CN116173211A - Yap蛋白的表达激活剂在制备治疗前列腺癌的药物中的用途 - Google Patents
Yap蛋白的表达激活剂在制备治疗前列腺癌的药物中的用途 Download PDFInfo
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Abstract
本发明属于抗肿瘤药物技术领域，具体涉及YAP蛋白的表达激活剂在制备治疗前列腺癌的药物中的用途。本研究发现YAP在雄激素受体(AR)阳性前列腺癌(PCa)中具有环境依赖性的肿瘤抑制功能，并表明YAP通过拮抗TEAD介导的AR信号抑制AR+PCa生长。药理抑制MST1/2或LATS1/2，或转基因激活YAP抑制了表达AR剪接变异体的PCa的生长。我们的研究发现了Hippo和AR信号通路之间意想不到的串扰，揭示了AR+PCa中YAP和TEAD之间的拮抗关系，并提示靶向Hippo信号通路可能为治疗耐药AR变体驱动的PCa提供了一个治疗机会。
Description
技术领域
本发明属于抗肿瘤药物技术领域，具体涉及YAP蛋白的表达激活剂在制备治疗前列腺癌的药物中的用途。
背景技术
前列腺癌(PCa)是美国第二大确诊的恶性肿瘤，每年约有3万人死于转移性前列腺癌。大多数前列腺癌是由雄激素受体(AR)驱动的，AR是癌症进展的主要驱动因素，通过雄激素剥夺和雄激素受体抑制是前列腺癌患者的首选治疗方法。然而，很大一部分接受治疗的患者最终会发展成去势抵抗性前列腺癌(CRPC)。尽管第二代抗雄激素药物如AR拮抗剂Enzalutamide可以延长转移性CRPC患者4-5个月的生存时间，但对这些药物的原发性或获得性耐药在接受治疗的患者中很常见。Enzalutamide耐药的常见机制包括AR扩增和缺乏配体结合域的AR变体的上调，如AR-v7。
Hippo肿瘤抑制通路是一种进化保守的信号通路，控制组织生长、器官大小、组织再生和癌症进展。在Hippo核心通路中，上游激酶Hippo(Hpo)/MST1/2磷酸化并激活下游激酶LATS1/2，导致转录效应子YAP/TAZ的磷酸化和失活。抑制Hippo信号通路可使YAP/TAZ转位至细胞核，与Hippo通路转录因子TEAD结合，调节靶基因表达。在包括肝癌、肺癌、结肠癌和卵巢癌在内的多种人类癌症中，YAP被扩增，其蛋白水平和核定位升高。此外，YAP在小鼠肝脏中过表达和敲除MST1/2或Hippo通路的其他上游成分会导致肝肿大，导致肝细胞癌的形成。这些观察结果导致了一种普遍的观点，即Hippo信号通路通过阻断YAP/TAZ的致癌潜能，发挥肿瘤抑制通路的作用。然而，近年来的研究表明，YAP在多种类型的癌症中具有抑癌作用，但是其作用机制因癌症类型而异，针对YAP在AR阳性PCa中的作用，尚属研究空白。
发明内容
基于以上问题，本发明提供了以下技术方案：
本发明提供了YAP蛋白的表达激活剂在制备治疗前列腺癌的药物中的用途，所述前列腺癌为雄激素受体阳性前列腺癌。
进一步的，所述YAP蛋白表达升高可以抑制雄激素受体的转录活性和雄激素受体阳性前列腺癌的生长。
更进一步的，所述YAP蛋白通过和雄激素受体竞争结合TEAD，抑制雄激素受体靶基因的表达，阻止TEAD促进雄激素受体信号传导发挥作用。
进一步的，述YAP蛋白的表达激活剂包括转基因表达盒，所述转基因表达盒包含YAP蛋白和/或其突变体的编码序列，所述突变体包括YAP-5SA和YAP-5SAS94A。
进一步的，所述YAP蛋白的表达激活剂包括Hippo信号通路抑制剂，所述抑制剂抑制YAP蛋白上游激酶的活性或表达，使YAP蛋白水平升高。
更进一步的，所述YAP蛋白上游激酶包括MST1/2激酶和LATS1/2激酶。
更进一步的，所述抑制剂包括靶向抑制MST1/2激酶和/或LATS1/2激酶的小分子化合物、siRNA或慢病毒。
更进一步的，靶向抑制MST1/2激酶的小分子化合物为XMU-MP-1，靶向抑制LATS1/2激酶的小分子化合物为TRULI。
本发明的有益效果：
本发明研究了Hippo信号在AR阳性PCa中的作用，并探讨了Hippo信号通过YAP调节AR信号活性的机制。我们发现抑制Hippo信号通路或异位激活YAP阻断AR转录程序和AR+PCa生长。我们发现了TEAD通过与AR形成复合物以增加其启动子/增强子的占有率来辅助AR信号传导的一种以前未发现的功能。我们证明YAP通过破坏AR-TEAD信号复合物抑制AR转录活性和AR与其靶启动子的结合。我们进一步表明，对Hippo信号通路的药理学抑制损害了体内抗治疗PCa的生长。因此，我们的研究揭示了Hippo信号在癌症中的非常规功能，并表明靶向Hippo通路可能是克服AR+PCa患者内分泌治疗耐药性的潜在策略。
附图说明
