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类型 基础研究 预答辩日期 2017-12-31
开始(开题)日期 2016-06-03 论文结束日期 2017-06-05
地点 东南大学李文正楼北225会议室 论文选题来源 国家自然科学基金项目     论文字数 5 (万字)
题目 CD44调控ERK,AKT及Hippo-YAP通路促进癌症发展的机制研究
主题词 癌症,CD44,MAPK-ERK通路,PI3K-AKT通路,Hippo-YAP通路
摘要 癌症是危害人类健康的头号杀手。在癌症的发生发展过程中,生长信号通路的持续激活至关重要,是癌细胞的重要特征之一。近些年的研究表明正常细胞通过接触抑制减弱生长信号通络来控制细胞数量,而癌细胞获得了逃避接触抑制的能力,从而可以维持生长信号通路的持续激活。而关于癌细胞逃避接触抑制的机制却不是十分清楚。PI3K-AKT通路,MAPK-ERK通路和Hippo-YAP通路与癌细胞增殖高度相关,研究表明在接触抑制的过程中,AKT,ERK1/2及YAP的活性受到抑制。CD44是广泛表达于细胞表面的糖蛋白,参与细胞与细胞之间的通讯。然而,关于CD44在接触抑制中的功能,在维持肿瘤干细胞干性中的作用以及对信号网络的调控机制还有待探讨。因此,我们想探明CD44对信号网络的调控作用及与接触抑制的关系,进一步分析其在细胞增殖,细胞周期和细胞干性维持中的功能。 首先,我们研究发现高密度培养下的癌细胞YAP水平明显降低,p-YAP/YAP升高,表明在癌细胞中,抑制生长的Hippo-YAP通路可以通过细胞接触正常激活;而p-AKT, p-ERK1/2水平升高,表明在癌细胞接触的过程中,促进生长的信号通路PI3K-AKT通路,MAPK-ERK通路会异常地激活。在高密度培养的条件下,通过小干扰RNA(siRNA)介导CD44敲低后,我们观察到在MDA-MB-435S和BT549两种癌细胞系中,p-YAP/YAP出现下降,说明CD44拮抗细胞接触过程中Hippo-YAP通路的激活;而Hippo-YAP通路上游分子NF2的磷酸化水平出现下降,Hippo-YAP通路中的核心激酶LATS1/2的活性出现显著上升,CD44可能通过调控NF2抑制Hippo-YAP通路的激活。此外,p-AKT, p-ERK1/2也出现了显著降低,说明细胞接触时异常的生长通路激活与CD44有关。这些结果表明,在细胞接触时CD44可以调节PI3K-AKT通路,MAPK-ERK通路,Hippo-YAP通路这三条通路逃避接触抑制。为了深入研究通路之间调控的机制,我们用PI3K-AKT通路抑制剂LY294002,MK-2206及MAPK-ERK通路抑制剂PD0325901分别处理MDA-MB-435S,发现MAPK-ERK通路及PI3K-AKT通路之间存在交互;同时,我们观察到siRNA介导的ERK1/2敲低会显著降低YAP的水平。进一步的研究发现,单独敲低ERK1会升高YAP的转录水平从而导致YAP蛋白水平升高,单独敲低ERK2会降低YAP稳定性而使YAP蛋白水平下降。此外,在51种乳腺癌细胞系中,ERK1的mRNA水平与YAP的mRNA水平有显著的负相关性,这种负相关也同时存在于ERK1与YAP的11个靶基因之间。临床分析显示,ERK1的高表达与乳腺癌患者良好的预后相关。最后,我们发现CD44的敲低会1.抑制细胞增殖,降低增殖相关基因PCNA的转录。2.改变细胞周期,缩短G1期,延长G2/M期,升高G1/S期关键蛋白cyclinE2,cyclinD1的转录,降低G2/M期关键蛋白CDK1的转录。3.降低癌细胞的干性,降低悬浮培养干细胞球的大小和数量,减弱细胞平板克隆形成能力,强烈抑制癌细胞在裸鼠中成瘤的能力,降低干性相关基因KLF4的转录和蛋白水平。 该研究的结果显示,在癌细胞中PI3K-AKT通路,MAPK-ERK通路的异常激活参与了癌细胞对接触抑制的逃避。在接触抑制的过程中,CD44作为上游的细胞黏附分子,参与对PI3K-AKT通路,MAPK-ERK通路及Hippo-YAP通路活性的调控,导致了癌细胞的接触抑制逃避。在这个过程中,CD44对接触抑制中的Hippo-YAP通路的激活起抑制作用,这是通过调控其NF2实现的。PI3K-AKT通路,MAPK-ERK通路及Hippo-YAP通路存在相互调控关系。MAPK-ERK通路的抑制会引起PI3K-AKT通路的反馈激活,而MAPK-ERK通路中的ERK1通过转录水平抑制YAP蛋白表达,ERK2通过操控蛋白稳定性升高YAP蛋白水平。此外CD44通过调节PI3K-AKT通路,MAPK-ERK通路及Hippo-YAP通路三条主要通路,在转录水平上控制细胞行为中的关键蛋白,参与细胞增殖,细胞周期的控制和细胞干性的维持,因此,是一个潜在的癌症治疗靶点。该研究不仅对揭示CD44在癌症的分子发病机理有重要科学意义,而且在进一步改善肿瘤治疗和控制方面有重要的现实意义。
英文题目 CD44 Regulates ERK, AKT and Hippo-YAP Pathway to Promote Cancer Development
英文主题词 Cancer,CD44,MAPK-ERK pathway,PI3K-AKT pathway,Hippo-YAP pathway
英文摘要 Cancer remains a leading cause of human death. During the development of cancer, growth related signaling pathways are continuously activated. Sustaining cell proliferation is thus considered one of six hallmarks in cancer. In dense populations of normal cells, the growth signaling could be diminished in response to cell-cell contact, however, whose inhibition is abolished in various types of cancer cells. Until recently, the mechanism basis for evading contact inhibition remains elusive. As ackonwledged, PI3K-AKT, MAPK-ERK and Hippo-YAP pathways are highly involved in maintaining fast growth of cancer cells. Recent