Etsuko Miyagi

Activation of Cancer Cell Migration and Invasion by Ectopic Synthesis of Coagulation Factor VII

Blood coagulation factor VII (fVII) is physiologically synthesized in the liver and released into the blood. Binding of fVII to tissue factor (TF) at sites of vascular injury triggers coagulation and hemostasis. TF/fVIIa complex formation on the surface of cancer cells plays important roles in cancer biology. Although fVII is synthesized by hepatocellular carcinoma, it remained unclear how TF/fVIIa complex formation and promigratory signaling can occur for most other cancers in extravascular locations. Here, we show by reverse transcription-PCR analysis that nonhepatic cancer cell lines constitutively express fVII mRNA and that endogenously synthesized fVIIa triggers coagulation activation on these cells. fVIIa expression in cancer cells is inducible under hypoxic conditions and hypoxia-inducible factor-2 alpha bound the promoter region of the FVII gene in chromatin immunoprecipitation analyses. Constitutive fVII expression in an ovarian cancer cell line enhanced both migration and invasion. Enhanced motility was blocked by anti-TF antibodies, factor Xa inhibition, and anti-protease-activated receptor-1 antibody treatment, confirming that TF/fVIIa stimulated migration by triggering cell signaling. This study shows that ectopic synthesis of fVII by cancer cells is sufficient to support proinvasive factor Xa-mediated protease-activated receptor-1 signaling and that this pathway is inducible under hypoxia.

High-Risk Ovarian Cancer Based on 126-Gene Expression Signature Is Uniquely Characterized by Downregulation of Antigen Presentation Pathway

Purpose: High-grade serous ovarian cancers are heterogeneous not only in terms of clinical outcome but also at the molecular level. Our aim was to establish a novel risk classification system based on a gene expression signature for predicting overall survival, leading to suggesting novel therapeutic strategies for high-risk patients. Experimental Design: In this large-scale cross-platform study of six microarray data sets consisting of 1,054 ovarian cancer patients, we developed a gene expression signature for predicting overall survival by applying elastic net and 10-fold cross-validation to a Japanese data set A (n = 260) and evaluated the signature in five other data sets. Subsequently, we investigated differences in the biological characteristics between high- and low-risk ovarian cancer groups. Results: An elastic net analysis identified a 126-gene expression signature for predicting overall survival in patients with ovarian cancer using the Japanese data set A (multivariate analysis, P = 4 × 10−20). We validated its predictive ability with five other data sets using multivariate analysis (Tothills data set, P = 1 × 10−5; Bonomes data set, P = 0.0033; Dressmans data set, P = 0.0016; TCGA data set, P = 0.0027; Japanese data set B, P = 0.021). Through gene ontology and pathway analyses, we identified a significant reduction in expression of immune-response–related genes, especially on the antigen presentation pathway, in high-risk ovarian cancer patients. Conclusions: This risk classification based on the 126-gene expression signature is an accurate predictor of clinical outcome in patients with advanced stage high-grade serous ovarian cancer and has the potential to develop new therapeutic strategies for high-grade serous ovarian cancer patients. Clin Cancer Res; 18(5); 1374–85. ©2012 AACR.

Uterine carcinosarcoma is derived from a single stem cell: An in vitro study

Expression of intermediate filaments (IFs) has been suggested to be a reliable marker for differentiating epithelial and non‐epithelial tumors. Moreover, the c‐erbB‐2 and p53 genes are considered to be involved relatively early in the process of human carcinogenesis. In order to elucidate the origin of uterine carcinosarcomas, we analyzed IF, c‐erbB‐2 and p53 expression in and the ultrastructural characteristics of clones derived from a human uterine‐carcinosarcoma cell line, EMTOKA. The expression of IFs and other proteins in the EMTOKA clones was identical to that in the EMTOKA cell line. It and its 7 clones all expressed cytokeratins 8, 17, 18 and 19, vimentin, epithelial membrane antigen, S‐100, myoglobin, type‐II collagen, α‐smooth‐muscle actin, placental alkaline phosphatase and epidermal‐growth‐factor receptor. The c‐erbB‐2 and p53 expression levels of all the cell types of the EMTOKA cell line and its clones were the same. Interestingly, an ultrastructural study showed that the EMTOKA cell line and its clones at early and late passages possessed the characteristics of epithelial cell types without either transitional forms between the epithelial and stromal components or differentiation into sarcomatous components. The results of this study lend particular support to the combination tumor hypothesis that a precursor (stem) cell gives rise both to epithelial and to mesenchymal components during the histogenesis of uterine carcinosarcoma, the epithelial component of which appears to be dominant, suggesting that the established cell lines derived from a common stem cell. Int. J. Cancer 72:821–827, 1997.