图1为前列腺癌中AR和YAP信号活性的反向相关性；A为正常前列腺组织与前列腺癌样本中AP、TAZ、AMOTL2、KLK2和KLK3的mRNA水平比较；B为前列腺癌数据集中AR信号和YAP信号通路靶基因表达的热图；C为前列腺癌数据集中AR信号和YAP信号之间的相关系数；D为TCGA前列腺癌数据集中AR特征(KLK2、KLK3、NKX3-1、FKBP5、CAMKK2、PMEPA1)和YAP特征(YAP1、TAZ、CTGF、CYR61、AMOTL2、ANKRD1)之间的相关性；E为前列腺癌患者中，YAP的表达水平与无病间隔(DFI)的相关性，F为TAZ的表达水平与无病间隔(DFI)的相关性。
图2为MST1/2抑制或YAP激活对AR+PCa的影响；A-D依次为通过慢病毒感染对照、YAP-WT或YAP-5SA构建体的LNCaP、C4-2、22RV1和R1-D567细胞的相对生长(顶部)或蛋白质印迹分析(底部)；E-H依次为XMU-MP-1或5μM Enzalutamide处理的LNCaP、C4-2、22RV1和R1-D567细胞的相对生长；I为XMU-MP-1处理的C4-2细胞用DAPI抗YAP抗体免疫染色，J为Western blot分析经XMU-MP-1处理的C4-2细胞的细胞质和细胞核中YAP的表达；K为YAPsiRNA处理的C4-2细胞中YAP的Western blot分析(顶部)，以及细胞相对生长(底部)；L为经siYAP、或siYAP+XMU-MP-1处理的C4-2细胞的相对生长；M-N为经siLATS1/2处理的C4-2细胞中相关蛋白的表达(M)和细胞的相对生长(N)；O-P为经TRULI处理的C4-2细胞的Westernblot分析(O)和细胞的相对生长(P)；Q-R为经siLATS1/2、siYAP或siLATS1/2+siYAP处理的C4-2细胞的Western blot分析(Q)和细胞的相对生长(R)；S-T为经慢病毒感染YAP-5SA构建体的LNCaP细胞在无DHT添加(S)和有DHT(T)添加的培养基中的相对生长。
图3为LATS1/2敲除对LNCaP和22RV1细胞生长的影响；A，C为用对照或LATS1/2siRNA处理的LNCaP(A)或22RV1(C)细胞中LATS1和YAP磷酸化的蛋白质印迹分析；B，D为用对照或LATS1/2siRNA处理的LNCaP(A)或22RV1(C)细胞的相对生长；E，F为在不存在或存在2μM XMU-MP-1或0.2μg/ml DOX的情况下，C4-2(E)或C4-2-Tet-O-YAP-5SA(F)的锚定独立生长。
图4为TEAD与AR形成复合物对AR信号传导的影响。A-C为TEAD1和TEAD4的蛋白质印迹分析(A)、TEAD1和TEAD4的相对mRNA水平以及指示的AR靶基因(B)和用两个独立的TEAD1/3/4siRNA或对照siRNA处理的C4-2细胞的相对生长(C)；D为表达对照或TEAD4构建体并用XMU-MP-1处理的C4-2细胞中所指示的AR靶基因的相对mRNA水平(左)和TEAD的蛋白质印迹分析(右)；E-F为用IgG、抗AR(E)、抗YAP(F)或抗TEAD4(G)抗体免疫沉淀C4-2细胞提取物，E和F中的星号表示IgG；H-J为用AR、TEAD和YAP构建体组合转染的HEK-293T细胞的白质印迹分析结果；K为GST或GST-TEAD4和Flag AR组合转染的HEK-293T细胞的体外结合测定结果；L为AR和TEAD结构示意图；M-R为AR和TEAD构建体转染的HEK-293T细胞的蛋白质印迹分析结果。
图5为TEAD敲除对AR靶基因表达和22RV1和R-D567 PCa生长的影响；A、D为在22RV1(A)和R-D567(D)中TEAD1蛋白质印迹分析，B、E为TEAD1、KLK2和KLK3在22RV1(B)和R-D567(E)的相对mRNA水平；C、F为22RV1(C)和R-D567(F)的相对生长；G为TEAD1、KLK2和KLK3在TEAD1过表达C4-2细胞中的相对mRNA水平。
图6为TEAD表达减少，YAP/TAZ耗竭对AR信号传导的抑制作用；A-C为用siYAP/TAZ处理18(A)、36(B)或72(C)小时的C4-2细胞中YAP和TEAD(左)的Western blot分析以及所示基因(右)的mRNA水平；D为在过表达TEAD4转基因并用siYAP/TAZ处理72小时的C4-2细胞中，YAP和TEAD(左)的蛋白质印迹分析以及所示基因(右)的mRNA水平；
图7为MST1/2抑制/YAP激活在在体内对耐药AR的抑制。A-F为雄性NOD scid gamma(NSG)小鼠携带稳定表达Tet-O-EGFP结构的C4-2肿瘤细胞作为对照，雄性NOD scid gamma(NSG)小鼠携带肿瘤，在指定的时间内每天注射含有DOX的PBS(20mg/kg)或PBS，结果显示治疗结束时肿瘤生长曲线(A,D)，肿瘤样本照片(B,E)，肿瘤重量量化(C,F)。
