Rictor在结肠肿瘤发展中多向性调控的研究
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摘要
第一部分PKD对Rictor的转录后调控机制的研究
目的和意义
Rictor (Rapamycin-insensitive companion of mTOR)是mTOR复合物2(mTORC2)中一个新确定的蛋白成员,参与调节mTORC2信号及其下游靶基因如Akt和SGK1。mTORC2作为mTOR信号通路中一个新的复合物,对其调控的分子及自身调控的报道研究仍不完善,且对Rictor上游的相关调控尚无文献报道。本研究拟检测蛋白激酶D (PKD)与Rictor的相互作用,从而探索PKD对Rictor的调控机制,并深入了解Rictor作为mTORC2中的成员在肿瘤发生发展中的作用,以期指导恶性肿瘤的生物靶向治疗。
材料和方法
1、主要材料
293T、HT29和HCT116细胞,G66976、G56983和CID755673, MG132,CHX,抗体(PKD1、PKD2、PKD3、Rictor、mTOR、Raptor、p-Akt和Akt),组织芯片,RNA提取试剂盒,逆转录试剂盒,免疫组织化学相关试剂,免疫印迹及免疫共沉淀相关试剂。
2、方法
2.1细胞培养
293T细胞使用含10%FBS的DMEM培养,HT29和HCT116细胞采用含10%FBS的McCoy's5A培养,所有细胞置于37℃,5%C02,湿度充分的恒温培养箱中。
2.2慢病毒的包备与稳定株的建立
将7×106个293T细胞分别种于100mm培养皿中培养过夜,按照说明书使用Lipfectmin2000转染相应的质粒入细胞中(shRNA、pPAX2和pMD2G各个质粒的转染比例为4：3：1),转染后6小时换新鲜培养液,48小时后收集细胞上清,离心过滤后即为病毒悬液。测试病毒滴度后取等量的病毒液置于待感染的细胞,并补等量的培养液培养48小时。根据细胞对puromycin的敏感度采用不同浓度的puromycin筛选1-2周后,Real-time PCR鉴定筛选稳定表达株。剩余病毒液分装后置于-70℃保存备用。
2.3细胞总RNA提取与Real-time PCR
1×106细胞种于6孔板中常规培养过夜。采用RNeasy Mini试剂盒提取总细胞RNA,紫外分光光度计定量后,取1μg总RNA用无RNA酶的纯净水补至10μl,加入预先配制好的10μl/反应的逆转录酶混合液中,上机25℃10min、37℃120min和85℃10sec,将总RNA逆转录生成cDNA。按Taqman基因表达检测方法检测PKD2, PKD3和Rictor的mRNA水平。
2.4蛋白样品制备及免疫印迹
转染后48小时或相应处理后按指定时间收集细胞。采用1%Triton裂解液常规冰上裂解细胞蛋白30min (20mM Tris-HCl (pH7.5),150mM NaCl,1mM Na2EDTA,1mM EGTA,1%Triton,2.5mM sodium pyrophosphate,1mM beta-glycerophosphate,1mM Na3VO4,1g/ml leupeptin),低温高速离心后检测蛋白浓度。加入上样缓冲液的蛋白裂解液加热至95℃、5min变性后,取等量的蛋白用MOPS SDS电泳缓冲液于4-12%Bis-Tris梯度胶电泳200V、60min分离蛋白,NuPAGE转移缓冲液中70V、120min将蛋白转移至预先用甲醇处理的PVDF膜。5%脱脂牛奶室温封闭1小时后分别采用特异性的一抗4℃孵育过夜。TBST洗膜,5分钟×3次；加入对应的二抗(α-Rabbit抗体1:5000; α-Mouse抗体1：4000)室温孵育1小时,TBST洗膜,于暗室中ECL或ECL plus显影曝光。
2.5免疫共沉淀
细胞分别转染相应的质粒48小时后,常规裂解细胞。定量后每组取500μg蛋白进行实验。首先每组蛋白使用20μl protein G PLUS凝胶珠4℃预孵育半小时,离心后弃去沉淀。取上清分别加入Myc抗体锚定的凝胶珠20μl或正常IgG作阴性对照,置于4℃360°旋转器上过夜孵育。次日,使用裂解液将凝胶珠充分洗净后加入等量的2×上样缓冲液混匀,加热至95℃、5min变性,所得样品全部上样,行Western Blot检测相应蛋白的表达水平。
2.6细胞增殖及克隆形成实验
将1×105个构建的空白对照、Rictor、PKD2、PKD3和PKD2&3稳定敲低表达细胞株种于6孔板中常规培养,每3天更换培养基,并于第1天和第7天分别用胰酶消化细胞成单细胞悬液,细胞仪(Beckman coulter)计数细胞数量,直接比较细胞增殖速度。另外,配制含0.8%软琼脂的培养基事先铺于60mm培养皿中,超净台中充分凝固。将等数量上述稳定表达株的单细胞重悬于含0.4%软琼脂的培养基中,铺于处理后的培养皿上,置于超净台中常温凝固。转移入37℃、5%CO2、湿度充分的恒温培养箱中培养10～12天后取出,甲醛固定,0.005%结晶紫染色后显微镜下计数细胞克隆形成数目。
2.7免疫组织化学
组织芯片置于58℃烤箱中孵育固定30min后,立即依次转移入二甲苯中5minx2次、95%乙醇3minx2次、双蒸水×2次,脱蜡至水。3%H202室温封闭组织切片10min,PBS缓冲液冲洗2次后,放入盛有抗原修复液0.01M枸橼酸钠缓冲溶液(pH=7.0)的耐高温容器,在微波炉里加热至沸腾后,断电,间隔5～10min,反复2次后停止冷却至室温；滴加正常血清封闭液,室温20mmin,甩去多余液体；分别滴加稀释后的Rictor、PKD2和PKD3抗体,每组均以抗体稀释液作为空白对照,4℃孵育过夜；PBS缓冲液5min×3次,滴加相应的生物素化二抗,室温孵育20min; PBS缓冲液5min×3次,DAB显色(镜下掌握显色程度及时间)；双蒸水洗,苏木素复染20sec,盐酸酒精分化1sec；乙醇脱水、二甲苯透明,中性树脂封片后镜检拍照。
2.8数据统计
Real-time PCR、细胞增殖、克隆形成实验结果用三次实验结果的x±SD表示,均采用两独立样本t检验分析,检验水准以P<0.05认为差异有统计学意义,以上分析使用SPSS13.0完成。
结果
1、PKD2和PKD3共同参与Rictor的调节
Go6976处理结肠癌细胞后Rictor的表达降低,而Go6983对Rictor的表达无明显影响；特异性PKD抑制剂CID755673也可抑制Rictor的表达,这就说明PKD可能参与Rictor的表达调控(图1-2.A)。但是采用shRNA分别抑制PKD三个亚型PKD1、PKD2和PKD3的表达,未能对Rictor的表达产生影响(图1-2.B)；同样的,在HT29中分别过表达PKD1和PKD2,对Rictor蛋白水平均无影响(图1-2.C)。当我们使用PKD3的腺病毒感染结肠癌细胞后发现,Rictor蛋白水平增加,且其下游靶基因Akt的磷酸化水平也同时增加(图1-2.D)。
综合以上结果,PKD抑制剂可抑制Rictor的表达,而单独抑制其中任意—个亚基均不能起到相同的作用,因此,PKD对Rictor的调控可能是通过两个或以上的PKD亚型同时起作用。将PKD2shRNA和PKD3shRNA同时感染HT29细胞建立稳定双重敲低表达株,免疫印迹验证成功敲低PKD2和PKD3的表达。此时检测Rictor的表达水平较空白对照组降低,同时Akt磷酸化水平也降低(图1-2.E)。因而我们推测,PKD对Rictor的调控主要是通过PKD2和PKD3两个亚型来实现的。
2、PKD2和PKD3能与Rictor相结合
Real-time PCR检测了PKD对Rictor mRNA水平的影响,GAPDH为内参,每组实验重复3次,结果取平均值,以两独立样本t检验分析数据。如图1-3.A所示,PKD2和PKD3双重敲低稳定表达细胞(PKD2&3sh)与空白对照细胞(Ctrlsh)中PKD2mRNA的表达分别为0.16±0.016和1.00±0.046,PKD3的表达分别为0.12±0.020和1.00±0.058,PKD2和PKD3mRNA水平差异具有统计学意义(t值分别为24.910和30.200,P值为0.000和0.000),提示稳定敲底表达株构建成功；两细胞中Rictor的表达分别为0.86±0.346和1.00±0.097,差异无统计学意义(t值为0.705,P值为0.520)；如图1-3.B所示,腺病毒过表达PKD3细胞(Ad-PKD3)与空白对照细胞(Ad-Ctrl) PKD3的表达为17.05±0.181和1.00±0.026,PKD3表达水平差异具有统计学意义(t值为-151.500,P值为0.000),Rictor的表达水平为4.81±0.297和4.27±0.524,差异无统计学意义(t值为-1.544,P值为0.198)。因此,PKD并不是从转录水平影响Rictor的表达。
接下来,我们想进一步验证PKD与Rictor蛋白间的相互作用。HCT116细胞共同转染Myc-Rictor和GST-PKD1或Myc-Rictor和GST-PKD2,免疫共沉淀实验发现PKD2可与Rictor相结合,IgG对照组无结合,但未检测到PKD1与Rictor的相互作用(图1-3.C)；另外,PKD3腺病毒高表达的细胞转染Myc-Rictor,用Myc (Myc-Rictor)标记的凝胶珠与蛋白孵育后,免疫印迹检测到了PKD3的表达,说明PKD3也可与Rictor蛋白相结合(图1-3.D)。这就进一步证实了我们的假设,PKD1不与Rictor蛋白结合,未直接参与Rictor的调节,而PKD2和PKD3则可通过与Rictor直接或间接的结合来调控其蛋白水平的表达。
3、Rictor通过泛素化途径降解,过表达PKD3可抑制Rictor的降解
基于以上结果得知,PKD对Rictor是转录后水平的调控,这就包括翻译水平和蛋白稳定性的调控。26蛋白酶体信号通路参与蛋白转录后调节,蛋白被泛素化修饰后即被蛋白酶体进一步的水解成多肽。MG132为一个常用的真核细胞蛋白酶体抑制剂,可透过细胞膜选择性的抑制蛋白酶体。如图1-4.A所示,使用MG132处理HT29细胞不同的时间,发现Rictor的表达水平增加,mTOR信号通路中的mTOR和Raptor分子水平也有所增加,同时伴有mTORC2下游靶分子Akt的磷酸化水平增加。随后,我们采用CHX(放线菌酮,蛋白合成抑制剂)抑制细胞新的蛋白的合成,发现Rictor蛋白的表达水平随着处理时间的延长而降低(图1-4.B)。因此,可以确定Rictor蛋白是通过26蛋白酶体通路来降解的。
为进一步明确PKD对Rictor蛋白的调控是否是通过26蛋白酶体途径来实现,我们设计了CHX追逐实验进行验证。CHX (cycloheximide)为一种蛋白合成抑制剂,可以抑制真核细胞蛋白的合成,常用于观察蛋白的稳定性。如图1-4.C所示,在对照细胞中,Rictor蛋白随CHX处理时间的延长而降低,说明Rictor蛋白处于正常的降解过程中；而在PKD3过表达的细胞中,CHX处理细胞几乎不引起Rictor蛋白的降解,说明过表达PKD3可增加Rictor蛋白的稳定性,抑制了Rictor蛋白的降解。
4、PKD和Rictor不影响结肠癌细胞的增殖
如图1-2.D和1-2.E所示,PKD可通过影响Rictor的表达进而改变Akt的磷酸化水平。众所周知,Akt在肿瘤细胞增殖、分化、凋亡和转移过程中起着重要的作用,且有报道指出,抑制mTOR可抑制肿瘤细胞的增殖。计数等量的各个稳定细胞株培养7天,分别于第1天和第7天计数细胞,以第7天与第1天细胞的增殖倍数作为结果比较。如图1-5.A,在HCT116和HT29中,Rictor敲低表达(Rictor sh)与空白对照(Ctrlsh)细胞的增殖速度分别为17.25±3.256、21.17±2.926和14.72±2.104、11.70±2.405,此次实验采用两样本t检验分析,组间差异无显著性(t值分别为1.552和1.633,P值分别为0.196和0.178)。然后我们观察PKD各个亚型对肿瘤细胞增殖的影响(图1-5.B和图1-5.C),HCT116和HT29对照细胞与PKD2或PKD3敲低表达细胞(PKD2sh或PKD3sh)的增值速度分别为27.79±3.150、30.61±2.706或29.08±0.995和50.16±12.928、43.88±4.638或35.22±3.846,采用两样本t检验差异无统计学意义(t值分别为-1.176、-0.647和0.792、1.919,P值分别为0.305、0.537和0.472、0.127)。HCT116和HT29对照细胞与双重敲低表达细胞(PKD2&3sh)的增值速度分别为27.17±7.029、30.31±5.108和10.45±1.433、13.24±3.476,采用两样本t检验差异无统计学意义(t值分别为-0.626和-1.285,P值分别为0.565和0.268)。
5、抑制PKD和Rictor降低结肠癌细胞的致瘤性
于是,我们采用软琼脂集落形成实验观察PKD和Rictor对肿瘤细胞致瘤性的影响(图1-6)。结果发现,空白对照细胞(Ctrlsh)与Rictor敲低表达细胞(Rictorsh)、空白对照细胞与PKD2&3双重敲低表达细胞(PKD2&3sh)的克隆形成数目分别为180.00±22.869、63.33±12.097和119.33±27.791、42.67±35.233,抑制Rictor和PKD2&3均能显著抑制结肠癌细胞的致瘤作用,差异具有统计学意义(t值为7.811和2.959,P值分别为0.001和0.042)。
6、PKD和Rictor在结肠癌原位及转移肿瘤中高表达
最后,我们检测了结肠正常组织、原位和肿瘤肝转移组织中PKD和Rictor的表达水平。图1-7分别显示了各个蛋白在组织中的表达,可见在结肠正常组织中,Rictor、PKD2和PKD3的表达水平较低或不表达,而在原位及肿瘤肝转移组织中,三者的表达水平均较高。原位肿瘤组织中Rictor和PKD2主要表达在细胞浆中,PKD3在细胞浆和细胞核中均有表达；而在肿瘤肝转移组织中三者在细胞核和细胞浆均有表达,随着肿瘤的进展程度,PKD和Rictor在细胞内聚集的位置发生了一定的改变,进一步提示Rictor、PKD2和PKD3均与结肠癌的进展有关。
结论
获得了蛋白激酶D (PKD)参与调节Rictor/mTOR信号通路的证据。过表达PKD3可增加Rictor的蛋白水平,而同时抑制PKD2和PKD3可降低Rictor蛋白的水平,两种调节均不影响Rictor的mRNA水平；PKD2和PKD3在结肠癌细胞中能与Rictor结合并增强其下游靶基因Akt的活性；PKD3可影响Rictor蛋白的稳定性；PKD和Rictor对结肠癌细胞的增殖无明显的影响,但可增加其致瘤性；PKD和Rictor在结肠原位和肝转移肿瘤组织中均高表达。以上结果均提示PKD在一定程度上参与了Rictor的转录后调控,从蛋白的修饰方面影响了Rictor蛋白的稳定性,从而参与Rictor/mTOR信号通路的调控；抑制PKD可下调Rictor蛋白的表达,并抑制结肠癌细胞的致瘤性,为肿瘤的生物靶向性治疗提供的一定的理论依据。
第二部分Rictor在mTOR非依赖的信号通路中的调控研究
目的和意义
Rictor (Rapamycin-insensitive companion of mTOR)作为mTORC2中的成员,可磷酸化并激活Akt,激活的Akt可诱导c-Myc和cyclin E的表达,从而对结肠癌细胞增殖和周期起重要的调节作用。本研究检测了Rictor在mTOR信号通路非依赖的调控的作用,与FBXW7相互作用作为一个E3复合物调节c-Myc和cyclin E蛋白的降解,并了解该调节在肿瘤发展及治疗中的作用。本课题发现了Rictor在结肠癌肿瘤中新的调控作用,对新的临床用药指导有着重要的意义。
材料和方法
1、主要材料
293T、SW620、HT29和HCT116细胞,MG132, CHX,抗体(ubiquitin、 c-Myc、cyclin E、Rictor、mTOR、Raptor p-Akt和Akt), RNA提取试剂盒,逆转录试剂盒,免疫印迹相关试剂,免疫共沉淀相关试剂。
2、方法
2.1细胞培养
