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.     

胃癌组织中Maspin 蛋白与血管内皮生长因子-C的表达及其相关性

 目的 探讨胃癌组织中Maspin蛋白与血管内皮生长因子-C（VEGF-C）的表达及其相关性。方法 收集哈尔滨医科大学附属肿瘤医院2002年～2003年间手术切除的胃癌组织石蜡标本61例,采用免疫组化技术检测胃癌组织中Maspin蛋白与血管内皮生长因子-C（VEGF-C）的表达情况,结合临床病理特征进行分析,并探讨两者蛋白表达水平的相关性。结果 Maspin蛋白的阳性表达率为50.8%（31/61）。淋巴结转移阳性组织中Maspin蛋白的阳性表达率明显低于淋巴结转移阴性的组织（32.6%vs94.4%,P=0.000）。且Maspin蛋白在组织分化较低,TNM分期较晚,周围有肿瘤浸润的病例中表达率显著降低（P分别为0.018,0.011和0.028）。VEGF-C在胃癌组织中的阳性表达率为86.9%（53/61）。淋巴结转移阳性组织中VEGF-C蛋白的阳性表达率明显高于淋巴结转移阴性的组织（95.3%vs66.7%,P=0.006）。Maspin蛋白与VEGF-C在胃癌组织的表达存在负相关（Spearmanr=-0.429,P〈0.05）。结论 Maspin蛋白在胃癌组织中低表达,胃癌的浸润转移能力增强可能与Maspin蛋白表达下调、缺失相关;VEGF-C的高表达与肿瘤淋巴结转移关系密切;Maspin蛋白与VEGF-C在胃癌组织中的表达呈明显负相关。     

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.     

Changes in P-glycoprotein expression in gastric carcinoma with respect to distant gastric mucosa may be influenced by p53

BACKGROUND: The effectiveness of some chemotherapeutic agents used to treat gastric carcinoma patients may be impaired by the presence of P-glycoprotein (P-gp) and the status of p53. A modulation of P-gp expression by p53 or other alterations during tumorigenesis have been reported. The authors analyzed P-gp expression in relation to p53 and histopathologic features in gastric carcinoma. METHODS: Forty-one resected gastric carcinomas and mucosa distant from the tumor were assessed for P-gp expression by immunohistochemistry with C494 and JSB-1 antibodies. p53 expression was also immunohistochemically assessed by DO7 antibody in tumor samples. P-gp and p53 expression were semiquantitatively analyzed according to the percentage of stained cells. Histologic type, grade, vessel invasion, and stage were also studied. RESULTS: Moderate or high P-gp expression was detected in gastric carcinoma in 29 cases (71%) and in gastric mucosa remote from the tumor in 36 cases (88%). This reduction in P-gp expression was observed in 22% of the carcinomas, all but 1 being p53 immunonegative tumors. Thus, 8 (42%) of the p53 immunonegative carcinomas showed a loss of P-gp expression compared with their distant gastric mucosa. All p53 immunopositive carcinomas coexpressed P-gp. No correlation between P-gp expression and histologic type, grade, vessel invasion, or stage was found. CONCLUSIONS: P-gp expression in gastric carcinomas is frequent and coexpression with p53 is found. The analysis of P-gp expression in carcinomas and distant mucosa show that it is not regulated by p53, but a loss of P-gp detected in some of these carcinomas is mainly associated with a lack of p53 protein accumulation.     

The paradoxical expression of maspin in ovarian carcinoma.

Maspin is a noninhibitory member of the serpin family that is down-regulated in breast carcinoma but overexpressed in pancreatic carcinoma. There are no published data regarding the role of maspin in ovarian carcinoma, which is the focus of the present study. Ovarian cell lines (normal and cancer) and tumors (80 invasive, 14 benign, and 10 low malignant potential) were evaluated for maspin expression and localization. Normal ovarian surface epithelial cells had low levels of maspin. Two of four ovarian cancer cell lines (OVCAR3 and SKOV3) expressed maspin, whereas the cell line EG had weak expression, and 222 had no detectable maspin. Subcellular fractionation studies revealed that the two maspin-positive ovarian cancer cell lines contained maspin in both the nuclear and cytosolic compartments. Wild-type maspin was transfected into the aggressive ovarian cancer cell lines SKOV3 and 222. The in vitro invasive activity of the maspin-transfected cell lines was 44-68% lower than respective controls. The histopathology analysis revealed that among the ovarian tumors examined, 57 (71%) were ranked positive for maspin. Thirty (37%) of the invasive tumors overexpressed maspin. Invasive cancers were more likely to have predominantly cytoplasmic staining compared with benign and low-malignant-potential tumors. Maspin overexpression was significantly associated with a high tumor grade (P = 0.004), the presence of ascites (P = 0.02), a lower likelihood of optimal surgical cytoreduction (P = 0.04), and a shorter duration of overall survival (median survival, 6.33 versus 2.67 years; P = 0.003). The Cox proportional hazards multivariate model revealed that maspin overexpression and high stage were independent predictors of survival. Thus, maspin was found to be overexpressed in a substantial proportion of ovarian tumors, which may serve as an adverse prognostic factor; however, its localization may provide new clues as to its activity and function. These paradoxical results may offer new insights regarding the role of maspin in ovarian cancer progression that may also impact diagnosis and treatment strategies.     

