IQGAP1 Knockdown Research Articles

In silico predicted compound targeting the IQGAP1-GRD domain selectively inhibits growth of human acute myeloid leukemia

Acute myeloid leukemia (AML) is fatal in the majority of adults. Identification of new therapeutic targets and their pharmacologic modulators are needed to improve outcomes. Previous studies had shown that immunization of rabbits with normal peripheral WBCs that had been incubated with fluorodinitrobenzene elicited high titer antibodies that bound to a spectrum of human leukemias. We report that proteomic analyses of immunoaffinity-purified lysates of primary AML cells showed enrichment of scaffolding protein IQGAP1. Immunohistochemistry and gene-expression analyses confirmed IQGAP1 mRNA overexpression in various cytogenetic subtypes of primary human AML compared to normal hematopoietic cells. shRNA knockdown of IQGAP1 blocked proliferation and clonogenicity of human leukemia cell-lines. To develop small molecules targeting IQGAP1 we performed in-silico screening of 212,966 compounds, selected 4 hits targeting the IQGAP1-GRD domain, and conducted SAR of the ‘fittest hit’ to identify UR778Br, a prototypical agent targeting IQGAP1. UR778Br inhibited proliferation, induced apoptosis, resulted in G2/M arrest, and inhibited colony formation by leukemia cell-lines and primary-AML while sparing normal marrow cells. UR778Br exhibited favorable ADME/T profiles and drug-likeness to treat AML. In summary, AML shows response to IQGAP1 inhibition, and UR778Br, identified through in-silico studies, selectively targeted AML cells while sparing normal marrow.

Retraction: Targeted Knockdown of IQGAP1 Inhibits the Progression of Esophageal Squamous Cell Carcinoma In Vitro and In Vivo.

Retraction: Targeted Knockdown of IQGAP1 Inhibits the Progression of Esophageal Squamous Cell Carcinoma In Vitro and In Vivo.

Identification of the interactome of the DP1 receptor for Prostaglandin D2: Regulation of DP1 receptor signaling and trafficking by IQGAP1

BackgroundMechanisms governing localization, trafficking and signaling of G protein-coupled receptors (GPCRs) are critical in cell function. Protein-protein interactions are determinant in these processes. However, there are very little interacting proteins known to date for the DP1 receptor for prostaglandin D2. MethodsWe performed LC-MS/MS analyses of the DP1 receptor interactome in HEK293 cells. To functionally validate our LC-MS/MS data, we studied the implications of the interaction with the IQGAP1 scaffold protein in the trafficking and signaling of DP1. ResultsIn addition to expected interacting proteins such as heterotrimeric G protein subunits, we identified proteins involved in signaling, trafficking, and folding localized in various cell compartments. Endogenous DP1-IQGAP1 co-immunoprecipitation was observed in colon cancer HT-29 cells. The interaction was augmented by DP1 agonist activation in HEK293 cells and GST-pulldown assays showed that IQGAP1 binds to intracellular loops 2 and 3 of DP1. Co-localization of the two proteins was observed by confocal microscopy at the cell periphery and in intracellular vesicles in the basal state. PGD2 treatment resulted in the redistribution of the DP1-IQGAP1 co-localization in the perinuclear vicinity. DP1 receptor internalization was promoted by overexpression of IQGAP1, while it was diminished by IQGAP1 knockdown with DsiRNAs. DP1-mediated ERK1/2 activation was augmented and sustained overtime by overexpression of IQGAP1 when compared to DP1 expressed alone. IQGAP1 knockdown decreased ERK1/2 activation by DP1 stimulation. Interestingly, ERK1/2 signaling by DP1 was increased when IQGAP2 was silenced, while it was impaired by IQGAP3 knockdown. ConclusionsOur findings define the putative DP1 interactome, a patho-physiologically important receptor, and validated the interaction with IQGAP1 in DP1 function. Our data also reveal that IQGAP proteins may differentially regulate GPCR signaling. General significanceThe identified putative DP1-interacting proteins open multiple lines of research in DP1 and GPCR biology in various cell compartments.