293T和SW620细胞使用含10%FBS的DMEM培养,HT29和HCT116采用含10%FBS的McCoy's5A培养,HCT116-FBXW7+/+和HCT116-FBXW7-/-同样培养于含10%FBS的McCoy's5A培养液中。所有细胞采用上述培养基置于37℃,5%CO2,湿度充分的恒温培养箱中。
2.2慢病毒的包备与稳定株的建立
将7×106个293T细胞分别种于100mm培养皿中培养过夜,按照说明书使用Lipfectmin2000转染相应的质粒入细胞中(shRNA、pPAX2和pMD2G各个质粒的转染比例为4：3：1),转染后6小时换新鲜培养液,48小时后收集细胞上清,离心过滤后即为病毒悬液。测试病毒滴度后取等量的病毒液置于待感染的细胞,并补等量的培养液培养48小时。根据细胞对puromycin的敏感度采用不同浓度的puromycin筛选1-2周后,Real-time PCR鉴定筛选稳定表达株。剩余病毒液分装后置于-70℃保存备用。
2.3细胞总RNA提取与RT-PCR
1×106细胞种于6孔板中常规培养过夜。采用RNeasy Mini试剂盒提取总细胞RNA,紫外分光光度计定量后,取1μg总RNA用无RNA酶的纯净水补至10μl,加入预先配制好的10μl/反应的逆转录酶混合液中,上机25℃10min、37℃120min和85℃10sec,将总RNA逆转录生成cDNA。根据靶基因序列及参考文献设计引物,Hotstart热启动酶扩增目的DNA片段,凝胶电泳分离后紫外灯观察拍照。
2.4蛋白样品制备及免疫印迹
转染后48小时或相应处理后按指定时间收集细胞。采用1%Triton裂解液常规冰上裂解细胞蛋白30min (20mM Tris-HCl (pH7.5),150mM NaCl,1mM Na2EDTA,1mM EGTA,1%Triton,2.5mM sodium pyrophosphate,1mM beta-glyceropho sphate,1mM Na3VO4,1g/ml leupeptin),低温高速离心后检测蛋白浓度。加入上样缓冲液的蛋白裂解液加热至95℃、5mmin变性后,取等量的蛋白用MOPS SDS电泳缓冲液于4～12%Bis-Tris梯度胶电泳200V、60min分离蛋白,NuPAGE转移缓冲液中70V、120min将蛋白转移至预先用甲醇处理的PVDF膜。5%脱脂牛奶室温封闭1小时后分别采用特异性的一抗4℃孵育过夜。TBST洗膜,5分钟×3次；加入对应的二抗(a-Rabbit抗体1:5000; a-Mouse抗体1：4000)室温孵育1小时,TBST洗膜,于暗室中ECL或ECL plus显影曝光。
2.5免疫共沉淀
细胞分别转染相应的质粒48小时后,分别采用1%Triton裂解液或0.3%CHAPS裂解液裂解细胞。定量后每组取500μg蛋白进行实验。首先每组蛋白使用20μl protein G PLUS凝胶珠4℃预孵育半小时,离心后弃去沉淀。取上清分别加入抗体1μg或抗体锚定的凝胶珠20μl,置于4℃360°旋转器上过夜孵育。次日,加入抗体的实验组再给予20μl protein G PLUS凝胶珠4℃旋转孵育4小时。使用相应裂解液将凝胶珠充分洗净后加入等量的上样缓冲液加热至95℃、5min变性,所得样品全部上样行Western Blot检测相应蛋白的表达水平。
2.6体内泛素化水平检测
蛋白泛素化水平检测时,将His-ubiquitin表达载体与指定的质粒共转染入细胞内,收集细胞4小时前使用MG132处理细胞。100μl0.3%CHAPS裂解液常规裂解细胞,加入10%SDS10μl混匀后加热至95℃、5min变性蛋白,再加入900μl0.3%CHAPS裂解液超声裂解20秒,离心后用免疫共沉淀方法检测细胞内靶蛋白的泛素化水平。
2.7数据统计
本部分未涉及统计学处理。
结果
1、Rictor调节c-Myc和cyclin E蛋白的表达水平
过去的研究充分表明,Akt的激活可增加c-Myc和cyclin E的表达。因而我们想进一步了解,mTORC2活性的抑制对c-Myc和cyclin E表达的影响。在稳定转染Rictor shRNA了的SW620和HT29细胞中,敲低Rictor并未明显影响到c-Myc和cyclin E的表达；然而,同样的细胞给予血清饥饿刺激后再检测c-Myc和cyclin E,发现抑制Rictor可明显增加两者的表达水平(图2-3.A),这就与之前的报道存在一定的差别。另外,我们使用MG132(一个特异性的细胞26蛋白酶体抑制剂,可抑制蛋白的降解)来处理血清饥饿后的细胞,结果发现,MG132能够逆转Rictor敲低所引起的c-Myc和cyclin E蛋白水平的增加(图2-3.B)。这就说明,26S蛋白酶体信号通路参与了Rictor的这一调节。
为了进一步验证我们的结果,使用Rictor的siRNA转染入细胞中瞬时压低Rictor的表达,收集蛋白前使用血清饥饿预处理细胞,发现siRNA瞬时抑制Rictor的表达后也可使c-Myc和cyclin E的表达增加(图2-3.C)。如图2-3.D所示,过表达Rictor同样可以抑制c-Myc和cyclin E蛋白水平的表达。有趣的是,在SW620Rictor shRNA稳定敲低表达细胞中,RT-PCR结果显示,Rictor低表达可抑制c-Myc和cyclin E的mRNA水平,与其对蛋白水平的调控相反(图2-3.E)。
综上所述,Rictor在无血清的培养状态下可调节c-Myc和cyclin E蛋白水平的表达。由于Akt磷酸化激活后导致c-Myc和cyclin E表达水平的增加,而此前有报道指出Rictor敲低表达可导致Akt的去磷酸化和失活表达,Akt表达水平的变化导致c-Myc和cyclin E mRNA及蛋白水平降低。故我们有理由相信,Rictor是通过mTORC2/Akt非依赖的信号通路来调节c-Myc和cyclin E蛋白的表达。
2、Rictor与FBXW7的相互作用
作为一个E3的组成部分,FBXW7可影响其靶基因c-Myc和cyclin E的泛素化修饰从而促进它们的降解。故我们推测Rictor是否通过与FBXW7相结合来进一步调控c-Myc和cyclin E。Myc-Rictor分别与Flag-FBXW7α、Flag-FBXW7p和Flag-FBXW7y共同转染入细胞中,免疫共沉淀结果显示,Rictor与FBXW7的三个亚型均有结合(图2-4.A)。由于mTOR也是FBXW7下游的靶基因之一,可与FBXW7结合后被泛素化修饰、降解,故我们需要进一步的实验鉴别Rictor与FBXW7的结合与mTOR/FBXW7的关系。我们构建了3个包含Rictor蛋白不同片段的表达载体(所有截短载体均包含碳末端),测序鉴定成功后分别与Flag-FBXW7a共同转染入细胞中。如图2-4.B所示,在3个Rictor截短表达载体及全长Rictor载体中,同时检测到FBXW7a和mTOR的结合表达。我们需要进一步的实验来鉴别Rictor/FBXW7复合物是否是通过mTOR而间接连接的。1%Triton裂解液可破坏mTORC2,而0.3%CHAPS裂解液则较缓和,能保持mTORC2的完整性。如图2-4.C所示,在使用1%Triton和0.3%CHAPS裂解液的两组实验中均检测到了Rictor与FBXW7a的相互作用,而Rictor与mTOR的相互作用仅能在使用0.3%CHAPS裂解液的实验组中检测到。这就说明,Rictor和FBXW7的相互作用是独立于mTORC2以外的。
由于我们所检测到的Rictor对c-Myc和cyclin E蛋白水平的调控是在无血清培养的条件下的,那么Rictor与FBXW7的作用是否也依赖于血清饥饿而存在呢?如图2-4.D所示,在血清培养和血清饥饿培养的转染细胞中,均检测到了Rictor与FBXW7的相互作用,说明Rictor/FBXW7复合物是不依赖于血清饥饿而存在的。
3、Rictor通过FBXW7调节c-Myc和cyclin E
基于以上实验结果,我们推测Rictor对c-Myc和cyclin E的调节可能是通过与两者特异性的E3-BXW7共同组成E3复合物来起作用的。因此,我们在野生型FBXW7+/+和缺失型FBXW7-/-细胞中分别建立Rictor shRNA稳定敲低表达株及空白对照株。如图2-5.A所示,在野生型(FBXW7+/+)HCT116细胞中敲低Rictor表达后可诱导c-Myc和cyclin E的表达,但在缺失型(FBXW7-/-)HCT116细胞中同样的处理未见c-Myc和cyclin E表达水平的变化。同时,HCT116control shRNA和Rictor shRNA稳定细胞株中分别转染空白质粒和Flag-FBXW7a表达载体,结果显示,FBXW7a在对照细胞中可降低c-Myc和cyclin E的蛋白水平,而在Rictor敲低表达的细胞中该作用明显减弱(图2-5.B)。
4、Rictor调节FBXW7的稳定性
Rictor与FBXW7结合后对其作为一个E3复合体有什么样的影响呢?首先,我们进行了RT-PCR实验比较Rictor敲低表达的稳定细胞株和阴性对照细胞株中FBXW7表达水平的差异,结果发现,抑制Rictor表达并未明显影响FBXW7mRNA的水平(图2-6.A),Rictor并没有从转录水平影响FBXW7的表达。然后,我们检测了FBXW7在体内的泛素化水平。如图2-6.B所示,在Rictor敲低表达的稳定细胞株中,Flag-FBXW7a蛋白泛素化水平明显较阴性对照细胞中高,证明抑制Rictor的表达可解聚Rictor/FBXW7复合体,进而促进了FBXW7a的降解。CHX能抑制真核细胞蛋白的合成,使用CHX可观察蛋白降解半衰期的时长,因此CHX追逐实验广泛被应用于蛋白稳定性的检测。图2-6.C显示,在Rictor敲低表达的稳定细胞株中,Flag-FBXW7a蛋白的稳定性明显较阴性对照细胞低,从而进一步验证了我们的假设。
5、Rictor/FBXW7启动c-Myc和cyclin E的泛素化
那么Rictor与FBXW7结合后是如何调节c-Myc和cyclin E的呢?由于FBXW7对c-Myc和cyclin E的调节是通过泛素化修饰来实现的,而且图2-3.E已经显示抑制Rictor表达可在mRNA水平上抑制c-Myc和cyclin E(与蛋白水平的变化不一致),因而我们有理由相信,Rictor在此对c-Myc和cyclin E的调节也是通过与FBXW7相互作用,从蛋白水平上修饰c-Myc和cyclin E来实现的。如图2-7.A和图2-7.C所示,在Rictor敲低表达的稳定细胞株中, c-Myc和cyclinE的泛素化水平明显降低。而且,在野生型HCT116细胞中,敲低Rictor可抑制c-Myc和cyclin E的泛素化；在缺失型(FBXW7-/-) HCT116细胞中,c-Myc和cyclin E的泛素化水平已经很低,抑制Rictor的表达不能进一步抑制两者的泛素化(图2-7.B和图2-7.D)。
6、Rictor降低c-Myc和cyclin E的稳定性
使用CHX追逐实验观察c-Myc和cyclin E的稳定性与Rictor表达的相关关系。如图2-8.A和图2-8.C所示,抑制Rictor的表达可延长c-Myc和cyclin E蛋白降解的半衰期。本研究中使用的Rictor shRNA序列针对的是Rictor的3'-UTR区,在Myc-Rictor表达载体上无作用位点,故可以使用Myc-Rictor表达载体在Rictor敲低表达稳定细胞中重新表达Rictor蛋白。如图2-8.B和图2-8.D所示,Myc-Rictor表达载体在Rictor敲低表达稳定细胞中表达良好,且Rictor的重新表达可缩短c-Myc和cyclin E蛋白的半衰期,从而促进c-Myc和cyclin E的降解。
7、Rictor能与USP28结合,但不参与USP28的去泛素化调节
USP28作为一个去泛素化特异蛋白,可与FBXW7相结合来平衡其下游的靶基因如c-Myc的泛素化与去泛素化修饰,从而影响c-Myc蛋白水平。如图2-9.A所示,在HT29中感染USP28shRNA建立稳定表达株,发现抑制USP28的表达可抑制c-Myc蛋白水平；在HCT116细胞中转染Flag-USP28表达载体,过表达USP28可增加c-Myc蛋白水平,抑制或增加USP28的表达均不影响cyclin E的表达,说明USP28能特异的将c-Myc去泛素化进而抑制其降解,这与前人的发现是一致的。那么,Rictor/FBXW7复合物中是否也存在USP28呢?结果发现,Rictor也能与USP28特异性相互结合,且mTOR没有参与该复合物的形成(图2-9. B)。Rictor是否参与USP28/FBXW7复合物对底物去泛素化的调节呢?在Rictor稳定敲低表达细胞和空白对照细胞中,免疫共沉淀检测FBXW7与USP28间的相互作用并没有受Rictor表达水平的影响(图2-9.C),且抑制Rictor的表达不能逆转USP28所致的c-Myc蛋白增加(图2-9.D),提示USP28与FBXW7的相互作用与Rictor无关,Rictor可通过FBXW7与USP28形成复合物,但Rictor的表达水平对USP28的去泛素化作用无明显的影响。
8、Rictor可能参与Rapamycin诱导的耐药
大量研究工作表明,Rapamycin是通过与mTORC1直接结合来抑制mTORC1信号通路的活性,但也有研究表明长时间Rapamycin的处理也可抑制Rictor的活性。Rapamycin可通过抑制mTOR信号通路来抑制肿瘤的生长,但并不是所有的肿瘤细胞都对Rapamycin的治疗有反应,有些肿瘤细胞就对Rapamycin不敏感,因此明确肿瘤细胞对其耐药的机制就显得尤为重要。如图2-10.A所示,Rapamycin处理HT29和SW620(Rapamycin不敏感细胞)24小时后,Rictor蛋白水平降低,而c-Myc、cyclin E和磷酸化的Akt水平增加。另外,Rapamycin对c-Myc和cyclin E的mRNA无明显影响或仅轻度抑制(图2-10.B),说明Rapamycin诱导的c-Myc和cyclin E增加与激活的Akt无关。以上结果说明Rapamycin可能通过抑制Rictor/FBXW7的活性来激活c-Myc和cyclin E的表达,从而诱导耐药的产生。
结论
发现了Rictor独立于mTOR信号通路以外的调控作用。Rictor可作为一个调节子与FBXW7相结合,从而参与调控与其相关的靶基因如c-Myc和cyclin E的降解；血清饥饿抑制了mTORC/Akt参与的c-Myc和cyclin E的正向调控,在此基础上,抑制Rictor可抑制两者的泛素化修饰,增加两者蛋白的稳定性,导致c-Myc和cyclin E蛋白水平的增加；在血清培养条件下,Rictor同样可与FBXW7形成复合物,且该复合物是独立于mTOR信号通路以外的；Rapamycin诱导的肿瘤细胞耐药性与Rictor的这一调控作用有关。以上结果均提示Rictor存在与mTOR不相关的调控作用,且其调控的结果可能与mTOR信号通路的调节起到相反的作用,因此,应用Rictor作为生物治疗的靶点时应全面考虑到其各个方面的作用,利用其在抗肿瘤治疗中有利的一面,同时避免或抑制其他副作用的产生。
Part one:Post-transcriptional regulation of Rictor by protein kinase D in human colorectal cancer cells
BACKGROUND
mTORC2consists of mTOR, mLST8, Rictor, Sinl and PROTOR/PRR5. mTORC2phosphorylates Akt at Ser473thus resulting in Akt activation, knockdown of Rictor blocks Ser473phosphorylation. Knockdown of Rictor leads to growth inhibition and induces apoptosis in colorectal cancer cells. However, the molecular mechanism(s) involved in the regulation of Rictor expression remains unclear.
