IQ-domain GTPase-activating Protein Research Articles

Pepsinogen C Interacts with IQGAP1 to Inhibit the Metastasis of Gastric Cancer Cells by Suppressing Rho-GTPase Pathway.

This study systematically explored the biological effects and mechanisms of PGC on gastric cancer (GC) cells in vitro and in vivo. The critical biological roles of PGC in GC were assessed via EdU staining, Hoechst staining, flow cytometry, mouse models, CCK-8, wound healing, transwell, and sphere-forming assays. The interaction study with IQ-domain GTPase-activating protein 1 (IQGAP1) was used by Liquid chromatography-mass spectrometry co-immunoprecipitation, immunofluorescence staining, CHX-chase assay, MG132 assay, and qRT-PCR. PGC inhibited the proliferation, viability, epithelial-mesenchymal transition, migration, invasion, and stemness of GC cells and promoted GC cell differentiation. PGC suppressed subcutaneous tumor growth and peritoneal dissemination in vivo. The interaction study found PGC inhibits GC cell migration and invasion by downregulating IQGAP1 protein and IQGAP1-mediated Rho-GTPase signaling suppression. In addition, PGC disrupts the stability of the IQGAP1 protein, promoting its degradation and significantly shortening its half-life. Moreover, the expression levels of PGC and IQGAP1 in GC tissues were significantly negatively correlated. PGC may act as a tumor suppressor in the development and metastasis of GC. PGC can downregulate its interacting protein IQGAP1 and inhibit the Rho-GTPase pathway, thereby participating in the inhibition of GC cell migration and invasion.

Suppression of Migration and Invasion by 4-Carbomethoxyl-10-Epigyrosanoldie E from the Cultured Soft Coral Sinularia sandensis through the MAPKs Pathway on Oral Cancer Cells.

The primary reason for cancer-related fatalities is metastasis. The compound 4-carbomethoxyl-10-epigyrosanoldie E, derived from the Sinularia sandensis soft coral species grown in cultures, exhibits properties that counteract inflammation. Moreover, it has been observed to trigger both apoptosis and autophagy within cancerous cells. This research focuses on examining the inhibitory impact of 4-carbomethoxyl-10-epigyrosanoldie E on the migration and invasion processes in Cal-27 and Ca9-22 oral cancer cell lines. To assess how this compound affects cell migration and invasion, the Boyden chamber assay was employed. Furthermore, Western blot analysis was utilized to explore the underlying molecular mechanisms. In a dose-dependent manner, 4-carbomethoxyl-10-epigyrosanoldie E notably decreased the levels of matrix metalloproteinase-2 (MMP-2) and MMP-9, along with urokinase-type plasminogen activator (uPA), in both Cal-27 and Ca9-22 cell lines. Conversely, it elevated the concentrations of tissue inhibitors of metalloproteinases-1 (TIMP-1) and TIMP-2. In addition, the treatment with this compound led to the inhibition of phosphorylation in extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). It also curtailed the expression of several key proteins including focal adhesion kinase (FAK), protein kinase C (PKC), growth factor receptor-bound protein 2 (GRB2), Rac, Ras, Rho A, mitogen-activated protein kinase kinase kinase 3 (MEKK3), and mitogen-activated protein kinase kinase 7 (MKK7). Furthermore, the expression levels of IQ-domain GTPase-activating protein 1 (IQGAP1) and zonula occludens-1 (ZO-1) were significantly reduced by the compound. The ability of 4-carbomethoxyl-10-epigyrosanoldie E to inhibit the migration and invasion of Cal-27 and Ca9-22 oral cancer cells was observed to be dose dependent. This inhibitory effect is primarily attributed to the suppression of MMP-2 and MMP-9 expression, as well as the downregulation of the mitogen-activated protein kinase (MAPK) signaling pathway.