IQGAP1: A Potential Therapeutic Target in Acute Myelogenous Leukemia (AML)

Introduction: While AML is phenotypically diverse, genome-wide sequencing studies indicate that the median number of non-synonymous mutations is eight (Science 339,1546, 2013) and that the same pathways are affected in tumors with distinct genetic alterations (Nature 453,1112-1116, 2008). These insights provided the impetus to immunoaffinity purify shared antigenic moieties in human AML and examine their prevalence as well as function.Methods: We employed a previously described method wherein immunization of rabbits with normal human peripheral blood cells whose surface charge had been modified by incubation with fluorodinitrobenzene (FDNB) elicited an antibody response that cross reacted against a broad range of human leukemias (Nature 232,197, 1971). Replicating the described methods WBCs from healthy volunteer donors were suspended at 1x107 cells per ml of PBS and mixed with equal volume of 0.86 mM FDNB for 12 to 15 minutes at room temperature. Three rabbits were immunized with FDNB-treated cells (experimental rabbits). A control rabbit was immunized with sham-treated cells (control rabbit). After complement inactivation, immune sera were absorbed against WBCs from healthy donors. Absorbed immune sera were tested in clinical AML samples by Western blotting. Immunoprecipitation of antigens from whole cell lysates of clinical AML samples was performed using IgG adsorbed on protein A/G Agarose beads. Liquid chromatography and mass spectrometry of the immunoprecipitated material was performed. Fold change in IQGAP1 expression in normal vs AML bone marrow was determined by qRT-PCR and from raw data from Gene Expression Omnibus at the NCBI. IQGAP1 expression was evaluated by immunohistochemistry in cell lines, bone marrow biopsies from AML patients and normal healthy volunteers. IQGAP1 expression was modulated by RNAi and the effect on proliferation and colony formation was measured in cell lines.Results: Western blotting of whole cell lysates of clinical AML samples revealed bands that were recognized by immune serum from experimental rabbits but not the control rabbit. Immuno-precipitation of antigens from whole cell lysates of clinical AML samples revealed the scaffolding protein IQGAP1 as being differentially recognized in independent experiments. We focused on IQGAP1 because it regulates multiple cellular functions including signaling pathways involved in proliferation and transformation and stimulates cell motility and invasion. It is overexpressed in colon cancer, pancreatic cancer, hepatocellular carcinoma, glioma, and other cancers. (Cell Signaling 21,1471-1678, 2009). Western blots of human AML samples probed with anti-IQGAP1 antibody revealed the predicted 190 kDa band, see figure. There was 3 to 9-fold greater expression of IQGAP1 expression in AML versus normal bone marrow observed by qRT-PCR. Analysis of raw data from Gene Expression Omnibus at the NCBI revealed fold change of -3.22636, p-value 2.62 x 10e-7 between normal bone marrow versus AML. Preliminary IHC studies showed strong membranous staining in three leukemia cell lines and bone marrow biopsies from primary AML bone marrow samples but weak partial staining in normal bone marrow. Knocking down expression of IQGAP1 in K562, MV4-11 and THP1 cell lines resulted in significant decrease in proliferation and colony formation as compared to effects of scramble control. Mean and SD of colonies per 2000 cells plated were for K562: naïve 311+/-46, IQGAP1 knockdown 37+/-12, scramble control 127+/-12, MV4-11: naïve 156+/37, IQGAP1 knockdown 55+/- 8, scramble control 147+/-16, and THP1 naïve 232+/-45, IQGAP1 knockdown 17+/-2.9, scramble control 150+/-24.Conclusions: 1. The scaffolding protein IQGAP1is overexpressed in AML compared to normal bone marrow. 2. Knocking down expression of IQGAP1 in AML cell lines resulted in significant decrease in proliferation and colony formation providing proof of concept that IQGAP1 is a potential therapeutic target in AML. [Display omitted] DisclosuresLiesveld:Onconova: Honoraria; Seattle Genetics: Honoraria.

Actin-binding protein, IQGAP1, regulates LPS-induced RPMVECs hyperpermeability and ICAM-1 upregulation via Rap1/Src signalling pathway

Pulmonary microvascular barrier dysfunction is a hallmark feature of acute lung injury (ALI). IQGAP1 is a ubiquitously expressed scaffolding protein known to regulate cancer metastasis, angiogenesis, and barrier stability. However, the function of IQGAP1 in lipopolysaccharide (LPS)-induced microvascular endothelial hyperpermeability remains poorly understood. In the present study, we demonstrated that IQGAP1 was markedly upregulated in LPS-induced ALI models and rat pulmonary microvascular endothelial cells (RPMVECs). Lentivirus-mediated knockdown of IQGAP1 significantly attenuated the formation of actin stress fibers, phosphorylation of myosin light chain (MLC), and disruption of VE-cadherin, thereby protecting the RPMVECs barrier failure from LPS damage. In addition, IQGAP1 depletion reduced the reactive oxygen species (ROS)-mediated increase in intracellular adhesion molecule-1 (ICAM-1) in RPMVECs stimulated with LPS. Mechanistically, we found that the upregulation of IQGAP1 affected the activity of Rap1 and the downstream phosphorylation of Src. In conclusion, these findings reveal an essential mechanism by which increased IQGAP1 in LPS-treated RPMVECs promotes barrier dysfunction and ICAM-1 upregulation, at least in part by regulating Rap1/Src signalling, indicating that IQGAP1 may be a potential therapeutic target to prevent endothelial hyperpermeability and inflammation in ALI.

