中心体蛋白70促进乳腺癌细胞的侵袭和转移

中心体蛋白70（centrosomal protein 70, CEP70）可通过介导内皮细胞的迁移影响血管新生,肿瘤的转移能力与肿瘤细胞的迁移密切相关,CEP70是否影响肿瘤细胞的侵袭转移尚不明确。结合前期淋巴结转移和未发生淋巴结转移原位乳腺癌组织的基因表达芯片的比较结果,本研究通过免疫组化染色,检测CEP70在淋巴结转移和未发生淋巴结转移的原位乳腺癌组织中的表达情况,以及real-time PCR和Western 印迹检测不同乳腺癌细胞系中CEP70的表达,结果提示,淋巴结转移患者的乳腺癌组织中CEP70强阳性的比例明显高于未发生淋巴结转移的乳腺癌组织,同时CEP70在侵袭能力强的乳腺癌细胞中表达较高。利用慢病毒转染构建CEP70稳定下调的MDA-MB-231细胞系,划痕实验以及侵袭转移的结果显示,下调CEP70的表达,可明显抑制MDA-MB-231细胞系的细胞迁移和侵袭能力。上述结果证明,CEP70的表达与乳腺癌的侵袭转移呈正相关,下调CEP70可抑制乳腺癌的侵袭转移,因此CEP70有望成为乳腺癌临床诊断及治疗的新靶点。     

生物钟蛋白TIMELESS对乳腺癌细胞增殖和侵袭转移的差异调控

生物钟蛋白TIMELESS是生物体周期节律相关的核心分子钟的一员。TIMELESS可以调节细胞的增殖和代谢,DNA损伤的识别和修复等。研究发现,TIMELESS的表达与肝癌、肺癌、结直肠癌和鼻咽癌等的发生发展明显关联。在乳腺癌中,TIMELESS的调节作用和机制仍不十分清晰。本文分别研究了TIMELESS在乳腺癌细胞增殖和转移方面的调控作用。通过细胞增殖实验发现,TIMELESS可明显促进乳腺癌细胞的增殖。克隆形成实验发现,在乳腺癌细胞ZR-75-30和T-47D中,TIMELESS过表达分别上调克隆数量约1.6倍和1.8倍。通过报告基因检测对雌激素信号通路的研究发现,TIMELESS和雌激素受体ERα(estrogen receptor)共表达与单转ERα相比,使得雌激素受体ERα的转录活性提高约3倍;而通过对于Basal型乳腺癌患者生存曲线分析发现,Timeless高表达与basal型乳腺癌患者的生存率正相关。基于此,我们进一步研究了TIMELESS对于乳腺癌细胞转移的调控作用。通过细胞划痕实验和F-肌动蛋白组装检测发现,TIMELESS抑制乳腺癌细胞的转移;同时,TIMELESS敲低提高了乳腺癌细胞的转移数量约1.6倍,并下调了上皮标志物E 钙黏着蛋白的表达,上调了间质标志物N-钙黏着蛋白表达。本研究提示,TIMELESS在调控乳腺癌的增殖和转移过程中存在差异调控,即TIMELESS促进乳腺癌细胞增殖,而抑制了乳腺癌细胞的转移。这为生物钟蛋白质调控乳腺癌的发生发展提供了分子基础,同时为乳腺癌的分型治疗提供了一定的理论依据。     

