Adaptor protein APS binds the NH2-terminal autoinhibitory domain of guanine nucleotide exchange factor Vav3 and augments its activity

The N-terminal calponin homology (CH) domain of Vav guanine nucleotide exchange factor is thought to serve a regulatory role in the autoinhibition, however, its precise function is not entirely clear. We found that the adaptor molecule APS could bind the CH domain of Vav3, a member of the vav proto-oncogene family. The binding of Vav3 and APS was apparently stabilized by the tyrosine phosphorylation of Vav3 by Lck, and the association of APS with Vav3 in turn enhanced the Lck-mediated phosphorylation of Vav3. Focus formation assays demonstrated that APS could increase the transforming activity of proto-Vav3. Further analyses revealed that the Vav3 CH domain could bind the pleckstrin homology (PH) domain of APS and that this binding was indispensable to enhance the transforming activity of Vav3. We present here a novel stimulatory mechanism of Vav3 in which APS directly relieves the autoinhibitory CH domain and furthermore enhances its tyrosine phosphorylation by Lck.     

ELMO1 and Dock180, a bipartite Rac1 guanine nucleotide exchange factor, promote human glioma cell invasion

A distinct feature of malignant gliomas is the intrinsic ability of single tumor cells to disperse throughout the brain, contributing to the failure of existing therapies to alter the progression and recurrence of these deadly brain tumors. Regrettably, the mechanisms underlying the inherent invasiveness of glioma cells are poorly understood. Here, we report for the first time that engulfment and cell motility 1 (ELMO1) and dedicator of cytokinesis 1 (Dock180), a bipartite Rac1 guanine nucleotide exchange factor (GEF), are evidently linked to the invasive phenotype of glioma cells. Immunohistochemical analysis of primary human glioma specimens showed high expression levels of ELMO1 and Dock180 in actively invading tumor cells in the invasive areas, but not in the central regions of these tumors. Elevated expression of ELMO1 and Dock180 was also found in various human glioma cell lines compared with normal human astrocytes. Inhibition of endogenous ELMO1 and Dock180 expression significantly impeded glioma cell invasion in vitro and in brain tissue slices with a concomitant reduction in Rac1 activation. Conversely, exogenous expression of ELMO1 and Dock180 in glioma cells with low level endogenous expression increased their migratory and invasive capacity in vitro and in brain tissue. These data suggest that the bipartite GEF, ELMO1 and Dock180, play an important role in promoting cancer cell invasion and could be potential therapeutic targets for the treatment of diffuse malignant gliomas.     

Guanine nucleotide exchange factor Dock7 mediates HGF-induced glioblastoma cell invasion via Rac activation

Background:

Glioblastoma multiforme (GBM), a highly invasive primary brain tumour, remains an incurable disease. Rho GTPases and their activators, guanine nucleotide exchange factors (GEFs), have central roles in GBM invasion. Anti-angiogenic therapies may stimulate GBM invasion via HGF/c-Met signalling. We aim to identify mediators of HGF-induced GBM invasion that may represent targets in a combination anti-angiogenic/anti-invasion therapeutic paradigm.

Methods:

Guanine nucleotide exchange factor expression was measured by microarray analysis and western blotting. Specific depletion of proteins was accomplished using siRNA. Cell invasion was determined using matrigel and brain slice assays. Cell proliferation and survival were monitored using sulforhodamine B and colony formation assays. Guanine nucleotide exchange factor and GTPase activities were determined using specific affinity precipitation assays.

Results:

We found that expression of Dock7, a GEF, is elevated in human GBM tissue in comparison with non-neoplastic brain. We showed that Dock7 mediates serum- and HGF-induced glioblastoma cell invasion. We also showed that Dock7 co-immunoprecipitates with c-Met and that this interaction is enhanced upon HGF stimulation in a manner that is dependent on the adaptor protein Gab1. Dock7 and Gab1 also co-immunoprecipitate in an HGF-dependent manner. Furthermore, Gab1 is required for HGF-induced Dock7 and Rac1 activation and glioblastoma cell invasion.

Conclusions:

Dock7 mediates HGF-induced GBM invasion. Targeting Dock7 in GBM may inhibit c-MET-mediated invasion in tumours treated with anti-angiogenic regimens.     

