GSTP1 promoter hypermethylation is an early event in breast carcinogenesis

Promoter hypermethylation in precursor lesions of the breast cancer may be biomarkers of cancer risk and targets for cancer chemoprevention. Pi-class glutathione-S-transferases (GSTP1) is inactivated by promoter hypermethylation in invasive breast cancers. However, little is known about epigenetic silencing of GSTP1 gene by promoter hypermethylation in precursor lesions. To determine the significance of GSTP1 promoter hypermethylation in breast carcinogenesis, methylation status of GSTP1 gene was studied by nested methylation-specific polymerase chain reaction, and GSTP1 expression was studied by immunohistochemistry in invasive ductal carcinoma (IDC), ductal carcinoma in situ (DCIS), usual ductal hyperplasia (UDH), and normal breast tissue. GSTP1 promoter hypermethylation was detected in 4/24 (16.7%) of UDH, 18/49 (36.7%) of DCIS, and 14/36 (38.9%) of IDC. No hypermethylation was detected in normal breast tissues. GSTP1 promoter hypermethylation was found to be progressively elevated during breast carcinogenesis (p < 0.01). GSTP1 promoter hypermethylation was associated with loss of GSTP1 expression (p < 0.01 for UDH, p < 0.001 for DCIS and IDC). Our results suggest that GSTP1 promoter hypermethylation is an early event in breast carcinogenesis and appears to functionally silence GSTP1 expression. GSTP1 promoter hypermethylation in the precursor lesions of breast cancer may be used as a target for cancer chemoprevention.     

Genistein inhibits DNA methylation and increases expression of tumor suppressor genes in human breast cancer cells

It has been previously demonstrated that genistein exhibits anticancer activity against breast cancer. However, the precise mechanisms underlying the anticancer effect of genistein, in particular the epigenetic basis, remain unclear. In this study, we investigated whether genistein could modulate the DNA methylation status and expression of cancer‐related genes in breast cancer cells. We treated MCF‐7 and MDA‐MB‐231 human breast cancer cells with genistein in vitro. We found that genistein decreased the levels of global DNA methylation, DNA methyltransferase (DNMT) activity and expression of DNMT1. Yet, the expression of DNMT3A and DNMT3B showed no significant change. Using molecular modeling, we observed that genistein might directly interact with the catalytic domain of DNMT1, thus competitively inhibiting the binding of hemimethylated DNA to the catalytic domain of DNMT1. Furthermore, genistein decreased DNA methylation in the promoter region of multiple tumor suppressor genes (TSGs) such as ataxia telangiectasia mutated (ATM), adenomatous polyposis coli (APC), phosphatase and tensin homolog (PTEN), mammary serpin peptidase inhibitor (SERPINB5), and increased the mRNA expression of these genes. However, we detected no significant changes in the DNA methylation status or mRNA expression of stratifin (SFN). These results suggest that the anticancer effect of genistein on breast cancer may be partly due to its ability to demethylate and reactivate methylation‐silenced TSGs through direct interaction with the DNMT1 catalytic domain and inhibition of DNMT1 expression. © 2014 Wiley Periodicals, Inc.     

GSTP1在前列腺癌组织中的表达及其甲基化检测

目的寻找前列腺癌患者血清中的生物标志物,为临床前列腺癌的早期无创性诊断提供依据。方法采用免疫组化SP染色法检测前列腺癌组织中GSTP1表达;运用甲基化特异性聚合酶链反应(MSP)方法检测前列腺癌患者癌组织和相应血清GSTP1基因启动子区5’端CpG岛甲基化程度。结果免疫组化结果:GSTP1在88例前列腺癌组仅有2例呈阳性反应且均为石蜡包埋标本;49例前列腺增生组均为阳性反应,其中38例呈现为强阳性反应。MSP检测结果:38例新鲜前列腺癌组织中,GSTP1基因高甲基化29例,此29例相应血清GSTP1基因高甲基化28例。对照组19例前列腺增生标本和对应的血清均没有检测到GSTP1基因甲基化。结论前列腺癌组织中GSTP1基因的表达与其启动子区甲基化程度呈负相关。前列腺癌患者的组织和血清中GSTP1甲基化检测结果相一致,检测血清中GSTP1的甲基化程度可反应癌组织中GSTP1基因的表达的情况。     