Protein kinase D (PKD), a serine/threonine kinase family that includes PKD1, PKD2and PKD3, has been implicated in the regulation of cell proliferation and apoptosis. Selective PKD inhibitor has shown potential inhibitory roles in certain cancer cell proliferation, cell migration, and invasion. Recently, PKD3has been shown to increase Akt phosphorylation at Ser473. The role of PKD and its downstream effectors with regard to CRC proliferation and metastasis are not known.
MATERIALS Go6976, G66983, MG132and cycloheximide (CHX) were purchased from Calbiochem (San Diego, CA). CID755673was from TOCRIS bioscience (Ellisville, Missouri). Antibodies against PKD2and PKD3were obtained from Abgent, Inc.(San Diego, CA). Antibodies against mTOR, Raptor, Rictor, PKD1, PKD2, PKD3, p-Akt, and Akt were obtained from Cell Signaling (Beverly, MA). GST-tagged PKD plasmids expressing wild-type PKD1and2were from Dr. Vivek Malhotra,(Universityof California, San Diego). Recombinant adenovirus expressing wild type (WT)-PKD3was kindly provided by Dr. Q. Jane Wang (University of Pittsburgh, Pittsburgh, PA). Tissue culture media and reagents were obtained from Invitrogen. Polyvinylidene difluoride (PVDF) membranes for Western blots were from Millipore Corp.(Bedford, MA). The enhanced chemiluminescence (ECL) system for Western immunoblot analysis was purchased from Amersham Biosciences.
METHODS
Cell culture and transfection
The human colon cancer cell lines, HT29and HCT116, were purchased from ATCC. HT29and HCT116cells were maintained in McCoy's5A supplemented with10%fetal bovine serum (FBS). PKD inhibitors were initially dissolved in dimethyl sulfoxide (DMSO) and compared with cells treated with DMSO at the same final concentration. HT29and HCT116cells were transfected with the siRNA duplexes and plasmids using electroporation (Gene Pulser; Bio-Rad, Hercules, CA) and lipofectamine2000(Invitrogen, Carlsbad, CA), respectively.
Stably Rictor knockdown HT29and HCT116cells were generated. Cells were infected with the control shRNA or shRNA to human Rictor lentivirus particles and stably expressing cells were selected with puromycin at a concentration of2.5μg/ml. The effective knockdown of Rictor was monitored by Western blot analysis.
RNA extraction and Real-time PCR
Total RNA was extracted and DNase-treated (RQ1, Promega). Synthesis of cDNA was performed with1μg of total RNA using the reagents in the Taqman Reverse Transcription Reagents Kit from ABI (#N8080234). Quantitative real time RT-PCR analysis was performed with an Applied Biosystems Prism7000HT Sequence
Detection System using TaqMan universal PCR master mix according to the manufacturer's specifications (Applied Biosystems Inc., Foster City, CA). The TaqMan probe and primers for human Rictor was purchased from Applied Biosystems. Human GAPDH gene was used as endogenous control. Each sample was run in triplicate. Average Ct values were calculated and normalized to Ct values for GAPDH. The results were graphed with the corresponding standard deviation indicated with error bars in the figures.
Protein preparation and Western blot analysis
Total protein (60μg) was resolved on a4-12%Bis-Tris gel and transferred to polyvinylidene difluoride (PVDF) membranes. Filters were incubated for1h at room temperature in blotting solution. Raptor, Rictor, mTOR, PKD1, PKD2, PKD3, p-Akt, Akt, and P-actin were detected with specific antibodies following blotting with a horseradish peroxidase-conjugated secondary antibody.
Immunoprecipitation
Cells were transfected with different plasmids as indicated,1%Triton lysis buffer was used. After pre-incubation with protein G PLUS-Agarose beads, equal amount of protein (500μg) were incubated with the indicated antibodies (1μg) using an end-to-end rotor overnight at4℃, followed by4h incubation with20μl of protein G PLUS-Agarose beads at4℃. Indicated buffer was used to wash the beads three times. Reactions were stopped by adding15μl of2×loading buffer. Samples were denatured by boiling for7min and separated by NuPAGE4-12%Bis-Tris gels.
Cell proliferation analyses
Equal numbers of cells were seeded onto24-well plates at a density of1×104per well in the appropriate culture medium with supplements. Cells carried with PKD shRNA or Rictor shRNA respectively were cultured for7days. Cells were trypsinized and counted using a cell counter (Beckman-Coulter).
Soft agar assay
Melt1.6%Agarose (www.lonza.com Cat.#50101) in distilled water in microwave, cool to40℃in a water bath, warm culture medium to40℃in water bath as well. Allow at least30minutes for temperature to equilibrate. Mix equal volumes of1.6% of Agar and medium to give0.8%Agar in medium. Add2ml/well of60mm dish, allow cooling down for30min. For plating, mix1ml of cell in medium and1ml0.8%Agar to a tube, mix gently and add2ml to each well (usually plate out in triplicate). Cool down in hood for30-60min. Add culture medium on the top agar for keeping humidity. Incubate assay at37℃in humidified incubator for10-14days. Stain plates with0.5ml of0.005%Crystal Violet (dilute with PBS) for>1hour, wash each well with PBS until every colonies could be seen clearly, count colonies using a dissecting microscope.
Immunoh istoch emical Analysis
Tissue microarrays containing normal and cancer tissues, A203(IV), were purchased from ISUABXIS through Accurate Chemical&Scientific Corporation. Sections were fixed to the slide by incubation in a dry oven at58℃for30min, and then sequentially transferred to xylene (5min,2changes),100%ethanol (3min,2changes),95%ethanol (3min,2changes) and rinsed with deionized water. Slides were allowed to cool at room temperature and rinsed twice with deionized water. Endogenous peroxidase was blocked by placing slides in3%H2O2/methanol block solution for10min, washed with deionized water, and placed in phosphate-buffered saline for5min. Slides were incubated with primary antibodies against human PKD1, PKD2, PKD3or Rictor overnight at4℃. Avidin-biotin peroxidase complex amplification and detection system (LSAB2, DAKO) with diaminobenzidine as chromagen was used. Negative controls (including no primary antibody or isotype matched mouse IgG) were used in each assessment.
Statistical analysis
The data of real time-PCR, proliferation and soft agar were expressed as the mean of three independent expression±S.D, evaluated with t-test. P<0.05was defined as different signicantly. All of these analyses were made using SPSS Version13(SPSS, Chicago, IL, USA).
RESULTS
PKD2and PKD3regulate expression of Rictor
In HT29and HCT116cells, both of Go6976(PKC and PKD inhibitors) and CID755673(PKD specific inhibitors) treatment inhibited expression of Rictor, while Go6983(PKC inhibitor) treatment had no affection on it (Fig.1-2A). Knocking down of PKD1, PKD2or PKD3individually could not inhibit Rictor expression (Fig.1-2B); overexpression of PKDl and PKD2did not increase Rictor (Fig.1-2C) as well. But we found that overexpressed PKD3using adenovirus increased expression of Rictor, with downstream target of Rictor-Akt S473phosphorylation increased at the same time (Fig.1-2D). Next, we set up PKD2and PKD3double knockdown stable cells, western blot analysis showed that both Rictor and p-Akt were decreased compared with control shRNA cells (Fig.1-2E).
PKD2and PKD3bind with Rictor
How do PKDs regulate expression of Rictor? First, we investigated if PKD regulated the mRNA level of Rictor by using real-time PCR. As shown in Fig.1-3A, no affection of Rictor mRNA was detected in PKD2and PKD3double knockdown cells. Similar result was gained in PKD3highly expression cells (Fig.1-3B), indicating the regulation of Rictor by PKD was post-transcriptional. Then, the interaction between PKD and Rictor was detected by Immunoprecipitation. Both of PKD2and PKD3bound with Rictor respectively, while no binding was investigated between PKD1and Rictor (Fig.1-3C and Fig.1-3D). Based on the data above, we hypothesized that PKD2and PKD3interacted with Rictor directly to regulate its protein expression.
Rictor is degradation by proteasomal pathway and overexpression of PKD3inhibits degradation of Rictor
Rictor is rapidly degraded by the ubiquitin-proteasome pathway. HT29cells were treated with MG132for the indicated times and subjected to Western blot analyses (Fig.1-4A). HT29were treated with CHX for the indicated times and Rictor levels were detected by Western analyses (Fig.1-4B).
Mosty importantly, we investigated the role of PKD3during Rictor degradation by CHX chase assay. As shown in Fig.1-4C, we found that the degradation of Rictor was decreased with highly expressed PKD3, demonstrating that overexpression of PKD3inhibited Rictor degradation and increased stability of Rictor protein.
PKD and Rictor do not affect CRC cells proliferation The previous data already showed that PKD affected Akt activities by regulating Rictor (Fig.1-2D and Fig.1-2E). So we was wandering that whether PKD and Rictor were involved in cell proliferation or not. After7days culture, cell numbers were counted by Beckman-Coulter, no difference was detected between control shRNA cells and PKD shRNA/Rictor shRNA cells individually (Fig1-5).
Inhibition of PKD and Rictor reduces tumorigenesis of CRC cells
Next, soft agar assay was used to investigate tumorigenesis ability in those cells carried with different shRNA. As shown in Fig.1-6, inhibition of either Rictor or PKD2&3decreased tumorigenesis ability of colorectal cancer cells.
Rictor as well as PKD2and PKD3are up-regulated in CRCs and liver metastasis
In Fig.1-7, we showed Immunohistochemical analysis of Rictor, PKD2and PKD3in CRC and liver metastasis sections. Rictor as well as PKD2and PKD3were up-regulated in primary CRCs and liver metastases compared with normal mucosa.
CONCLUSIONS
1. Inhibition of PKD2and PKD3decreases Rictor protein expression in CRC cells; consistently, overexpression of PKD3increases Rictor as well.
2. PKD2and PKD3bind with Rictor individually; Rictor is a target of26S proteasomal degradation in CRC cells and PKD3stabilzes Rictor protein
3. No affection on proliferation is detected while knocking down of both Rictor and PKD, while Rictor and PKD up-regulates tumorigenesis of CRC cells.
4. Rictor as well as PKD2and PKD3are up-regulated in primary CRCs and liver metastases compared with normal mucosa, implying a potential role of PKDs upstream of the Rictor signaling pathway in the pathogenesis of CRC.
