表没食子儿茶素没食子酸酯抑制乳腺癌细胞增殖和迁移

表没食子儿茶素没食子酸酯（epigallocatechin-3-gallate, EGCG）是茶叶活性物质主要成分.EGCG可预防或治疗多种肿瘤.本文旨在探讨EGCG对人乳腺癌MDA-MB-231细胞增殖、凋亡及迁移力的作用及其机制. 经EGCG处理后,通过流式细胞术及噻唑蓝（MTT）法发现,EGCG使MDA-MB-231细胞周期阻滞,细胞凋亡数量上升,明显抑制乳腺癌细胞的存活率. EGCG处理后,MDA-MB-231细胞的形态由正常的纤维状变为鹅卵石状. 免疫荧光染色及免疫印迹结果表明,其上皮细胞标志物表达量增加,而间质标志物表达量下降. 通过划痕实验发现,EGCG明显抑制了细胞迁移能力. 本文揭示了EGCG通过抑制乳腺癌MDA-MB-231细胞周期进程,促进间质-上皮转化,抑制乳腺癌细胞增殖和迁移.     

分泌蛋白YKL-40增强肿瘤细胞的上皮间质样转化

目的:分泌糖蛋白YKL-40在多种晚期肿瘤病人的血液中显著升高,提示YKL-40蛋白的血浓度是肿瘤恶变的生物标志物。本课题研究YKL-40重组蛋白和过表达YKL-40肿瘤细胞对肿瘤细胞的上皮间质样转化的作用。方法:构建YKL-40过表达的纤维状乳腺癌细胞系MDA-MB-231和非纤维状结肠癌细胞系HCT-116,观察细胞形态学变化,收集细胞和细胞培养液用于Western Blot(WB)检测YKL-40和上皮间质转化标记蛋白Vimentin和N-cadherin。观察重组蛋白YKL-40对原代MDA-MB-231细胞在无血清条件下的细胞存活影响;另外,用细胞存活试剂盒检测YKL-40过表达HCT-116细胞在无血清的培养液中细胞存活情况。最后,用细胞侵袭试验检测YKL-40过表达MDA-MB-231细胞的侵袭力,并用WB和Zymography来测定细胞分泌MMP9蛋白的表达和酶活性。结果:YKL-40过表达增强MDA-MB-231细胞的形态向上皮间质样转化,并显著提高Vimentin、N-cadherin蛋白的表达,但对HCT-116细胞无法诱导上皮间质样转化。在无血清培养基培养条件下,YKL-40可以增强两种细胞的存活能力,并且YKL-40过表达的MDA-MB-231细胞增强了细胞的侵袭能力,促进了MMP9蛋白表达和蛋白活性。结论:YKL-40可以增加肿瘤细胞的存活力,增强纤维状细胞向上皮间质样转化;并且,YKL-40增加MMP9蛋白表达和活性,增强细胞侵袭力。YKL-40是间质样肿瘤细胞EMT的增强子,此发现为抑制肿瘤恶变提供新靶点。     

Lung Epithelial Cells Induce Both Phenotype Alteration and Senescence in Breast Cancer Cells

Purpose

The lung is one of the most common sites of breast cancer metastasis. While metastatic seeding is often accompanied by a dormancy-promoting mesenchymal to epithelial reverting transitions (MErT), we aimed to determine whether lung epithelial cells can impart this phenotype on aggressive breast cancer cells.

Methods

Co-culture experiments of normal lung epithelial cell lines (SAEC, NHBE or BEAS-2B) and breast cancer cell lines (MCF-7 or MDA-MB-231) were conducted. Flow cytometry analysis, immunofluorescence staining for E-cadherin or Ki-67 and senescence associated beta-galactosidase assays assessed breast cancer cell outgrowth and phenotype.

