DOCK2 is involved in the host genetics and biology of severe COVID-19.

Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge1-5. Here we conducted a genome-wide association study (GWAS) involving 2,393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3,289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target.

Liquiritigenin alleviates doxorubicin-induced chronic heart failure via promoting ARHGAP18 and suppressing RhoA/ROCK1 pathway.

Chronic heart failure (CHF) is one of the most common chronic diseases with increasing incidence and mortality. Liquiritigenin (LQG) is shown to protect mice from cardiotoxicity. However, its underlying mechanism remains unclear. Our study aimed to reveal the role of ARHGAP18 in LQG-mediated cardioprotective effects in CHF. In the current study, CHF cell model and rat model were established by the application of doxorubicin (DOX). The reactive oxygen species (ROS) level and cell apoptosis were determined by flow cytometry. The cardiac function of rats was evaluated by measuring left ventricular systolic pressure, left ventricular end diastolic pressure, and serum level of lactate dehydrogenase and brain natriuretic peptide. The expression of active RhoA was elevated and that of ARHGAP18 was decreased in DOX-induced CHF cell model. ARHGAP18 could reduce DOX-induced RhoA activation, ROS elevation, and cell apoptosis. Meanwhile, the knockdown of ARHGAP18 could promote the activation of RhoA, the level of ROS, and the rate of cell apoptosis, which could be reversed by the application of RhoA inhibitor. LQG promoted the expression of ARHGAP18 and exerted similar effects of ARHGAP18 in CHF cell model. The application of LQG could also reverse the effects mediated by ARHGAP18 knockdown. Moreover, LQG significantly improved cardiac function and ameliorated DOX-induced cardiotoxicity of CHF rats. In conclusion, LQG could alleviate DOX-induced CHF via promoting ARHGAP18 and suppressing RhoA/ROCK1 pathway. LQG was a potential agent for CHF treatment.

Downregulation of Rap1GAP Expression Activates the TGF-ß/Smad3 Pathway to Inhibit the Expression of Sodium/Iodine Transporter in Papillary Thyroid Carcinoma Cells.

OBJECTIVE: Rap1GAP is considered a tumor suppressor gene, but its regulatory mechanism in papillary thyroid cancer (PTC) has not been clearly elucidated. The aim of this study was to explore whether the regulation between Rap1GAP and sodium/iodine transporter (NIS) in tumorigenesis of PTC is mediated by TGF-ß1. METHODS: Western blotting (WB) and quantitative reverse-transcription polymerase chain reaction were performed to analyze the relationships between TGF-ß1 concentration and NIS expression. After transfecting BCPAP cells with siRNAs, the Rap1GAP interference model was successfully established. Then, the expression and nuclear localization of TGF-ß1 and pathway-related proteins were detected. Flow cytometry was applied to analyze cell apoptosis and cycle. WB was performed to detect apoptotic-related proteins. Wound healing and transwell assays were used to measure cell migration and invasion. EDU was performed to detect cell proliferative activity. RESULTS: The results suggested that TGF-ß1 could significantly inhibit the expression of NIS in both mRNA and protein levels. In BCPAP cells transfected with siRNA-Rap1GAP, the expression levels of TGF-ß1, Foxp3, and p-Smad3 were significantly increased. By applying immunofluorescence assay, the nuclear localizations of TßR-1 and p-Smad3 were found to be activated. Moreover, anti-TGF-ß1 can reverse the decrease in NIS expression caused by downregulation of Rap1GAP. Additionally, the knockdown of Rap1GAP could alter the cell apoptosis, cycle, migration, invasion, and proliferation of BCPAP. CONCLUSION: The downregulation of Rap1GAP expression can activate the TGF-ß/Smad3 pathway to inhibit NIS expression and alter the tumor cell functions of PTC.

Cytohesin-2 mediates group I metabotropic glutamate receptor-dependent mechanical allodynia through the activation of ADP ribosylation factor 6 in the spinal cord.

