关键词： KRAS4B   Ras isoforms   K-Ras   H-Ras   N-Ras   Plasma membrane   Calcium   Proliferation   Phosphorylated tyrosine motif   Drug discovery  

摘要： Calmodulin (CaM) binds only oncogenic KRas, but not HRas or NRas, and thus contributes only to KRAS-driven cancers. How CaM interacts with KRas and how it boosts KRAS cancers are among the most coveted aims in cancer biology. Here we address this question, and further ask: Are there proteins that can substitute for CaM in HRAS- and NRAS-driven cancers? Can scaffolding protein IQGAP1 be one? Data suggest that formation of a CaM-KRas-PI3K alpha ternary complex promotes full PI3K alpha activation, and thereby potent PI3K alpha/Akt/mTOR proliferative signaling. CaM binds PI3K alpha at the cSH2 and nSH2 domains of its regulatory p85 subunit;the WW domain of IQGAP1 binds cSH2. This raises the question whether IQGAP1, together with an oncogenic Ras isoform, can partially activate PI3K alpha. Activated, membrane-bound PI3K alpha generates PIPS. CaM shuttles Akt to the plasma membrane;CaM's release and concomitant phosphoinositide binding stimulates Akt activation. Notably, IQGAP1 directly interacts with, and helps juxtapose, PI3K alpha and Akt as well as mTOR. Our mechanistic review aims to illuminate CaM's actions, and help decipher how oncogenic Ras isoforms not only KRas4B can activate the PI3K alpha/Akt/mTOR pathway at the membrane and innovate drug discovery, including blocking the PI3K alpha IQGAP1 interaction in HRAS- and NRAS-driven cancers.

关键词： Calcium   Drug discovery   H-Ras   K-Ras   KRAS4B   N-Ras   Phosphorylated tyrosine motif   Plasma membrane   Proliferation   Ras isoforms  

摘要： Calmodulin (CaM) binds only oncogenic KRas, but not HRas or NRas, and thus contributes only to KRAS -driven cancers. How CaM interacts with KRas and how it boosts KRAS cancers are among the most coveted aims in cancer biology. Here we address this question, and further ask: Are there proteins that can substitute for CaM in HRAS - and NRAS -driven cancers? Can scaffolding protein IQGAP1 be one? Data suggest that formation of a CaM–KRas–PI3Kα ternary complex promotes full PI3Kα activation, and thereby potent PI3Kα/Akt/mTOR proliferative signaling. CaM binds PI3Kα at the cSH2 and nSH2 domains of its regulatory p85 subunit; the WW domain of IQGAP1 binds cSH2. This raises the question whether IQGAP1, together with an oncogenic Ras isoform, can partially activate PI3Kα. Activated, membrane-bound PI3Kα generates PIP 3 . CaM shuttles Akt to the plasma membrane; CaM's release and concomitant phosphoinositide binding stimulates Akt activation. Notably, IQGAP1 directly interacts with, and helps juxtapose, PI3Kα and Akt as well as mTOR. Our mechanistic review aims to illuminate CaM's actions, and help decipher how oncogenic Ras isoforms – not only KRas4B – can activate the PI3Kα/Akt/mTOR pathway at the membrane and innovate drug discovery, including blocking the PI3Kα–IQGAP1 interaction in HRAS - and NRAS -driven cancers. [ABSTRACT FROM AUTHOR]

关键词： IQGAP1   malignant transformation   metastasis-associated protein 1   nasopharyngeal carcinoma   p53  

摘要： Nasopharyngeal carcinoma (NPC) is frequently seen in Chinese, especially the population that resides in southeast China. Metastasis-associated protein 1 (MTA1) is a chromatin modifier and plays a role in tumor cell metastasis. IQGAP1 is a ubiquitously expressed protein that contributes to cytoskeleton remodeling. This study aimed to investigate the role of MTA1 and IQGAP1 in NPC malignant transformation. MTA1 and IQGAP1 expression in NPC (n=43) and control tissues (n=31) were detected using qRT-PCR, immunoblot, and immunohistochemistry. MTA1 was overexpressed in CNE-1 and CNE-2 cell line by pcDNA3.1/MTA1 transfection. Dominant-negative p53 was transfected to inhibit p53 activity. si-IQGAP1 or dominant-negative IQGAP1 (IQGAP1GRD) was used to suppress IQGAP1 activity. Cell proliferation was measured by CKK-8 assay. Cell migration was evaluated by Transwell assay. The results showed that MTA1 and IQGAP1 were highly expressed in NPC tissues compared with the controls. Forced expression of MTA1 accelerated cell proliferation and migration and upregulated IQGAP1 expression in a p53-independent way. Knockdown of IQGAP1 or transfection of dominant-negative IQGAP1 impeded tumor cell proliferation and migration as well as PI3K/Akt signaling induced by MTA1. In conclusion, MTA1 participates in NPC malignant transformation via regulating IQGAP1 expression and PI3K/Akt signaling pathway.