MicroRNA-124 inhibits hepatic stellate cells inflammatory cytokines secretion by targeting IQGAP1 through NF-κB pathway

Liver fibrosis is a health concern that leads to organ failure mediated via production of inflammatory cytokines and fibrotic biomarkers. To date, there was no direct approved antifibrotic therapy, and current treatment was mainly the removal of the causative factor. Recent studies demonstrated that aberrant expression of miR-124 was involved in the progression of various liver diseases including hepatocellular carcinoma (HCC). However, whether miR-124 could function as a transcriptional regulator in the inflammatory cytokines secretion of liver fibrosis remains unclear. In this study, we demonstrated that the expression of miR-124 was downregulated in liver fibrosis tissues and TNF-α-induced LX-2 cells, concomitant with the upregulated expression of IQGAP1, suggesting that miR-124 and IQGAP1 might be associated with the development of inflammation in liver fibrosis. Therefore, we demonstrated that the overexpression of miR-124 and knockdown of IQGAP1 could lead to the downregulation of TNF-α, IL-1β and IL-6. While knockdown of miR-124 or overexpression of IQGAP1 showed reversed results. Moreover, dual luciferase reporter assays demonstrated that miR‐124 specifically targeted the 3′‐UTR of IQGAP1, and thus inhibited the expression of IQGAP1. Mechanistically, we found that the expression changes of miR-124 and IQGAP1 could be involved in inhibition or activation of NF-κB signaling pathway in response to TNF-α. In conclusion, these results indicated that miR-124 plays a crucial role in TNF-α-induced LX-2 cells via regulating NF-κB signaling pathway.

CircRNA circ‐IQGAP1 Knockdown Alleviates Interleukin‐1β‐Induced Osteoarthritis Progression via Targeting miR‐671‐5p/TCF4

ObjectiveTo explore the function of circular RNA IQ motif‐containing GTPase‐activating protein 1 (circ‐IQGAP1) in interleukin (IL)‐1β‐induced osteoarthritis (OA) model and to explore whether circ‐IQGAP1 can modulate microRNA‐671‐5p (miR‐671‐5p) and transcription factor 4 (TCF4) to regulate chondrocyte apoptosis, inflammatory injury, and extracellular matrix degradation.MethodsThe cartilage tissues were collected from 32 OA patients or normal subjects. Human chondrocyte CHON‐001 cells were challenged via different doses of IL‐1β for 24 hours. CHON‐001 cells were transfected with circ‐IQGAP1 overexpression vector, TCF4 overexpression vector, small interfering RNA (siRNA) for circ‐IQGAP1, miR‐671‐5p mimic, miR‐671‐5p inhibitor or corresponding negative controls. Circ‐IQGAP1, miR‐671‐5p and TCF4 abundances in cartilage tissues or CHON‐001 cells were examined via quantitative reverse transcription polymerase chain reaction (qRT‐PCR) or western blot. Cell viability was investigated by 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide (MTT). Cell apoptosis was measured by flow cytometry. The inflammatory injury was analyzed by the secretion levels of inflammatory cytokines (IL‐6, IL‐8 and tumor necrosis factor‐α [TNF‐α]) by enzyme‐linked immunosorbent assay (ELISA). The extracellular matrix degradation was evaluated by expression of aggrecan and matrix metalloproteinase 13 (MMP13) via western blot. The target relationship of miR‐671‐5p and circ‐IQGAP1 or TCF4 was analyzed via dual‐luciferase reporter and RNA immunoprecipitation (RIP) analyses.ResultsCirc‐IQGAP1 abundance was enhanced in the cartilage tissues from OA patients compared with normal subjects (n = 32), and its expression was increased in CHON‐001 cells after treatment of IL‐1β in a dose‐dependent pattern. MiR‐671‐5p expression was decreased in the cartilage tissues from OA patients (n = 32) and IL‐1β‐challenged CHON‐001 cells. MiR‐671‐5p expression was negatively associated with circ‐IQGAP1 level in OA patients. Circ‐IQGAP1 silence mitigated IL‐1β‐caused chondrocyte viability reduction, apoptosis promotion, secretion of inflammatory cytokine (IL‐6, IL‐8 and TNF‐α), and extracellular matrix degradation (reduction of aggrecan and increase of MMP13). MiR‐671‐5p was targeted and inhibited via circ‐IQGAP1. MiR‐671‐5p knockdown attenuated the influence of circ‐IQGAP1 interference on IL‐1β‐caused chondrocyte apoptosis, inflammatory injury, and extracellular matrix degradation. TCF4 was targeted via miR‐671‐5p, and TCF4 expression was increased in the cartilage tissues from OA patients (n = 32) and IL‐1β‐challenged CHON‐001 cells. TCF4 abundance in OA patients was negatively correlated with miR‐671‐5p expression. MiR‐671‐5p overexpression alleviated IL‐1β‐mediated chondrocyte apoptosis, inflammatory injury, and extracellular matrix degradation via decreasing TCF4 expression. Circ‐IQGAP1 silence reduced TCF4 expression via regulating miR‐671‐5p in IL‐1β‐challenged CHON‐001 cells.ConclusionCirc‐IQGAP1 knockdown attenuated IL‐1β‐caused chondrocyte apoptosis, inflammatory injury, and extracellular matrix degradation. Circ‐IQGAP1 could regulate miR‐671‐5p/TCF4 axis to modulate IL‐1β‐caused chondrocyte damage. Circ‐IQGAP1 might act as a new target for the treatment of OA.

