【作者】 胡欣；

【导师】 顾健人；

【作者基本信息】 复旦大学 ， 病原生物学， 2008， 博士

【摘要】 癌症可能是现代社会面临的最严重的挑战之一。到2020年,由于癌症而死亡的患者将达到一千万人。所以现代医学的一项重要的目标就是提高癌症的诊断和治疗水平,从而减低癌症的发病率和死亡率。近期的研究提示癌症的进程可能是一种系统性疾病,其中涉及大量相关基因表达和功能的改变。多项研究表明,神经系统的组成成分和功能的改变参与到了多种类型癌症的发生或进展密切相关。肝癌(hepatic cancer)包括肝细胞肝癌(hepatocellular carcinoma,HCC)、胆管细胞癌(cholangiocarcinoma)和肝母细胞瘤(Hepatoblastoma),是一类恶性程度极高、预后极差的恶性肿瘤。本实验室前期大规模功能基因筛选中发现了一批与肝癌生长密切相关的基因,其中就包含一些与神经系统相关的细胞因子和相关受体。本文所展开的关于睫状神经营养因子(Ciliary Neurotrophic Factor,CNTF)及其受体(Ciliary Neurotrophic Factor Receptor,CNTFR)系统在肝癌细胞中的表达、信号转导和功能等方面的研究是对前期工作的延续和深入。睫状神经营养因子(CNTF)首次被发现是作为一种能促进鸡的睫状神经节生长的神经营养因子,其蛋白质结构属于白介素-6(IL-6)家族。早期研究发现,CNTF在神经系统中具有重要的作用,其功能包括:促进神经元的存活,促进神经元的分化以及防止受损神经元的退变等等。近期实验发现,CNTF能具有减肥作用,其机制可能是通过激活类似leptin信号途径:作用于中枢神经系统产生摄食抑制功能;作用于外周代谢器官来实现对能量代谢的调控。最新研究发现,睫状神经营养因子能够在ob/ob小鼠中起到促进脂肪酸的氧化,降低细胞胰岛素的抵抗的作用。睫状神经营养因子受体(CNTFR)作为CNTF的受体属于IL-6受体家族的一员,是由睫状神经营养因子受体α亚基(CNTFRα),白血病抑制因子受体(LIFR)的β亚基与gp130组成的。其中LIFR和gp130为细胞中广泛存在的跨膜蛋白,主要起传递跨膜信号的作用。CNTFRα为受体的核心亚基,能特异性地与CNTF结合,从而激活下游信号通路。在神经系统及其它外周组织中,CNTF激活的相关下游信号通路可能包括JAK/STAT,PI3K/Akt,MAPK和AMPK等。但是睫状神经营养因子及其受体系统在肝脏中的表达,下游信号通路和相关生物学功能等相关领域还尚为未知。我们的实验首先发现睫状神经营养因子受体α亚基在肝脏中表达的;CNTFRα在正常肝,肝癌组织和相对应的癌旁肝组织中存在异质性表达;并在肝细胞来源的肝癌细胞株中有表达。同时,我们也发现CNTFRα在胎肝组织,肝母细胞瘤组织,以及肝母细胞瘤来源的肝癌细胞株HepG2中不存在表达。此外,我们发现CNTFR的另两个亚基LIFR和gp130,以及广泛地分布于各类肝组织和肝癌细胞株中。根据以上结果我们分别选择了SMMC-7721(CNTFRα+/LIFR+/gp130+)和HepG2(CNTFRα-/LIFR+/gp130+)作为两种肝癌细胞模型来研究CNTFRα的作用及其下游的信号通路。我们分别检测了CNTFRα阳性、阴性细胞株和相应的siRNA-CNTFRα作用后,JAK/STAT,PI3K/Akt,MAPK和AMPK信号途径的激活水平。实验发现细胞受到CNTF刺激后,细胞CNTFRα的表达决定了下游四条信号通路不同的激活水平。结论是:PI3K/Akt和MAPK的激活依赖于CNTFRα的表达;JAK/STAT3的激活部分依赖于CNTFRα的表达;而AMPK的激活不依赖于CNTFRα的表达。我们的实验发现,CNTF能够促进肝细胞摄取葡萄糖,而且这个过程与CNTFRα的表达无关。同时CNTF与肝细胞的结合并不仅仅限于CNTFRα阳性细胞的结果也提示可能存在一种CNTFRα非依赖的机制来解释这种现象。进一步实验发现,PI3K/Akt和AMPK信号通路的激活可能参与到CNTF刺激引起的葡萄糖转运蛋白4(GLUT4)的细胞内转位,从而导致葡萄糖摄取的增加。在CNTFRα阳性细胞中,PI3K/Akt和AMPK的激活同时参与了这个过程;而在CNTFRα阴性细胞中,AMPK起了主要作用。另外,我们在对AMPK激活的过程的分析中发现,AMPK的激活受到了MAPK途径激活的抑制。这个过程是发生在CNTF刺激的早期。这里我们分别运用了MEK1的抑制剂和持续激活的MEK1质粒的转染来达到抑制或是激活MAPK途径的效果,从而来检测AMPK途径的激活水平。实验证明CNTF引起的AMPK的激活在刺激发生的初期受到了MAPK途径激活的抑制。我们在以前的实验基础上,进一步证明了敲除CNTFRα能够抑制肝癌细胞的生长。现在,我们进一步发现CNTF对于细胞生长的作用在对于细胞周期的调控。CNTF能够帮助细胞从G2/M期的阻滞中恢复进入细胞周期。这种现象只存在于CNTFRα阳性的细胞株。考虑到CNTF对于葡萄糖代谢的影响,我们意外地发现CNTF对于细胞周期的影响还依赖于培养环境中葡萄糖的水平:CNTF对于细胞周期方面的这种作用在正常血糖环境范围(3-6mM)中比较明显,而在高糖和低糖环境中的作用则不太明显。我们继续发现,细胞在不同浓度的葡萄糖培养环境中对CNTF的敏感性也不同,其中在高糖培养环境中CNTF对下游JAK/STAT3和PI3K/Akt途径的激活更为明显。由于常规培养基均含有较高浓度的葡萄糖,上述结果给予我们一些有意义的启示,提示我们培养条件中葡萄糖的水平在研究过程中的重要性,从而可以避免某些重要的生物学现象被忽略。关于这里的分子机制还需要以后进一步的深入研究。综上所述,我们的实验揭示了CNTFRα在肝癌细胞中的表达决定了CNTF所激活的下游多条相关信号通路的激活的调节及其相关的生物学功能,从而为研究CNTF和CNTFR系统对于肝脏的生理和病理过程的作用提供了研究基础,也为研究肝脏代谢疾病及肝癌的研究提供了新的启示。更多还原

【Abstract】 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.更多还原