Targeting ARF1-IQGAP1 interaction to suppress colorectal cancer metastasis and vemurafenib resistance

IntroductionAcquired resistance to BRAF inhibitor vemurafenib is frequently observed in metastatic colorectal cancer (CRC), and it is a thorny issue that results in treatment failure. As adaptive responses for vemurafenib treatment, a series of cellular bypasses are response for the adaptive feedback reactivation of ERK signaling, which warrant further investigation. ObjectivesWe identified ARF1 (ADP-ribosylation factor 1) as a novel regulator of both vemurafenib resistance and cancer metastasis, its molecular mechanism and potential inhibitor were investigated in this study. MethodsDIA-based quantitative proteomics and RNA-seq were performed to systematic analyze the profiling of vemurafenib-resistant RKO cells (RKO-VR) and highly invasive RKO cells (RKO-I8), respectively. Co‑immunoprecipitation assay was performed to detect the interaction of ARF1 and IQGAP1 (IQ-domain GTPase activating protein 1). An ELISA-based drug screen system on FDA-approved drug library was established to screen the compounds against the interaction of ARF1-IQGAP1.The biological functions of ARF1 and LY2835219 were determined by transwell, western blotting, Annexin V-FITC/PI staining and in vivo experimental metastasis assays. ResultsWe found that ARF1 strongly interacted with IQGAP1 to activate ERK signaling in VR and I8 CRC cells. Deletion of IQGAP1 or inactivation of ARF1 (ARF-T48S) restored the invasive ability induced by ARF1. As ARF1-IQGAP1 interaction is essential for ERK activation, we screened LY2835219 as novel inhibitor of ARF1-IQGAP1 interaction, which inactivated ERK signaling and suppressed CRC metastasis and vemurafenib-resistance in vitro and in vivo with no observed side effect. Furthermore, LY2835219 in combined treatment with vemurafenib exerted significantly inhibitory effect on ARF1-mediated cancer metastasis than used independently. ConclusionThis study uncovers that ARF1-IQGAP1 interaction-mediated ERK signaling reactivation is critical for vemurafenib resistance and cancer metastasis, and that LY2835219 is a promising therapeutic agent for CRC both as a single agent and in combination with vemurafenib.

The role of Type III secretion system in the pathogenesis of Pseudomonas aeruginosa microbial keratitis.

Pseudomonas aeruginosa is the most commonly isolated Gram-negative pathogen causing sight-threatening microbial keratitis (MK). Contact lens wear is the most significant risk factor associated with pseudomonal MK. Understanding the pathogenesis of MK due to P. aeruginosa and its interactions with contact lenses is crucial in preventing these often rapidly progressive and highly antibiotic-resistant infections. Bacterial virulence factor Type III secretion system (T3SS) has significant interplays between contact lens material, antibiotic sensitivity, disinfectant selectivity, and bacterial cell invasion. Depending on the T3SS exotoxins produced, P. aeruginosa strains are divided into cytotoxic or invasive strains. Cytotoxic strains are relatively resistant to commercial disinfectants, while invasive strains are more antibiotic resistant. Therefore, contact lens wearers are more predisposed to cytotoxic P. aeruginosa infections, and patients with trauma or previous surgery are more prone to infection by invasive strains. Previous studies with mutant P. aeruginosa strains unable to produce T3SS exotoxins were more susceptible to disinfectants and less able to adhere to soft contact lenses, indicating an essential role of T3SS in bacterial virulence. Invasion of P. aeruginosa intracellularly was found to be associated with control of scaffold protein IQ-domain GTPase-activating protein 1 (IQGAP1) and human corneal epithelial cell tight junctions. Knockdown of IQGAP1 strengthened tight junctions that prevented intracellular survival of invasive P. aeruginosa strains and enhanced corneal epithelial cell survival. These novel findings of the vital role of T3SS in the pathogenesis of pseudomonal MKs will provide new guidelines in both prevention and treatment of this common eye-blinding infection.

Correlation of pterygium severity with IQ-domain GTPase-activating protein 1 (IQGAP1) and mast cells

IQ-domain GTPase-activating protein 1 (IQGAP1) is a multidomain scaffold protein that is involved in cytoskeleton dynamics and tumor metastasis. Although the role of IQGAP1 in various cancers had been reported, the function of IQGAP1 in pterygium has not been studied. In this study, surgically excised pterygium and control conjunctival tissue from cataract patients were analysed by immunohistochemistry, confocal microscopy, and Western blot for IQGAP1 expression, mast cell counts, and microvascular count. Pterygium was clinically divided into mild and severe types according to Tan's classification and Kim's criteria based on translucency and vascularity of the tissue. Greater clinical severity of pterygium was associated with higher expression of IQGAP1 expression. Compared to normal conjunctival tissue, severe pterygium had significantly higher IQGAP1 expression (P = 0.005), which strongly correlated to the number of microvessels (P = 0.003) and mast cells (P = 0.01). Confocal microscopy revealed IQGAP1 colocalization with mast cell and CD31. IQGAP1 expression was higher in the pterygium body compared to the head. In conclusion, the level of IQGAP1 expression was found to be correlated to the clinical severity of pterygium. Mast cells were identified in pterygium and is suspected to be involved in promoting fibrovascular invasion.