Results

Co-culture of the breast cancer cells with the normal lung cells had different effects on the epithelial and mesenchymal carcinoma cells. The epithelial MCF-7 cells were increased in number but still clustered even if in a slightly more mesenchymal-spindle morphology. On the other hand, the mesenchymal MDA-MB-231 cells survived but did not progressively grow out in co-culture. These aggressive carcinoma cells underwent an epithelial shift as indicated by cuboidal morphology and increased E-cadherin. Disruption of E-cadherin expressed in MDA-MB-231 using shRNA prevented this phenotypic reversion in co-culture. Lung cells limited cancer cell growth kinetics as noted by both (1) some of the cells becoming larger and positive for senescence markers/negative for proliferation marker Ki-67, and (2) Ki-67 positive cells significantly decreasing in MDA-MB-231 and MCF-7 cells after co-culture.

Conclusions

Our data indicate that normal lung epithelial cells can drive an epithelial phenotype and suppress the growth kinetics of breast cancer cells coincident with changing their phenotypes.     

MiR-5047在乳腺癌细胞增殖迁移中的作用及表达调控*

探讨miR-5047在乳腺癌细胞中的表达及其在乳腺癌细胞增殖和迁移中的作用,并明确地西他滨在miR-5047表达调控中的作用。通过实时荧光定量PCR(qRT-PCR)检测人乳腺癌细胞系和正常乳腺上皮细胞MCF10A中miR-5047的表达水平;将miR-5047模拟物(mimic),阴性对照(NC)分别转染至MDA-MB-231和MCF7细胞,经平板克隆实验、MTT实验、划痕愈合实验检测乳腺癌细胞的增殖和迁移能力,通过qRT-PCR和Western blot检测相关基因表达及蛋白水平。使用浓度5 μmol/L和10 μmol/L的地西他滨分别处理MDA-MB-231和MCF-7细胞,经qRT-PCR检测不同浓度和处理时间条件下地西他滨对miR-5047表达的影响。同时,通过形态观察和Western blot检测地西他滨对乳腺癌细胞上皮间质转化的影响。与正常乳腺上皮细胞MCF-10A相比,miR-5047在乳腺癌细胞中表达均显著下调。miR-5047过表达可显著抑制乳腺癌细胞的增殖和迁移,促进上皮细胞标志物E-cadherin的表达,抑制间质细胞标志物Vimentin的表达。不同浓度地西他滨处理MDA-MB-231和MCF7细胞后,miR-5047表达均增强,且10 μmol/L作用48 h效果最显著。地西他滨可诱导MDA-MB-231细胞向上皮样转变。miR-5047在乳腺癌细胞系中表达显著下调,过表达miR-5047可抑制乳腺癌细胞的增殖和迁移,地西他滨可促进乳腺癌细胞中miR-5047的表达,并诱导细胞向上皮样转变。     

Indole-3-carbinol-induced death in cancer cells involves EGFR downregulation and is exacerbated in a 3D environment

Indole-3-carbinol (I3C) is a promising anticancer dietary compound, which inhibits breast cancer in animal models. The objective of the current study was to characterize I3C-induced cell death in a panel of human breast tumorigenic cells (MCF7, MDA-MB-468, MDA-MB-231 and HBL100) in comparison with normal fibroblasts. Since epithelial cells are protected from cell death by a three-dimensional environment, 3D cell culture (collagen I gel and spheroids) was employed to investigate susceptibility to I3C. Cell viability in the presence of 256 μM I3C, a concentration close to the physiologically achievable range, was in the order fibroblasts = HBL100>MDA-MB-231>MCF7>MDA-MB-468 in monolayer culture. However, 3D culture conditions increased the susceptibility of MCF7 and MDA-MB-468 cancer cells towards I3C. I3C induced cell death in breast cancer MCF7, MDA-MB-468 and MDA-MB–231 cells via the mitochondrial apoptotic pathway. I3C significantly reduced levels of epidermal growth factor receptor (EGFR) in MDA-MB-468 after 6 h and in MDA-MB-231 and HBL100 cells after 30 h. Downregulation of EGFR in MDA-MB468 and MDA-MB-231 cells using an EGFR inhibitor resulted in apoptosis. EGFR modulation using EGF or an EGFR inhibitor markedly influenced viability and response to I3C in MDA-MB-468 cells in 3D conditions. EGFR expression was modulated by 3D conditions. Therefore, I3C-induced EGFR reduction in these cells is likely to be responsible for I3C-induced apoptosis.     

