关键词： 食管癌   顺铂   紫杉醇   耐药   IQGAP1  

摘要： 目的:1.体外建立人食管癌顺铂耐药KYSE150/CDDP、人食管癌紫杉醇耐药KYSE150/PTX细胞系。2.观察顺铂和紫杉醇耐药对人食管癌细胞恶性表型的影响。3.探讨影响顺铂和紫杉醇耐药的分子机制。方法:1.采用体外低剂量间歇诱导+浓度递增的方式,分别使用顺铂、紫杉醇为诱导药物,建立人食管癌顺铂耐药KYSE150/CDDP细胞系、人食管癌紫杉醇耐药KYSE150/PTX细胞系,显微镜下拍照记录顺铂和紫杉醇诱导后KYSE150细胞的变化。MTT法测细胞对顺铂、紫杉醇和5-氟尿嘧啶的耐受程度。2.实验检测顺铂和紫杉醇耐药对食管癌细胞的影响,MTT法及细胞克隆形成实验检测增殖、DAPI染色法检测凋亡、粘附实验及划痕愈合实验检测粘附及迁移、体外小管形成实验检测小管形成。3.利用Western blot检测耐药细胞及亲代KYSE150细胞中Caspase-3、E-cadherin及MMP-2、VEGF、IQGAP1蛋白的表达水平。4.探讨IQGAP1基因干扰能否增强KYSE150/CDDP和KYSE150/PTX细胞的化疗敏感性。首先构建及鉴定IQGAP1基因短发卡RNA干扰质粒,然后稳定转染KYSE150/CDDP细胞、KYSE150/PTX细胞,通过Western blot鉴定IQGAP1蛋白的表达水平,最后通过上述实验方法检测IQGAP1对耐药细胞化疗敏感性、增殖、抗凋亡、粘附和迁移能力的影响,并采用Western blot法检测MMP-2蛋白及ERK通路的变化。结果:1.历时10个月,建立人食管癌顺铂耐药KYSE150/CDDP细胞系及人食管癌紫杉醇耐药KYSE150/PTX细胞系,显微镜下观察耐药细胞形态,发现KYSE150细胞对CDDP、PTX产生稳定耐药后并未发现细胞形态上的改变。MTT测得顺铂耐药KYSE150/CDDP细胞对顺铂耐药指数(RI)为6.175,紫杉醇耐药KYSE150/PTX细胞对紫杉醇RI为5.226,且两株耐药细胞有交叉耐药现象。2.实验结果显示,与亲代KYSE150细胞相比,KYSE150/CDDP细胞和KYSE150/PTX细胞生长速度减慢、增殖能力也降低、UV照射30 s后的凋亡细胞数减少、细胞间聚集能力减弱及分离能力增强(粘附力减弱)、划痕24 h后无细胞区域更小、形成的小管数更多。*** blot结果显示:与亲代KYSE150细胞相比,KYSE150/CDDP细胞和KYSE150/PTX细胞的Procaspase-3、MMP-2、VEGF、IQGAP1蛋白表达水平均增高,而E-cadherin蛋白表达水平降低。4.经测序证实IQGAP1基因短发卡RNA干扰质粒构建成功,荧光显微镜下观察绿色荧光蛋白(GFP)显示,实验组与对照组转染效率分别为(76±5.780)%和(83±3.851)%,RT-PCR和Western blot结果也表明构建的IQGAP1干扰载体能显著抑制KYSE150细胞中IQGAP1基因的表达;经G418筛选构建IQGAP1干扰的稳转细胞系及阴性对照细胞系,分别命名为K/D/sh RNA及K/D/Control、K/P/sh RNA及K/P/Control,Western blot结果显示IQGAP1干扰组细胞的IQGAP1蛋白表达水平较空载体转染组显著降低,提示IQGAP1基因干扰的稳转耐药细胞系构建成功;干扰IQGAP1基因的表达使耐药细胞的药物耐受性降低、抗凋亡能力减弱、细胞间粘附力增强而迁移能力减弱。Western blot检测发现,与K/D/Control细胞相比,K/D/sh RNA细胞中MMP-2、p-ERK水平降低。结论:1.成功建立人食管癌顺铂耐药KYSE150/CDDP细胞系及人食管癌紫杉醇耐药KYSE150/PTX细胞系,且获得顺铂及紫杉醇抗性后,细胞增殖能力减弱,抗凋亡、迁移及血管形成能力增强,细胞间粘附能力降低,提示这些改变可能是顺铂和紫杉醇耐药影响临床治疗效果的重要原因。*** blots结果提示IQGAP1、Caspase-3、E-cadherin、MMP-2、VEGF基因参与了食管癌顺铂和紫杉醇耐药机制的形成。3.成功构建IQGAP1基因干扰载体,并利用该重组质粒在KYSE150/CDDP、KYSE150/PTX细胞中构建了干扰IQGAP1基因表达的稳转细胞系,IQGAP1干扰后提高了耐药细胞的药物敏感性及细胞间粘附力,减慢了细胞的增殖,降低了耐药细胞的抗凋亡及迁移能力。而IQGAP1干扰可能通过ERK通路活化逆转食管癌顺铂耐药。

关键词： microRNA-124   IQ motif containing GTPase activating protein 1   colorectal cancer   extracellular signal regulated kinase 1/2   beta-catenin  

