Adult hepatic progenitor (oval) cells are facultative stem cells in liver, which participate in a range of human liver diseases, including hepatocellular carcinoma (HCC). However, the molecular pathways regulating the expansion and differentiation of these cells are poorly understood. We show that active Wnt/B-catenin signaling occurs preferentially within the oval cell population, and forced expression of constitutively active B-catenin mutant promotes expansion of the oval cell population in the regenerated liver. More importantly, we identify a subpopulation of less differentiated progenitor-like cells in HCC cell lines and primary HCC tissues, which are defined by expression of the hepatic progenitor marker OV6 and endowed with endogenously active Wnt/B-catenin signaling. These OV6 + HCC cells possess a greater ability to form tumor in vivo and show a substantial resistance to standard chemotherapy compared with OV6 À tumor cells. The fraction of tumor cells expressing OV6 is enriched after Wnt pathway activation, whereas inhibition of B-catenin signaling leads to a decrease in the proportion of OV6 + cells. In addition, the chemoresistance of OV6 + HCC progenitor-like cells can be reversed by lentivirus-delivered stable expression of micro-RNA targeting B-catenin. These results highlight the importance of the Wnt/B-catenin pathway in activation and expansion of oval cells in normal rodent models and human HCCs. OV6 + tumor cells may represent the cellular population that confers HCC chemoresistance, and therapies targeted to the Wnt/B-catenin signaling may provide a specific method to disrupt this resistance mechanism to improve overall tumor control with chemotherapy. [Cancer Res 2008;68(11):4287-95]

Purpose: Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is overexpressed in many types of cancer. Herein, we aimed to investigate its expression pattern, clinical significance, and biological function in hepatocellular carcinoma (HCC).Experimental Design: MAP4K4 expression was examined in 20 fresh HCCs and corresponding nontumor liver tissues. Immunohistochemistry for MAP4K4 was performed on additional 400 HCCs, of which 305 (76%) were positive for hepatitis B surface antigens. The clinical significance of MAP4K4 expression was analyzed. MAP4K4 downregulation was performed in HCC cell lines HepG2 and Hep3B with high abundance of MAP4K4, and the effects of MAP4K4 silencing on cell proliferation in vitro and tumor growth in vivo were evaluated. Quantitative real-time PCR arrays were employed to identify the MAP4K4-regulated signaling pathways.Results: MAP4K4 was aberrantly overexpressed in HCCs relative to adjacent nontumor liver tissues. This overexpression was significantly associated with larger tumor size, increased histologic grade, advanced tumor stage, and intrahepatic metastasis, as well as worse overall survival and higher early recurrence rate. Knockdown of the MAP4K4 expression reduced cell proliferation, blocked cell cycle at S phase, and increased apoptosis. The antitumor effects of MAP4K4 silencing were also observed in vivo, manifested as retarded tumor xenograft growth. Furthermore, multiple tumor progression-related signaling pathways including JNK, NFkB, and toll-like receptors were repressed by MAP4K4 downregulation.Conclusions: MAP4K4 overexpression is an independent predictor of poor prognosis of HCC patients, and inhibition of its expression might be of therapeutic significance.

Chemical neurotransmission occurs at chemical synapses and endocrine glands, but up to now there was no means for direct monitoring of neurotransmitter exocytosis fluxes and their precise kinetics from inside an individual synapse. The fabrication of a novel finite conical nanoelectrode is reported perfectly suited in size and electrochemical properties for probing amperometrically inside what appears to be single synapses and monitoring individual vesicular exocytotic events in real time. This allowed obtaining direct and important physiological evidences which may yield important and new insights into the nature of synaptic communications.

BackgroundPancreatic cancer is one of the most lethal cancers worldwide. The aim of this study was to determine the expression pattern, clinical significance, and biological functions of Wnt5a in pancreatic cancer.MethodsImmunohistochemistry was performed to examine Wnt5a expression in 134 surgically resected pancreatic adenocarcinoma and adjacent normal pancreatic tissues. Associations of Wnt5a expression with clinicopathological factors and cancer-specific survival were analyzed. The effects of Wnt5a overexpression or silencing on the invasiveness and epithelial-to-mesenchymal transition (EMT) of pancreatic cancer cells were studied. Silencing of β-catenin by small interfering RNA was done to determine its role in the Wnt5a-mediated tumor phenotype.ResultsThe percentage of Wnt5a positive expression showed a bell-shaped pattern in pancreatic cancer tissues, peaking in well-differentiated carcinomas. The median cancer-specific survival was comparable between patients with positive versus negative expression of Wnt5a. Overexpression of Wnt5a promoted the migration and invasion of pancreatic cancer cells, whereas Wnt5a depletion had an inhibitory effect. In an orthotopic pancreatic cancer mouse model, Wnt5a overexpression resulted in increased invasiveness and metastasis, coupled with induction of EMT in tumor cells. Treatment with recombinant Wnt5a elevated the nuclear β-catenin level in pancreatic cancer cells, without altering the Ror2 expression. Targeted reduction of β-catenin antagonized exogenous Wnt5a-induced EMT and invasiveness in pancreatic cancer cells.ConclusionUpregulation of Wnt5a promotes EMT and metastasis in pancreatic cancer models, which involves activation of β-catenin-dependent canonical Wnt signaling. These findings warrant further investigation of the clinical relevance of Wnt5 upregulation in pancreatic cancer.