图8为YAP-5SAS94A或YAP-5SA过表达对体内C4-2 PCa生长的影响。A-C为携带表达Tet-O-YAP5SAS94A的C4-2肿瘤的雄性NOD scid gamma(NSG)小鼠在指定的时间段内每天腹腔注射含DOX(20mg/kg)PBS或PBS，显示了治疗结束时的肿瘤生长曲线(A)、肿瘤样品照片(B)和肿瘤重量定量(C)。D-E为用PBS或DOX处理22天的小鼠的表达Tet-O-YAP-5SA或Tet-O-YAP-5AS94A的异种移植C4-2肿瘤中AR(D)或YAP靶基因(E)的相对mRNA水平；F-G为PBS或DOX处理22天的小鼠的表达Tet-O-YAP5SA(F)或Tet-O-YAP-5SAS94A(G)的异种移植C4-2肿瘤中YAP的蛋白质印迹分析。
图9为XMU-MP-1在小鼠中的耐受性结果。A-C为携带C-42(A)、22RV1(B)或R1-D567肿瘤的小鼠的体重(左)、肝脏重量/体重(中)和脾脏重量/体重/体重(右)。
图10为XMU-MP-1和Enzalutamide对异种移植中22RV1肿瘤生长的影响；A-F为携带22RV1肿瘤的雄性NOD scid gamma(NSG)小鼠每天用XMUMP-1(15mg/kg)、恩扎鲁胺(10mg/kg)或赋形剂治疗指定时间，结果显示了治疗结束时的肿瘤生长曲线(A)、肿瘤样品照片(B)、肿瘤重量(C)、体重(D)、肝脏重量/体重(E)和脾脏重量/重量(F)。
图11为Hippo和AR信号串扰的工作模型。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不应理解为本发明的限制。如未特殊说明，下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段，下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明披露了YAP蛋白的表达激活剂在制备治疗前列腺癌的药物中的用途，所述前列腺癌为雄激素受体阳性前列腺癌。
为了证明上述用途的可行性，进行了如下验证：
1、材料与方法
1.1材料
DNA结构：YAP-WT和YAP-5SA为市售，YAP-5SAS94A是由YAP-5SA通过PCR诱变产生的。对于慢病毒感染，将YAP-WT、YAP-5SA、TEAD4、TEAD4m和TEAD1克隆到pLVXIRES-ZSgreen载体中。为了构建Tet-O-EGFP，将GFP、YAP-5SA和YAP-5SAS94A的编码序列亚克隆到pTet-O-Ngn2-puro(Addgene plasmid,Cat.No 52047)，其中T2A序列被UBC启动子取代。巨细胞病毒rtTA3 Hygro(Addgene plasmid,Cat.No.26730)用于生成Doxinducible细胞系。人体TEAD4结构物购自Origene。(https://www.origene.com/；RC219686).用PCR扩增TEAD4全长和缺失结构，PCR产物亚克隆到pcDNA3.1-FLAG载体上。TEAD1构建物通过PCR扩增，亚克隆到p3xflag-CMV-10载体上。AR-WT结构来自Addgene(pCMV-hAR,Addgene plasmid,Cat.No89078)。用PCR扩增ARFL、缺失突变体和剪接变异体构建，PCR产物亚克隆到pCMV-HA或pCMV-myc载体中。
细胞：LNCaP、C4-2、22RV1和HEK-293T细胞最初从美国典型菌种保藏中心(ATCC)中获得。R1-D567是由之前的研究生成的(Nyquist et al,2013；Sramkoski et al,1999).LNCaP、C4-2、22RV1和R1-D567细胞用RPMI1640+2mM L-谷氨酰胺和10％胎牛血清维持。HEK-293T用Dulbecco's Modified Eagle's培养基培养，该培养基含有4,5g/L葡萄糖和4mmL-谷氨酰胺(DMEM,Gibco,Cat.No.11965092)，添加10％胎牛血清对于包装慢病毒，HEK-293T细胞通过PolyJet(SignaGen laboratories,Cat)转染表达载体和包装载体。48小时后，用标准方法收集病毒进行细胞系感染。
试剂：使用的试剂有:XMU-MP-1(MCE,Cat.No.HY-100526),TRULI(MCE,Cat.Cat.No.:HY-138489),Enzalutamide(MCE,Cat.No.HY-70002),5α-DHT-D3(DHT；SigmaCat:D077),Doxycycline(Dox)(Sigma,Cat.No.33429),Hygromycin B(Sigma,Cat.No.H3274),Puromycin(Sigma,Cat.No.P9620),MG-132(Calbiochem,Cat.No.474790)。
