摘要： 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.

关键词： IQ结构域三磷酸鸟苷酶激活蛋白1   胰腺癌细胞   增殖   凋亡  

摘要： 目的分析下调IQ结构域三磷酸鸟苷酶激活蛋白(IQGAP)1对胰腺癌细胞增殖、凋亡的影响及作用机制。方法人胰腺癌HPAF-Ⅱ细胞分为对照组、过表达组、沉默组。对照组不做任何处理,过表达组为人胰腺癌细胞做IQGAP1过表达质粒转染,沉默组为人胰腺癌细胞做IQGAP1沉默质粒转染。鉴定IQGAP1转染效率,分析沉默IQGAP1基因对胰腺癌细胞的增殖抑制作用、凋亡及Wnt/β-连环蛋白(catenin)通路蛋白的影响,并分析其作用机制。结果过表达组IQGAP1基因表达量显著高于对照组,沉默组IQGAP1基因表达量显著低于对照组(P<0.05),说明IQGAP1基因转染成功。过表达组细胞增殖率显著高于对照组,沉默组细胞增殖率显著低于对照组、过表达组(P<0.05)。过表达组细胞凋亡率显著低于对照组,沉默组细胞凋亡率显著高于对照组、过表达组(P<0.05)。过表达组Wnt、β-catenin蛋白表达量显著低于对照组,细胞外信号调节激酶(ERK)、B细胞淋巴瘤-2相关X蛋白(Bax)、细胞外信号调节激酶的激酶(MEK)蛋白表达量显著高于对照组,沉默组Wnt、β-catenin蛋白表达量显著高于对照组,ERK、Bax、MEK蛋白表达量显著低于对照组(P<0.05);相比于过表达组,沉默组Wnt、β-catenin蛋白表达量显著较高,ERK、Bax、MEK蛋白表达量显著较低(P<0.05)。结论下调IQGAP1基因可以抑制胰腺癌细胞增殖,促进其凋亡,其机制可能与激活Wnt/β-catenin通路相关。

关键词： circAnk3   Glutamate receptors   IQ motif containing GTPase activating protein 1   miR-7080-3p   Psychiatric disorder   Synaptic plasticity  

摘要： BACKGROUND: Circular RNAs are highly enriched in the synapses of the mammalian brain and play important roles in neurological function by acting as molecular sponges of microRNAs. circAnk3 is derived from the 11th intron of the ankyrin-3 gene, Ank3 , a strong genetic risk factor for neuropsychiatric disorders;however, the function of circAnk3 remains elusive. In this study, we investigated the function of circAnk3 and its downstream regulatory network for target genes in the hippocampus of mice. METHODS: The DNA sequence from which circAnk3 is generated was modified using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9) technology, and neurobehavioral tests (anxiety and depression -like behaviors, social behaviors) were performed in circAnk3 + /- mice. A series of molecular and biochemical assays were used to investigate the function of circAnk3 as a microRNA sponge and its downstream regulatory network for target genes. RESULTS: circAnk3 + /- mice exhibited both anxiety -like behaviors and social deficits. circAnk3 was predominantly located in the cytoplasm of neuronal cells and functioned as a miR-7080-3p sponge to regulate the expression of Iqgap1 . Inhibition of miR-7080-3p or restoration of Iqgap1 in the hippocampus ameliorated the behavioral deficits of circAnk3 + /- mice. Furthermore, circAnk3 deficiency decreased the expression of the NMDA receptor subunit GluN2a and impaired the structural plasticity of dendritic synapses in the hippocampus. CONCLUSIONS: Our results reveal an important role of the circAnk3/miR-7080-3p/IQGAP1 axis in maintaining the structural plasticity of hippocampal synapses. circAnk3 might offer new insights into the involvement of circular RNAs in neuropsychiatric disorders.