乳腺癌发生过程中NOEY2基因启动子区甲基化及mRNA表达

目的 探讨乳腺癌发生过程中抑癌基因NOEY2启动子区甲基化状态及其对mRNA表达的影响。方法 应用甲基化特异性PCR及双亚硫酸钠基因测序技术检测MCF10模型中乳腺增生细胞系MCF10A、癌前细胞系MCF10AT、导管内癌细胞系MCFIODCIS．com、浸润癌细胞系MCF10CA1a、MCF10CA1d、MCF10CA1h及正常乳腺组织中NOEY2基因启动子区CpG岛Ⅰ甲基化状态,然后用RT-PCR和实时PCR技术检测上述样品的mRNA表达水平。结果 MCF10模型的增生细胞系、癌前细胞系、导管内癌细胞系、浸润癌细胞系均发生该基因启动子区CpG岛Ⅰ高度甲基化;与正常乳腺组织相比,上述细胞系mRNA表达显著减少。结论 NOEY2基因启动子区高度甲基化及相应的mRNA表达减少是乳腺癌发生过程中的早期事件,与乳腺癌发生有关,可能成为早期诊断乳腺癌的潜在分子生物学标记。     

乳腺癌发生模型MCF10各细胞系中WT1基因启动子区甲基化及mRNA表达研究

目的通过观察乳腺癌发生模型MCF10中WT1基因启动子区甲基化状态和mRNA表达水平,探讨该基因在乳腺癌发生中的作用。方法应用甲基化特异性PCR及双亚硫酸钠基因测序技术检测MCF10模型的乳腺增生细胞系MCF10A、癌前细胞系MCF10AT、导管内癌细胞系MCF10DCIS．com、浸润癌细胞系（MCF10CAla、MCF10CA1d、MCF10CA1h）、经典乳腺癌细胞系MCF7及正常乳腺组织中WT1基因启动子区甲基化状态,然后用逆转录．聚合酶链反应（RT—PCR）和即时定量PCR技术检测上述样品的mRNA表达水平。结果在MCF10模型的增生细胞系MCF10A、癌前细胞系MCF10AT、导管内癌细胞系MCF10DCIS．com、浸润癌细胞系（MCF10CAla、MCF10CA1d、MCF10CA1h）、经典乳腺癌细胞系MCF7中,WT1基因启动子均处于高度甲基化状态。与正常乳腺组织相比,WT1基因mRNA在MCFl0模型的增生细胞系、癌前细胞系、导管内癌细胞系、浸润癌细胞系和经典乳腺癌细胞系MCF7中的表达均有不同程度的增加（MCF10A、MCF10AT、MCF10CAla、MCF10CA1d、MCF10CA1h、MCF10DCIS、MCF7的WT1基因mRNA表达量分别是正常乳腺组织3．23、1．94、4．20、1．53、4．20、4．35、28．69倍）。结论乳腺癌发生过程中WnmRNA的表达不被启动子甲基化所抑制;可WT1mRNA过表达出现于乳腺癌发生的早期阶段,提示该基因在乳腺癌发生中起作用。     

Alu序列甲基化水平与两种乳腺癌细胞系转移能力高度相关

目的 探讨Alu序列甲基化与乳腺癌转移潜能的关系.方法 用亚硫酸氢盐修饰联合限制性内切酶分析法(combined bisulfite restriction analysis,COBRA)、亚硫酸氢盐修饰结合直接测序法(bisulfite sequencing,BSP)检测两株转移能力不同的乳腺癌细胞系MCF7和MDA-MB-435S中Alu甲基化状态,每个样品挑取10个克隆测序.结果 MCF7和MDA-MB-435S中Alu甲基化水平均明显低于报道的正常人体细胞Alu甲基化水平,但MCF7中Alu的甲基化水平明显高于MDA-MB-435S.同时,Alu甲基化位点在基因组中分布不均匀.结论 乳腺癌的转移潜能可能与Alu序列的去甲基化以及去甲基化位点的分布相关,值得进一步探讨.     