Ca2+-Dependent Switch of Calmodulin Interaction Mode with Tandem IQ Motifs in the Scaffolding Protein IQGAP1.

IQ domain GTPase-activating scaffolding protein 1 (IQGAP1) mediates cytoskeleton, cell migration, proliferation, and apoptosis events. Calmodulin (CaM) modulates IQGAP1 functions by binding to its four tandem IQ motifs. Exactly how CaM binds the IQ motifs and which functions of IQGAP1 CaM regulates and how are fundamental mechanistic questions. We combine experimental pull-down assays, mutational data, and molecular dynamics simulations to understand the IQ-CaM complexes with and without Ca2+ at the atomic level. Apo-CaM favors the IQ3 and IQ4 motifs but not the IQ1 and IQ2 motifs that lack two hydrophobic residues for interactions with apo-CaM's hydrophobic pocket. Ca2+-CaM binds all four IQ motifs, with both N- and C-lobes tightly wrapped around each motif. Ca2+ promotes IQ-CaM interactions and increases the amount of IQGAP1-loaded CaM for IQGAP1-mediated signaling. Collectively, we describe IQ-CaM binding in atomistic detail and feature the emergence of Ca2+ as a key modulator of the CaM-IQGAP1 interactions.

Knockdown of IQGAP-1 Enhances Tight Junctions and Prevents P. aeruginosa Invasion of Human Corneal Epithelial Cells

ABSTRACT Purpose To determine the role of IQ-domain GTPase-activating protein1 (IQGAP-1) in tight junctions of human corneal epithelial cells (HCECs) and its effect against P. aeruginosa (PAK) invasion. Material and Methods Primary human corneal epithelial cells (HCECs), immortalized HCECs, and IQGAP-1 RNA knockdown HCECs (siHCECs) were used. Confocal microscopy, transepithelial electrical resistance (TER), trypan blue exclusion assay and gentamicin invasion assay were done. Results In primary and immortalized HCECs, IQGAP-1 co-localized with zonular occludin-1 (ZO-1) and actin. Enhanced actin and ZO-1 aggregation were seen in siHCECs. IQGAP-1 knockdown significantly increased TER of immortalized HCECs (P < .0001). Cell viability after PAK infection increased for siHCECs for up to 4 h after infection. PAK intracellular invasion was significantly lowered by 50% in siHCECs at 1 h post-infection. Conclusion IQGAP-1 knockdown increased the strength and integrity of tight junctions and may provide an early protective effect against P. aeruginosa invasion.

Downregulation of IQGAP1 inhibits epithelial-mesenchymal transition via the HIF1α/VEGF-A signaling pathway in gastric cancer.

As an oncogene, IQ-domain GTPase-activating protein 1 (IQGAP1) regulates the epithelial-mesenchymal transition (EMT) of several cancers, such as breast cancer, thyroid cancer, and esophageal squamous cell carcinoma. However, the role of the scaffold protein IQGAP1 on EMT in gastric cancer remains unclear. Therefore, the present work was performed to address the question. Our results showed that IQGAP1 expression is upregulated in human gastric cancer specimens and cell lines. Furthermore, IQGAP1 knockdown inhibited the migratory ability of gastric cancer cells and reduced the expression of mesenchymal phenotype markers, including Slug, β-catenin, Snail, Vimentin, and N-cadherin, as well as vascular endothelial growth factor-A (VEGF-A) secretion in gastric cancer cells. Conversely, IQGAP1 downregulation increased the epithelial phenotype marker E-cadherin. Furthermore, IQGAP1 silencing not only downregulated hypoxia-inducible transcription factor 1α (HIF1α) but also limited its translocation from the cytosol to the nucleus. Collectively, our results indicated that EMT was regulated by IQGAP1, which was associated with VEGF-A, since other data demonstrated that HIF1α was involved in VEGF-A expression. Therefore, we speculated that IQGAP1 regulated EMT of gastric cancer partially via the HIF1α/VEGF-A signaling pathway. IQGAP1 may serve as an effective therapeutic biomarker for gastric cancer.