Part two:Rictor regulates FBXW7-dependent c-Myc and cyclin E degradation in colorectal cancer cells
BACKGROUND:
Rictor (Rapamycin-insensitive companion of mTOR) forms a complex with mTOR and phosphorylates and activates Akt. Activation of Akt induces expression of c-Myc and cyclin E, which are overexpressed in colorectal cancer and play an important role in colorectal cancer cell proliferation. Here, we show that Rictor associates with FBXW7to form an E3complex participating in the regulation of c-Myc and cyclin E degradation. The Rictor/FBXW7complex is biochemically distinct from the previously reported mTORC2and can be immunoprecipitated independently of mTORC2. Moreover, knocking down of Rictor in serum-deprived colorectal cancer cells results in the decreased ubiquitination and increased protein levels of e-Myc and cyclin E while overexpression of Rictor induces the degradation of c-Myc and cyclin E proteins. Genetic knockout of FBXW7blunts the effects of Rictor, suggesting that Rictor regulation of c-Myc and cyclin E requires FBXW7.
MATERIALS
MG132and cycloheximide (CHX) were purchased from Calbiochem (San Diego, CA). Rabbit monoclonal anti-Rictor antibody, used for both immunoblotting and immunoprecipitation, was purchased from Bethyl Laboratories (Montgomery, TX). Rabbit anti-mTOR, rabbit anti-phospho-Akt (Ser473), mouse anti-Ubiquitin, rabbit anti-USP28and rabbit anti-myc-tag antibodies were obtained from Cell Signaling (Beverly, MA). Mouse monoclonal anti-Flag-tag and anti-β-actin antibodies, and non-targeting control shRNA and Rictor shRNA lentiviral particles were from Sigma (St. Louis, MO). Protein G PLUS-Agarose beads, rabbit anti-cyclin E and mouse anti-Aktl antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-c-Myc antibody was from Epitomics (Burlingame, CA). The plasmids encoding c-Myc, myc-tagged Rictor and myc-tagged mTOR were from Addgene (Cambridge, MA). The plasmid encoding Flag-tagged FBXW7a was kindly provided by Dr. Bruce E. Clurman (Fred Hutchinson Cancer Research Center, Seattle, WA). Human Rictor and non-targeting control siRNA
METHODS
Cell culture and transfection
The human CRC cell lines SW620, HT29and HCT116were from ATCC (Manassas, VA). SW620cells were maintained in DMEM supplemented with10%of FBS. HT29and HCT116cells were cultured in McCoy's5A supplemented with10%of FBS. Wild-type and FBXW7-/-HCT116cells, kindly provided by Dr. Bert Vogelstein (The Johns Hopkins University School of Medicine, Baltimore, MD), were maintained in McCoy's5A with10%FBS. SW620and HT29cells were transfected with the siRNA duplexes and plasmids using electroporation (Gene Pulser; Bio-Rad, Hercules, CA) and lipofectamine2000(Invitrogen, Carlsbad, CA), respectively.
Stable Rictor knockdown SW620, HT29and HCT116cells were generated. Cells were infected with the control shRNA or shRNA to human Rictor lentivirus particles and stably expressing cells were selected with puromycin at a concentration of2.5μg/ml. The effective knockdown of Rictor was monitored by Western blot.
RNA extraction and RT-PCR
Total RNA was extracted and DNase-treated (RQ1, Promega). Synthesis of cDNA was performed with1μg of total RNA using the reagents in the Taqman Reverse Transcription Reagents Kit from ABI (#N8080234). Reverse transcriptional PCR was performed by Hotstart DNA polymerase (Qiagen, Hilden, Germany), forward and reverse primers, and deoxynucleoside triphosphates in a final volume of25μl. The amplification product was of the expected sizes.
Protein preparation and Western blot analysis
Cells were collected at48h after transfection or at the indicated time points after treatment. Equal amounts of cell lysates were resolved on a4-12%Bis-Tris gel and transferred to polyvinylidene fluoride membranes. Membranes were blocked by5%non-fat milk for1h at room temperature. Rictor, mTOR, phospho-Akt (Ser473), Akt1, c-Myc, cyclin E, Myc-tag, Flag-tag and β-actin were detected with specific antibodies following blotting with a horseradish peroxidase-conjugated secondary antibody and visualized using a chemiluminescence detection system.
Immunoprecipitation and in vivo ubiquitination analysis
Cells were transfected with different plasmids as indicated,1%Triton lysis buffer or CHAPS lysis buffer was used. After pre-incubation with protein G PLUS-Agarose beads, equal amount of protein (500μg) were incubated with the indicated antibodies (1μg) using an end-to-end rotor overnight at4℃, followed by4h incubation with20μl of protein G PLUS-Agarose beads at4℃. Indicated buffer was used to wash the beads three times. Reactions were stopped by adding15μl of2×loading buffer. Samples were denatured by boiling for7min and separated by NuPAGE4-12%Bis-Tris gels. For in vivo ubiquitination analysis, His-ubiquitin plasmid was co-transfected with the indicated plasmids. Transfected cells were incubated with MG132(20μM) in serum free medium for4h before harvesting.
Statistical analysis
There is no statistical analysis in this section.
RESULTS
Rictor regulates protein expression of c-Myc and cyclin E
Activation of Akt increases c-Myc and cyclin E expression. Since inhibition of mTORC2by knockdown of Rictor inhibits Akt activation, we were interested to know whether Rictor regulates c-Myc and cyclin E expression. Human colorectal cancer cells, SW620and HT29, were transfected with shRNA targeting Rictor and stable cell lines were established. Knockdown of Rictor did not obviously affect the expression of c-Myc and cyclin E; however, with serum starvation, knockdown of Rictor increased protein expression of c-Myc and cyclin E (Fig.2-3A). In addition, treatment with MG132, a specific cell-permeable proteasome inhibitor, attenuated the increases of c-Myc and cyclin E protein expression resulting from Rictor knockdown (Fig.2-3B), indicating that the26S proteasome pathway was involved in this regulation. To further confirm our findings, we used Rictor siRNA containing a different sequence from the Rictor shRNA to decrease Rictor expression. Consistently, knockdown of Rictor by transient transfection with siRNA targeting Rictor resulted in a significant increase of c-Myc and cyclin E protein expression (Fig.2-3C). To further determine the role of Rictor in the regulation of c-Myc and cyclin E protein expression, SW620and HT29cells were transiently transfected with a myc-tagged Rictor plasmid and the transfected cells were serum starved for24h before harvesting. As shown in Fig.2-3D, overexpression of Rictor decreased c-Myc and cyclin E protein levels in both SW620and HT29cells, suggesting that Rictor regulates the protein levels of c-Myc and cyclin E associated with serum deprivation. Interestingly, RT-PCR assay showed that knockdown of Rictor inhibited mRNA level of c-Myc and cyclin E (Fig.2-3E). Considering the increases of c-Myc and cyclin E protein expression by Akt activation and knockdown of Rictor resulting in the dephosphorylation and inhibition of Akt, our results demonstrate that Rictor regulates c-Myc and cyclin E protein in an mTORC2/Akt pathway independent fashion.
Rictor interacts with FBXW7without mTOR
Degradation of c-Myc and cyclin E was targeted by FBXW7as an E3component. And it has been shown that Rictor forms a complex with cullinl to degrade SGK1protein. To determined whether Rictor interacts with FBXW7. As shown in Fig2-4A, Myc-Rictor constructs were co-transfected with Flag-FBXW7a, Flag-FBXW7p and Flag-FBXW7y individually. The interactions between Rictor and three FBXW7isforms were detected. FBXW7a is expressed at a much higher level than FBXW7P and FBXW7y in most human cell lines, and usually plays a major role as an E3ligase to the downstream targets. Therefore, we transfected HCT116cells with Flag-FBXW7a together with Myc-Rictor and the interactions between FBXW7and Rictor were detected. Two different lysis buffers were used in this assay:1%Triton and CHAPS lysis buffers. CHAPS buffer was used as a mild buffer to keep mTORC2intact while1%Triton was used to dissociate mTORC2. As shown in Fig.2-4C, Flag-FBXW7a was clearly copurified when Rictor was immunoprecipitated from cell lysates extracted with both1%Triton lysis buffer and CHAPS buffer, but the interaction between mTOR and Rictor was only observed in the lysates extracted using CHAPS buffer. Although it has been reported that FBXW7interacts with mTOR to promote its ubiquitination and degradation, our results indicate that Rictor forms a complex with FBXW7independent of mTORC2. Furthermore, the binding between Rictor and FBXW7is not affected by serum-deprivation (Fig2-4D).
Rictor interacts with FBXW7to regulate c-Myc and cyclinE
We next determine whether Rictor participates in FBXW7-dependent regulation of c-Myc and cyclin E degradation, we generated Rictor shRNA stable cell lines based on wild-type and FBXW7-/-HCT116cells. As shown in Fig.2-5A, knockdown of Rictor induced the expression of c-Myc and cyclin E in wild type HCT116cells but not in FBXW7-/-cells. These data suggest that Rictor regulation of c-Myc and cyclin E is FBXW7dependent. To further demonstrate the role of Rictor in the FBXW7E3ligase complex, HCT116control shRNA and Rictor shRNA stable cell lines were transfected with empty vector (control) or Flag-FBXW7a plasmid and c-Myc and cyclin E protein levels were analyzed (Fig.2-5B). Overexpression of FBXW7αdecreased the protein levels of c-Myc and cyclin E as expected. However, this degradation by FBXW7a was significantly attenuated in Rictor shRNA cells, demonstrating a role of Rictor, bound with FBXW7a as a part of E3complex, to induce the degradation of c-Myc and cyclin E.
Rictor regulates stability of FBXW7
Rictor didn't affect the mRNA level of FBXW7by RT-PCR assays (Fig.2-6A), which means Rictor regulates FBXW7through post-transcriptional regulation. Next, in vivo ubiquitination assay was performed to investigate the ubiquitination of FBXW7with or without Rictor expression. As expected in Fig.2-6B, the ubiquitination of FBXW7a was diminished in Rictor knockdown cells compared with control cells. Moreover, in Rictor stably knockdown cells, the stability of FBXW7αwas decreased; futher confirmed our hypothesis (Fig.2-6C).
Rictor/FBXW7promotes c-Myc and cyclin E ubiquitination
FBXW7degrades c-Myc and cyclin E through the ubiquitination of c-Myc and cyclin E proteins. To address whether Rictor promotes the ubiquitination of c-Myc and cyclin E, in vivo ubiquitination assays were performed. c-Myc or cyclin E and His-ubiquitin were expressed by transient transfection. Knockdown of Rictor reduced the ubiquitination of both c-Myc (Fig.2-7A) and cyclin E (Fig.2-7C). As expected, the ubiquitination of c-Myc and cyclin E was diminished in FBXW7-/-HCT116cells compared with wild-type and, importantly, Rictor-dependent regulation of c-Myc and cyclin E ubiquitination was also blunted in FBXW-/-HCT116cells (Fig.2-7B and Fig.2-7D). Taken together, these data indicate a role of Rictor in the regulation of c-Myc and cyclin E protein ubiquitination and degradation.
Rictor reduces the stability of c-Myc and cyclin E
To determine whether Rictor regulates the stability of c-Myc and cyclin E, we next performed CHX chase assays. Time-course experiments showed that the half-lives of c-Myc (Fig.2-8A) and cyclin E (Fig.2-8C) were prolonged in HCT116cells with Rictor shRNA compared with HCT116cells with non-targeting control shRNA. The sequence of Rictor shRNA used in this study was designed to target the3'-UTR region of Rictor and would not affect the expression of transfected myc-Rictor plasmid. Therefore, Rictor protein levels can be rescued in Rictor shRNA stable cell lines with myc-Rictor overexpression. Indeed, transfection of HCTl16Rictor shRNA stable cells with myc-Rictor plasmid decreased the half-life of c-Myc (Fig.2-8B) and cyclin E (Fig.2-8D). Our results demonstrate that Rictor is required for the integrity of the FBXW7E3complex to promote the degradation of c-Myc and cyclin E.
Rictor binds with USP28through FBXW7
USP28is a deubquitin specific protein, which can bind with FBXW7to deubiquitinate its downstreams. c-Myc, as one of USP28specific targets, was decreased with USP28knockdown in HT29; consistently, overexpression of USP28in HCT116induced c-Myc protein level (Fig.2-9A). Rictor bound with USP28via FBXW7(Fig.2-9B), but Rictor did not have affect on the deubiquitination regulation of c-Myc by USP28(Fig.2-9C and Fig.2-9D).
Rictor/FBXW7may mediate Rapamycin resistance in CRC
Rictor activity was also inhibited by long-time exposure of Rapamycin. Some of cancer cells were rapamycin sensitive, while some of them were rapamycin resistant and the mechanisms of rapamycin resistance is still unclear. As shown in Fig.2-10A,24hours exposure of rapamycin in rapamycin resistant cells HT29and S W620led to the decrease of Rictor, with increase of c-Myc, cyclin E and Akt phosphorylation at the same time. And no increase of c-Myc, cyclin E mRNA level was detected in the same cell lines with rapamycin treatment (Fig.2-10B), indicating rapamycin induced c-Myc and cyclin E was mTORC2/Akt pathway independent. Based on these data, we hypothesize that rapamycin resistance is mediated by increased c-Myc and cyclin E via Rictor/FBXW7regulation.
CONCLUSIONS
1. Our findings identify Rictor as an important component of FBXW7E3ligase complex participating in the regulation of c-Myc and cyclin E protein ubiquitination and degradation.
2. Our results suggest that elevated growth factor signaling may contribute to decrease Rictor/FBXW7-mediated ubiquitination of c-Myc and cyclin E, thus leading to accumulation of cyclin E and c-Myc in colorectal cancer cells.
3. Our study figures out a possible mechanism of Rapmycin resistance in colorectal cancer cell, which yields another role of Rictor during tumor progression.