生物钟蛋白TIMELESS对乳腺癌细胞增殖和侵袭转移的差异调控

生物钟蛋白TIMELESS是生物体周期节律相关的核心分子钟的一员。TIMELESS可以调节细胞的增殖和代谢,DNA损伤的识别和修复等。研究发现,TIMELESS的表达与肝癌、肺癌、结直肠癌和鼻咽癌等的发生发展明显关联。在乳腺癌中,TIMELESS的调节作用和机制仍不十分清晰。本文分别研究了TIMELESS在乳腺癌细胞增殖和转移方面的调控作用。通过细胞增殖实验发现,TIMELESS可明显促进乳腺癌细胞的增殖。克隆形成实验发现,在乳腺癌细胞ZR-75-30和T-47D中,TIMELESS过表达分别上调克隆数量约1.6倍和1.8倍。通过报告基因检测对雌激素信号通路的研究发现,TIMELESS和雌激素受体ERα(estrogen receptor)共表达与单转ERα相比,使得雌激素受体ERα的转录活性提高约3倍;而通过对于Basal型乳腺癌患者生存曲线分析发现,Timeless高表达与basal型乳腺癌患者的生存率正相关。基于此,我们进一步研究了TIMELESS对于乳腺癌细胞转移的调控作用。通过细胞划痕实验和F-肌动蛋白组装检测发现,TIMELESS抑制乳腺癌细胞的转移;同时,TIMELESS敲低提高了乳腺癌细胞的转移数量约1.6倍,并下调了上皮标志物E 钙黏着蛋白的表达,上调了间质标志物N-钙黏着蛋白表达。本研究提示,TIMELESS在调控乳腺癌的增殖和转移过程中存在差异调控,即TIMELESS促进乳腺癌细胞增殖,而抑制了乳腺癌细胞的转移。这为生物钟蛋白质调控乳腺癌的发生发展提供了分子基础,同时为乳腺癌的分型治疗提供了一定的理论依据。     

Cyclin B1 Suppresses Colorectal Cancer Invasion and Metastasis by Regulating E-Cadherin

Cyclin B1, a mitotic cyclin, has been implicated in malignances. However, its contribution to colorectal cancer invasion and metastasis are still not well understood. Here, we demonstrated that the invasion and metastasis of colorectal cancer is regulated by Cyclin B1. Overexpression of Cyclin B1 was observed in colorectal cancer tissues, but this elevated expression was negatively associated with lymph node metastasis, distant metastasis stage, and TNM stage. The Kaplan-Meier survival analysis proved that low Cyclin B1 expression was associated with poor overall survival of patients with colorectal cancer. Inhibition of Cyclin B1 in colorectal cancer cells enhanced the cell migration and invasion of three different colorectal cancer cell lines. In studying the possible mechanism by which Cyclin B1 suppresses colorectal cancer invasion and metastasis, we observed that suppression of Cyclin B1 decreased the expression of E-cadherin protein level. Our findings suggest that Cyclin B1 could suppress the invasion and metastasis of colorectal cancer cells through regulating E-cadherin expression, which enables the development of potential intervention strategies for colorectal cancer.     

窖蛋白-1、基质金属蛋白酶-2与乳腺肿瘤的侵袭和转移

窖蛋白(caveolin)是分子量为21～24 kD的整合膜蛋白,是胞膜窖(caveolae)的标志性结构分子,其家族成员窖蛋白-1(caveolin-1,Cav-1)参与细胞内许多重要的生命活动.近来研究发现,窖蛋白-1与乳腺上皮细胞转化及乳腺癌的发生密切相关.基质金属蛋白酶(matrix metalloproteinases,MMPs)是基质降解代谢的主要酶类,几乎能降解细胞外基质和基底膜的所有成分,其家族成员明胶酶A(MMP-2)在乳腺癌的浸润和转移过程中起重要作用.新近发现,窖蛋白-1与基质金属蛋白酶-2在胞膜窖中共定位,窖蛋白-1通过抑制基质金属蛋白酶-2的激活来抑制乳腺癌的侵袭和转移,起到肿瘤抑制因子的作用.本文对窖蛋白-1与基质金属蛋白酶-2各自在乳腺肿瘤侵袭转移中的作用及两者关系的研究进行综述.     