Endothelial Calmodulin and CaMKII Play a Role in Leukocyte Transmigration

Inflammation results in the dilation of blood vessels and recruitment of circulating leukocytes into damaged tissues for repair. A transient increase in endothelial cytosolic free calcium (↑[Ca2+]i) is required for leukocyte transendothelial migration (TEM). We have also found that IQ‐domain GTPase‐activating protein 1 (IQGAP1) is essential for TEM. Specifically, the calmodulin‐binding isoleucine‐glutamine (IQ) motif domain is critical for its function during TEM. Calmodulin (CaM) is an intracellular calcium‐binding protein, however, its role in TEM is undescribed. Therefore, we hypothesized that it is involved in the endothelial calcium signaling cascade essential for supporting leukocyte transmigration.In endothelial cells IQGAP1 knockdown reduced TEM substantially. Re‐expressing IQGAP1 truncation mutants containing both the actin‐binding and IQ‐motif domains rescued TEM but truncation mutants lacking the IQ motif domain but did not support TEM.Human endothelial cells treated with the calmodulin inhibitor trifluoperazine (TFP) demonstrated a marked reduction in leukocyte TEM. Only 13% of leukocytes transmigrated through endothelial monolayers pretreated with 100 mM TFP compared to 84% TEM in control, untreated monolayers (p&lt;0.05). We also looked at the role of calmodulin kinase II (CaMKII). Treating endothelial monolayers with 10 mM KN‐93, a CaMKII inhibitor, substantially reduced leukocyte TEM. Here 22% of leukocytes transmigrated which was significantly reduced compared to control TEM rates (p&lt;0.05). Calmodulin pulldowns also showed that without the IQ‐motif domain IQGAP1 cannot substantially interact with calmodulin further suggesting a link between endothelial calcium, IQGAP1 and CaM.Current experiments focus on inhibiting CaM and CaMKII in endothelial cells using genetically encoded selective inhibitory peptides and inducible endothelial‐specific CaMKII‐deficient mice. Identifying the mechanism by which endothelial Ca2+ influx mediates TEM will provide novel insight into the signaling and also provide potential targets for anti‐inflammatory therapy.Support or Funding InformationR01 HL046849, R01 HL064774, F30 HL134202This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of extracellular matrix degradation and metastatic spread by IQGAP1 through endothelin-1 receptor signalling in ovarian cancer

The invasive phenotype of serous ovarian cancer (SOC) cells is linked to the formation of actin-based protrusions, invadopodia, operating extracellular matrix (ECM) degradation and metastatic spread. Growth factor receptors might cause engagement of integrin-related proteins, like the polarity protein IQ-domain GTPase-activating protein 1 (IQGAP1), to F-actin core needed for invadopodia functions. Here, we investigated whether IQGAP1 forms a signalosome with endothelin-1 (ET-1)/β-arrestin1 (β-arr1) network, as signal-integrating module for adhesion components, cytoskeletal remodelling and ECM degradation. In SOC cells, ET-1 receptor (ET-1R) activation, besides altering IQGAP1 expression and localization, coordinates the binding of IQGAP1 with β-arr1, representing a “hotspot” for ET-1R-induced invasive signalling. We demonstrated that the molecular interaction of IQGAP1 with β-arr1 affects relocalization of focal adhesion components, as vinculin, and cytoskeleton dynamics, through the regulation of invadopodia-related pathways. In particular, ET-1R deactivates Rac1 thereby promoting RhoA/C activation for the correct functions of invasive structures. Silencing of either IQGAP1 or β-arr1, or blocking ET-1R activation with a dual antagonist macitentan, prevents matrix metalloproteinase (MMP) activity, invadopodial function, transendothelial migration and cell invasion. In vivo, targeting ET-1R/β-arr1 signalling controls the process of SOC metastasis, associated with reduced levels of IQGAP1, as well as other invadopodia effectors, such as vinculin, phospho-cortactin and membrane type 1-MMP. High expression of ETAR/β-arr1/IQGAP1 positively correlates with poor prognosis, validating the clinical implication of this signature in early prognosis of SOC. These data establish the ET-1R-driven β-arr1/IQGAP1 interaction as a prerequisite for the dynamic integration of pathways in fostering invadopodia and metastatic process in human SOC.

Pivotal Pathogenic and Biomarker Role of Chlamydia Pneumoniae in Neurovascular Diseases.