Group I metabotropic glutamate receptors (mGluRs), mGluR1 and mGluR5, in the spinal cord are implicated in nociceptive transmission and plasticity through G protein-mediated second messenger cascades leading to the activation of various protein kinases such as extracellular signal-regulated kinase (ERK). In this study, we demonstrated that cytohesin-2, a guanine nucleotide exchange factor for ADP ribosylation factors (Arfs), is abundantly expressed in subsets of excitatory interneurons and projection neurons in the superficial dorsal horn. Cytohesin-2 is enriched in the perisynapse on the postsynaptic membrane of dorsal horn neurons and forms a protein complex with mGluR5 in the spinal cord. Central nervous system-specific cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia in inflammatory and neuropathic pain models. Pharmacological blockade of cytohesin catalytic activity with SecinH3 similarly reduced mechanical allodynia and inhibited the spinal activation of Arf6, but not Arf1, in both pain models. Furthermore, cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia and ERK1/2 activation following the pharmacological activation of spinal mGluR1/5 with 3,5-dihydroxylphenylglycine (DHPG). The present study suggests that cytothesin-2 is functionally associated with mGluR5 during the development of mechanical allodynia through the activation of Arf6 in spinal dorsal horn neurons.

The Molecular Basis of COVID-19 Pathogenesis, Conventional and Nanomedicine Therapy.

In late 2019, a new member of the Coronaviridae family, officially designated as "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2), emerged and spread rapidly. The Coronavirus Disease-19 (COVID-19) outbreak was accompanied by a high rate of morbidity and mortality worldwide and was declared a pandemic by the World Health Organization in March 2020. Within the Coronaviridae family, SARS-CoV-2 is considered to be the third most highly pathogenic virus that infects humans, following the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV). Four major mechanisms are thought to be involved in COVID-19 pathogenesis, including the activation of the renin-angiotensin system (RAS) signaling pathway, oxidative stress and cell death, cytokine storm, and endothelial dysfunction. Following virus entry and RAS activation, acute respiratory distress syndrome develops with an oxidative/nitrosative burst. The DNA damage induced by oxidative stress activates poly ADP-ribose polymerase-1 (PARP-1), viral macrodomain of non-structural protein 3, poly (ADP-ribose) glycohydrolase (PARG), and transient receptor potential melastatin type 2 (TRPM2) channel in a sequential manner which results in cell apoptosis or necrosis. In this review, blockers of angiotensin II receptor and/or PARP, PARG, and TRPM2, including vitamin D3, trehalose, tannins, flufenamic and mefenamic acid, and losartan, have been investigated for inhibiting RAS activation and quenching oxidative burst. Moreover, the application of organic and inorganic nanoparticles, including liposomes, dendrimers, quantum dots, and iron oxides, as therapeutic agents for SARS-CoV-2 were fully reviewed. In the present review, the clinical manifestations of COVID-19 are explained by focusing on molecular mechanisms. Potential therapeutic targets, including the RAS signaling pathway, PARP, PARG, and TRPM2, are also discussed in depth.

Tumor suppressor gene DLC1: Its modifications, interactive molecules, and potential prospects for clinical cancer application.

Deleted in liver cancer 1 (DLC1) is a recognized tumor suppressor gene that negatively regulates Rho family proteins by hydrolyzing the active GTP-bound state to its inactive GDP-bound state. Active Rho proteins play a positive role in tumorigenesis. Numerous in vitro and in vivo experiments have shown that DLC1 is downregulated or inactivated in various solid tumors, which may be due to the following five reasons: genomic deletion, epigenetic modification and ubiquitin-dependent proteasomal degradation may cause DLC1 underexpression; phosphorylation at the post-translation level may cause DLC1 inactivation; and failure to localize at focal adhesions (FAs) may prevent DLC1 from exerting full activity. All of the causes could be attributed to molecular binding. Experimental evidence suggests that direct or indirect targeting of DLC1 is feasible for cancer treatment. Therefore, elucidating the interaction of DLC1 with its binding partners might provide novel targeted therapies for cancer. In this review, we summarized the binding partners of DLC1 at both the gene and protein levels and expounded a variety of anticancer drugs targeting DLC1 to provide information about DLC1 as a cancer diagnostic indicator or therapeutic target.

