NUAK1通过影响F-actin聚合促进乳腺癌的侵袭转移

本课题组先前研究证明NUAK1/ARK5参与乳腺癌、胶质瘤侵袭转移,但机制尚不清楚.本文证明,NUAK1通过影响F-actin聚合促进乳腺癌的侵袭转移.应用小RNA干扰技术敲除乳腺癌细胞系MDA-MB-231中的NUAK1,用G418进行稳定筛选,并用Western印迹进行蛋白质表达检测,结果显示,成功敲除MDA-MB-231细胞中的NUAK1;采用Transwell侵袭实验检测NUAK1在乳腺癌细胞侵袭转移中的作用,结果表明,NUAK1被干扰的SiNUAK1/MDA-231细胞的侵袭能力明显减弱;应用免疫荧光法对细胞的F-actin进行荧光染色,半定量F-actin聚合分析结果显示,IGF-1在转染空载的细胞组（Scr/MDA-231）能诱导肌动蛋白短暂的聚合反应,而在敲除NUAK1的细胞组（SiNUAK1/MDA-231）肌动蛋白的聚合显著减少;用细胞因子IGF-1刺激乳腺癌细胞观察cofilin磷酸化,结果显示,在敲除NUAK1的细胞组（SiNUAK1/MDA-231）,IGF-1诱导的cofilin的磷酸化明显受抑制.上述结果表明,NUAK1能通过促进F-actin的聚合从而影响乳腺癌细胞的侵袭转移.     

沉默ST3Gal III抑制MDA-MB-231细胞黏附和侵袭能力

我们前期研究表明α2,3-唾液酸水平与乳腺癌侵袭转移密切相关。人α2,3-唾液酸转移酶（ST3Gal Ⅲ）可催化合成细胞表面的α2,3-唾液酸,并在乳腺癌组织中高表达,此酶活性与肿瘤转移潜能密切相关,但其机制尚未阐明。本研究中我们将继续探讨ST3Gal Ⅲ在对乳腺癌转移关键步骤粘附和侵袭中的作用。构建特异靶向ST3Gal Ⅲ的短发夹RNA（shRNA）序列的慢病毒载体,采用细胞转染沉默乳腺癌MDA-MB-231细胞的ST3Gal Ⅲ,经实时定量PCR及Western印迹检测转染后细胞ST3Gal Ⅲ mRNA及蛋白表达,验证构建了稳定下调ST3Gal Ⅲ表达的两个细胞克隆,分别记作shRNA-2、shRNA-4。细胞表面α2,3-唾液酸是ST3Gal Ⅲ下游产物,可代表酶活性。流式细胞术分析结果证实,shRNA-2、shRNA-4细胞表面α2,3-唾液酸的含量显著降低（P<0.05）。细胞黏附、细胞迁移及侵袭能力等功能学检测结果表明,shRNA细胞黏附能力及侵袭能力明显降低（P<0.05）。β1整合素表达与肿瘤侵袭能力获取密切相关。本研究中,沉默ST3Gal Ⅲ可抑制β1整合素表达（P<0.05）。这些结果提示,ST3Gal Ⅲ在乳腺癌转移关键步骤黏附和侵袭中具有重要作用,沉默ST3Gal Ⅲ抑制MDA MB-231细胞黏附和侵袭能力,其作用机制可能是通过下调β1整合素表达。此研究从新的视角认识了乳腺癌转移的机制,并可能提供乳腺癌转移治疗的新靶点。     

