星形胶质细胞瘤IDH1突变对TAZ蛋白表达的影响

李宁宁　熊　佶　汪　寅　朱静静　刘　颖△

(1复旦大学基础医学院病理学系　上海　20032; 2 复旦大学附属华山医院病理科　上海　200040)



星形胶质细胞瘤IDH1突变对TAZ蛋白表达的影响

李宁宁1熊佶2汪寅2朱静静2刘颖1△

(1复旦大学基础医学院病理学系上海20032;2复旦大学附属华山医院病理科上海200040)

目的研究星形胶质细胞瘤异柠檬酸脱氢酶1 (isocitrate dehydrogenase 1,IDH1)突变对TAZ (transcriptional coactivator with PDZ-binding motif)蛋白的影响,并探讨相关机制。方法采用稳定转染突变型IDH1-132H (132H)及野生型IDH1-132R (WT)的胶质母细胞瘤 (U87MG)细胞,通过Western blot法检测细胞内TAZ蛋白表达;免疫组化法检测14例IDH1突变阴性和7例IDH1突变阳性的人脑胶质母细胞瘤组织样本中TAZ蛋白及其胞质结合蛋白14-3-3e的表达差异;qRT-PCR观察两组细胞TAZ在mRNA水平有无差异;Western blot方法检测Hippo信号通路核心激酶LATS1及磷酸化TAZ蛋白的表达,以及14-3-3e蛋白的表达。结果Western blot结果表明 IDH1突变的星形胶质细胞瘤中TAZ蛋白表达降低;人体胶质细胞瘤组织的免疫组织化学结果与Western blot一致,证实了IDH1突变的星形胶质细胞瘤中TAZ胞浆结合蛋白14-3-3e表达升高;qRT-PCR发现IDH1突变细胞TAZ mRNA水平相比野生型细胞表达明显降低;IDH1突变细胞中,LATS1以及TAZ磷酸化水平升高且细胞14-3-3e蛋白表达升高。结论星形胶质细胞瘤IDH1突变导致TAZ mRNA及蛋白水平表达降低,并通过活化Hippo 信号通路影响TAZ蛋白的磷酸化水平从而影响TAZ表达。

星形胶质细胞瘤;IDH1突变;TAZ蛋白;Hippo 信号通路;14-3-3e蛋白

胶质细胞瘤是中枢神经系统最常见的恶性肿瘤,而胶质母细胞瘤 (glioblastoma,GBM)是其中侵袭性最高、预后最差的一种类型[1]。由于分子生物学技术的进步,对肿瘤的诊断逐步向可靠、实用的分子诊断方向发展[2]。异柠檬酸脱氢酶1 (isocitrate dehydrogenase 1,IDH1)突变是星形胶质细胞瘤诊断中一个非常关键且热门的分子标记物,在肿瘤中发现的IDH1-R132H突变,表现为编码IDH1蛋白132位氨基酸由精氨酸变为组氨酸[3]。有研究统计,R132H在Ⅱ～Ⅲ级胶质瘤中的发生率约80%,在GBM患者中约12%,现普遍认为其与肿瘤侵袭性及患者预后密切相关[4],但具体机制仍有待进一步研究。

Hippo信号通路最早在果蝇中发现,参与调控细胞的生长、增殖、凋亡,它与器官大小和组织再生密切相关。当遗传或环境因素导致其调节机制失效,可使细胞数目增多、器官增大,从而导致肿瘤发生[5]。TAZ (transcriptional coactivator with PDZ-binding motif)蛋白和YAP (yes-associated protein)是同源蛋白,两者都是Hippo信号通路下游的致癌性转录共激活因子[6],TAZ可在细胞核内与转录因子结合调控下游基因表达,进而参与细胞内的信号转导、细胞增殖与凋亡以及组织器官稳态的调控[7]。在TAZ入核发挥作用的过程中受到Hippo信号通路的负性调控,即TAZ受到Hippo信号通路尤其是激酶LATS的调控而磷酸化,磷酸化的TAZ可与其胞质中的结合蛋白14-3-3结合而滞留在细胞质中[8],使TAZ降解增多、活性降低[9]。有研究发现TAZ蛋白在胶质瘤中呈高表达,其表达情况与肿瘤侵袭性及肿瘤级别正相关,且发现TAZ蛋白的表达与肿瘤预后呈负相关,这提示TAZ可能在胶质瘤的发生和发展中起重要作用[10]。肿瘤内在的生物学特性,特别是分子水平的差异很可能与肿瘤的发生、发展及预后有密切关系。本研究首次将胶质细胞瘤IDH1突变与TAZ蛋白的表达联系起来,致力于发现胶质细胞瘤发生IDH1突变后其生物学行为发生改变的分子机制,从而为临床提供个体化治疗的理论依据。

