis (5-fold) and cell death (6-fold). In ARF6 knockouts, effects of NAV2729 (5 µM) were widely reduced, including lacking or minor effects on contractions (0% inhibition across all three knockout clones), actin (18%) and proliferation (13%), and lacking increases of apoptosis and cell death. Viability was reduced by NAV2729 with an IC50 of 3.3 µM across all three ARF6 control clones, but of 4.5-8.2 µM in ARF6 knockouts. In conclusion, ARF6 promotes prostate smooth muscle contraction and proliferation of stromal cells. Both are inhibited by NAV2729, which showed high specificity for ARF6 up to 5 µM and represents an attractive compound in the context of BPH. Considering the relevance of smooth muscle-based diseases, shared roles of ARF6 in other smooth muscle types merit further investigation. SIGNIFICANCE STATEMENT: By knockout of ARF6 in prostate stromal cells, this study demonstrates the involvement of ARF6 in promotion of prostate smooth muscle contraction and stromal growth, and defines concentration ranges for their ARF6-specific inhibition by NAV2729. Besides the context of benign prostatic hyperplasia and lower urinary tract symptoms, analog ARF6 functions in contraction and growth appear possible in other smooth muscle-rich organs, which merits further attention considering the high clinical relevance of smooth muscle-based diseases.

The small GTPase ARF6 regulates GABAergic synapse development.

ADP ribosylation factors (ARFs) are a family of small GTPases composed of six members (ARF1-6) that control various cellular functions, including membrane trafficking and actin cytoskeletal rearrangement, in eukaryotic cells. Among them, ARF1 and ARF6 are the most studied in neurons, particularly at glutamatergic synapses, but their roles at GABAergic synapses have not been investigated. Here, we show that a subset of ARF6 protein is localized at GABAergic synapses in cultured hippocampal neurons. In addition, we found that knockdown (KD) of ARF6, but not ARF1, triggered a reduction in the number of GABAergic synaptic puncta in mature cultured neurons in an ARF activity-dependent manner. ARF6 KD also reduced GABAergic synaptic density in the mouse hippocampal dentate gyrus (DG) region. Furthermore, ARF6 KD in the DG increased seizure susceptibility in an induced epilepsy model. Viewed together, our results suggest that modulating ARF6 and its regulators could be a therapeutic strategy against brain pathologies involving hippocampal network dysfunction, such as epilepsy.

Dissecting the localization of lipopolysaccharide-responsive and beige-like anchor protein (LRBA) in the endomembrane system.

INTRODUCTION AND OBJECTIVES: LRBA deficiency is caused by loss of LRBA protein expression, due to either homozygous or compounds heterozygous mutations in LRBA. LRBA deficiency has been shown to affect vesicular trafficking and autophagy. To date, LRBA has been observed in the cytosol, Golgi apparatus and some lysosomes in LPS-stimulated murine macrophages. The objectives of the present study were to study the LRBA localization in organelles involved in vesicular traffic, phagocytosis, and autophagy in mononuclear phagocytes (MP). MATERIALS AND METHODS: We analyzed LRBA colocalization with different endosomes markets using confocal microscopy in MP. We used the autophagy inhibitors to determine the role of LRBA in formation, maturation or degradation of the autophagosome. RESULTS: LRBA intracellular trafficking depends on the activity of the GTPase ADP ribosylation factor-1 (ARF) in MP. LRBA was identified in early, late endosomes but did not colocalize strongly with lysosomal markers. Although LRBA appears not to be recruited during the phagocytic cargo uptake, it greatly colocalized with the microtubule-associated protein 1A/1B-light chain 3 (LC3) under a steady state and this decreased after the induction of autophagy flux. Although the use of inhibitors of lysosome fusion did not restore the LRBA/LC3 colocalization, inhibitors of either early to late endosomes trafficking or PI3K pathway did. CONCLUSIONS: Taken together, our results show that LRBA is located in endomembrane system vesicles, mainly in the early and late endosomes. Although LRBA appears not to be involved in the phagocytic uptake, it is recruited in the early steps of the autophagy flux.

