Expression Of ADP-ribosylation Factor 6 Research Articles

Abstract 3926: ARF6-dependent endocytic trafficking of the interferon-gamma receptor drives adaptive immune resistance and response to immune checkpoint blockade

Abstract Interferon-gamma (IFNγ)-driven adaptive immune resistance (AIR) in cancer begins at the plasma membrane where cytokine signaling is initiated. Cytotoxic CD8+ T cells secrete IFNγ, eliciting tumor-intrinsic IFNγ signaling and expression of immunosuppressive genes, which are critical for effective therapeutic response to anti-PD-1 and anti-CTLA-4 immune checkpoint blockade (ICB). Although the IFNγ receptor (IFNγR) is expressed in all cell types, its surface density can determine a cell’s responsiveness to ligand. Mechanistic control over this process remains a mystery. Herein we report that the endocytic trafficking protein ADP-Ribosylation Factor 6 (ARF6) is critical for IFNγ-driven AIR during primary tumor progression and in the setting of ICB therapy. The IFNγR1 subunit of the receptor is constitutively internalized in a ligand-independent manner. ARF6 is necessary for endocytic recycling of IFNγR1 and determines the steady-state surface density of the receptor. Loss of ARF6 results in lysosomal degradation of the receptor and controls IFNγR1 protein levels in melanoma, non-small cell lung cancer, colorectal cancer, and triple-negative breast cancer. IFNγ activates ARF6 in parallel with JAK1-STAT1 signaling, suggesting a positive feedback loop that allows tumor cells to dynamically respond to immune attack. Silencing ARF6 reduces tumor intrinsic IFNγ signaling and downstream expression of immunosuppressive genes. In murine melanoma, loss of ARF6 causes resistance to systemic ICB therapy. Likewise, low expression of ARF6 in patient tumors correlates with inferior outcomes with ICB. Our data provide new mechanistic insights into tumor immune escape, defined by ARF6-dependent adaptive immunity, and support that ARF6-dependent endomembrane trafficking of the IFNγ receptor shapes clinical outcomes of ICB therapy. Private Information Citation Format: Yinshen Wee, Junhua Wang, Emily C. Wilson, Coulson P. Rich, Aaron Rogers, Zhongzong Tong, Evelyn DeGroot, Y.N. Vashisht Gopal, Michael Davies, Huseyin Atakan Ekiz, Joshua K.H. Tay, Chris Stubben, Kenneth M. Boucher, Juan M. Oviedo, Keke C. Fairfax, Matthew A. Williams, Sheri L. Holmen, Roger K. Wolff, Allie H. Grossmann. ARF6-dependent endocytic trafficking of the interferon-gamma receptor drives adaptive immune resistance and response to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3926.

Genetic Analysis of Differentially Expressed Genes Associated with the Pathogenesis of Autism Spectrum Disorder

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder present at birth and affects 1 in 36 children in the US. It causes symptoms like intellectual disability, lack of communication skills, delayed development of coordination and movement, and more. ASD is a spectrum disorder, and its complexity makes early diagnosis in children difficult as there are no known biomarkers that cause ASD. This project aims to identify genetic factors that may be associated with ASD. By splitting samples in the dataset accessed from the GEO database into a test group with ASD and a control group, this study used a statistical t-test to identify the top 250 differentially expressed genes with p-values < 0.05. These genes were entered in the STRING database to create a map of gene interactions and identify top pathways and biological processes. Analysis of pathways and biological processes led to findings that the differentially expressed gene ARF6 (ADP Ribosylation Factor 6) was active in all three pathways: RAS Signaling, Phospholipase D Signaling, and Salmonella Infection Pathways and played important roles in cellular functions. The experiment validated my hypothesis by providing evidence for a significant difference in gene expressions between ASD individuals and neurotypical individuals. ARF6 was found to be downregulated and involved in pathways and biological processes associated with ASD like immune and cell signaling dysregulation. The implication is If the ARF6 gene factor can be controlled to regulate ARF6 expression, the mutations that contribute to ASD can be suppressed through multiple pathways.

Addiction to Small Gtpase ARF6-Mediated Sphingolipid Homeostasis By AML but Not the Host

