Epigenetic silencing of maspin gene expression in human breast cancers

Maspin is a tumor suppressor whose expression is lost in many advanced breast cancers. Maspin has been shown to inhibit cell motility, invasion and metastasis; however, its precise role in normal mammary epithelium remains to be elucidated. Although expression of maspin mRNA is low or absent in most human breast cancer cells, the maspin gene is rarely re-arranged or deleted. We hypothesized that aberrant cytosine methylation and chromatin condensation of the maspin promoter participates in the silencing of maspin expression during neoplastic progression. To test this hypothesis, we compared cultured normal human mammary epithelial cells (HMECs) to 9 cultured human breast cancer cell lines. HMECs expressed maspin mRNA and displayed a completely non-methylated maspin gene promoter with an open chromatin structure. In contrast, 7 of 9 breast cancer cell lines had no detectable maspin expression and 6 of these 7 maspin-negative breast cancer cell lines also displayed an aberrant pattern of cytosine methylation of the maspin promoter. Interestingly, the maspin promoter was completely methylated in maspin-negative normal peripheral blood lymphocytes. This indicates that the maspin promoter is not a functional CpG island and that cytosine methylation of this region may contribute to normal tissue-restricted gene expression. Chromatin accessibility studies with MCF-7 cells, which lack maspin expression and have a methylated maspin promoter, showed a closed chromatin structure compared with HMECs. Moreover, maspin gene expression could be re-activated in MCF-7 cells by treatment with 5-aza-2;-deoxycytidine, a DNA demethylating agent. Thus, aberrant cytosine methylation and heterochromatinization of the maspin promoter may silence maspin gene expression, thereby contributing to the progression of human mammary cancer.     

Expression and regulation of tumor suppressor gene maspin in human bladder cancer

Maspin is a member of serine protease inhibitor family with tumor suppressing activity for breast and prostate cancers, acting at the level of tumor invasion and metastasis. However, there have been no published data regarding the role of maspin in human bladder cancer. We evaluated maspin expression in 65 series of bladder cancer samples (22 transurethral resection (TUR) and 43 radical cystectomy) and studied the regulatory mechanism of maspin gene activation in bladder cancer cells. Maspin expression was immunohistochemically detected in four (18.2%) patients with TUR and 22 (51.2%) patients with radical cystectomy whereas no expression was observed in normal transitional cells located at tumor-free area in bladder. The maspin expression was significantly correlated with the development of muscle invasive bladder cancer (P=0.00008). Using a luciferase reporter system, maspin promoter activity was induced in the maspin-positive bladder cancer cell lines as well as maspin-negative RT4 cells. Furthermore, treatment with the DNA methyltransferase inhibitor, 5-aza-2' deoxycytidine, and histone deacetylase inhibitor, trichostatin A, led to re-expression of maspin in RT4 cells. Our results indicate that maspin may contribute to bladder cancer development and that DNA methylation and histone deacetylation may be important for regulating maspin gene activation in bladder cancer cells.     

Aberrant maspin expression in human endometrial cancer

Maspin, a mammary serine protease inhibitor, was originally reported as a tumor suppressor gene in breast cancer. The purpose of the present study was to examine maspin expression and evaluate its clinicopathological significance in endometrial cancer. We examined maspin expression immunohistochemically in 41 cases with endometrioid adenocarcinoma. DNA methylation status at the maspin promoter region was determined by the methylation-specific polymerase chain reaction method. Aberrant maspin expression was observed in 27 (66%) of 41 endometrioid adenocarcinomas but not in normal endometrial glands. Maspin immunoreactivity of the tumor cells varied in incidence and density among tumors. Positive staining was correlated significantly with the presence of squamous differentiation (presence vs absence = 11/11 [100%] vs 16/30 [53%], P < 0.05), and nuclear subcellular localization of maspin protein was also significantly associated with squamous differentiation (nuclear positive vs nuclear negative = 6/11 [54%] vs 2/30 [6.7%], P < 0.05). An inverse correlation between their immunoreactivity and methylation status was observed (P < 0.01). Three of the four cell lines established from endometrioid adenocarcinomas overexpressed maspin mRNA and its protein product. In a maspin-negative cell line, maspin expression was induced by treatment with 5-aza-2'-deoxycytidine, a DNA demethylating agent. There was no significant correlation between maspin expression and any clinicopathlogical data. These findings suggest that maspin induced by DNA demethylation at the promoter region may contribute to squamous differentiation of tumor cells in endometrioid adenocarcinomas.     