siRNA：YAP沉默的序列为:siYAP_1(SEQ ID NO.1)：5’-CAC CUA UCA CUC UCG AGAU-3’和siYAP_2(SEQ ID NO.2)5’-GCU CAU UCC UCU CCA GCU U-3’.TEAD1/3/4沉默序列为:siTEAD1/3/4_1(SEQ ID NO.3)：5’-AUG AUC AAC UUCA UCC ACA AG-3’和siTEAD1/3/4_2(SEQ ID NO.4)：5’-GAU CAA CUU CAU CCA CAA GCU-3’.AR沉默的序列为:siAR_1(SEQ IDNO.5)5’-CCA UCU UUC UGA AUG UCC U-3’和siAR_2(SEQ ID NO.6)：5’-CAG GAA UUC CUGUGC AUG A-3’.阴性对照的序列为:siNC(SEQ ID NO.7)：5’-UUC UCC GAA CGU GUC ACG U-3’.LATS1/2siRNA购自Sigma,siLATS1的siRNA ID为SASI_Hs01_00046128和SASI_Hs01_00046129,siLATS2的siRNA ID为SASI_Hs01_00158803和SASI_Hs01_00158806。RNAi MAX试剂(Invitrogen Cat.No.13778150)转染siRNA。通过实时PCR和免疫印迹法验证敲除效率。
1.2方法
(1)免疫印迹分析(Immunoblot analysis)
收集细胞，用含有1M Tris pH8.0、5M NaCl、1M NaF、0.1M Na3VO4、1％NP-40、10％甘油和0.5M EDTA (pH 8.0)的裂解液裂解细胞。蛋白质经SDA-聚丙烯酰胺凝胶电泳(PAGE)分离后电转移到PVDF膜上。用TBST清洗膜，与一抗孵育2小时。然后用TBST洗涤三次，用二抗孵育2小时，TBST洗涤三次后，用ECL系统检测(Cytiva,Cat.No.RPN2105)。
(2)免疫共沉淀分析(Co-immunoprecipitation assay)
按照标准方案进行免疫沉淀。C4-2细胞裂解物与AR(Sigma,Cat.No.A9853)、YAP(Santa Cruz,Cat.No.SC101199)、TEAD1(BD Transduction Laboratories,Cat.No.610922)、TEAD4(Santa Cruz,Cat.No.sc-101184)抗体或小鼠IgG抗体(作为阴性对照)孵育过夜，蛋白A树脂固定沉淀。结合蛋白用蛋白印迹法分析。在过表达实验中，HEK-293T细胞转染5μg表位标记的AR(全长或缺失突变体)和TEAD4(全长或缺失突变体)质粒，在10cm培养皿中不存在或不存在Flag-YAP。将细胞裂解物与针对表位标签的抗体孵育过夜，然后用蛋白A树脂固定和沉淀。通过免疫印迹法分析结合蛋白。
(3)GST下拉分析(GST pull down assay)
GST-TEAD4构建体是通过将全长人TEAD4亚克隆到pGEX-4T1载体(Sigma-Aldrich)中而产生的。GST-TEAD4融合蛋白在大肠杆菌BL21(DE3)中表达，并在4℃下使用谷胱甘肽-4B琼脂糖凝胶(GE Healthcare)纯化。为了纯化Flag YAP和Flag AR，HEK-293A细胞分别用Flag YAP和Flag AR构建体转染，生长48小时，并在裂解缓冲液中收获(1M Tris pH8.0,5MNaCl,1M NaF,0.1M Na3VO4,1％ NP-40,10％glycerol).将细胞提取物与M2(抗Flag)抗体(Sigma-Aldrich)在4℃下孵育过夜，然后用蛋白A树脂(Pierson)固定并沉淀2小时。
然后洗涤免疫沉淀剂，并根据制造商的说明用Flag肽(Sigma-Aldrich)洗脱结合蛋白。将Flag YAP和Flag AR与纯化的GSTTEAD4在4℃下孵育过夜，然后固定化并用谷胱甘肽-4B Sepharose沉淀2小时.用抗Flag(Sigma，Cat.No.F3165)和抗GST(Santa Cruz，Cat.No.SC-138)抗体通过免疫印迹分析结合的蛋白质。
(4)免疫荧光分析(Immunofluorescence assay)
C4-2细胞用PBS中的4％多聚甲醛固定10分钟，用0.2％Triton X-100(Sigma,Cat.No.T8787)渗透5分钟，并用PBS的5％BSA阻断1小时。使用兔抗AR抗体(Cellsignaling,Cat.No.5153)和小鼠抗YAP单克隆抗体(Santa Cruz,Cat.No.SC-101199)，然后使用Cy2-和Cy3-缀合的二级抗体(Jackson ImmunoResearch).作为阴性对照，样品与不含一级抗体的二级抗体一起孵育。图像由Zeiss LSM510共焦显微镜拍摄。使用ImageJ对获取的图片进行进一步处理和组装。
