ANXA8 down-regulation by EGF-FOXO4 signaling is involved in cell scattering and tumor metastasis of cholangiocarcinoma.

BACKGROUND & AIMS: The sarcomatoid change in cholangiocarcinoma (CC) contributes to more aggressive intrahepatic spread and widespread metastasis. Therefore, the aim of this study was to identify the molecular mechanisms of CC metastasis during tumor progression and sarcomatoid change. METHODS: Using the subtraction suppression hybridization (SSH) method, we identified altered expression of the candidate gene ANXA8 and epidermal growth factor receptor (EGFR) in sarcomatoid CC cells. We assessed ANXA8 expression during the progression of CC in cells and tissues and examined its functional significance by performing in vitro cell experiments and using in vivo animal models. RESULTS: ANXA8 is highly expressed in human and hamster CCs but is down-regulated with tumor dedifferentiation. ANXA8 is transcriptionally down-regulated by epidermal growth factor (EGF), which is correlated with the morphologic changes of the epithelial-to-mesenchymal transition (EMT) in the CC cells. Furthermore, ectopic ANXA8 reverses the morphology of cells, and this is associated with focal adhesion kinase expression and altered F-actin dynamics. EGFR and its downstream targets, phosphatidylinositol-3-kinase and Akt, are linked to the phosphorylation of FOXO4, which leads to the inhibition of ANXA8 transcription. In addition, an in vitro cell invasion assay and in vivo spontaneous metastasis assay reveal that ANXA8 inhibits the cell migratory and metastatic characteristics of CC cells. CONCLUSIONS: These findings suggest that FOXO4 and ANXA8 play key roles in growth factor-mediated tumor progression and metastasis during the EMT change in CC.

Identification of cystatin B as a potential serum marker in hepatocellular carcinoma.

PURPOSE: The poor survival rate of hepatocellular carcinoma (HCC) is in part due to the inability to diagnose patients at an early stage. Therefore, the aim of this study was to search for candidate serum marker for HCC and to test their ability to distinguish a HCC from benign liver disease. EXPERIMENTAL DESIGN: Genome-wide analysis by a microarray in 40 HCC patients was done between HCC and paired nontumor liver tissues. Expression of cystatin B (CSTB) was examined by mRNA expression analysis and immunohistochemistry. The serum CSTB levels were measured using a sandwich ELISA method in four groups, including normal healthy subjects (group 1, n = 52) and patients with noncirrhotic chronic hepatitis (group 2, n = 53), cirrhosis (group 3, n = 43), and HCC (group 4, n = 62). RESULTS: Microarray and statistical analyses identified 248 genes that were expressed differently between HCC and nontumor liver tissues. One of them, CSTB, was expressed preferentially in the HCCs compared with the nontumor tissues, 36 of 45 specimens (80%) by Northern blot and semiquantitative reverse transcription-PCR analyses. The serum CSTB level was much higher in HCC patients than in those with nonmalignant chronic liver disease (groups 2 and 3; P < 0.0001). The receiver operating characteristic curve indicated 5.34 ng/mL to be the optimal value for CSTB, and the sensitivity and specificity for this CSTB value were 85.5% (95% confidence interval, 74.2-93.1%) and 53.1% (95% confidence interval, 42.7-63.4%), respectively, in distinguishing between patients with HCC and those with nonmalignant chronic liver disease. CONCLUSION: CSTB is specifically overexpressed in most HCCs and is also elevated in the serum of a large proportion of HCC patients. CSTB or the combination of CSTB and alpha-fetoprotein may be a useful marker for diagnosing patients with HCC with a high sensitivity.

Identification of molecular markers for the oncogenic differentiation of hepatocellular carcinoma.

The aim of this study was to identify molecular markers associated with oncogenic differentiation in hepatocellular carcinoma (HCC). Using an unsupervised clustering method with a cDNA microarray, HCC (T) gene expression profiles and corresponding non-tumor tissues (NT) from 40 patients were analyzed. Of total 217 genes, 72 were expressed preferentially in HCC tissues. Among 186 differentially regulated genes, there were molecular chaperone and tumor suppressor gene clusters in the Edmondson grades I and II (GI/II) subclass compared with the liver cirrhosis (LC) subclass. The Edmondson grades III and IV (GIII/IV) subclass with a poor survival (P=0.0133) contained 122 differentially regulated genes with a cluster containing various metastasis- and invasion-related genes compared with the GI/II subclass. Immunohistochemical analysis revealed that ANXA2, one of the 72 genes preferentially expressed in HCC, was over-expressed in the sinusoidal endothelium and in malignant hepatocytes in HCC. The genes identified in the HCC subclasses will be useful molecular markers for the genesis and progression of HCC. In addition, ANXA2 might be a novel marker for tumor angiogenesis in HCC.

NF-Y-dependent cyclin B2 expression in colorectal adenocarcinoma.

PURPOSE: Cyclin B2, a G(2)-M cyclin, is overexpressed in colorectal adenocarcinomas compared with the normal mucosa. This study examined the level of cyclin B2 overexpression according to the histologic findings and investigated the mechanism(s) and clinical implications of cyclin B2 overexpression in colorectal adenocarcinomas. EXPERIMENTAL DESIGN: The immunoreactivity of the polyclonal antibodies to cyclin B2 was determined in colorectal cancer cells. The transcriptional regulation of cyclin B2 by NF-Y was analyzed using an in vitro transfection assay and an in vivo chromatin immunoprecipitation assay. The proliferative activity of the colorectal cancer cells in relation to cyclin B2 overexpression was further examined. RESULTS: The cytoplasmic distribution of cyclin B2 immunoreactivity was positive in 42 of 65 (64.6%) cases of colorectal adenocarcinoma, and the level was similar regardless of the histologic type. A dominant-negative form of NF-YA effectively inhibited the cyclin B2 promoter activity, and NF-Y was found to bind three conserved CCAAT boxes in the cyclin B2 promoter in colorectal adenocarcinoma cells. Tumor cells with a higher functional cyclin B2 activity grew faster than those with a lower activity. Furthermore, there was a correlation between the cells showing immunoreactivity to cyclin B2 and those containing the proliferating cell nuclear antigen, a G1-S cyclin, which is also downstream of NF-Y in colorectal adenocarcinoma cells. CONCLUSIONS: Cyclin B2 seems to be a molecular marker of a colorectal adenocarcinoma and that its up-regulation and coordinate expression of the other cell cycle-related genes by NF-Y might contribute to tumor cell proliferation by accelerating cell cycle progression.