5-((7-Chloro-6-fluoro-1h-indol-3-yl) methyl)-3-methylimidazolidine-2,4-dione as a RIP1 inhibitor protects LPS/D-galactosamine-induced liver failure.

AIMS: Necroptosis, an inflammatory form of regulated necrosis mediated by receptor-interacting kinase 1 (RIP1), RIP3, and pseudokinase mixed lineage kinase domain-like protein (MLKL) is extensively implicated in liver inflammatory disease. Thus identification small-molecule inhibitor of necroptosis has emerged as a potential therapeutic strategy to prevent liver damage. In this study, we identified 5-((7-chloro-6-fluoro-1 h-indol-3-yl) methyl)-3-methylimidazolidine-2,4-dione (F-nec) as a novel potent necroptosis inhibitor. MAIN METHODS: To find out the potent chemical inhibitors of necroptosis, human monocytic U937 cells were treated with a combination of tumor necrosis factor alpha (TNFα) and a pan-caspase inhibitor z-VAD-fmk. LPS and D-galactosamine (LPS/GalN) were further employed to simulate acute liver failure to explore therapeutic potency of F-nec in vivo. In addition, a specific inhibitor of c-Jun NH (2)-terminal kinases (JNK) SP600125 and its activator anisomycin are used to elucidate its mechanisms in acute liver failure therapy. Necroptosis pathway related proteins were tested by western blot. KEY FINDINGS: In this study, we identified F-nec as a novel potent RIP1 inhibitor which efficiently blocked TNFα-induced necroptosis in human and mice cells. Furthermore, pre-treatment of F-nec could prevent hepatic necrosis by reducing RIP1-mediated necroptosis also effectively ameliorated LPS/GalN induced acute liver failure by attenuating cell death signaling-stimulated JNK pathway activation and then suppressing JNK-triggered inflammation. SIGNIFICANCE: Altogether, this study demonstrates that F-nec is a potent inhibitor of RIP1 and highlights its great potential for use in the treatment of RIP1-driven inflammatory liver diseases.

DOCK7 protects against replication stress by promoting RPA stability on chromatin.

RPA is a critical factor for DNA replication and replication stress response. Surprisingly, we found that chromatin RPA stability is tightly regulated. We report that the GDP/GTP exchange factor DOCK7 acts as a critical replication stress regulator to promote RPA stability on chromatin. DOCK7 is phosphorylated by ATR and then recruited by MDC1 to the chromatin and replication fork during replication stress. DOCK7-mediated Rac1/Cdc42 activation leads to the activation of PAK1, which subsequently phosphorylates RPA1 at S135 and T180 to stabilize chromatin-loaded RPA1 and ensure proper replication stress response. Moreover, DOCK7 is overexpressed in ovarian cancer and depleting DOCK7 sensitizes cancer cells to camptothecin. Taken together, our results highlight a novel role for DOCK7 in regulation of the replication stress response and highlight potential therapeutic targets to overcome chemoresistance in cancer.

RLIP depletion induces apoptosis associated with inhibition of JAK2/STAT3 signaling in melanoma cells.