(5)病毒感染和瞬时转染(Virus infection and transient transfection)
病毒感染时，表达载体与包装载体(psPAX2 and pMD2.G)一起通过PolyJet转染HEK-293T细胞.(SignaGen laboratories,Cat.No.SL100688).48h后，收集培养基上清液，用0.45μm过滤器过滤。含病毒上清4℃保存细胞感染。前列腺癌细胞在新鲜培养基中培养，然后与聚苯醚一起用慢病毒感染一夜(Sigma,Cat.No.H9268)。
(6)RNA提取和RT-qPCR分析(RNA extraction and RT-qPCR analysis)
按照常规方法进行。
(7)染色质免疫沉淀(ChIP-qPCR)
细胞交联是通过添加最终浓度为1％的甲醛10min或2mM DSG交联剂(CovaChem,Cat.No.13301)室温1小时，然后用1％甲醛二次固定(Pierce,Cat.No.28908)加热10分钟，加入甘氨酸淬火随后，用冷PBS冲洗细胞并进行细胞裂解。细胞提取物进行超声处理。离心后，细胞提取物与制备的AR/TEAD抗体dynabeads室温孵育1小时，4℃孵育1小时，在洗涤缓冲液中洗涤5次，65℃洗脱缓冲液中去交联ChIP过夜。
(8)染色质免疫共沉淀-测序和数据分析(ChIP-seq experiment and dataanalysis)
ChIP-Seq库使用KAPA HTP Library Preparation Kit
平台(KAPA,KR0426)根据制造商协议生成。简单地说，将ChIP DNA与多重适配器连接，通过PCR扩增生成文库，经过纯化和大小选择后，在安捷伦2100生物分析仪上进行文库检测。
样品量化、归一化和汇集后，最终的样品使用75bp高输出测序试剂盒在IlluminaHiSeq 2500上运行，在UTSW下一代测序核心进行单端测序。使用“Bowtie2”对Reads进行修剪并与人类基因组(hg19)对齐，随后使用“Samtools”对Reads进行排序。峰的识别采用MACS2 v2.1.1,p值截止值1e-5。在进行峰值调用后，从峰值文件中去除ENCODE人类黑名单区域。使用'HOMER'v4.9进行峰值重叠分析、motif富集分析和峰值注释。ChIP-seq信号轨迹由“DeepTools”生成，并通过RPKM进行规范化。ChIP-seq数据存入基因表达综合(GEO)数据库(评估号:GSE208606)。利用综合基因组学观察软件(IGV)对信号轨迹进行可视化分析。ChIP-seq峰值子集的热图和信号图是使用“DeepTools”生成的。
(9)RNA测序和数据分析(RNA sequencing and data analysis)
全基因组基因表达分析(Vehicle vs XMU-MP-1治疗组，对照组vs Dox治疗组)基于华大基因(北京基因组研究所)和UTSW下一代测序核心的RNA测序平台。使用Qiagen RNA提取试剂盒(Qiagen；Cat:74104)根据制造商的说明。细胞RNA被发送到华大基因(https://www.bgi.com)进行RNA测序。RNA用Agilent2100生物分析仪(Agilent RNA 6000Nano Kit)进行质量检测，RNA完整性编号大于9，用于文库构建。总RNA按照BGISEQ-500平台协议进行文库构建。用BGISEQ-500平台对文库进行测序。对于对照组和DOX治疗组RNA-seq，细胞RNA被发送到UTSW下一代测序核心，用Agilent 2100生物分析仪进行质量访问，用TruSeq链mRNA样品制备试剂盒建立文库。库样本在标准化和池化之前用Qubit量化，然后使用SBS v3试剂在Illumina HiSeq 2500上运行。然后使用STAR比对器将FASTQ测序文件与hg19人类基因组进行比对，并保留唯一的映射读数以供进一步分析.RNA序列数据保存在基因表达综合(GEO)数据库中(评估编号:GSE186177和GSE217580)。差异表达基因(DEGs)的路径分析(FPKM>5,P值<0.05,Fold change>1.5)使用metscape(https://metascape.org)进行，热图由数据分析和可视化免费在线平台http://www.bioinformatics.com.cn绘制。RNA-seq数据的基因集富集分析使用Hallmark Androgen Response和Cordenonsi YAP保守签名的基因集，并从分子签名数据库v7下载。GSEA采用GSEA 4.1.0软件实现，参数默认。使用R中的'ggplot2'包生成火山图(阈值P<0.05，倍数变化>1.5)。
(10)TCGA前列腺癌数据集分析(Analysis of TCGAprostate cancer data sets)
