2810

SFN

stratifin;SFN;stratifin

14-3-3 protein sigma|14-3-3 sigma|epithelial cell marker protein 1

1p36.11

protein-coding

BACKGROUND: Numerous hypermethylated genes have been reported in breast cancer, and the silencing of these genes plays an important role in carcinogenesis, tumor progression and diagnosis. These hypermethylated promoters are very rarely found in normal breast. It has been suggested that aberrant hypermethylation may be useful as a biomarker, with implications for breast cancer etiology, diagnosis, and management. The relationship between primary neoplasm and metastasis remains largely unknown. There has been no comprehensive comparative study on the clinical usefulness of tumor-associated methylated DNA biomarkers in primary breast carcinoma and metastatic breast carcinoma. The objective of the present study was to investigate the association between clinical extension of breast cancer and methylation status of estrogen receptor1 (ESR1) and stratifin (14-3-3-sigma) gene promoters in disease-free and metastatic breast cancer patients. METHODS: We studied two cohorts of patients: 77 patients treated for breast cancer with no signs of disease, and 34 patients with metastatic breast cancer. DNA was obtained from serum samples, and promoter methylation status was determined by using DNA bisulfite modification and quantitative methylation-specific PCR. RESULTS: Serum levels of methylated gene promoter 14-3-3-sigma significantly differed between Control and metastatic Breast cancer groups (P < 0.001), and between Disease-Free and metastatic Breast cancer groups (P < 0.001). The ratio of the 14-3-3-sigma level before the first chemotherapy cycle to the level just before administration of the second chemotherapy cycle was defined as the Biomarker Response Ratio [BRR]. We calculated BRR values for the "continuous decline" and "rise-and-fall" groups. Subsequent ROC analysis showed a sensitivity of 75% (95% CI: 47.6 - 86.7) and a specificity of 66.7% (95% CI: 41.0 - 86.7) to discriminate between the groups for a cut-off level of BRR = 2.39. The area under the ROC curve (Z = 0.804 +/- 0.074) indicates that this test is a good approach to post-treatment prognosis. CONCLUSIONS: The relationship of 14-3-3-sigma with breast cancer metastasis and progression found in this study suggests a possible application of 14-3-3-sigma as a biomarker to screen for metastasis and to follow up patients treated for metastatic breast cancer, monitoring their disease status and treatment response.

PURPOSE: Cholangiocarcinoma (CCA) is a lethal malignancy that afflicts thousands of patients worldwide. Stratifin has been shown to participate in mediating G2 arrest in several cancers, and cells that express stratifin could contribute to the chemo- and radioresistance of cancers and poor prognosis. However, the clinical impact of stratifin on clinicopathological features of mass forming (MF)-CCA is still unclear. METHODS: Seventy-eight patients with MF-CCA who had undergone hepatectomy were selected for an immunohistochemical study of stratifin. Sixteen clinicopathological variables were used for the survival analyses. RESULTS: Seventy-eight MF-CCA patients (36 men and 42 women) were studied. Cytoplasmic immunostaining with membrane prominence was found in 52.6% (41/78) of patients with MF-CCA after hepatectomy; this was significantly associated with elevated carcinoembryonic antigen (CEA) levels. During the median follow-up duration of 13.6 months, the 5-year overall survival (OS) rate was 14.9%. Univariate analysis showed that an absence of clinical symptoms, better nutritional status, lower alkaline phosphatase, smaller tumor, negative lymph node metastasis, negative stratifin staining, and curative hepatic resection were associated with favorable OS rate for MF-CCA patients after hepatectomy. Multivariate Cox proportional hazard analysis showed that the absence of clinical symptoms, negative lymph node metastasis, and curative hepatectomy independently predicted MF-CCA patients with favorable OS rate after hepatectomy. CONCLUSIONS: Overexpression of stratifin was significantly associated with elevated CEA levels in patients with MF-CCA. The favorable OS for MF-CCA patients depends on the absence of clinical symptoms, negative lymph node metastasis, and curative hepatectomy.

OBJECTIVE: To investigate the expression of 14-3-3 sigma and heat shock protein 27 (HSP27) in colorectal carcinoma(CRC) tissue and its clinical significance. METHODS: The expression of 14-3-3 sigma and HSP27 was detected by immunohistochemical staining in 50 pathologically verified CRC cases. The association of clinical data with 14-3-3 sigma and HSP27 expression was examined. RESULTS: The positive expression rate of 14-3-3 sigma was 10% in normal control mucosa and 58% in CRC tissue (P<0.01). The positive expression rate of HSP27 was 16% in normal control mucosa and 54% in CRC tissue (P<0.01). No correlation between 14-3-3 sigma and HSP27 expression was found in CRC tissue (P>0.05). The expression of 14-3-3 sigma was associated with patient age, tumor diameter and lymph node metastasis (LNM) (P<0.05), but not with gender, tumor differentiation or serous membrane invasion (P>0.05). The expression of HSP27 was associated with LNM (P<0.05), but not with gender, age, differentiation, tumor diameter or serous membrane invasion (P>0.05). CONCLUSION: The abnormal expression of 14-3-3 sigma and HSP27 is significantly associated with LNM in CRC.