Background Cancer cells' unique metabolic demand has emerged as a promising therapeutic target in Acute Myeloid Leukemia (AML). The vesicular trafficking and signaling gatekeeper, ADP-Ribosylation Factor 6 (ARF6), belongs to the RAS superfamily of small GTPases and has been shown to regulate the homeostasis of lipids such as PIP2, phosphatidic acids, and cholesterol. Here we describe AML cells' unusual addiction to ARF6-mediated sphingolipid homeostasis and its clinical significance. Experiments and Results ARF6 knockdown (KD) or CRISPR-mediated ARF6 knockout (KO) inhibited the proliferation of AML cell lines, THP-1 and MV4-11, as well as the colony formation of primary AML CD34 + cells. Surprisingly, Tamoxifen-induced whole body KO of Arf6 in adult Arf6 f/f;Rosa-CreERT 2 mice led to healthy and grossly normal mice with a normal lifespan. Adult mice lacking ARF6 show no abnormality in complete blood count, urine test, or histology. Hence, ARF6 is genetically indispensable for AML, but not for the host. To confirm AML's addition to ARF6 in vivo, we introduced Arf6 KO in an MLL-AF9-induced AML model in C57BL6/J mice. We are currently following up with the disease progression between Arf6-KO AML group and control AML group, and expect to obtain results before the 2023 ASH meeting. After discovering that ARF6 is genetically necessary for AML but not needed for the host, we analyzed its clinical significance. Since ARF6 mutations are not detected in AML samples, we focused on analyzing its expression. With univariate analysis, we show that high ARF6 expression (top 50%) is significantly associated with worse overall survival (OS) and worse disease-free survival (DFS) of AML patients in the TCGA-NEJM-2013 (TCGA) cohort (N=200) and the Münchner Leukämielabor (MLL) cohort (N=579). In multi-variate analysis, high ARF6 expression is significantly associated with worse OS in the BeatAML cohort (N=562) and shows a trend towards worse OS and DFS in the TCGA cohort. Following a hypothesis-driven approach, we investigated the role of ARF6 in regulating sphingolipid homeostasis by HPLC-MS lipidomic analyses of AML cells (THP-1) with or without ARF6 depletion. Lipidomic profiles revealed that ARF6 KD led to an increase of the overall ceramide/sphingomyelin ratio, suggesting a defect in sphingolipid homeostasis. We then analyzed the expression of sphingolipid metabolic enzymes and observed that ARF6 KD downregulated sphingomyelin synthase 1 and 2 (SGMS1 and SGMS2) expression, hindering ceramide-to-sphingomyelin conversion. Supporting this, we found a positive linear correlation between ARF6 and SGMS1/2 expression among the patients in TCGA cohort, confirming their correlation. High expression of the ARF6 effectors SGMS1/2 is also associated with worse OS and DFS in the TCGA cohort and the MLL cohort in univariate analysis, resembling that of ARF6. Downstream of ceramide accumulation, ARF6 KD inhibited the pro-survival AKT pathway, confirming the pro-apoptotic roles of ceramide in cancers. The anti-proliferative effect of ARF6 depletion can be rescued by sphingomyelinase inhibitor GW4869 (blocking ceramide production) or by expressing a constitutively active form of AKT (AKT1 E17K). To pharmacologically target ARF6, we developed two generations of ARF6 small molecular inhibitor pro-drugs, named NAV-2729 and A6-4471. NAV-2729 and A6-4471 showed high specificity to ARF6 without inhibiting other small GTPases, with in vitro IC 50 in the lower μM range. Both NAV-2729 and A6-4471 inhibited AML cell line proliferation and reduced primary AML cell colony formation in lower μM range. Cord blood normal CD34 + cells are not sensitive to ARF6 inhibitors, but non-specific toxicity was observed at higher concentrations. When tested in vivo with THP-1 xenografts in NSG mice, both inhibitors reduced circulating AML cells without obvious toxicity to the host. Conclusion ARF6 is necessary for AML proliferation and its higher expression is associated with worse patient survival. However, ARF6 is dispensable for normal host physiology. Mechanistically, ARF6 maintains critical ceramide/sphingomyelin balance in AML by regulating SGMS1/2 expression. Pharmacologically inhibiting ARF6 suppressed AML proliferation in vitro and in vivo in a genotype-agnostic fashion. Our study implicates ARF6 as a potentially actionable and safe target in AML.

ADP Ribosylation Factor 6 Relieves Airway Inflammation and Remodeling by Inhibiting Ovalbumin Induced-Epithelial Mesenchymal Transition in Experimental Asthma, Possibly by Regulating of E2F Transcription Factor 8

ABSTRACT Background Childhood asthma is a major global health concern. ADP-ribosylation factor 6 (ARF6) is a low-molecular-weight GTPase; however, its role in childhood asthma remains unclear. Methods Ovalbumin (OVA)-challenged neonatal mice and transforming growth factor-β1 (TGF-β1)-induced BEAS-2B cells were used as in vivo and in vitro models of childhood asthma, respectively. Results Upon OVA stimulation, ARF6 expression was upregulated in the lung tissue. Neonatal mice administered SehinH3 (an ARF6 inhibitor) exhibited improved pulmonary pathological injury, along with reduced inflammatory cell infiltration in the lungs and cytokine release in bronchial alveolar lavage fluid and serum (interleukin [IL]−3, IL−5, IL−13, IgE, and OVA-specific IgE). SehinH3 treatment restrained epithelial – mesenchymal transition (EMT) in the lungs of asthmatic mice, as evidenced by increased E-cadherin and decreased N-cadherin and α-smooth muscle actin expression. Different TGF-β1 exposures to BEAS-2B cells induced a time- and dose-dependent increase in ARF6 expression in vitro. Upon TGF-β1 stimulation, ARF6 knockdown repressed EMT and SehinH3 treatment caused similar results in BEAS-2B cells. The transcription factor E2F8 is involved in diverse biological functions and its increased expression was confirmed in vivo and in vitro. Dual-luciferase assays confirmed that E2F8 binds to the ARF6 promoter and promotes its transcriptional activity. In vitro results revealed that E2F8 silencing suppressed EMT, whereas rescue experiments showed that ARF6 overexpression partly reversed these phenomena. Conclusion Our study showed that ARF6 is associated with childhood asthma progression and may be positively regulated by E2F8. These results provide insight into the pathogenesis and treatment of childhood asthma.