目的和意义
Rictor (Rapamycin-insensitive companion of mTOR)是mTOR复合物2(mTORC2)中一个新确定的蛋白成员,参与调节mTORC2信号及其下游靶基因如Akt和SGK1。mTORC2作为mTOR信号通路中一个新的复合物,对其调控的分子及自身调控的报道研究仍不完善,且对Rictor上游的相关调控尚无文献报道。本研究拟检测蛋白激酶D (PKD)与Rictor的相互作用,从而探索PKD对Rictor的调控机制,并深入了解Rictor作为mTORC2中的成员在肿瘤发生发展中的作用,以期指导恶性肿瘤的生物靶向治疗。
材料和方法
1、主要材料
293T、HT29和HCT116细胞,G66976、G56983和CID755673, MG132,CHX,抗体(PKD1、PKD2、PKD3、Rictor、mTOR、Raptor、p-Akt和Akt),组织芯片,RNA提取试剂盒,逆转录试剂盒,免疫组织化学相关试剂,免疫印迹及免疫共沉淀相关试剂。
2、方法
2.1细胞培养
293T细胞使用含10%FBS的DMEM培养,HT29和HCT116细胞采用含10%FBS的McCoy's5A培养,所有细胞置于37℃,5%C02,湿度充分的恒温培养箱中。
2.2慢病毒的包备与稳定株的建立
将7×106个293T细胞分别种于100mm培养皿中培养过夜,按照说明书使用Lipfectmin2000转染相应的质粒入细胞中(shRNA、pPAX2和pMD2G各个质粒的转染比例为4：3：1),转染后6小时换新鲜培养液,48小时后收集细胞上清,离心过滤后即为病毒悬液。测试病毒滴度后取等量的病毒液置于待感染的细胞,并补等量的培养液培养48小时。根据细胞对puromycin的敏感度采用不同浓度的puromycin筛选1-2周后,Real-time PCR鉴定筛选稳定表达株。剩余病毒液分装后置于-70℃保存备用。
2.3细胞总RNA提取与Real-time PCR
1×106细胞种于6孔板中常规培养过夜。采用RNeasy Mini试剂盒提取总细胞RNA,紫外分光光度计定量后,取1μg总RNA用无RNA酶的纯净水补至10μl,加入预先配制好的10μl/反应的逆转录酶混合液中,上机25℃10min、37℃120min和85℃10sec,将总RNA逆转录生成cDNA。按Taqman基因表达检测方法检测PKD2, PKD3和Rictor的mRNA水平。
2.4蛋白样品制备及免疫印迹
转染后48小时或相应处理后按指定时间收集细胞。采用1%Triton裂解液常规冰上裂解细胞蛋白30min (20mM Tris-HCl (pH7.5),150mM NaCl,1mM Na2EDTA,1mM EGTA,1%Triton,2.5mM sodium pyrophosphate,1mM beta-glycerophosphate,1mM Na3VO4,1g/ml leupeptin),低温高速离心后检测蛋白浓度。加入上样缓冲液的蛋白裂解液加热至95℃、5min变性后,取等量的蛋白用MOPS SDS电泳缓冲液于4-12%Bis-Tris梯度胶电泳200V、60min分离蛋白,NuPAGE转移缓冲液中70V、120min将蛋白转移至预先用甲醇处理的PVDF膜。5%脱脂牛奶室温封闭1小时后分别采用特异性的一抗4℃孵育过夜。TBST洗膜,5分钟×3次；加入对应的二抗(α-Rabbit抗体1:5000; α-Mouse抗体1：4000)室温孵育1小时,TBST洗膜,于暗室中ECL或ECL plus显影曝光。
2.5免疫共沉淀
细胞分别转染相应的质粒48小时后,常规裂解细胞。定量后每组取500μg蛋白进行实验。首先每组蛋白使用20μl protein G PLUS凝胶珠4℃预孵育半小时,离心后弃去沉淀。取上清分别加入Myc抗体锚定的凝胶珠20μl或正常IgG作阴性对照,置于4℃360°旋转器上过夜孵育。次日,使用裂解液将凝胶珠充分洗净后加入等量的2×上样缓冲液混匀,加热至95℃、5min变性,所得样品全部上样,行Western Blot检测相应蛋白的表达水平。
2.6细胞增殖及克隆形成实验
将1×105个构建的空白对照、Rictor、PKD2、PKD3和PKD2&3稳定敲低表达细胞株种于6孔板中常规培养,每3天更换培养基,并于第1天和第7天分别用胰酶消化细胞成单细胞悬液,细胞仪(Beckman coulter)计数细胞数量,直接比较细胞增殖速度。另外,配制含0.8%软琼脂的培养基事先铺于60mm培养皿中,超净台中充分凝固。将等数量上述稳定表达株的单细胞重悬于含0.4%软琼脂的培养基中,铺于处理后的培养皿上,置于超净台中常温凝固。转移入37℃、5%CO2、湿度充分的恒温培养箱中培养10～12天后取出,甲醛固定,0.005%结晶紫染色后显微镜下计数细胞克隆形成数目。
2.7免疫组织化学
组织芯片置于58℃烤箱中孵育固定30min后,立即依次转移入二甲苯中5minx2次、95%乙醇3minx2次、双蒸水×2次,脱蜡至水。3%H202室温封闭组织切片10min,PBS缓冲液冲洗2次后,放入盛有抗原修复液0.01M枸橼酸钠缓冲溶液(pH=7.0)的耐高温容器,在微波炉里加热至沸腾后,断电,间隔5～10min,反复2次后停止冷却至室温；滴加正常血清封闭液,室温20mmin,甩去多余液体；分别滴加稀释后的Rictor、PKD2和PKD3抗体,每组均以抗体稀释液作为空白对照,4℃孵育过夜；PBS缓冲液5min×3次,滴加相应的生物素化二抗,室温孵育20min; PBS缓冲液5min×3次,DAB显色(镜下掌握显色程度及时间)；双蒸水洗,苏木素复染20sec,盐酸酒精分化1sec；乙醇脱水、二甲苯透明,中性树脂封片后镜检拍照。
2.8数据统计
Real-time PCR、细胞增殖、克隆形成实验结果用三次实验结果的x±SD表示,均采用两独立样本t检验分析,检验水准以P<0.05认为差异有统计学意义,以上分析使用SPSS13.0完成。
结果
1、PKD2和PKD3共同参与Rictor的调节
Go6976处理结肠癌细胞后Rictor的表达降低,而Go6983对Rictor的表达无明显影响；特异性PKD抑制剂CID755673也可抑制Rictor的表达,这就说明PKD可能参与Rictor的表达调控(图1-2.A)。但是采用shRNA分别抑制PKD三个亚型PKD1、PKD2和PKD3的表达,未能对Rictor的表达产生影响(图1-2.B)；同样的,在HT29中分别过表达PKD1和PKD2,对Rictor蛋白水平均无影响(图1-2.C)。当我们使用PKD3的腺病毒感染结肠癌细胞后发现,Rictor蛋白水平增加,且其下游靶基因Akt的磷酸化水平也同时增加(图1-2.D)。
综合以上结果,PKD抑制剂可抑制Rictor的表达,而单独抑制其中任意—个亚基均不能起到相同的作用,因此,PKD对Rictor的调控可能是通过两个或以上的PKD亚型同时起作用。将PKD2shRNA和PKD3shRNA同时感染HT29细胞建立稳定双重敲低表达株,免疫印迹验证成功敲低PKD2和PKD3的表达。此时检测Rictor的表达水平较空白对照组降低,同时Akt磷酸化水平也降低(图1-2.E)。因而我们推测,PKD对Rictor的调控主要是通过PKD2和PKD3两个亚型来实现的。
2、PKD2和PKD3能与Rictor相结合
Real-time PCR检测了PKD对Rictor mRNA水平的影响,GAPDH为内参,每组实验重复3次,结果取平均值,以两独立样本t检验分析数据。如图1-3.A所示,PKD2和PKD3双重敲低稳定表达细胞(PKD2&3sh)与空白对照细胞(Ctrlsh)中PKD2mRNA的表达分别为0.16±0.016和1.00±0.046,PKD3的表达分别为0.12±0.020和1.00±0.058,PKD2和PKD3mRNA水平差异具有统计学意义(t值分别为24.910和30.200,P值为0.000和0.000),提示稳定敲底表达株构建成功；两细胞中Rictor的表达分别为0.86±0.346和1.00±0.097,差异无统计学意义(t值为0.705,P值为0.520)；如图1-3.B所示,腺病毒过表达PKD3细胞(Ad-PKD3)与空白对照细胞(Ad-Ctrl) PKD3的表达为17.05±0.181和1.00±0.026,PKD3表达水平差异具有统计学意义(t值为-151.500,P值为0.000),Rictor的表达水平为4.81±0.297和4.27±0.524,差异无统计学意义(t值为-1.544,P值为0.198)。因此,PKD并不是从转录水平影响Rictor的表达。
接下来,我们想进一步验证PKD与Rictor蛋白间的相互作用。HCT116细胞共同转染Myc-Rictor和GST-PKD1或Myc-Rictor和GST-PKD2,免疫共沉淀实验发现PKD2可与Rictor相结合,IgG对照组无结合,但未检测到PKD1与Rictor的相互作用(图1-3.C)；另外,PKD3腺病毒高表达的细胞转染Myc-Rictor,用Myc (Myc-Rictor)标记的凝胶珠与蛋白孵育后,免疫印迹检测到了PKD3的表达,说明PKD3也可与Rictor蛋白相结合(图1-3.D)。这就进一步证实了我们的假设,PKD1不与Rictor蛋白结合,未直接参与Rictor的调节,而PKD2和PKD3则可通过与Rictor直接或间接的结合来调控其蛋白水平的表达。
3、Rictor通过泛素化途径降解,过表达PKD3可抑制Rictor的降解
基于以上结果得知,PKD对Rictor是转录后水平的调控,这就包括翻译水平和蛋白稳定性的调控。26蛋白酶体信号通路参与蛋白转录后调节,蛋白被泛素化修饰后即被蛋白酶体进一步的水解成多肽。MG132为一个常用的真核细胞蛋白酶体抑制剂,可透过细胞膜选择性的抑制蛋白酶体。如图1-4.A所示,使用MG132处理HT29细胞不同的时间,发现Rictor的表达水平增加,mTOR信号通路中的mTOR和Raptor分子水平也有所增加,同时伴有mTORC2下游靶分子Akt的磷酸化水平增加。随后,我们采用CHX(放线菌酮,蛋白合成抑制剂)抑制细胞新的蛋白的合成,发现Rictor蛋白的表达水平随着处理时间的延长而降低(图1-4.B)。因此,可以确定Rictor蛋白是通过26蛋白酶体通路来降解的。
为进一步明确PKD对Rictor蛋白的调控是否是通过26蛋白酶体途径来实现,我们设计了CHX追逐实验进行验证。CHX (cycloheximide)为一种蛋白合成抑制剂,可以抑制真核细胞蛋白的合成,常用于观察蛋白的稳定性。如图1-4.C所示,在对照细胞中,Rictor蛋白随CHX处理时间的延长而降低,说明Rictor蛋白处于正常的降解过程中；而在PKD3过表达的细胞中,CHX处理细胞几乎不引起Rictor蛋白的降解,说明过表达PKD3可增加Rictor蛋白的稳定性,抑制了Rictor蛋白的降解。
4、PKD和Rictor不影响结肠癌细胞的增殖
如图1-2.D和1-2.E所示,PKD可通过影响Rictor的表达进而改变Akt的磷酸化水平。众所周知,Akt在肿瘤细胞增殖、分化、凋亡和转移过程中起着重要的作用,且有报道指出,抑制mTOR可抑制肿瘤细胞的增殖。计数等量的各个稳定细胞株培养7天,分别于第1天和第7天计数细胞,以第7天与第1天细胞的增殖倍数作为结果比较。如图1-5.A,在HCT116和HT29中,Rictor敲低表达(Rictor sh)与空白对照(Ctrlsh)细胞的增殖速度分别为17.25±3.256、21.17±2.926和14.72±2.104、11.70±2.405,此次实验采用两样本t检验分析,组间差异无显著性(t值分别为1.552和1.633,P值分别为0.196和0.178)。然后我们观察PKD各个亚型对肿瘤细胞增殖的影响(图1-5.B和图1-5.C),HCT116和HT29对照细胞与PKD2或PKD3敲低表达细胞(PKD2sh或PKD3sh)的增值速度分别为27.79±3.150、30.61±2.706或29.08±0.995和50.16±12.928、43.88±4.638或35.22±3.846,采用两样本t检验差异无统计学意义(t值分别为-1.176、-0.647和0.792、1.919,P值分别为0.305、0.537和0.472、0.127)。HCT116和HT29对照细胞与双重敲低表达细胞(PKD2&3sh)的增值速度分别为27.17±7.029、30.31±5.108和10.45±1.433、13.24±3.476,采用两样本t检验差异无统计学意义(t值分别为-0.626和-1.285,P值分别为0.565和0.268)。
5、抑制PKD和Rictor降低结肠癌细胞的致瘤性
于是,我们采用软琼脂集落形成实验观察PKD和Rictor对肿瘤细胞致瘤性的影响(图1-6)。结果发现,空白对照细胞(Ctrlsh)与Rictor敲低表达细胞(Rictorsh)、空白对照细胞与PKD2&3双重敲低表达细胞(PKD2&3sh)的克隆形成数目分别为180.00±22.869、63.33±12.097和119.33±27.791、42.67±35.233,抑制Rictor和PKD2&3均能显著抑制结肠癌细胞的致瘤作用,差异具有统计学意义(t值为7.811和2.959,P值分别为0.001和0.042)。
6、PKD和Rictor在结肠癌原位及转移肿瘤中高表达
最后,我们检测了结肠正常组织、原位和肿瘤肝转移组织中PKD和Rictor的表达水平。图1-7分别显示了各个蛋白在组织中的表达,可见在结肠正常组织中,Rictor、PKD2和PKD3的表达水平较低或不表达,而在原位及肿瘤肝转移组织中,三者的表达水平均较高。原位肿瘤组织中Rictor和PKD2主要表达在细胞浆中,PKD3在细胞浆和细胞核中均有表达；而在肿瘤肝转移组织中三者在细胞核和细胞浆均有表达,随着肿瘤的进展程度,PKD和Rictor在细胞内聚集的位置发生了一定的改变,进一步提示Rictor、PKD2和PKD3均与结肠癌的进展有关。
结论
获得了蛋白激酶D (PKD)参与调节Rictor/mTOR信号通路的证据。过表达PKD3可增加Rictor的蛋白水平,而同时抑制PKD2和PKD3可降低Rictor蛋白的水平,两种调节均不影响Rictor的mRNA水平；PKD2和PKD3在结肠癌细胞中能与Rictor结合并增强其下游靶基因Akt的活性；PKD3可影响Rictor蛋白的稳定性；PKD和Rictor对结肠癌细胞的增殖无明显的影响,但可增加其致瘤性；PKD和Rictor在结肠原位和肝转移肿瘤组织中均高表达。以上结果均提示PKD在一定程度上参与了Rictor的转录后调控,从蛋白的修饰方面影响了Rictor蛋白的稳定性,从而参与Rictor/mTOR信号通路的调控；抑制PKD可下调Rictor蛋白的表达,并抑制结肠癌细胞的致瘤性,为肿瘤的生物靶向性治疗提供的一定的理论依据。
第二部分Rictor在mTOR非依赖的信号通路中的调控研究
目的和意义
Rictor (Rapamycin-insensitive companion of mTOR)作为mTORC2中的成员,可磷酸化并激活Akt,激活的Akt可诱导c-Myc和cyclin E的表达,从而对结肠癌细胞增殖和周期起重要的调节作用。本研究检测了Rictor在mTOR信号通路非依赖的调控的作用,与FBXW7相互作用作为一个E3复合物调节c-Myc和cyclin E蛋白的降解,并了解该调节在肿瘤发展及治疗中的作用。本课题发现了Rictor在结肠癌肿瘤中新的调控作用,对新的临床用药指导有着重要的意义。
材料和方法
1、主要材料
293T、SW620、HT29和HCT116细胞,MG132, CHX,抗体(ubiquitin、 c-Myc、cyclin E、Rictor、mTOR、Raptor p-Akt和Akt), RNA提取试剂盒,逆转录试剂盒,免疫印迹相关试剂,免疫共沉淀相关试剂。
2、方法
2.1细胞培养
293T和SW620细胞使用含10%FBS的DMEM培养,HT29和HCT116采用含10%FBS的McCoy's5A培养,HCT116-FBXW7+/+和HCT116-FBXW7-/-同样培养于含10%FBS的McCoy's5A培养液中。所有细胞采用上述培养基置于37℃,5%CO2,湿度充分的恒温培养箱中。