材 料 和 方 法

细胞培养 人胶质母细胞瘤细胞系U87MG (美国ATCC公司),稳定转染突变型IDH1-132H突变及空载对照,分别标记为IDH1-132H和IDH1-WT两株细胞 (复旦大学IBS熊跃教授惠赠),用含10%FBS (美国Gibco公司) 的DMEM高糖培养基 (美国Gibco公司)培养,细胞培养在37 ℃恒温培养箱中,培养室内通入5% CO2以维持细胞培养液的酸碱度平衡。

临床样本样本来自复旦大学附属华山医院神经外科2012—2015年的手术石蜡组织,依据中枢神经系统肿瘤诊断标准 (Word Health Organization,2000)诊断为GBM,14例IDH1阴性,7例IDH1阳性 (IDH1-R132H特异性抗体,德国Dianova公司,internal clone H09,稀释度1∶100),所有人体组织学样本获取及使用过程获复旦大学基础医学院伦理委员会批准。

Western blot 运用Western blot方法检测132H及WT细胞中TAZ (TAZ兔抗,美国CST公司,货号8418,稀释度1∶1 000),磷酸化TAZ [兔抗P-TAZ (SER89),美国Santa Cruz公司,货号sc-17610-R,稀释度1∶200],LATS1 (兔抗LATS1,美国CST公司,货号3477,稀释度1∶1 000)以及14-3-3e (兔抗14-3-3e,美国CST公司,货号9635,稀释度1∶1 000)蛋白的表达。内参选用GAPDH (鼠抗GAPDH,美国Santa Cruz公司,货号sc-166574,稀释度1∶2 000),二抗 (羊抗小鼠/兔二抗,美国Proteintech公司,货号SA00001-1/2,稀释度1∶1 000),将细胞裂解后提取细胞蛋白并定量,转膜后与相应蛋白抗体孵育过夜,第2天加二抗并显色。

免疫组化TAZ单克隆抗体 (鼠抗TAZ,美国BD PharmingenTM公司,货号560235,稀释度1∶200),14-3-3e (兔抗14-3-3e,美国CST公司,货号9635,稀释度1∶1 000),人脑GBM石蜡组织样本,采用辣根过氧化物酶法检测TAZ蛋白的表达情况。

qRT-PCR运用qRT-PCR法检测132H和WT两株细胞中TAZ mRNA的表达。将细胞PBS洗净后加入预冷的Trizol提取RNA,逆转录成cDNA后进行qRT-PCR。10 μL PCR体系包括:SYBR Premix EX Taq 5 μL,ROX Reference Dye 0.2 μL,上下游引物各0.2 μL,cDNA模板1 μL,ddH2O 3.4 μL。PCR反应条件为:95 ℃ 40 s,95 ℃ 35 s,60 ℃ 1 min,循环40次。

统计学处理 Alphaview SA软件对Western blot 的结果进行灰度分析,Graphpad 6.1软件对所有数据进行统计处理统计处理。采用双侧t检验,P<0.05为差异有统计学意义。

结　　　果

TAZ蛋白在132H和WT细胞中的表达为了检测星形胶质细胞瘤IDH1突变与TAZ蛋白之间的关系,采用稳定转染突变型IDH1-132H及野生型IDH1-132R的胶质母细胞瘤 (U87MG)细胞,通过Western blot 法检测TAZ蛋白的表达情况。IDH1-R132H组细胞TAZ蛋白表达较IDH1-WT组低,差异有统计学意义 (图1)。

人脑石蜡组织切片TAZ蛋白表达水平采用免疫组化法检测TAZ蛋白及其胞质结合蛋白14-3-3e在人体组织样本中的表达情况。将TAZ免疫组化标记结果按12分法进行半定量分析。染色强度分为0～3分,阴性标记为0分,弱阳性标记为1分,中等强度标记为2分,强阳性标记为3分,阳性细胞百分比对应值:将阳性的细胞数<5%算为0分,5%～25%为1分,26%～50%为2分,51%～75%为3分,>75%为4分。然后将染色强度×阳性细胞百分比对应的值作为评分,最高分为12分。免疫组化结果显示132H突变型GBM病例组TAZ蛋白表达较野生型GBM病例组明显降低,而14-3-3e蛋白的表达则明显升高,差异均具有统计意义 (图2,放大倍数为10×40倍)。

The expression of TAZ protein decreased in IDH1 mutated cells (132H) compared with that of control cells (WT);A:Western blot;B:The ratio of gray value.(1)P<0.05.