EBP50通过Wnt3a/β-catenin信号通路抑制乳腺癌细胞的侵袭和转移

EBP50通过抑制LIMK/cofilin信号通路和PI3K/Akt/m TOR/MMP信号通路抑制乳腺癌细胞的侵袭和转移。然而,EBP50是否可以通过其他机制抑制乳腺癌细胞的侵袭和转移尚未可知。本研究发现,EBP50能通过Wnt3a/β-catenin信号通路影响乳腺癌细胞的侵袭和转移。Western印迹结果显示,EBP50在低转移细胞株MCF-7和正常乳腺细胞株MCF-10A中高表达,而在高转移细胞株MDA-MB-231低表达。采用RNAi技术将小RNA质粒瞬时转染乳腺癌细胞株MCF-7,同时将质粒pc DNA3.1-EBP50转入乳腺癌细胞株MDA-MB-231。Western印迹结果显示,Si EBP50/MCF-7细胞组的EBP50表达水平明显下调,MDA231/EBP50细胞组的EBP50表达水平明显上调。Transwell侵袭实验和划痕实验结果显示,用Wnt3a刺激后,Si EBP50/MCF-7细胞组体外迁移和侵袭能力明显增强,MDA231/EBP50细胞组体外迁移和侵袭能力明显减弱。Western印迹结果显示,与未用Wnt3a或同时用Wnt3a和抑制剂Dkk1刺激的相比,Si EBP50/MCF-7细胞组上皮-钙粘蛋白(Ecadherin)下调,波形蛋白(vimentin)上调,细胞核中β-联蛋白(β-catenin)的表达水平升高,而MDA231/EBP50细胞组上皮-钙粘蛋白上调,波形蛋白下调,细胞核中β-联蛋白表达下降。上述结果表明,EBP50通过Wnt3a/β-catenin信号通路影响β-联蛋白的转核,抑制EMT的发生,进而抑制乳腺癌细胞的侵袭和转移。     

吞噬细胞运动蛋白1通过NF-κB/snail信号通路促进肺腺癌细胞A549的上皮-间质转化

吞噬细胞运动蛋白1(engulfment and cell motility 1,ELMO1)在许多恶性肿瘤的侵袭转移中发挥重要作用,但其在肺腺癌侵袭转移中的研究相对较少。本研究旨在探讨ELMO1在肺腺癌上皮-间质转化(epithelial-mesenchymal transition,EMT)中的作用,为临床预防肺腺癌的侵袭和转移提供理论和实验依据。Western印迹结果显示,ELMO1在人正常肺上皮细胞BEAS-2B中的蛋白质表达水平低,而在人肺腺癌细胞A549中表达水平高。si ELMO1/A549细胞组中ELMO1的表达水平明显降低。用白细胞介素-8(interleukin-8,IL-8)刺激肺腺癌细胞A549 24 h后,趋化运动实验结果显示,IL-8浓度为100 ng/m L时为最适刺激浓度,此时肺腺癌细胞A549具有最强的趋化运动能力,增高或降低IL-8的浓度时细胞的趋化运动能力均下降。Transwell侵袭实验结果显示,用IL-8刺激24 h后,si ELMO1/A549细胞相比于Scr/A549细胞组的侵袭转移能力明显减弱。Western印迹结果显示,与未用IL-8刺激或用IL-8和抑制剂BAY11-7082同时刺激的相比,si ELMO1/A549细胞较Scr/ELMO1A549细胞组E-cadherin蛋白的表达上调,Vimentin蛋白的表达下调,p-IκBα、细胞核中snail的蛋白质表达水平均降低。综上所述,ELMO1可以通过NF-κB信号通路影响snail的转核,从而促进肺腺癌细胞A549的上皮-间质转化。     