摘要： Precise mitotic spindle orientation is essential for both cell fate and tissue organization while defects in this process are associated with tumorigenesis and other diseases. In most animal cell types, the dynein motor complex is anchored at the cell cortex and exerts pulling forces on astral microtubules to position the spindle. The actin-binding protein MISP controls spindle orientation and mitotic progression in human cells. However, the exact underlying mechanism remains to be elucidated. Here we report that MISP interacts with the multidomain scaffolding protein IQGAP1. We further show that MISP binds to the active form of Cdc42 through IQGAP1. Depletion of MISP promotes increased accumulation of IQGAP1 at the cell cortex and a decrease in its Cdc42-binding capacity leading to reduced active Cdc42 levels. Interestingly, overexpression of IQGAP1 can rescue mitotic defects caused by MISP downregulation including spindle misorientation, loss of astral microtubules and prolonged mitosis and also restores active Cdc42 levels. Importantly, we find that IQGAP1 acts downsteam of MISP in regulating astral microtubule dynamics and the localization of the dynactin subunit p150(glued) that is crucial for proper spindle positioning. We propose that MISP regulates IQGAP1 and Cdc42 to ensure proper mitotic progression and correct spindle orientation.

关键词： 肝细胞肝癌   血管生成拟态   IQGAP1   转移   干性  

摘要： 目的:检测IQGAP1在肝细胞肝癌(hepatocellular carcinoma,HCC)组织中的表达并研究其对肝癌细胞血管生成拟态形成能力的影响。方法:采用免疫组织化学法检测2001年1月至2009年1月天津医科大学肿瘤医院180例肝癌组织中IQGAP1的表达,采用CD31/PAS双染法检测肝癌中血管生成拟态的情况,并分析IQGAP1与血管生成拟态之间的相关性;将IQGAP1过表达质粒和干扰质粒分别转染至肝癌细胞系Hep G2和SMMC7721中,使用Western blot法检测转染后Hep G2和SMMC7721细胞中干性相关蛋白CD133、CD44、Sox2和ALDH1的表达情况;细胞功能学实验检测IQGAP1对迁移侵袭、增殖、管道形成能力的影响。结果:免疫组织化学结果显示IQGAP1定位于细胞膜或细胞质,其表达与肿瘤分级、转移和血管生成拟态形成相关(P<0.05)。在Hep G2细胞中上调IQGAP1,增强了Hep G2细胞迁移侵袭、增殖、管道行成能力,促进了干性相关蛋白CD133、CD44、Sox2和ALDH1的表达;在SMMC7721细胞中下调IQGAP1,抑制了SMMC7721细胞迁移侵袭、增殖、管道行成能力(P<0.05),降低了上述干性相关蛋白的表达。结论:IQGAP1表达升高促进了HCC的恶性生物学行为,IQGAP1可能通过诱导HCC干性来促进血管生成拟态形成。

关键词： β连环蛋白   含IQ基序的GTP激酶活化蛋白1   有丝分裂原激活蛋白激酶通路   细胞增殖  

摘要： 本文主要研究β连环蛋白(β-catenin)和含IQ基序的GTP激酶活化蛋白1(IQGAP1)的相互调控,及其在肠癌细胞增殖中的功能。利用细胞转染技术改变肠癌细胞SW1116中的β-catenin或者IQGAP1表达后,通过蛋白质印迹法分别检测SW1116细胞中IQGAP1和β-catenin的表达情况。利用CCK-8细胞增殖实验检测干扰下调IQGAP1对β-catenin促进SW1116细胞增殖的影响。结果发现在SW1116细胞中干扰β-catenin可以下调IQGAP1表达,过表达β-catenin可以上调IQGAP1并加强有丝分裂原激活蛋白激酶(MAPK)通路;干扰IQGAP1在SW1116细胞中反而引起β-catenin上升;同时CCK-8细胞增殖实验发现干扰IQGAP1减弱了β-catenin的促肠癌细胞增殖作用。本研究表明β-catenin与IQGAP1形成负向反馈调控环路,在调控肠癌细胞的增殖中发挥着重要功能。