The monoamine oxidase-A inhibitor clorgyline promotes a mesenchymal-to-epithelial transition in the MDA-MB-231 breast cancer cell line

Monoamine oxidase-A (MAO-A) dysfunction has been historically associated with depression. Recently, depression as well as altered MAO-A expression have both been associated with a poor prognosis in cancers, although the mechanism involved remains ambiguous. For example, MAO-A mRNA is repressed across cancers, yet MAO-A protein and levels of serotonin, a substrate of MAO-A implicated in depression, are paradoxically increased in malignancies, including breast cancer.The effect of clorgyline (CLG), a selective inhibitor of MAO-A, on malignant behaviour, expression of transitional markers, and biochemical correlates was examined in two human breast carcinoma cell lines, i.e. the epithelial, oestrogen receptor (ER)-positive MCF-7 cell line and the post-EMT (mesenchymal), ER-negative MDA-MB-231 cell line.CLG exerted little effect on malignant behaviour in MCF-7 cells, but inhibited proliferation and anchorage-independent growth, and increased invasiveness and active migration of MDA-MB-231 cells. CLG induced the expression of the mesenchymal marker vimentin in MCF-7 cells, but not in MDA-MB-231 cells. In contrast, CLG induced the epithelial protein marker E-cadherin in both cell lines, with a more robust effect in MDA-MB-231 cells (where a nuclear E-cadherin signal was also detected). This effect appears to be independent of any canonical Snai1-mediated regulation of E-cadherin mRNA expression. CLG interfered with the β-catenin/[phospho]GSK-3β complex as well as the E-cadherin/β-catenin complex in both cell lines cells, but, again, the effect was more robust in MDA-MB-231 cells. Parallel studies revealed a general lack of effect of CLG on the ER-negative, epithelial Au565 breast cancer cell line. Thus, any effect of CLG on metastatic behaviours appears to rely on the cell's EMT status rather than on the cell's ER status.These data suggest that inactivation of MAO-A triggers a mesenchymal-to-epithelial transition in MDA-MB-231 cells via a non-canonical mechanism. This potentially implicates an MAO-A-sensitive step in advanced breast cancer and should be borne in mind when considering pharmacological treatment options for co-morbid depression in breast cancer patients.     

Withaferin A-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species

Withaferin A (WA), a promising anticancer constituent of Ayurvedic medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 and MCF-7 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction, but the mechanism of cell death is not fully understood. We now demonstrate, for the first time, that WA-induced apoptosis is mediated by reactive oxygen species (ROS) production due to inhibition of mitochondrial respiration. WA treatment caused ROS production in MDA-MB-231 and MCF-7 cells, but not in a normal human mammary epithelial cell line (HMEC). The HMEC was also resistant to WA-induced apoptosis. WA-mediated ROS production as well as apoptotic histone-associated DNA fragment release into the cytosol was significantly attenuated by ectopic expression of Cu,Zn-superoxide dismutase in both MDA-MB-231 and MCF-7 cells. ROS production resulting from WA exposure was accompanied by inhibition of oxidative phosphorylation and inhibition of complex III activity. Mitochondrial DNA-deficient Rho-0 variants of MDA-MB-231 and MCF-7 cells were resistant to WA-induced ROS production, collapse of mitochondrial membrane potential, and apoptosis compared with respective wild-type cells. WA treatment resulted in activation of Bax and Bak in MDA-MB-231 and MCF-7 cells, and SV40 immortalized embryonic fibroblasts derived from Bax and Bak double knockout mouse were significantly more resistant to WA-induced apoptosis compared with fibroblasts derived from wild-type mouse. In conclusion, the present study provides novel insight into the molecular circuitry of WA-induced apoptosis involving ROS production and activation of Bax/Bak.     