A NAV2729-sensitive mechanism promotes adrenergic smooth muscle contraction and growth of stromal cells in the human prostate.

Voiding symptoms in benign prostatic hyperplasia (BPH) are driven by prostate smooth muscle contraction and prostate growth. Smooth muscle contraction in the prostate and other organs critically depends on activation of the small monomeric GTPase RhoA and probably Rac1. A role of another GTPase, ADP-ribosylation factor 6 (ARF6), for smooth muscle contraction has been recently suggested by indirect evidence but remains to be proven for any organ. Here, we report effects of NAV2729, an inhibitor with assumed specificity for ARF6, in human prostate tissues and cultured prostate stromal cells (WPMY-1). NAV2729 (5 µm) inhibited neurogenic and α1-adrenergic contractions of human prostate tissues. Contractions induced by endothelin-1, by the thromboxane A2 agonist U46619, or by high molar KCl were not inhibited. Correlation analyses suggested up-regulation of prostatic ARF6 expression with increasing degree of BPH, as ARF6 expression increased with the content of prostate-specific antigen (PSA) of prostate tissues. NAV2729 inhibited ARF6 activity but not other GTPases (ARF1, RhoA, Rac1) in prostate tissues and in WPMY-1 cells. Proliferation of WPMY-1 cells was inhibited concentration-dependently by NAV2726, as reflected by decreased viability, 5-ethynyl-2'-deoxyuridine (EdU) assay, colony formation assay, and expression of Ki-67. Silencing of ARF6 expression mimicked effects of NAV2729 on viability and in the EdU assay. Effects of NAV2729 on viability and proliferation were attenuated in cells with silenced ARF6 expression. Our findings suggest that a NAV2729-sensitive mechanism promotes adrenergic contraction and stromal cell growth in the human prostate, which is probably ARF6-mediated. Similar actions in other organs and urodynamic effects of NAV2729 appear possible.

Chemically Modified Antisense Oligonucleotide Against ARL4C Inhibits Primary and Metastatic Liver Tumor Growth.

ADP-ribosylation factor-like 4c (ARL4C) is identified as a small GTP-binding protein, which is expressed by Wnt and EGF signaling and plays an important role in tubulogenesis of cultured cells and the ureters. ARL4C is little expressed in adult tissues, but it is highly expressed in lung cancer and colorectal cancer and shown to represent a molecular target for cancer therapy based on siRNA experiments. This study revealed that ARL4C is highly expressed in primary hepatocellular carcinoma (HCC) tumors and colorectal cancer liver metastases, and that ARL4C expression is associated with poor prognosis for these cancers. Chemically modified antisense oligonucleotides (ASO) against ARL4C effectively reduced ARL4C expression in both HCC and colorectal cancer cells and inhibited proliferation and migration of these cancer cells in vitro ARL4C ASOs decreased the PIK3CD mRNA levels and inhibited the activity of AKT in HCC cells, suggesting that the downstream signaling of ARL4C in HCC cells is different from that in lung and colon cancer cells. In addition, subcutaneous injection of ARL4C ASO was effective in reducing the growth of primary HCC and metastatic colorectal cancer in the liver of immunodeficient mice. ARL4C ASO accumulated in cancer cells more efficiently than the surrounding normal cells in the liver and decreased ARL4C expression in the tumor. These results suggest that ARL4C ASO represents a novel targeted nucleic acid medicine for the treatment of primary and metastatic liver cancers.

Humanized anti-CD123 antibody facilitates NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) of Hodgkin lymphoma targets via ARF6/PLD-1.