关键词： Epithelial-to-mesenchymal transition   Invasion   IQGAP1   Laryngeal squamous cell carcinoma (LSCC)  

摘要： Background: 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. Methods: 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. Results: 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. Conclusions: 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.

关键词： 糖尿病肾病   流行病学   跨膜蛋白   发病机制  

摘要： 本文分为以下几个部分进行探讨：　　第一部分 糖尿病肾病的流行病学　　目的：　　本研究通过回顾性的分析山东省内37家医院的肾穿刺病理结果来系统的研究继发性肾小球疾病的病因尤其糖尿病肾病在继发性肾小球疾病中的变化趋势。　　方法：　　在这个横断面研究中，对于临床上已明确诊断为糖尿病肾病的患者，未再行肾穿刺活检术。自2010年6月到2015年5月共纳入6017例患者。剔除82个不合格的样本，共纳入5935个合格的样本。其中，1038(17.5％)例患者被诊断为继发性肾小球疾病。根据行肾穿刺的时间以1年为一个时间间隔共分为5个时期，根据纳入患者年龄分为四个组别。　　结果：　　在1038例继发性肾小球疾病患者中，525例(50.6％)为男性，513例(49.4％)为女性。患者平均年龄为38.05±17.05岁。5个时期中分别纳入92、184、270、219和273例患者，平均年龄为41.0±15.6岁、36.1±16.2岁、36.5±16.5岁、37.2±17.5岁和40.6±17.9岁。　　在纳入的继发性肾小球疾病患者中，糖尿病肾病所占的比例(224，21.6％)略低于狼疮性肾炎(287，27.6％)和过敏性紫癜性肾炎(225，21.7％)，其他依次为高血压肾病(155，14.9％)、乙肝相关性肾炎(95，9.2％)、肾淀粉样变(45，4.3％)、系统性血管炎肾损害(2，0.2％)、肾病综合征出血热(1，0.1％)、尿酸性肾病(1，0.1％)、血栓性微血管病(1，0.1％)、肥胖相关性肾病(1，0.1％)和抗肾小球基底膜肾炎(1，0.1％)。　　根据年度分层，发现糖尿病肾病的比例从第1年度到第3年度有增加趋势，在第3年度成为继发性肾小球疾病最主要的病理类型，占继发性肾小球疾病的29.3％。狼疮性肾炎在第1、2、4、5四个年度是继发性肾小球疾病最常见的病理类型。乙肝相关性肾炎所占比例则逐年下降，其比例从第2年度的14.7％下降至第5年度的5.1％。肾淀粉样变的比例从第1年度的2.2％增加到第5年度的7.0％。　　根据年龄分层，糖尿病肾病的比例随年龄增长而明显增加。在儿童患者、青年患者、中年患者及老年患者中分别占0.6％、18.2％、31.7％和40.7％，成为中老年患者中最常见的继发性肾小球疾病的病理类型。同样，肾淀粉样变的患病率随年龄增加而增加，而过敏性紫癜性肾炎则呈现相反的走势。　　根据年龄分层，在儿童患者中，最常见的病理类型是过敏性紫癜性肾炎(72.3％)，其他依次为狼疮性肾炎(24.5％)、乙肝相关性肾炎(1.3％)、糖尿病肾病(0.6％)、高血压肾病(0.6％)和肾淀粉样变(0.6％)。在青年患者中，排在首位的病理类型是狼疮性肾炎(37.3％)，其他依次为糖尿病肾病(18.2％)、高血压肾病（17.6％）、过敏性紫癜性肾炎(12.4％)、乙肝相关性肾炎(12.4％)和肾淀粉样变(1.0％)。在中年患者中，排在首位的是糖尿病肾病(31.7％)，狼疮性肾炎居第二位(20.7％)，其他依次为高血压肾病(19.2％)、过敏性紫癜性肾炎(12.2％)、乙肝相关性肾炎(8.9％)和肾淀粉样变(7.0％)。糖尿病肾病和肾淀粉样变的比例在老年患者中分别占第一位(40.7％)和第二位(16.9％)，其他依次为过敏性紫癜性肾炎(13.6％)、高血压肾病(13.6％)、狼疮性肾炎(7.6％)和乙肝相关性肾炎(6.8％)。　　结论：　　我们的研究表明，糖尿病肾病的患病率随年龄增加而增加，在45岁以上的中老年患者中，成为最常见的继发性肾小球疾病的病理类型。在总人群中，糖尿病肾病的比例仅次于狼疮性肾炎和过敏性紫癜性肾炎，是继发性肾小球疾病中第三位的肾穿刺的病因。对于临床上明确诊断为糖尿病肾病的患者未再行肾穿刺活检术，可能导致糖尿病肾病的患病率被低估。　　第二部分:IQGAP1介导nephrin内化在糖尿病肾病发病机制中的研究　　目的：本研究旨在探讨IQGAP1在糖尿病肾病中的作用及分子机制，明确IQGAP1介导nephrin内化在糖尿病足细胞损伤中的作用机制，为糖尿病肾病的机制研究提供新的理论依据。　　方法：　　流程一　　21只6-8周雄性SPF级C57小鼠，随机分组，手术切除一侧肾脏，链脲佐菌素（streptozotocin，STZ）腹腔注射，1型糖尿病肾病模型造模成功后，连续喂养12周，定期监测血糖、尿蛋白/肌酐比值和体重等指标，确定造模是否成功。PAS染色观察肾脏组织病理学改变，透射电镜观察足细胞超微结构变化，免疫荧光检测IQGAP1、nephrin在肾小球的表达及分布。Western blotting检测IQGAP1在两组小鼠肾小球内的表达水平，免疫共沉淀检测IQGAP1与nephrin的相互作用。　　流程二　　体外培养条件永生性小鼠足细胞，Wes