Association of genetic variants in the Rho guanine nucleotide exchange factor AKAP13 with familial breast cancer

The A-kinase anchor protein 13 (AKAP13, alias BRX and lbc) tethers cAMP-dependent protein kinase to its subcellular environment and catalyses Rho GTPases activity as a guanine nucleotide exchange factor. The crucial role of members of the Rho family of GTPases in carcinogenesis is well established and targeting Rho proteins with antineoplastic compounds has become a major effort in the fight against cancer. Thus, genetic alterations within the candidate cancer susceptibility gene AKAP13 would be expected to provoke a constitutive Rho signalling, thereby facilitating the development of cancer. Here, we analysed the potential impact of four polymorphic non-conservative amino acid exchanges (Arg494Trp, Lys526Gln, Asn1086Asp and Gly2461Ser) in AKAP13 on familial breast cancer. We performed a case-control study using genomic DNA of BRCA1/2 mutation-negative German female index patients from 601 unrelated families, among a subset of 356 high-risk families, and 1053 German female unrelated controls. The newfound Lys526Gln polymorphism revealed a significant association with familial breast cancer (OR = 1.58, 95% CI = 1.07-2.35) and an even stronger association with high-risk familial breast cancer (OR = 1.85, 95% CI = 1.19-2.88). Haplotype analyses were in line with genotype results displaying a similar significance as analyses of individual polymorphisms. Due to the pivotal role of AKAP13 in the Rho GTPases signalling network, this variant might affect the susceptibility to other cancers as well.     

siRNA-mediated down-regulation of survivin inhibits bladder cancer cell growth

Survivin is recognized as a general target in cancer therapy because of its selective overexpression in the majority of tumors. In bladder cancer (BCa), its expression correlates with tumor grade, recurrence risk and survival. In this study, we compared the therapeutic efficiency of two survivin specific small interfering RNA (siRNA) constructs, SVV284 and SVV094, to inhibit the growth of five human BCa cell lines (EJ28, 5637, J82, RT112, RT4). In a period between 24 to 72 h after siRNA SVV284 transfection, EJ28 and 5637 showed a significant reduction (up to 47%) of viability. For both cell lines cell cycle analysis and quantitation of apoptosis revealed both a specific G2/M arrest and an induction of apoptosis, as well as the occurrence of multinucleated cells. The cell lines EJ28, 5637 and J82 exhibited a prolonged duplication time up to 1.4-fold at 72 h after treatment. Furthermore, in these three cell lines the mRNA and protein expression quantified by real time RT-PCR and ELISA was reduced by at least 50% and up to 99%, respectively. However, RT112 and RT4 cells did not show an effective down-regulation of survivin expression. In comparison to siRNA SVV284, the treatment with siRNA SVV094 exhibited less inhibitory effects on cell growth and survivin expression in all BCa cell lines tested. In summary, the results suggest that the anti-survivin siRNA treatment might represent a suitable therapeutic approach to selectively inhibit growth of BCa cells in addition to commonly applied therapy schemes.     

Vav3对人胃癌细胞株BGC823血管生成基因的调节作用及意义

背景与目的：研究发现Vav3基因在多种恶性肿瘤中表达异常,但Vav3与胃癌血管生成的关系尚不明确。本研究通过抑制内源性Vav3,观察其对胃癌细胞血管生成基因表达的影响,并探讨其机制和意义。方法：荧光定量RT-PCR及蛋白质印迹法(Western blot)检测人胃癌细胞株BGC823、胃上皮细胞株GES-1中Vav3的表达;合成针对Vav3的小干扰RNA(small interference RNA,siRNA),并转染胃癌细胞株BGC823;MTT法检测转染前后胃癌细胞的活性;检测转染前后BGC823血管生成相关基因血管内皮生长因子-A(vascular endothelial growth factor-A,VEGF-A)、VEGF-C、VEGF-D、血管生成素-2(angiogenin-2,Ang-2)、血管生成抑制蛋白-1(vasohibin-1)表达的变化。结果：Vav3在胃癌BGC823细胞中的表达明显高于胃上皮细胞株GES-1(P<0.01);Vav3-siRNA转染BGC823细胞后,Vav3表达明显受到抑制(P均<0.01);MTT结果显示Vav3-siRNA转染后,BGC823活性明显降低(P<0.05);转染后B G C 8 2 3 中VEGF-C和Ang-2表达均较转染前降低(P均<0 . 0 5 ) , 而vasohibin-1表达则升高(P均<0.05)。结论：Vav3对部分胃癌细胞血管生成相关基因有调控作用,并可能通过这种作用促进肿瘤血管生成。     