Expression of the tumor suppressor gene Maspin in human pancreatic cancers.

The tumor suppressor gene maspin, a unique member of the serpin superfamily, inhibits cell motility, invasion, and metastasis in breast and prostate cancers. Maspin is expressed in normal human mammary and prostate epithelial cells but down-regulated during cancer progression. In this study, we analyzed the expression of maspin in various human cancer cells by means of Northern blot and immunohistochemistry. Maspin gene expression proved to be up-regulated in pancreatic cancer. Maspin expression was not detected in any of 6 gastric cancers, 4 melanomas, or 6 of 7 breast cancer cell lines examined. In contrast, 5 of 9 pancreatic cancer cell lines showed maspin expression, although maspin expression was not detected in normal pancreatic tissue. Furthermore, maspin was expressed in 23 of 24 tumor specimens obtained from pancreatic cancer patients as well as all high-grade precancerous lesions (PanIN3 and intraductal carcinoma extension). In contrast, no expression was observed in normal and low-grade precancerous lesions. Our results show that maspin is a new factor associated with pancreatic cancer. In addition, the detection of maspin in pancreatic tumor tissues and its lack of expression in all normal pancreatic tissues suggests that maspin may be a useful marker of primary human pancreatic cancer.     

Clinicopathological significance and molecular regulation of maspin expression in ductal adenocarcinoma of the pancreas

We evaluated the biological relevance of maspin expression in pancreatic ductal adenocarcinoma and studied regulatory mechanisms of maspin gene activation in pancreatic carcinoma cell lines. Maspin expression was immunohistochemically detected in a series of 57 pancreatic ductal adenocarcinomas, 51 (90%) of which were classified as high-expressers. In lymph node metastases, maspin expression was somewhat decreasingly found in 39/49 (80%). Maspin high-expressers showed predominantly a low histological grade (p=0.013). Moreover, maspin expression was found in two mixed ductal-endocrine carcinomas, but not in 10 endocrine tumors and the surrounding normal pancreatic tissues. Using a luciferase reporter system, maspin promoter activity was induced in the maspin-positive pancreatic cancer cell lines as well as maspin-negative PANC-1 cells. Additionally, treatment with the DNA methyltransferase inhibitor, 5-aza-2' deoxycytidine, and histone deacetylase inhibitor, trichostatin A, led to re-expression of maspin mRNA in PANC-1 cells. Our results indicate that maspin expression is up-regulated in most if not all pancreatic ductal adenocarcinomas and may be related to the development and differentiation, and that DNA methylation and histone deacetylation may suppress maspin gene activation in pancreatic cancer cells.     

Regulating the tumor suppressor gene maspin in breast cancer cells: a potential mechanism for the anticancer properties of tamoxifen.

PURPOSE: Mammary epithelial cells and the majority of breast cancer tumors require estrogen for continued growth. Antiestrogen therapy alone, or in combination with other drugs, has long been a common procedure for breast cancer treatment and prophylaxis. Thus, there is a critical need to elucidate the mechanism(s) of action of antiestrogen treatment, especially for patients who are at risk of breast cancer development or who are currently receiving hormone therapy. In this study, we examined the ability of hormones to regulate the expression of a tumor suppressor gene, maspin, which is a serine protease inhibitor (serpin) that plays an important role in mammary gland development and is silenced during breast cancer progression. Specifically, our hypothesis tested the clinical efficacy of tamoxifen to regulate maspin expression. EXPERIMENTAL DESIGN: We used maspin promoter luciferase reporter plasmids that were transfected into normal human mammary epithelial (HMEC1331) and MCF-7 breast cancer cells, followed by determination of the effect of hormones and their antagonists on maspin promoter activity. At the protein level, cytosolic fractions from both cell types before and after hormone treatment were subjected to Western blot analysis to determine maspin level. RESULTS AND CONCLUSIONS: Our studies revealed that the antiestrogen tamoxifen induces maspin promoter activity. Interestingly, antiandrogen flutamide could also induce maspin in both cell lines tested. These observations were further confirmed in patient tissues. These novel findings provide a new mechanism of action for tamoxifen under normal and pathological conditions. More significantly, these findings could have a potential impact on future therapeutic intervention strategies for breast cancer.     