Impact of cancer stem cell markers Nestin and Sox2 on cell migration of epithelial and mesenchymal pancreatic cancer cells

Breast cancer (BC) is common cancer in female globally. Sevoflurane (SEV) has been reported to inhibit the metastasis of multiple cancers, including glioma, colorectal cancer, and hepatocellular carcinoma. However, the role of SEV in the metastasis of BC cells remains poorly understood.Transwell migration and invasion assays were performed to detect the migration and invasion of BC cells. Western blot assay was carried out to measure epithelial-mesenchymal transition (EMT)-related proteins in BC cells, including E-cadherin, N-cadherin, and fibronectin. Quantitative real-time polymerase chain reaction was conducted to determine the enrichment of miR-139-5p and ADP-ribosylation factor 6 (ARF6) in BC tissues and cells. The protein expression of ARF6 in BC tissues and cells was measured by western blot assay. The target of miR-139-5p was predicted by starBase software, and the target relationship between miR-139-5p and ARF6 in BC cells was confirmed by dual-luciferase reporter assay.SEV suppressed the migration, invasion, and EMT of BC cells, especially in the high-concentration SEV group. The level of miR-139-5p was lower in BC tissues and cells than that in paired normal tissues and normal mammary epithelial cells MCF-10A. MiR-139-5p was upregulated in BC cells treated with SEV. ARF6 was upregulated in BC tissues and cells compared with that in corresponding normal tissues and normal mammary epithelial cells MCF-10A. SEV reduced the mRNA and protein expression of ARF6 in BC cells. The accumulation of ARF6 or the interference of miR-139-5p reversed the suppressive effects of SEV treatment on the migration, invasion, and EMT of BC cells. MiR-139-5p bound to ARF6 and inversely modulated the level of ARF6 in BC cells. The transfection of si-ARF6 attenuated the promoting effects of miR-139-5p depletion on the migration, invasion, and EMT of BC cells treated with SEV.SEV suppressed the migration, invasion, and EMT of BC cells through downregulating the abundance of ARF6 by upregulating miR-139-5p. The miR-139-5p/ARF6 axis might be a promising target for the treatment of BC.

XIST sponges miR-320d to promote chordoma progression by regulating ARF6

BackgroundLong non-coding RNAs (lncRNAs) have been demonstrated to play important roles in various tumors, including chordoma. The purpose of this study was to investigate the role and mechanism of lncRNA X-inactive specific transcript (XIST) in chordoma. MethodsRNA levels and protein levels were measured by real-time quantitative polymerase chain reaction (RT‑qPCR) and western blot assay, respectively. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2′-deoxyuridine (EdU) assay and colony formation assay. Tanswell assay was used to examine cell migration and invasion. Cellular glycolysis was examined via the measurement of extracellular acidification rate (ECAR) and lactate production. The interaction between microRNA-320d (miR-320d) and XIST or ADP-ribosylation factor 6 (ARF6) was predicted by bioinformatics analysis and verified by a dual-luciferase reporter and RNA-pull down assays. The xenograft tumor model was used to explore the biological function of XIST in vivo. ResultsXIST was overexpressed in chordoma tissues. XIST knockdown suppressed chordoma cell proliferation, migration, invasion, and glycolysis. XIST acted as a sponge of miR-320d. Moreover, miR-320d overexpression inhibited the proliferation, migration, invasion, and glycolysis of chordoma cells. ARF6 was a direct target of miR-320d, and XIST upregulated ARF6 expression via sponging miR-320d. Furthermore, overexpression of ARF6 reversed the inhibitory effects of XIST knockdown on chordoma cell proliferation, migration, invasion, and glycolysis. Importantly, XIST silencing blocked xenograft tumor growth in vivo. ConclusionXIST knockdown inhibited chordoma progression via regulating the miR-320d/ARF6 axis, providing a novel insight into chordoma pathogenesis.

CCL18 promotes breast cancer progression by exosomal miR-760 activation of ARF6/Src/PI3K/Akt pathway

The small GTPase ADP-ribosylation factor 6 (ARF6) mediates chemokine (C-C motif) ligand 18 (CCL18)-induced activation of breast cancer (BC) metastasis through its downstream effector AMAP1. However, the molecular mechanisms underlying CCL18 up-regulating ARF6 remain largely unclear. Here, microRNAs (miRNAs) that target ARF6 were predicted and selected in high metastatic BC cells treated with CCL18. Next, we assessed the role of exosomal miR-760 in vitro and in vivo. We further analyzed the expression of ARF6, AMAP1, and phosphorylated (p)-AMAP1 in tumor and adjacent normal tissues. We first observed that CCL18 increased the expression of ARF6 and p-AMAP1 and activated the Src/phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. ARF6 knockdown significantly impaired CCL18-induced malignant cellular behaviors and the Src/PI3K/Akt signaling pathway. Next, ARF6 was confirmed as a target gene of miR-760 in exosomes derived from CCL18-stimulated high metastatic BC cells. Moreover, recipient MCF-7 cells could effectively uptake these miR-760-rich exosomes that significantly promoted proliferation, tumor growth in vivo, migration, invasion, and chemoresistance by activating ARF6-mediated Src/PI3K/Akt signaling and the epithelial-mesenchymal transition (EMT) pathway. Together, our results support that exosomal miR-760 secreted by CCL18-stimulated high metastatic BC cells promoted the malignant behaviors in low metastatic BC cells by up-regulating the ARF6-mediated Src/PI3K/Akt signaling pathway.

