关键词： cancer   cell signaling   extracellular-signal-regulated kinase (ERK)   mitogen-activated protein kinase (MAPK)   protein complex   protein kinase   protein-protein interaction   scaffold protein   IQGAP1  

摘要： Mitogen-activated protein kinase (MAPK) scaffold proteins, such as IQ motif containing GTPase activating protein 1 (IQGAP1), are promising targets for novel therapies against cancer and other diseases. Such approaches require accurate information about which domains on the scaffold protein bind to the kinases in the MAPK cascade. Results from previous studies have suggested that the WW domain of IQGAP1 binds to the cancer-associated MAPKs ERK1 and ERK2, and that this domain might thus offer a new tool to selectively inhibit MAPK activation in cancer cells. The goal of this work was therefore to critically evaluate which IQGAP1 domains bind to ERK1/2. Here, using quantitative in vitro binding assays, we show that the IQ domain of IQGAP1 is both necessary and sufficient for binding to ERK1 and ERK2, as well as to the MAPK kinases MEK1 and MEK2. Furthermore, we show that the WW domain is not required for ERK-IQGAP1 binding, and contributes little or no binding energy to this interaction, challenging previous models of how WW-based peptides might inhibit tumorigenesis. Finally, we show that the ERK2-IQGAP1 interaction does not require ERK2 phosphorylation or catalytic activity and does not involve known docking recruitment sites on ERK2, and we obtain an estimate of the dissociation constant (K-d) for this interaction of 8 m. These results prompt a re-evaluation of published findings and a refined model of IQGAP scaffolding.

关键词： Melanoma   Melanoma cells   Cutaneous melanoma   MAPK signaling cascades   Skin tumors   Cancer treatment   Protein expression   Immunoblotting  

摘要： Canine oral mucosal melanoma is an aggressive malignant neoplasm and is characterized by local infiltration and a high metastatic potential. The disease progression is similar to that of human oral melanomas. Whereas human cutaneous melanoma is primarily driven by activating mutations in Braf (60%) or Nras (20%), human mucosal melanoma harbors these mutations much less frequently. This makes therapeutic targeting and research modeling of the oral form potentially different from that of the cutaneous form in humans. Similarly, research has found only rare Nras mutations and no activating Braf mutations in canine oral melanomas, but they are still reliant on MAPK signaling. IQGAP1 is a signaling scaffold that regulates oncogenic ERK1/2 MAPK signaling in human Ras- and Raf-driven cancers, including melanomas. To investigate whether IQGAP1 is a potential target in canine melanoma, we examined the expression and localization of IQGAP1 in primary canine melanomas and canine oral melanoma cell lines obtained from the University of California-Davis. Using CRISPR/Cas9 knockout of IQGAP1, we examined effects on downstream ERK1/2 pathway activity and assayed proliferation of cell lines when treated with a peptide that blocks the interaction between IQGAP1 and ERK1/2. We observed that canine IQGAP1 is expressed and localizes to a similar extent in both human and canine melanoma by qPCR, Western blot, and immunofluorescence. Deletion of IQGAP1 reduces MAPK pathway activation in cell lines, similar to effects seen in human Braf (V600E) cell lines. Additionally, we demonstrated reduced proliferation when these cells are treated with a blocking peptide in vitro.

摘要： ERK signaling and Akt signaling are inversely correlated in some cancers. Yet, the precise molecular mechanism for cross-inhibition remains unclear. In this issue of The EMBO Journal, Pan et al (2017) show that when Akt is on, its phosphorylated cytoplasmic substrate FOXO1 turns off ERK activity by reshaping the Ras-ERK scaffold IQGAP1.

关键词： FOXO1   AKT   MAPK   chemoresistance   cancer  

摘要： Nuclear FOXO proteins act as tumor suppressors by transcriptionally activating genes involved in apoptosis and cell cycle arrest, and these anticancer functions are inhibited by AKT-induced phosphorylation and cytoplasmic sequestration of FOXOs. We found that, after AKT-mediated phosphorylation at serine 319, FOXO1 binds to IQGAP1, a hub for activation of the MAPK pathway, and impedes IQGAP1-dependent phosphorylation of ERK1/2 (pERK1/2). Conversely, decreased FOXO1 expression increases pERK1/2 in cancer cell lines and correlates with increased pERK1/2 levels in patient specimens and disease progression. Treatment of cancer cells with PI3K inhibitors or taxane causes FOXO1 localization in the nucleus, increased expression of pERK1/2, and drug resistance. These effects are reversed by administering a small FOXO1-derived phospho-mimicking peptide inhibitor in vitro and in mice. Our results show a tumor suppressor role of AKT-phosphorylated FOXO1 in the cytoplasm and suggest that this function of FOXO1 can be harnessed to overcome chemoresistance in cancer.

关键词： colorectal cancer   scaffold proteins   AmotL2   FKBP51   IQGAP1   metastasized liver   FOLFOX   oxaliplatin   pericytes   telocytes  

摘要： Scaffold proteins play pivotal roles in the regulation of signaling pathways, integrating external and internal stimuli to various cellular outputs. We report the pattern of cellular and subcellular expression of scaffoldins angiomotin-like 2 (AmotL2), FK506 binding protein 5 (FKBP51) and IQ motif containing GTPase-activating protein 1 (IQGAP1) in colorectal cancer (CRC) and metastases in liver resected after oxaliplatin-based chemotherapy (CT). Positive immunostaining for the three scaffoldins was found in most cells in healthy colon, tumor, healthy liver and metastasized liver. The patterns of expression of AmotL2, FKBP51 and IQGAP1 show the greatest variability in immune system cells and neurons and glia cells and the least in blood vessel cells. The simultaneous subcellular localization in tumor cells and other cell types within the tumor suggest an involvement of these three scaffoldins in cancer biology, including a role in Epithelial Mesenchymal Transition. The display in differential localization and quantitative expression of AmotL2, FKBP51, and IQGAP1 could be used as biomarkers for more accurate tumor staging and as potential targets for anti-cancer therapeutics by blocking or slowing down their interconnecting functions. Tough further research needs to be done in order to improve these assessments.