studies indicated that these three pathways also contribute to contact inhibition in densely cultured cells. CD44 is a ubiquitously expressed glycoprotein that functions in cell-cell communication. However, the function of CD44 in contact inhibition,maintenance of cancer cell stemness and how CD44 integrating multiple pathways have yet to be fully established. Herein, this work further explored the signaling networks regulated by CD44 and its role in contact inhibition evasion, cell growth, cell cycle as well as maintenance of stemness. In the present study, western blot results indicated that high-dense culture of MDA-MB-435S cells would lead to activation of Hippo-YAP pathway with an increase of p-YAP/YAP ratio. In addition, the phosphorylation level of AKT and ERK1/2 were also elevated, indicating an abnormal activation of PI3K-AKT and MAPK-ERK signaling pathways towards contact inhibition. To explore the role of CD44 in contact inhibition, protein levels of AKT, ERK1/2, YAP and their phosphorylation status were measured in MDA-MB-435S and BT549 cells which treated with CD44 siRNA. Decrease of AKT, ERK1/2 phosphorylation level and increase of phospho-YAP level were observed indicating a common upstream regulator of CD44 to these three pathways. Importantly, NF2, a well-known contact inhibition mediator and CD44 interacting protein, was activated in response to CD44 silencing. The phosphorylation level of LATS1/2, a core component of Hippo-YAP pathway, was dropped in CD44-silenced MDA-MB-435S cells. These data suggested that CD44 participated in evading contact inhibition of cancer cells via regulating a network including MAPK-ERK pathway, PI3K-AKT pathway and Hippo-YAP pathway. To further evaluate the interplay among PI3K-AKT, MAPK-ERK and Hippo-YAP pathway, protein levels of core components of these pathways were detected in cells treated with vehicle or LY294002 (PI3K inhibitor) or MK2206 (AKT inhibitor) or PD0325901 (MAPK inhibitor). Inhibition of MAPK-ERK pathway would result in an compensatory activation of PI3K-AKT pathway. The interplay between MAPK-ERK pathway and Hippo-YAP pathway was far more complex, while siRNA mediated knockdown of ERK1 induced an increase of YAP protein level through transcriptional activation, ERK2 silencing destablized YAP to reduce YAP protein level. Moreover, transcription level of ERK1 was negatively correlated with YAP and 11 target genes of YAP in transcriptome of 51 breast cancer cell lines analyzed. Additionally, higher ERK1 expression was associated with better clinical outcome in breast cancer patients. Finally, cell behaviors in cells treated with CD44 siRNA was studied. Silencing of CD44 would generate changes as followed: 1. inhibition of cell proliferation with a decrease of PCNA mRNA level. 2. cell cycle perturbation with prolonged G2/M phase and shortened G1 phase, elevated transcription of key G1/S checkpoint protein Cyclin D1 and Cyclin E2, transcriptional inactivation of CDK1. 