关键词： Hepatocellular carcinoma   Transfection   Small interfering RNA   Cell migration   Cell proliferation   Hyperexpression techniques   Differentiated tumors   Metastasis  

摘要： 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 beta-catenin and regulates beta-catenin expression in hepatocellular carcinoma (HCC) cells. The expression levels of IQGAP1 and beta-catenin and their associations have a positive correlation with cell metastasis ability in several HCC cell lines. The up-regulation of IQGAP1 and beta-catenin improves cell proliferation and migration ability of HCC cells, whereas the knockdown of IQGAP1 by small interfering RNA can decrease beta-catenin expression, which results in the reduction of cell proliferation and migration ability in vitro. In addition, a significantly higher expression of IQGAP1 and beta-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 beta-catenin contribute to HCC progression by promoting cell proliferation and migration.

关键词： Extracellular vesicles   Liquid chromatography-tandem mass spectrometry   Colorectal cancer   Proteomics   14-3-3   STAT1   IQGAP1   Raf-1   prohibitin   NF-kappa B   Luciferase reporter assay  

摘要： Background: Extracellular vesicles (EVs) are secreted from many cells, carrying cargoes including proteins and nucleic acids. Research has shown that EVs play a role in a variety of biological processes including immunity, bone formation and recently they have been implicated in promotion of a metastatic phenotype. Methods: EVs were isolated from HCT116 colon cancer cells, 1459 non-malignant colon fibroblast cells, and tumor and normal colon tissue from a patient sample. Co-cultures were performed with 1459 cells and malignant vesicles, as well as HCT116 cells and non-malignant vesicles. Malignant phenotype was measured using soft agar colony formation assay. Co-cultures were also analyzed for protein levels using mass spectrometry. The importance of 14-3-3 zeta/delta in transfer of malignant phenotype was explored using siRNA. Additionally, luciferase reporter assay was used to measure the transcriptional activity of NF-kappa B. Results: This study demonstrates the ability of EVs derived from malignant colon cancer cell line and malignant patient tissue to induce the malignant phenotype in non-malignant colon cells. Similarly, EVs derived from non-malignant colon cell lines and normal patient tissue reversed the malignant phenotype of HCT116 cells. Cells expressing an EV-induced malignant phenotype showed increased transcriptional activity of NF-kappa B which was inhibited by the NF-kappa B inhibitor, BAY117082. We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs. Conclusions: Evidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis. Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer.

关键词： SCAFFOLD proteins   CXCR4 receptors   CANCER cells   T cells   CELLULAR signal transduction   ENDOSOMES   EXTRACELLULAR signal-regulated kinases   MITOGEN-activated protein kinases  

摘要： IQ motif containing GTPase-activating protein 1 (IQGAP1) is a cytoskeleton-interacting scaffold protein. CXCR4 is a chemokine receptor that binds stromal cell derived factor-1 (SDF-1;also known as CXCL12). Both IQGAP1 and CXCR4 are overexpressed in cancer cell types, yet it was unclear whether these molecules functionally interact. Here, we show that depleting IQGAP1 in Jurkat T leukemic cells reduced CXCR4 expression, disrupted trafficking of endocytosed CXCR4 via EEA-1(+) endosomes, and decreased efficiency of CXCR4 recycling. SDF-1 induced cell migration and activation of extracellular signal-regulated kinases 1 and 2 (ERK) MAPK were strongly inhibited, even when forced overexpression restored CXCR4 levels. Similar results were seen in KMBC and HEK293 cells. Exploring the mechanism, we found that SDF-1 treatment induced IQGAP1 binding to a-tubulin and localization to CXCR4-containing endosomes and that CXCR4-containing EEA-1(+) endosomes were abnormally located distal from the microtubule (MT)-organizing center (MTOC) in IQGAP1-deficient cells. Thus, IQGAP1 critically mediates CXCR4 cell surface expression and signaling, evidently by regulating EEA-1(+) endosome interactions with MTs during CXCR4 trafficking and recycling. IQGAP1 may similarly promote CXCR4 functions in other cancer cell types.