癌睾丸抗原TFDP3与乳腺癌上皮间质化(EMT)的相关性研究

目的:本研究探讨了癌睾丸抗原TFDP3与乳腺癌细胞上皮间质化(epithelial-mesenchymal transition,EMT)的关系。方法:本研究中选取了乳腺癌细胞系(MCF-10A,MCF-7,SK-BR-3和MDA-MB-231)作为研究对象,通过Western Blot的方法筛选获得了TFDP3低水平表达的乳腺癌细胞株。进一步通过质粒转染的方式构建TFDP3过表达的细胞系模型,观察TFDP3在EMT中的作用。结果:TFDP3在MCF-10A及SK-BR-3中不表达,在间质化程度较高的MDA-MB-231中高水平表达,而在上皮化程度较高的MCF-7中的低水平表达。MCF-7中过表达TFDP3后,上皮细胞标记分子E-cadherin表达下调,而间质细胞标记分子N-cadherin、Snail、Twist及细胞骨架蛋白Vimentin表达上调。结论:TFDP3可以促进乳腺癌细胞发生EMT。     

Epithelial-Stromal Interactions in Human Breast Cancer: Effects on Adhesion,Plasma Membrane Fluidity and Migration Speed and Directness

Interactions occurring between malignant cells and the stromal microenvironment heavily influence tumor progression. We investigated whether this cross-talk affects some molecular and functional aspects specifically correlated with the invasive phenotype of breast tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration) by co-culturing mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB-231>MCF-7) with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer-associated fibroblasts, CAFs) in 2D or 3D (nodules) cultures. Confocal immunofluorescence analysis of the epithelial adhesion molecule E-cadherin on frozen nodule sections demonstrated that NFs and CAFs, respectively, induced or inhibited its expression in MCF-7 cells. An increase in the mesenchymal adhesion protein N-cadherin was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of cancer cells. CAFs, in turn, promoted N-cadherin up-regulation in MDA-MB-231 cells and its de novo expression in MCF-7 cells. Beyond promotion of “cadherin switching”, another sign of the CAF-triggered epithelial-mesenchymal transition (EMT) was the induction of vimentin expression in MCF-7 cells. Plasma membrane labeling of monolayer cultures with the fluorescent probe Laurdan showed an enhancement of the membrane fluidity in cancer cells co-cultured with NFs or CAFs. An increase in lipid packing density of fibroblast membranes was promoted by MCF-7 cells. Time-lapsed cell tracking analysis of mammary cancer cells co-cultured with NFs or CAFs revealed an enhancement of tumor cell migration velocity, even with a marked increase in the directness induced by CAFs.Our results demonstrate a reciprocal influence of mammary cancer and fibroblasts on various adhesiveness/invasiveness features. Notably, CAFs'' ability to promote EMT, reduction of cell adhesion, increase in membrane fluidity, and migration velocity and directness in mammary cancer cells can be viewed as an overall progression- and invasion-promoting effect.     

Small GTPase Rho regulates R-cadherin through Dia1/profilin-1

R-cadherin is a member of the classical cadherins. Though much is known about E-cadherin in adherens junction formation in epithelial cells, the role of R-cadherin in epithelial cells remains elusive. This study examines regulation of R-cadherin adherens junctions by the small GTPase Rho and its downstream effectors in MDA-MB-231 breast cancer cells, MDA-MB-231 cells stably expressing the N-terminus of c-Cbl, and MCF10A normal breast epithelial cells. We find that the small GTPase Rho regulates R-cadherin adherens junction formation via Dia1 (also known as p140mDia) and profilin-1-mediated signaling pathway. The role played by Rho in regulating R-cadherin is underscored by the fact that constitutively active RhoA(Q63L) induces R-cadherin junction formation in MDA-MB-231 cells. Importantly, R-cadherin adherens junction formation facilitates a mesenchymal to epithelial-like transition in MDA-MB-231 cells. Additionally, our data suggest an inverse relationship between EGFR signaling and R-cadherin adherens junction formation. Taken together, results from this study indicate that R-cadherin is a critical regulator of epithelial phenotype.     