CD123 (IL-3Rα) is frequently expressed by malignant Hodgkin lymphoma (HL) cells. Naked monoclonal antibodies (mAb) against HL lack clinical benefit, partially due to absence of natural killer (NK) cells in the tumor microenvironment. Here we show that the combination of a fully humanized anti-CD123 mAb (CSL362) and high-affinity Fcγ-receptor NK-92 cells (haNK) effectively target and kill HL cells in vitro. First, we confirmed high expression of CD123 in 2 of the 3 HL cell lines (KM-H2 and L-428), and its absence in NK cells. Cytotoxicity of haNK cells against CD123-positive HL cells was significantly higher in the presence of CSL362. This was also shown with IL-15-activated primary NK cells, although haNK cells showed a 10.87-fold lower estimated half-maximal stimulatory effective concentration (EC50). CSL362 facilitated a significant increase in the expression of CD107a, intracellular IFN-γ and TNF-α and enhanced expression of c-JUN, PLD-1, and ARF6 by NK cells. Inhibition of the ARF6-PLD-1 axis (NAV2729), but not of the MAPK pathway (U0126), completely abrogated CSL362-facilitated antibody-dependent cell-mediated cytotoxicity (ADCC) in haNK and activated primary NK cells. Our results support CD123 as an immunotherapeutic target for HL and the combination of NK cells and CSL362 as a treatment strategy for HL.

HGF-induced migration depends on the PI(3,4,5)P3-binding microexon-spliced variant of the Arf6 exchange factor cytohesin-1.

Differential inclusion or skipping of microexons is an increasingly recognized class of alternative splicing events. However, the functional significance of microexons and their contribution to signaling diversity is poorly understood. The Met receptor tyrosine kinase (RTK) modulates invasive growth and migration in development and cancer. Here, we show that microexon switching in the Arf6 guanine nucleotide exchange factor cytohesin-1 controls Met-dependent cell migration. Cytohesin-1 isoforms, differing by the inclusion of an evolutionarily conserved three-nucleotide microexon in the pleckstrin homology domain, display differential affinity for PI(4,5)P2 (triglycine) and PI(3,4,5)P3 (diglycine). We show that selective phosphoinositide recognition by cytohesin-1 isoforms promotes distinct subcellular localizations, whereby the triglycine isoform localizes to the plasma membrane and the diglycine to the leading edge. These data highlight microexon skipping as a mechanism to spatially restrict signaling and provide a mechanistic link between RTK-initiated phosphoinositide microdomains and Arf6 during signal transduction and cancer cell migration.

CLIC4/Arf6 Pathway.

RATIONALE: Increased expression of CLIC4 (chloride intracellular channel 4) is a feature of endothelial dysfunction in pulmonary arterial hypertension, but its role in disease pathology is not fully understood. OBJECTIVE: To identify CLIC4 effectors and evaluate strategies targeting CLIC4 signaling in pulmonary hypertension. METHODS AND RESULTS: Proteomic analysis of CLIC4-interacting proteins in human pulmonary artery endothelial cells identified regulators of endosomal trafficking, including Arf6 (ADP ribosylation factor 6) GTPase activating proteins and clathrin, while CLIC4 overexpression affected protein regulators of vesicular trafficking, lysosomal function, and inflammation. CLIC4 reduced BMPRII (bone morphogenetic protein receptor II) expression and signaling as a result of Arf6-mediated reduction in gyrating clathrin and increased lysosomal targeting of the receptor. BMPRII expression was restored by Arf6 siRNA, Arf inhibitor Sec7 inhibitor H3 (SecinH3), and inhibitors of clathrin-mediated endocytosis but was unaffected by chloride channel inhibitor, indanyloxyacetic acid 94 or Arf1 siRNA. The effects of CLIC4 on NF-κB (nuclear factor-kappa B), HIF (hypoxia-inducible factor), and angiogenic response were prevented by Arf6 siRNA and SecinH3. Sugen/hypoxia mice and monocrotaline rats showed elevated expression of CLIC4, activation of Arf6 and NF-κB, and reduced expression of BMPRII in the lung. These changes were established early during disease development. Lung endothelium-targeted delivery of CLIC4 siRNA or treatment with SecinH3 attenuated the disease, reduced CLIC4/Arf activation, and restored BMPRII expression in the lung. Endothelial colony-forming cells from idiopathic pulmonary hypertensive patients showed upregulation of CLIC4 expression and Arf6 activity, suggesting potential importance of this pathway in the human condition. CONCLUSIONS: Arf6 is a novel effector of CLIC4 and a new therapeutic target in pulmonary hypertension.