High-resolution genomic profiles of breast cancer cell lines assessed by tiling BAC array comparative genomic hybridization

A BAC-array platform for comparative genomic hybridization was constructed from a library of 32,433 clones providing complete genome coverage, and evaluated by screening for DNA copy number changes in 10 breast cancer cell lines (BT474, MCF7, HCC1937, SK-BR-3, L56Br-C1, ZR-75-1, JIMT1, MDA-MB-231, MDA-MB-361, and HCC2218) and one cell line derived from fibrocystic disease of the breast (MCF10A). These were also characterized by gene expression analysis and found to represent all five recently described breast cancer subtypes using the "intrinsic gene set" and centroid correlation. Three cell lines, HCC1937 and L56BrC1 derived from BRCA1 mutation carriers and MDA-MB-231, were of basal-like subtype and characterized by a high frequency of low-level gains and losses of typical pattern, including limited deletions on 5q. Four estrogen receptor positive cell lines were of luminal A subtype and characterized by a different pattern of aberrations and high-level amplifications, including ERBB2 and other 17q amplicons in BT474 and MDA-MB-361. SK-BR-3 cells, characterized by a complex genome including ERBB2 amplification, massive high-level amplifications on 8q and a homozygous deletion of CDH1 at 16q22, had an expression signature closest to luminal B subtype. The effects of gene amplifications were verified by gene expression analysis to distinguish targeted genes from silent amplicon passengers. JIMT1, derived from an ERBB2 amplified trastuzumab resistant tumor, was of the ERBB2 subtype. Homozygous deletions included other known targets such as PTEN (HCC1937) and CDKN2A (MDA-MB-231, MCF10A), but also new candidate suppressor genes such as FUSSEL18 (HCC1937) and WDR11 (L56Br-C1) as well as regions without known genes. The tiling BAC-arrays constitute a powerful tool for high-resolution genomic profiling suitable for cancer research and clinical diagnostics.     

乳腺癌发生模型MCF10各细胞系中APC基因启动子区甲基化及mRNA表达检测

目的探讨乳腺癌发生模型MCF10中抑癌基因APC启动子区甲基化状态及其对mRNA表达的影响。方法应用甲基化特异性聚合酶链反应（MSP）及双亚硫酸钠基因测序技术检测MCF10模型的乳腺增生细胞系MCF10A、癌前细胞系MCF10AT、导管内癌细胞系MCF10DCIS.com、浸润癌细胞系MCF10CA1a、MCF10CA1d、MCF10CA1h及经典乳腺癌细胞系MCF-7和正常乳腺组织中APC基因启动子1A甲基化状态,然后用逆转录聚合酶链反应（RT-PCR）和实时PCR技术检测上述样品的mRNA表达水平。结果在MCF10模型的增生细胞系、癌前细胞系、导管内癌细胞系、浸润癌细胞系中,APC基因启动子1A处于低甲基化状态;与正常乳腺组织相比,各细胞系APC基因mRNA表达无明显减少,MCF10AT、MCF10CA1d、MCF10CA1h、MCF10DCIS．com的mRNA表达分别减少0．27、0．96、1．78、2．70、2．03倍,MCF10A和MCF-7分别增加0．02和0．33倍）。结论MCF10模型中乳腺癌的发生发展过程与APC基因启动子区异常甲基化无关。     