Chlamydia Pneumoniae (C. Pn) is an obligatory intracellular bacterium that is associated with respiratory tract infections like pneumonia, pharyngitis and bronchitis. It has also been implicated in cerebrovascular (stroke) as well as cardiovascular diseases. The most possible pathway via which C. Pn elicits its pathogenesis could be via activation of Human Vascular Smooth Muscle Cells (VSMCs) proliferation resulting in the stimulation of Toll-Like Receptor-4 (TLR-4) and/or phospho-44/42(p44/p42) Mitogen-Activated Protein Kinases (MAPK). It is also established that tyrosine phosphorylation of IQ domain GTPase-activating protein 1 (IQGAP1) also contributed to C. Pn infection-triggered Vascular Endothelial Cell (VEC) movements via the SRC tyrosine kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) resulting in angiogenesis. It is also proven that restricted inflammatory cell infiltrates as well as apoptosis have been linked to C. Pn or C. Pn-specific proteins in atherosclerotic plaques of patients with stroke. It is further an evidence that C. Pn enters the cerebral vasculature during the initial infection and worsen atherosclerosis either directly or indirectly. Chronic, persistent C. Pn infection is also capable of triggering the secretion of Chlamydial Heat Shock Protein 60 (cHSP60) in the vessel wall resulting in augmentation of inflammation. C. Pn also aids in the activation of explicit cell-intermediated immunity within plaques. Macrophages in the carotid plaques co-exist with CD4+ lymphocytes which are capable of triggering the release of pro-inflammatory cytokines resulting in the augmentation of atherogenic development during C. Pn infection. C. Pn actively participated in the modification of both histones H3 and H4 during chromatin analysis via the interleukin 8(IL-8) gene facilitator as well as conscription of nuclear factor kappa-B(NF-κB) or NF- κB/p65 complex and polymerase II (Pol II). This review, therefore, focuses on the crucial involvement of C. Pn in the pathogenesis of cerebrovascular events.

Overexpression of IQGAP1 promotes the angiogenesis of esophageal squamous cell carcinoma through the AKT and ERK‑mediated VEGF‑VEGFR2 signaling pathway

Angiogenesis is crucial for the progression of esophageal squamous cell carcinoma (ESCC). Anti‑angiogenesis by targeting important molecules has been considered as one of the most promising and efficient strategy for cancer therapy. Recent studies have demonstrated that the IQ‑domain GTPase activating protein1 (IQGAP1) plays critical roles in tumorigenesis and cancer progression. We previously reported that IQGAP1 is overexpressed in ESCC, and IQGAP1 knockdown can decrease cell proliferation and metastasis ability invitro and invivo. However, the effects of IQGAP1 on the angiogenesis of ESCC and its underlying mechanisms remain unknown. In the present study, we found that IQGAP1 overexpression promoted tumor angiogenesis confirmed by human umbilical vascular endothelial cell (HUVEC) tube formation invitro and chicken embryo chorioallantoic membrane (CAM) assay invivo. Moreover, IQGAP1 overexpression in ESCC cells increased expression of vascular endothelial growth factor (VEGF) and phosphorylation of vascular endothelial growth factor receptor2 (VEGFR2). Meanwhile, we found that levels of AKT and ERK phosphorylation were upregulated in IQGAP1‑overexpressing cells. Importantly, IQGAP1‑knockdown cells showed the opposing results. Furthermore, AKT and ERK inhibitors not only significantly decreased VEGF expression and VEGFR2 phosphorylation in IQGAP1‑overexpressing cells, but also abolished the pro‑angiogenic effect of IQGAP1 overexpression on angiogenesis in the HUVEC tube formation and chicken embryo CAM assay. Taken together, this evidence confirms that IQGAP1 overexpression promotes tumor angiogenesis via the AKT and ERK‑mediated VEGF‑VEGFR2 signaling pathway in ESCC, and IQGAP1 may be an attractive therapeutic target for cancer anti‑angiogenesis treatment.

Over-expression of IQGAP1 indicates poor prognosis in head and neck squamous cell carcinoma.