2.2慢病毒的包备与稳定株的建立
将7×106个293T细胞分别种于100mm培养皿中培养过夜,按照说明书使用Lipfectmin2000转染相应的质粒入细胞中(shRNA、pPAX2和pMD2G各个质粒的转染比例为4：3：1),转染后6小时换新鲜培养液,48小时后收集细胞上清,离心过滤后即为病毒悬液。测试病毒滴度后取等量的病毒液置于待感染的细胞,并补等量的培养液培养48小时。根据细胞对puromycin的敏感度采用不同浓度的puromycin筛选1-2周后,Real-time PCR鉴定筛选稳定表达株。剩余病毒液分装后置于-70℃保存备用。
2.3细胞总RNA提取与RT-PCR
1×106细胞种于6孔板中常规培养过夜。采用RNeasy Mini试剂盒提取总细胞RNA,紫外分光光度计定量后,取1μg总RNA用无RNA酶的纯净水补至10μl,加入预先配制好的10μl/反应的逆转录酶混合液中,上机25℃10min、37℃120min和85℃10sec,将总RNA逆转录生成cDNA。根据靶基因序列及参考文献设计引物,Hotstart热启动酶扩增目的DNA片段,凝胶电泳分离后紫外灯观察拍照。
2.4蛋白样品制备及免疫印迹
转染后48小时或相应处理后按指定时间收集细胞。采用1%Triton裂解液常规冰上裂解细胞蛋白30min (20mM Tris-HCl (pH7.5),150mM NaCl,1mM Na2EDTA,1mM EGTA,1%Triton,2.5mM sodium pyrophosphate,1mM beta-glyceropho sphate,1mM Na3VO4,1g/ml leupeptin),低温高速离心后检测蛋白浓度。加入上样缓冲液的蛋白裂解液加热至95℃、5mmin变性后,取等量的蛋白用MOPS SDS电泳缓冲液于4～12%Bis-Tris梯度胶电泳200V、60min分离蛋白,NuPAGE转移缓冲液中70V、120min将蛋白转移至预先用甲醇处理的PVDF膜。5%脱脂牛奶室温封闭1小时后分别采用特异性的一抗4℃孵育过夜。TBST洗膜,5分钟×3次；加入对应的二抗(a-Rabbit抗体1:5000; a-Mouse抗体1：4000)室温孵育1小时,TBST洗膜,于暗室中ECL或ECL plus显影曝光。
2.5免疫共沉淀
细胞分别转染相应的质粒48小时后,分别采用1%Triton裂解液或0.3%CHAPS裂解液裂解细胞。定量后每组取500μg蛋白进行实验。首先每组蛋白使用20μl protein G PLUS凝胶珠4℃预孵育半小时,离心后弃去沉淀。取上清分别加入抗体1μg或抗体锚定的凝胶珠20μl,置于4℃360°旋转器上过夜孵育。次日,加入抗体的实验组再给予20μl protein G PLUS凝胶珠4℃旋转孵育4小时。使用相应裂解液将凝胶珠充分洗净后加入等量的上样缓冲液加热至95℃、5min变性,所得样品全部上样行Western Blot检测相应蛋白的表达水平。
2.6体内泛素化水平检测
蛋白泛素化水平检测时,将His-ubiquitin表达载体与指定的质粒共转染入细胞内,收集细胞4小时前使用MG132处理细胞。100μl0.3%CHAPS裂解液常规裂解细胞,加入10%SDS10μl混匀后加热至95℃、5min变性蛋白,再加入900μl0.3%CHAPS裂解液超声裂解20秒,离心后用免疫共沉淀方法检测细胞内靶蛋白的泛素化水平。
2.7数据统计
本部分未涉及统计学处理。
结果
1、Rictor调节c-Myc和cyclin E蛋白的表达水平
过去的研究充分表明,Akt的激活可增加c-Myc和cyclin E的表达。因而我们想进一步了解,mTORC2活性的抑制对c-Myc和cyclin E表达的影响。在稳定转染Rictor shRNA了的SW620和HT29细胞中,敲低Rictor并未明显影响到c-Myc和cyclin E的表达；然而,同样的细胞给予血清饥饿刺激后再检测c-Myc和cyclin E,发现抑制Rictor可明显增加两者的表达水平(图2-3.A),这就与之前的报道存在一定的差别。另外,我们使用MG132(一个特异性的细胞26蛋白酶体抑制剂,可抑制蛋白的降解)来处理血清饥饿后的细胞,结果发现,MG132能够逆转Rictor敲低所引起的c-Myc和cyclin E蛋白水平的增加(图2-3.B)。这就说明,26S蛋白酶体信号通路参与了Rictor的这一调节。
为了进一步验证我们的结果,使用Rictor的siRNA转染入细胞中瞬时压低Rictor的表达,收集蛋白前使用血清饥饿预处理细胞,发现siRNA瞬时抑制Rictor的表达后也可使c-Myc和cyclin E的表达增加(图2-3.C)。如图2-3.D所示,过表达Rictor同样可以抑制c-Myc和cyclin E蛋白水平的表达。有趣的是,在SW620Rictor shRNA稳定敲低表达细胞中,RT-PCR结果显示,Rictor低表达可抑制c-Myc和cyclin E的mRNA水平,与其对蛋白水平的调控相反(图2-3.E)。
综上所述,Rictor在无血清的培养状态下可调节c-Myc和cyclin E蛋白水平的表达。由于Akt磷酸化激活后导致c-Myc和cyclin E表达水平的增加,而此前有报道指出Rictor敲低表达可导致Akt的去磷酸化和失活表达,Akt表达水平的变化导致c-Myc和cyclin E mRNA及蛋白水平降低。故我们有理由相信,Rictor是通过mTORC2/Akt非依赖的信号通路来调节c-Myc和cyclin E蛋白的表达。
2、Rictor与FBXW7的相互作用
作为一个E3的组成部分,FBXW7可影响其靶基因c-Myc和cyclin E的泛素化修饰从而促进它们的降解。故我们推测Rictor是否通过与FBXW7相结合来进一步调控c-Myc和cyclin E。Myc-Rictor分别与Flag-FBXW7α、Flag-FBXW7p和Flag-FBXW7y共同转染入细胞中,免疫共沉淀结果显示,Rictor与FBXW7的三个亚型均有结合(图2-4.A)。由于mTOR也是FBXW7下游的靶基因之一,可与FBXW7结合后被泛素化修饰、降解,故我们需要进一步的实验鉴别Rictor与FBXW7的结合与mTOR/FBXW7的关系。我们构建了3个包含Rictor蛋白不同片段的表达载体(所有截短载体均包含碳末端),测序鉴定成功后分别与Flag-FBXW7a共同转染入细胞中。如图2-4.B所示,在3个Rictor截短表达载体及全长Rictor载体中,同时检测到FBXW7a和mTOR的结合表达。我们需要进一步的实验来鉴别Rictor/FBXW7复合物是否是通过mTOR而间接连接的。1%Triton裂解液可破坏mTORC2,而0.3%CHAPS裂解液则较缓和,能保持mTORC2的完整性。如图2-4.C所示,在使用1%Triton和0.3%CHAPS裂解液的两组实验中均检测到了Rictor与FBXW7a的相互作用,而Rictor与mTOR的相互作用仅能在使用0.3%CHAPS裂解液的实验组中检测到。这就说明,Rictor和FBXW7的相互作用是独立于mTORC2以外的。
由于我们所检测到的Rictor对c-Myc和cyclin E蛋白水平的调控是在无血清培养的条件下的,那么Rictor与FBXW7的作用是否也依赖于血清饥饿而存在呢?如图2-4.D所示,在血清培养和血清饥饿培养的转染细胞中,均检测到了Rictor与FBXW7的相互作用,说明Rictor/FBXW7复合物是不依赖于血清饥饿而存在的。
3、Rictor通过FBXW7调节c-Myc和cyclin E
基于以上实验结果,我们推测Rictor对c-Myc和cyclin E的调节可能是通过与两者特异性的E3-BXW7共同组成E3复合物来起作用的。因此,我们在野生型FBXW7+/+和缺失型FBXW7-/-细胞中分别建立Rictor shRNA稳定敲低表达株及空白对照株。如图2-5.A所示,在野生型(FBXW7+/+)HCT116细胞中敲低Rictor表达后可诱导c-Myc和cyclin E的表达,但在缺失型(FBXW7-/-)HCT116细胞中同样的处理未见c-Myc和cyclin E表达水平的变化。同时,HCT116control shRNA和Rictor shRNA稳定细胞株中分别转染空白质粒和Flag-FBXW7a表达载体,结果显示,FBXW7a在对照细胞中可降低c-Myc和cyclin E的蛋白水平,而在Rictor敲低表达的细胞中该作用明显减弱(图2-5.B)。
4、Rictor调节FBXW7的稳定性
Rictor与FBXW7结合后对其作为一个E3复合体有什么样的影响呢?首先,我们进行了RT-PCR实验比较Rictor敲低表达的稳定细胞株和阴性对照细胞株中FBXW7表达水平的差异,结果发现,抑制Rictor表达并未明显影响FBXW7mRNA的水平(图2-6.A),Rictor并没有从转录水平影响FBXW7的表达。然后,我们检测了FBXW7在体内的泛素化水平。如图2-6.B所示,在Rictor敲低表达的稳定细胞株中,Flag-FBXW7a蛋白泛素化水平明显较阴性对照细胞中高,证明抑制Rictor的表达可解聚Rictor/FBXW7复合体,进而促进了FBXW7a的降解。CHX能抑制真核细胞蛋白的合成,使用CHX可观察蛋白降解半衰期的时长,因此CHX追逐实验广泛被应用于蛋白稳定性的检测。图2-6.C显示,在Rictor敲低表达的稳定细胞株中,Flag-FBXW7a蛋白的稳定性明显较阴性对照细胞低,从而进一步验证了我们的假设。
5、Rictor/FBXW7启动c-Myc和cyclin E的泛素化
那么Rictor与FBXW7结合后是如何调节c-Myc和cyclin E的呢?由于FBXW7对c-Myc和cyclin E的调节是通过泛素化修饰来实现的,而且图2-3.E已经显示抑制Rictor表达可在mRNA水平上抑制c-Myc和cyclin E(与蛋白水平的变化不一致),因而我们有理由相信,Rictor在此对c-Myc和cyclin E的调节也是通过与FBXW7相互作用,从蛋白水平上修饰c-Myc和cyclin E来实现的。如图2-7.A和图2-7.C所示,在Rictor敲低表达的稳定细胞株中, c-Myc和cyclinE的泛素化水平明显降低。而且,在野生型HCT116细胞中,敲低Rictor可抑制c-Myc和cyclin E的泛素化；在缺失型(FBXW7-/-) HCT116细胞中,c-Myc和cyclin E的泛素化水平已经很低,抑制Rictor的表达不能进一步抑制两者的泛素化(图2-7.B和图2-7.D)。
6、Rictor降低c-Myc和cyclin E的稳定性
使用CHX追逐实验观察c-Myc和cyclin E的稳定性与Rictor表达的相关关系。如图2-8.A和图2-8.C所示,抑制Rictor的表达可延长c-Myc和cyclin E蛋白降解的半衰期。本研究中使用的Rictor shRNA序列针对的是Rictor的3'-UTR区,在Myc-Rictor表达载体上无作用位点,故可以使用Myc-Rictor表达载体在Rictor敲低表达稳定细胞中重新表达Rictor蛋白。如图2-8.B和图2-8.D所示,Myc-Rictor表达载体在Rictor敲低表达稳定细胞中表达良好,且Rictor的重新表达可缩短c-Myc和cyclin E蛋白的半衰期,从而促进c-Myc和cyclin E的降解。
7、Rictor能与USP28结合,但不参与USP28的去泛素化调节
USP28作为一个去泛素化特异蛋白,可与FBXW7相结合来平衡其下游的靶基因如c-Myc的泛素化与去泛素化修饰,从而影响c-Myc蛋白水平。如图2-9.A所示,在HT29中感染USP28shRNA建立稳定表达株,发现抑制USP28的表达可抑制c-Myc蛋白水平；在HCT116细胞中转染Flag-USP28表达载体,过表达USP28可增加c-Myc蛋白水平,抑制或增加USP28的表达均不影响cyclin E的表达,说明USP28能特异的将c-Myc去泛素化进而抑制其降解,这与前人的发现是一致的。那么,Rictor/FBXW7复合物中是否也存在USP28呢?结果发现,Rictor也能与USP28特异性相互结合,且mTOR没有参与该复合物的形成(图2-9. B)。Rictor是否参与USP28/FBXW7复合物对底物去泛素化的调节呢?在Rictor稳定敲低表达细胞和空白对照细胞中,免疫共沉淀检测FBXW7与USP28间的相互作用并没有受Rictor表达水平的影响(图2-9.C),且抑制Rictor的表达不能逆转USP28所致的c-Myc蛋白增加(图2-9.D),提示USP28与FBXW7的相互作用与Rictor无关,Rictor可通过FBXW7与USP28形成复合物,但Rictor的表达水平对USP28的去泛素化作用无明显的影响。
8、Rictor可能参与Rapamycin诱导的耐药
大量研究工作表明,Rapamycin是通过与mTORC1直接结合来抑制mTORC1信号通路的活性,但也有研究表明长时间Rapamycin的处理也可抑制Rictor的活性。Rapamycin可通过抑制mTOR信号通路来抑制肿瘤的生长,但并不是所有的肿瘤细胞都对Rapamycin的治疗有反应,有些肿瘤细胞就对Rapamycin不敏感,因此明确肿瘤细胞对其耐药的机制就显得尤为重要。如图2-10.A所示,Rapamycin处理HT29和SW620(Rapamycin不敏感细胞)24小时后,Rictor蛋白水平降低,而c-Myc、cyclin E和磷酸化的Akt水平增加。另外,Rapamycin对c-Myc和cyclin E的mRNA无明显影响或仅轻度抑制(图2-10.B),说明Rapamycin诱导的c-Myc和cyclin E增加与激活的Akt无关。以上结果说明Rapamycin可能通过抑制Rictor/FBXW7的活性来激活c-Myc和cyclin E的表达,从而诱导耐药的产生。
结论
发现了Rictor独立于mTOR信号通路以外的调控作用。Rictor可作为一个调节子与FBXW7相结合,从而参与调控与其相关的靶基因如c-Myc和cyclin E的降解；血清饥饿抑制了mTORC/Akt参与的c-Myc和cyclin E的正向调控,在此基础上,抑制Rictor可抑制两者的泛素化修饰,增加两者蛋白的稳定性,导致c-Myc和cyclin E蛋白水平的增加；在血清培养条件下,Rictor同样可与FBXW7形成复合物,且该复合物是独立于mTOR信号通路以外的；Rapamycin诱导的肿瘤细胞耐药性与Rictor的这一调控作用有关。以上结果均提示Rictor存在与mTOR不相关的调控作用,且其调控的结果可能与mTOR信号通路的调节起到相反的作用,因此,应用Rictor作为生物治疗的靶点时应全面考虑到其各个方面的作用,利用其在抗肿瘤治疗中有利的一面,同时避免或抑制其他副作用的产生。
Part one:Post-transcriptional regulation of Rictor by protein kinase D in human colorectal cancer cells
BACKGROUND
mTORC2consists of mTOR, mLST8, Rictor, Sinl and PROTOR/PRR5. mTORC2phosphorylates Akt at Ser473thus resulting in Akt activation, knockdown of Rictor blocks Ser473phosphorylation. Knockdown of Rictor leads to growth inhibition and induces apoptosis in colorectal cancer cells. However, the molecular mechanism(s) involved in the regulation of Rictor expression remains unclear.