关键词： Scaffold proteins   Lymphocytes   Signaling   IQGAP1  

摘要： The intracellular spatiotemporal organization of signaling events is critical for normal cellular function. In response to environmental stimuli, cells utilize highly organized signaling pathways that are subject to multiple layers of regulation. However, the molecular mechanisms that coordinate these complex processes remain an enigma. Scaffolding proteins (scaffolins) have emerged as critical regulators of signaling pathways, many of which have well-described functions in immune cells. IQGAP1, a highly conserved cytoplasmic scaffold protein, is able to curb, compartmentalize, and coordinate multiple signaling pathways in a variety of cell types. IQGAP1 plays a central role in cell-cell interaction, cell adherence, and movement via actin/tubulin-based cytoskeletal reorganization. Evidence also implicates IQGAP1 as an essential regulator of the MAPK and Wnt/beta-catenin signaling pathways. Here, we summarize the recent advances on the cellular and molecular biology of IQGAP1. We also describe how this pleiotropic scaffolin acts as a true molecular puppeteer, and highlight the significance of future research regarding the role of IQGAP1 in immune cells. (C) 2015 Elsevier Ltd. All rights reserved.

关键词： FRET: Forster (or fluorescence) resonance energy transfer   E%: energy-transfer efficiency   quantitative FRET analysis   actin nucleation   N-WASP   Rac1   Cdc42   IQGAP1   ROI: region of interest   PFRET: processed FRET or spectral bleedthrough-corrected FRET  

摘要： Experiments using live cell 3-color Forster (or fluorescence) resonance energy transfer (FRET) microscopy and corresponding in vitro biochemical reconstitution of the same proteins were conducted to evaluate actin filament nucleation. A novel application of 3-color FRET data is demonstrated, extending the analysis beyond the customary energy-transfer efficiency (E%) calculations. MDCK cells were transfected for coexpression of Teal-N-WASP/Venus-IQGAP1/mRFP1-Rac1, Teal-N-WASP/Venus-IQGAP1/mRFP1-Cdc42, CFP-Rac1/Venus-IQGAP1/mCherry-actin, or CFP-Cdc42/Venus-IQGAP1/mCherry-actin, and with single-label equivalents for spectral bleedthrough correction. Using confirmed E% as an entry point, fluorescence levels and related ratios were correlated at discrete accumulating levels at cell peripheries. Rising ratios of CFP-Rac1:Venus-IQGAP1 were correlated with lower overall actin fluorescence, whereas the CFP-Cdc42:Venus-IQGAP1 ratio correlated with increased actin fluorescence at low ratios, but was neutral at higher ratios. The new FRET analyses also indicated that rising levels of mRFP1-Cdc42 or mRFP1-Rac1, respectively, promoted or suppressed the association of Teal-N-WASP with Venus-IQGAP1. These 3-color FRET assays further support our in vitro results about the role of IQGAP1, Rac1, and Cdc42 in actin nucleation, and the differential impact of Rac1 and Cdc42 on the association of N-WASP with IQGAP1. In addition, this study emphasizes the power of 3-color FRET as a systems biology strategy for simultaneous evaluation of multiple interacting proteins in individual live cells. (c) 2015 International Society for Advancement of Cytometry

关键词： 乳腺肿瘤   基底细胞样型   细胞分裂周期蛋白42(Cdc42)   支架蛋白1(IQGAP1)  

摘要： [目的]分析细胞分裂周期蛋白42(Cdc42)和支架蛋白1(IQGAP1)与基底细胞样型乳腺癌(basal-like breast cancer,BLBC)临床病理特征之间的关系及二者之间的相关性,探讨Cdc42和IQGAP1在BLBC发生、发展及侵袭转移中的作用。[方法 ]采用免疫组化方法检测60例BLBC、45例非基底细胞样型乳腺癌(non-basal-like breast carcinoma,Non-BLBC)和35例癌旁正常乳腺组织中Cdc42和IQGAP1的表达。[结果]Cdc42在BLBC中的表达明显高于Non-BLBC和癌旁正常乳腺组织(P<0.0125),IQGAP1在BLBC和Non-BLBC中的表达明显高于癌旁正常乳腺组织(P<0.0125);BLBC中Cdc42的表达与淋巴结转移和TNM分期密切相关(P<0.05),IQGAP1的表达与肿瘤大小和淋巴结转移密切相关(P<0.05),并且两者在BLBC中的表达呈正相关关系(r=0.638,P<0.05)。[结论]Cdc42和IQGAP1可能是促进肿瘤的局部侵袭和远处转移的重要因素,在BLBC的发生发展中起着重要的作用。

关键词： IQGAP1   Ovarian cancer   Cancer stem cell-like cells   Differentiation   Invasion  

摘要： Background Wide 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. Methods Spheroid 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. Results Ovarian 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. Conclusions Increased IQGAP1 expression during the differentiation of CSC-LCs is involved in an aggressive cell behavior, which may contribute to metastasis of ovarian cancer.