The incidence of malignant melanoma, a neoplasm of melanocytic cells, is increasing rapidly. The lymph nodes are often the first site of metastasis and can herald systemic dissemination, which is almost uniformly fatal. RLIP, a multi-specific ATP-dependent transporter that is over-expressed in several types of cancers, plays a central role in cancer cell resistance to radiation and chemotherapy. RLIP appears to be necessary for cancer cell survival because both in vitro cell culture and in vivo animal tumor studies show that the depletion or inhibition of RLIP causes selective toxicity to malignant cells. RLIP depletion/inhibition triggers apoptosis in cancer cells by inducing the accumulation of endogenously formed glutathione-conjugates. In our in vivo studies, we administered RLIP antibodies or antisense oligonucleotides to mice bearing subcutaneous xenografts of SKMEL2 and SKMEL5 melanoma cells and demonstrated that both treatments caused significant xenograft regression with no apparent toxic effects. Anti-RLIP antibodies and antisense, which respectively inhibit RLIP-mediated transport and deplete RLIP expression, showed similar tumor regressing activities, indicating that the inhibition of RLIP transport activity at the cell surface is sufficient to achieve anti-tumor activity. Furthermore, RLIP antisense treatment reduced levels of RLIP, pSTAT3, pJAK2, pSrc, Mcl-1 and Bcl2, as well as CDK4 and cyclin B1, and increased levels of Bax and phospho 5' AMP-activated protein kinase (pAMPK). These studies indicate that RLIP serves as a key effector in the survival of melanoma cells and is a valid target for cancer therapy. Overall, compounds that inhibit, deplete or downregulate RLIP will function as wide-spectrum agents to treat melanoma, independent of common signaling pathway mutations.

AraC-FdUMP[10] Is a Next-Generation Fluoropyrimidine with Potent Antitumor Activity in PDAC and Synergy with PARG Inhibition.

AraC-FdUMP[10] (CF10) is a second-generation polymeric fluoropyrimidine that targets both thymidylate synthase (TS), the target of 5-fluorouracil (5-FU), and DNA topoisomerase 1 (Top1), the target of irinotecan, two drugs that are key components of FOLFIRNOX, a standard-of-care regimen for pancreatic ductal adenocarcinoma (PDAC). We demonstrated that F10 and CF10 are potent inhibitors of PDAC cell survival (in multiple cell lines including patient-derived lines) with IC50s in the nanomolar range and are nearly 1,000-fold more potent than 5-FU. The increased potency of CF10 relative to 5-FU correlated with enhanced TS inhibition and strong Top1 cleavage complex formation. Furthermore, CF10 displayed single-agent activity in PDAC murine xenografts without inducing weight loss. Through a focused drug synergy screen, we identified that combining CF10 with targeting the DNA repair enzyme, poly (ADP-ribose) glycohydrolase, induces substantial DNA damage and apoptosis. This work moves CF10 closer to a clinical trial for the treatment of PDAC. IMPLICATIONS: CF10 is a promising polymeric fluoropyrimidine with dual mechanisms of action (i.e., TS and Top1 inhibition) for the treatment of PDAC and synergizes with targeting of DNA repair. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/4/565/F1.large.jpg.

DOCK2 contributes to endotoxemia-induced acute lung injury in mice by activating proinflammatory macrophages.

Dedicator of cytokinesis 2 (DOCK2), an atypical Rac activator, has important anti-inflammatory properties in blepharitis, enteric bacterial infection and colitis. However, the roles of DOCK2 in macrophage activation and acute lung injury (ALI) are still poorly elucidated. In vitro studies demonstrated that DOCK2 was essential for the nucleotide-sensing Toll-like receptor (TLR) 4-mediated inflammatory response in macrophages. We also confirmed that exposure of macrophages to LPS induced Rac activation through a TLR4-independent, DOCK2-dependent mechanism. Phosphorylation of IκB kinase (IKK) ß and nuclear translocation of transcription factor nuclear factor kappa B (NF-κB) were impaired in Ad-shDOCK2-expressing macrophages, resulting in a decreased inflammatory response. Similar results were obtained when EHop-016 (a Rac inhibitor) was used to treat uninfected macrophages. In summary, these data indicate that the DOCK2-Rac signaling pathway acts in parallel with TLR4 engagement to control IKKß activation for inflammatory cytokine release. Next, we investigated whether pharmacological inhibition of DOCK2 protects against endotoxemia-induced lung injury in mice. Treatment with 4-[3'-(2″-chlorophenyl)-2'-propen-1'-ylidene]-1-phenyl-3,5-pyrazolidinedione (CPYPP), a small-molecule inhibitor of DOCK2, reduced the severity of lung injury, as indicated by decreases in the lung injury score and myeloperoxidase (MPO) activity. Moreover, CPYPP attenuated LPS-induced proinflammatory cytokine release in mice. Our studies suggest that inhibition of DOCK2 may suppress LPS-induced macrophage activation and that DOCK2 may be a novel target for treating endotoxemia-related ALI.