关键词： cancer metastasis   Rho GTPases   endothelial adhesion   β-integrin   signaling   shear stress   IQGAP1   NWASP   cell-cell adhesion  

摘要： Attachment of circulating tumor cells to the endothelial cells (ECs) lining blood vessels is a critical step in cancer metastatic colonization, which leads to metastatic outgrowth. Breast and prostate cancers are common malignancies in women and men, respectively. Here, we observe that 01-integrin is required for human prostate and breast cancer cell adhesion to ECs under shear -stress conditions in vitro and to lung blood vessel ECs in vivo. We identify IQGAP1 and neural Wiskott-Aldrich syndrome protein (NWASP) as regulators of 01-integrin transcription and protein expression in prostate and breast cancer cells. IQGAP1 and NWASP depletion in cancer cells decreases adhesion to ECs in vitro and retention in the lung vasculature and metastatic lung nodule formation in vivo. Mechanistically, NWASP and IQGAP1 act downstream of Cdc42 to increase 01-integrin expression both via extracellular signal -regulated kinase (ERK)/focal adhesion kinase signaling at the protein level and by myocardin-related transcription factor/ serum response factor (SRF) transcriptionally. Our results identify IQGAP1 and NWASP as potential therapeutic targets to reduce early metastatic dissemination.

关键词： ANGPTL4   Cancer-associated fibroblasts   Chemosensitivity   Mitochondria   Prostate cancer  

摘要： Introduction: Cancer-associated fibroblasts (CAFs) are a critical component of the tumor microenvironment, being implicated in enhancing tumor growth and fostering drug resistance. Nonetheless, the mechanisms underlying their function in prostate cancer (PCa) remain incompletely understood, which is essential for devising effective therapeutic strategies. Objectives: The main objective of this study was to explore the mechanisms by which CAFs mediate PCa growth and chemoresistance. Methods: We validated through data analysis and experimentation that CAFs significantly impact PCa cell proliferation and chemoresistance. Subsequently, we conducted a comprehensive proteomic analysis of the conditioned media from CAFs and PCa cells and identified angiopoietin-like protein 4 (ANGPTL4) as a key factor. We employed ELISA and multiplex immunofluorescence assays, all of which indicated that ANGPTL4 was primarily secreted by ***, we conducted metabolomics analysis, GST pull-down assays, Co-IP, and other experiments to explore the specific molecular mechanisms of ANGPTL4 and its precise effects on PCa cells. Through drug screening, we identified Quercetin 3-O-(6′-galactopyranosyl)-β-D-galactopyranoside (QGGP) as an effective inhibitor of CAFs function. Finally, we thoroughly assessed the therapeutic potential of QGGP both as a monotherapy and in combination with docetaxel in PCa cells. Results: We discovered that the extracrine factor ANGPTL4 is primarily expressed in CAFs in PCa. When ANGPTL4 binds to IQ motif-containing GTPase-activating protein 1 (IQGAP1) on the PCa cell membrane, it activates the Raf-MEK-ERK-PGC1α axis, promoting mitochondrial biogenesis and OXPHOS metabolism, and thereby facilitating PCa growth and chemoresistance. Furthermore, virtual and functional screening strategies identified QGGP as a specific inhibitor of IQGAP1 that promotes its degradation. Combined with docetaxel treatment, QGGP can reverse the effects of CAFs and improve the responsivenes

