Significance of E-cadherin/beta-catenin complex and cyclin D1 in breast cancer

E-cadherin and beta-catenin are important epithelial adhesion molecules in normal epithelium. Loss of E-cadherin - beta-catenin adhesion is an important step in the progression of many epithelial malignancies. beta-catenin plays also a role in intracellular signaling and can function as an oncogene when binds to the T-cell factor 4 (Tcf4)-binding site in the promotor region of cyclin D1 and transactivates genes after translocation to the nucleus. We evaluated the immunohistochemical expression pattern of E-cadherin, beta-catenin in relationship with cyclin D1 overexpression, tumor stage, clinicopathologic parameters and patient survival in 128 mammary infiltrating duct carcinomas. The expression of E-cadherin/beta-catenin complex and beta-catenin/cyclin D1 double staining with confocal scanning laser microscope was evaluated. There were aberrant expressions in 78% of E-cadherin, 79% of beta-catenin, and 66% of cyclin D1 in breast cancer. There was correlation of aberrant expression of E-cadherin or beta-catenin with lymph node metastasis, survival rate, and survival length. However, there was no correlation of cyclin D1 overexpression with aberrant expression of E-cadherin or beta-catenin. No death was found in normal expression of beta-catenin, however lowest survival (50%) was found in nuclear beta-catenin expression. There was correlation of overexpression of cyclin D1 with survival rate and survival length. The highest survival rate and survival length were found in membranous normal beta-catenin expression group, however significant decrement of survival length was found in the groups of aberrant expression one or both of E-cadherin or/and beta-catenin. These results suggest that aberrant expression of E-cadherin, beta-catenin, and cyclin D1 may be involved in tumor metastasis, and analysis of the degree or the pattern of E-cadherin, beta-catenin, cyclin D1, and E-cadherin/beta-catenin complex may be good prognostic markers of mammary infiltrating duct carcinoma.     

RGS5在乳腺癌组织和细胞系中的表达和定位

目的:探讨G蛋白信号转导调节蛋白5（RGS5）在人乳腺癌细胞系中的表达。方法:提取人正常乳腺上皮细胞HBL100,人乳腺癌细胞系MCF-7,ZR-75-1,ZR-75-30,MDA-MB-231,HCC1937的总蛋白和总RNA,提取细胞的总蛋白和总RNA进行蛋白免疫印迹和实时定量PCR检测RGS5的表达。共聚焦激光扫描显微镜观察RGS5胞内定位;免疫组化检测RGS5在乳腺肿瘤组织中表达情况。结果:MDA-MB-231细胞系中RGS5基因表达上调,MCF7和ZR-75-1细胞系中RGS5蛋白表达提高。结论:RGS5在乳腺癌细胞的高表达可能参与其生长和转移,揭示RGS5可能是人乳腺肿瘤的治疗靶点。     

E-cadherin and beta-catenin expression in breast medullary carcinomas.

The initial step of cancer invasion and metastasis is the escape of tumour cells from the primary site, involving disruption of normal cell-cell adhesion and E-cadherin (E-cad) and beta-catenin (beta-cat) down-regulation, as shown in various types of human malignancies including breast carcinomas. Medullary carcinomas are high grade and poorly differentiated tumours with syncytial typical pattern, and prognosis unexpectedly better than that in high grade breast carcinomas. In a series of 55 breast typical medullary carcinomas diagnosed according to the strict use of Ridolfi et al (Cancer 40: 1365-1385, 1977) criteria, E-cad and beta-cat were investigated using quantitative (SAMBA 2005 system) immunocytochemical assays on frozen sections. Results were compared to that obtained on paraffin sections and in a series (n=55) of grade 3 ductal carcinomas. It was shown that medullary carcinomas significantly (p<0.001) expressed more E-cad and beta-cat than grade 3 ductal carcinomas. E-cad and beta-cat correlated with high expression of P53, of c-erbB, and of Ki-67 antigens, and with lack of hormone receptors antigenic sites (p<0.001). It was concluded that favourable prognosis and syncytial pattern of typical breast medullary carcinomas likely results, at least partly, from a particular expression of cell-cell adhesion molecules, significantly limiting tumour growth and efficiently mastering the tumour cell dissemination, opposing to high proliferative activity (grade 3).     