Aberrant methylation of the maspin promoter is an early event in human breast cancer

The maspin gene functions as a tumor suppressor in human breasts, and its expression is frequently lost during breast cancer progression. In vitro models of human breast cancer indicate that the loss of maspin expression is closely linked to aberrant methylation of the maspin promoter. We conducted a study on 30 archival ductal carcinoma in situ (DCIS) specimens to determine if aberrant methylation of the maspin promoter occurred in vivo, and whether it occurred early in breast cancer evolution. Healthy tissue obtained from reduction mammoplasty was used as normal control. Results from immunohistochemical analysis indicate that maspin expression is lost in a substantial fraction of DCIS specimens (57%). Bisulfite sequencing of DNA isolated from laser capture-microdissected normal and neoplastic ducts showed that loss of maspin expression was often, but not always, linked to aberrant methylation of the maspin promoter, suggesting that other mechanisms, in addition to aberrant methylation, participate and/or cooperate to silence maspin gene expression. Taken together, these results indicate that aberrant methylation of the maspin promoter is an early event in human breast cancer.     

Tissue microarray analysis of maspin expression and its reverse correlation with mutant p53 in various tumors

Maspin is a member of serpin family with tumor suppressing activity. Initially identified from normal mammary epithelial cells, maspin expression was down-regulated in breast tumor cells by both in vitro assay and by immunostaining of clinical specimen from breast cancer patients. Recently, maspin research has been advanced to clinical research aimed at correlating the tumor progression with the expression level of maspin in breast cancers. However, due to the variation and large sample sizes, no comparison study of maspin expression has been done using various normal and tumor samples. The tissue microarray is a technique recently developed for the standardization and high-throughput screening of clinical markers. We have used the tissue microarray to examine the maspin expression in various normal tissues and cancers. Our data indicated that maspin was expressed at different level in most of human tissues in the array. However, maspin expression was consistently down-regulated during tumor progression. There were no obvious correlation between maspin expression and tumor grades, nor was there any correlation with the age of patients. Since wild-type p53 was found to activate maspin promoter in vitro, we examined weather there was a connection between p53 level and maspin expression in vivo. Our data indicate that maspin expression inversely correlates with mutant p53 level in majority of cancer, suggesting maspin is likely a p53 target gene in vivo.     

Selenium disrupts estrogen signaling by altering estrogen receptor expression and ligand binding in human breast cancer cells

Cancer prevention studies suggest that selenium is effective in reducing the incidence of cancers including prostate, colon, and lung cancers. Previous reports showed that selenium inhibits premalignant human breast MCF-10AT1 and MCF10AT3B cell growth in vitro and reduces mammary tumor incidence after exposure to carcinogens in tumor models. Because estrogen is critical to the development and differentiation of estrogen target tissues, including the breast, the present study was designed to examine the effect of selenium on estrogen receptor (ER) expression and activation using methylseleninic acid (MSA), an active form of selenium in vitro. Selenium decreased the levels of expression of ERalpha mRNA and protein and reduced the binding of labeled estradiol to estrogen receptor in MCF-7 cells. Selenium inhibited the trans-activating activity of estrogen receptor in MCF-7 cells expressing functional estrogen receptor using a luciferase reporter construct linked to estrogen responsive element. Selenium decreased the binding of estrogen receptor to the estrogen responsive element site using an electrophoretic mobility gel shift assay. Selenium suppressed estrogen induction of the endogenous target gene c-myc. In contrast to the effect on ERalpha in MCF-7 cells, selenium increased ERbeta mRNA expression in MDA-MB231 human breast cancer cells. Thus, differential regulation of ERalpha and ERbeta in breast cancer cells may represent a novel mechanism of selenium action and provide a rationale for selenium breast cancer prevention trial.     