IQGAP1 causes choroidal neovascularization by sustaining VEGFR2-mediated Rac1 activation.

Loss of visual acuity in neovascular age-related macular degeneration (nAMD) occurs when factors activate choroidal endothelial cells (CECs) to transmigrate the retinal pigment epithelium into the sensory retina and develop into choroidal neovascularization (CNV). Active Rac1 (Rac1GTP) is required for CEC migration and is induced by different AMD-related stresses, including vascular endothelial growth factor (VEGF). Besides its role in pathologic events, Rac1 also plays a role in physiologic functions. Therefore, we were interested in a method to inhibit pathologic activation of Rac1. We addressed the hypothesis that IQGAP1, a scaffold protein with a Rac1 binding domain, regulates pathologic Rac1GTP in CEC migration and CNV. Compared to littermate Iqgap1+/+, Iqgap1-/- mice had reduced volumes of laser-induced CNV and decreased Rac1GTP and phosphorylated VEGFR2 (p-VEGFR2) within lectin-stained CNV. Knockdown of IQGAP1 in CECs significantly reduced VEGF-induced Rac1GTP, mediated through p-VEGFR2, which was necessary for CEC migration. Moreover, sustained activation of Rac1GTP induced by VEGF was eliminated when CECs were transfected with an IQGAP1 construct that is unable to bind Rac1. IQGAP1-mediated Src activation was involved in initiating Rac1 activation, CEC migration, and tube formation. Our findings indicate that CEC IQGAP1 interacts with VEGFR2 to mediate Src activation and subsequent Rac1 activation and CEC migration. In addition, IQGAP1 binding to Rac1GTP results in sustained activation of Rac1, leading to CEC migration toward VEGF. Our study supports a role of IQGAP1 and the interaction between IQGAP1 and Rac1GTP to restore CECs quiescence and, therefore, prevent vision-threatening CNV in nAMD.

The scaffold protein IQGAP1 is crucial for extravasation and metastasis

IQGAP1 is a scaffold protein involved in a range of cellular activities, including migration, invasion, adhesion and proliferation. It is also oncogenic in a variety of cancers, promoting primary tumor growth and invasiveness. However, the role of IQGAP1 in tumor progression and metastasis remains unclear. In this study, we use both knockdown and knockout of IQGAP1 to investigate its role in the metastatic cascade of both melanoma and breast cancer cells in vivo. We find that reduction of IQGAP1 expression decreases the formation of both spontaneous and experimental metastases, without limiting primary or metastatic tumor growth. Furthermore, IQGAP1 knockout significantly inhibits extravasation of tumor cells from circulation, possibly involving invadopodial function. By expressing mutant forms of IQGAP1 in a knockout context, we also determine that IQGAP1’s pro-metastatic functions are dependent on multiple domains and functions. These data demonstrate that IQGAP1 is crucial for metastasis in vivo through regulation of extravasation and suggest that it may represent a valid therapeutic target for inhibiting metastasis.