Long Non-Coding RNA MDFIC-7 Promotes Chordoma Progression Through Modulating the miR-525-5p/ARF6 Axis.

BackgroundChordoma, an extremely rare malignant tumor, remains difficult to be cured because of its strong local invasiveness and high recurrence rate. Long non-coding RNAs (lncRNAs) have been demonstrated to play multiple roles in various cancers. The purpose of this study was to investigate the modulatory function of lncRNA MDFIC-7 in chordoma and to elucidate its underlying mechanisms.MethodsQuantitative real-time polymerase chain reaction was performed to detect the expression of lncRNA MDFIC-7 in tumor tissues and adjacent nontumorous tissues collected from 15 chordoma patients, as well as in chordoma cell lines. Gene silencing and overexpression experiments were carried out by RNA interference and lentiviral transduction. The effect of lncRNA MDFIC-7 on the proliferation of chordoma cells was evaluated by cell counting kit-8 assay, colony formation assay and xenograft tumor experiments. RNA immunoprecipitation and dual luciferase reporter assays were conducted to evaluate the binding between lncRNA MDFIC-7 and miRNA-525-5p and the interaction between miR-525-5p and the 3′ untranslated region of ADP-ribosylation factor 6 (ARF6) mRNA. The glycolytic capacity and mitochondrial function of chordoma cells were measured by the Seahorse Bioscience XF96 Extracellular Flux Analyzer.ResultsThe expression of lncRNA MDFIC-7 was higher in chordoma tumor tissues than in adjacent non-tumor tissues. Downregulation of lncRNA MDFIC-7 reduced colony formation and cell proliferation in chordoma cells and decreased xenograft tumor growth in a nude mouse model. Moreover, lncRNA MDFIC-7 knockdown attenuated the Warburg effect in chordoma cells and xenograft tumors. LncRNA MDFIC-7 knockdown elevated miR-525-5p levels and decreased ARF6 expressions. Overexpression of ARF6 reversed the inhibitory effect of lncRNA MDFIC-7 knockdown on cell proliferation and the Warburg effect in chordoma cells and xenograft tumors. Mechanistically, lncRNA MDFIC-7, as a molecular sponge of miR-525-5p, negatively regulated miR-525-5p expression and promoted the gene expression of ARF6, a miR-525-5p target.ConclusionOur findings demonstrate that lncRNA MDFIC-7 acts as a molecular sponge to competitively bind to miR-525-5p and promote expression of ARF6. The lncRNA MDFIC-7/miR-525-5p/ARF6 axis regulates chordoma progression and the Warburg effect in chordoma, suggesting that lncRNA MDFIC-7 and miR-525-5p could be promising therapeutic targets for the treatment of chordoma.

MiR-28-3p inhibits prostate cancer cell proliferation, migration and invasion, and promotes apoptosis by targeting ARF6.

Previous studies have reported that the expression levels of microRNA (miR)-28-3p are downregulated in prostate cancer (PCa) compared with those in adjacent normal tissues. However, to the best of our knowledge, the function and underlying mechanisms of miR-28-3p in PCa have not been reported. The present study aimed to explore the role of miR-28-3p and its mechanism in the development of PCa. In the present study, miR-28-3p and ADP-ribosylation factor 6 (ARF6) expression levels were analyzed using reverse transcription-quantitative PCR (RT-qPCR). Cell proliferation, colony formation, apoptosis, migration and invasion were determined using Cell Counting Kit-8, colony forming, flow cytometry and Transwell assays, respectively. The association between miR-28-3p and ARF6 was investigated using a dual luciferase reporter assay. ARF6, Rac1, Erk1/2 and phosphorylated (p)-Erk1/2 protein expression levels were analyzed using western blotting. The results of the present study revealed that miR-28-3p expression levels were downregulated, whereas ARF6 expression levels were upregulated in PCa cell lines (LNCaP, 22Rv-1, PC-3 and DU145) compared with those in the normal prostate line RWPE-1. The overexpression of miR-28-3p promoted cell apoptosis, and inhibited cell proliferation, colony formation, migration and invasion. However, the knockdown of miR-28-3p exerted the opposite results. The results of the dual luciferase reporter assays, RT-qPCR and western blotting indicated that ARF6 was a target gene of miR-28-3p. Finally, rescue experiments demonstrated that ARF6 overexpression attenuated the effects of the miR-28-3p mimic by upregulating Rac1 and p-Erk1/2 expression in PCa cells. In conclusion, these findings indicated that miR-28-3p may inhibit the biological behaviors of PCa cells by targeting ARF6, and therefore may represent a novel therapeutic candidate for PCa.

Association of high gene expression levels of ARF6 with the immune microenvironment and prediction of poor outcomes.