图1IDH1-132H和IDH1-WT两株细胞中TAZ蛋白的表达

Fig 1The expression of TAZ protein in IDH1-132H and IDH1-WT cells

qRT-PCRqRT-PCR检测两株细胞中TAZ mRNA的表达,以β-actin为内参,RQ值采用2-Δct的方法计算,132H细胞TAZ在mRNA水平降低,差异有统计学意义 (图3)。

Western blotTAZ最初是作为14-3-3蛋白的底物被发现的,其结合需要TAZ第89位丝氨酸的磷酸化,TAZser 89在YAP中对应的相同位置是YAPser 127位点,这两个位点恰好符合14-3-3蛋白其中的一个最佳结合序列[11]。Hippo信号通路通过调节丝氨酸/苏氨酸蛋白激酶LATS,使TAZ的第89位丝氨酸 (YAP的第127位丝氨酸)发生磷酸化,从而产生14-3-3蛋白的结合序列,TAZ与14-3-3e蛋白结合滞留胞质内或者直接降解,最终使TAZ活性及表达量均有所降低[12-13]。为了进一步验证IDH1突变是否影响Hippo信号对TAZ的调控,我们采用Western blot检测IDH1突变对Hippo信号通路活化状态以及TAZ胞浆结合蛋白14-3-3e表达的影响。Western blot结果显示在IDH1-132H细胞中LATS1表达高于WT细胞,差异有统计学意义;p-TAZ (ser89)、p-YAP (ser127)及14-3-3e蛋白表达明显升高,差异均具有统计学意义 (图4)。这一结果表明IDH1突变后,Hippo信号通路明显活化,导致TAZ与胞质中14-3-3e结合增多且降解增加。

A:HE staining of IDH1 mutated GBM samples (a) and wild type GBM samples (b);The expression of TAZ was decreased in IDH1 mutated samples (c) compared with that of control samples (d);on the contrary,the expression of 14-3-3e was increased in IDH1 mutated samples (e,f);B,C showed the IHC staining density of TAZ and 14-3-3e separately.(1)P<0.01;(2)P<0.05.

图2人胶质母细胞瘤组织中TAZ及14-3-3e免疫组化染色

Fig 2Immunohistochemistry staining of TAZ and 14-3-3e in human glioblastoma tissue

qRT-PCR showed that TAZ mRNA was also decreased after IDH1 mutation.(1)P<0.01.

图3IDH1-132H和IDH1-WT两株细胞TAZ mRNA表达

Fig 3The relative mRNA expression of TAZ in IDH1-132H and IDH1-WT cells

讨　　　论

脑胶质细胞瘤作为颅内最常见的肿瘤,近年来发病率仍有上升的趋势[14],肿瘤呈广泛浸润性生长,单纯手术难以完全切除,即使结合术后放化疗,其术后复发率仍然很高,患者预后很差,生活质量受到严重影响。2008年Parsons等[4]首次发现IDH1突变的存在,并发现IDH1突变在胶质瘤的形成及诊断中发挥重要作用。IDH1突变也可见于急性髓系白血病 (acute myeloid leukemia,AML)、软骨肉瘤等[15-16]。IDH1究竟以何种机制导致胶质瘤的生物学行为发生改变仍需要进一步探索。

研究普遍认为IDH1突变的高级别胶质瘤相比于同级别的野生型侵袭性降低[17]。事实上,IDH1突变状态能否作为高级别胶质瘤的独立预后因子仍存在很大争议[3,18]。Hartmann等[19]的研究甚至表明,伴IDH1突变的Ⅳ级GBM预后比无IDH1突变的Ⅲ级间变性星形细胞瘤要好。因此,IDH1突变后胶质瘤的生物学行为,包括肿瘤的侵袭性、转移性以及预后仍然需要进一步探索。