TSG-6 promotes Cancer Cell aggressiveness in a CD44-Dependent Manner and Reprograms Normal Fibroblasts to create a Pro-metastatic Microenvironment in Colorectal Cancer

Tumor necrosis factor α stimulated gene 6 (TSG-6), a 30-KD secretory protein, plays an essential role in modulating inflammatory responses and extracellular matrix remodeling. However, little is known regarding the role of TSG-6 in human cancers. Here, we investigated the mechanism of action and the role of TSG-6 in colorectal cancer (CRC) metastasis. We found that TSG-6 was highly expressed in tumor tissues and was associated with poor prognosis and metastasis in CRC. Mechanistically, TSG-6 overexpression in CRC cells resulted in ERK activation and epithelial-mesenchymal transition by means of stabilizing CD44 and facilitating the CD44-EGFR complex formation on the cell membrane. Consequently, this resulted in the promotion of tumor migration and invasion both in vitro and in vivo. Notably, our data showed that CRC cells secreted TSG-6 could trigger a paracrine activation of JAK2-STAT3 signaling and reprogram normal fibroblasts into cancer-associated fibroblasts, which exhibited upregulation of pro-metastatic cytokines (CCL5 and MMP3) and higher movement ability. In animal models, the co-injection of cancer cells and TSG6-reprogrammed fibroblasts led to a significant increase in tumor metastasis. Our findings indicated that TSG-6 overexpression in CRC cells could promote cancer metastasis in both an autocrine and paracrine manner. Therefore, targeting TSG-6 might be a potential therapeutic strategy for the treatment of metastatic CRC.     

CD44v6 Is a Marker of Constitutive and Reprogrammed Cancer Stem Cells Driving Colon Cancer Metastasis

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MicroRNA-2 Suppresses Lewis Lung Cancer Cells Proliferation,Invasion, and Migration in Tumor-Bearing Mice

We sought to find the biological effects of MicroRNA-2 in suppressing Lewis lung cancer cells proliferation, invasion, and migration in tumor-bearing mice. MicroRNA-2 was transfected into Lewis lung cancer cells of tumor-bearing mice by gene transient transfection technique and these Lewis-microRNA-2 cells were taken as MicroRNA transfection group. At the same time, Lewis cells were taken as control group and Lewis-EGFP cells as empty plasmid group. The growth curves of cells in the three groups were drawn by manual counting method, while the invasiveness of cells in the three groups was compared by transmembrane cell invasion assay. The three kinds of cells were seeded into BALB/Nude SPF level nude mice to detect the formation of tumors and the number of metastases by Xenograft experiments. The result showed that the MicroRNA transfection group has the lowest vitality of cells proliferation, fewest cells passed through matrigel matrix protein layer, and lowest cells invasive rate. Mice with Lewis-microRNA-2 cells apparently had a longer time of tumor formation. The average tumor mass and the number of metastases were significantly lower than the other two groups. MicroRNA-2 significantly inhibited Lewis lung cancer cell proliferation, invasion and migration in tumor-bearing mice, which may be associated with the regulation of target genes PLK1 and TGF-β.     

Blockade of Fas Signaling in Breast Cancer Cells Suppresses Tumor Growth and Metastasis via Disruption of Fas Signaling-initiated Cancer-related Inflammation

Mechanisms for cancer-related inflammation remain to be fully elucidated. Non-apoptotic functions of Fas signaling have been proposed to play an important role in promoting tumor progression. It has yet to be determined if targeting Fas signaling can control tumor progression through suppression of cancer-related inflammation. In the current study we found that breast cancer cells with constitutive Fas expression were resistant to apoptosis induction by agonistic anti-Fas antibody (Jo2) ligation or Fas ligand cross-linking. Higher expression of Fas in human breast cancer tissue has been significantly correlated with poorer prognosis in breast cancer patients. To determine whether blockade of Fas signaling in breast cancer could suppress tumor progression, we prepared an orthotopic xenograft mouse model with mammary cancer cells 4T1 and found that blockade of Fas signaling in 4T1 cancer cells markedly reduced tumor growth, inhibited tumor metastasis in vivo, and prolonged survival of tumor-bearing mice. Mechanistically, blockade of Fas signaling in cancer cells significantly decreased systemic or local recruitment of myeloid derived suppressor cells (MDSCs) in vivo. Furthermore, blockade of Fas signaling markedly reduced IL-6, prostaglandin E2 production from breast cancer cells by impairing p-p38, and activity of the NFκB pathway. In addition, administration of a COX-2 inhibitor and anti-IL-6 antibody significantly reduced MDSC accumulation in vivo. Therefore, blockade of Fas signaling can suppress breast cancer progression by inhibiting proinflammatory cytokine production and MDSC accumulation, indicating that Fas signaling-initiated cancer-related inflammation in breast cancer cells may be a potential target for treatment of breast cancer.     