GDP-Bound Rab27a Dissociates from the Endocytic Machinery in a Phosphorylation-Dependent Manner after Insulin Secretion

Glucose-stimulated insulin secretion is controlled by both exocytosis and endocytosis in pancreatic β-cells. Although endocytosis is a fundamental step to maintain cellular responses to the secretagogue, the molecular mechanism of endocytosis remains poorly defined. We have previously shown that in response to high concentrations of glucose, guanosine 5'-diphosphate (GDP)-bound Rab27a is recruited to the plasma membrane where IQ motif-containing guanosine 5'-triphosphatase (GTPase)-activating protein 1 (IQGAP1) is expressed, and that complex formation promotes endocytosis of secretory membranes after insulin secretion. In the present study, the regulatory mechanisms of dissociation of the complex were investigated. Phosphorylation of IQGAP1 on serine (Ser)-1443, a site recognized by protein kinase Cε (PKCε), inhibited the interaction of GDP-bound Rab27a with IQGAP1 in a Cdc42-independent manner. Glucose stimulation caused a translocation of PKCε from the cytosol to the plasma membrane. In addition, glucose-induced endocytosis was inhibited by the knockdown of IQGAP1 with small interfering RNA (siRNA). However, the expression of the non-phosphorylatable or phosphomimetic form of IQGAP1 could not rescue the inhibition, suggesting that a phosphorylation-dephosphorylation cycle of IQGAP1 is required for endocytosis. These results suggest that IQGAP1 phosphorylated by PKCε promotes the dissociation of the IQGAP1-GDP-bound Rab27a complex in pancreatic β-cells, thereby regulating endocytosis of secretory membranes following insulin secretion.

Endothelial IQGAP1 regulates leukocyte transmigration by directing the LBRC to the site of diapedesis.

Transendothelial migration (TEM) of leukocytes across the endothelium is critical for inflammation. In the endothelium, TEM requires the coordination of membrane movements and cytoskeletal interactions, including, prominently, recruitment of the lateral border recycling compartment (LBRC). The scaffold protein IQGAP1 was recently identified in a screen for LBRC-interacting proteins. Knockdown of endothelial IQGAP1 disrupted the directed movement of the LBRC and substantially reduced leukocyte TEM. Expression of truncated IQGAP1 constructs demonstrated that the calponin homology domain is required for IQGAP1 localization to endothelial borders and that the IQ domain, on the same IQGAP1 polypeptide, is required for its function in TEM. This is the first reported function of IQGAP1 requiring two domains to be present on the same polypeptide. Additionally, we show for the first time that IQGAP1 in the endothelium is required for efficient TEM in vivo. These findings reveal a novel function for IQGAP1 and demonstrate that IQGAP1 in endothelial cells facilitates TEM by directing the LBRC to the site of TEM.

Endothelial Cell IQGAP1 is Required to Support Efficient Leukocyte Transmigration both In Vitro and In Vivo

Inflammation is the body's fundamental response to tissue insult and damage. When operating correctly, it allows the organism to fight infection, repair injuries, and return the body to homeostasis. When the response is incorrectly directed, whether insufficient, excessive, or against an improper target, it contributes to and even directly causes numerous human pathologies. One of the critical components of inflammation is the recruitment of leukocytes to the target site. This process is a carefully choreographed series of sequential steps that leads to the net efflux of leukocytes out of the blood stream and into tissue. In the culminating and putative committed stage, the leukocyte squeezes between adjacent endothelial cells in a process commonly referred to as transendothelial migration (TEM). TEM is a critical step that requires several intercellular interactions and thus represents an attractive therapeutic target for regulating aberrant inflammation. During TEM, endothelial cells direct the lateral border recycling compartment (LBRC), a sub‐junctional membrane network, to the site of leukocyte migration to facilitate its passage. All manipulations that affect the function and delivery of the LBRC substantially reduce leukocyte efflux.To better understand the molecular components and function of the LBRC, we recently conducted a screen for LBRC‐interacting proteins and identified IQGAP1. IQGAP1 is a large multi‐domain cytoplasmic protein that is reported to bind several signaling proteins in other systems. Knockdown of endothelial cell IQGAP1 disrupted the directed movement of the LBRC and substantially reduced leukocyte TEM. To determine which domains of IQGAP1 are required for its function in TEM, we expressed a series of truncated constructs and examined their ability to rescue the knockdown of endogenous IQGAP1. We found that the actin‐binding domain of IQGAP1 is required for its localization to endothelial borders. Furthermore, both the actin‐binding domain and the IQ‐motifs are required for its function in TEM. We extended these studies in vivo by generating bone marrow chimeras, in which IQGAP1 knockout animals were reconstituted with wild‐type leukocytes. These studies show that IQGAP1 in endothelium is required for efficient TEM in vivo in two different inflammatory models. Together these findings detail a novel function for IQGAP1 in TEM that involves both its actin‐binding domain and IQ motifs which together coordinate the directed movement of the LBRC to the site of TEM.Support or Funding InformationFunding Support: R01HL046849, R01HL064774This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Scaffolding Protein IQGAP1 Interacts with NLRC3 and Inhibits Type I IFN Production.