使用http://ualcan.path.uab.edu/在线工具分析YAP、TAZ、AMOTL2、KLK3、KLK2在前列腺癌组织和正常组织中的表达。AR与YAP靶基因的相关性分析使用MORPHEUS(https://software.broadinstitute.org/morphe us/)生成基因相关性热图，使用cBioprotal(http://www.cbioportal.org/)对489例前列腺腺癌(TCGA,panccancer Atlas)样本进行基因间spearman相关性分析。YAP签名与AR签名的Pearson相关性分析，采用R软件gsa包(选择参数为方法＝“ssGSEA”)分析TCGA前列腺癌数据集各样本AR签名和YAP签名的富集评分。无病期生存Kaplan-Meier分析来自Xena(https://xenabrowser.net/)在线工具
(11)细胞增殖试验(Cell pliration ssay)
LNCaP、C4-2、22RV1和R1-D567细胞在24孔板中转染siYAP、siTEAD1/3/4、siLATS1/2或scramble sirna。转染12h后计数细胞数，将10000个细胞接种到96孔板中。YAP/TAZ敲除实验中，C4-2细胞在12孔板中转染siYAP/TAZ或scramble siRNA,24h后计数10000个细胞播种到96孔板中.在96孔板中，用1-5μM XMU-MP-1、5μM TRULI或20μM Enzalutamide处理5000-10000个细胞。在指定的时间点测量相对细胞活力。在激素消耗条件下，计数5000个LNCaP细胞，在96孔板中种子培养24h，然后用rmi-1640添加10％脱炭FBS(Gibco,Cat.No.A3382101)治疗前过夜。使用WST-1(Sigma Aldrich,Cat.No.5015944001)测定细胞数。
(12)非锚定生长试验(Anchorage-Independent Growth Assays)
将C4-2或C4-2-tet-o-yap-5sa细胞以5000细胞/mL的密度镀于含0.4％琼脂糖的完整培养基(含或不含2μM XMU-MP-1或0.2μg/mL DOX)，底层由含1％琼脂糖的培养基组成。细胞在4℃下孵育10分钟，然后置于37℃培养箱中。每4天，在培养皿上滴加3滴完全培养基(含或不含2μM XMU-MP-1或0.2μg/mL DOX)。培养细胞2～3周后，吸取培养皿上的额外液体，用生长培养基中100μg/mL氯化碘硝四唑(Sigma,Cat.No.I8377)染色。细胞培养板在分析前孵育一夜。
(13)异种移植瘤模型(Xenograft tumor models)
所有动物实验的程序都经过德克萨斯大学西南医学院IACUC的审查和批准。实验采用6周龄雄性NOD scid gamma(NSG)小鼠，将肿瘤细胞(1×106细胞悬浮于含50％Matrigel的100ul PBS中)皮下植入小鼠背侧。当肿瘤移植瘤达到平均体积约100mm3(长×宽/2)时，将小鼠随机分为实验治疗组(6-8只/组)。XMU-MP-1(5％葡萄糖溶于水)每日腹腔注射，剂量为3～15mg/kg体重，疗程为指示时间，对照组给予溶剂注射。每天腹腔注射PBS中的Dox,20mg/kg体重。使用数字卡尺测量肿瘤大小。在研究结束时，处死小鼠，采集肝脏、脾脏和肿瘤并称重。
2、结果
2.1前列腺癌中AR和YAP信号活性的反向相关性
通过分析TCGA数据，我们发现与52个正常组织相比，497个PCa样本中YAP和TAZ及其靶基因AMOTL2的表达水平更低，而AR靶基因KLK2和KLK3的表达水平更高(图1A)。在AR活性高的PCa样品中，YAP信号传导活性与AR信号传导活性呈负相关，YAP和YAP标志靶基因(包括AMOTL2、CTGF和CYR61)的表达水平表示YAP信号转导活性，而KLK2和KLK3的表达水平反映了这一点(图1B-D)。此外，高水平的YAP/TAZ表达与PCa患者的良好预后相关(图1E-F)。
2.2MST1/2抑制或YAP激活抑制AR+Pca生长
YAP-5SA的表达均抑制4种AR+PCa细胞的生长(图2A-D)。C4-2和22RV1细胞对YAP激活相对更敏感，因为YAP-WT的过表达也抑制了它们的增殖，尽管与YAP-5SA相比效果较差(图2B,C)。小分子化合物XMU-MP-1处理细胞，使Hippo信号失活，该化合物特异性抑制MST1/2激酶活性，也抑制了LNCaP、C4-2、22RV1和R1-D567癌细胞的生长，C4-2对Enzalutamide的敏感性低于LNCaP，而22RV1和R1-D567均对Enzalutamide耐药(图2E-H)。经XMU-MP-1处理的C4-2核YAP水平升高(图2I-J)。敲除YAP后，C4-2细胞的生长略有增加，XMU-MP-1的生长抑制作用得到部分缓解(图2K-L)。这表明XMU-MP-1至少部分通过激活YAP抑制了C4-2的生长。通过敲低LATS1使用小分子LATS1/2激酶抑制剂TRULI，阻断Hippo信号通路，也可以抑制LNCaP、C4-2和22RV1癌细胞的生长(图2M-P，图3A-D)。此外，LATS1/2KD对C4-2细胞生长的抑制被YAP RNAi部分逆转(图2Q-R)。我们还发现XMU-MP-1和YAP5SA抑制了C4-2细胞的独立生长(图3E-F)。因此，通过转基因过表达激活YAP或阻断Hippo信号通路均可抑制AR+PCa的体外生长。