IQ-domain GTPase-activating protein 1 (IQGAP1) is associated with the development and progression of many human cancers. We aimed to investigate the expression and clinicopathological significances of IQGAP1 in head and neck squamous cell carcinoma (HNSCC). In this study, immunohistochemical staining of IQGAP1, co-inhibitory immune checkpoint molecules and macrophage markers were performed in human HNSCC samples to analyze the expression and correlation with clinicopathological characteristics. Immunoreactivity of IQGAP1 was also detected in immunocompetent mouse HNSCC tissue. We found that IQGAP1 expression level was significantly increased in human HNSCC compared with dysplasia and normal mucosa, and the expression of IQGAP1 in HNSCC was positively associated with advanced lymph node status. Besides, our data indicated that patients with higher IQGAP1 expression exhibited poor overall survival compared with patients with lower IQGAP1 expression. Furthermore, this study demonstrated that IQGAP1 expression was positively associated with TIM3, Galectin-9 (TIM3 ligand), B7H4, macrophage markers CD68 and CD163. In conclusion, these findings suggest that over-expression of IQGAP1 in human HNSCC may indicate poor prognosis.

Exploring the Role of Calmodulin and Calcium Signaling in Leukocyte Transmigration

Inflammation results in the dilation of local blood vessels, the activation of endothelium by cytokines and the recruitment of circulating leukocytes into areas of tissue damage. A transient increase in endothelial cytosolic free calcium (Ca2+) is required for leukocyte transendothelial migration (TEM) and transient receptor potential canonical 6 (TRPC6) has been found to mediate this required Ca2+ influx. Furthermore, we have also found that IQ‐domain GTPase‐activating protein 1 (IQGAP1) is essential for TEM. Specifically, the actin‐binding domain and the calmodulin‐binding isoleucine‐glutamine (IQ) domain of IQGAP1 are critical for its function during TEM. In endothelial cells re‐expression of IQGAP1 truncation mutants lacking the N‐terminal actin‐binding domain did not localize to endothelial borders and did not support TEM. Re‐expression of truncated IQGAP1 constructs containing the actin‐binding domain and the IQ domain localized to cell borders and supported TEM while constructs containing the actin‐binding domain but lacking the IQ domain localized to the cell borders but did not restore TEM.Calmodulin (CaM) is a small, highly conserved intracellular calcium‐binding protein, which has been shown to interact with IQGAP1 through the IQ domain. It has also been previously found to regulate TRPC channel activation; however, not much is known regarding its role in TEM. Therefore, we hypothesized that it may be involved in the calcium signaling cascade within endothelial cells essential for supporting leukocyte transmigration.Human endothelial cells treated with calmodulin inhibitor trifluoperazine (TFP) demonstrated a marked reduction in leukocyte TEM. Endothelial monolayers pretreated with 100 mM TFP for 1 hour drastically reduced leukocyte TEM. Only about 13% of leukocytes were able to transmigrate compared to 84% TEM in control, untreated monolayers (p=0.0025).After identifying the potential role of calmodulin, we generated CaM shRNA to deplete endogenous CaM within endothelial cells, wildtype Flag‐tagged CaM re‐expression constructs and CaM calcium‐binding mutants that we are currently in the process of testing. Identifying the specific mechanism by which endothelial Ca2+ influx through TRPC6 mediates the events of TEM will provide novel insight into the signaling involved in TEM and also provide potential therapeutic targets for anti‐inflammatory therapy.Support or Funding InformationF30HL134202, 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.

IQGAP1 silencing suppresses the malignant characteristics of laryngeal squamous cell carcinoma cells.

Laryngeal squamous cell carcinoma (LSCC) has a poor prognosis due to recurrence and metastasis. IQ-domain GTPase-activating protein 1 (IQGAP1), a scaffold protein, plays an important role in tumorigenesis and malignant development. In this study, we aimed to explore the role of IQGAP1 in LSCC. Expression of IQGAP1 in human LSCC specimens was assessed by immunohistochemistry. We also evaluated the roles of IQGAP1 in cell proliferation, migration and invasion and epithelial-to-mesenchymal transition (EMT) in Hep-2 cells. The expression of IQGAP1 protein was significantly up-regulated in LSCC tissues compared with normal laryngeal tissues (p = 0.002). Furthermore, the knockdown of IQGAP1 in Hep-2 cells inhibited cell growth, migration and invasion. Moreover, we found that IQGAP1 silencing reversed EMT. These results show for the first time that IQGAP1 is up-regulated in LSCC tissues and plays an important role in LSCC cell proliferation and invasiveness, which indicates that IQGAP1 could work as an oncogene and may serve as a promising molecular target for treatment of LSCC.

IQ-domain GTPase-activating protein 1 promotes the malignant phenotype of invasive ductal breast carcinoma via canonical Wnt pathway.