关键词： 肝细胞肝癌   血管生成拟态   LOXL2   IQGAP1   转移  

摘要： 研究目的:肝细胞肝癌(Hepatocellular carcinoma,HCC)是临床上最常见的恶性肿瘤之一,其发病率逐年上升,现已成为世界范围内第三大最常见的癌症死亡原因。肿瘤血管生成是肿瘤生长、侵袭、转移的重要步骤,因此抗血管治疗已成为当前防治研究的热点。血管生成拟态(Vasculogenic mimicry,VM)是一种不同于内皮依赖性血管的特殊的肿瘤血液供应模式,其存在与肿瘤的不良预后相关,但其发生的机制尚不明确。本研究采用免疫组化方法检测了赖氨酰氧化酶样蛋白2(LOXL2)、含有IQ模体的Ras GTP酶活化蛋白1(IQGAP1)、VM在201例HCC组织中的表达情况,分别分析LOXL2、IQGAP1与VM及临床资料的关系,并探讨LOXL2与IQGAP1的相关性。通过细胞转染、免疫印迹等细胞实验研究LOXL2、IQGAP1对HCC细胞迁移、侵袭、增殖及形成VM能力的影响,研究其促进HCC发生发展的机制,从而为HCC的靶向治疗提供新的分子靶点。研究方法:1.本研究收集了经手术切除的肝细胞肝癌组织标本201例,以上标本均来自天津医科大学肿瘤医院、天津医科大学总医院,所取标本均经病理医师确诊。采用免疫组化和CD31/PAS双染,检测LOXL2、IQGAP1的表达水平和VM的存在情况。分别分析LOXL2、IQGAP1的表达与HCC临床病理因素、VM及其与患者生存预后的关系,并分析LOXL2与IQGAP1之间的相关性。2.应用Western blot检测LOXL2在肝癌细胞系中的本底表达,筛选出本底低表达和高表达的细胞系。采用质粒转染的方法调节LOXL2的表达,构建并筛选出相应的稳定转染细胞系。运用划痕、迁移、侵袭、三维培养实验检测LOXL2对肝癌细胞迁移、侵袭和VM形成能力的影响。Western blot鉴定转染效率并检测VM相关分子VE-vadherin、MMP2、MMP9的表达变化,并采用免疫荧光法验证。3.采用质粒转染的方法,将IQGAP1下调质粒转入上述LOXL2上调稳转细胞系中;将IQGAP1上调质粒转入上述LOXL2下调稳转细胞系中。划痕、迁移、侵袭、三维培养、免疫印迹实验检测IQGAP1对于LOXL2功能恢复的影响。免疫共沉淀实验检测LOXL2与IQGAP1的关系。4.应用Western blot检测IQGAP1在肝癌细胞系中的表达情况,筛选出本底低表达和高表达IQGAP1的细胞系。采用质粒转染的方法调节IQGAP1的表达,构建转染细胞系。运用划痕、迁移、侵袭、三维培养、平板克隆、软琼脂克隆和MTT增殖实验检测IQGAP1对肝癌细胞迁移、侵袭、VM形成以及增殖能力的影响。采用Western blot法鉴定转染效率并检测VM相关分子VE-vadherin、MMP2、MMP9的表达变化。研究结果:1.201例HCC组织免疫组化结果显示:LOXL2在肝癌细胞的胞质和胞核中表达,IQGAP1在肝癌细胞的胞质和胞膜中表达。LOXL2和IQGAP1在HCC组织中高表达,与肿瘤分级、转移、VM相关,且LOXL2与IQGAP1的表达呈正相关。生存分析显示LOXL2、IQGAP1阳性患者的预后均较差。2.在肝癌细胞系中,筛选出LOXL2高表达细胞系Bel7402和低表达细胞系Hep G2。在Bel7402中转染LOXL2下调质粒,出现了IQGAP1、VE-cadherin、MMP2、MMP9的表达降低,迁移、侵袭、管道形成能力均减弱。在Hep G2中转染LOXL2上调质粒,出现了IQGAP1、VE-cadherin、MMP2、MMP9的表达升高,迁移、侵袭、管道形成能力均增强。3.在Bel7402下调LOXL2细胞系中,转染IQGAP1上调质粒,出现了IQGAP1、VE-cadherin、MMP2、MMP9的表达升高,迁移、侵袭、管道形成能力均增强。