Sensing of cytosolic nucleotides is a critical initial step in the elaboration of type I IFN. One of several upstream receptors, cyclic GMP-AMP synthase, binds to cytosolic DNA and generates dicyclic nucleotides that act as secondary messengers. These secondary messengers bind directly to stimulator of IFN genes (STING). STING recruits TNFR-associated NF-κB kinase-binding kinase 1 which acts as a critical node that allows for efficient activation of IFN regulatory factors to drive the antiviral transcriptome. NLRC3 is a recently characterized nucleotide-binding domain, leucine-rich repeat containing protein (NLR) that negatively regulates the type I IFN pathway by inhibiting subcellular redistribution and effective signaling of STING, thus blunting the transcription of type I IFNs. NLRC3 is predominantly expressed in lymphoid and myeloid cells. IQGAP1 was identified as a putative interacting partner of NLRC3 through yeast two-hybrid screening. In this article, we show that IQGAP1 associates with NLRC3 and can disrupt the NLRC3-STING interaction in the cytosol of human epithelial cells. Furthermore, knockdown of IQGAP1 in THP1 and HeLa cells causes significantly more IFN-β production in response to cytosolic nucleic acids. This result phenocopies NLRC3-deficient macrophages and fibroblasts and short hairpin RNA knockdown of NLRC3 in THP1 cells. Our findings suggest that IQGAP1 is a novel regulator of type I IFN production, possibly via interacting with NLRC3 in human monocytic and epithelial cells.

Chlamydia pneumoniae infection promotes vascular endothelial cell angiogenesis through an IQGAP1-related signaling pathway

Chlamydia pneumoniae (C. pneumoniae) infection plays a potential role in angiogenesis. However, it is still an enigma how C. pneumoniae is involved in this process. Therefore, we investigated the effect of C. pneumoniae infection on angiogenesis, and then explored the roles of IQGAP1-related signaling in C. pneumoniae infection-induced angiogenesis. C. pneumoniae infection significantly enhanced angiogenesis as assessed by the tube formation assay possibly by inducing vascular endothelial cell (VEC) migration in the wound healing and Transwell migration assays. Subsequently, immunoprecipitation, Western blot and tube formation assay results showed that the phosphorylation of both IQGAP1 and N-WASP was required for the angiogenesis induced by C. pneumoniae infection. Our co-immunoprecipitation study revealed that IQGAP1 physically associated with N-WASP after C. pneumoniae infection of VECs. Actin polymerization assay further showed that in C. pneumoniae-infected VECs, both IQGAP1 and N-WASP were recruited to filamentous actin, and shared some common compartments localized at the leading edge of lamellipodia, which was impaired after the depletion of IQGAP1 by using the small interference RNA. Moreover, the knockdown of IQGAP1 also significantly decreased N-WASP phosphorylation at Tyr256 induced by C. pneumoniae infection. We conclude that C. pneumoniae infection promotes VEC migration and angiogenesis presumably through the IQGAP1-related signaling pathway.

Knockdown of IQGAP1 inhibits proliferation and epithelial-mesenchymal transition by Wnt/β-catenin pathway in thyroid cancer.