Value of 18F-FDG PET in the detection of peritoneal carcinomatosis

PurposePeritoneal carcinomatosis can be difficult to diagnose using computed tomography (CT). The purpose of this study was to evaluate the role of 2-(fluorine 18) fluoro-2-deoxy-d-glucose (FDG) positron emission tomography (PET) in the detection of peritoneal carcinomatosis.MethodsWe reviewed the CT and FDG PET radiological reports and clinical charts of 18 patients with peritoneal carcinomatosis and 17 cancer patients without peritoneal carcinomatosis. We also assessed FDG PET scans from 20 healthy volunteers as a baseline study. The maximum standardised uptake values (SUVmax) over peritoneal lesions in cancer patients and over the area of most intense intestinal uptake in healthy volunteers and cancer patients without peritoneal carcinomatosis were measured.ResultsThe sensitivity and positive predictive value (PPV) of combined FDG PET and CT were superior to those of CT alone for the detection of peritoneal lesions (sensitivity: 66.7% vs 22.2%, p<0.025; PPV: 92.3% vs 50.0%, p<0.05). The most frequent pattern of FDG uptake in patients with peritoneal carcinomatosis was abnormally intense focal uptake near the abdominal wall. An SUVmax threshold of 5.1 produced a diagnostic accuracy of combined FDG PET and CT of 78%. The additional information provided by FDG PET allowed a more accurate diagnosis in 14 patients (40.0%), and led to alteration of the therapeutic strategy in five (14.3%) of the enrolled cancer patients.ConclusionWe found that use of an intra-abdominal FDG uptake cut-off value for SUVmax of >5.1 assists in the diagnosis of peritoneal carcinomatosis. FDG PET may play an important role in the clinical management of patients with suspected peritoneal carcinomatosis.

Specific expression of PP5/TFPI-2 mRNA by syncytiotrophoblasts in human placenta as revealed by in situ hybridization

Placental protein 5 (PP5) is a placenta-derived glycoprotein with serine proteinase-inhibiting activity. To date its physiological functions have not been well elucidated. Recently, cDNA sequence analysis revealed that PP5 belongs to the Kunitz-type proteinase inhibitor family and it is identical to tissue factor pathway inhibitor-2 (TFPI-2), homologous to TFPI. Northern blot analysis demonstrated that placental tissue is extremely rich in the transcripts. This study localized PP5/TFPI-2 mRNA in placental tissues at three different gestational periods using in situ hybridization. PP5/TFPI-2 mRNA was specifically detected in syncytiotrophoblast at any gestational period examined, suggesting that syncytiotrophoblast is the principal production site of PP5/TFPI-2 in developing placental tissues. This mRNA expression pattern of PP5/TFPI-2 is quite different from that of TFPI, which is mainly found in vascular endothelial cells. The results indicated possible roles of PP5/TFPI-2 in the trophoblast differentiation and in the maintenance of intervillous blood flow. Also, Northern analysis demonstrated no or little expression of PP5/TFPI-2 in four choriocarcinoma cell lines, in contrast to its abundant expression in syncytiotrophoblast.

Self-production of tissue factor-coagulation factor VII complex by ovarian cancer cells

Background:Thromboembolic events are a major complication in ovarian cancer patients. Tissue factor (TF) is frequently overexpressed in ovarian cancer tissue and correlates with intravascular thrombosis. TF binds to coagulation factor VII (fVII), changing it to its active form, fVIIa. This leads to activation of the extrinsic coagulation cascade. fVII is produced by the liver and believed to be supplied from blood plasma at the site of coagulation. However, we recently showed that ovarian cancer cells express fVII transcripts under normoxia and that this transcription is inducible under hypoxia. These findings led us to hypothesise that ovarian cancer cells are intrinsically associated with TF-fVIIa coagulation activity, which could result in thrombosis.Methods:In this study, we examined whether ectopically expressed fVII could cause thrombosis by means of immunohistochemistry, RT–PCR, western blotting and flow cytometry.Results:Ectopic fVII expression occurs frequently in ovarian cancers, particularly in clear cell carcinoma. We further showed that ovarian cancer cells express TF-fVIIa on the cell surface under normoxia and that this procoagulant activity is enhanced by hypoxic stimuli. Moreover, we showed that ovarian cancer cells secrete microparticles (MPs) with TF-fVIIa activity. Production of this procoagulant secretion is enhanced under hypoxia.Conclusion:These results raise the possibility that cancer cell-derived TF-fVIIa could cause thrombotic events in ovarian cancer patients.