3092 Background: ADP-ribosylation factor 6 ( ARF6) is a small GTPase in the RAS superfamily, which regulates membrane trafficking, remodeling and tumor progression. Preclinical study shows that TP53 and KRAS cooperatively activate the ARF6-AMAP1 pathway which serves as a link by which pancreatic driver mutations promote tumor invasion, PD-L1 dynamics and immune evasion properties in pancreatic ductal adenocarcinoma (PDAC). The clinical impact of ARF6 on cancer progression and prognosis remains unclear. Methods: A total of 2,948 PDAC samples were analyzed using next-generation sequencing of RNA (whole transcriptome, NovaSeq) and DNA (NextSeq, 592 genes or NovaSeq, whole exome sequencing) and immunohistochemistry (IHC) (Caris Life Sciences, Phoenix, AZ). QuantiSeq (Finotello 2019, Genome Medicine) was used to quantify immune cell infiltration. Overall survival (OS) was obtained from insurance claims, and Kaplan-Meier estimates were calculated for molecularly defined cohorts. Significance was determined as p values adjusted for multiple correction ( q) of <.05. Results: Median ARF6 expression was higher in metastases (33.69 transcriptions per million) compared to primary/local tumors (27.59, q<.05). Specific metastatic sites showed higher expression than did primary tumors ( q<.05 for liver and p<.05 for skin, bone and lymph nodes). Dividing into quartiles by ARF6 expression (the highest expression quartile, QH; the lowest, QL), KRAS mutations were significantly more prevalent in QH than QL (93.4 vs 87.2%, q<.05), and TP53 mutations had similar trends (81.0% in QH vs 74.7% in QL, p=.0078). The mutation rates of KDM6A, FANCD2 and TFEB amplifications trended higher in QH than QL; the STK11 mutation rate tended to be lower in QH ( p<.05). PD-L1 expression by IHC was significantly higher in QH than QL (20.9 vs 13.1%); immune checkpoint genes by RNA expression: IFNG, IDO1, PDCD1G2, CD274, PDCD1 and PDCD2L were significantly higher in QH than QL (all q<.05). Macrophages, neutrophils, NK cells, fibroblasts and endothelial cells were more abundant in QH than QL (all q<.05); whereas CD4+ and CD8+ T cells were lower in QH ( q<.05), and monocytes had similar trends ( p<.05). High expression of ARF6 was significantly associated with unfavorable outcomes in OS (HR = 1.83, 95% CI [1.51–2.22], p<.0001); the effect on OS was seen when primary (HR = 1.47, [1.06–2.05], p=.02) and metastatic tumors (HR = 0.608, [1.29–2.10], p<.0001) were investigated separately. Conclusions: This is the first report showing that high gene expression of ARF6 in PDAC indicates a different immune profile, is enriched in cancer metastases, and is associated with poor survival. Our results provide the first clinical evidence supporting the ARF6 pathway as a major downstream target of KRAS and TP53 mutations promoting immune evasion, suggesting ARF6 is a novel marker for prognosis and a potential target for immune therapeutic strategies in PDAC.

Identification of miR-145 as a regulator of the cardiomyocyte inflammatory response and oxidative stress under hyperglycemia.

The current study aimed to explore the effects of microRNA (miR)-145 on the inflammatory response and oxidative stress (OS) in high glucose (HG)-induced cardiomyocytes, as well as the specific mechanism underlying this action. H9c2 cells were treated with 33 mmol/l glucose (HG group) or cotreated with 24.5 mmol/l mannitol and 5.5 mmol/l glucose (hypertonic group), and the expression levels of miR-145 and ADP ribosylation factor 6 (ARF6) were detected. The cells were transfected with pcDNA3.1-ARF6, miR-145 mimics or corresponding negative controls prior to the assessment of cell survival rate. Levels of lactate dehydrogenase (LDH), reactive oxygen species (ROS) and malondialdehyde (MDA), as well as the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and the levels of IL-6, TNF-α and monocyte chemoattractant protein-1 (MCP-1) were subsequently determined. The apoptotic rate of H9c2 cells was examined by flow cytometry. The interaction between miR-145-ARF6 was predicted and confirmed by luciferase reporter assays. In the HG group, miR-145 expression was significantly decreased and ARF6 expression significantly increased compared with controls. Furthermore, the levels of inflammatory factors (IL-6, TNF-α and MCP-1), LDH, ROS and MDA were significantly elevated in the HG group compared with controls. Significantly decreased SOD, CAT and GPx activities and significantly increased numbers of apoptotic cells were observed in the HG group compared with controls. The cells transfected with miR-145 mimics exhibited significantly decreased LDH, ROS and MDA levels, significantly increased antioxidant enzyme activities and significantly decreased apoptotic rates compared with controls, while the opposite results were observed in cells transfected with pcDNA3.1-ARF6. Moreover, co-transfection with miR-145 mimics and pcDNA3.1-ARF6 exacerbated the inflammatory response and OS injury in HG-induced cardiomyocytes compared with cells transfected with miR-145 mimics alone. Furthermore, miR-145 negatively targeted ARF6. miR-145 attenuated the HG-induced inflammatory response and OS injury in cardiomyocytes by negatively regulating ARF6, which may contribute to providing a theoretical basis for the treatment of diabetic cardiomyopathy.