芹菜素对MDA-MB-231细胞增殖及侵袭转移的影响

目的:研究芹菜素对乳腺癌MDA-MB-231细胞增殖及侵袭转移能力的影响,探讨其作用 机制.方法:芹菜素处理MDA-MB-231细胞,MTT法及流式法检测细胞增殖、粘附和周期变化 ;Millicell小室检测新生血管形成、侵袭转移能力的改变;免疫细胞化学检测Bcl-2、Bax 、cyclinB1、VEGF、MMP-9、E-cd蛋白的表达差异.结果:芹菜素明显抑 制MDA-MB-231细胞增殖,诱导细胞凋,G2/M期细胞比例由4.79%增至36.12%(P＜0.05 );细胞的黏附、新生血管形成 能力均显著降低;侵袭转移穿膜细胞数明显低于对照组,分别由88.67、106.33降至45.67、 68(P＜0.05);Bcl-2、cyclinB1、VEGF、MMP-9蛋白表达明显降低;Bax,E-cd的表达则增 加.结论:芹菜素有效抑制MDA-MB-231细胞增殖,阻滞细胞于G2/M期, 诱导细胞凋亡;抑制 细胞粘附、新生血管形成、侵袭与转移的能力,其机制与抑制Bcl-2、cyclinB1、VEGF、MMP 9表达,促进Bax、E-cd表达有关.     

BAG3 蛋白Ser187位点磷酸化诱导人甲状腺癌FRO 细胞上皮间质转化

Bcl-2相关抗凋亡蛋白3（Bcl-2 associated athanogene 3,BAG3）是BAG家族的重要成员,调节肿瘤细胞的粘附、迁移和侵袭,促进恶性肿瘤的复发和转移.本室前期工作证明,PKCδ可催化BAG3的Ser187位点磷酸化.本文研究BAG3蛋白磷酸化修饰对甲状腺癌FRO 细胞EMT表型转化的影响.稳定转染野生型WT-BAG3、模拟磷酸化型S187D-BAG3、阻碍磷酸化型S187A-BAG3 FRO细胞后,观察细胞形态的变化.结果显示,稳定转染模拟磷酸化型S187D-BAG3引起甲状腺癌FRO细胞呈现明显的间质细胞形态.实时PCR 和Western印迹,结果显示,稳定表达S187D-BAG3显著上调间质细胞标记物N-cadherin和波形蛋白mRNA与蛋白质在FRO细胞的表达,但下调上皮细胞标记物E-cadherin的mRNA和蛋白质的表达.同时,免疫荧光结果显示,稳定过表达S187D-BAG3的FRO细胞,E-cadherin和β-catenin出现向核周的内化.本文结果提示,BAG3蛋白磷酸化修饰可诱导甲状腺癌FRO细胞上皮间质转化.     

MiR-150-5p通过靶向调控Rab1A抑制乳腺癌细胞MDA-231的上皮-间质转化

先前的研究表明,miR-150-5p发挥抑癌基因的作用,调控肿瘤细胞的侵袭与转移。然而,关于其在乳腺癌细胞侵袭与转移中的机制尚不明确。本实验旨在研究miR-150-5p负向调控Rab1A在乳腺癌细胞上皮-间质转化（epithelial-mesenchymal transition,EMT）中的作用。双荧光素酶的结果显示,miR-150-5p可负向调控Rab1A。荧光定量PCR (qRT-PCR) 结果显示,miR-150-5p在乳腺癌细胞MCF-7及MDA-MB-231（MDA-231）中的表达水平明显低于正常乳腺上皮细胞MCF-10A; 在MDA-231中过表达miR-150-5p后,qRT-PCR结果显示,Rab1A mRNA的表达水平明显降低。Western印迹结果显示,过表达miR-150-5p后,miR-150-5p组细胞中的Rab1A、波形蛋白(vimentin)及N-钙黏着蛋白(N-cadherin)的表达水平相对于对照组（NC）细胞明显降低,而E-钙黏着蛋白(E-cadherin)的表达水平明显增加。Transwell侵袭和划痕实验显示,与miR-150-5p+Con组细胞相比,miR-150-5p+Rab1A组细胞的侵袭和迁移能力明显增加。qRT-PCR结果显示,miR-150-5p+Rab1A组细胞的Rab1A mRNA表达水平明显增加。Western印迹结果显示,miR-150-5p+Rab1A组细胞中的波形蛋白、N-钙黏着蛋白表达水平明显增加, 而E-钙黏着蛋白表达明显降低,过表达Rab1A后显著逆转了miR-150-5p对EMT的影响。综上所述,miR-150-5p可以通过负向调控Rab1A抑制EMT,进而抑制乳腺癌细胞的侵袭和迁移。     