Loss of Family with Sequence Similarity 13, Member A Exacerbates Pulmonary Fibrosis Potentially by Promoting Epithelial to Mesenchymal Transition.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease with poor prognosis due to limited clinical treatment options. IPF is characterized by the augmented deposition of extracellular matrix driven by myofibroblasts, and the epithelial-mesenchymal transition (EMT) has been known to play an essential role in the mechanism of pulmonary fibrosis. Previous genome-wide association study identified Fam13a as one of genes that showed genetic link with IPF and chronic obstructive pulmonary disease. Here, we analyzed the role of Fam13a in the pathogenesis of pulmonary fibrosis using Fam13a-deficient mice. We found that Fam13a was down-regulated in mouse lungs of bleomycin-induced pulmonary fibrosis model. Of note, genetic deletion of Fam13a exacerbated the lung fibrosis induced by bleomycin in association with enhanced EMT in mice. Moreover, silencing of Fam13a accelerated EMT induced by TGF-ß and TNF-α in alveolar epithelial cells, accompanied by increased active ß-catenin and its nuclear accumulation. Our data revealed a crucial role of Fam13a in the development of pulmonary fibrosis potentially through inhibiting EMT, and thus Fam13a has a therapeutic potential in the treatment of IPF.

Quantitative proteomic analysis reveals that the Rap1/MAPK/ERK pathway is inhibited through selenomethionine strengthening antioxidant activity.

To investigate the influence on the proteome of chicken skeletal muscles of Selenomethionine (SeMet) use, 36 chicks were fed with SeMet feeding for 35 days. A total of 72 1-day old broiler chicks were randomly allocated into two groups (n = 36/group): the control group (C group), the SeMet supplemented group (SeMet group). The Selenium (Se) concentrations of skeletal muscles from the chicks with basal diet (negative control group) and SeMet feeding were found to be 0.01 mg/kg and 0.40 mg/kg, respectively. The skeletal muscles from the two groups were investigated using isobaric Tags for Relative and Absolute Quantitation (iTRAQ), coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. This proteomic analysis identified proteins that were differentially expressed between the two groups. A total of 3564 proteins from the SeMet and the control (C) groups at 35 days were analyzed. 86 proteins were found by iTRAQ to be differentially expressed in the SeMet group, including 38 up-regulated proteins and 48 down-regulated proteins. These differential proteins were later identified as being mainly involved in antioxidant and enzyme-regulating activities. Fluorescent quantitative PCR(qPCR) and Western blot analyse proved to be consistent with the results of iTRAQ identification. The differentially expressed proteins (DEPs) identified in our work could be specific biomarkers related to SeMet intake in chicks. SeMet intake may strengthen antioxidant activity through Rap1/mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinase (ERK) signal pathways.

RIP1 kinase mediates angiogenesis by modulating macrophages in experimental neovascularization.

Inflammation plays an important role in pathological angiogenesis. Receptor-interacting protein 1 (RIP1) is highly expressed in inflammatory cells and is known to play an important role in the regulation of apoptosis, necroptosis, and inflammation; however, a comprehensive description of its role in angiogenesis remains elusive. Here, we show that RIP1 is abundantly expressed in infiltrating macrophages during angiogenesis, and genetic or pharmacological inhibition of RIP1 kinase activity using kinase-inactive RIP1K45A/K45A mice or necrostatin-1 attenuates angiogenesis in laser-induced choroidal neovascularization, Matrigel plug angiogenesis, and alkali injury-induced corneal neovascularization in mice. The inhibitory effect on angiogenesis is mediated by caspase activation through a kinase-independent function of RIP1 and RIP3. Mechanistically, infiltrating macrophages are the key target of RIP1 kinase inhibition to attenuate pathological angiogenesis. Inhibition of RIP1 kinase activity is associated with caspase activation in infiltrating macrophages and decreased expression of proangiogenic M2-like markers but not M1-like markers. Similarly, in vitro, catalytic inhibition of RIP1 down-regulates the expression of M2-like markers in interleukin-4-activated bone marrow-derived macrophages, and this effect is blocked by simultaneous caspase inhibition. Collectively, these results demonstrate a nonnecrotic function of RIP1 kinase activity and suggest that RIP1-mediated modulation of macrophage activation may be a therapeutic target of pathological angiogenesis.