Identification of a unique epigenetic sub-microenvironment in prostate cancer

The glutathione S-transferase P1 (GSTP1) gene promoter is methylated in tumour cells in more than 90% of prostate carcinomas. Recently, GSTP1 promoter methylation was identified in tumour-associated stromal cells in addition to the tumour epithelium. To define the extent and location of stromal methylation, epigenetic mapping using pyrosequencing quantification of GSTP1 promoter methylation and an anatomical three-dimensional reconstruction of an entire human prostate specimen with cancer were performed. Normal epithelium and stroma, tumour epithelium, and tumour-associated stromal cells were laser capture-microdissected from multiple locations throughout the gland. As expected, the GSTP1 promoter in both normal epithelium and normal stromal cells distant from the tumour was not methylated and the tumour epithelium showed consistently high levels of promoter methylation throughout. However, tumour-associated stromal cells were found to be methylated only in a localized and distinct anatomical sub-field of the tumour, revealing the presence of an epigenetically unique microenvironment within the cancer. Morphologically, the sub-field consisted of typical, non-reactive stroma, representing a genomic alteration in cells that appeared otherwise histologically normal. Similar epigenetic anatomical mapping of a control prostate gland without cancer showed low background methylation levels in all cell types throughout the specimen. These data suggest that stromal cell methylation can occur in a distinct sub-region of prostate cancer and may have implications for understanding tumour biology and clinical intervention.     

4株乳腺癌细胞中内皮素受体B基因的甲基化状态及对MCF-7细胞增殖的影响

目的 探讨内皮素受体B（EDNRB）基因在4株乳腺癌细胞中的表达、甲基化状态以及恢复EDNRB表达对MCF-7细胞增殖的影响。 方法 采用甲基化特异性PCR（MS-PCR）和亚硫酸盐测序法（BSP）分析4株乳腺癌细胞中EDNRB的甲基化状态;反转录聚合酶链反应（RT-PCR）检测EDNRB mRNA的表达水平;采用四甲基偶氮唑蓝（MTT）法和集落形成实验检测EDNRB表达恢复对MCF-7细胞增殖的影响。 结果 EDNRB在乳腺癌细胞MCF-7和ZR-75-1中表达缺失,并呈高甲基化状态;而在EDNRB表达最高的MDA-MB-231细胞中其启动子呈低甲基化,表明乳腺癌细胞中EDNRB基因启动子甲基化状态与其表达成负相关。5-氮杂胞苷（5-Aza-CR）能够反转EDNRB基因的表达,EDNRB表达恢复后MCF-7细胞的增殖受到抑制。 结论 EDNRB基因启动子区CpG岛频繁甲基化可能在乳腺癌发生发展中发挥重要作用,EDNRB有望成为乳腺癌早期诊断的新的分子标志物。     

Down-regulation of BRMS1 by DNA hypermethylation and its association with metastatic progression in triple-negative breast cancer

Breast cancer metastasis suppressor 1 (BRMS1) is a metastasis suppressor gene in several solid tumors. However, the expression and function of BRMS1 in triple-negative breast cancer (TNBC) have not been reported. In this study, we found that BRMS1 was down-regulation in breast cancer cell lines and primary TNBC, while decreased expression of BRMS1 mRNA was significantly associated with lymph node metastasis. And this down-regulation was found to be in accordance with aberrant methylation of the gene. Hypermethylation of the gene was observed in 53.4% (62/116) of the TNBC primary breast carcinomas, while it was found in only 24.1% (28/116) of the corresponding nonmalignant tissues. In addition, BRMS1 expression was restored in MDA-MB-231 after treatment with the demethylating agent, 5-aza-2-deoxycytidine (5-Aza-dC), and demethylation of the highly metastatic cells MDA-MB-231 induced invasion suppression of the cells. Furthermore, the suppression of BRMS1 by siRNA transfection enhanced cancer cells invasion. Collectively, our results suggest that the aberrant methylation of BRMS1 frequently occurs in the down-regulation of BRMS1 in TNBC and that it may play a role in the metastasis of breast cancer.     

PI3K/Akt与染料木黄酮增敏阿霉素体外化疗人乳腺癌 MDA-MB-453 细胞的关系研究

目的 观察染料木黄酮(GEN)对HER2/neu高表达人乳腺癌MDA-MB-453细胞PI3K/Akt信号途径的抑制作用,探讨其在GEN增敏阿霉素(DOX)体外化疗乳腺癌MDA-MB-453细胞中的作用.方法 GEN、PI3K特异性抑制剂渥曼青霉素(WT)和DOX单独或联合处理体外培养的MDA-MB-453细胞,免疫细胞化学法检测HER-2/neu蛋白表达,免疫印迹法检测总Akt和磷酸化Akt蛋白(p-Akt)表达.结果 GEN对MDA-MB-453细胞HER2/neu和总Akt蛋白没有明显影响,但可明显降低p-Akt蛋白水平;同时WT也能明显降低p-Akt蛋白,并显著增强DOX抑制MDA-MB-453细胞生长的作用.结论 GEN增敏DOX体外化疗乳腺癌MDA-MB-453细胞株可能与其抑制PI3K/Akt信号途径有关.     