beta-catenin has emerged as a key regulator of Wnt signaling pathway, which plays an important role in the development and progression of various cancers. Its accumulation in nucleus of the esophagus squamous epithelium might be the crucial step for the carcinogenesis of esophageal squamous cell carcinoma (ESCC). To detect the proteins correlated with beta-catenin function, we used the established cell lines of pGen-3-con (Eca109 cells transfected by control vector) and pGen-3-CTNNB1 (Eca109 cells transfected by beta-catenin siRNA) as cell models for further analysis. Two-dimensional gel electrophoresis technology was performed to separate the proteins of pGen-3-con and pGen-3-CTNNB1 cell lines, respectively. The differential protein spots were analyzed by software analysis, subjected to in-gel digestion, and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Consequently, 13 differentially expressed proteins between the two cell lines were identified, of which 14-3-3sigma, prohibitin, and nm23-H1 were further verified by western blotting and quantitative real-time reverse transcriptase-polymerase chain reaction. Then, the tissue microarray and immunohistochemical analysis were employed to research their relationship in ESCC and their corresponding normal mucosa tissues. The upregulation of prohibitin or the downregulation of 14-3-3sigma and nm23-H1 proteins was significantly associated with the proliferation, invasion depth, and lymph node metastasis of ESCC. There were statistically significant correlations between the expression of beta-catenin and the three proteins. The results presented here might provide potential protein markers to elucidate the mechanism of beta-catenin-mediated biologic characteristics for ESCC.

In this study, we aim to screen metastasis-related proteins in human lung squamous carcinoma (LSC) using laser capture microdissection and a proteomic approach. Twenty two differential proteins were identified from pooled microdissected primary LSC and matched lymph node (LN) metastatic tissues. expression of the differential protein 14-3-3 sigma was determined by Western blotting and immunohistochemistry. In cell invasion assay, down-regulated 14-3-3 sigma by siRNA increased in vitro invasive ability of HTB-182 and A549 cells, up-regulation of 14-3-3 sigma by pcDNA3.0/14-3-3 sigma decreased in vitro invasive ability of HTB-182 and A549 cells. The data suggest that 14-3-3 sigma is a potential LN metastasis-related protein in LSC, and its dysregulation might play an important role in the LN metastatic process of LSC.

14-3-3 sigma (sigma) is a negative regulator of the cell cycle and contributes to G2 arrest. Lack of its expression due to hypermethylation of CpG islands has been reported in some carcinomas. A recent study showed that 14-3-3 sigma was down-regulated through proteolysis by estrogen-responsive finger protein (Efp). Here, we investigated the expression of 14-3-3 sigma, hormone receptors, Efp and p53 in 86 cases of endometrial adenocarcinoma and 46 cases of normal or non-neoplastic endometria by means of immunohistochemistry and methylation-specific polymerase chain reaction. In normal endometrium, 14-3-3 sigma was overexpressed in the mid- to late-secretory phase due to hypomethylation. In endometrial adenocarcinoma, 14-3-3 sigma expression was low in low grade endometrioid adenocarcinoma due to hypermethylation, and increased significantly with increasing histological grade due to hypomethylation. 14-3-3 sigma expression inversely correlated with estrogen receptor alpha, progesterone receptor and Efp, and positively correlated with myometrial invasion and lymph node metastasis. These results suggest that 14-3-3 sigma was one of the menstrual cycle-related proteins regulated by epigenetic methylation, and its expression was influenced by epigenetic methylation or hormone receptors in progression of endometrial adenocarcinoma, and therefore was more than just a cell-cycle regulator.

14-3-3 sigma, the downstream target of p53, is a negative regulator of cell cycle G2-M phase checkpoint in response to DNA damage. Our previous comparative proteomics study showed that 14-3-3 sigma was downregulated or lost in nasopharyngeal carcinoma (NPC) tissue compared with non-cancerous nasopharyngeal epithelial tissue (NNET). In this study, we further investigated for the epigenetic mechanism of 14-3-3 sigma inactivation. Methylation-specific PCR showed 14-3-3 sigma promoter methylation in 100% of analyzed NPC cell lines (4/4) but not in immortalized human nasopharyngeal epithelial cell line NP69. Treatment of the four NPC cell lines with the methyltransferase inhibitor 5-aza-2'-dC resulted in the demethylation and upregulation of 14-3-3 sigma. In tissues, 14-3-3 sigma promoter methylation occurred at a higher frequency in NPC, 63/75 (84%), compared to adjacent NNET, 7/25 (28%), and fully methylated 14-3-3 sigma promoter was detected in NPC but not in any of adjacent NNET. RT-PCR, Western blotting, and immunohistochemistry showed that 14-3-3 sigma expression was downregulated or lost in NPC with methylation, and there was a negative correlation between the expression levels and methylation statuses of 14-3-3 sigma gene. In addition, the patients with methylated 14-3-3 sigma presented a higher frequency of lymph node and distant metastasis, and an advanced clinical stage, and overexpression of 14-3-3 sigma in NPC cell line 5-8F with high metastatic potential was able to inhibit its in vitro invasive ability. Our data are the first to show that 14-3-3 sigma is frequently inactivated by promoter methylation in NPC and this aberrant methylation correlates with lymph node and distant metastasis.

