E-cadherin-mediated cell-cell adhesion prevents invasiveness of human carcinoma cells

The ability of carcinomas to invade and to metastasize largely depends on the degree of epithelial differentiation within the tumors, i.e., poorly differentiated being more invasive than well-differentiated carcinomas. Here we confirmed this correlation by examining various human cell lines derived from bladder, breast, lung, and pancreas carcinomas. We found that carcinoma cell lines with an epithelioid phenotype were noninvasive and expressed the epithelium-specific cell-cell adhesion molecule E-cadherin (also known as Arc-1, uvomorulin, and cell-CAM 120/80), as visualized by immunofluorescence microscopy and by Western and Northern blotting, whereas carcinoma cell lines with a fibroblastoid phenotype were invasive and had lost E-cadherin expression. Invasiveness of these latter cells could be prevented by transfection with E-cadherin cDNA and was again induced by treatment of the transfected cells with anti-E-cadherin mAbs. These findings indicate that the selective loss of E-cadherin expression can generate dedifferentiation and invasiveness of human carcinoma cells, and they suggest further that E-cadherin acts as an invasion suppressor.     

N-Cadherin Promotes Adhesion Between Invasive Breast Cancer Cells and the Stroma

Calcium-dependent cell adhesion molecules (cadherins) are involved in maintaining the epithelial structure of a number of tissues including the mammary gland. In breast and other tumor types, loss of E-cadherin expression has been seen in high grade tumors and correlates with increased invasiveness. Here we show high levels of expression of N-cadherin in the most invasive breast cancer cell lines which was inversely correlated with their expression of E-cadherin. A stromal cell line also expressed N-cadherin in accordance with its fibroblastic morphology. N-cadherin localized to areas of cell-cell contact in all cells that expressed it. Calcium-dependent intercellular adhesion of N-cadherin-expressing breast cancer and stromal cells was specifically inhibited by an anti N-cadherin monoclonal antibody. In addition, N-cadherin promoted the interaction of invasive breast cancer cells with mammary stromal cells: in contrast, E-cadherin expressing cell lines did not co-aggregate with stromal cells. The combined results suggest a functional role for N-cadherin in cohesion of breast tumor cells which, in addition promotes their interaction with the surrounding stromal cells, thereby facilitating invasion and metastasis.     

N-Cadherin Promotes Adhesion Between Invasive Breast Cancer Cells and the Stroma

Calcium-dependent cell adhesion molecules (cadherins) are involved in maintaining the epithelial structure of a number of tissues including the mammary gland. In breast and other tumor types, loss of E-cadherin expression has been seen in high grade tumors and correlates with increased invasiveness. Here we show high levels of expression of N-cadherin in the most invasive breast cancer cell lines which was inversely correlated with their expression of E-cadherin. A stromal cell line also expressed N-cadherin in accordance with its fibroblastic morphology. N-cadherin localized to areas of cell-cell contact in all cells that expressed it. Calcium-dependent intercellular adhesion of N-cadherin-expressing breast cancer and stromal cells was specifically inhibited by an anti N-cadherin monoclonal antibody. In addition, N-cadherin promoted the interaction of invasive breast cancer cells with mammary stromal cells: in contrast, E-cadherin expressing cell lines did not co-aggregate with stromal cells. The combined results suggest a functional role for N-cadherin in cohesion of breast tumor cells which, in addition promotes their interaction with the surrounding stromal cells, thereby facilitating invasion and metastasis.     

Cell-cell contacts mediated by E-cadherin (uvomorulin) restrict invasive behavior of L-cells

L-cells were cotransfected with plasmids coding for mouse E-cadherin (uvomorulin) and the neophosphotransferase gene, and stable transfectants expressing E-cadherin at the cell surface were selected and cloned. Control transfection was done with the neophosphotransferase gene alone. The invasive migration of transfected and untransfected L-cells into three-dimensional collagen gels was then analyzed. L-cells not expressing E-cadherin migrated efficiently into the gels, whereas invasion of the E-cadherin-expressing L-cells was restricted in a cell density dependent manner. At sparse density, when the cells exhibited little cell-cell contacts, no difference was observed between the level of invasion of the cadherin-expressing cells and the control cells. However, with increasing cell density, decreasing amounts of the cadherin-expressing cells but increasing amounts of the control cells migrated into the gels. At confluent density hardly any cadherin-expressing cells were able to migrate into the gels. The inhibition of the invasion of the cadherin-expressing cells could be reverted if confluent cells were cultured in the presence of monoclonal antibodies against E-cadherin. Since the expression of E-cadherin did not influence the invasive mobility of single cells, these results indicate that E-cadherin-mediated cell-cell contacts inhibited invasive cellular migration. Time-lapse videoscopy and studies of cell migration from a monolayer into a cell-free area demonstrated that the restricted invasion could be explained by contact inhibition of cell movement of the cadherin-expressing cells.     