YAP5SA在激素消耗条件下促进LNCaP细胞生长，但在激素消耗培养基中重新加入双氢睾酮(DHT)时抑制LNCaP细胞生长(图2S,T)，这表明YAP抑制LNCaP细胞生长取决于AR信号状态。
2.3YAP调节AR的转录活性
为了进一步确定YAP如何调节AR的转录活性，我们生成了稳定表达诱导型YAP-5SA(Tet-O-YAP-5SA)的C4-2细胞系。DOX诱导YAP5SA10或16小时可抑制AR与靶启动子/增强子的结合和AR靶基因表达，但对AR蛋白水平几乎没有影响(图7I-K)。因此，YAP抑制AR与目标启动子/增强子的结合，这可能随后导致AR退化。为了确定YAP是否通过直接与AR结合来抑制AR，我们进行了CoIP实验。然而，我们未能观察到AR与YAP-5sa或内源性YAP的关联。我们的结论是，YAP通过与其他蛋白质结合来抑制AR。在Hippo典型信号通路中，YAP与通路转录因子TEAD形成复合物，调控Hippo靶基因表达。为了确定YAP是否依赖于与TEAD的结合而抑制AR，我们生成了稳定表达TEAD结合缺陷形式的YAP-5SA(Tet-O-YAP-5SAS94A)的C4-2细胞系。DOX诱导YAP-5SAS94A表达10或16小时，既不抑制AR与靶启动子/增强子的结合，也不影响AR靶基因的表达(图7L-N)。因此，YAP通过与TEAD结合抑制AR的转录活性。
2.4TEAD与AR形成复合物，促进AR活性和AR+PCa生长
TEAD转录因子家族包含四个成员(TEAD1,TEAD2,TEAD3和TEAD4)，它们通常冗余或相加地调节Hippo途径靶基因的表达。为了确定TEAD在AR+PCa中的作用，我们使用两个独立的siRNAs(siTEAD1/3/4_1和siTEAD1/3/4_2)同时敲除TEAD1、TEAD3和TEAD4，它们的目标是这些TEAD家族成员中的共享序列。TEAD1/3/4基因敲低抑制了C4-2、22RV1和R-D567细胞AR靶基因的表达，抑制AR+PCa细胞的生长(图4A-C；图5A-F)。另一方面，TEAD4或TEAD1过表达增加了C4-2细胞AR靶基因的表达，缓解了XMU-MP-1对AR靶基因表达的抑制(图4D；图5G)，说明TEAD正调控AR介导的转录，与YAP相反。CoIP实验表明，在C4-2细胞中，内源性AR与TEAD1和TEAD4相互作用，而与YAP不相互作用；内源性YAP与TEAD1和TEAD4形成复合物，而与AR不相互作用(图4E-F)。内源性TEAD4在C4-2细胞中与AR或YAP形成复合物(图4G)。当这些蛋白在HEK-293T细胞中共表达时，也得到了类似的结果(图4H-J)。在体外结合试验中，重组GST-TEAD4融合蛋白拉低了免疫纯化的Flag-AR而不是GST(图4K)。这些结果表明AR/TEAD和YAP/TEAD形成了不同的蛋白质复合物。缺失映射显示，TEAD的c端半域(YBD:YAP结合域)和AR的n端结构域(NTD)介导了TEAD与AR之间的联系(图4L-N)。22RV1和R1-D567细胞分别表达AR间接变体AR_V7和AR_V567ES，两者的配体结合域(LBD)被删除，但包含完整的NTD(图4L)。与AR通过其NTD与TEAD结合一致，我们发现AR_V7和AR_V567ES在HEK-293T细胞中共同表达时与TEAD4或TEAD1形成复合物(图4O-R)。这些结果表明，TEAD通过与AR的物理相互作用影响AR活性和AR+PCa生长。
2.5YAP/TAZ双重耗竭抑制TEAD表达减少的AR信号传导
先前的研究表明，shRNA耗尽YAP抑制了LnCaP和C4-2细胞中AR靶基因的表达。而我们的研究表明siRNA敲除YAP略微增加了C4-2细胞的AR靶基因表达。我们推测，不同的结果可能与YAP耗竭的程度和/或持续时间有关。用siYAP/TAZ处理18小时的C4-2细胞显示AR靶基因的表达略有增加，而用siYAP/TAZ处理更长时间(36或72小时)的细胞显示AR目标基因的表达减少(图6A-C)。然而，我们也注意到，TEAD蛋白水平以及TEAD1和TEAD4mRNA水平也随着YAP/TAZ的长期耗竭而下调(图6A-C)。此外，YAP/TAZ耗竭细胞中TEAD4的过度表达部分挽救了AR靶基因的表达(图6D)，这表明YAP/TAZ耗竭至少部分由于TEAD表达减少而下调AR靶基因表达。因此，通过维持TEAD表达，YAP/TAZ的基础水平是最佳AR途径活性所必需的。