G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration,Invasion, and Metastasis

Triple negative breast cancer (TNBC) is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3) is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis.     

Gonadotropin-Releasing Hormone Agonists Sensitize,and Resensitize,Prostate Cancer Cells to Docetaxel in a p53-Dependent Manner

Gonadotropin-releasing hormone (GnRH) receptors are expressed in prostate cancer, specifically in the most aggressive stage of the tumor (castration-resistant prostate cancer, CRPC) for which the standard treatment, docetaxel-based chemotherapy, can only improve the median survival time by few months. We previously showed that GnRH agonists exert an antitumor activity in CRPC cells; however, a link between GnRH receptors and the apoptotic machinery remains to be defined. Aim of this study was to evaluate whether, in CRPC cells, GnRH agonists might affect the expression/activity of apoptosis-related proteins and might sensitize, or resensitize, cancer cells to chemotherapeutics. We demonstrated that, in p53-positive DU145 cells, GnRH agonists: a) increase the expression of the proapoptotic protein Bax; this effect is mediated by the phosphorylation (activation) of p53, triggered by the p38 MAPK; b) potentiate the antiproliferative/proapoptotic activity of docetaxel; c) resensitize docetaxel-resistant cells to the antitumor activity of the cytotoxic drug. These data indicate that GnRH agonists sensitize and, more importantly, resensitize DU145 CRPC cells to chemotherapy in a p53-dependent manner. To confirm the crucial role of p53 in the activity of GnRH agonists, experiments were performed in p53-null PC3 cells. We found that GnRH agonists fail to increase Bax expression and do not potentiate the cytotoxic activity of docetaxel. These results may provide a rationale for novel combination treatment strategies, especially for docetaxel-resistant CRPC patients expressing a functional p53 protein.     

基于生物信息学分析研究miR-182-5p通过靶向调控EP300促进乳腺癌细胞的侵袭和转移

近期研究表明,miR-182-5p对多种癌症的侵袭和转移具有重要作用,但其在乳腺癌侵袭转移中的研究相对较少。本研究通过网上在线microRNA分析工具下载乳腺癌组织及正常乳腺组织表达比较的数据集,分析发现在GSE4589、GSE38167、GSE61438等3个数据库中,在乳腺癌组织中存在26个相同的microRNA,其中8个上调,而我们实验验证发现hsa-miR-182在8例病理组织中的表达上调差异最显著(P=0.001),选定目的基因hsa-miR-182;qRT-PCR检测细胞中miR-182-5p的表达,结果显示,与MCF-10A相比,miR-182-5p在MDA-MB-231、T47D、MDA-MB-453、MCF-7中表达上调(P<0.05);转染miR-182-5p干扰质粒,qRT-PCR检测细胞中miR-182-5p的表达情况。结果显示,miR-182-5p表达显著降低(P=0.003),提示转染成功;Transwell侵袭结果显示,MDAMB-231细胞敲低miR-182-5p,与对照组相比,体外侵袭能力明显降低(P=0.002);Western印迹检测转染miR-182-5p干扰质粒时,MDA-MB-231中上皮-间质转化(epithelial-mesenchymal transition,EMT)相关标志物的表达情况,结果显示,与对照组相比,敲低miR-182-5p使细胞中上皮-钙黏着蛋白(E-cadherin)表达上调,神经-钙黏着蛋白(N-cadherin)、波形蛋白(vimentin)表达下调。为研究探讨miR-182-5p的靶蛋白,采用在线预测软件预测可能与miR-182-5p结合的靶蛋白,cytoscape构建蛋白质互作网络图并筛选出hub基因;双荧光素酶结果证实,miR-182-5p可与EP300靶向结合(P=0.001);采用qRT-PCR、Western印迹检测转染miR-182-5p干扰质粒后EP300在mRNA及蛋白质水平的表达,结果显示,与对照组相比,在敲低miR-182-5p组中EP300在mRNA及蛋白质的表达上调(P=0.001)。综上所述,miR-182-5p可靶向调节EP300,促进乳腺癌细胞的侵袭与转移。     