Chemical synaptic transmission is central to the brain functions. In this regard, real-time monitoring of chemical synaptic transmission during neuronal communication remains a great challenge. In this work, in vivo-like oriented neural networks between superior cervical ganglion (SCG) neurons and their effector smooth muscle cells (SMC) were assembled in a microfluidic device. This allowed amperometric detection of individual neurotransmitter release events inside functional SCG-SMC synapse with carbon fiber nanoelectrodes as well as recording of postsynaptic potential using glass nanopipette electrodes. The high vesicular release activities essentially involved complex events arising from flickering fusion pores as quantitatively established based on simulations. This work allowed for the first time monitoring in situ chemical synaptic transmission under conditions close to those found in vivo, which may yield important and new insights into the nature of neuronal communications.

The spontaneous variation of blood pressure is defined as "blood pressure variability" (BPV). The chronic sinoaortic-denervated (SAD) rat is a model of high BPV without sustained hypertension. Little is known about vascular remodeling in this model. In the present study, we examined blood pressure, vascular remodeling, and aortic angiotensin II concentration in chronic SAD rats in separate experiments. In experiment 1, intra-arterial blood pressure was continuously recorded in conscious unrestrained rats. The 16-week SAD rats had a significant increase in BPV and no change in the mean level of blood pressure over a 24-h period. In experiment 2, we measured structural changes of seven kinds of arteries by histologic method and computer image analysis and functional changes of thoracic aortas by isolated artery preparation. Structural remodeling after 16-week sinoaortic denervation was characterized by increase in wall thickness, wall area, and ratio of wall thickness to internal diameter, with different changes in internal diameter and external diameter in different arteries, indicating that arterial structural remodeling expresses itself mainly as vascular growth. This vascular growth might be caused by medial smooth muscle cell growth and collagen accumulation. Aortic contraction induced by norepinephrine was potentiated, whereas aortic relaxation induced by acetylcholine was attenuated after sinoaortic denervation. In experiment 3, plasma and aortic angiotensin II concentrations were determined by radioimmunoassay. The former remained unchanged, whereas the latter was significantly increased in 10-week SAD rats. It is concluded that in rats chronic sinoaortic denervation can produce vascular remodeling that might be related to increased BPV and an activated tissue renin-angiotensin system.

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Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of human cancer worldwide. In the present study, we investigated the diagnostic and biological significance of microRNA-194 (miR-194) in PDAC. miRNA expression profiling of human PDACs and adjacent normal pancreatic tissues identified a total of 16 genes including miR-194 with >1.15-fold expression changes (8 overexpressed and 8 underexpressed). Quantitative real-time polymerase chain reaction (PCR) revealed elevation of serum miR-194 levels were significantly greater in PDAC patients than in duodenal adenocarcinoma patients and healthy controls. Receiver operating characteristic analysis demonstrated that serum miR-194 had a sensitivity of 54.3% and a specificity of 57.5% for discriminating PDAC patients from healthy controls. Combined analysis of the 3 groups yielded a sensitivity of 84.0 and a specificity of 75.0% for the combined detection of miR-192 and miR-194 in the diagnosis of PDAC. Ectopic expression of miR-194 in PANC-1 pancreatic cancer cells enhanced cell proliferation, migration and colony formation, which was coupled with decreased expression of the tumor suppressor DACH1. miR-194 overexpression increased tumor growth and local invasion and suppressed the expression of DACH1 in an orthotopic pancreatic cancer mouse model. In conclusion, upregulation of miR-194 contributes to tumor growth and progression in PDAC, possibly through suppression of DACH1. However, serum miR-194 has a low capacity for detection of PDAC. Combined detection of serum miR-192 and miR-194 levels may serve as a sensitive diagnostic biomarker for PDAC.