Immunohistochemical expression of E-cadherin and beta-catenin in the normal and malignant human endometrium: an inverse correlation between E-cadherin and nuclear beta-catenin expression

BACKGROUND: The E-cadherin/beta-catenin complex plays a crucial role in epithelial cell-cell adhesion and in the maintenance of tissue architecture. We previously reported aberrant expression of beta-catenin in endometrial carcinomas. However, the expression and correlation of E-cadherin and beta-catenin in normal and malignant endometrial tissues are not fully understood. MATERIALS AND METHODS: Immunohistochemical expression of E-cadherin and beta-catenin was detected in 30 cases of normal endometrium and 73 cases of endometrial carcinoma. RESULTS: In the normal endometrium, the expression of E-cadherin and cytoplasmic beta-catenin in glandular cells was predominantly observed in the proliferative phase, and decreased in the secretory phase. In endometrial carcinomas, the expression of E-cadherin and cytoplasmic beta-catenin decreased compared to that in the normal proliferative endometrial glands. The expression of E-cadherin and cytoplasmic beta-catenin tended to be reduced in histologically high-grade tumors compared to low-grade tumors. Nuclear expression of beta-catenin was observed in the glandular cells in the late proliferative and early secretory phases, as well as in high-grade endometrial carcinomas. Interestingly, nuclear beta-catenin expression was associated with the loss of E-cadherin expression in normal and carcinoma cells, indicating an inverse correlation. CONCLUSION: The cyclic expression of E-cadherin and beta-catenin in the normal endometrium suggests that the adhesion complex may act to maintain the endometrial architectures. In addition, nuclear beta-catenin expression associated with loss of E-cadherin expression may be involved in the acquisition of aggressive biological behavior, especially in high-grade tumors.     

Preclinical studies of gemcitabine and trastuzumab in breast and lung cancer cell lines

Overexpression of the HER2/neu oncogene and receptor protein has been reported in 20%-30% of patients with breast cancer and is associated with a poor prognosis. HER2/neu expression in breast cancer patients assessed by fluorescence in situ hybridization or immunohistochemistry is a predictor for response to trastuzumab, a humanized monoclonal antibody against the HER2/neu cell-surface protein. Data regarding HER2/neu expression in lung cancer are more limited, and there is little information regarding HER2/neu expression and response to trastuzumab alone or in combination with chemotherapeutic agents. Gemcitabine is an active agent against non-small-cell lung cancer (NSCLC) and has demonstrated activity in breast cancer as well. In vitro modified tetrazolium salt growth assays were performed to determine whether the combination of trastuzumab/gemcitabine produced synergistic or additive effects on breast and lung cancer cell lines. The effects of trastuzumab alone, gemcitabine alone, and the trastuzumab/gemcitabine combination was evaluated on 4 NSCLC cell lines, 1 small-cell lung cancer (SCLC) cell line, and 2 breast cancer cell lines. HER2/neu surface protein expression was assessed by fluorescence flow cytometry and immunohistochemistry. Fluorescence in situ hybridization analysis was used to study gene expression. Trastuzumab treatment alone resulted in growth inhibition in all cell lines expressing HER2/neu and the inhibitive effect correlated with the level of cell surface HER2/neu protein expression. Treatment with gemcitabine alone resulted in growth inhibition in both breast and NSCLC cell lines. A synergistic growth inhibition effect was seen with the trastuzumab/ gemcitabine combination as indicated by combination index values < 1. The degree of synergy observed did not directly correlate with the level of surface protein expression, as synergy was seen even in cancer cell lines expressing low levels of HER2/neu. No treatment effect was seen in the SCLC cell line, which did not express HER2/neu. These preclinical studies indicate a need to study the clinical synergistic effects of the gemcitabine/trastuzumab combination in breast cancer and NSCLC patients whose tumors overexpress HER2/ neu.     