BackgroundThyroid cancer is the most common endocrine malignant disease with a high incidence rate. The expression of IQGAP1 is upregulated in various cancers, including thyroid cancer. However, the role and underlying mechanism of IQGAP1 in thyroid cancer are still not clear.Materials and methodsThe expression of IQGAP1 in thyroid cancer tissues and cells was determined by reverse transcription polymerase chain reaction and Western blot analysis. Cells were transfected with different siRNAs using Lipofectamine 2000 or were treated with various concentrations of XAV939. The effects of IQGAP1 knockdown on proliferation and epithelial–mesenchymal transition (EMT) of thyroid cancer cells were determined by MTT assay and Western blot analysis. Animal experiments were performed to investigate the effects of IQGAP1 knockdown on the growth of tumors in vivo.ResultsHigh IQGAP1 expression is found in thyroid cancer tissues and cells. Knockdown of IQGAP1 had inhibitory effects on cell proliferation and EMT, as well as on the Wnt/β-catenin pathway. Additionally, inactivation of the Wnt/β-catenin pathway by XAV939 or si-β-catenin suppressed cell proliferation and EMT. Furthermore, suppression of the Wnt/β-catenin pathway reversed the positive effects of pcDNA-IQGAP1 on cell proliferation and EMT in vitro. Moreover, downregulation of IQGAP1 suppressed tumor growth and EMT in SW579 tumor xenografts through the Wnt/β-catenin pathway in vivo.ConclusionOur study demonstrated that knockdown of IQGAP1 inhibited cell proliferation and EMT through blocking the Wnt/β-catenin pathway in thyroid cancer.

HECTD1 controls the protein level of IQGAP1 to regulate the dynamics of adhesive structures

BackgroundCell migration including collective cell movement and individual cell migration are crucial factors in embryogenesis. During the spreading/migration of cells, several types of adhesive structures physically interacting with the extracellular matrix (ECM) or with another cell have been described and the formation and maturation of adhesion structures are coordinated, however the molecular pathways involved are still not fully understood.ResultsWe generated a mouse embryonic fibroblast line (MEF) from homozygous mutant (Hectd1R/R, Hectd1Gt(RRC200)) mouse of the E3 ubiquitin ligase for inhibin B receptor (Hectd1). Detailed examination of cell motion on MEF cells demonstrated that loss of Hectd1 resulted in accelerated cell spreading and migration but impaired directionality of migration. In Hectd1R/R cells paxillin and zyxin were largely mis-localized, whereas their expression levels were unchanged. In addition the formation of focal adhesions (FAs) was impaired and the focal complexes (FXs) were increased. We further identified HECTD1 as a key regulator of IQGAP1. IQGAP1 co-localized together with HECTD1 in the leading edge of cells. HECTD1 interacted with IQGAP1 and regulated its degradation through ubiquitination. Over-expression of IQGAP1 in control MEF phenocopied the spreading and migration defects of Hectd1R/R cells. In contrast, siRNA-mediated knockdown of IQGAP1 rescued the defects in cellular movement of Hectd1R/R cells.ConclusionsThe E3 ligase activity of Hectd1 regulates the protein level of IQGAP1 through ubiquitination and therefore mediates the dynamics of FXs including the recruitment of paxillin and actinin. IQGAP1 is one of the effectors of HECTD1.

Deletion of IQGAP1 promotes Helicobacter pylori-induced gastric dysplasia in mice and acquisition of cancer stem cell properties in vitro.

Helicobacter pylori infection is responsible for gastric carcinogenesis but host factors are also implicated. IQGAP1, a scaffolding protein of the adherens junctions interacting with E-cadherin, regulates cellular plasticity and proliferation. In mice, IQGAP1 deficiency leads to gastric hyperplasia. The aim of this study was to elucidate the consequences of IQGAP1 deletion on H. pylori-induced gastric carcinogenesis.Transgenic mice deleted for iqgap1 and WT littermates were infected with Helicobacter sp., and histopathological analyses of the gastric mucosa were performed. IQGAP1 and E-cadherin expression was evaluated in gastric tissues and in gastric epithelial cell lines in response to H. pylori infection. The consequences of IQGAP1 deletion on gastric epithelial cell behaviour and on the acquisition of cancer stem cell (CSC)-like properties were evaluated. After one year of infection, iqgap1+/- mice developed more preneoplastic lesions and up to 8 times more gastro-intestinal neoplasia (GIN) than WT littermates. H. pylori infection induced IQGAP1 and E-cadherin delocalization from cell-cell junctions. In vitro, knock-down of IQGAP1 favoured the acquisition of a mesenchymal phenotype and CSC-like properties induced by H. pylori infection.Our results indicate that alterations in IQGAP1 signalling promote the emergence of CSCs and gastric adenocarcinoma development in the context of an H. pylori infection.