Sphingolipids inhibit endosomal recycling of nutrient transporters by inactivating ARF6.

Endogenous sphingolipids (ceramide) and related synthetic molecules (FTY720, SH-BC-893) reduce nutrient access by decreasing cell surface expression of a subset of nutrient transporter proteins. Here, we report that these sphingolipids disrupt endocytic recycling by inactivating the small GTPase ARF6. Consistent with reported roles for ARF6 in maintaining the tubular recycling endosome, MICAL-L1-positive tubules were lost from sphingolipid-treated cells. We propose that ARF6 inactivation may occur downstream of PP2A activation since: (1) sphingolipids that fail to activate PP2A did not reduce ARF6-GTP levels; (2) a structurally unrelated PP2A activator disrupted tubular recycling endosome morphology and transporter localization; and (3) overexpression of a phosphomimetic mutant of the ARF6 GEF GRP1 prevented nutrient transporter loss. ARF6 inhibition alone was not toxic; however, the ARF6 inhibitors SecinH3 and NAV2729 dramatically enhanced the killing of cancer cells by SH-BC-893 without increasing toxicity to peripheral blood mononuclear cells, suggesting that ARF6 inactivation contributes to the anti-neoplastic actions of sphingolipids. Taken together, these studies provide mechanistic insight into how ceramide and sphingolipid-like molecules limit nutrient access and suppress tumor cell growth and survival.

Frequent overexpression of AMAP1, an Arf6 effector in cell invasion, is characteristic of the MMTV-PyMT rather than the MMTV-Neu human breast cancer model.

BACKGROUND: The small GTPase Arf6 and its downstream effector AMAP1 (also called ASAP1/DDEF1) constitute a signaling pathway promoting cell invasion, in which AMAP1 interacts with several different proteins, including PRKD2, EPB41L5, paxillin, and cortactin. Components of this pathway are often overexpressed in human breast cancer cells, to be correlated with poor prognosis of the patients, whereas overexpression of the Arf6 pathway did not correlate with the four main molecular classes of human breast tumors. In this pathway, receptor tyrosine kinases, including EGFR and Her2, activate Arf6 via GEP100. MMTV-PyMT mice and MMTV-Neu mice are well-established models of human breast cancer, and exhibit the early dissemination and the lung metastasis, by utilizing protein tyrosine phosphorylation for oncogenesis. PyMT-tumors and Neu-tumors are known to have overlapping gene expression profiles, which primarily correspond to the luminal B-type of human mammary tumors, although they differ in the time necessary for tumor onset and metastasis. Given the common usage of protein tyrosine phosphorylation, as well as the frequent use of these animal models for studying breast cancer at the molecular level, we here investigated whether mammary tumors in these mouse models utilize the Arf6-based pathway for invasion. METHODS: Expression levels of Arf6, AMAP1, and GEP100 were analyzed in PyMT-tumors and Neu-tumors by western blotting. Expression of Arf6 and AMAP1 was also analyzed by immunohistochemistry. The involvement of AMAP1 in invasion, and the possible correlation of its high expression levels with cancer mesenchymal properties were also investigated. RESULTS: We found that PyMT-tumors, but not Neu-tumors, frequently overexpress AMAP1 and use it for invasion, whereas both types of tumors expressed Arf6 and GEP100 at different levels. High levels of the AMAP1 expression among PyMT-tumor cells were frequently correlated with loss of the epithelial marker CK8 and also with expression of the mesenchymal marker vimentin both at the primary sites and at sites of the lung metastases. CONCLUSIONS: PyMT-tumors appear to frequently utilize the Arf6-based invasive machinery, whereas Neu-tumors do not. Our results suggest that MMTV-PyMT mice, rather than MMTV-Neu mice, are useful to study the Arf6-based mammary tumor malignancies, as a representative model of human breast cancer.

Deficiency of primary cilia in kidney epithelial cells induces epithelial to mesenchymal transition.