BACKGROUND: In vitro cell culture experiments with primary cells have reported that cell proliferation is retarded in the presence of ambient compared to physiological O(2) levels. cancer is primarily a disease of aberrant cell proliferation, therefore, studying cancer cells grown under ambient O(2) may be undesirable. To understand better the impact of O(2) on the propagation of cancer cells in vitro, we compared the growth potential of a panel of ovarian cancer cell lines under ambient (21%) or physiological (3%) O(2). PRINCIPAL FINDINGS: Our observations demonstrate that similar to primary cells, many cancer cells maintain an inherent sensitivity to O(2), but some display insensitivity to changes in O(2) concentration. Further analysis revealed an association between defective G2/M cell cycle transition regulation and O(2) insensitivity resultant from overexpression of 14-3-3 sigma. Targeting 14-3-3 sigma overexpression with RNAi restored O(2) sensitivity in these cell lines. Additionally, we found that metastatic ovarian tumors frequently overexpress 14-3-3 sigma, which in conjunction with phosphorylated RB, results in poor prognosis. CONCLUSIONS: cancer cells show differential proliferative sensitivity to changes in O(2) concentration. Although a direct link between O(2) insensitivity and metastasis was not determined, this investigation showed that an O(2) insensitive phenotype in cancer cells to correlate with metastatic tumor progression.

14-3-3sigma proteins regulate numerous cellular processes that are important to cancer development. One of its biological roles involves G2 cell-cycle arrest following DNA damage. It has also been reported that the loss of 14-3-3sigma expression via CpG methylation may contribute to malignant transformation by impairing the G2 cell-cycle checkpoint function, thereby allowing an accumulation of genetic defects. However, how the CpG methylation-dependent silencing mechanism works in relation to promoter methylation associated with methyl-CpG-binding proteins (MeCPs) is still unclear. To better understand the mechanism, we first examined the methylation status of the 14-3-3sigma promoter-associated CpG islands and 14-3-3sigma gene expression in a subset of prostate cancer cell lines using methylation-specific PCR (MSP), an HhaI-based DNA methylation assay, and reverse transcription-PCR (RT-PCR). We found that the 14-3-3sigma expression is lost in LNCaP and Tramp-C1 prostate cancer cell lines and that this expression is restored after treatment with epigenetic silencing modifiers 5-aza-2'-deoxycytidine (5-aza) and trichostatin A (TSA). These results imply transcriptional silencing via promoter-associated CpG methylation. Chromatin immunoprecipitation analysis revealed that methyl-CpG-binding protein 2 (MBD2) is associated preferentially to the methylated CpG island in the 14-3-3sigma promoter in LNCaP and Tramp-C1 cells but not in 14-3-3sigma-expressing PC3 and DU145 cells, which contain an unmethylated CpG island in the 14-3-3sigma promoter region. The 14-3-3sigma gene silencing because of CpG methylation correlates with binding of MBD2. In addition, the activation of 14-3-3sigma gene expression by a combination of 5-aza and TSA also involves the release of the MBD2 from the 14-3-3sigma promoter-methylated CpG island in LNCaP and Tramp-C1 cells. Furthermore, MBD2 knockdown by siRNA stimulated 14-3-3sigma expression in LNCaP cells. We also investigated whether the loss of 14-3-3sigma expression in LNCaP and Tramp-C1 cells affects cell proliferation by MTT assays. Interestingly, we observed that 14-3-3sigma-inactivated LNCaP and Tramp-C1 cells had markedly decreased cell proliferation and protein expression of proliferation cell nuclear antigen (PCNA) after restoration of 14-3-3sigma expression with 5-aza and TSA treatment. On the other hand, the same treatment did not significantly affect 14-3-3sigma-active PC3 and DU145 cells, which normally express 14-3-3sigma. Finally, 14-3-3sigma knockdown by siRNA resulted in increased proliferation in PC3 and DU145 cells. These findings suggest that the transcriptional silencing of the 14-3-3sigma gene is caused by promoter CpG island methylation associated with MBD2, and that this may play an important role in prostate cancer progression during the invasive and metastatic stages of the disease.