摘要： MicroRNA (miRNA/miR)-124 is a miRNA, which exerts tumor suppressive effects but is frequently absent in tumors. Although it has been validated to target oncogenic genes such as signal transducer and activator of transcription 3, forkhead box Q1, and Slug, the mechanistic link between miR-124 and potential target genes that contribute to tumor progression, is yet to be investigated. IQ motif containing GTPase activating protein 1 (IQGAP1) is a scaffold protein that participates in protein-protein interactions and integrating diverse signaling pathways. Previous studies suggest that overexpression of IQGAP1 enhances activity of mitogen activated protein kinase 1 and -catenin signaling cascades to facilitate tumor progression. The present study aimed to identify the regulative link between miR-124 and IQGAP1 in colorectal cancer (CRC). It was demonstrated that IQGAP1 was aberrantly overexpressed in CRC tissues and cell lines. Knockdown of IQGPA1 by introducing short hairpin-IQGAP1 lentivirus inhibited CRC cell growth and colony formation ability, and simultaneously suppressed phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and -catenin expression. Furthermore, it was demonstrated that miR-124 was silenced in CRC. Restoration of miR-124 in CRC cells impeded cell growth and colony formation ability. The direct binding of miR-124 to the 3untranslated region of IQGAP1 mRNA was confirmed using a luciferase reporter gene assay. Importantly, downregulation of IQGAP1 expression was observed in miR-124-restoration cells with simultaneous reduction of phosphorylated-ERK1/2 and -catenin. In conclusion, the present study describes a potential mechanism underlying the miR-124/IQGAP1 link in CRC progression. Silencing of miR-124 may depress IQGAP1 expression, leading to increased activity of ERK1/2 and -catenin signaling.

关键词： molecular scaffolds   receptor tyrosine kinases   receptors   signaling  

摘要： Axl is a tyrosine kinase receptor that is important for hematopoiesis, the innate immune response, platelet aggregation, engulfment of apoptotic cells and cell survival. Binding of growth arrest-specific protein 6 (Gas6) activates Axl signaling, but the mechanism of inactivation of the Axl receptor is poorly understood. In the present study, we show that IQGAP1 modulates Axl signaling. IQGAP1 is a scaffold protein that integrates cell signaling pathways by binding several growth factor receptors and intracellular signaling molecules. Our in vitro analysis revealed a direct interaction between the IQ domain of IQGAP1 and Axl. Analysis by both immunoprecipitation and proximity ligation assays demonstrated an association between Axl and IQGAP1 in cells and this interaction was decreased by Gas6. Unexpectedly, reducing IQGAP1 levels in cells significantly enhanced the ability of Gas6 to stimulate both Axl phosphorylation and activation of Akt. Moreover, IQGAP1 regulates the interaction of Axl with the epidermal growth factor receptor. Our data identify IQGAP1 as a previously undescribed suppressor of Axl and provide insight into regulation of Axl function.

关键词： IQ-domain GTPase-activating protein 1   Head and neck squamous cell carcinoma   Prognosis   Transgenic mice   Tissue microarray  

摘要： 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.

摘要： Despite major advances in targeted melanoma therapies, drug resistance limits their efficacy. Long noncoding RNAs (lncRNAs) are transcriptome elements that do not encode proteins but are important regulatory molecules. LncRNAs have been implicated in cancer development and response to different therapeutics and are thus potential treatment targets;however, the majority of their functions and molecular interactions remain unexplored. In this study, we identify a novel cytoplasmic intergenic lincRNA (MIRAT), which is upregulated following prolonged MAPK inhibition in NRAS mutant melanoma and modulates MAPK signaling by binding to the MEK scaffold protein IQGAP1. Collectively, our results present MIRAT's direct modulatory effect on the MAPK pathway and highlight the relevance of cytoplasmic lncRNAs as potential targets in drug resistant cancer.

关键词： CDC42   IL-6   IQGAP1   JAK2   Janus kinase 2   STAT3   cell migration   interleukin 6   metastasis   pancreatic cancer   small GTPase  

摘要： Inflammation is a major driver of tumor progression and metastasis, although the mechanisms by which proinflammatory cytokines drive metastatic invasion are unknown. Interleukin-6 (IL-6) is a potent proinflammatory cytokine that is elevated in individuals with pancreatic cancer (PDAC), is required for PDAC progression in mice, and increases tumor cell invasion in vitro. Here, we provide insights into the mechanisms by which IL-6 activates tumor cell invasion. We found that IL-6 stimulation rapidly and robustly activates the small GTPase cell division cycle 42 (CDC42) in human PDAC cells and promotes the formation of premigratory filopodia. The CDC42 activation was required for IL-6-induced invasion as blocking CDC42 activity rendered the cells insensitive to IL-6's proinvasive effects. Loss of Janus kinase 2 (JAK2) or signal transducer and activator of transcription 3 (STAT3) prevented IL-6-mediated CDC42 activation, indicating that IL-6 activates CDC42 through both JAK2 and STAT3. However, the rapid activation of CDC42 suggested that this activation may be distinct from canonical STAT3-mediated transcriptional activation. Importantly, we observed an interaction between STAT3 and IQ motif-containing GTPase-activating protein 1 (IQGAP1), a scaffolding platform that binds CDC42. STAT3 colocalized with CDC42 and IQGAP1 at the plasma membrane, suggesting cross-talk between IL-6-mediated STAT3 signaling and CDC42 activation. These results suggest that IL-6 promotes metastatic invasion, at least partially, through CDC42 and that, along with its pleiotropic effects on tumor growth and progression, IL-6 signaling also activates proinvasive GTPase signaling, priming tumor cells for metastatic invasion.

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.