关键词： IQGAP1   氧化应激   mtDNA   焦亡   动脉粥样硬化  

摘要： 研究背景 动脉粥样硬化（Atheroscleros,AS）是一种慢性炎症性疾病,简单来说一般包括三个病理过程—脂质沉积、炎症浸润、泡沫细胞形成最终导致血管内径变窄,是世界上最常见的引起死亡的原因之一。AS的形成与内皮细胞,单核细胞,巨噬细胞以及平滑肌细胞的功能有着密切关系,在形成早期,血液和动脉内膜之间内皮单层发生改变,这是AS的起始部位;且内皮细胞易受高脂血症、氧化应激、趋化因子、DNA异位等损伤模式分子的影响引起炎症反应,这进一步导致内环境的紊乱促进了内皮细胞功能活化。然而,目前药物治疗AS仍然具有一定的局限性。越来越多的研究表明抑制内皮细胞焦亡可能成为治疗AS的一种新思路,但其具体发生机制仍不清楚。焦亡是一种不同于细胞凋亡和坏死的程序性细胞死亡过程,与炎症小体的形成密切相关并伴随着促炎因子的大量释放。IQGAP蛋白家族存在于许多生物体中,已在人类中鉴定出三种IQGAP1、IQGAP2和IQGAP3,其中IQGAP1在组织中广泛表达,是最具特征的IQGAP蛋白质,在真核生物中具有进化保守性,有助于形成调节细胞骨架动力学、细胞内信号传导和细胞间相互作用的复合物;同时,在调节细胞粘附、信号传导和迁移中起重要作用。随着研究的逐渐深入,日益增多的证据表明IQGAP1作用于心肌细胞影响心脏重塑,心室壁变薄和收缩力下降;同时对血管生成过程中内皮细胞的增殖和迁移有着重要作用,这与线粒体活性氧（mt ROS）的产生有关。重要的是,随着mt ROS在细胞内累积可能会导致线粒体损伤进而发生Caspase-1-NLRP3经典途径焦亡。新的发现表明线粒体损伤导致线粒体DNA（mtDNA）泄露到细胞质,激活细胞质中DNA感受器cGAS-STING,并触发分子链反应以诱导I型干扰素（IFN-I）,启动和维持炎症反应。在心血管领域中发现mtDNA激活的cGAS-STING信号在心肌细胞焦亡中同样被广泛关注,但在AS中未有确切报道。我们的探索性研究IQGAP1是否通过调节线粒体功能导致mtDNA泄露激活cGAS-STING信号通路诱导内皮细胞焦亡以及特异性小分子抑制剂RU.521和C-176的治疗作用,以期对AS的发生机制和治疗提供了一个新的视野。 研究目的 1.研究动脉粥样硬化模型中IQGAP1,cGAS,STING对内皮细胞焦亡的影响。 2.阐明IQGAP1对动脉粥样硬化进程中活性氧ROS影响及其部分调控机制。 3.检测线粒体损伤后细胞质中ds DNA泄露并进行定量和组织定位分析。 4.明确IQGAP1在线粒体损伤过程中调节mtDNA释放激活DNA感受器cGAS-STING信号通路。 5.小分子抑制剂RU.521和C-176通过特异性阻断cGAS和STING信号通路,从而抑制IQGAP1促进的动脉粥样硬化,探索可能存在的的药物靶点。 研究方法 1.购买40只ApoE-/-C57BL/J和40只Ldlr-/-C57BL/J,两种小鼠各随机分为四组正常饮食组（Cont）,高脂饮食组（HFD）,高脂饮食+AAV-Sh RNA-IQGAP1（AAV-Si-IQGAP1）组,高脂饮食+AAV-Sh RNA-NC（AAV-NC）组。在适应一周后注射腺病毒及其对照,两种小鼠分别使用相对应的高脂饲料连续喂养12周,每间隔两周记录小鼠体重。使用HE,油红切片,油红大体染色对主动脉组织进行病理检测并检测血清生化指标LDL,TC,TG,HDL。使用IHC、IF和Westernblot观察焦亡相关分子NLRP3,GSDMD-N,Caspase-1和cGAS,STING信号通路蛋白表达,qRT-PCR观察炎症因子IL-1β,IL-18和DNA感受器cGAS,STING以及抗氧化应激GPX-1,SOD-1 m RNA的表达情况。 2.在小鼠组织中通过线粒体追踪标记Mito-Tracker与ds DNA抗体进行双重定位染色,观察mtDNA的释放情况的定性分析。同时,对小鼠主动脉组织进行利用mtDNA提取试剂盒进行q-PCR定量分析。利用TUNEL染色观察主动脉组织内皮细胞焦亡情况,通过透射电镜观察线粒体损伤。 3.在体外通过棕榈酸（PA,0.3mmol/L）刺激人脐静脉内皮细胞（HUVECs）模拟内皮细胞损伤模型。通过Mito SOX染色,DCFH-DA流式细胞术检测PA诱导HUVECs细胞中活性氧（ROS）的产生。通过Western blot观察PA诱导的HUVECs中焦亡相关蛋白NLRP3,GSDMD-N,Caspase-1,IL-1β,IL-18的表达情况,扫描电镜观察焦亡细胞膜“打孔”现象。 4.在体外分别构建IQGAP1的过表达质粒和阴性对照（pc DNA3.1-3x Flag-IQGAP1,pc DNA3.1-3x Flag-NC）,小干扰RN

关键词： Hepatocellular carcinoma   PD-L1   M2 macrophages   CD8(+)T cells   Immune escape   MISP   IQGAP1   STAT3  