Brajendra Tripathi: Keeping an eye out for translational research.

Tripathi investigates how the tumor suppressor DLC1 is regulated by oncogenic kinases.

DEPDC1 drives hepatocellular carcinoma cell proliferation, invasion and angiogenesis by regulating the CCL20/CCR6 signaling pathway.

DEP domain containing 1 (DEPDC1) functions as an oncogene in hepatocellular carcinoma (HCC). However, the underlying mechanism of DEPDC1 remains largely unknown. The present study revealed that DEPDC1 knockdown inhibited HCC cell proliferation, colony formation and invasion in vitro and suppressed the growth of HCC xenografts in vivo. Furthermore, DEPDC1 overexpression promoted HCC cell proliferation, colony formation and invasion. DNA microarray, reverse transcription­quantitative­PCR and western blotting results demonstrated that DEPDC1 knockdown in Huh­7 significantly inhibited the expression of chemokine (C­C motif) ligand 20 (CCL20) and chemokine (C­C motif) receptor 6 (CCR6). In addition, the expression of CCL20 and CCR6 were upregulated in HCC tissues and cell lines, and were positively correlated with DEPDC1 expression. CCL20 or CCR6 knockdown via small interfering RNA reversed the effects of DEPDC1 overexpression in HCC cells. Furthermore, it was revealed that conditioned medium from DEPDC1 upregulated Li­7 and Hep3B cells led to angiogenesis in vitro, whereas CCL20 knockdown in Li­7 and Hep3B cells or CCR6 knockdown in human umbilical vein endothelial cells reversed the angiogenic effect of DEPDC1 overexpression. In conclusion, DEPDC1 facilitated cell proliferation, invasion and angiogenesis via the CCL20/CCR6 pathway in HCC.

ARHGAP15 regulates lung cancer cell proliferation and metastasis via the STAT3 pathway.

OBJECTIVE: Lung cancer, which is typically diagnosed at later stages, is a leading cause of cancer death among both males and females given its highly invasive and rapidly metastasizing nature. Rho GTPase activating protein 15 (ARHGAP15) is a member of the RhoGAP family and functions in multiple biological processes, such as cell proliferation and migration. However, the effect of ARHGAP15 in lung cancer and its underlying molecular mechanisms remain unclear. PATIENTS AND METHODS: In this study, immunohistochemistry and Real Time PCR were performed to detect ARHGAP15 expression in lung cancer tissues and cells. Proliferation, transwell, and Western blot assays were further performed to explore the role and underlying mechanism of ARHGAP15 in lung cancer. RESULTS: Reduced ARHGAP15 expression was observed in lung cancer tissues and cells. In vitro upregulation of ARHGAP15 in lung cancer cells strongly suppressed cell proliferation, migration, and invasion and was accompanied by reduced matrix metalloproteinase-2 (MMP2), MMP9, vascular endothelial growth factor (VEGF) expression, and the phosphorylation of the signal transducer and activator of transcription-3 (p-STAT3). In contrast, interleukin-6 (IL-6) had the opposite effect and the induction of IL-6 was counteracted by ARHGAP15 upregulation. In addition, the proliferation, migration, and cell invasion induced by ARHGAP15 silencing were potentially inhibited by the STAT3 inhibitor AG490 (100 µM), MMP2, MMP9, VEGF, and p-STAT3 levels decreased. CONCLUSIONS: These results suggest that ARGFAP15 suppressed the proliferation and metastasis of lung cancer cells, which may occur through the inhibition of MMP2, MMP9, and VEGF expression via the STAT3 pathway inactivation.