PI3K/Akt与染料木黄酮增敏阿霉素体外化疗人乳腺癌 MDA-MB-453 细胞的关系研究

目的观察染料木黄酮（GEN）对HER2／neu高表达人乳腺癌MDA-MB-453细胞P13K／Akt信号途径的抑制作用，探讨其在GEN增敏阿霉素（DOX）体外化疗乳腺癌MDA-MB-453细胞中的作用。方法GEN、P13K特异性抑制剂渥曼青霉素（WT）和DOX单独或联合处理体外培养的MDA-MB-453细胞，免疫细胞化学法检测HER-2／neu蛋白表达，免疫印迹法检测总Akt和磷酸化Akt蛋白（p-Akt）表达。结果GEN对MDA-MB-453．细胞HER2／neu和总Akt蛋白没有明显影响，但可明显降低p-Akt蛋白水平；同时WT也能明显降低p-Akt蛋白，并显著增强DOX抑制MDA-MB-453细胞生长的作用。结论GEN增敏DOX体外化疗乳腺癌MDA-MB-453细胞株可能与其抑制P13K／Akt信号途径有关。     

乳腺癌发生模型MCF10各阶段细胞系中TIMP3基因启动子区甲基化分析

目的探讨抑癌基因TIMP3失活与乳腺癌发生和进展的关系。方法用甲基化特异性PCR技术和亚硫酸盐测序技术检测乳腺癌发生模型MCF10的增生细胞系MCF10A、癌前细胞系MCF10AT、导管内癌细胞系MCF10DCIS.com、浸润癌细胞系MCF10CA1a和转移癌细胞系MCF10CA1d、MCF10CA1h中TIMP3启动子区甲基化状态。结果甲基化特异性PCR分析显示,在上述各细胞系中,TIMP3启动子区均呈高度甲基化状态。亚硫酸盐测序显示,在上述各细胞系中,测序区内的68个CG位点几乎全部发生了甲基化,且甲基化累及了绝大部分等位基因。结论TIMP3基因启动子区甲基化在乳腺癌的发生和进展中起重要作用,可能成为早期诊断乳腺癌和判断乳腺癌预后的分子生物学标记。     

乳腺癌中印记基因SLC22A18/TSSC5/BWR1A启动子区甲基化及mRNA表达的研究

目的： 研究印记基因SLC22A18启动子区甲基化对乳腺侵润性导管癌组织中的SLC22A18 mRNA表达的影响，探讨其与临床病理特征之间的关系。方法：实时荧光定量逆转录聚合酶链反应（real-time quantitative RT-PCR）方法检测40例乳腺侵润性导管癌及其癌旁组织中SLC22A18 mRNA的表达，甲基化特异性聚合酶链反应(MSP)检测SLC22A18基因启动子区的甲基化状态。检测DNA甲基转移酶抑制剂5-氮杂-2′-脱氧胞苷（5-aza-2′-deoxycytidine，5-aza-dc）和组蛋白去乙酰化酶抑制剂曲古霉素A（TSA）作用于乳腺癌细胞株MDA-MB-231后，对SLC22A18基因启动子区DNA甲基化和mRNA表达的影响。结果：SLC22A18在40例乳腺侵润性导管癌中mRNA表达量低于癌旁组织（0.12±0.10 vs 0.69±1.05，P＜0.01）；40例乳腺侵润性导管癌及对应癌旁组织中，SLC22A18启动子区的甲基化发生率分别为75％和37.5%，差异有统计学意义（P＜0.01）；在40例乳腺侵润性导管癌组织中，甲基化组SLC22A18 mRNA表达量低于非甲基化组（0.11±0.08 vs 0.24±0.18，P＜0.01）。5-aza-dc和5-aza-dc/TSA能不同程度逆转乳腺癌细胞株MDA-MB-231中SLC22A18基因的甲基化状态，并上调SLC22A18基因表达。结论：SLC22A18基因甲基化与乳腺癌发生有一定的关联，SLC22A18基因表达下调与其启动子区CpG岛异常甲基化相关。     