TPST1表达在CXCR4诱导的乳腺癌细胞侵袭中的作用研究

C-X-C趋化因子受体4(CXCR4)是乳腺癌细胞运动的关键调节因子。CXCR4的功能性表达与乳腺癌的恶性进展密切相关。酪氨酸硫酸化转移酶1(tyrosylprotein sulfotransferase 1,TPST1)是CXCR4蛋白翻译后酪氨酸硫酸化修饰的一个关键酶。本研究将探索TPST1在CXCR4调节乳腺癌细胞侵袭过程中的作用机制。利用定量PCR,免疫组织化学和蛋白质免疫印迹等试验技术检测乳腺癌组织和细胞系中CXCR4和TPST1的mRNA和蛋白表达水平。RNA干扰,趋化试验和侵袭试验用于检测TPST1对于CXCR4诱导的乳腺癌细胞侵袭的影响。研究发现CXCR4蛋白在乳腺癌转移淋巴结组织中呈高表达(P=0. 0016)。CXCR4在乳腺癌转移淋巴结组织中的高表达与肿瘤浸润深度密切相关(P=0. 026)。TPST1与CXCR4蛋白表达在乳腺癌原发组织和配对转移淋巴结组织中均呈显著正相关(P=0. 009; P=0. 006)。TPST1在高度恶性乳腺癌MDA-MB-231细胞中呈高表达,在低度恶性乳腺癌MCF-7细胞中弱表达,而两者CXCR4表达基本相同。小RNA干扰降低TPST1的表达后,下调了乳腺癌MDA-MB-231细胞对于CXCR4配体即基质细胞衍生因子1α(stromal cell-derived factor 1 alpha,SDF-1α)的运动反应性,进而降低CXCR4诱导的MDA-MB-231细胞迁移和侵袭能力。综上,在CXCR4诱导的乳腺癌细胞侵袭过程中,TPST1表达对于CXCR4功能性活化至关重要,TPST1可能作为潜在的抗CXCR4药物治疗乳腺癌恶性进展的联合靶点。     

miR-145通过靶向吞噬和细胞活力蛋白1抑制乳腺癌细胞侵袭

吞噬和细胞活力蛋白1（engulfment and cell motility protein 1,ELMO1）可以促进多种癌细胞的侵袭和转移,但ELMO1的表达是否受miRNA的调控鲜有研究。本研究旨在探讨miR-145与ELMO1表达的相关性,以及miR-145通过结合ELMO1的mRNA对乳腺癌侵袭的影响。通过TargetScan (http://www.targetscan.org/)靶基因预测软件预测与ELMO1的3′UTR结合的miR-145。荧光素酶结果证实两者互补结合。Transwell侵袭结果显示,miR-145组和siELMO1+miR-145组MDA-231乳腺癌细胞穿膜数较对照组分别降低40%（P＜0.05）和79%（P＜0.05）。siELMO1+miR-145组和siELMO1组细胞穿膜数则无显著差异（P>0.05）。结果提示,miR-145通过与ELMO1的mRNA结合抑制细胞侵袭。qRT-PCR显示,低侵袭的MCF-7乳腺癌细胞miR-145的表达量较高侵袭的MDA-435细胞高80%（P＜0.05）,较MDA-231乳腺癌细胞高75%（P＜0.05）,即miR-145与癌细胞侵袭能力呈负相关。Western印迹结果表明,miR-145组ELMO1表达量低于阴性对照组,miR-145 抑制组ELMO1表达量高于抑制剂NC组（P＜0.05）,证明miR-145抑制ELMO1的表达。qRT-PCR显示,过表达miR-145后ELMO1 mRNA含量与对照组无显著差异（P>0.05）。结果提示,miR-145对ELMO1的调控作用通过抑制其翻译实现。F-肌动蛋白聚合实验表明,miR-145组和阴性对照组于20 s和60 s时F-肌动蛋白聚合结果存在明显区别（P＜0.05）。Western 印迹结果表明,miR-145组活化的Rac1表达量较阴性对照组降低60%（P<0.05）,抑制剂NC组活化的Rac1较miR-145 抑制组降低55%（P<0.05）;miR-145组磷酸化的整合素β1较对照组于15 min时降低42%（P<0.05）,于30 min时降低31%（P<0.05）。由此得出的miR-145过表达显著促进乳腺癌细胞F-肌动蛋白聚合、Rac1活化和整合素β1磷酸化结论。综上所述,miR-145通过靶向ELMO1的 mRNA抑制ELMO1翻译,从而抑制乳腺癌的侵袭。     