IQ-domain GTPase-activating protein 1 is a scaffolding protein with multidomain which plays a role in modulating dishevelled (Dvl) nuclear translocation in canonical Wnt pathway. However, the biological function and mechanism of IQ-domain GTPase-activating protein 1 in invasive ductal carcinoma (IDC) remain unknown. In this study, we found that IQ-domain GTPase-activating protein 1 expression was elevated in invasive ductal carcinoma, which was positively correlated with tumor grade, lymphatic metastasis, and poor prognosis. Coexpression of IQ-domain GTPase-activating protein 1 and Dvl in the nucleus and cytoplasm of invasive ductal carcinoma was significantly correlated but not in the membrane. Postoperative survival in the patients with their coexpression in the nucleus and cytoplasm was obviously lower than that without coexpression. The positive expression rates of c-myc and cyclin D1 were significantly higher in the patients with nuclear coexpression of Dvl and IQ-domain GTPase-activating protein 1 than that with cytoplasmic coexpression, correlating with poor prognosis. IQ-domain GTPase-activating protein 1 significantly enhanced cell proliferation and invasion in invasive ductal carcinoma cell lines by interacting with Dvl in cytoplasm to promote Dvl nuclear translocation so as to upregulate the expression of c-myc and cyclin D1. Collectively, our data suggest that IQ-domain GTPase-activating protein 1 may promote the malignant phenotype of invasive ductal carcinoma via canonical Wnt signaling, and it could be used as a potential prognostic biomarker for breast cancer patients.

Identifying Key Domains in Endothelial IQGAP1 Critical for Leukocyte Transmigration

Inflammation results in the dilation of local blood vessels, the activation of endothelium by cytokines and the recruitment of circulating leukocytes into areas of tissue damage. The majority of leukocytes undergo transendothelial migration (TEM) by using a unique endothelial cell membrane system referred to as the lateral border recycling compartment (LBRC). IQ‐domain GTPase‐activating protein 1 (IQGAP1) was identified as an LBRC‐associated protein and subsequently found to be essential for TEM. IQGAP1 is a scaffolding protein comprised of six major interaction domains. It has been reported to interact productively with over 135 cellular proteins. In endothelial cells IQGAP1 partially localizes to cell borders and knocking down its expression prevents TEM. However, it is not known which of the many proteins it is known to interact with carries out its role in TEM. Since these proteins bind to specific IQGAP1 domains, as a first step toward understanding the mechanism by which IQGAP1 regulates TEM, we explored which of its six protein interactions domains were critical for its function. We knocked down IQGAP1 in endothelial cells and re‐expressed truncated forms of IQGAP1 with specific regions deleted to identify the regions (and thereby the potential binding partners) that are critical for TEM. We show that the actin‐binding domain and the calmodulin‐binding isoleucine‐glutamine (IQ) domain of IQGAP1 are critical for its function during TEM.Human endothelial cells in which &gt;80% of endogenous IQGAP1 was depleted by shRNA did not support TEM; re‐expression of wild‐type IQGAP1 restored TEM to control levels. Re‐expression of IQGAP1 with a GFP‐tagged construct after knockdown also showed IQGAP1 enrichment around transmigrating leukocytes. Re‐expression of IQGAP1 truncation mutants lacking the N‐terminal actin‐binding domain did not localize to endothelial borders and did not support TEM. Re‐expression of truncated IQGAP1 constructs containing the actin‐binding domain and the IQ domain localized to cell borders and supported TEM. Constructs containing the actin‐binding domain but lacking the IQ domain localized to the cell borders but did not restore TEM.After identifying the IQ domain as having a critical role in TEM, we generated specific point mutations in this domain within the full‐length construct. In particular, the isoleucine (I)‐glutamine (Q) motif after which the IQ domain is named has been identified in the literature as having an essential role in IQGAP1 function and we are currently testing whether they are also important in TEM. Using these constructs in future studies will help identify the role of the IQ domain in TEM and its interacting partner(s). Identifying the binding partners of IQGAP1 that are critical for inflammation will provide clues to its mechanism of action and potential therapeutic targets for anti‐inflammatory therapy.Support or Funding InformationFunded by R01 HL046849, R01 HL064774, Alpha Omega Alpha Carolyn Kuckein Student Research Fellowship

The Overexpression of IQGAP1 and β-Catenin Is Associated with Tumor Progression in Hepatocellular Carcinoma In Vitro and In Vivo.