Primary cilium is a microtubule-based non-motile organelle that plays critical roles in kidney pathophysiology. Our previous studies revealed that the lengths of primary cilia decreased upon renal ischemia/reperfusion injury and oxidative stress, and restored with recovery. Here, we tested the hypothesis that lack of primary cilium causes epithelial to mesenchymal transition (EMT) of kidney tubule cells. We investigated the alteration of length of primary cilia in TGF-ß-induced EMT via visualization of primary cilia by fluorescence staining against acetylated α-tubulin. EMT was determined by measuring mesenchymal protein expression using quantitative PCR and indirect fluorescence staining. As a result, TGF-ß treatment decreased ciliary length along with EMT. To test whether defect of primary cilia trigger onset of EMT, cilia formation was disturbed by knock down of ciliary protein using siRNA along with/without TGF-ß treatment. Knock down of Arl13b and Ift20 reduced cilia elongation and increased expression of EMT markers such as fibronectin, α-SMA, and collagen III. TGF-ß-induced EMT was greater as well in Arl13b and Ift20-knock down cells compared to control cells. Taken together, deficiency of primary cilia trigger EMT and exacerbates it under pro-fibrotic signals.

MiR-221-3p targets ARF4 and inhibits the proliferation and migration of epithelial ovarian cancer cells.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer. Although molecular diagnostic tools and targeted therapies have been developed over the past few decades, the survival rate is still rather low. Numerous researches suggest that some microRNAs (miRNAs) are key regulators of tumor progression. Among those miRNAs that has attracted much attention for their multiple roles in human cancers, the function of miR-221-3p in EOC has not been elucidated. Herein, we examined the expression of miR-221-3p in EOC patients and cell lines. Our data revealed that higher expression of miR-221-3p was linked to better overall survival in EOC patients. In-vitro experiments indicated that miR-221-3p inhibited EOC cell proliferation and migration. By performing subsequent systematic molecular biological and bioinformatic analyses, we found ADP-ribosylation factor (ARF) 4 is one of the putative target genes, the direct binding relationship was further confirmed by dual-luciferase reporter assay. Finally, a distinct gene expression between miR-221-3p and ARF4 in EOC group and normal group was identified, and the negative correlation between their expression levels in EOC specimens was further confirmed. Taken together, our research uncovered the tumor suppressive role of miR-221-3p in EOC and directly targeted ARF4, suggesting that miR-221-3p might be a novel potential candidate for clinical prognosis and therapeutics of EOC.

Identification of ARL4C as a Peritoneal Dissemination-Associated Gene and Its Clinical Significance in Gastric Cancer.

BACKGROUND: In gastric cancer (GC), peritoneal dissemination (PD) occurs frequently and is incurable. In this study, we aimed to identify PD-associated genes in GC. METHODS: We identified a PD-associated gene using three GC datasets: highly disseminated peritoneal GC cell lines, the Singapore dataset and The Cancer Genome Atlas (TCGA) dataset. We assessed the clinicopathological significance of the gene expression using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and performed immunohistochemical analysis for the gene in our patient cohort. We also performed survival analyses of the gene in our patient cohort, the Singapore dataset and the GSE62254 datasets. Moreover, gene set enrichment analysis (GSEA) was performed using the Singapore and TCGA datasets. Finally, in vitro experiments such as invasion/migration assays, immunofluorescence staining of actin filaments, epidermal growth factor (EGF) treatment analysis, and gene expression analysis were conducted using three gene-knockdown GC cell lines (AGS, 58As9, MKN45). RESULTS: ADP-ribosylation factor-like 4c (ARL4C) was identified as a PD-associated gene, and immunohistochemical analysis showed that ARL4C was overexpressed in GC cells. High ARL4C expression was associated with the depth of invasion (p < 0.01) and PD (p < 0.05) and was a poor prognostic factor (p < 0.05) in our patient cohort, the Singapore dataset and the GSE62254 dataset. ARL4C expression positively correlated with the epithelial-mesenchymal transition (EMT) gene set in GSEA. Moreover, ARL4C knockdown reduced invasion/migration capacity, SLUG expression, and the formation of lamellipodia or filopodia in AGS and 58As9 cells. Finally, EGF treatment increased ARL4C expression in MKN45 cells. CONCLUSIONS: ARL4C was associated with PD and was a poor prognostic factor in GC, possibly through promoting invasive capacity by activation of both EMT and motility.