miR-145通过靶向吞噬和细胞活力蛋白1抑制乳腺癌细胞侵袭

吞噬和细胞活力蛋白1（engulfment and cell motility protein 1,ELMO1）可以促进多种癌细胞的侵袭和转移,但ELMO1的表达是否受miRNA的调控鲜有研究。本研究旨在探讨miR-145与ELMO1表达的相关性,以及miR-145通过结合ELMO1的mRNA对乳腺癌侵袭的影响。通过TargetScan (http://www.targetscan.org/)靶基因预测软件预测与ELMO1的3′UTR结合的miR-145。荧光素酶结果证实两者互补结合。Transwell侵袭结果显示,miR-145组和siELMO1+miR-145组MDA-231乳腺癌细胞穿膜数较对照组分别降低40%（P＜0.05）和79%（P＜0.05）。siELMO1+miR-145组和siELMO1组细胞穿膜数则无显著差异（P>0.05）。结果提示,miR-145通过与ELMO1的mRNA结合抑制细胞侵袭。qRT-PCR显示,低侵袭的MCF-7乳腺癌细胞miR-145的表达量较高侵袭的MDA-435细胞高80%（P＜0.05）,较MDA-231乳腺癌细胞高75%（P＜0.05）,即miR-145与癌细胞侵袭能力呈负相关。Western印迹结果表明,miR-145组ELMO1表达量低于阴性对照组,miR-145 抑制组ELMO1表达量高于抑制剂NC组（P＜0.05）,证明miR-145抑制ELMO1的表达。qRT-PCR显示,过表达miR-145后ELMO1 mRNA含量与对照组无显著差异（P>0.05）。结果提示,miR-145对ELMO1的调控作用通过抑制其翻译实现。F-肌动蛋白聚合实验表明,miR-145组和阴性对照组于20 s和60 s时F-肌动蛋白聚合结果存在明显区别（P＜0.05）。Western 印迹结果表明,miR-145组活化的Rac1表达量较阴性对照组降低60%（P<0.05）,抑制剂NC组活化的Rac1较miR-145 抑制组降低55%（P<0.05）;miR-145组磷酸化的整合素β1较对照组于15 min时降低42%（P<0.05）,于30 min时降低31%（P<0.05）。由此得出的miR-145过表达显著促进乳腺癌细胞F-肌动蛋白聚合、Rac1活化和整合素β1磷酸化结论。综上所述,miR-145通过靶向ELMO1的 mRNA抑制ELMO1翻译,从而抑制乳腺癌的侵袭。     

Reduction of Akt2 expression inhibits chemotaxis signal transduction in human breast cancer cells