Inhibition of invasion and metastasis of gastric cancer cells through snail targeting artificial microRNA interference

The zinc-finger factor Snai1 plays an important role in the down-regulation of E-cadherin expression and in the induction of epithelial-mesenchymal transition (EMT) during cancer progression. In gastric cancer tissues, we noted that Snail is abnormally high expressed and is remarkably associated with the lymph node metastasis. Using a plasmid containing newly synthesized artificial microRNA (amiRNA), we transfected gastric cancer cells to block Snail expression. Both Snail protein and mRNA levels were significantly decreased in stably transfected cells, while protein and mRNA expression of E-cadherin was up-regulated. In addition, migration and invasion potential were significantly decreased after knockdown of Snail.     

Cytoskeletal modification of Rho guanine nucleotide exchange factor activity: identification of a Rho guanine nucleotide exchange factor as a binding partner for Sept9b, a mammalian septin

Small GTPase Rho and septin family proteins are thought to be related to tumorigenesis. We have identified a Rho-guanine nucleotide exchange factor (GEF) as a binding partner for a mammalian septin Sept9b using yeast two-hybrid screening. We termed this molecule septin-associated RhoGEF (SA-RhoGEF). Molecular dissection analyses indicated that the C-terminal area of SA-RhoGEF exhibited binding to the N-terminal variable region of Sept9b. SA-RhoGEF was found by immunoprecipitation analysis to associate with septin complexes in REF52 fibroblast cells, maybe through direct interaction with Sept9b. Immunofluorescence analyses revealed the colocalization of SA-RhoGEF and Sept9b along with actin stress fibers in REF52 cells, and their colocalization along stress fibers was most likely to depend on their mutual interaction. In transient expression analyses, Sept9b inhibited SA-RhoGEF-dependent Rho activation in COS7 and HeLa cells. SA-RhoGEF and its fragments expressed in REF52 cells altered endogenous septin filament structures. To our knowledge, SA-RhoGEF is the first molecule providing a link between septins and Rho signaling.     

Inhibition of fibroblast growth factor receptor 2 attenuates proliferation and invasion of pancreatic cancer

The alternative splicing of the extracellular domain of fibroblast growth factor receptor (FGFR)‐2 generates the IIIb and IIIc isoforms. Expression of FGFR‐2 IIIb correlates with vascular endothelial growth factor‐A (VEGF‐A) expression and venous invasion of pancreatic ductal adenocarcinoma (PDAC). By contrast, FGFR‐2 IIIc expression correlates with faster development of liver metastasis after surgery, and increased proliferation rates and invasion of the cancer. In this study, we analyzed the expression and roles of total FGFR‐2 (both isoforms) to determine the effectiveness of FGFR‐2‐targeting therapy for PDAC. Immunohistochemically, FGFR‐2 was highly expressed in 25/48 (52.1%) PDAC cases, and correlated with advanced stage cancer. In FISH analysis, FGFR2 was amplified in 3/7 PDAC cell lines. We stably transfected an FGFR‐2 shRNA targeting the IIIb and IIIc isoforms into FGFR2‐amplified PDAC cells. The proliferation rates, migration, and invasion of FGFR‐2‐shRNA‐transfected cells were lower than those of control cells in vitro. In response to FGF‐2, FGFR‐2‐shRNA‐transfected cells showed decreased phosphorylation of ERK compared with control cells. The FGFR‐2‐shRNA‐transfected cells also expressed lower levels of vascular endothelial growth factor‐A than control cells, and formed smaller s.c. tumors in nude mice. These findings suggest that FGFR‐2 is a therapeutic target for inhibition in PDAC.     