Epidermal Growth Factor Induces Internalization But Not Degradation of the Epidermal Growth Factor Receptor in a Human Breast Cancer Cell Line

Abstract

Metabolism of the epidermal growth factor (EGF) receptor was studied in the MDA-MB-231 human breast cancer cell line. As in normal fibroblasts the EGF receptor from MDA-MB-231 cells was synthesized from a M_r =160,000 precursor and tunicamycin treatment of cells resulted in accumulation of a M_r =130,000 polypeptide. Unlike normal fibroblasts in which a M_r =170,000 mature form of the EGF receptor was found, MDA-MB-231 cells contained a M_r =172,000 mature form. Addition of EGF to MDA-MB-231 cells led to rapid internalization of EGF receptors, however, internalization did not affect receptor half-life and receptors did not recycle to the cell surface. EGF receptors could be visualized by immunofluorescence and remained sequestered in intracellular membranous structures following internalization. EGF was degraded slowly by MDA-MB-231 cells relative to degradation of EGF by normal cells. A high endogenous level of in vivo phosphorylation of threonine 654 of the EGF receptor was found in MDA-MB-231 cells and treatment of cells with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) further stimulated phosphorylation of this residue. EGF induced receptor internalization resulted in dephosphorylation of threonine 654. The significance of these unusual properties of EGF receptor metabolism in MDA-MB-231 cells is discussed.     

Specific expression of the human voltage-gated proton channel Hv1 in highly metastatic breast cancer cells, promotes tumor progression and metastasis

The newly discovered human voltage-gated proton channel Hv1 is essential for proton transfer, which contains a voltage sensor domain (VSD) without a pore domain. We report here for the first time that Hv1 is specifically expressed in the highly metastatic human breast tumor tissues, but not in poorly metastatic breast cancer tissues, detected by immunohistochemistry. Meanwhile, real-time RT-PCR and immunocytochemistry showed that the expression levels of Hv1 have significant differences among breast cancer cell lines, MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-453, T-47D and SK-BR-3, in which Hv1 is expressed at a high level in highly metastatic human breast cancer cell line MDA-MB-231, but at a very low level in poorly metastatic human breast cancer cell line MCF-7. Inhibition of Hv1 expression in the highly metastatic MDA-MB-231 cells by small interfering RNA (siRNA) significantly decreases the invasion and migration of the cells. The intracellular pH of MDA-MB-231 cells down-regulated Hv1 expression by siRNA is obviously decreased compared with MDA-MB-231 with the scrambled siRNA. The expression of matrix metalloproteinase-2 and gelatinase activity in MDA-MB-231 cells suppressed Hv1 by siRNA were reduced. Our results strongly suggest that Hv1 regulates breast cancer intracellular pH and exacerbates the migratory ability of metastatic cells.     

Role of TRPV4 in matrix stiffness-induced expression of EMT-specific LncRNA

Molecular and Cellular Biochemistry - Human triple negative breast cancer cells, MDA-MB-231, show typical epithelial to mesenchymal transition associated with cancer progression. Mitochondria play...     

Livin promotes progression of breast cancer through induction of epithelial–mesenchymal transition and activation of AKT signaling

