Novel biomarkers are needed for early detection and progression evaluation of hepatocellular carcinoma (HCC). The purpose of this study was to identify useful biomolecular markers for HCC. The 26 genes that encode membrane or secretory proteins were identified from cDNA microarray data. We further examined the expression of EFNA1 and its receptor EphA2 and determined their biological implications during the development and progression of HCC. The EFNA1 mRNA was overexpressed in most HCCs as compared with its expression in corresponding nontumor tissues (36 out of 40 cases, 90%), but EphA2 expression was noted in only half of the HCC tissues (20 of 40 cases, 50%). In most of the hepatoma cell lines, the EFNA1 protein expression was positively associated with alpha-fetoprotien (AFP) expression but inversely associated with EphA2 expression. Furthermore, EFNA1 levels were detectable in the supernatant of the cultured hepatoma cells and in the serum of patients with HCC. In contrast, EphA2 expression was prominent in highly invasive hepatoma cells, and its overexpression was significantly correlated with decreased differentiation (r 5 0.0248, p < 0.010) and poor survival (p 5 0.0453) for HCC patients. EFNA1 and EphA2 may be useful serum markers for the detection of HCC development and progression, respectively.Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and it is known to be the third most common cause of cancer-related mortality. 1 The incidence of HCC is increasing in Western countries such as Europe and the Unites States. 2 One of the reasons for the high mortality is that HCC tumors are commonly detected at a stage when curative resection is no longer feasible because of intrahepatic and extrahepatic metastasis. The diagnosis of HCC currently relies on observation of a liver mass in radiology imaging studies such as ultrasonography, computed tomography scanning or magnetic resonance imaging. However, the diagnosis of small lesions is relatively inaccurate. 3 The only approach to screen for the presence of HCC in a high risk-population is the combination of serum alpha-fetoprotein (AFP) level determination and ultrasonography. 4,5 However, the AFP level test has low sensitivity and specificity, particularly in patients with smaller HCCs. Several biomarkers such as Desgamma carboxyprothrombin (DCP), lens cularis agglutininreactive AFP and glypican-3 (GPC3) have yet to be validated for their abilities to detect an early HCC. 6 Therefore, there is an urgent need to identify adequate biochemical markers for early detection and to evaluate the progression of HCC. Genome-wide microarray analysis offers a systemic approach for obtaining comprehensive information on the transcriptional profiles of HCC. Using microarray technology, we previously determined the molecular nature of multistep hepatocarcinogenesis and the specific genetic changes associated with the oncogenic differentiation of HCC. 7 In our previous study, we used a modified analytical approach to identify several molecular marke...

BackgroundThe molecular mechanisms of CC (cholangiocarcinoma) oncogenesis and progression are poorly understood. This study aimed to determine the genome-wide expression of genes related to CC oncogenesis and sarcomatous transdifferentiation.MethodsGenes that were differentially expressed between CC cell lines or tissues and cultured normal biliary epithelial (NBE) cells were identified using DNA microarray technology. Expressions were validated in human CC tissues and cells.ResultsUsing unsupervised hierarchical clustering analysis of the cell line and tissue samples, we identified a set of 342 commonly regulated (>2-fold change) genes. Of these, 53, including tumor-related genes, were upregulated, and 289, including tumor suppressor genes, were downregulated (<0.5 fold change). Expression of SPP1, EFNB2, E2F2, IRX3, PTTG1, PPARγ, KRT17, UCHL1, IGFBP7 and SPARC proteins was immunohistochemically verified in human and hamster CC tissues. Additional unsupervised hierarchical clustering analysis of sarcomatoid CC cells compared to three adenocarcinomatous CC cell lines revealed 292 differentially upregulated genes (>4-fold change), and 267 differentially downregulated genes (<0.25 fold change). The expression of 12 proteins was validated in the CC cell lines by immunoblot analysis and immunohistochemical staining. Of the proteins analyzed, we found upregulation of the expression of the epithelial-mesenchymal transition (EMT)-related proteins VIM and TWIST1, and restoration of the methylation-silenced proteins LDHB, BNIP3, UCHL1, and NPTX2 during sarcomatoid transdifferentiation of CC.ConclusionThe deregulation of oncogenes, tumor suppressor genes, and methylation-related genes may be useful in identifying molecular targets for CC diagnosis and prognosis.

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Eph receptor 2 (EphA2) overexpression is frequently accompanied by the loss of its cognate ligand during tumor progression. However, the molecular mechanism of this ligandindependent promotion of tumor by EphA2 remains unclear in highly malignant and fatal cholangiocarcinoma (CC). We examined the biological role of EphA2 in tumor growth and metastasis in CC tissues and cells according to the degree of differentiation and we explored the downstream signaling pathways of EphA2. Growth factor-mediated EphA2 overexpression itself leads to the activation of the mammalian target of rapamycin complex 1 (mTORC1) and extracellular signal-regulated kinase (ERK) pathways through ligandindependent activation of EphA2 (phosphorylation of S897). An in vitro soft agar assay and in vivo orthotopic or subcutaneous tumor model showed that EphA2 enhanced colony formation and accelerated tumor growth, and which seemed to be mainly associated with Akt (T308)/mTORC1 activation. Aberrant expression and activation of EphA2 was also associated with poorer differentiation and higher metastatic ability. Enhanced metastatic ability was also observed in an orthotopic tumor model or lung metastasis model, correlating with Pyk2(Y402)/c-Src/ERK activation in addition to activation of the canonical Raf/ MEK/ERK pathway. The mTORC1 and Raf/Pyk2 pathways also appeared to affect each other. These results suggest that growth factor-mediated EphA2 might be involved in tumor growth and metastasis through activation of the mTORC1 and Raf/Pyk2 pathways. Therapeutic strategies that target EphA2 and its downstream effectors may be useful to control CC. (HEPATOLOGY 2013;57:2248-2260

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