鸟苷酸交换因子ARHGEF 10功能鉴定及其与肿瘤恶性表型间的关系研究

目的 Dbl家族鸟苷交换因子(GEFs)是Rho家族蛋白发生恶性转化的主要调控单位,它通过使Rho蛋白从无活性的GDP形式转换为GTP形式的Rho蛋白而发挥作用。本研究对一个GEF分子-ARHGEF 10进行了结构和功能上的初步探究,对该分子在肿瘤发展过程中扮演的角色进行讨论。方法 Realtime PCR对ARHGEF 10在人体42种正常组织中的表达情况进行了测定;GST-pulldown技术对ARHGEF 10的体内GEF活性进行了检测;双荧光素酶报告基因检测技术对下游小分子进行转录因子活性检测;应用免疫荧光双染标记法完成了高表达ARHGEF 10对正常细胞骨架形态的影响;在细胞表型实验中分别使用CCK8法、Transwell法及软琼脂克隆形成实验检测了高表达ARHGEF 10对细胞增殖侵袭迁移及体外成瘤能力的影响。结果 生物信息学分析结果显示ARHGEF10分子量为156kD,具有典型的Dbl家族分子结构域,在肺组织中表达量最高,能够促使正常成纤维细胞中的应力纤维(Stress fiber)增多,同时促进细胞增殖、侵袭及克隆形成,体外转录因子活性检测发现该基因可能与JAK/STAT通路有关。结论 ARHGEF 10是一个典型的鸟苷交换因子家族分子,能激活Rho家族分子RhoA,具有明显的癌基因特征。     

Insulin-like growth factor binding protein 2 promotes ovarian cancer cell invasion

Background  

Insulin-like growth factor binding protein 2 (IGFBP2) is overexpressed in ovarian malignant tissues and in the serum and cystic fluid of ovarian cancer patients, suggesting an important role of IGFBP2 in the biology of ovarian cancer. The purpose of this study was to assess the role of increased IGFBP2 in ovarian cancer cells.     

Effect of hepatocyte growth factor on invasion of prostate cancer cell lines

Hepatocyte growth factor (HGF) was suggested to play an important role in the regulation of mitogenesis, motogenesis, angiogenesis, migration and invasion for various types of cells, and acts through a specific membrane receptor encoded by c-met proto-oncogene. However, the mechanism of the effect of HGF on tumor invasion of prostate cancer cells remains unclear. We investigated the effect of HGF on the invasion of PC-3 and DU-145 prostate cancer cells through a reconstituted basement membrane (Matrigel), the haptotactic migration to fibronectin substrate, the expression of protein and mRNA for matrix metalloproteinases (MMP)-1 and -9, membrane-type 1-MMP (MT1-MMP), urokinase-type plasminogen activator (u-PA) and its receptor (uPAR). HGF increased both Matrigel invasion and haptotactic migration of prostate cancer cells. Furthermore, HGF also increased the production of MMP-1 and -9, MT1-MMP, u-PA and uPAR of these cells. These results suggested that HGF increased the invasive potential of prostate cancer cells probably through enhancement of cell motility and the production of MMPs and u-PA.     

Inhibition of cancer cell invasion and metastasis by genistein

Genistein is a small, biologically active flavonoid that is found in high amounts in soy. This important compound possesses a wide variety of biological activities, but it is best known for its ability to inhibit cancer progression. In particular, genistein has emerged as an important inhibitor of cancer metastasis. Consumption of genistein in the diet has been linked to decreased rates of metastatic cancer in a number of population-based studies. Extensive investigations have been performed to determine the molecular mechanisms underlying genistein’s antimetastatic activity, with results indicating that this small molecule has significant inhibitory activity at nearly every step of the metastatic cascade. Reports have demonstrated that, at high concentrations, genistein can inhibit several proteins involved with primary tumor growth and apoptosis, including the cyclin class of cell cycle regulators and the Akt family of proteins. At lower concentrations that are similar to those achieved through dietary consumption, genistein can inhibit the prometastatic processes of cancer cell detachment, migration, and invasion through a variety of mechanisms, including the transforming growth factor (TGF)-β signaling pathway. Several in vitro findings have been corroborated in both in vivo animal studies and in early-phase human clinical trials, demonstrating that genistein can both inhibit human cancer metastasis and also modulate markers of metastatic potential in humans, respectively. Herein, we discuss the variety of mechanisms by which genistein regulates individual steps of the metastatic cascade and highlight the potential of this natural product as a promising therapeutic inhibitor of metastasis.     