Sevoflurane Suppresses the Migration, Invasion, and Epithelial-Mesenchymal Transition of Breast Cancer Cells Through the miR-139-5p/ARF6 Axis

BackgroundBreast cancer (BC) is common cancer in female globally. Sevoflurane (SEV) has been reported to inhibit the metastasis of multiple cancers, including glioma, colorectal cancer, and hepatocellular carcinoma. However, the role of SEV in the metastasis of BC cells remains poorly understood. MethodsTranswell migration and invasion assays were performed to detect the migration and invasion of BC cells. Western blot assay was carried out to measure epithelial-mesenchymal transition (EMT)-related proteins in BC cells, including E-cadherin, N-cadherin, and fibronectin. Quantitative real-time polymerase chain reaction was conducted to determine the enrichment of miR-139-5p and ADP-ribosylation factor 6 (ARF6) in BC tissues and cells. The protein expression of ARF6 in BC tissues and cells was measured by western blot assay. The target of miR-139-5p was predicted by starBase software, and the target relationship between miR-139-5p and ARF6 in BC cells was confirmed by dual-luciferase reporter assay. ResultsSEV suppressed the migration, invasion, and EMT of BC cells, especially in the high-concentration SEV group. The level of miR-139-5p was lower in BC tissues and cells than that in paired normal tissues and normal mammary epithelial cells MCF-10A. MiR-139-5p was upregulated in BC cells treated with SEV. ARF6 was upregulated in BC tissues and cells compared with that in corresponding normal tissues and normal mammary epithelial cells MCF-10A. SEV reduced the mRNA and protein expression of ARF6 in BC cells. The accumulation of ARF6 or the interference of miR-139-5p reversed the suppressive effects of SEV treatment on the migration, invasion, and EMT of BC cells. MiR-139-5p bound to ARF6 and inversely modulated the level of ARF6 in BC cells. The transfection of si-ARF6 attenuated the promoting effects of miR-139-5p depletion on the migration, invasion, and EMT of BC cells treated with SEV. ConclusionsSEV suppressed the migration, invasion, and EMT of BC cells through downregulating the abundance of ARF6 by upregulating miR-139-5p. The miR-139-5p/ARF6 axis might be a promising target for the treatment of BC.

ARF6-Rac1 signaling-mediated neurite outgrowth is potentiated by the neuronal adaptor FE65 through orchestrating ARF6 and ELMO1.

Ras-related C3 botulinum toxin substrate 1 (Rac1) is a member of the Rho family of GTPases that functions as a molecular switch to regulate many important cellular events including actin cytoskeleton remodeling during neurite outgrowth. Engulfment and cell motility 1 (ELMO1)-dedicator of cytokinesis 1 (DOCK180) is a bipartite guanine nucleotide exchange factor (GEF) complex that has been reported to activate Rac1 on the plasma membrane (PM). Emerging evidence suggests that the small GTPase ADP ribosylation factor 6 (ARF6) activates Rac1 via the ELMO1/DOCK180 complex. However, the exact mechanism by which ARF6 triggers ELMO1/DOCK180-mediated Rac1 signaling remains unclear. Here, we report that the neuronal scaffold protein FE65 serves as a functional link between ARF6 and ELMO1, allowing the formation of a multimeric signaling complex. Interfering with formation of this complex by transfecting either FE65-binding-defective mutants or FE65 siRNA attenuates both ARF6-ELMO1-mediated Rac1 activation and neurite elongation. Notably, the PM trafficking of ELMO1 is markedly decreased in cells with suppressed expression of either FE65 or ARF6. Likewise, this process is attenuated in the FE65-binding-defective mutants transfected cells. Moreover, overexpression of FE65 increases the amount of ELMO1 in the recycling endosome, an organelle responsible for returning proteins to the PM, whereas knockout of FE65 shows opposite effect. Together, our data indicates that FE65 potentiates ARF6-Rac1 signaling by orchestrating ARF6 and ELMO1 to promote the PM trafficking of ELMO1 via the endosomal recycling pathway, and thus, promotes Rac1-mediated neurite outgrowth.

MicroRNA-145 suppresses cell migration and invasion in upper tract urothelial carcinoma by targeting ARF6.

ADP-ribosylation factor 6 (ARF6) is a well-studied protein that is involved in multiple biological functions including cell migration and invasion. The mechanism by which ARF6 regulates the migration and invasion of upper tract urothelial carcinoma (UTUC) is still unknown. MiR-145-5p is a tumor suppressor microRNA, which is downregulated in several cancer types. We aimed to elucidate the molecular mechanism underlying the regulation of ARF6 by miR-145-5p in UTUC. ARF6 expression was observed to be higher in UTUC tissues than paired adjacent normal tissues. A reverse correlation between ARF6 and miR-145-5p was found in UTUC tissues. MiR-145-5p inhibited ARF6 expression by directly targeting its 3'-UTR. The functional studies indicated that ARF6 expression reversed the miR-145-5p-reduced tumor cell migration and invasion. Notably, miR-145-5p reduced MMP2, N-cadherin, FAK and MMP7, and elevated E-cadherin protein levels in vitro; however, the above effects were reversed by ARF6. Further, the expression of epithelial-to-mesenchymal transition (EMT) markers and cell invasion was suppressed by knocking down MMP7 in UTUC cells. These findings suggest that miR-145-5p may suppress UTUC cell motility and invasion by targeting ARF6/MMP7 through EMT.