在Hep G2上调LOXL2细胞系中,转染IQGAP1下调质粒,出现了IQGAP1、VE-cadherin、MMP2、MMP9的表达降低,迁移、侵袭、管道形成能力均减弱。Co-IP结果显示LOXL2可以与IQGAP1相结合。LOXL2可能是通过调控IQGAP1的表达来影响肝癌细胞侵袭、迁移及VM形成的能力。4.在肝癌细胞系中,筛选出IQGAP1高表达细胞系SMMC7721和低表达细胞系Hep G2。在SMMC7721中转染IQGAP1下调质粒,出现了VE-cadherin、MMP2、MMP9的表达降低,迁移、侵袭、管道形成能力均减弱。平板克隆、软琼脂克隆、MTT增殖实验显示,IQGAP1的下调抑制了SMMC7721细胞的增殖能力。在Hep G2中转染IQGAP1上调质粒,出现了VE-cadherin、MMP2、MMP9的表达升高,迁移、侵袭、管道形成能力均增强。平板克隆、软琼脂克隆、

关键词： IQGAP1   Colorectal carcinoma   SUMOylation   SUMO1  

摘要： IQGAP1 is a conserved multifunctional protein implicated in tumorigenesis. An aberrant expression of IQGAP1 widely exists in many cancers, but the SUMOylation modification of IQGAP1 in carcinogenesis is unknown by now. Here we first time explore biological functions of IQGAP1 SUMOylation in promoting colorectal cancer progression in vitro and in vivo. The expression of IQGAP1 and its SUMOylation level are both increased in human colorectal carcinoma (CRC) cells and tissues. IQGAP1 is mainly SUMOylated by SUMO1 at the K1445 residue, which could stabilize IQGAP1 by reducing protein ubiquitination. IQGAP1 SUMOylation improves CRC cell growth, cell migration and tumorigenesis in vivo through activating the phosphorylation of ERK, MEK and AKT. While the SUMOylation site mutation at K1445 of IQGAP1 greatly reduces CRC cell proliferation, migration ability and tumor growth of CRC-xenograft mice by suppressing phosphorylation of ERK, MEK and AKT. Our findings discover the IQGAPl SUMOylation is a novel regulatory mechanism to enhance tumorigenesis and development of CRC in vitro and in vivo. (c) 2017 Elsevier B.V. All rights reserved.