有研究表明TAZ作为一个原癌基因,可以促进细胞增殖、迁移、转化,从而促进肿瘤的发生[20]。TAZ蛋白是Hippo信号通路下游最主要的效应器,Hippo通路通过激活激酶LATS后直接使 TAZ发生磷酸化,产生14-3-3蛋白的结合位点。14-3-3蛋白分布于胞质中,与TAZ结合后会促进TAZ滞留在胞质,并且促进其降解而失活[21]。本研究发现IDH1突变的胶质瘤中TAZ蛋白低表达,并在人胶质瘤组织样本中加以验证;本研究进一步探讨了TAZ蛋白表达降低的机制:首先发现TAZ在mRNA水平也有所降低,证明其转录水平的低表达;同时也发现在IDH1突变的细胞中Hippo信号通路中的LATS表达有明显改变,IDH1突变的细胞中TAZ的胞质结合蛋白14-3-3e表达明显升高。这些结果表明IDH1突变的星形胶质细胞瘤通过Hippo信号通路的核心激酶影响TAZ与其胞浆结合蛋白的结合,最终影响TAZ蛋白表达。综上所述,TAZ转录水平上低表达以及蛋白降解的增加导致了TAZ蛋白总量在IDH1突变的细胞中表达明显下降。

The levels of P-TAZ (ser89),14-3-3e,LATS1 and P-YAP (127) were higher expressed in IDH1-132H cells than those in IDH1-WT cells (A) and the ratio of their gray values (B).(1)P<0.05.

图4P-TAZ (ser89)、14-3-3e、 LATS1及P-YAP (127)蛋白的表达

Fig 4The expression levels of P-TAZ (ser89),14-3-3e,LATS1 and P-YAP (127) protein

目前证实IDH1突变可以产生两种酶活性的改变:野生型IDH1功能的下降——即依赖NADP+的催化异柠檬酸转化为α-KG (α-ketoglutarate)减少,以及新催化功能的获得——即还原α-KG生成D-2-HG (D-2-Hydroxyglutarate)增多、同时伴有NADPH的氧化,最终导致α-KG水平下降,D-2-HG水平明显升高[22-25],而正常人体其含量很低。现多认为D-2-HG是一种肿瘤性代谢产物,由于D-2-HG与α-KG结构的相似性,导致其可竞争性抑制多种α-KG依赖的酶,从而引起人体代谢以及表观遗传学的改变[1]。哺乳动物的Hippo信号通路受到精密调节并与许多信号通路之间存在密切联系,从而共同调控细胞数量及器官大小。有研究发现Hippo信号通路与Wnt、CD44、shh、Notch[26-29]等信号通路密切相关。因此,IDH1突变后究竟以何种机制引发TAZ转录水平下降及Hippo信号通路的活化,尚待进一步的研究。

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E-mail:yliu@shmu.edu.cn

Effects of mutated IDH1 on TAZ protein in human astrocytoma

LI Ning-ning1,XIONG Ji2,WANG Yin2,ZHU Jing-jing2,LIU Ying1△

(1DepartmentofPathology,SchoolofBasicMedicalSciences,FudanUniversity,Shanghai200032,China;2DepartmentofPathology,HuashanHospital,FudanUniversity,Shanghai200040,China)

ObjectiveTo investigate the effects of mutated isocitrate dehydrogenase 1 (IDHl) on TAZ (transcriptional coactivator with PDZ-binding motif protein) expression in human astrocytoma and to explore the relevant mechanisms.MethodsGlioblastoma (GBM) U87MG cells transfected with mutated IDH1-132H and wild type IDH1-132R (WT) were used to detected the TAZ protein expression by Western blot;The expression of TAZ protein and its cytoplasmic binding protein 14-3-3e were investigated in a cohort of GBM cases,which including 14 IDH1 wild type cases and 7 IDH1 mutated cases.Invitro,qRT-PCR was used to detect TAZ mRNA expression;Western blot was used to detect the levels of LATS1,phosphorylated TAZ and 14-3-3e protein.ResultsWe found that the expression of TAZ protein was decreased in IDH1 mutated cells compared with those of IDH1 wild type cells.This result was further verifiedinvivoby using immunohistochemistry to detect the expression of TAZ in human GBM samples with or without IDH1 mutation.Immunohistochemistry also showed that TAZ cytoplasmic binding protein 14-3-3e was increased;qRT-PCR showed that TAZ mRNA level was decreased in IDH1 mutated cells;and subsequent Western blot showed the levels of LATS1,TAZ phosphorylation and the 14-3-3e were increased in IDH1 mutant cells.ConclusionsIDH1 mutation in gliomacould reduce TAZ mRNA and its protein expression,increase the phosphorylation level of TAZ protein by activating Hippo signaling pathway,thereby furtherinhibit TAZ expression.

astrocytoma;IDH1 mutation;TAZ protein;Hippo signaling pathway;14-3-3e protein

R36, R739.4

Adoi: 10.3969/j.issn.1672-8467.2016.04.001

2016-02-19;编辑:王蔚)

国家自然科学基金面上项目(81272796)

*This work was supported by the General Program of National Natural Science Foundation of China (81272796).