Protein kinase Cζ PKCζ mediates cancer cell chemotaxis by regulating cytoskeleton rearrangement and cell adhesion. In the research for its upstream regulator, we investigated the role of Akt2 in chemotaxis and metastasis of human breast cancer cells. Reduction of Akt2 expression by siRNA inhibited chemotaxis of MDA-MB-231, T47D, and MCF7 cells, three representative human breast cancer cells. Expression of a wild type Akt2 in siRNA transfected cells rescued the phenotype. EGF-induced integrin β1 phosphorylation was dampened, consistent with defects in adhesion. Phosphorylation of LIMK and cofilin, a critical step of cofilin recycle and actin polymerization, was also impaired. Thus, Akt2 regulates both cell adhesion and cytoskeleton rearrangement during chemotaxis. Depletion of Akt2 by siRNA impaired the activation of PKCζ while inhibition of PKCζ did not interfere with EGF induced phosphorylation of Akt. Furthermore, EGF induced co-immunoprecipitation between PKCζ and Akt2, but not Akt1, suggesting that a direct interaction between PKCζ and Akt2 in chemotaxis. Protein levels of integrin β1, LIMK, cofilin, and PKCζ didn't alter, suggesting that Akt2 does not regulate the expression of these signaling molecules. In a Severe Combine Immunodeficiency mouse model, Akt2 depleted MDA-MB-231 cells showed a marked reduction in metastasis to mouse lungs, demonstrating the biological relevancy of Akt2 in cancer metastasis in vivo. Taken together, our results suggest that Akt2 directly mediates EGF-induced chemotactic signaling pathways through PKCζ and its expression is critical during the extravasation of circulating cancer cells.     

miR-125a-5p通过靶向调控GAB2抑制乳腺癌细胞的迁移能力

miR-125a-5p可负性调节GAB2表达,抑制胶质瘤细胞的侵袭和转移。本研究旨在证明miR-125a-5p抑癌作用的普遍性,即miR-125a-5p是否可通过靶向抑制GAB2抑制乳腺癌细胞的迁移。荧光素酶实验结果显示,miR-125a-5p可特异识别GAB2的3′-UTR,抑制报告酶的表达。荧光定量PCR结果揭示,与正常乳腺上皮细胞MCF-10A比较,miR-125a-5p在乳腺癌细胞MDA231和MCF-7中的表达明显降低;与迁移能力相对较低的MCF-7细胞比较,miR-125a-5p在迁移能力较高的MDA231细胞中的表达量更低。Western 印迹结果证明,与空载体（对照）和anti-miR125a 5p转染细胞比较,转染miR-125a-5p明显抑制GAB2蛋白在乳腺癌细胞中的表达。Transwell结果显示,与空载体转染的对照细胞比较,转染miR-125a-5p的乳腺癌细胞穿过基质胶的细胞数明显减少;相反,转染anti-miR125a-5p的细胞穿过基质胶的细胞数却明显增多。上述结果提示,miR-125a-5p在正常的乳腺细胞中高表达,而在乳腺癌细胞中低表达,其表达水平与癌细胞的迁移能力和GAB2表达呈反向关系。本研究结果还提示,miR-125a-5p通过靶向负调控GAB2抑制乳腺癌细胞的迁移能力。总之,本研究证明,miR-125a-5p在肿瘤中发挥抑癌作用。     

Downregulation of cPLA2γ expression inhibits EGF-induced chemotaxis of human breast cancer cells through Akt pathway

Phospholipids play an important role in mediating cell migration. In the present study, we investigated the role of cPLA₂γ in chemotaxis of human breast cancer cells. Inhibition of cPLA₂γ expression by small interference RNA severely inhibits EGF-induced chemotaxis in a dose-dependent manner in MDA-MB-231, MCF-7, T47D and ZR-75-30 cells. Furthermore, silencing cPLA₂γ expression also impaired directional migration, adhesion and invasion in MDA-MB-231 cells. In addition, we investigated the molecular mechanism by which cPLA₂γ regulated migration. Knockdown of cPLA₂γ suppressed the phosphorylation of Akt at both Thr308 and Ser473. Phosphorylation of PKCζ, downstream of Akt, was also dampened. Knockdown of cPLA₂γ also impaired the phosphorylation of integrin β1 and cofilin, key regulators of cell adhesion and actin polymerization, respectively. Taken together, our results suggest that cPLA₂γ plays an important role in cancer cell chemotaxis.     