NCAM-induced focal adhesion assembly: a functional switch upon loss of E-cadherin

Loss of expression of the cell-cell adhesion molecule E-cadherin is a hallmark of epithelial-mesenchymal transition (EMT) in development and in the progression from epithelial tumours to invasive and metastatic cancers. Here, we demonstrate that the loss of E-cadherin function upregulates expression of the neuronal cell adhesion molecule (NCAM). Subsequently, a subset of NCAM translocates from fibroblast growth factor receptor (FGFR) complexes outside lipid rafts into lipid rafts where it stimulates the non-receptor tyrosine kinase p59(Fyn) leading to the phosphorylation and activation of focal adhesion kinase and the assembly of integrin-mediated focal adhesions. Ablation of NCAM expression during EMT inhibits focal adhesion assembly, cell spreading and EMT. Conversely, forced expression of NCAM induces epithelial cell delamination and migration, and high NCAM expression correlates with tumour invasion. These results establish a mechanistic link between the loss of E-cadherin expression, NCAM function, focal adhesion assembly and cell migration and invasion.     

L1-mediated colon cancer cell metastasis does not require changes in EMT and cancer stem cell markers

Aberrant activation of Wnt/β-catenin signaling is common in most sporadic and inherited colorectal cancer (CRC) cells leading to elevated β-catenin/TCF transactivation. We previously identified the neural cell adhesion molecule L1 as a target gene of β-catenin/TCF in CRC cells. Forced expression of L1 confers increased cell motility, invasion, and tumorigenesis, and the induction of human CRC cell metastasis to the liver. In human CRC tissue, L1 is exclusively localized at the invasive front of such tumors in a subpopulation of cells displaying nuclear β-catenin. We determined whether L1 expression confers metastatic capacities by inducing an epithelial to mesenchymal transition (EMT) and whether L1 cosegregates with cancer stem cell (CSC) markers. We found that changes in L1 levels do not affect the organization or expression of E-cadherin in cell lines, or in invading CRC tissue cells, and no changes in other epithelial or mesenchymal markers were detected after L1 transfection. The introduction of major EMT regulators (Slug and Twist) into CRC cell lines reduced the levels of E-cadherin and induced fibronectin and vimentin, but unlike L1, Slug and Twist expression was insufficient for conferring metastasis. In CRC cells L1 did not specifically cosegregate with CSC markers including CD133, CD44, and EpCAM. L1-mediated metastasis required NF-κB signaling in cells harboring either high or low levels of endogenous E-cadherin. The results suggest that L1-mediated metastasis of CRC cells does not require changes in EMT and CSC markers and operates by activating NF-κβ signaling.     

Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis

E- and N-cadherin are calcium-dependent cell adhesion molecules that mediate cell-cell adhesion and also modulate cell migration and tumor invasiveness. The loss of E-cadherin-mediated adhesion has been shown to play an important role in the transition of epithelial tumors from a benign to an invasive state. However, recent evidence indicates that another member of the cadherin family, N-cadherin, is expressed in highly invasive tumor cell lines that lacked E-cadherin expression. These findings have raised the possibility that N-cadherin contributes to the invasive phenotype. To determine whether N-cadherin promotes invasion and metastasis, we transfected a weakly metastatic and E-cadherin-expressing breast cancer cell line, MCF-7, with N-cadherin and analyzed the effects on cell migration, invasion, and metastasis. Transfected cells expressed both E- and N-cadherin and exhibited homotypic cell adhesion from both molecules. In vitro, N-cadherin-expressing cells migrated more efficiently, showed an increased invasion of Matrigel, and adhered more efficiently to monolayers of endothelial cells. All cells produced low levels of the matrix metalloproteinase MMP-9, which was dramatically upregulated by treatment with FGF-2 only in N-cadherin-expressing cells. Migration and invasion of Matrigel were also greatly enhanced by this treatment. When injected into the mammary fat pad of nude mice, N-cadherin-expressing cells, but not control MCF-7 cells, metastasized widely to the liver, pancreas, salivary gland, omentum, lung, lymph nodes, and lumbar spinal muscle. The expression of both E- and N-cadherin was maintained both in the primary tumors and metastatic lesions. These results demonstrate that N-cadherin promotes motility, invasion, and metastasis even in the presence of the normally suppressive E-cadherin. The increase in MMP-9 production by N-cadherin-expressing cells in response to a growth factor may endow them with a greater ability to penetrate matrix protein barriers, while the increase in their adherence to endothelium may improve their ability to enter and exit the vasculature, two properties that may be responsible for metastasis of N-cadherin-expressing cells.     