Family-wide Analysis of the Inhibition of Arf Guanine Nucleotide Exchange Factors with Small Molecules: Evidence of Unique Inhibitory Profiles.

Arf GTPases and their guanine nucleotide exchange factors (ArfGEFs) are major regulators of membrane traffic and organelle structure in cells. They are associated with a variety of diseases and are thus attractive therapeutic targets for inhibition by small molecules. Several inhibitors of unrelated chemical structures have been discovered, which have shown their potential in dissecting molecular pathways and blocking disease-related functions. However, their specificity across the ArfGEF family has remained elusive. Importantly, inhibitory responses in the context of membranes, which are critical determinants of Arf and ArfGEF cellular functions, have not been investigated. Here, we compare the efficiency and specificity of four structurally distinct ArfGEF inhibitors, Brefeldin A, SecinH3, M-COPA, and NAV-2729, toward six ArfGEFs (human ARNO, EFA6, BIG1, and BRAG2 and Legionella and Rickettsia RalF). Inhibition was assessed by fluorescence kinetics using pure proteins, and its modulation by membranes was determined with lipidated GTPases in the presence of liposomes. Our analysis shows that despite the intra-ArfGEF family resemblance, each inhibitor has a specific inhibitory profile. Notably, M-COPA is a potent pan-ArfGEF inhibitor, and NAV-2729 inhibits all GEFs, the strongest effects being against BRAG2 and Arf1. Furthermore, the presence of the membrane-binding domain in Legionella RalF reveals a strong inhibitory effect of BFA that is not measured on its GEF domain alone. This study demonstrates the value of family-wide assays with incorporation of membranes, and it should enable accurate dissection of Arf pathways by these inhibitors to best guide their use and development as therapeutic agents.

Commensal microbiota-induced microRNA modulates intestinal epithelial permeability through the small GTPase ARF4.

The intestinal tract contains many commensal bacteria that modulate various physiological host functions. Dysbiosis of commensal bacteria triggers dysfunction of the intestinal epithelial barrier, leading to the induction or aggravation of intestinal inflammation. To elucidate whether microRNA plays a role in commensal microbiome-dependent intestinal epithelial barrier regulation, we compared transcripts in intestinal epithelial cells (IECs) from conventional and germ-free mice and found that commensal bacteria induced the expression of miR-21-5p in IECs. miR-21-5p increased intestinal epithelial permeability and up-regulated ADP ribosylation factor 4 (ARF4), a small GTPase, in the IEC line Caco-2. We also found that ARF4 expression was up-regulated upon suppression of phosphatase and tensin homolog (PTEN) and programmed cell death 4 (PDCD4), which are known miR-21-5p targets, by RNAi. Furthermore, ARF4 expression in epithelial cells of the large intestine was higher in conventional mice than in germ-free mice. ARF4 suppression in the IEC line increased the expression of tight junction proteins and decreased intestinal epithelial permeability. These results indicate that commensal microbiome-dependent miR-21-5p expression in IECs regulates intestinal epithelial permeability via ARF4, which may therefore represent a target for preventing or managing dysfunction of the intestinal epithelial barrier.

Roles of Arf6 in cancer cell invasion, metastasis and proliferation.

ADP-ribosylation factor 6 (Arf6), a member of small GTPases ADP-ribosylation factor (Arf) family, expresses widely in mammalian cells and mainly regulates the functions of membrane traffic and actin remodeling. Current studies indicated that the activation and high expression of Arf6 protein may be significantly correlated with the invasion and metastasis of several tumors, such as breast cancer, pancreatic cancer, lung cancer, etc. Meanwhile, the ability of tumor invasion and metastasis can be suppressed when Arf6 activity is blocked by the inhibitors or small-interfering RNAs of Arf6. To explore the precisely potential mechanisms between Arf6 and the process of tumor invasion, metastasis and proliferation, we concludes the functions and potential signaling pathways of Arf6 in tumor cells and provides an overview about clinical prospects of Arf6 in the screening, diagnosis, treatment and evaluation of prognosis of neoplasms.