Expression of S100A4, E-cadherin,alpha- and beta-catenin in breast cancer biopsies

In 66 breast cancer biopsies, the expression of the Ca(2+)-binding protein S100A4, E-cadherin, alpha- and beta-catenin was examined by immunohistochemistry, and the results were related to clinical and pathological parameters. High levels of S100A4 were found to significantly correlate with histological grade (P=0.030) and loss of oestrogen receptor (P=0.046), but not to the time interval between surgery and development of distant metastasis (P=0.51) or to patient survival (P=0.89). Loss of E-cadherin expression, associated with altered cell-cell adhesion, showed a highly significant association to overall survival (P=0.020) and metastasis-free period (P=0.0052). In multivariate analysis, only lymph node involvement was a more significant predictor of patient demise. No association was found between expression of S100A4 and any single member of the cadherin-catenin complex, but a trend (P=0.053) towards reduced expression of one or several of these proteins and S100A4 immunoreactivity was observed. In conclusion, although our results suggest an association between S100A4 expression and an aggressive tumour phenotype, no relationship to overall survival was found. Deregulation of E-cadherin expression, however, was of high prognostic significance.     

p57kip2蛋白在乳腺癌组织和细胞系中的表达和定位

目的：研究p57kip2在人乳腺癌组织和细胞系中的表达和定位.方法：免疫印迹法检测人乳腺肿瘤细胞系中p57kip2的表达.人乳腺肿瘤组织进行免疫组化染色,检测p57kip2在组织中的表达.激光共聚焦扫描显微镜检测内源p57kip2在MCF-7细胞中的定位.结果：p57kip2在乳腺癌细胞系内表达,免疫组化染色分析和激光共聚焦扫描显微镜证实p57kip2主要定位于细胞核内.结论：p57kip2在乳腺癌细胞内表达,p57kip2主要定位于乳腺癌组织细胞核内,可能是参与肿瘤细胞基因转录调控和蛋白表达的重要蛋白之一.     

肺癌细胞中RASSF4的高表达抑制肺癌细胞的增殖和侵袭能力

目的:探讨RASSF4在肺癌中的增殖和侵袭能力。方法:向肺癌细胞系H460和A549中导入RASSF4的cDNA质粒后,通过MTT、克隆形成实验、基质胶侵袭实验、流式细胞术等方法检测肺癌细胞的生长和侵袭能力。结果:RASSF4能够通过下调MMP2、MMP9和cyclinD1的表达,抑制肺癌细胞的侵袭、增殖及克隆形成能力。结论:RASSF4在肺癌细胞中通过抑制细胞生长及侵袭能力发挥重要的肿瘤抑制功能。     

Expression of E-cadherin and beta-catenin in human non-small cell lung cancer and the clinical significance.

E-cadherin, a calcium-dependent cell-cell adhesion molecule, plays a key role in the maintenance of tissue integrity. The function of this molecule is partly mediated by alpha-/beta-/gamma-catenin. Loss or dysfunction of E-cadherin is associated with an invasive phenotype. We analyzed the expression of E-cadherin and beta-catenin in human lung cancer to determine the relationship to clinicopathological factors and prognosis. E-cadherin and beta-catenin expressions were evaluated in 331 lung cancer tissues in a immunohistochemical analysis. Reduced E-cadherin expression was evident in 138 (42%), and reduced beta-catenin expression was noted in 122 (37%). Reduced E-cadherin expression significantly correlated with lymph nodes metastasis (P = 0.0199). E-cadherin expression significantly correlated with increasing histological differentiation (P = 0.0403). Although reduced E-cadherin did not correlate with the prognosis (P = 0.0652), reduced beta-catenin expression did significantly correlate with a poor prognosis (P = 0.0001). When both were reduced, there was a significant unfavorable prognosis compared with either the reduced expression (P = 0.0493) and preserved expression (P = 0.0003). Multivariate analysis showed a significantly lower survival rate for patients with reduced beta-catenin (P < 0.0001). We interpret these data to mean that dysfunction of the cell-cell adhesion molecule has a role in the progression of lung cancer and that analysis of E-cadherin and beta-catenin expression can provide clinically important evidence on which to base treatment.     