摘要： Background: Hepatocellular carcinoma (HCC) is a prevailing cancer affecting human health. M2 macrophages are essential in mediating immune responses in tumors. This study investigated the action of M2 macrophages in immune escape of HCC. Methods: Mitotic spindle positioning (MISP), IQ motif containing GTPase activating protein 1 (IQGAP1) and programmed cell death-1 (PD-L1) levels in primary HCC/tumor-adjacent tissues were determined by Western blot, followed by correlation analysis. M2 macrophage and CD3(+)CD8(+)T cell percentages were estimated by flow cytometry. Hep3B and HepG2 cells were treated with M2 macrophage conditioned medium (M2-CM) and M2 macrophage-derived extracellular vesicles (M2-EVs) and/or co-cultured with CD8(+)T cells, followed by assessment of cell viability and apoptosis. TNF- alpha and INF-gamma levels were measured by ELISA. MISP and IQGAP1 over-expression plasmids were transfected into HCC cells to explore their role in immune escape. The interactions among MISP, IQGAP1, STAT3, and PD-L1 were analyzed by co-immunoprecipitation. The mechanism of M2-EVs in HCC immune escape was verified in nude mice. Results: MISP/IQGAP1/PD-L1 were upregulated in HCC tissues. MISP negatively-correlated with IQGAP1/PD-L1 and IQGAP1 positively-correlated with PD-L1. M2 macrophages were reduced but CD8(+)T cells were increased in HCC tissues with high MISP expression. M2-CM or M2-EVs inhibited the killing ability of CD8(+)T cells, increased HCC cell viability, impeded HCC cell apoptosis, induced CD8(+)T cell apoptosis, downregulated TNF- alpha and INF-gamma, and upregulated PD-L1. M2-EVs facilitated HCC cell immune escape by potentiating IQGAP1 nuclear trans-location and activating STAT3 phosphorylation through MISP downregulation. In vivo experiments further verified the action of M2-EVs through MISP. Conclusion: M2-EVs promote HCC cell immune escape by upregulating PD-L1 through the MISP/IQGAP1/STAT3 axis.

关键词： Atherosclerosis   IQGAP1   Oxidative stress   MtDNA sensors   Pyroptosis  

摘要： Atherosclerosis (AS) is the most common cardiovascular disease and has limited therapeutic options. IQ motifcontaining GTPase-activating protein 1 (IQGAP1) is an important scaffolding protein regulating mitochondrial function influencing endothelial cell activity. Evidence suggests that mitochondrial damage can lead to leakage of mtDNA into the cytoplasm to activate the DNA sensor cGAS-STING to mediate pyroptosis. However, whether IQGAP1 induces NLRP3-mediated endothelial cell pyroptosis by regulating mitochondrial function and activating the DNA sensor cGAS-STING, and its underlying mechanisms remain unclear. In vivo, ApoE-/- C57BL/J and Ldlr-/- C57BL/J mice were pre-injected with adeno-associated virus (AAV) by the tail vein to specifically silence IQGAP1 expression and were fed a high-fat diet (HFD) for 12 weeks. IQGAP1 knockdown reduced mtDNA release and decreased the expression of DNA receptors and pyroptosis-related molecules as determined by immunohistochemistry and immunofluorescence. In vitro, palmitic acid (0.3 mmol/L) was incubated with human umbilical vein endothelial cells (HUVECs) for 24 h. Overexpression of IQGAP1 in HUVECs, flow cytometry, and mitochondrial superoxide staining revealed increased levels of ROS. Moreover, the mitochondrial tracker with dsDNA co-localization showed the release of mtDNA into the cytoplasm increased, which activated the DNA receptor cGAS-STING. Protein blotting and TUNEL staining revealed that IQGAP1 promoted NLRP3mediated pyroptosis. Furthermore, cGAS or STING small-molecule inhibitors RU.521 or C-176 reverse IQGAP1-promoted HUVECs from undergoing NLRP3-mediated pyroptosis. These results suggest that IQGAP1 promotes oxidative stress and mtDNA release, activates the DNA sensor cGAS-STING, and leads to NLRP3mediated pyroptosis. The present study provides new insights into the mechanisms underlying AS and identifies new pharmacological targets for treatment.