The Japanese Guideline for Cervical Cancer Screening

Cervical cancer is the 11th leading cause of death from cancer for females in Japan. In 2005, there were 2486 deaths from cervical cancer, accounting for 1.8% of the total number of cancer deaths in Japan. Cervical cancer screening using conventional cytology has been conducted worldwide. The guideline for cervical cancer screening was developed based on the established method. The efficacies of conventional and liquid-based cytology, human papillomavirus testing alone and two combination methods were evaluated. On the basis of the balance of the benefits and harms, recommendations for population-based and opportunistic screening were formulated. Five methods of cervical cancer screening were evaluated. On the basis of the analytic framework involving key questions, 3450 articles published from January 1985 to October 2007 were selected using MEDLINE and other methods. After the systematic literature review, 66 articles were confirmed. The results of 33 studies were consistent, and the evidence was sufficient to evaluate the effect of conventional cytology screening. The accuracy of liquid-based cytology was almost equal to that of conventional cytology. Although human papillomavirus testing and combination methods showed high sensitivity, no study has evaluated the reduction in mortality from cervical cancer. Except for the possibility of overdiagnosis, no serious adverse effects of cervical cancer screening were found. Cervical cancer screening using conventional and liquid-based cytology is recommended for population-based and opportunistic screening due to sufficient evidence. Cervical cancer screening using either human papillomavirus testing alone or two combination methods is not recommended for population-based screening due to insufficient evidence.

Characterization of Matrix-degrading Proteinases and Their Inhibitors Secreted by Human Gynecological Carcinoma Cells

Matrix‐degrading proteinases secreted by tumor cells play crucial roles in tumor cell invasion and metastasis. Serum‐free conditioned media of 7 human gynecological carcinoma cell lines were examined for proteinases and their inhibitors by using gelatin zymography, reverse zymograpby and immunoblotting. All of three ovarian adenocarcinoma cell lines secreted urokinase‐type plasminogen activator. Among them, a mucinous cystadenocarcinoma cell line also secreted tissue‐type plasminogen activator, plasmin‐like enzyme and trypsinogen. On the other hand, two ovarian undifferentiated carcinoma cell lines mainly secreted gelatinase A or B. A choriocarcinoma cell line secreted multiple metalloproteinases in the highest amount, whereas an endometrial adenocarcinoma cell line (HEC‐1) derived from an early clinical stage hardly secreted any gelatinolytic enzyme. The five high proteinases producers hardly secreted the corresponding inhibitors, such as tissue inhibitor of metalloproteinases (TIMP)‐1,‐2 or plasminogen activator inhibitor‐1. In contrast to these highly malignant cell lines, a poor proteinase producer, HEC‐1, secreted a large amount of TIMPs. Therefore, an enhanced proteolytic tendency appears to be associated with gynecological cancer cells established from highly malignant tumors.

Hepatocyte Nuclear Factor-4-Independent Synthesis of Coagulation Factor VII in Breast Cancer Cells and Its Inhibition by Targeting Selective Histone Acetyltransferases

Tissue factor/coagulation factor VII (fVII) complex formation on the surface of cancer cells plays important roles in cancer biology, such as cell migration and invasion, angiogenesis, and antiapoptotic effects. We recently found that various cancer cells ectopically synthesize fVII, resulting in activation of cell motility and invasion. Here, we characterized mechanisms of hepatic and ectopic fVII (FVII) gene expression to identify molecular targets enabling selective inhibition of the ectopic expression. Unlike hepatic expression, hepatocyte nuclear factor-4 binding to the promoter is not required for ectopic FVII expression, although Sp1 binding is essential. Furthermore, we found novel nuclear targets of basal hepatocytic and ectopic FVII expression. Notably, histone acetyltransferases p300 and cyclic AMP–responsive element binding protein–binding protein (CBP) are exclusively recruited to the promoter region of the FVII gene specifically in breast cancer cells. We further show that curcumin, a dietary compound, can selectively inhibit ectopic fVII expression by targeting p300/CBP activity. These results suggest a strategy to inhibit ectopic fVII-induced tumor progression without impairment of the physiologic hemostatic process. (Mol Cancer Res 2009;7(12):1928–36)

Prolactin inhibits apoptosis of ovarian carcinoma cells induced by serum starvation or cisplatin treatment

Prolactin, a peptide hormone essential for the development and function of reproductive organs, is involved in development of breast, prostate and colorectal cancers. However, the role of prolactin on the carcinogenesis of ovarian carcinomas is unclear. In this study, we show that mRNA of prolactin receptor is expressed in 5 out of 9 ovarian carcinoma cell lines, 15 out of 17 cases of surgical samples and all of normal ovarian surface epithelium, while prolactin transcript is detected only in 1 ovarian carcinoma cell line and in 1 of the surgical samples. For the prolactin receptor‐positive ovarian carcinoma cells, exogenous prolactin did not affect the proliferation but markedly inhibited apoptosis. Therefore, actual cell growth was enhanced by prolactin in a dose‐dependent manner. The blocking of prolactin receptor by antibody severely impaired the antiapoptotic and growth‐promoting effects of prolactin. Interestingly, the cisplatin‐induced cell death of the prolactin receptor‐positive cells was significantly inhibited by pretreatment with prolactin. These findings indicate a responsiveness of ovarian carcinomas to prolactin and suggest that the prolactin/prolactin receptor system may be a new therapeutic target of ovarian carcinomas.