摘要： IQ motif containing GTPase activating protein 1 (IQGAP1) is a scaffold protein, which is aberrantly expressed in several tumor types and is closely associated with the development, metastasis and prognosis of cancer. Several studies have demonstrated that IQGAP1 has broad prospects in the basic and clinical research of tumors. The present study aimed to explore the effects of IQGAP1‑small interfering (si) NA on the proliferation and metastasis of U251 and U373 glioma cell lines, which markedly expressed IQGAP1. The human glioma cell lines (U251 and U373) were transfected with siRNA and transfection efficacy was confirmed by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis. Cell proliferation was detected using the Cell Counting kit‑8, and cell metastasis capabilities were detected using cell adhesion, migration and invasion assays. In addition, the expression levels of several tumor‑associated genes were determined by RT‑qPCR and western blotting. The results indicated that IQGAP1 was expressed at higher levels in glioma tissues compared with in normal brain tissues. IQGAP1‑siRNA significantly inhibited cell proliferation, and cell adhesion, migration and invasion. Furthermore, the expression levels of matrix metalloproteinase (MMP)2, Snail, MMP9, fibronectin 1 and Twist were suppressed, and E‑cadherin was upregulated in response to siRNA‑IQGAP1. The present study identified the function of IQGAP1 in glioma cell biology, and indicated that it may be considered a novel target for glioma treatment.

关键词： Akt PKB   insulin   insulin receptor   insulin receptor substrate 1 (IRS-1)   insulin resistance   mitogen-activated protein kinase (MAPK)   phosphatidylinositide 3-kinase (PI 3-kinase)   IQGAP1  

摘要： Insulin binds to the insulin receptor (IR) and induces tyrosine phosphorylation of the receptor and insulin receptor substrate-1 (IRS-1), leading to activation of the PKB/Akt and MAPK/ERK pathways. IQGAP1 is a scaffold protein that interacts with multiple binding partners and integrates diverse signaling cascades. Here we show that IQGAP1 associates with both IR and IRS-1 and influences insulin action. In vitro analysis with pure proteins revealed that the IQ region of IQGAP1 binds directly to the intracellular domain of IR. Similarly, the phosphotyrosine-binding domain of IRS-1 mediates a direct interaction with the C-terminal tail of IQGAP1. Consistent with these observations, both IR and IRS-1 co-immunoprecipitated with IQGAP1 from cells. Investigation of the functional effects of the interactions revealed that in the absence of IQGAP1, insulin-stimulated phosphorylation of Akt and ERK, as well as the association of phosphatidylinositol 3-kinase with IRS-1, were significantly decreased. Importantly, loss of IQGAP1 results in impaired insulin signaling and glucose homeostasis in vivo. Collectively, these data reveal that IQGAP1 is a scaffold for IR and IRS-1 and implicate IQGAP1 as a participant in insulin signaling.

摘要： 2118-Pos

关键词： 甲状腺癌   miRNA   靶基因   增殖  

摘要： 目的探讨miR-124对甲状腺癌细胞的作用机制。方法通过RT-PCR检测人甲状腺癌细胞K1、BCPAP、TPC-1和人甲状腺细胞Nthy-ori 3-1中miR-124的表达水平。MTT法检测甲状腺癌细胞K1转染miR-124 mimics、mimics control后的细胞存活率。根据靶基因预测软件The miRBase、Target Scan、Pic Tar预测miR-124的靶基因IQGAP1,构建wt-IQGAP1和mut-IQGAP1,采用双荧光素酶活性检测鉴定靶基因的正确性。Western印迹检测转染miR-124 mimics、mimics control后甲状腺癌细胞K1中IQGAP1的表达情况。甲状腺癌细胞K1中转染si IQGAP1和si IQGAP1 control,用MTT检测细胞增殖能力,Western印迹检测细胞中IQGAP1蛋白表达含量。结果甲状腺癌细胞K1、BCPAP、TPC-1中的miR-124的相对表达量与甲状腺细胞相比差异显著(P<0.05),三种甲状腺癌细胞中K1细胞的表达量最低。转染后12 h内,空白组、miR-124 mimics组和mimics control组的甲状腺癌K1细胞的存活数量差异不显著(P>0.05),从转染后12 h开始,miR-124 mimics组与对照组、mimics control组相比,甲状腺癌细胞生长抑制明显。预测miR-124的靶基因可能为IQGAP1,转染miR-124 mimics野生型IQGAP1中荧光素酶活性比突变型IQGAP1中荧光素酶活性显著降低,差异显著(P<0.05)。mimics control和wt-IQGAP1和mut-IQGAP1共转染组比较无明显差异(P>0.05)。转染miR-124 mimics组甲状腺癌K1细胞IQGAP1蛋白明显下降,miR-124负调控IQGAP1的表达。抑制甲状腺癌K1细胞中IQGAP1基因,细胞增殖能力减弱,细胞中IQGAP1蛋白表达减弱。结论 miR-124在甲状腺癌细胞中表达下调,miR-124可以抑制甲状腺癌细胞的增殖分化,其通过负调控靶基因IQGAP1抑制癌细胞增殖分化。