ApoE4 Alters ABCA1 Membrane Trafficking in Astrocytes.

The APOE ε4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease (AD). ApoE protein aggregation plays a central role in AD pathology, including the accumulation of β-amyloid (Aβ). Lipid-poor ApoE4 protein is prone to aggregate and lipidating ApoE4 protects it from aggregation. The mechanisms regulating ApoE4 aggregation in vivo are surprisingly not known. ApoE lipidation is controlled by the activity of the ATP binding cassette A1 (ABCA1). ABCA1 recycling and degradation is regulated by ADP-ribosylation factor 6 (ARF6). We found that ApoE4 promoted greater expression of ARF6 compared with ApoE3, trapping ABCA1 in late-endosomes and impairing its recycling to the cell membrane. This was associated with lower ABCA1-mediated cholesterol efflux activity, a greater percentage of lipid-free ApoE particles, and lower Aβ degradation capacity. Human CSF from APOE ε4/ε4 carriers showed a lower ability to induce ABCA1-mediated cholesterol efflux activity and greater percentage of aggregated ApoE protein compared with CSF from APOE ε3/ε3 carriers. Enhancing ABCA1 activity rescued impaired Aβ degradation in ApoE4-treated cells and reduced both ApoE and ABCA1 aggregation in the hippocampus of male ApoE4-targeted replacement mice. Together, our data demonstrate that aggregated and lipid-poor ApoE4 increases ABCA1 aggregation and decreases ABCA1 cell membrane recycling. Enhancing ABCA1 activity to reduce ApoE and ABCA1 aggregation is a potential therapeutic strategy for the prevention of ApoE4 aggregation-driven pathology.SIGNIFICANCE STATEMENT ApoE protein plays a key role in the formation of amyloid plaques, a hallmark of Alzheimer's disease (AD). ApoE4 is more aggregated and hypolipidated compared with ApoE3, but whether enhancing ApoE lipidation in vivo can reverse ApoE aggregation is not known. ApoE lipidation is controlled by the activity of the ATP binding cassette A1 (ABCA1). In this study, we demonstrated that the greater propensity of lipid-poor ApoE4 to aggregate decreased ABCA1 membrane recycling and its ability to lipidate ApoE. Importantly, enhancing ABCA1 activity to lipidate ApoE reduced ApoE and ABCA1 aggregation. This work provides critical insights into the interactions among ABCA1, ApoE lipidation and aggregation, and underscores the promise of stabilizing ABCA1 activity to prevent ApoE-driven aggregation pathology.

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.

Abstract 5157: ARF6 controls WNT5A receptor internalization to promote uveal melanoma invasion and metastasis

Abstract Introduction: Uveal melanoma is the most common primary ocular malignancy and there is currently no effective treatment for metastatic uveal melanoma largely because of the lack of understanding of the molecular mechanisms underlying this cancer. Although activating oncogenic mutations in GNAQ and GNA11 are present in 95% of uveal melanoma tumors, virtually nothing is known about the molecular mechanisms that drive oncogenesis in the remaining 5% of uveal melanomas that possess only wild type Gαq alleles and the molecular and cellular mechanisms that promote metastasis have not yet been discovered. This latter deficit in knowledge is extremely vexing, given that metastatic disease causes death in most uveal melanoma patients. We have recently shown that ADP-ribosylation factor 6 (ARF6), a small GTPase, is an immediate downstream effector of oncogenic Gαq and controls the entire major signaling pathways known to drive Gαq-mediated tumor formation and growth in uveal melanoma cells that have an activating mutation in Gαq. Moreover, in cutaneous melanoma, stimulation of the receptor by WNT5A activates ARF6 and controls metastasis via the release of β-catenin from N-cadherin and β-catenin's subsequent nuclear transport. Previous studies have also demonstrated that ARF6 plays a role in the internalization of various classes of membrane receptors including receptor tyrosine kinase (RTK) and G protein coupled receptor (GPCR) to control signal transduction, and WNT5A and its receptor promote tumor invasion and metastasis via receptor internalization in human cancers. Based on these intriguing findings, we postulate that ARF6 might be functioning to control tumor invasion and metastasis in uveal melanomas. Experimental Procedures: To test our hypothesis, we conducted cell invasion assays on uveal melanoma cell lines while targeting ARF6 expression by siRNA or ARF6 activity by pharmacological inhibition. We then proceeded with cell fractionation to look for any differences in the properties of subcellular components in treated cells compared to untreated cells. Unpublished findings: Here we show that WNT5A is expressed at high levels in Gαq wild type uveal melanoma cells and that WNT5A activates ARF6 through ROR2 receptor. When we block ARF6 expression by knockdown or ARF6 activity with a small molecule inhibitor, uveal melanoma invasion is significantly reduced. Notably, ARF6 is necessary for WNT5A-mediated ROR2 internalization. Conclusions: This work indicates that ARF6 functions in the internalization of WNT5A receptor to activate the signaling pathways that drive invasion and metastasis in uveal melanoma. These results would suggest that targeting ARF6 activation might be an effective therapy for the treatment of all uveal melanomas, regardless of their mutational status for Gαq. Citation Format: Donghan Shin, Coulson P. Rich, Lehi Acosta, Jackson R. Richards, Jae Hyuk Yoo, Allie H. Grossmann, Zongzhong Tong, Alan L. Mueller, Weiquan Zhu, Dean Y. Li, Shannon J. Odelberg. ARF6 controls WNT5A receptor internalization to promote uveal melanoma invasion and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5157.