Protein kinase D (PKD), a serine/threonine kinase family that includes PKD1, PKD2and PKD3, has been implicated in the regulation of cell proliferation and apoptosis. Selective PKD inhibitor has shown potential inhibitory roles in certain cancer cell proliferation, cell migration, and invasion. Recently, PKD3has been shown to increase Akt phosphorylation at Ser473. The role of PKD and its downstream effectors with regard to CRC proliferation and metastasis are not known.
MATERIALS Go6976, G66983, MG132and cycloheximide (CHX) were purchased from Calbiochem (San Diego, CA). CID755673was from TOCRIS bioscience (Ellisville, Missouri). Antibodies against PKD2and PKD3were obtained from Abgent, Inc.(San Diego, CA). Antibodies against mTOR, Raptor, Rictor, PKD1, PKD2, PKD3, p-Akt, and Akt were obtained from Cell Signaling (Beverly, MA). GST-tagged PKD plasmids expressing wild-type PKD1and2were from Dr. Vivek Malhotra,(Universityof California, San Diego). Recombinant adenovirus expressing wild type (WT)-PKD3was kindly provided by Dr. Q. Jane Wang (University of Pittsburgh, Pittsburgh, PA). Tissue culture media and reagents were obtained from Invitrogen. Polyvinylidene difluoride (PVDF) membranes for Western blots were from Millipore Corp.(Bedford, MA). The enhanced chemiluminescence (ECL) system for Western immunoblot analysis was purchased from Amersham Biosciences.
METHODS
Cell culture and transfection
The human colon cancer cell lines, HT29and HCT116, were purchased from ATCC. HT29and HCT116cells were maintained in McCoy's5A supplemented with10%fetal bovine serum (FBS). PKD inhibitors were initially dissolved in dimethyl sulfoxide (DMSO) and compared with cells treated with DMSO at the same final concentration. HT29and HCT116cells were transfected with the siRNA duplexes and plasmids using electroporation (Gene Pulser; Bio-Rad, Hercules, CA) and lipofectamine2000(Invitrogen, Carlsbad, CA), respectively.
Stably Rictor knockdown HT29and HCT116cells were generated. Cells were infected with the control shRNA or shRNA to human Rictor lentivirus particles and stably expressing cells were selected with puromycin at a concentration of2.5μg/ml. The effective knockdown of Rictor was monitored by Western blot analysis.
RNA extraction and Real-time PCR
Total RNA was extracted and DNase-treated (RQ1, Promega). Synthesis of cDNA was performed with1μg of total RNA using the reagents in the Taqman Reverse Transcription Reagents Kit from ABI (#N8080234). Quantitative real time RT-PCR analysis was performed with an Applied Biosystems Prism7000HT Sequence
Detection System using TaqMan universal PCR master mix according to the manufacturer's specifications (Applied Biosystems Inc., Foster City, CA). The TaqMan probe and primers for human Rictor was purchased from Applied Biosystems. Human GAPDH gene was used as endogenous control. Each sample was run in triplicate. Average Ct values were calculated and normalized to Ct values for GAPDH. The results were graphed with the corresponding standard deviation indicated with error bars in the figures.
Protein preparation and Western blot analysis
Total protein (60μg) was resolved on a4-12%Bis-Tris gel and transferred to polyvinylidene difluoride (PVDF) membranes. Filters were incubated for1h at room temperature in blotting solution. Raptor, Rictor, mTOR, PKD1, PKD2, PKD3, p-Akt, Akt, and P-actin were detected with specific antibodies following blotting with a horseradish peroxidase-conjugated secondary antibody.
Immunoprecipitation
Cells were transfected with different plasmids as indicated,1%Triton lysis buffer was used. After pre-incubation with protein G PLUS-Agarose beads, equal amount of protein (500μg) were incubated with the indicated antibodies (1μg) using an end-to-end rotor overnight at4℃, followed by4h incubation with20μl of protein G PLUS-Agarose beads at4℃. Indicated buffer was used to wash the beads three times. Reactions were stopped by adding15μl of2×loading buffer. Samples were denatured by boiling for7min and separated by NuPAGE4-12%Bis-Tris gels.
Cell proliferation analyses
Equal numbers of cells were seeded onto24-well plates at a density of1×104per well in the appropriate culture medium with supplements. Cells carried with PKD shRNA or Rictor shRNA respectively were cultured for7days. Cells were trypsinized and counted using a cell counter (Beckman-Coulter).
Soft agar assay
Melt1.6%Agarose (www.lonza.com Cat.#50101) in distilled water in microwave, cool to40℃in a water bath, warm culture medium to40℃in water bath as well. Allow at least30minutes for temperature to equilibrate. Mix equal volumes of1.6% of Agar and medium to give0.8%Agar in medium. Add2ml/well of60mm dish, allow cooling down for30min. For plating, mix1ml of cell in medium and1ml0.8%Agar to a tube, mix gently and add2ml to each well (usually plate out in triplicate). Cool down in hood for30-60min. Add culture medium on the top agar for keeping humidity. Incubate assay at37℃in humidified incubator for10-14days. Stain plates with0.5ml of0.005%Crystal Violet (dilute with PBS) for>1hour, wash each well with PBS until every colonies could be seen clearly, count colonies using a dissecting microscope.
Immunoh istoch emical Analysis
Tissue microarrays containing normal and cancer tissues, A203(IV), were purchased from ISUABXIS through Accurate Chemical&Scientific Corporation. Sections were fixed to the slide by incubation in a dry oven at58℃for30min, and then sequentially transferred to xylene (5min,2changes),100%ethanol (3min,2changes),95%ethanol (3min,2changes) and rinsed with deionized water. Slides were allowed to cool at room temperature and rinsed twice with deionized water. Endogenous peroxidase was blocked by placing slides in3%H2O2/methanol block solution for10min, washed with deionized water, and placed in phosphate-buffered saline for5min. Slides were incubated with primary antibodies against human PKD1, PKD2, PKD3or Rictor overnight at4℃. Avidin-biotin peroxidase complex amplification and detection system (LSAB2, DAKO) with diaminobenzidine as chromagen was used. Negative controls (including no primary antibody or isotype matched mouse IgG) were used in each assessment.
Statistical analysis
The data of real time-PCR, proliferation and soft agar were expressed as the mean of three independent expression±S.D, evaluated with t-test. P<0.05was defined as different signicantly. All of these analyses were made using SPSS Version13(SPSS, Chicago, IL, USA).
RESULTS
PKD2and PKD3regulate expression of Rictor
In HT29and HCT116cells, both of Go6976(PKC and PKD inhibitors) and CID755673(PKD specific inhibitors) treatment inhibited expression of Rictor, while Go6983(PKC inhibitor) treatment had no affection on it (Fig.1-2A). Knocking down of PKD1, PKD2or PKD3individually could not inhibit Rictor expression (Fig.1-2B); overexpression of PKDl and PKD2did not increase Rictor (Fig.1-2C) as well. But we found that overexpressed PKD3using adenovirus increased expression of Rictor, with downstream target of Rictor-Akt S473phosphorylation increased at the same time (Fig.1-2D). Next, we set up PKD2and PKD3double knockdown stable cells, western blot analysis showed that both Rictor and p-Akt were decreased compared with control shRNA cells (Fig.1-2E).
PKD2and PKD3bind with Rictor
How do PKDs regulate expression of Rictor? First, we investigated if PKD regulated the mRNA level of Rictor by using real-time PCR. As shown in Fig.1-3A, no affection of Rictor mRNA was detected in PKD2and PKD3double knockdown cells. Similar result was gained in PKD3highly expression cells (Fig.1-3B), indicating the regulation of Rictor by PKD was post-transcriptional. Then, the interaction between PKD and Rictor was detected by Immunoprecipitation. Both of PKD2and PKD3bound with Rictor respectively, while no binding was investigated between PKD1and Rictor (Fig.1-3C and Fig.1-3D). Based on the data above, we hypothesized that PKD2and PKD3interacted with Rictor directly to regulate its protein expression.
Rictor is degradation by proteasomal pathway and overexpression of PKD3inhibits degradation of Rictor
Rictor is rapidly degraded by the ubiquitin-proteasome pathway. HT29cells were treated with MG132for the indicated times and subjected to Western blot analyses (Fig.1-4A). HT29were treated with CHX for the indicated times and Rictor levels were detected by Western analyses (Fig.1-4B).
Mosty importantly, we investigated the role of PKD3during Rictor degradation by CHX chase assay. As shown in Fig.1-4C, we found that the degradation of Rictor was decreased with highly expressed PKD3, demonstrating that overexpression of PKD3inhibited Rictor degradation and increased stability of Rictor protein.
PKD and Rictor do not affect CRC cells proliferation The previous data already showed that PKD affected Akt activities by regulating Rictor (Fig.1-2D and Fig.1-2E). So we was wandering that whether PKD and Rictor were involved in cell proliferation or not. After7days culture, cell numbers were counted by Beckman-Coulter, no difference was detected between control shRNA cells and PKD shRNA/Rictor shRNA cells individually (Fig1-5).
Inhibition of PKD and Rictor reduces tumorigenesis of CRC cells
Next, soft agar assay was used to investigate tumorigenesis ability in those cells carried with different shRNA. As shown in Fig.1-6, inhibition of either Rictor or PKD2&3decreased tumorigenesis ability of colorectal cancer cells.
Rictor as well as PKD2and PKD3are up-regulated in CRCs and liver metastasis
In Fig.1-7, we showed Immunohistochemical analysis of Rictor, PKD2and PKD3in CRC and liver metastasis sections. Rictor as well as PKD2and PKD3were up-regulated in primary CRCs and liver metastases compared with normal mucosa.
CONCLUSIONS
1. Inhibition of PKD2and PKD3decreases Rictor protein expression in CRC cells; consistently, overexpression of PKD3increases Rictor as well.
2. PKD2and PKD3bind with Rictor individually; Rictor is a target of26S proteasomal degradation in CRC cells and PKD3stabilzes Rictor protein
3. No affection on proliferation is detected while knocking down of both Rictor and PKD, while Rictor and PKD up-regulates tumorigenesis of CRC cells.
4. Rictor as well as PKD2and PKD3are up-regulated in primary CRCs and liver metastases compared with normal mucosa, implying a potential role of PKDs upstream of the Rictor signaling pathway in the pathogenesis of CRC.
Part two:Rictor regulates FBXW7-dependent c-Myc and cyclin E degradation in colorectal cancer cells
BACKGROUND:
Rictor (Rapamycin-insensitive companion of mTOR) forms a complex with mTOR and phosphorylates and activates Akt. Activation of Akt induces expression of c-Myc and cyclin E, which are overexpressed in colorectal cancer and play an important role in colorectal cancer cell proliferation. Here, we show that Rictor associates with FBXW7to form an E3complex participating in the regulation of c-Myc and cyclin E degradation. The Rictor/FBXW7complex is biochemically distinct from the previously reported mTORC2and can be immunoprecipitated independently of mTORC2. Moreover, knocking down of Rictor in serum-deprived colorectal cancer cells results in the decreased ubiquitination and increased protein levels of e-Myc and cyclin E while overexpression of Rictor induces the degradation of c-Myc and cyclin E proteins. Genetic knockout of FBXW7blunts the effects of Rictor, suggesting that Rictor regulation of c-Myc and cyclin E requires FBXW7.