乳腺癌组织中Maspin蛋白表达与maspin基因启动子甲基化关系的研究

背量与目的:探讨乳腺癌与maspin基因失活之间的关系.材料与方法:采用免疫组织化学方法检测30例乳腺癌患者癌组织、癌近旁组织和癌远旁组织中Maspin蛋白的表达情况,用亚硫酸氢钠测序法检测乳腺癌组织maspin启动子CpG岛的甲基化情况.结果:乳腺癌组织中的Maspin蛋白表达明显低于癌近旁组织(P<0.05)和癌远旁组织(P<0.01),乳腺癌组织maspin基因启动子CpG岛甲基化率为98.72%.结论:乳腺癌的发生与nmspin基因失活关系密切,启动子异常甲基化可能是maspin基因在乳腺癌中失活的重要途径.     

Maspin gene expression is a significant prognostic factor in resected non-small cell lung cancer (NSCLC). Maspin in NSCLC

Maspin is a member of the serpin (serine protease inhibitor) superfamily, and some experimental studies revealed a potential tumor suppressor activity of maspin. To reveal clinical significance of maspin status in non-small cell lung cancer (NSCLC), we quantitatively evaluated maspin gene expression in lung primary tumors cut from a total of 55 resected NSCLC patients. Maspin expression in squamous cell carcinoma (Sq) was significantly higher than that in adenocarcinoma (Ad, p=0.011). Five-year overall survival rates of maspin-high and maspin-low patients were 67.7 and 41.4%, respectively, demonstrating a significant favorable prognosis of maspin-high patients (log-rank, p=0.042). A multivariate analysis confirmed that high maspin expression was an independent and significant factor to predict a favorable overall survival (p=0.031). These results suggested that maspin expression was significantly increased in Sq than in Ad, and that increased maspin expression was a significant factor to predict a favorable prognosis in resected NSCLC.     

Genistein sensitizes inhibitory effect of tamoxifen on the growth of estrogen receptor-positive and HER2-overexpressing human breast cancer cells

Although tamoxifen (TAM) is used for the front-line treatment and prevention of estrogen receptor-positive (ER+) breast tumors, nearly 40% of estrogen-dependent breast tumors do not respond to TAM treatment. Moreover, the positive response is usually of short duration, and most tumors eventually develop TAM-resistance. Overexpression of HER2 gene is associated with TAM-resistance of breast tumor, and suppression of HER2 expression enhances the TAM activity. Soy isoflavone genistein has been shown to have anti-cancer activities and suppress expression of HER2 and ERalpha. The objective of this study was to test the hypothesis that genistein may sensitize the response of ER+ and HER2-overexpressing breast cancer cells to TAM treatment. The combination treatment of TAM and genistein inhibited the growth of ER+/HER2-overexpressing BT-474 human breast cancer cells in a synergistic manner in vitro. Determination of cellular markers indicated that this synergistic inhibitory effect might be contributed in part from combined effects on cell-cycle arrest at G(1) phase and on induction of apoptosis. Further determination of the molecular markers showed that TAM and genistein combination synergistically induced BT-474 cell apoptosis in part by synergistic downregulation of the expression of survivin, one of the apoptotic effectors, and downregulation of EGFR, HER2, and ERalpha expression. Our research may provide a novel approach for the prevention and/or treatment of TAM insensitive/resistant human breast cancer, and warrants further in vivo studies to verify the efficacy of genistein and TAM combination on the growth of ER+/HER2-overexpressing breast tumors and to elucidate the in vivo mechanisms of synergistic actions.