The guanine nucleotide exchange factor Tiam1 increases colon carcinoma growth at metastatic sites in an orthotopic nude mouse model

Alterations in migration and adhesion are critical to invasion and metastasis. To examine signaling pathways important for colon tumor metastasis, cells of increased migratory potential from the low migratory SW480 human colorectal carcinoma parental cell line were biologically selected by serial migration through modified Boyden chambers. Several sublines were obtained with statistically significantly increased migration relative to the parental cell line. One highly migratory population was single-cell cloned and characterized. The migratory clones exhibit a four- to five-fold increase in protein and mRNA expression of T-lymphoma invasion and metastasis gene 1 (Tiam1), a guanine nucleotide exchange factor. To determine directly the role of Tiam1 in the migration of these migratory sublines, the parental SW480 cell line was transfected with a plasmid encoding the Tiam1 protein, and single cell clones were established. Ectopic expression of Tiam1 in these clones led to morphologic changes identical to biologically selected clones and increased migration. Finally, the implantation of clones that overexpress Tiam1 into the cecum of athymic mice resulted in tumor growth in the spleen, liver, and lung, whereas parental cells do not form tumors by this route of injection. These results demonstrate that overexpression of Tiam1 contributes to the metastatic phenotype of colon cancer cells.     

EZH2 knockdown suppresses the growth and invasion of human inflammatory breast cancer cells

Introduction

Inflammatory breast cancer (IBC) is the most metastatic variant of breast cancer with the poorest survival in all types of breast cancer patients and presently therapeutic targets for IBC are very limited. Enhancer of zeste homolog 2 (EZH2) is frequently expressed in human IBC and its expression positively correlates with worse clinical outcome. However, the molecular basis for EZH2 promoting IBC has not been explored. Here, we investigated the functional role of EZH2 in IBC cells by examining the effects of its knockdown on the formation of tumor spheroids and invasion of these cells in vitro and in vivo in an orthotopic xenograft model.

Methods

SUM149 and a new IBC cell line-FC-IBC-02 derived from pleural effusion fluid of an IBC patient were used in this study. Specific knockdown of EZH2 was performed using short hairpin RNA (shRNA) specific to the human EZH2 gene. Cell growth and the formation of tumor spheroids were examined in vitro. The effects of EZH2 knockdown on IBC cell migration and invasion were examined by a Boyden chamber assay. For the in vivo tumor growth studies, IBC cells were orthotopically transplanted into the mammary fat pads of immunodeficient mice.

Results

The results showed that EZH2 is expressed at higher levels in human IBC cell lines compared with normal human mammary epithelial cells, and the knockdown of EZH2 expression significantly suppressed cell growth and tumor spheroid formation of human IBC cells in vitro. In addition, EZH2 knockdown inhibited the migration and invasion of IBC cells. Significantly, EZH2 knockdown suppressed the angiogenesis and tumor growth of IBC cells in vivo.

Conclusions

Our results provide direct evidence that EZH2 is critical for the formation of tumor spheroids and invasion of human IBC cells and could be a potential target for developing novel therapeutic strategies for human IBC.     

Down-regulation of hepatoma-derived growth factor inhibits anchorage-independent growth and invasion of non-small cell lung cancer cells