The IQ-domain GTPase-activating protein 1 (IQGAP1) is a multifunctional scaffold protein, which interacts with diverse proteins to regulate cell adhesion and cell migration. The abnormal expression of IQGAP1 widely exists in many cancers, but biological roles of IQGAP1 cooperation with its interacting proteins to involve in tumorigenesis remain to clarify. In this study, we have found that IQGAP1 interacts with β-catenin and regulates β-catenin expression in hepatocellular carcinoma (HCC) cells. The expression levels of IQGAP1 and β-catenin and their associations have a positive correlation with cell metastasis ability in several HCC cell lines. The up-regulation of IQGAP1 and β-catenin improves cell proliferation and migration ability of HCC cells, whereas the knockdown of IQGAP1 by small interfering RNA can decrease β-catenin expression, which results in the reduction of cell proliferation and migration ability in vitro. In addition, a significantly higher expression of IQGAP1 and β-catenin also usually exists in human HCC tissues, especially their overexpression is clinicopathologically associated with tumor malignancy. Generally the overexpression and interactions of IQGAP1 and β-catenin contribute to HCC progression by promoting cell proliferation and migration.

IQGAP1 modulates the proliferation and invasion of thyroid cancer cells in response to estrogen.

Thyroid cancer is an endocrine malignancy with a high incidence rate, which is affected by female hormones, particularly estrogens, in its growth and progression. IQ-domain GTPase-activating protein 1 (IQGAP1) is overexpressed in a range of types of cancer and is reported to interact with estrogen receptor α (ERα) in breast cancer cells. However, the association between IQGAP1 and ERα in thyroid cancer cells remains to be elucidated. In this study, the role of IQGAP1 in thyroid cancer cells was examined. The expression of IQGAP1 (190 kDa) was analyzed using western blot analysis, which indicated that IQGAP1 was overexpressed in thyroid cancer tissues and FTC133 cells. However, IQGAP1 knockdown in the FTC133 cells led to a significant downregulation in ERα transcriptional activity, cell proliferation, cell adhesion and cell invasion under 17β-estradiol (E2) conditions. Furthermore, ERα knockdown inhibited the enhanced protein expression levels of phosphorylated ERK1/2 and cyclin D1, which were induced by the overexpression of IQGAP1. Co-immunoprecipitation was also performed in thyroid cancer cells and the results suggested that IQGAP1 directly interacted with ERα in the FTC133 cells and the co-transfected COS-7 cells. Taken together, these findings revealed that IQGAP1 may directly interact with ERα and serve as a signal integrator, mediating ERα transcriptional activity, cell proliferation and cell invasion during the progression of thyroid cancer.

IQGAP1 Is Involved in Enhanced Aggressive Behavior of Epithelial Ovarian Cancer Stem Cell-Like Cells During Differentiation.

BackgroundWide metastasis is one of characteristics of ovarian cancer. Cancer stem cells, as a source in cancer invasion and metastasis, possess powerful potential of differentiation. Scaffolding IQ domain GTPase-activating protein 1 (IQGAP1) plays a key role in the invasion and metastasis of cancer cells, but IQGAP1’s role in cancer stem cells including ovarian cancer was unclear.MethodsSpheroid culture with serum-free medium was used for enriching ovarian cancer stem cell-like cells (CSC-LCs) from 3AO cell line, and a medium with 10% fetal bovine serum was used to induce the differentiation of CSC-LCs. Immunofluorescence was for detecting the stem markers OCT4 and SOX2. The quantitative real-time-polymerase chain reaction and Western blotting were performed to determine the messenger RNA and protein expression of IQGAP1, respectively. The capacity of cell invasion was evaluated by transwell chamber assay.ResultsOvarian CSC-LCs obtained through spheroid culture showed irregularly elongated appearance, CD24 negative, and OCT4 and SOX2 positive. IQGAP1 expression was decreased in ovarian CSC-LCs compared with parental 3AO cells, but increased de novo during the differentiation of CSC-LCs. Knockdown of IQGAP1 by specific small interfering RNA remarkably weakened invasion capacity of 2-day differentiated ovarian CSC-LCs.ConclusionsIncreased IQGAP1 expression during the differentiation of CSC-LCs is involved in an aggressive cell behavior, which may contribute to metastasis of ovarian cancer.