Maspin nuclear localization is linked to favorable morphological features in pulmonary adenocarcinoma

Maspin, a mammary homologue of Serine Protease Inhibitors, has been shown to inhibit tumor progression and metastasis. Recently, its biological functions have been linked to its subcellular localization. Specifically, a nuclear, opposed to a combined nuclear and cytoplasmic localization has been associated with increased survival in human malignancies, including non-small cell lung cancer (NSCLC). However, it is not known whether transformation affects maspin expression during lung carcinogenesis, and whether its subcellular localization correlates with the morphological features of NSCLC. To address these questions, we studied maspin expression in a model of transformation of bronchial epithelial cells and in resected NSCLC. We found that decreased maspin accompanied chemical transformation of normal immortalized bronchial epithelial cells BEAS 2B. Immunohistochemistry revealed maspin expression to be virtually universal in NSCLC, occurring in 72/77 Adenocarcinoma (ACa), and 46/46 squamous cell carcinoma (SqCCa). SqCCa showed almost exclusively a combined nuclear-cytosolic stain. In contrast, nuclear maspin, but not combined nuclear-cytoplasmic maspin significantly correlated with low histological grade, lower proliferative rate, absence of invasion, and negative p53 stain in ACa. These data support the hypothesis that nuclear localization of maspin may stratify subtypes of NSCLC with favorable clinical-pathological features.     

Maspin在非小细胞肺癌中的表达及与p53的相关性

目的探讨maspin在非小细胞肺癌中的表达及其与p53的关系。方法对99例非小细胞肺癌组织maspin及p53蛋白的表达情况通过免疫组化法进行检测,分析其临床病理意义及相关性。结果 maspin蛋白表达与肿瘤病理类型、肿瘤分化程度、淋巴结状态及临床分期相关（P〈0.05）。而p53蛋白的表达则与临床分期、淋巴结状态相关（P〈0.05）。maspin与p53蛋白的表达呈负相关（γ=-0.180）,但无统计学意义（P=0.075）。结论 Maspin及p53蛋白的表达在非小细胞肺癌的发生、发展过程中起重要作用,但本研究未发现两者具有统计学意义的相关性。     

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.     

Maspin expression in early oral tongue cancer and its relation to expression of mutant-type p53 and vascular endothelial growth factor (VEGF)

Even though oral tongue cancer is generally diagnosed at an early stage, the prognosis is poor due to frequent recurrence. Therefore, it is important to identify factors predictive of recurrence and to treat aggressively those patients with a high probability of recurrence. The relationship between angiogenesis and recurrence in tongue cancer has been widely investigated but no consensus has been reached. Mutant-type p53 and VEGF are known to be related to angiogenesis, and maspin is a potent angiogenic inhibitor but its role in tongue cancer has scarcely been examined. We observed the expression of maspin, mutant-type p53 and VEGF by immunohistochemistry in 33 patients with stages I and II oral tongue cancer. And the relationships between maspin, mutant-type p53, VEGF expression and recurrence were analyzed. Maspin and VEGF displayed a cytoplasmic staining pattern and mutant-type p53 a nuclear pattern. None of expression of maspin, mutant-type p53, and VEGF was significantly correlated with tumor recurrence (p=0.34, 0.56, and 0.33, respectively) and survival. Maspin expression was negatively correlated with both mutant-type p53 expression (p=0.02), and VEGF expression (p=0.01). There was no correlation between age, sex, clinical staging, and recurrence. In conclusion, the expression of maspin is not related to recurrence of early stage oral tongue cancer. It is inversely correlated with that of mutant-type p53 and of VEGF, suggesting that the maspin gene is a mutant-type p53 target in vivo and may contribute to regulate VEGF expression.     

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.     

Pleiotrophic inhibition of pericellular urokinase-type plasminogen activator system by endogenous tumor suppressive maspin.

Maspin is a novel serine protease inhibitor with tumor suppressive activity, inhibiting tumor invasion and metastasis. To date, the underlying molecular mechanism of maspin remains elusive. Recombinant maspin has been shown to specifically inhibit cell surface-associated urokinase-type plasminogen activator (uPA) and fibrinogen-bound tissue-type plasminogen activator. However, the role of endogenous maspin in plasminogen activation is totally unknown. To address this issue, we generated stable maspin-expressing transfectants using prostate carcinoma cells DU145 as the parental cell line. We report here that endogenous maspin exerts pleiotropic inhibitory effects on the pericellular uPA system. Maspin expression led to a significantly reduced level of cell surface-bound uPA and uPA receptor proteins without altering the steady-state levels of the respective mRNAs. Treatment with receptor-associated protein (RAP), a specific inhibitor of low-density lipoprotein receptor-related protein, lead to a significantly increased level of secreted uPA and cell surface uPAR in maspin transfectants but not in the mock control cells. A combination of enzymatic and molecular analyses revealed that maspin inhibits the cell surface-mediated plasminogen activation by forming an SDS-resistant complex with cell surface-bound uPA. In addition, maspin expression led to a dramatic reduction in the release of active uPA, both high molecular weight and the low molecular weight, into the conditioned culture medium. Consistently, the conditioned medium of maspin transfectant clones had a significantly reduced activity in converting plasminogen to plasmin. The inhibitory effect of maspin on pericellular uPA correlates with significantly decreased cell invasion potential and motility in vitro. The maspin-neutralizing antibody (Abs4A) reversed the subdued invasive potential of maspin transfectant cells in a dose-dependent manner. In summary, this study provides the first evidence that endogenous maspin is a potent inhibitor of pericellular uPA. Furthermore, our results support a current hypothesis that maspin blocks tumor invasion and motility by inhibiting localized pericellular proteolysis.