3. reduced cancer cell stemness with a reduced size/number of manmosphere, tumor volume of nude mice, plate colony number, KLF4 protein and mRNA levels. In summary, this study indicated that abnormal activation of MAPK-ERK pathway and PI3K-AKT pathway contribute to contact inhibition evasion in cancer cells. During contact inhibition, CD44 functions as an upstream regulator to activate PI3K-AKT pathway, MAPK-ERK pathway and inhibit Hippo-YAP pathway. The regulation of Hippo-YAP pathway by CD44 was realized via interaction with NF2. In addition, crosstalk amongst PI3K-AKT pathway, MAPK-ERK pathway and Hippo-YAP pathway was discovered in cancer cells. Inhibition of MAPK-ERK pathway was accompanied by activation of PI3K-AKT pathway. Importantly, ERK1 and ERK2, core kinases of MAPK-ERK pathway, regulated YAP in two different mechanisms that have not been discovered before. ERK1 repressed YAP transcription and ERK2 protected YAP from degradation. Additionally, as a common upstream regulator, CD44 participated in regulating cell proliferation, cell cycle and cancer cell stemness through transcriptional regulation of key genes in these processes. This study not only discovered the molecular mechanism of CD44 during cancer development, but also facilitated further improvement of cancer therapy.
学术讨论
主办单位时间地点报告人报告主题
东南大学 2017年5月26日 南京市玄武区四牌楼2号 顾宇 The regulation of the critical period plasticity in mouse visual cortex
南京师范大学 2014年10月24日 南京市栖霞区文苑路1号 余诗奕 A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCFβ-TRCP
南京师范大学 2016年5月8号 南京市栖霞区文苑路1号 余诗奕 A Preclinical Model for ERα-Positive Breast Cancer Points to the Epithelial Microenvironment as Determinant of Luminal Phenotype and Hormone Response
东南大学 2015年11月20日 南京市玄武区四牌楼2号 余诗奕 A miR-130a-YAP positive feedback loop promotes organ
东南大学 2013年12月5日 南京市玄武区四牌楼2号 戴振华 器官移植免疫耐受的诱导策略及其免疫调节机制
东南大学 2015年1月20日 南京市玄武区四牌楼2号 郝睿 HDAC6: a deacetylase integrating all aspects of protein
东南大学 2016年12月12日 南京市玄武区四牌楼2号 寿天德 Stream-preferred modulation of feedback projections from the higher areas to lower areas in the visual cortex
东南大学 2015年5月12日 南京市玄武区四牌楼2号 余诗奕 E-cadherin mediates contact inhibition of proliferation through Hippo signaling-pathway components
     
学术会议
会议名称时间地点本人报告本人报告题目
第五届国际乳腺癌干细胞高峰论坛 2016年10月9日 上海奉贤区 Targeting HSP90-HDAC6 regulating network implicates precision treatment of breast cancer
江苏省生物化学与分子生物学学会青年委员会学术会议 2014年10月19日 南京市栖霞区文苑路1号 CD44 functions as an upstream regulator of a signaling network in breast cancer
     
代表作
论文名称
Adhesion glycoprotein CD44 functions as an upstream regulator of a network connecting ERK, AKT and H
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
林承棋 正高 教授 博导 东南大学
潘玉峰 正高 教授 博导 东南大学
樊红 正高 教授 博导 东南大学
马飞 正高 教授 博导 南京师范大学
刘平 正高 教授 博导 南京师范大学
      
答辩秘书信息
姓名职称工作单位备注
史兴娟 副高 讲师 东南大学