MATERIALS
MG132and cycloheximide (CHX) were purchased from Calbiochem (San Diego, CA). Rabbit monoclonal anti-Rictor antibody, used for both immunoblotting and immunoprecipitation, was purchased from Bethyl Laboratories (Montgomery, TX). Rabbit anti-mTOR, rabbit anti-phospho-Akt (Ser473), mouse anti-Ubiquitin, rabbit anti-USP28and rabbit anti-myc-tag antibodies were obtained from Cell Signaling (Beverly, MA). Mouse monoclonal anti-Flag-tag and anti-β-actin antibodies, and non-targeting control shRNA and Rictor shRNA lentiviral particles were from Sigma (St. Louis, MO). Protein G PLUS-Agarose beads, rabbit anti-cyclin E and mouse anti-Aktl antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-c-Myc antibody was from Epitomics (Burlingame, CA). The plasmids encoding c-Myc, myc-tagged Rictor and myc-tagged mTOR were from Addgene (Cambridge, MA). The plasmid encoding Flag-tagged FBXW7a was kindly provided by Dr. Bruce E. Clurman (Fred Hutchinson Cancer Research Center, Seattle, WA). Human Rictor and non-targeting control siRNA
METHODS
Cell culture and transfection
The human CRC cell lines SW620, HT29and HCT116were from ATCC (Manassas, VA). SW620cells were maintained in DMEM supplemented with10%of FBS. HT29and HCT116cells were cultured in McCoy's5A supplemented with10%of FBS. Wild-type and FBXW7-/-HCT116cells, kindly provided by Dr. Bert Vogelstein (The Johns Hopkins University School of Medicine, Baltimore, MD), were maintained in McCoy's5A with10%FBS. SW620and HT29cells were transfected with the siRNA duplexes and plasmids using electroporation (Gene Pulser; Bio-Rad, Hercules, CA) and lipofectamine2000(Invitrogen, Carlsbad, CA), respectively.
Stable Rictor knockdown SW620, HT29and HCT116cells were generated. Cells were infected with the control shRNA or shRNA to human Rictor lentivirus particles and stably expressing cells were selected with puromycin at a concentration of2.5μg/ml. The effective knockdown of Rictor was monitored by Western blot.
RNA extraction and RT-PCR
Total RNA was extracted and DNase-treated (RQ1, Promega). Synthesis of cDNA was performed with1μg of total RNA using the reagents in the Taqman Reverse Transcription Reagents Kit from ABI (#N8080234). Reverse transcriptional PCR was performed by Hotstart DNA polymerase (Qiagen, Hilden, Germany), forward and reverse primers, and deoxynucleoside triphosphates in a final volume of25μl. The amplification product was of the expected sizes.
Protein preparation and Western blot analysis
Cells were collected at48h after transfection or at the indicated time points after treatment. Equal amounts of cell lysates were resolved on a4-12%Bis-Tris gel and transferred to polyvinylidene fluoride membranes. Membranes were blocked by5%non-fat milk for1h at room temperature. Rictor, mTOR, phospho-Akt (Ser473), Akt1, c-Myc, cyclin E, Myc-tag, Flag-tag and β-actin were detected with specific antibodies following blotting with a horseradish peroxidase-conjugated secondary antibody and visualized using a chemiluminescence detection system.
Immunoprecipitation and in vivo ubiquitination analysis
Cells were transfected with different plasmids as indicated,1%Triton lysis buffer or CHAPS lysis buffer was used. After pre-incubation with protein G PLUS-Agarose beads, equal amount of protein (500μg) were incubated with the indicated antibodies (1μg) using an end-to-end rotor overnight at4℃, followed by4h incubation with20μl of protein G PLUS-Agarose beads at4℃. Indicated buffer was used to wash the beads three times. Reactions were stopped by adding15μl of2×loading buffer. Samples were denatured by boiling for7min and separated by NuPAGE4-12%Bis-Tris gels. For in vivo ubiquitination analysis, His-ubiquitin plasmid was co-transfected with the indicated plasmids. Transfected cells were incubated with MG132(20μM) in serum free medium for4h before harvesting.
Statistical analysis
There is no statistical analysis in this section.
RESULTS
Rictor regulates protein expression of c-Myc and cyclin E
Activation of Akt increases c-Myc and cyclin E expression. Since inhibition of mTORC2by knockdown of Rictor inhibits Akt activation, we were interested to know whether Rictor regulates c-Myc and cyclin E expression. Human colorectal cancer cells, SW620and HT29, were transfected with shRNA targeting Rictor and stable cell lines were established. Knockdown of Rictor did not obviously affect the expression of c-Myc and cyclin E; however, with serum starvation, knockdown of Rictor increased protein expression of c-Myc and cyclin E (Fig.2-3A). In addition, treatment with MG132, a specific cell-permeable proteasome inhibitor, attenuated the increases of c-Myc and cyclin E protein expression resulting from Rictor knockdown (Fig.2-3B), indicating that the26S proteasome pathway was involved in this regulation. To further confirm our findings, we used Rictor siRNA containing a different sequence from the Rictor shRNA to decrease Rictor expression. Consistently, knockdown of Rictor by transient transfection with siRNA targeting Rictor resulted in a significant increase of c-Myc and cyclin E protein expression (Fig.2-3C). To further determine the role of Rictor in the regulation of c-Myc and cyclin E protein expression, SW620and HT29cells were transiently transfected with a myc-tagged Rictor plasmid and the transfected cells were serum starved for24h before harvesting. As shown in Fig.2-3D, overexpression of Rictor decreased c-Myc and cyclin E protein levels in both SW620and HT29cells, suggesting that Rictor regulates the protein levels of c-Myc and cyclin E associated with serum deprivation. Interestingly, RT-PCR assay showed that knockdown of Rictor inhibited mRNA level of c-Myc and cyclin E (Fig.2-3E). Considering the increases of c-Myc and cyclin E protein expression by Akt activation and knockdown of Rictor resulting in the dephosphorylation and inhibition of Akt, our results demonstrate that Rictor regulates c-Myc and cyclin E protein in an mTORC2/Akt pathway independent fashion.
Rictor interacts with FBXW7without mTOR
Degradation of c-Myc and cyclin E was targeted by FBXW7as an E3component. And it has been shown that Rictor forms a complex with cullinl to degrade SGK1protein. To determined whether Rictor interacts with FBXW7. As shown in Fig2-4A, Myc-Rictor constructs were co-transfected with Flag-FBXW7a, Flag-FBXW7p and Flag-FBXW7y individually. The interactions between Rictor and three FBXW7isforms were detected. FBXW7a is expressed at a much higher level than FBXW7P and FBXW7y in most human cell lines, and usually plays a major role as an E3ligase to the downstream targets. Therefore, we transfected HCT116cells with Flag-FBXW7a together with Myc-Rictor and the interactions between FBXW7and Rictor were detected. Two different lysis buffers were used in this assay:1%Triton and CHAPS lysis buffers. CHAPS buffer was used as a mild buffer to keep mTORC2intact while1%Triton was used to dissociate mTORC2. As shown in Fig.2-4C, Flag-FBXW7a was clearly copurified when Rictor was immunoprecipitated from cell lysates extracted with both1%Triton lysis buffer and CHAPS buffer, but the interaction between mTOR and Rictor was only observed in the lysates extracted using CHAPS buffer. Although it has been reported that FBXW7interacts with mTOR to promote its ubiquitination and degradation, our results indicate that Rictor forms a complex with FBXW7independent of mTORC2. Furthermore, the binding between Rictor and FBXW7is not affected by serum-deprivation (Fig2-4D).
Rictor interacts with FBXW7to regulate c-Myc and cyclinE
We next determine whether Rictor participates in FBXW7-dependent regulation of c-Myc and cyclin E degradation, we generated Rictor shRNA stable cell lines based on wild-type and FBXW7-/-HCT116cells. As shown in Fig.2-5A, knockdown of Rictor induced the expression of c-Myc and cyclin E in wild type HCT116cells but not in FBXW7-/-cells. These data suggest that Rictor regulation of c-Myc and cyclin E is FBXW7dependent. To further demonstrate the role of Rictor in the FBXW7E3ligase complex, HCT116control shRNA and Rictor shRNA stable cell lines were transfected with empty vector (control) or Flag-FBXW7a plasmid and c-Myc and cyclin E protein levels were analyzed (Fig.2-5B). Overexpression of FBXW7αdecreased the protein levels of c-Myc and cyclin E as expected. However, this degradation by FBXW7a was significantly attenuated in Rictor shRNA cells, demonstrating a role of Rictor, bound with FBXW7a as a part of E3complex, to induce the degradation of c-Myc and cyclin E.
Rictor regulates stability of FBXW7
Rictor didn't affect the mRNA level of FBXW7by RT-PCR assays (Fig.2-6A), which means Rictor regulates FBXW7through post-transcriptional regulation. Next, in vivo ubiquitination assay was performed to investigate the ubiquitination of FBXW7with or without Rictor expression. As expected in Fig.2-6B, the ubiquitination of FBXW7a was diminished in Rictor knockdown cells compared with control cells. Moreover, in Rictor stably knockdown cells, the stability of FBXW7αwas decreased; futher confirmed our hypothesis (Fig.2-6C).
Rictor/FBXW7promotes c-Myc and cyclin E ubiquitination
FBXW7degrades c-Myc and cyclin E through the ubiquitination of c-Myc and cyclin E proteins. To address whether Rictor promotes the ubiquitination of c-Myc and cyclin E, in vivo ubiquitination assays were performed. c-Myc or cyclin E and His-ubiquitin were expressed by transient transfection. Knockdown of Rictor reduced the ubiquitination of both c-Myc (Fig.2-7A) and cyclin E (Fig.2-7C). As expected, the ubiquitination of c-Myc and cyclin E was diminished in FBXW7-/-HCT116cells compared with wild-type and, importantly, Rictor-dependent regulation of c-Myc and cyclin E ubiquitination was also blunted in FBXW-/-HCT116cells (Fig.2-7B and Fig.2-7D). Taken together, these data indicate a role of Rictor in the regulation of c-Myc and cyclin E protein ubiquitination and degradation.
Rictor reduces the stability of c-Myc and cyclin E
To determine whether Rictor regulates the stability of c-Myc and cyclin E, we next performed CHX chase assays. Time-course experiments showed that the half-lives of c-Myc (Fig.2-8A) and cyclin E (Fig.2-8C) were prolonged in HCT116cells with Rictor shRNA compared with HCT116cells with non-targeting control shRNA. The sequence of Rictor shRNA used in this study was designed to target the3'-UTR region of Rictor and would not affect the expression of transfected myc-Rictor plasmid. Therefore, Rictor protein levels can be rescued in Rictor shRNA stable cell lines with myc-Rictor overexpression. Indeed, transfection of HCTl16Rictor shRNA stable cells with myc-Rictor plasmid decreased the half-life of c-Myc (Fig.2-8B) and cyclin E (Fig.2-8D). Our results demonstrate that Rictor is required for the integrity of the FBXW7E3complex to promote the degradation of c-Myc and cyclin E.
Rictor binds with USP28through FBXW7
USP28is a deubquitin specific protein, which can bind with FBXW7to deubiquitinate its downstreams. c-Myc, as one of USP28specific targets, was decreased with USP28knockdown in HT29; consistently, overexpression of USP28in HCT116induced c-Myc protein level (Fig.2-9A). Rictor bound with USP28via FBXW7(Fig.2-9B), but Rictor did not have affect on the deubiquitination regulation of c-Myc by USP28(Fig.2-9C and Fig.2-9D).
Rictor/FBXW7may mediate Rapamycin resistance in CRC
Rictor activity was also inhibited by long-time exposure of Rapamycin. Some of cancer cells were rapamycin sensitive, while some of them were rapamycin resistant and the mechanisms of rapamycin resistance is still unclear. As shown in Fig.2-10A,24hours exposure of rapamycin in rapamycin resistant cells HT29and S W620led to the decrease of Rictor, with increase of c-Myc, cyclin E and Akt phosphorylation at the same time. And no increase of c-Myc, cyclin E mRNA level was detected in the same cell lines with rapamycin treatment (Fig.2-10B), indicating rapamycin induced c-Myc and cyclin E was mTORC2/Akt pathway independent. Based on these data, we hypothesize that rapamycin resistance is mediated by increased c-Myc and cyclin E via Rictor/FBXW7regulation.
CONCLUSIONS
1. Our findings identify Rictor as an important component of FBXW7E3ligase complex participating in the regulation of c-Myc and cyclin E protein ubiquitination and degradation.
2. Our results suggest that elevated growth factor signaling may contribute to decrease Rictor/FBXW7-mediated ubiquitination of c-Myc and cyclin E, thus leading to accumulation of cyclin E and c-Myc in colorectal cancer cells.
3. Our study figures out a possible mechanism of Rapmycin resistance in colorectal cancer cell, which yields another role of Rictor during tumor progression.
引文
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3. Hara K, Maruki Y, Long X, et al. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 2002; 110:177-89.
4. Kim DH, Sarbassov DD, Ali SM, et al. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 2002;110:163-75.
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8. Sancak Y, Thoreen CC, Peterson TR, et al. PRAS40 is an insulin-regulated inhibitor of the mTORCl protein kinase. Mol Cell 2007;25:903-15.
9. Vander Haar E, Lee SI, Bandhakavi S, et al. Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat Cell Biol 2007;9:316-23.
10. Hilti N, Baumann D, Schweingruber AM, et al. Gene ste20 controls amiloride sensitivity and fertility in Schizosaccharomyces pombe. Curr Genet 1999;35:585-92.
11. Loewith R, Jacinto E, Wullschleger S, et al. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol Cell 2002;10:457-68.
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14. Valverde AM, Sinnett-Smith J, Van Lint J, et al. Molecular cloning and characterization of protein kinase D:a target for diacylglycerol and phorbol esters with a distinctive catalytic domain. Proc Natl Acad Sci U S A 1994;91:8572-6.
15. Sturany S, Van Lint J, Muller F, et al. Molecular cloning and characterization of the human protein kinase D2. A novel member of the protein kinase D family of serine threonine kinases. J Biol Chem 2001;276:3310-8.
16. Hayashi A, Seki N, Hattori A, et al. PKCnu, a new member of the protein kinase C family, composes a fourth subfamily with PKCmu. Biochim Biophys Acta 1999;1450:99-106.
17. Wang QJ. PKD at the crossroads of DAG and PKC signaling. Trends Pharmacol Sci 2006;27:317-23.
18. Iglesias T, Rozengurt E. Protein kinase D activation by mutations within its pleckstrin homology domain. J Biol Chem 1998;273:410-6.
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