2.6YAP在体内抑制激素治疗耐药的AR变体
AR的变化包括扩增、点突变和缺乏配体结合域的AR变体(AR-vs)都与抗激素治疗耐药有关。AR-Vs被认为具有组成性活性，并对Enzalutamide等抗AR抑制剂具有耐药性，因为它们失去了c端LBD。最常见的AR-V是AR-V7，它包含外显子1、2、3和一个隐外显子3b，编码后的蛋白质缺少LBD。我们的体外研究表明，抑制MST1/2或激活YAP可以降低CRPC细胞(C4-2)或AR剪接变异表达细胞(22Rv1和R1-D557)的生长，而Enzalutamide不能抑制22Rv1和R1-D557的生长(图2E-H)。为了确定YAP激活是否能在体内抑制这些抗激素治疗耐药PCa细胞的生长，我们建立了含有表达Tet-O-EGFP(对照)、Tet-O-YAP-5SA或Tet-O-YAP-5SAS94A的C4-2细胞和表达Tet-O-YAP-5SA的22RV1细胞的异种移植模型。当肿瘤生长到100mm3后，小鼠分别接受DOX或PBS治疗，持续3周。我们发现，DOX诱导的YAP-5SA在异种移植瘤中几乎完全阻断了C4-2和22RV1肿瘤的生长，而S94A突变则大大降低了YAP抑制C4-2肿瘤生长的能力(图7A-F；图8A-C)，提示YAP能有效抑制AR+前列腺癌在体内的生长，其作用方式取决于其与TEAD的结合。值得注意的是，YAP-5SAS94A在异种移植瘤中仍然轻微抑制C4-2肿瘤生长(图8A-C)，可能是因为S94A突变并没有完全消除YAP-TEAD的相互作用，延长YAP-5SAS94A的表达可能仍然能够抑制AR信号。事实上，对表达YAP-5SAS94a的肿瘤进行3周的检查显示，AR靶基因被抑制，而YAP靶基因被激活，尽管与表达YAP-5SA的肿瘤相比，效果不那么显著(图8D-G)。接下来，我们确定XMU-MP-1对MST1/2的药理抑制是否能抑制体内耐药PCs的生长。携带C4-2细胞的异种移植物分别用两种不同剂量的XMU-MP-1(3mg/kg或6mg/kg/d)或对照剂处理25天。如图7G-I所示，XUM-MP-1显著抑制肿瘤生长，且呈剂量依赖性。我们还发现XMU-MP-1在表达AR变异体V7(22RV1)和V567ES(R1-D567)的异种移植瘤中可以抑制肿瘤生长，尽管其抑制C4-2肿瘤生长的效果不如XMU-MP-1(图7J-O)。与之前XMU-MP-1在小鼠中耐受性良好的研究一致。我们发现XMU-MP-1治疗后小鼠体重和肝脏大小正常，但脾脏在高剂量XMU-MP-1治疗后略有增加(图9)，我们还用XMU-MP1或Enzalutamide同时治疗22RV1荷瘤小鼠，发现Enzalutamide对22RV1肿瘤生长几乎没有影响，而XMU-MP-1可以显著抑制肿瘤生长(图10A-F)。
以上结果表明，TEAD是AR的关键辅助因子，是AR+PCa的主要致癌驱动因素。TEAD和AR的结合促进了AR的转录活性。YAP和AR竞争TEAD，YAP与TEAD的结合破坏了TEAD和AR之间的协同作用，导致AR与目标启动子/增强子分离，随后发生蛋白酶体介导的降解，AR靶基因表达减少，AR+PCa生长受到抑制(图11)。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
Claims (8)
1.YAP蛋白的表达激活剂在制备治疗前列腺癌的药物中的用途，其特征在于，所述前列腺癌为雄激素受体阳性前列腺癌。
2.根据权利要求1所述的用途，其特征在于，所述YAP蛋白表达升高可以抑制雄激素受体的转录活性和雄激素受体阳性前列腺癌的生长。
3.根据权利要求2所述的用途，其特征在于，所述YAP蛋白通过和雄激素受体竞争结合TEAD，抑制雄激素受体靶基因的表达，阻止TEAD促进雄激素受体信号传导发挥作用。
4.根据权利要求1所述的用途，其特征在于，所述YAP蛋白的表达激活剂包括转基因表达盒，所述转基因表达盒包含YAP蛋白和/或其突变体的编码序列，所述突变体包括YAP-5SA和YAP-5SAS94A。
5.根据权利要求1所述的用途，其特征在于，其特征在于，所述YAP蛋白的表达激活剂包括Hippo信号通路抑制剂，所述抑制剂抑制YAP蛋白上游激酶的活性或表达，使YAP蛋白水平升高。
6.根据权利要求5所述的用途，其特征在于，其特征在于，所述YAP蛋白上游激酶包括MST1/2激酶和LATS1/2激酶。
7.根据权利要求6所述的用途，其特征在于，所述抑制剂包括靶向抑制MST1/2激酶和/或LATS1/2激酶的小分子化合物、siRNA或慢病毒。
8.根据权利要求7所述的用途，其特征在于，靶向抑制MST1/2激酶的小分子化合物为XMU-MP-1，靶向抑制LATS1/2激酶的小分子化合物为TRULI。
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