Involvement of integrins in fimbriae-mediated binding and invasion by Porphyromonas gingivalis

Interaction between the major fimbriae of Porphyromonas gingivalis and gingival epithelial cells is important for bacterial adhesion and invasion. In this study, we identified integrins as an epithelial cell cognate receptor for P. gingivalis fimbriae. Immunoprecipitation and direct binding assays revealed a physical association between recombinant fimbrillin and beta1 integrins. In vitro adhesion and invasion assays demonstrated inhibition of binding and invasion of P. gingivalis by beta1 integrin antibodies. In contrast, invasion of a fimbriae-deficient mutant of P. gingivalis was not affected by integrin antibodies. Infection of gingival epithelial cells with wild-type P. gingivalis induced tyrosine phosphorylation of the 68 kDa focal adhesion protein paxillin, whereas the fimbriae-deficient mutant failed to evoke similar changes. Interestingly, activation of paxillin was not accompanied by an increase in the phosphorylation of focal adhesion kinase (FAK). These results provide evidence that P. gingivalis fimbriae promote adhesion to gingival epithelial cells through interaction with beta1 integrins, and this association represents a key step in the induction of the invasive process and subsequent cell responses to P. gingivalis infection.     

Tumor cell invasion is promoted by activation of protease activated receptor-1 in cooperation with the alpha vbeta 5 integrin

The first prototype of the protease activated receptor (PAR) family, the thrombin receptor PAR1, plays a central role both in the malignant invasion process of breast carcinoma metastasis and in the physiological process of placental implantation. The molecular mechanism underlying PAR1 involvement in tumor invasion and metastasis, however, is poorly defined. Here we show that PAR1 increases the invasive properties of tumor cells primarily by increased adhesion to extracellular matrix components. This preferential adhesion is accompanied by the cytoskeletal reorganization of F-actin toward migration-favoring morphology as detected by phalloidin staining. Activation of PAR1 increased the phosphorylation of focal adhesion kinase and paxillin, and the induced formation of focal contact complexes. PAR1 activation affected integrin cell-surface distribution without altering their level of expression. The specific recruitment of alpha(v)beta(5) to focal contact sites, but not of alpha(v)beta(3) or alpha(5)beta(1), was observed by immunofluorescent microscopy. PAR1 overexpressing cells showed selective reciprocal co-precipitation with alpha(v)beta(5) and paxillin but not with alpha(v)beta(3) that remained evenly distributed under these conditions. This co-immunoprecipitation failed to occur in cells containing the truncated form of PAR1 that lacked the entire cytoplasmic portion of the receptor. Thus, the PAR1 cytoplasmic tail is essential for conveying the cross-talk and recruiting the alpha(v)beta(5) integrin. While PAR1 overexpressing cells were invasive in vitro, as reflected by their migration through a Matrigel barrier, invasion was further enhanced by ligand activation of PAR1. Moreover, the application of anti-alpha(v)beta(5) antibodies specifically attenuated this PAR1 induced invasion. We propose that the activation of PAR1 may lead to a novel cooperation with the alpha(v)beta(5) integrin that supports tumor cell invasion.     