An EP4 Antagonist ONO-AE3-208 Suppresses Cell Invasion,Migration, and Metastasis of Prostate Cancer

EP4 is one of the prostaglandin E2 receptors, which is the most common prostanoid and is associated with inflammatory disease and cancer. We previously reported that over-expression of EP4 was one of the mechanisms responsible for progression to castration-resistant prostate cancer, and an EP4 antagonist ONO-AE3-208 in vivo suppressed the castration-resistant progression regulating the activation of androgen receptor. The aim of this study was to analyze the association of EP4 with prostate cancer metastasis and the efficacy of ONO-AE3-208 for suppressing the metastasis. The expression levels of EP4 mRNA were evaluated in prostate cancer cell lines, LNCaP, and PC3. EP4 over-expressing LNCaP was established, and their cell invasiveness was compared with the control LNCaP (LNCaP/mock). The in vitro cell proliferation, invasion, and migration of these cells were examined under different concentrations of ONO-AE3-208. An in vivo bone metastatic mouse model was constructed by inoculating luciferase expressing PC3 cells into left ventricle of nude mice. Their bone metastasis was observed by bioluminescent imaging with or without ONO-AE3-208 administration. The EP4 mRNA expression levels were higher in PC3 than in LNCaP, and EP4 over-expression of LNCaP cells enhanced their cell invasiveness. The in vitro cell invasion and migration were suppressed by ONO-AE3-208 in a dose-dependent manner without affecting cell proliferation. The in vivo bone metastasis of PC3 was also suppressed by ONO-AE3-208 treatment. EP4 expression levels were correlated with prostate cancer cell invasiveness and EP4 specific antagonist ONO-AE3-208 suppressed cell invasion, migration, and bone metastasis, indicating that it is a potential novel therapeutic modality for the treatment of metastatic prostate cancer.     

MicroRNA-493 Suppresses Tumor Growth,Invasion and Metastasis of Lung Cancer by Regulating E2F1

miRNAs have been proposed to be key regulators of progression and metastasis in cancer. However, an understanding of their roles and molecular mechanisms is needed to provide deeper insights for better therapeutic opportunities. In this study we investigated the role and mechanism of miR-493 in the development and progression of nonsmall-cell lung cancer (NSCLC). Our data indicated that the expression of miR-493 was markedly reduced in pulmonary carcinoma. The ectopic expression of miR-493 impaired cell growth and invasion in vitro and in vivo. Mechanically, miR-493 commonly directly targeted E2F1, which resulted in a robust reduction of the expression of mRNA and protein. This effect, in turn, decreased the growth, invasion and metastasis of lung cancer cells. Our findings highlight the importance of miR-493 dysfunction in promoting tumor progression, and implicate miR-493 as a potential therapeutic target in lung cancer.     

Effects of Osthole on Migration and Invasion in Breast Cancer Cells

Osthole, a natural coumarin derivative, is extracted from the fruit of Cnidium monnieri Cusson. Breast cancer is one of the most commonly diagnosed cancers and the leading cause of death in women. Recent studies have shown that Osthole has anti-tumor activity. However, the effects of Osthole on the migration and invasion of cancer cells have not yet been reported. Here, we found that Osthole is effective in inhibiting the migration and invasion of breast cancer cells by wound healing and transwell assays. Luciferase and zymography assays revealed that Osthole effectively inhibits matrix metalloproteinase-2 promoter and enzyme activity, which might be one of the causes that lead to the inhibition of migration and invasion by Osthole. This is the first report on the inhibitory function of Osthole in migration and invasion in breast cancer cells. Our findings indicate a need for further evaluation of Osthole in breast cancer chemotherapy and chemoprevention.