CD44+/CD24-细胞在乳腺癌组织及细胞系中的数量与分布

目的 检测CD44+/CIY24-细胞在乳腺癌组织及细胞系中的分布及数量,探讨其与乳腺癌常用标志物表达和乳腺癌分子亚型的关系.方法 采用免疫组织化学SP双染及单染法,分别检测了60例乳腺浸润性导管癌中的CD44及C1724的共表达情况和ER、PR、HER2、人雌激素诱导蛋白PS2、bcl-2、nm23的单独表达情况,同时检测了三种乳腺痛细胞系(MCF-7、MDA-MB-468及MDA-MB-231)中CD44及CD24的表达情况.结果 不同病例标本中CD44+/C1724-细胞的数量差异较大,分布无明显规律,总阳性率为65.0%;CD44+/CD24-细胞数量与患者年龄、肿瘤大小、淋巴结转移情况及ER、PR、HER-2、人雌激素诱导蛋白PS2、bcl-2、nm23表达情况无关(P均>0.05);CD44+/CD24-细胞数量与乳腺癌分子亚型无关.CD44+/CIY24-细胞在MCF-7、MDA-MB-468及MDA-MB-231细胞系中的比例分别为<1%、5%及>80%.结论 CD44+/CD24-细胞存在于部分乳腺癌组织及细胞系中,其数量及分布与乳腺癌的分子亚型和临床病理参数无直接关系.     

Clinical and functional significance of loss of caveolin-1 expression in breast cancer-associated fibroblasts

Loss of caveolin-1 (Cav-1) expression in breast cancer-associated fibroblasts (CAFs) is predictive of poor prognosis in breast cancer, but its function has not been established. Our study tested the hypotheses that loss of Cav-1 expression in breast fibroblasts was associated with poor prognosis in breast cancer, through promotion of breast cancer cell invasion. Cav-1 stromal expression was immunohistochemically assessed in 358 breast cancers. Cav-1 expression in primary breast fibroblasts was analysed by western blot. Modified Boyden chamber assays determined fibroblast ability to promote invasion of breast cancer cells. The impact of siRNA silencing of Cav-1 in fibroblasts was evaluated using invasion assays and 3D co-culture assays. Loss of Cav-1 expression in breast stroma was significantly associated with decreased breast cancer-specific and disease-free survival (p = 0.01). Mean survival was 72 months (Cav-1(+) group) versus 29.5 months (Cav-1(-) group). This was confirmed in multivariate analysis. Cav-1 expression was significantly decreased in CAFs compared to normal fibroblasts (p = 0.01) and was associated with increased invasion-promoting capacity. Cav-1 siRNA-treated fibroblasts promoted significantly increased invasion of MDA-MB-468 and T47D breast cancer cells from 27% (control) to 67% (p = 0.006) and from 37% to 56%, respectively (p = 0.01). 3D co-cultures of MDA-MB-468 cells with myoepithelial cells led to the formation of organized cohesive structures when cultured with conditioned media from fibroblasts but resulted in a disorganized appearance in the presence of conditioned media from Cav-1 siRNA-treated fibroblasts, accompanied by loss of E-cadherin expression in tumour cells. Our data confirm that loss of stromal Cav-1 in breast cancer predicts poor outcome. At a functional level, Cav-1-deficient CAFs are capable of significantly increasing the invasive capacity of breast cancer cells.     