Metastasis from human breast cancer cell lines

Summary Immunodeficient animals, principally nude mice, when used in appropriately designed studies have been shown to be useful for the experimental analysis of human breast cancer metastasis. As with many other human tumors, the implantation of breast cancer cells into an anatomically appropriate tissue (the mammary fatpad) results in increased tumor take and incidence of metastasis for certain cell lines compared with subcutaneous injection. Testing a number of widely available human breast cancer cell lines identified the MDA-MB-435 cell line as the most metastatic, producing lung and lymph node metastases in a high proportion of nude and severe combined immunodeficient (SCID) mice after injection in the mammary fatpad. Mixing human breast cancer cells with normal fibroblasts or with Matrigel also increases the tumor incidence and growth rates in nude mice. Different routes of injection can be used to assess the ability of human breast cancer cells to form metastatic lesions in the lungs (i.v. injection), the liver (injection in the spleen), the brain (direct or intracarotid artery injection) and the bone marrow and bone (injection into the left ventricle of the heart). These different approaches demonstrate the potential of experimental studies of human breast cancer growth and metastasis using immunodeficient mice; this model is valuable for experiments that test the role of metastasis-associated genes and the efficacy of novel forms of therapy.     

黑色素瘤抗原基因A1在乳腺癌组织和四株乳腺癌细胞株中的表达

目的研究乳腺癌组织和细胞株中Mage A1基因的表达情况，为Mage A1基因编码蛋白用于乳腺癌免疫治疗提供依据。方法采用RT—PCR检删33例乳腺癌组织和4株常用乳腺癌细胞株MCF-7、Sk-Br-3，MDA-MB-435s和TM40D中Mage A1基因的表达。结果33例乳腺癌组织中12％(4／33)Mage—A1阳性，细胞株MCF-7和Sk-Br-3中Mage-A1阳性表达，MDA-MB-435s和TM40D中Mage A1基因的表达阴性。结论不同的乳腺癌细胞表达Mage A1基因有差别，Mage A1蛋白可望成为乳腺癌免疫治疗的靶抗原。     

Analysis of nuclear nestin localization in cell lines derived from neurogenic tumors

Nestin is a class VI intermediate filament protein expressed in the cytoplasm of stem and progenitor cells in the mammalian CNS during development. In adults, nestin is present only in a small subset of cells and tissues, including the subventricular zone of the adult mammalian brain, where neurogenesis occurs. Nestin expression has also been detected under such pathological conditions as ischemia, inflammation, and brain injury, as well as in various types of human solid tumors and their corresponding cell lines. Furthermore, nestin was recently found in the nuclei of glioblastoma, neuroblastoma, and angiosarcoma cells and it was proved to interact directly with the nuclear DNA in neuroblastoma cells. Here, we perform the first study of the intracellular distribution of nestin in cell lines derived from neurogenic tumors. Using immunodetection methods, we examined nestin expression in tumor-derived cell lines obtained from 11 patients with neuroblastoma, medulloblastoma, or glioblastoma multiforme. Besides its standard cytoplasmic localization, nestin was present in the nuclei of two neuroblastoma cell lines and one medulloblastoma cell line. Nestin was only present in the nuclei of cells with diffuse cytoplasmic staining for this protein, and the proportion of cells positive for nestin in nuclei, as well as the intensity of staining, varied. The presence of nestin in the nuclei was confirmed by both transmission electron microscopy and Western blotting. Our results indicate that the presence of nestin in the nuclei of tumor cells is not very rare, especially under in vitro conditions.     

Bisphosphonates induce apoptosis in human breast cancer cell lines

Breast cancer has a prodigious capacity to metastasize to bone. In women with advanced breast cancer and bone metastases, bisphosphonates reduce the incidence of hypercalcaemia and skeletal morbidity. Recent clinical findings suggest that some bisphosphonates reduce the tumour burden in bone with a consequent increase in survival, raising the possibility that bisphosphonates may have a direct effect on breast cancer cells. We have investigated the in vitro effects of bisphosphonates zoledronate, pamidronate, clodronate and EB 1053 on growth, viability and induction of apoptosis in three human breast cancer cell lines (MDA-MB-231, Hs 578T and MCF-7). Cell growth was monitored by crystal violet dye assay, and cell viability was quantitated by MTS dye reduction. Induction of apoptosis was determined by identification of morphological features of apoptosis using time-lapse videomicroscopy, identifying morphological changes in nucleis using Hoechst staining, quantitation of DNA fragmentation, level of expression of bcl-2 and bax proteins and identification of the proteolytic cleavage of Poly (ADP)-ribose polymerase (PARP). All four bisphosphonates significantly reduced cell viability in all three cell lines. Zoledronate was the most potent bisphosphonate with IC50 values of 15, 20 and 3 microM respectively in MDA-MB-231, MCF-7 and Hs 578T cells. Corresponding values for pamidronate were 40, 35 and 25 microM, whereas clodronate and EB 1053 were more than two orders of magnitude less potent. An increase in the proportion of cells having morphological features characteristic of apoptosis, characteristic apoptotic changes in the nucleus, time-dependent increase in the percentage of fragmented chromosomal DNA, down-regulation in bcl-2 protein and proteolytic cleavage of PARP, all indicate that bisphosphonates have direct anti-tumour effects on human breast cancer cells.     