Vimentin affects the mobility and invasiveness of prostate cancer cells

A significant proportion of prostate cancer patients treated with curative intent go on to develop advanced disease. At a fundamental biological level, very little is known about what makes the disease aggressive and metastatic. Observational pathology reports and experimental data suggest that epithelial-mesenchymal transition is involved in prostate cancer invasiveness. Here, we investigated vimentin expression of prostate cancer cells, and explored the potential mechanism of vimentin promoting prostate cancer cells invasion. Vimentin expression was not detected in well differentiated tumors or in moderately differentiated tumors, but the majority of poorly differentiated cancers (5/11 with negative bone scan, 11/14 bone with positive scan) and bone metastases (8/8) had high vimentin expression in tumor cells. Downregulation of vimentin expression in PC-3 cells by transfection with antisense-vimentin led to a significant decrease in tumor cells motility and invasive activity. Furthermore, the expression of E-cadherin was inversely associated with expression of vimentin. Our results suggest that vimentin affects prostate cancer cells motility and invasiveness.     

E-cadherin和Cathepsin D的表达与乳腺导管癌的侵袭和淋巴结转移的关系

目的　观察上皮性钙粘素 (E cadherin)和组织蛋白酶D (CathepsinD)在乳腺导管癌中的表达并分析其与肿瘤侵袭及腋下淋巴结转移的关系。方法　应用免疫组织化学方法检测E cadherin和CathepsinD在乳腺导管癌组织中的表达。结果　乳腺导管原位癌 (carcinomainsitu ,CIS)组织中E cadherin的表达与浸润性导管癌 (infiltratingductalcarcinoma ,IDC)相比无明显差异 (P >0 0 5 )。乳腺导管癌腋下淋巴结阴性组 (nodenegativeductalcarcinoma,NNDC)中E cadherin的表达与腋下淋巴结阳性组 (nodepositiveductalcarcinoma ,NPDC)相比差异不明显 (P >0 0 5 )。乳腺导管癌间质中CathepsinD的表达CIS与IDC相比差异显著 (P <0 0 1) ,NNDC与NPDC相比差异显著 (P <0 0 5 ) ;而在癌细胞中CathepsinD的表达在上述两组中差异均不明显 (P >0 0 5 )。结论　E cadherin在乳腺导管癌的表达与肿瘤的侵袭及淋巴结转移无相关关系。CathepsinD在乳腺导管癌间质的表达与肿瘤的侵袭及淋巴结转移密切相关 ,可作为临床判定肿瘤恶性程度的一个参考指标。     

E-cadherin 在肿瘤治疗中的研究进展

E-cadherin 参与形成细胞间黏附性连接,是胚胎发育过程中的一个关键因子。越来越多的研究表明,E-cadherin 在肿瘤的发生发 展过程中也发挥了至关重要的作用。在生物体内,E-cadherin 的表达和功能受到多个水平、多重因素的调控,而 E-cadherin 又可以影响 多条重要信号通路的活性,参与到多种生理病理过程中。E-cadherin 下调造成细胞间黏附性连接减少、极性减弱,细胞由上皮样转变为间 质样,这一变化是上皮间质转化（EMT）的重要标志之一。E-cadherin 与多种肿瘤的发生有一定的相关性。同时 E-cadherin 下调所引起 的 EMT 促进肿瘤细胞的迁移运动,肿瘤细胞侵袭力增强,促进转移的发生。近年来,大量研究关注到 E-cadherin 对肿瘤细胞的耐药及干 细胞特性的获得都有影响。综述 E-cadherin 在肿瘤发生发展中的作用,探讨以 E-cadherin 为靶点的肿瘤治疗的现状及展望。     

Progression of Oral Squamous Cell Carcinoma Accompanied with Reduced E-Cadherin Expression but Not Cadherin Switch

The cadherin switch from E-cadherin to N-cadherin is considered as a hallmark of the epithelial-mesenchymal transition and progression of carcinomas. Although it enhances aggressive behaviors of adenocarcinoma cells, the significance and role of cadherin switch in squamous cell carcinomas (SCCs) are largely controversial. In the present study, we immunohistochemically examined expression of E-cadherin and N-cadherin in oral SCCs (n = 63) and its implications for the disease progression. The E-cadherin-positive carcinoma cells were rapidly decreased at the invasive front. The percentage of carcinoma cells stained E-cadherin at the cell membrane was reduced in parallel with tumor dedifferentiation (P<0.01) and enhanced invasion (P<0.01). In contrast, N-cadherin-positive cells were very limited and did not correlate with the clinicopathological parameters. Mouse tongue tumors xenotransplantated oral SCC cell lines expressing both cadherins in vitro reproduced the reduction of E-cadherin-positive carcinoma cells at the invasive front and the negligible expression of N-cadherin. These results demonstrate that the reduction of E-cadherin-mediated carcinoma cell-cell adhesion at the invasive front, but not the cadherin switch, is an important determinant for oral SCC progression, and suggest that the environments surrounding carcinoma cells largely affect the cadherin expression.     