Pro-adhesive and chemotactic activities of thrombospondin-1 for breast carcinoma cells are mediated by alpha3beta1 integrin and regulated by insulin-like growth factor-1 and CD98

Thrombospondin-1 (TSP1) is a matricellular protein that displays both pro- and anti-adhesive activities. Binding to sulfated glycoconjugates mediates most high affinity binding of soluble TSP1 to MDA-MB-435 cells, but attachment and spreading of these cells on immobilized TSP1 is primarily beta1 integrin-dependent. The integrin alpha3beta1 is the major mediator of breast carcinoma cell adhesion and chemotaxis to TSP1. This integrin is partially active in MDA-MB-435 cells but is mostly inactive in MDA-MB-231 and MCF-7 cells, which require beta1 integrin activation to induce spreading on TSP1. Integrin-mediated cell spreading on TSP1 is accompanied by extension of filopodia containing beta1 integrins. TSP1 binding activity of the alpha3beta1 integrin is not stimulated by CD47-binding peptides from TSP1 or by protein kinase C activation, which activate alphavbeta3 integrin function in the same cells. In MDA-MB-231 but not MDA-MB-435 cells, this integrin is activated by pertussis toxin, whereas serum, insulin, insulin-like growth factor-1, and ligation of CD98 increase activity of this integrin in both cell lines. Serum stimulation is accompanied by increased surface expression of CD98, whereas insulin-like growth factor-1 does not increase CD98 expression. Thus, the pro-adhesive activity of TSP1 for breast carcinoma cells is controlled by several signals that regulate activity of the alpha3beta1 integrin.     

Anandamide inhibits adhesion and migration of breast cancer cells

The endocannabinoid system regulates cell proliferation in human breast cancer cells. We reasoned that stimulation of cannabinoid CB1 receptors could induce a non-invasive phenotype in breast metastatic cells. In a model of metastatic spreading in vivo, the metabolically stable anandamide analogue, 2-methyl-2'-F-anandamide (Met-F-AEA), significantly reduced the number and dimension of metastatic nodes, this effect being antagonized by the selective CB1 antagonist SR141716A. In MDA-MB-231 cells, a highly invasive human breast cancer cell line, and in TSA-E1 cells, a murine breast cancer cell line, Met-F-AEA inhibited adhesion and migration on type IV collagen in vitro without modifying integrin expression: both these effects were antagonized by SR141716A. In order to understand the molecular mechanism involved in these processes, we analyzed the phosphorylation of FAK and Src, two tyrosine kinases involved in migration and adhesion. In Met-F-AEA-treated cells, we observed a decreased tyrosine phosphorylation of both FAK and Src, this effect being attenuated by SR141716A. We propose that CB1 receptor agonists inhibit tumor cell invasion and metastasis by modulating FAK phosphorylation, and that CB1 receptor activation might represent a novel therapeutic strategy to slow down the growth of breast carcinoma and to inhibit its metastatic diffusion in vivo.     

A novel role of ERK5 in integrin‐mediated cell adhesion and motility in cancer cells via Fak signaling

In metastatic cancer, high expression levels of vitronectin (VN) receptors (integrins), FAK, and ERK5 are reported. We hypothesized that integrin‐mediated ERK5 activation via FAK may play a pivotal role in cell adhesion, motility, and metastasis. ERK5 and FAK phosphorylation when metastatic MDA‐MB‐231 and PC‐3 cells were plated on VN was enhanced. Further experiments showed co‐immunoprecipitation of integrins β1, αVβ3, or αVβ5 with ERK5 and FAK. To gain better insight into the mechanism of ERK5, FAK, and VN receptors in cell adhesion and motility, we performed loss‐of‐function experiments using integrin blocking antibodies, and specific mutants of FAK and ERK5. Ectopic expression of dominant negative ERK5/AEF decreased ERK5 and FAK (Y397) phosphorylation, cell adhesion, and haptotactic motility (micromotion) on VN. Additionally, DN FAK expression attenuated ERK5 phosphorylation, cell adhesion, and motility. This study documents the novel finding that in breast and prostate cancer cells, ERK5 is a critical target of FAK in cell adhesion signaling. Using different cancer cells, our experiments unveil a novel mechanism by which VN receptors and FAK could promote cancer metastasis via ERK5 activation. J. Cell. Physiol. 219: 152–161, 2009. © 2008 Wiley‐Liss, Inc.