关键词： biology   IQGAP1   IQGAP2   IQGAP3   therapeutics  

摘要： IQGAP scaffold proteins are evolutionarily conserved in eukaryotes and facilitate the formation of complexes that regulate cytoskeletal dynamics, intracellular signaling, and intercellular interactions. Fungal and mammalian IQGAPs are implicated in cytokinesis. IQGAP1, IQGAP2, and IQGAP3 have diverse roles in vertebrate physiology, operating in the kidney, nervous system, cardiovascular system, pancreas, and lung. The functions of IQGAPs can be corrupted during oncogenesis and are usurped by microbial pathogens. Therefore, IQGAPs represent intriguing candidates for novel therapeutic agents. While modulation of the cytoskeletal architecture was initially thought to be the primary function of IQGAPs, it is now clear that they have roles beyond the cytoskeleton. This review describes contributions of IQGAPs to physiology at the organism level.

关键词： Actin cytoskeleton   IQ domain GTPase-activating protein 1   Nephrin   Podocyte  

摘要： Increasing data has shown that the cytoskeletal reorganization of podocytes is involved in the onset of proteinuria and the progression of glomerular disease. Nephrin behaves as a signal sensor of the slit diaphragm to transmit cytoskeletal signals to maintain the unique structure of podocytes. However, the nephrin signaling cascade deserves further study. IQGAP1 is a scaffolding protein with the ability to regulate cytoskeletal organization. It is hypothesized that IQGAP1 contributes to actin reorganization in podocytes through interaction with nephrin. IQGAP1 expression and IQGAP1-nephrin colocalization in glomeruli were progressively decreased and then gradually recovered in line with the development of foot process fusion and proteinuria in puromycin aminonucleoside-injected rats. In cultured human podocytes, puromycin aminonudeoside-induced disruption of F-actin and disorders of migration and spreading were aggravated by IQGAP1 siRNA, and these effects were partially restored by a wild-type IQGAP1 plasmid. Furthermore, the cytoskeletal disorganization stimulated by cytochalasin D in COS7 cells was recovered by cotransfection with wild-type IQGAP1 and nephrin plasmids but was not recovered either by single transfection of the wild-type IQGAP1 plasmid or by cotransfection of mutant IQGAP1 [Delta 1443(S -> A)] and wild-type nephrin plasmids. Co-immunoprecipitation analysis using lysates of COS7 cells overexpressing nephrin and each derivative-domain molecule of IQGAP1 demonstrated that the poly-proline binding domain and RasGAP domain in the carboxyl terminus of IQGAP1 are the target modules that interact with nephrin. Collectively, these findings showed that activated IQGAP1, as an intracellular partner of nephrin, is involved in actin cytoskeleton organization and functional regulation of podocytes. (c) 2015 Elsevier Inc. All rights reserved.

关键词： Insulin   Exocytosis   Endocytosis   Rab27a   IQGAP1   Coronin   3   Glucose   Small   GTPase  

摘要： The small GTPase Rab27 a is a member of the Rab family that is involved in membrane trafficking in various kinds of ***27 a has GTP-and GDP-bound forms,and their interconversion regulates intracellular signaling ***,only a GTP-bound GTPase binds its specific effectors with the resulting downstream signals controlling specific cellular *** previously identified novel Rab27a-interacting ***,some of these proteins interacted with GDP-bound Rab27 *** present study reviews recent progress in our understanding of the roles of Rab27 a and its effectors in the secretory *** pancreatic β-cells,GTP-bound Rab27 a regulates insulin secretion at the preexocytotic stages via its GTP-specific effectors such as Exophilin8/Slac2-c/My RIP and Slp4/*** stimulation causes insulin *** stimulation also converts Rab27 a from its GTP-to its GDP-bound ***-bound Rab27 a interacts with GDP-specific effectors and controls endocytosis of the secretory ***,Rab27 a cycling between GTP-and GDP-bound forms synchronizes with the recycling of secretory membrane to re-use the membrane and keep the β-cell volume constant.