We recently reported that a high level of hepatoma-derived growth factor (HDGF) expression in tumors correlates with a high incidence of tumor relapse or distant metastasis and shortened survival time in patients with non-small cell lung cancer (NSCLC). However, the mechanisms of the HDGF-associated aggressive biological behavior are unknown. In this study, we knocked down HDGF expression in NSCLC cells to determine the biological consequences. Transfection with HDGF-specific small interfering RNA (siRNA) resulted in down-regulation of HDGF expression in four NSCLC cell lines. Down-regulation of HDGF resulted in no detectable effect on anchorage-dependent cell growth as determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, a microelectronic cell sensor system, and flow cytometry. In contrast, cells transfected with HDGF-siRNA grew more slowly and formed significantly fewer colonies in soft agar than did cells treated with LipofectAMINE alone or transfected with negative control siRNA. In an in vitro invasion assay, significantly fewer cells transfected with HDGF-siRNA than cells treated with LipofectAMINE alone were able to invade across a Matrigel membrane barrier. In an in vivo mouse model, A549 cells treated with HDGF-siRNA grown significantly slower than the cells treated with LipofectAMINE alone or negative control siRNA. Morphologically, HDGF-siRNA-treated tumors exhibited markedly reduced blood vessel formation and increased necrosis, whereas the Ki67 labeling indices were similar in tumors treated with controls. Our results suggest that HDGF is involved in anchorage-independent growth, cell invasion, and formation of neovasculature of NSCLC. These qualities may contribute to the HDGF-associated aggressive biological behavior of NSCLC.     

Activation of Rac1 and the exchange factor Vav3 are involved in NPM-ALK signaling in anaplastic large cell lymphomas

The majority of anaplastic large cell lymphomas (ALCLs) express the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein, which is oncogenic due to its constitutive tyrosine kinase activity. Transformation by NPM-ALK not only increases proliferation, but also modifies cell shape and motility in both lymphoid and fibroblastic cells. We report that the Rac1 GTPase, a known cytoskeletal regulator, is activated by NPM-ALK in ALCL cell lines (Karpas 299 and Cost) and transfected cells (lymphoid Ba/F3 cells, NIH-3T3 fibroblasts). We have identified Vav3 as one of the exchange factors involved in Rac1 activation. Stimulation of Vav3 and Rac1 by NPM-ALK is under the control of Src kinases. It involves formation of a signaling complex between NPM-ALK, pp60(c-src), Lyn and Vav3, in which Vav3 associates with tyrosine 343 of NPM-ALK via its SH2 domain. Moreover, Vav3 is phosphorylated in NPM-ALK positive biopsies from patients suffering from ALCL, demonstrating the pathological relevance of this observation. The use of Vav3-specific shRNA and a dominant negative Rac1 mutant demonstrates the central role of GTPases in NPM-ALK elicited motility and invasion.     

Activation of gef-h1, a guanine nucleotide exchange factor for RhoA, by DNA transfection

Several oncogenes isolated by the NIH/3T3 transformation assay, i.e., dbl, dbs, lbc, lfc, lsc, net, ost and tim, contain a Dbl homology (DH) and a pleckstrin-homology (PH) domain and act as GEFs (guanine nucleotide exchange factors) for Rho-like GTPases. In a search for genes with oncogenic potential in DNA from the monocytic leukaemia cell line U937, we identified an amino-terminal truncated form of gef-h1, a gene encoding a GEF for RhoA. These data support the idea that a systematic search for mutations and/or deletions of GEFs in human cancer is promising.     

Various effects of hepatoma-derived growth factor on cell growth, migration and invasion of breast cancer and prostate cancer cells

Hepatoma-derived growth factor (HDGF) has been implicated in the growth and metastasis of various types of human cancer, but the role of HDGF expression in prostate cancer or breast cancer has not been documented. To assess the role of HDGF in the proliferation, migration and invasion by prostate and breast cancer cells, HDGF expression in DU145 and MCF7 cells was knocked down using siRNA, and the effect of such knockdown was assessed by MTS and [3H]-thymidine incorporation Transwell assays. Moreover, we identified differentially expressed genes that might mediate the HDGF-induced cellular effects. Our results demonstrate that down-regulation of HDGF expression significantly reduces the proliferation of both DU145 and MCF7 cells. However, down-regulation of HDGF expression in DU145 inhibited cell migration and invasion, but in MCF7 cells it stimulated cell migration and invasion. This differential effect might result from the differential induction of PIK3R1 or SERPINE1 in the two cell lines upon HDGF-siRNA treatment. In conclusion, HDGF may participate in the pathogenesis of prostate and breast cancer by promoting cell growth and it may be a therapeutic target for these cancers.