Rab35 protein regulates evoked exocytosis of endothelial Weibel-Palade bodies.

Weibel-Palade bodies (WPB) are secretory organelles of endothelial cells that undergo evoked exocytosis following intracellular Ca2+ or cAMP elevation, thereby supplying the vasculature with factors controlling hemostasis. Several cytosolic and membrane-associated proteins, including the Rab family members Rab3, Rab15, and Rab27a, have been implicated in regulating the acute exocytosis of WPB. Here, we carried out a genome-wide screen to identify Rab pathways affecting WPB exocytosis. Overexpression of a specific subset of Rab GTPase-activating proteins (RabGAPs) inhibited histamine-evoked, Ca2+-dependent WPB exocytosis, presumably by inactivating the target Rab GTPases. Among these RabGAPs, we concentrated on TBC1D10A and showed that the inhibitory effect depends on its GAP activity. We confirmed that Rab35 was a target Rab of TBC1D10A in human endothelial cells; Rab35 interacted with TBC1D10A, and expression of the GAP-insensitive Rab35(Q67A) mutant rescued the inhibitory effect of TBC1D10A overexpression on WPB exocytosis. Furthermore, knockdown of Rab35 and expression of a dominant-negative Rab35 mutant both inhibited histamine-evoked secretion of the WPB cargos von Willebrand factor and P-selectin. Pulldown and co-immunoprecipitation experiments identified the ArfGAP with coiled-coil, Ank repeat, and pleckstrin homology domain-containing protein ACAP2 as an Rab35 effector in endothelial cells, and depletion as well as overexpression approaches revealed that ACAP2 acts as a negative regulator of WPB exocytosis. Interestingly, a known ACAP2 target, the small GTPase Arf6, supported histamine-evoked WPB exocytosis, as shown by knockdown and overexpression of a dominant-negative Arf6 mutant. Our data identify Rab35 as a novel regulator of WPB exocytosis, most likely acting through the downstream effectors ACAP2 and Arf6.

Machineries regulating the activity of the small GTPase Arf6 in cancer cells are potential targets for developing innovative anti-cancer drugs.

The Small GTPase ADP-ribosylation factor 6 (Arf6) functions as the molecular switch in cellular signaling pathways by cycling between GDP-bound inactive and GTP-bound active form, which is precisely regulated by two regulators, guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Numerous studies have shown that these machineries play critical roles in tumor angiogenesis/growth and cancer cell invasion/metastasis through regulating the cycling of Arf6. Here, we summarize accumulating knowledge for involvement of Arf6 GEFs/GAPs and small molecule inhibitors of Arf6 signaling/cycling in cancer progression, and discuss possible strategies for developing innovative anti-cancer drugs targeting Arf6 signaling/cycling.

ARF6 Is an Actionable Node that Orchestrates Oncogenic GNAQ Signaling in Uveal Melanoma.

Activating mutations in Gαq proteins, which form the α subunit of certain heterotrimeric G proteins, drive uveal melanoma oncogenesis by triggering multiple downstream signaling pathways, including PLC/PKC, Rho/Rac, and YAP. Here we show that the small GTPase ARF6 acts as a proximal node of oncogenic Gαq signaling to induce all of these downstream pathways as well as ß-catenin signaling. ARF6 activates these diverse pathways through a common mechanism: the trafficking of GNAQ and ß-catenin from the plasma membrane to cytoplasmic vesicles and the nucleus, respectively. Blocking ARF6 with a small-molecule inhibitor reduces uveal melanoma cell proliferation and tumorigenesis in a mouse model, confirming the functional relevance of this pathway and suggesting a therapeutic strategy for Gα-mediated diseases.