The inhibitor of apoptosis proteins (IAP) are closely correlated with proliferation, apoptosis, motility, and metastasis. Livin is the most recently identified IAP, and its role in breast progression remains unknown. In our study, analyses of 50 patients with breast cancer revealed that the positive expression rate of Livin was higher in breast cancer tissues (62%) relative to that in adjacent (35%) and normal tissues (25%). Livin expression in breast cancer correlated with the clinical stage and axillary lymph node metastasis and could be used as a prognostic marker. Our in vitro experiment revealed that Livin was highly expressed in high-invasive MDA-MB-231 cells as compared to low-invasive cells (MCF-7). Suppression of Livin by short-hairpin RNA reduced the Livin expression of MDA-MB-231 cells and subsequently inhibited tumor cell growth, proliferation, and colony formation and induced tumor cell apoptosis, motility, migration, and invasion. Overexpression of Livin in MCF7 cells resulted in increased migration and invasion capabilities of the cells without affecting proliferation and apoptosis. In addition, epithelial–mesenchymal transition (EMT) was induced by Livin expression in breast cancer cell lines. The high level of phosphorylated AKT in MDA-MB-231 cells was suppressed by Livin knockdown. Further, Livin-induced migration and invasion could be abolished by either the application of the phosphoinositide-3-kinase inhibitor LY294002 or knockdown of AKT expression using small-interfering RNA. In conclusion, Livin serves as an independent prognostic indicator for breast cancer. Livin expression promotes breast cancer metastasis through the activation of AKT signaling and induction of EMT in breast cancer cells both in vitro and in vivo.     

Downregulation of stromal BRCA1 drives breast cancer tumor growth via upregulation of HIF-1α, autophagy and ketone body production

Our recent studies have mechanistically demonstrated that cancer-associated fibroblasts (CAFs) produce energy-rich metabolites that functionally support the growth of cancer cells. Also, several authors have demonstrated that DNA instability in the tumor stroma greatly contributes to carcinogenesis. To further test this hypothesis, we stably knocked-down BRCA1 expression in human hTERT-immortalized fibroblasts (shBRCA1) using an shRNA lentiviral approach. As expected, shBRCA1 fibroblasts displayed an elevated growth rate. Using immunofluorescence and immunoblot analysis, shBRCA1 fibroblasts demonstrated an increase in markers of autophagy and mitophagy. Most notably, shBRCA1 fibroblasts also displayed an elevation of HIF-1α expression. In accordance with these findings, shBRCA1 fibroblasts showed a 5.5-fold increase in ketone body production; ketone bodies function as high-energy mitochondrial fuels. This is consistent with the onset of mitochondrial dysfunction in BRCA1-deficient fibroblasts. Conversely, after 48 h of co-culturing shBRCA1 fibroblasts with a human breast cancer cell line (MDA-MB-231 cell), mitochondrial activity was enhanced in these epithelial cancer cells. Interestingly, our preclinical studies using xenografts demonstrated that shBRCA1 fibroblasts induced an ~2.2-fold increase in tumor growth when co-injected with MDA-MB-231 cells into nude mice. We conclude that a BRCA1 deficiency in the tumor stroma metabolically promotes cancer progression, via ketone production.     

Downregulation of stromal BRCA1 drives breast cancer tumor growth via upregulation of HIF-1α, autophagy and ketone body production

Our recent studies have mechanistically demonstrated that cancer-associated fibroblasts (CAFs) produce energy-rich metabolites that functionally support the growth of cancer cells. Also, several authors have demonstrated that DNA instability in the tumor stroma greatly contributes to carcinogenesis. To further test this hypothesis, we stably knocked-down BRCA1 expression in human hTERT-immortalized fibroblasts (shBRCA1) using an shRNA lentiviral approach. As expected, shBRCA1 fibroblasts displayed an elevated growth rate. Using immunofluorescence and immunoblot analysis, shBRCA1 fibroblasts demonstrated an increase in markers of autophagy and mitophagy. Most notably, shBRCA1 fibroblasts also displayed an elevation of HIF-1α expression. In accordance with these findings, shBRCA1 fibroblasts showed a 5.5-fold increase in ketone body production; ketone bodies function as high-energy mitochondrial fuels. This is consistent with the onset of mitochondrial dysfunction in BRCA1-deficient fibroblasts. Conversely, after 48 h of co-culturing shBRCA1 fibroblasts with a human breast cancer cell line (MDA-MB-231 cell), mitochondrial activity was enhanced in these epithelial cancer cells. Interestingly, our preclinical studies using xenografts demonstrated that shBRCA1 fibroblasts induced an ~2.2-fold increase in tumor growth when co-injected with MDA-MB-231 cells into nude mice. We conclude that a BRCA1 deficiency in the tumor stroma metabolically promotes cancer progression, via ketone production.     