应用亚硫酸氢盐修饰后PCR联合TA克隆测序对E-cadherin启动子区甲基化水平的检测

E-cadherin是一种细胞粘附因子,通过增强细胞之间的粘附而起到抑制肿瘤转移的作用.Ecadherin基因启动子区的高甲基化是导致其在众多肿瘤细胞中表达下调甚至缺失的主要原因之一.本实验首先抽提SGC-7901细胞(胃腺癌细胞)、A549细胞(肺腺癌细胞)、MCF-7细胞(乳腺癌细胞)等3个肿瘤细胞株的全基因组DNA,然后对抽提的DNA进行亚硫酸氢盐修饰和纯化回收,根据修饰后的DNA序列设计引物并对其进行PCR扩增.然后将PCR扩增产物与pUC-T TA载体连接并转化入感受态大肠杆菌DH5α中进行培养,对筛选出的含有阳性重组子的菌落进行测序.测序结果显示,3个肿瘤细胞株的E-cadherin基因启动子区的CpG岛都呈现了高度的甲基化,亚硫酸氢盐的修饰效率达到了99.2%.综上研究表明,亚硫酸氢盐修饰后PCR(BSP)联合TA克隆测序可以对肿瘤细胞某基因启动子区CpG岛的甲基化水平进行精确量化,研究所使用的3个肿瘤细胞株均可作为研究肿瘤细胞E-cadherin基因甲基化的细胞模型.     

Loss of E-cadherin promotes the growth,invasion and drug resistance of colorectal cancer cells and is associated with liver metastasis

The recent studies indicated that the epithelial cell adhesion molecule E-cadherin is a well-recognized molecule that is important in cell adhesion. To further investigate the molecular basis of this notion, we used small-interfering RNA to inhibit E-cadherin function and found that loss of E-cadherin promoted Colorectal cancer cell growth, invasion and drug resistance through induction of β-catenin nuclear translocation and epithelial-to-mesenchymal transition. Further analysis of E-cadherin expression with clinicopathologic parameters showed that E-cadherin expression decreased in Colorectal cancer patients who developed liver metastasis (P = 0.043). These findings indicate that E-cadherin loss in tumors contributes to progression and metastatic dissemination. Thus, E-cadherin can act as a central modulator of the cell biological phenotypes and a potential prognostic marker in Colorectal cancer.     

miR-208-Induced Epithelial to Mesenchymal Transition of Pancreatic Cancer Cells Promotes Cell Metastasis and Invasion

The aim of this study was to investigate the role of miR-208 in the invasion and metastasis of pancreatic cancer cells and the underlying molecular mechanism. miR-208 mimic, miR-208 inhibitor and NC were transfected into pancreatic cancer cell line Bxpc3 using liposome. Transwell invasion and scratch assays were used to test cell migratory and invasive abilities. Western blotting and quantitative PCR methods were used to detect E-cadherin, fibronectin and vimentin protein and mRNA expression in pancreatic cancer cell line BxPC3 after transfection by miR-208 mimic, miR-208 inhibitor and NC. Transwell invasion and scratch assays showed that after overexpressing miR-208, pancreatic cancer cell line BxPC3 exhibited enhanced in vitro migratory and invasive abilities, while after downregulating miR-208 expression, cell migratory and invasive abilities were decreased. Western blotting and quantitative PCR showed that after overexpressing miR-208, expression of E-cadherin, an epithelial cell marker, was decreased and expression of fibronectin and vimentin, interstitial cell markers, was increased in pancreatic cancer cell line BxPC3; however, after inhibiting miR-208, increased E-cadherin expression and decreased fibronectin and vimentin expression were observed in pancreatic cancer cell line BxPC3. After overexpressing miR-208, p-AKT and p-GSK-3β expression was altered by activating AKT/GSK-3β/snail signaling pathway. miR-208 induces epithelial to mesenchymal transition of pancreatic cancer cell line BxPC3 by activating AKT/GSK-3β/snail signaling pathway and thereby promotes cell metastasis and invasion.