Glutathione S-transferase pi (GSTP1) hypermethylation in prostate cancer: review 2007

Prostatic carcinoma is characterised by the silencing of the pi-class glutathione S-transferase gene (GSTP1), which encodes a detoxifying enzyme. The silencing of GSTP1 results from aberrant methylation at the CpG island in the promoter-5' and occurs in the vast majority of cases of high-grade prostatic intraepithelial neoplasia (PIN) and prostate cancers. We review the potential novel role of GSTP1 and its related expression in prostate cancer. The loss of expression (silencing) of the GSTP1 gene is the most common (>90%) genetic alteration reported to date in prostate cancer. Quantitative methylation-specific PCR assays allow detection of GSTP1 methylation in prostate biopsies and may improve the sensitivity of cancer detection. Advances in the epigenetic characterisation of prostate cancer have enabled the development of DNA methylation assays that may soon be used in diagnostic testing of serum and tissue for prostate cancer. Inhibition of aberrant promoter methylation could theoretically prevent carcinogenesis.     

Mutational, epigenetic and expressional analyses of caveolin-1 gene in breast cancers

Caveolin-1, an integral membrane protein of caveolae found in many cell types, has been suggested as a candidate tumor suppressor. However, the mechanism underlying caveolin-1 decreased expression is not fully understood. The purpose of this study is to investigate the role of aberrant promoter methylation in the regulation of caveolin-1 gene in breast cancer correlated with clinical findings. We used methylation specific PCR, direct sequencing and immunohistochemistry stain methods to explore the role of caveolin-1 gene in the development of breast cancer. We demonstrated that 14 of 55 cases (25.5%) and 4 of 55 cases (7.3%) had methylated CpG-island on caveolin-1 promoter in cancerous and non-cancerous cells, respectively. The frequency of aberrant promoter methylation of breast cancer tissues was significant higher than non-cancerous tissues (p<0.05). There were four types of methylation pattern of caveolin-1 gene in the breast cancer tissues. No mutation but one polymorphism GAC right curved arrow GAT at codon 82 was found in the whole exonic sequences of caveolin-1 gene. The methylation status of caveolin-1 gene had no clear relationship with age, cell grade, stage of tumor, and status of estrogen receptor, p53 and c-erbB2 in the breast cancer tissues. However, in breast tissue with aberrant promoter methylation of caveolin-1 gene, the presence of progesterone receptor showed borderline statistic difference compared to unmethylated promoter (p=0.11). Immunohistochemistry demonstrated that expression of caveolin-1 gene correlated with aberrant promoter methylation status in sporadic breast cancer tissues. Our findings suggest that aberrant promoter methylation of caveolin-1 gene is associated with inactivation of expression. This process occurs in the precancerous stage and may play an important role in the development of breast cancer.     

Ezrin and BCAR1/p130Cas mediate breast cancer growth as 3-D spheroids

CAS proteins and Ezrin, Radixin, Moesin (ERM) family members act as intracellular scaffolds and are involved in interactions with the cytoskeleton, respectively. Both protein families have previously been associated with metastasis and poor prognosis in cancer. Our group recently reported on the overexpression of EZR/VIL2 and BCAR1 and their protein products in breast carcinoma effusions compared to primary breast carcinoma. In the present study, the role of these two proteins was studied in semi-normal MCF10A cells and metastatic MDA-MB-231 breast carcinoma cells cultured in tri-dimensional (3-D) conditions that were hypothesized to reproduce the in vivo conditions of breast cancer metastasis. MCF10A cells formed spheroid-shaped colonies without any Matrigel invasion, while MDA-MB-231 cells displayed an invasive phenotype and showed satellite projections that bridged multiple cell colonies in 3-D culture. E-cadherin was expressed in MCF10A, but not in MDA-MB-231 cells. The temporal expression of ezrin and BCAR1/p130Cas at the mRNA and protein level differed in the two cell lines upon 3-D culturing on Matrigel. Upregulation of BCAR1/p130cas was observed in the transition of MDA-MB-231 from attached to detached culture. Silencing of Ezrin and p130Cas in MDA-MB-231 cells by short hairpin RNA resulted in decreased invasive potential, and p130Cas silencing further resulted in smaller spheroid/colony formation. Our data show that MCF10A and MDA-MB-231 cells differ in their ability to form spheroids, in expression of E-cadherin and in the expression of Ezrin and BCAR1/p130Cas in 3-D cultures on Matrigel, suggesting a role in tumor progression in breast carcinoma.