Toll-like receptor 9 mediates CpG oligonucleotide-induced cellular invasion

Toll-like receptor 9 (TLR9) belongs to the innate immune system and recognizes microbial and vertebrate DNA. We showed previously that treatment with the TLR9-agonistic ODN M362 (a CpG sequence containing oligonucleotide) induces matrix metalloproteinase-13-mediated invasion in TLR9-expressing human cancer cell lines. Here, we further characterized the role of the TLR9 pathway in this process. We show that CpG oligonucleotides induce invasion in macrophages from wild-type C57/B6 and MyD88 knockout mice and in human MDA-MB-231 breast cancer cells lacking MyD88 expression. This effect was significantly inhibited in macrophages from TLR9 knockout mice and in human MDA-MB-231 breast cancer cells stably expressing TLR9 small interfering RNA or dominant-negative tumor necrosis factor receptor-associated factor 6 (TRAF6). Sequence modifications to the CpG oligonucleotides that targeted the stem loop and other secondary structures were shown to influence the invasion-inducing effect in MDA-MB-231 cells. In contrast, methylation of the cytosine residues of the parent CpG oligonucleotide did not affect the TLR9-mediated invasion compared with the unmethylated parent CpG oligonucleotide. Finally, expression of TLR9 was studied in clinical breast cancer samples and normal breast epithelium with immunohistochemistry. TLR9 staining localized in epithelial cells in both cancer and normal samples. The mean TLR9 staining intensity was significantly increased in the breast cancer cells compared with normal breast epithelial cells. In conclusion, our results suggest that TLR9 expression is increased in breast cancer and CpG oligonucleotide-induced cellular invasion is mediated via TLR9 and TRAF6, independent of MyD88. Further, our findings suggest that the structure and/or stability of DNA may influence the induction of TLR9-mediated invasion in breast cancer.     

Integrin α2β1 Mediates Tyrosine Phosphorylation of Vascular Endothelial Cadherin Induced by Invasive Breast Cancer Cells

The molecular mechanisms that regulate the endothelial response during transendothelial migration (TEM) of invasive cancer cells remain elusive. Tyrosine phosphorylation of vascular endothelial cadherin (VE-cad) has been implicated in the disruption of endothelial cell adherens junctions and in the diapedesis of metastatic cancer cells. We sought to determine the signaling mechanisms underlying the disruption of endothelial adherens junctions after the attachment of invasive breast cancer cells. Attachment of invasive breast cancer cells (MDA-MB-231) to human umbilical vein endothelial cells induced tyrosine phosphorylation of VE-cad, dissociation of β-catenin from VE-cad, and retraction of endothelial cells. Breast cancer cell-induced tyrosine phosphorylation of VE-cad was mediated by activation of the H-Ras/Raf/MEK/ERK signaling cascade and depended on the phosphorylation of endothelial myosin light chain (MLC). The inhibition of H-Ras or MLC in endothelial cells inhibited TEM of MDA-MB-231 cells. VE-cad tyrosine phosphorylation in endothelial cells induced by the attachment of MDA-MB-231 cells was mediated by MDA-MB-231 α₂β₁ integrin. Compared with highly invasive MDA-MB-231 breast cancer cells, weakly invasive MCF-7 breast cancer cells expressed lower levels of α₂β₁ integrin. TEM of MCF-7 as well as induction of VE-cad tyrosine phosphorylation and dissociation of β-catenin from the VE-cad complex by MCF-7 cells were lower than in MDA-MB-231 cells. These processes were restored when MCF-7 cells were treated with β₁-activating antibody. Moreover, the response of endothelial cells to the attachment of prostatic (PC-3) and ovarian (SKOV3) invasive cancer cells resembled the response to MDA-MB-231 cells. Our study showed that the MDA-MB-231 cell-induced disruption of endothelial adherens junction integrity is triggered by MDA-MB-231 cell α₂β₁ integrin and is mediated by H-Ras/MLC-induced tyrosine phosphorylation of VE-cad.