YY1-Induced Upregulation of Long Noncoding RNA ARAP1-AS1 Promotes Cell Migration and Invasion in Colorectal Cancer Through the Wnt/ß-Catenin Signaling Pathway.

Introduction: It has been reported that long noncoding RNAs (lncRNAs) are crucial regulators in progression of human cancers, including colorectal cancer (CRC). However, the function of lncRNA ARAP1 antisense RNA 1 (ARAP1-AS1) in CRC remains unclear. Aim: The aim of this study was to investigate the function and molecular mechanism of lncRNA ARAP1-AS1 in CRC. Results: ARAP1-AS1 was highly expressed in CRC tissues and cell lines. ARAP1-AS1 knockdown suppressed cell migration, invasion, and epithelial-mesenchymal transition (EMT). YY1 transcription factor (YY1) enhanced the transcription activity of ARAP1-AS1. The YY1/ARAP1-AS1 axis promoted CRC cell migration and invasion. YY1/ARAP1-AS1 could regulate the Wnt/ß-catenin signaling pathway. Conclusions: This study revealed that YY1-induced upregulation of ARAP1-AS1 promoted cell migration, invasion, and EMT process in CRC through the Wnt/ß-catenin signaling pathway.

FLT3-ITD cooperates with Rac1 to modulate the sensitivity of leukemic cells to chemotherapeutic agents via regulation of DNA repair pathways.

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm, and patients with an internal tandem duplication (ITD) mutation of the FMS-like tyrosine kinase-3 (FLT3) receptor gene have a poor prognosis. FLT3-ITD interacts with DOCK2, a G effector protein that activates Rac1/2. Previously, we showed that knockdown of DOCK2 leads to decreased survival of FLT3-ITD leukemic cells. We further investigated the mechanisms by which Rac1/DOCK2 activity affects cell survival and chemotherapeutic response in FLT3-ITD leukemic cells. Exogenous expression of FLT3-ITD led to increased Rac1 activity, reactive oxygen species, phosphorylated STAT5, DNA damage response factors and cytarabine resistance. Conversely, DOCK2 knockdown resulted in a decrease in these factors. Consistent with the reduction in DNA damage response factors, FLT3-ITD cells with DOCK2 knockdown exhibited significantly increased sensitivity to DNA damage response inhibitors. Moreover, in a mouse model of FLT3-ITD AML, animals treated with the CHK1 inhibitor MK8776 + cytarabine survived longer than those treated with cytarabine alone. These findings suggest that FLT3-ITD and Rac1 activity cooperatively modulate DNA repair activity, the addition of DNA damage response inhibitors to conventional chemotherapy may be useful in the treatment of FLT3-ITD AML, and inhibition of the Rac signaling pathways via DOCK2 may provide a novel and promising therapeutic target for FLT3-ITD AML.

Targeting RalGAPα1 in skeletal muscle to simultaneously improve postprandial glucose and lipid control.

How insulin stimulates postprandial uptake of glucose and long-chain fatty acids (LCFAs) into skeletal muscle and the mechanisms by which these events are dampened in diet-induced obesity are incompletely understood. Here, we show that RalGAPα1 is a critical regulator of muscle insulin action and governs both glucose and lipid homeostasis. A high-fat diet increased RalGAPα1 protein but decreased its insulin-responsive Thr735-phosphorylation in skeletal muscle. A RalGAPα1Thr735Ala mutation impaired insulin-stimulated muscle assimilation of glucose and LCFAs and caused metabolic syndrome in mice. In contrast, skeletal muscle-specific deletion of RalGAPα1 improved postprandial glucose and lipid control. Mechanistically, these mutations of RalGAPα1 affected translocation of insulin-responsive glucose transporter GLUT4 and fatty acid translocase CD36 via RalA to affect glucose and lipid homeostasis. These data indicated RalGAPα1 as a dual-purpose target, for which we developed a peptide-blockade for improving muscle insulin sensitivity. Our findings have implications for drug discovery to combat metabolic disorders.