IQGAP1 Binds to Yes-associated Protein (YAP) and Modulates Its Transcriptional Activity

During development, the Hippo signaling pathway regulates key physiological processes, such as control of organ size, regeneration, and stem cell biology. Yes-associated protein (YAP) is a major transcriptional co-activator of the Hippo pathway. The scaffold protein IQGAP1 interacts with more than 100 binding partners to integrate diverse signaling pathways. In this study, we report that IQGAP1 binds to YAP and modulates its activity. IQGAP1 and YAP co-immunoprecipitated from cells. In vitro analysis with pure proteins demonstrated a direct interaction between IQGAP1 and YAP. Analysis with multiple fragments of each protein showed that the interaction occurs via the IQ domain of IQGAP1 and the TEAD-binding domain of YAP. The interaction between IQGAP1 and YAP has functional effects. Knock-out of endogenous IQGAP1 significantly increased the formation of nuclear YAP-TEAD complexes. Transcription assays were performed with IQGAP1-null mouse embryonic fibroblasts and HEK293 cells with IQGAP1 knockdown by CRISPR/Cas9. Quantification demonstrated that YAP-TEAD-mediated transcription in cells lacking IQGAP1 was significantly greater than in control cells. These data reveal that IQGAP1 binds to YAP and modulates its co-transcriptional function, suggesting that IQGAP1 participates in Hippo signaling.

Role of IQGAP1 in endothelial barrier enhancement caused by OxPAPC.

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (OxPAPC) attenuates agonist-induced endothelial cell (EC) permeability and increases pulmonary endothelial barrier function via enhancement of both the peripheral actin cytoskeleton and cell junctions mediated by Rac1 and Cdc42 GTPases. This study evaluated the role for the multifunctional Rac1/Cdc42 effector and regulator, IQGAP1, as a molecular transducer of the OxPAPC-mediated EC barrier enhancing signal. IQGAP1 knockdown in endothelial cells by gene-specific siRNA abolished OxPAPC-induced enlargement of VE-cadherin-positive adherens junctions, suppressed peripheral accumulation of actin polymerization regulators, namely cortactin, N-WASP and Arp3, and attenuated remodeling of the peripheral actin cytoskeleton. Inhibition of OxPAPC-induced barrier enhancement by IQGAP1 knockdown was due to suppressed Rac1 and Cdc42 activation. Expression of an IQGAP1 truncated mutant showed that the GTPase regulatory domain (GRD) of IQGAP1 was essential for the OxPAPC-induced membrane localization of cortactin, adherens junction proteins VE-cadherin and p120-catenin as well as for EC permeability response. IQGAP1knockdown attenuated the protective effect of OxPAPC against thrombin-induced cell contraction, cell junction disruption and EC permeability. These results demonstrate for the first time the role of IQGAP1 as a critical transducer of OxPAPC-induced Rac1/Cdc42 signaling to the actin cytoskeleton and adherens junctions which promotes cortical cytoskeletal remodeling and EC barrier protective effects of oxidized phospholipids.

MALAT1 promotes the proliferation and invasion of thyroid cancer cells via regulating the expression of IQGAP1.

Increasing evidence indicated that metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) acted as a key regulator in the proliferation and invasion of several cancers. However, the function of MALAT1 in the development of thyroid cancer has not been experimentally established. The expression of MALAT1 and IQGAP1 in thyroid cancer tissues and cells were detected by quantitative real-time PCR and western blot. The effects of MALAT1 and IQGAP1 on the cell proliferation and invasion of thyroid cancer cells were detected with a 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium 4 (MTT) assay and a Transwell assay, respectively. FTC-133 or SW1736 transfected with si-MALAT1 or pcDNA-MALAT1 were injected subcutaneously into 4-week-olds BALB/c mice to examine the impact of MALAT1 on the tumor development of thyroid cancer in vivo. In this study, we discovered the higher level of MALAT-1 and expression of IQGAP1 in thyroid cancer tissues and in thyroid cancer cells compared to that in the control. MTT and Transwell assay showed that the proliferation and invasion of FTC-133 cells with MALAT-1 knockdown were inhibited. Moreover, MALAT-1 could upregulate the expression of IQGAP1 in thyroid cancer cells. In addition, IQGAP1 knockdown reversed the decreasing cell proliferation and invasion of thyroid cancer induced by MALAT-1 overexpression. Finally, the study in vivo verified that MALAT-1 promoted the tumor growth of thyroid cancer. Our study indicated that MALAT1 promoted the proliferation and invasion of thyroid cancer cells via regulating the expression of IQGAP1.