Peripheral-type benzodiazepine receptor (PBR) in human breast cancer: correlation of breast cancer cell aggressive phenotype with PBR expression, nuclear localization, and PBR-mediated cell proliferation and nuclear transport of cholesterol

Aberrant cell proliferation and increased invasive and metastatic behavior are hallmarks of the advancement of breast cancer. Numerous studies implicate a role for cholesterol in the mechanisms underlying cell proliferation and cancer progression. The peripheral-type benzodiazepine receptor (PBR) is an Mr 18,000 protein primarily localized to the mitochondria. PBR mediates cholesterol transport across the mitochondrial membranes in steroidogenic cells. A role for PBR in the regulation of tumor cell proliferation has also been shown. In this study, we examined the expression, characteristics, localization, and function of PBR in a battery of human breast cancer cell lines differing in their invasive and chemotactic potential as well as in several human tissue biopsies. Expression of PBR ligand binding and mRNA was dramatically increased in the highly aggressive cell lines, such as MDA-231, relative to nonaggressive cell lines, such as MCF-7. PBR was also found to be expressed at high levels in aggressive metastatic human breast tumor biopsies compared with normal breast tissues. Subcellular localization with both antibodies and a fluorescent PBR drug ligand revealed that PBR from the MDA-231 cell line as well as from aggressive metastatic human breast tumor biopsies localized primarily in and around the nucleus. This localization is in direct contrast to the largely cytoplasmic localization seen in MCF-7 cells, normal breast tissue, and to the typical mitochondrial localization seen in mouse tumor Leydig cells. Pharmacological characterization of the receptor and partial nucleotide sequencing of PBR cDNA revealed that the MDA-231 PBR is similar, although not identical, to previously described PBR. Addition of high affinity PBR drug ligands to MDA-231 cells increased the incorporation of bromodeoxyuridine into the cells in a dose-dependent manner, suggesting a role for PBR in the regulation of MDA-231 cell proliferation. Cholesterol uptake into isolated MDA-231 nuclei was found to be 30% greater than into MCF-7 nuclei. High-affinity PBR drug ligands regulated the levels of cholesterol present in MDA-231 nuclei but not in MCF-7. In addition, the PBR-dependent MDA-231 cell proliferation was found to highly correlate (r = -0.99) with the PBR-mediated changes in nuclear membrane cholesterol levels. In conclusion, these data suggest that PBR expression, nuclear localization, and PBR-mediated cholesterol transport into the nucleus are involved in human breast cancer cell proliferation and aggressive phenotype expression, thus participating in the advancement of the disease.     

HMG-I/Y in human breast cancer cell lines

The HMG-I/Y gene encodes the HMG-I and -Y architectural, chromatin binding proteins originally identified based on their association with chromosomal DNA. HMG-I/Y proteins bind to AT-rich regions in chromosomal DNA and alter gene expression. Increased HMG-I/Y protein expression also correlates with neoplastic transformation. Previous work from our laboratory has shown that HMG-I/Y is a direct c-Myc target gene involved in neoplastic transformation in Burkitt's lymphoma. We also observed that HMG-I/Y proteins have several oncogenic properties. In this report, we show that HMG-I/Y proteins are increased in several human breast cancer cell lines compared to a human breast cell line derived from normal breast cells. Decreasing HMG-I/Y proteins using an antisense ribozyme approach inhibits transformation in human breast cancer cells, suggesting that HMG-I/Y is important for the transformed phenotype observed in these cells. In addition, increased expression of the HMG-I isoform in normal human breast cells leads to transformation. These results suggest that HMG-I/Y is an oncogene important in the pathogenesis of human breast cancer. Although additional studies with animal models are needed, the antisense experiments, which result in blocking transformation suggest that this approach may have therapeutic potential in patients with breast cancer characterized by increased HMG-I/Y expression.