The Study Of Ciliary Neurotrophic Factor And Its Receptor System In Hepatic Cancer Cells

Cancer represents one of the biggest problems for modern societies.By 2020,the global cancer deaths could reach 10 million.An important goal of life science research is to improve tumor diagnostics and anti-cancer treatment options to alleviate cancer-related morbidity and mortality.Recent studies revealed that cancer is a systemic disease due to an evolutionary process which involved in a large variety of deviations of oncogenes and tumor-suppressor genes.Moreover,some reports provided some evidences that the alterations of some neurotransmitter-related components might be associated with the process of cancer initiation and progression.Hepatic cancer,including hepatocellular carcinoma,cholangiocarcinoma and hepatoblastoma,is one of the most prevalent and lethal cancer in China.Our previous study of a large-scale complementary DNA transfection screening showed that some cytokines,neural transmitters or receptors are related to the proliferation of hepatocellular carcinoma cells.This work provided us some clues that ciliary neurotrophic factor receptorαsubunit(CNTFRα) might have some positive effect on hepatocellular carcinoma cell growth.In the present study,we investigated the existence of ciliary neurotrophic factor(CNTF) and its receptor(CNTFR) system in liver and liver cancer cells,explored its downstream signaling pathways and functional roles in hepatic cancer cells.Ciliary neurotrophic factor was first denoted as a neuron-nurturing factor in chick ciliary ganglion neurons more than two decades ago,and was subsequently defined as a member of the IL-6 cytokine family.In the past decades,CNTF has been shown to promote the differentiation of sympathetic neurons and glial progenitor cells into astrocytes,and promote the survival of a variety of neuronal cells such as sensory, motor,hippocampal and cerebral neurons.A recent study revealed that CNTF can suppress food intake and induce weight loss via a leptin-like mechanism in ob/ob mice.Moreover,CNTF increases metabolic rate and energy expenditure of peripheral metabolic organs,independent of the signaling from the brain.It was further demonstrated that CNTF can enhance fatty-acid oxidation in muscle and reduced insulin resistance in obese,diabetic mice.CNTF exerts its biological functions through its receptor CNTFR to activate multiple downstream signaling pathways.CNTFR is a tripartite complex composed of three subunits:CNTF receptorαsubunit(CNTFRα),gp130,and leukemia inhibitory factor receptor(LIFR).CNTFRα,the specificαsubunit for CNTF,is anchored to the membrane by a glycosyl-phosphatidylinositol linkage without a cytoplasmic domain. Binding of CNTF to CNTFRαinduces heterodimerization of the trans-membrane subunits(gp130 and LIFR) to transduce cell signals via the Janus kinase(JAK)/signal transducers of activated transcription(STAT) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase(ERK) signaling pathways.In addition, phosphotidyl inositol-3'-phosphate-kinase(PI3K)/Akt participates in the survival response of neurons to CNTE CNTF plays dual roles on the activation of AMP-activated protein kinase(AMPK).In the central neuronal system,CNTF inhibits AMPK activity in the hypothalamus to promote leptin-sensitive neurogenesis in the arcuate nuclei,and reduces neuropeptide Y(NPY) gene expression;in the periphery tissues,CNTF accelerates fat oxidation via an AMPK-dependent mechanism in skeletal muscle.However,the function of CNTF/CNTFRαin human liver cancer cells and their underlying molecular mechanisms have not been elucidated.In this study,we demonstrate that CNTFRαis expressed heterogeneously in normal human liver,hepatocellular carcinoma(HCC) and corresponding non-cancerous liver tissues,but not in fetal liver and hepatoblastoma specimens.Here,we chose the CNTFRα+/CNTFRα- cell models of hepatic origin to study multiple downstream pathways in hepatic cancer cells.The data presented here shows that exogenous CNTF can activate multiple signaling pathways in CNTFRα+ or CNTFRα- hepatic cancer cell lines,and we proposed a model to differentiate the four CNTF-induced signaling pathways into CNTFRαdependent or independent processes,including JAK/STAT3,MAPK/ERK,PI3K/Akt and AMPK signaling pathways(Fig.8).Based on our observations,the activation of MAPK/ERK,PI3K/Akt,and JAK/STAT3 pathways are dependent or partially dependent on the presence of CNTFRα,while the AMPK pathway is activated in a CNTFRαindependent manner.Moreover,our results demostrated that CNTF-triggered PI3K/Akt and AMPK signaling pathways also contribute to glucose uptake in hepatic cancer cells,and induce the translocation of GLUT4 from cytosol form pool to the plasma membrane.In CNTFRα~+ cells,the activation of PI3K/Akt was an immediate and persistent response; while AMPK activation had a lag phase.In the CNTFRα~- or CNTFRα-silenced cells, AMPK activation was immediate,whereas no activation of PI3K/Akt was observed.Furthermore,we found that CNTF-induced MAPK activation could suppress AMPK activity in the early phase of CNTF stimulation,as revealed by using specific inhibitors,RNA interference and constitutively active plasmids.Our previous study has provided some clues that knock-down of CNTFRαcould impair the proliferation of hepatocellular carcinoma cells.Here,we further demonstrated that the protective role of CNTF against cell-cycle arrest is dependent on the presence of CNTFRα,and is modulated by the glucose concentration of the culture medium.We also found that the relative higher glucose concentration could make CNTFRα~+ cells more sensitive to the CNTF stimulation in JAK/STAT3 and PI3K/Akt pathway.The reason for this phenomenon is presently unknown.Since the conventially used culture medium contains high concentration of glucose,these interesting results remind us of the importance of the glucose concentration in culture medium,which has been previously ignored for other in vitro studies of cytokines or growth factors involved in glucose metabolism in cancer biology.Conclusion:Our results demonstrate the importance of CNTFRα-mediated downstream signaling pathways and their functional implications in hepatic cancer cells,thus highlighting a better understanding of the biological roles of CNTFRαin human liver abnormalities,including metabolic diseases and hepatocarcinogenesis.