Progesterone and calcitriol reduce invasive potential of endometrial cancer cells by targeting ARF6, NEDD9 and MT1-MMP.

Previously, we have demonstrated that progesterone and calcitriol synergistically inhibit growth of endometrial and ovarian cancer by enhancing apoptosis and causing cell cycle arrest. Metastasis is the main reason of mortality in cancer patients. Activation of ADP-Ribosylation Factor 6 (ARF6), Neural Precursor cell expressed Developmentally Downregulated 9 (NEDD9), and Membrane-Type-1 Matrix Metalloproteinase (MT1-MMP) have been implicated in promoting tumor growth and metastasis. We examined the effects of progesterone, calcitriol and progesterone-calcitriol combination on metastasis promoting proteins in endometrial cancer. Expression of ARF6, NEDD9, and MT1-MMP was enhanced in advanced-stage endometrial tumors and in cancer cell lines compared to normal tissues and immortalized EM-E6/E7-TERT endometrial epithelial cells. Knockdown of these proteins significantly inhibited the invasiveness of the cancer cells. The expression levels of all three proteins was reduced with progesterone and progesterone-calcitriol combination treatment, whereas calcitriol alone showed no effect on their expression but moderately decreased MT1-MMP activity. Fluorescence microscopy showed membrane expression of MT1-MMP in vehicle and calcitriol-treated endometrial cancer cells. However, progesterone and calcitriol-progesterone combination treatment revealed MT1-MMP in the cytoplasm. Furthermore, progesterone and calcitriol reduced the activity of MT1-MMP, MMP-9, and MMP-2. In addition, invadopodia regulatory proteins were attenuated in both progesterone and progesterone-calcitriol combination treated cells as well as in MT1-MMP knockdown cells. Thus, targeting the aberrant MT1-MMP signaling with progesterone-calcitriol may be a novel approach to impede MT1-MMP mediated cancer dissemination and may have therapeutic benefits for endometrial cancer patients.

ARF6 mediates nephrin tyrosine phosphorylation-induced podocyte cellular dynamics.

ADP-ribosylation factor 6 (ARF6) is a small GTPase necessary for regulating cellular structure, motility, and vesicle trafficking. In several cellular systems, ARF6 was shown to regulate actin dynamics in coordination with Rac1, a Rho small GTPase. We examined the function of ARF6 in the kidney podocyte because Rac1 was implicated in kidney diseases involving this cell. We found that ARF6 expression was enriched in human podocytes and that it modulated podocyte cytoskeletal dynamics through a functional interaction with nephrin, an intercellular junction protein necessary for podocyte injury-induced signaling requiring activation by tyrosine phosphorylation of its cytoplasmic domain. ARF6 was necessary for nephrin activation-induced ruffling and focal adhesion turnover, possibly by altering Rac1 activity. In podocyte-specific Arf6 (ARF6_PodKO) knockout mice, ARF6 deficiency did not result in a spontaneous kidney developmental phenotype or proteinuria after aging. However, ARF6_PodKO mice exhibited distinct phenotypes in two in vivo glomerular injury models. In the protamine sulfate perfusion model, which induced acute podocyte effacement, ARF6_PodKO mice were protected from podocyte effacement. In the nephrotoxic serum nephritis model, which induced immune-complex mediated injury, ARF6_PodKO mice exhibited aggravated proteinuria. Together, these observations suggest that while ARF6 is necessary for nephrin tyrosine phosphorylation-induced cytoskeletal dynamics in cultured podocytes, ARF6 has pleotropic podocyte roles in vivo, where glomerular injury-specific mechanisms might activate distinct signaling pathways that dictate whether ARF6 activity is beneficial or deleterious for maintaining the integrity of the glomerular filtration barrier.

ARF6, induced by mutant Kras, promotes proliferation and Warburg effect in pancreatic cancer

Though significant progress has been made in the availability of diagnostic techniques and treatment strategies, pancreatic cancer remains a disease of high mortality rates. Therefore, there is an urgent need for a better understanding of the molecular mechanisms that governs the oncogenesis and metastasis process of pancreatic cancer. In our study, by using the Cancer Genome Atlas (TCGA) dataset analysis, we demonstrated that the small guanosine triphosphatase (GTPase) ADP-ribosylation factor 6 (ARF6) serves as a biomarker for predicting prognosis of pancreatic cancer. In vitro studies demonstrated that silencing ARF6 expression reduced cell proliferation and attenuated the Warburg effect. Moreover, we observed that ARF6 was a downstream target of Kras/ERK signaling pathway, and the strong correlation of expression between Kras and ARF6 in the TCGA dataset further confirmed this observation. Taken together, our novel findings suggest ARF6, a target of mutant Kras, may promote pancreatic cancer development by enhancing the Warburg effect.